Sample records for yeast maximizes recombinant

  1. Yeast synthetic biology for the production of recombinant therapeutic proteins.

    PubMed

    Kim, Hyunah; Yoo, Su Jin; Kang, Hyun Ah

    2015-02-01

    The production of recombinant therapeutic proteins is one of the fast-growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic post-translational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several nonconventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  2. Monitoring Recombination During Meiosis in Budding Yeast.

    PubMed

    Owens, Shannon; Tang, Shangming; Hunter, Neil

    2018-01-01

    Homologous recombination is fundamental to sexual reproduction, facilitating accurate segregation of homologous chromosomes at the first division of meiosis, and creating novel allele combinations that fuel evolution. Following initiation of meiotic recombination by programmed DNA double-strand breaks (DSBs), homologous pairing and DNA strand exchange form joint molecule (JM) intermediates that are ultimately resolved into crossover and noncrossover repair products. Physical monitoring of the DNA steps of meiotic recombination in Saccharomyces cerevisiae (budding yeast) cultures undergoing synchronous meiosis has provided seminal insights into the molecular basis of meiotic recombination and affords a powerful tool for dissecting the molecular roles of recombination factors. This chapter describes a suit of electrophoretic and Southern hybridization techniques used to detect and quantify the DNA intermediates of meiotic recombination at recombination hotspots in budding yeast. DSBs and recombination products (crossovers and noncrossovers) are resolved using one-dimensional electrophoresis and distinguished by restriction site polymorphisms between the parental chromosomes. Psoralen cross-linking is used to stabilize branched JMs, which are resolved from linear species by native/native two-dimensional electrophoresis. Native/denaturing two-dimensional electrophoresis is employed to determine the component DNA strands of JMs and to measure the processing of DSBs. These techniques are generally applicable to any locus where the frequency of recombination is high enough to detect intermediates by Southern hybridization. © 2018 Elsevier Inc. All rights reserved.

  3. Recombinant protein subunit vaccine synthesis in microbes: a role for yeast?

    PubMed

    Bill, Roslyn M

    2015-03-01

    Recombinant protein subunit vaccines are formulated using protein antigens that have been synthesized in heterologous host cells. Several host cells are available for this purpose, ranging from Escherichia coli to mammalian cell lines. This article highlights the benefits of using yeast as the recombinant host. The yeast species, Saccharomyces cerevisiae and Pichia pastoris, have been used to optimize the functional yields of potential antigens for the development of subunit vaccines against a wide range of diseases caused by bacteria and viruses. Saccharomyces cerevisiae has also been used in the manufacture of 11 approved vaccines against hepatitis B virus and one against human papillomavirus; in both cases, the recombinant protein forms highly immunogenic virus-like particles. Advances in our understanding of how a yeast cell responds to the metabolic load of producing recombinant proteins will allow us to identify host strains that have improved yield properties and enable the synthesis of more challenging antigens that cannot be produced in other systems. Yeasts therefore have the potential to become important host organisms for the production of recombinant antigens that can be used in the manufacture of subunit vaccines or in new vaccine development. © 2014 Royal Pharmaceutical Society.

  4. Comparison of Yeasts as Hosts for Recombinant Protein Production.

    PubMed

    Vieira Gomes, Antonio Milton; Souza Carmo, Talita; Silva Carvalho, Lucas; Mendonça Bahia, Frederico; Parachin, Nádia Skorupa

    2018-04-29

    Recombinant protein production emerged in the early 1980s with the development of genetic engineering tools, which represented a compelling alternative to protein extraction from natural sources. Over the years, a high level of heterologous protein was made possible in a variety of hosts ranging from the bacteria Escherichia coli to mammalian cells. Recombinant protein importance is represented by its market size, which reached $1654 million in 2016 and is expected to reach $2850.5 million by 2022. Among the available hosts, yeasts have been used for producing a great variety of proteins applied to chemicals, fuels, food, and pharmaceuticals, being one of the most used hosts for recombinant production nowadays. Historically, Saccharomyces cerevisiae was the dominant yeast host for heterologous protein production. Lately, other yeasts such as Komagataella sp., Kluyveromyces lactis , and Yarrowia lipolytica have emerged as advantageous hosts. In this review, a comparative analysis is done listing the advantages and disadvantages of using each host regarding the availability of genetic tools, strategies for cultivation in bioreactors, and the main techniques utilized for protein purification. Finally, examples of each host will be discussed regarding the total amount of protein recovered and its bioactivity due to correct folding and glycosylation patterns.

  5. Ends-in Vs. Ends-Out Recombination in Yeast

    PubMed Central

    Hastings, P. J.; McGill, C.; Shafer, B.; Strathern, J. N.

    1993-01-01

    Integration of linearized plasmids into yeast chromosomes has been used as a model system for the study of recombination initiated by double-strand breaks. The linearized plasmid DNA recombines efficiently into sequences homologous to the ends of the DNA. This efficient recombination occurs both for the configuration in which the break is in a contiguous region of homology (herein called the ends-in configuration) and for ``omega'' insertions in which plasmid sequences interrupt a linear region of homology (herein called the ends-out configuration). The requirements for integration of these two configurations are expected to be different. We compared these two processes in a yeast strain containing an ends-in target and an ends-out target for the same cut plasmid. Recovery of ends-in events exceeds ends-out events by two- to threefold. Possible causes for the origin of this small bias are discussed. The lack of an extreme difference in frequency implies that cooperativity between the two ends does not contribute to the efficiency with which cut circular plasmids are integrated. This may also be true for the repair of chromosomal double-strand breaks. PMID:8307337

  6. A Genome-Wide Map of Mitochondrial DNA Recombination in Yeast

    PubMed Central

    Fritsch, Emilie S.; Chabbert, Christophe D.; Klaus, Bernd; Steinmetz, Lars M.

    2014-01-01

    In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome

  7. A genome-wide map of mitochondrial DNA recombination in yeast.

    PubMed

    Fritsch, Emilie S; Chabbert, Christophe D; Klaus, Bernd; Steinmetz, Lars M

    2014-10-01

    In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome

  8. Recombinant yeast with improved ethanol tolerance and related methods of use

    DOEpatents

    Gasch, Audrey P [Madison, WI; Lewis, Jeffrey A [Madison, WI

    2012-05-15

    The present invention provides isolated Elo1 and Mig3 nucleic acid sequences capable of conferring increased ethanol tolerance on recombinant yeast and methods of using same in biofuel production, particularly ethanol production. Methods of bioengineering yeast using the Elo1 and, or, Mig3 nucleic acid sequences are also provided.

  9. Recombination walking: genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination.

    PubMed Central

    Miller, A M; Savinelli, E A; Couture, S M; Hannigan, G M; Han, Z; Selden, R F; Treco, D A

    1993-01-01

    Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unordered) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8367472

  10. Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors.

    PubMed

    Ducrot, Cécile; Lefebvre, Olivier; Landrieux, Emilie; Guirouilh-Barbat, Josée; Sentenac, André; Acker, Joel

    2006-04-28

    Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.

  11. Nonmutagenic carcinogens induce intrachromosomal recombination in dividing yeast cells.

    PubMed

    Schiestl, R H

    1993-12-01

    A large number of animal and human carcinogens without apparent genotoxic activity exist (nonmutagenic carcinogens) that are difficult or impossible to detect with the currently used short-term tests. Because of the association of carcinogenesis with genome rearrangement, a system selecting for intrachromosomal recombination (DEL recombination) that results in genome rearrangement has been constructed in the yeast Saccharomyces cerevisiae. Because DEL recombination is under different genetic control than interchromosomal recombination and meiotic recombination, it is probably due to a different mechanism. It has been found that DEL recombination is readily inducible by 10 mutagenic carcinogens and 17 nonmutagenic carcinogens that are not detectable (false negatives) with the Ames assay. In addition, three out of four mutagens that do not cause cancer (false positives in the Ames assay) do not induce DEL recombination. DEL recombination is inducible by UV only in dividing cells but not in cells synchronized in the G1 or G2 phase of the cell cycle. Interchromosomal recombination, on the other hand, is inducible in G1 but not in G2. The nonmutagenic carcinogens induce DEL recombination only in actively growing cells, which may give some indication as to their mechanism. Further characterization of the mechanism involved in induction of DEL recombination may contribute to the understanding of the biological activity of nonmutagenic carcinogens.

  12. Damage-induced ectopic recombination in the yeast Saccharomyces cerevisiae.

    PubMed

    Kupiec, M; Steinlauf, R

    1997-06-09

    Mitotic recombination in the yeast Saccharomyces cerevisiae is induced when cells are irradiated with UV or X-rays, reflecting the efficient repair of damage by recombinational repair mechanisms. We have used multiply marked haploid strains that allow the simultaneous detection of several types of ectopic recombination events. We show that inter-chromosomal ectopic conversion of lys2 heteroalleles and, to a lesser extent, direct repeat recombination (DRR) between non-tandem repeats, are increased by DNA-damaging agents; in contrast, ectopic recombination of the naturally occurring Ty element is not induced. We have tested several hypotheses that could explain the preferential lack of induction of Ty recombination by DNA-damaging agents. We have found that the lack of induction cannot be explained by a cell cycle control or by an effect of the mating-type genes. We also found no role for the flanking long terminal repeats (LTRs) of the Ty in preventing the induction. Ectopic conversion, DRR, and forward mutation of artificial repeats show different kinetics of induction at various positions of the cell cycle, reflecting different mechanisms of recombination. We discuss the mechanistic and evolutionary aspects of these results.

  13. Mitochondrial genetics in Bakers' yeast: a molecular mechanism for recombinational polarity and suppressiveness.

    PubMed

    Perlman, P S; Birky, C W

    1974-11-01

    Recombinational polarity and suppressiveness are two well-known but puzzling cytoplasmic genetic phenomena in bakers' yeast, Saccharomyces cerevisiae. Little progress has been made in characterizing the underlying molecular mechanisms of these phenomena. In this paper we describe a molecular model for recombinational polarity that is compatible with the available genetic evidence. The model stresses the role of small deletions and excision/repair processes in otherwise canonical recombinational events. According to the model, both phenomena require recombination and may share mechanistic elements.

  14. Effects of Temperature on the Meiotic Recombination Landscape of the Yeast Saccharomyces cerevisiae.

    PubMed

    Zhang, Ke; Wu, Xue-Chang; Zheng, Dao-Qiong; Petes, Thomas D

    2017-12-19

    Although meiosis in warm-blooded organisms takes place in a narrow temperature range, meiosis in many organisms occurs over a wide variety of temperatures. We analyzed the properties of meiosis in the yeast Saccharomyces cerevisiae in cells sporulated at 14°C, 30°C, or 37°C. Using comparative-genomic-hybridization microarrays, we examined the distribution of Spo11-generated meiosis-specific double-stranded DNA breaks throughout the genome. Although there were between 300 and 400 regions of the genome with high levels of recombination (hot spots) observed at each temperature, only about 20% of these hot spots were found to have occurred independently of the temperature. In S. cerevisiae , regions near the telomeres and centromeres tend to have low levels of meiotic recombination. This tendency was observed in cells sporulated at 14°C and 30°C, but not at 37°C. Thus, the temperature of sporulation in yeast affects some global property of chromosome structure relevant to meiotic recombination. Using single-nucleotide polymorphism (SNP)-specific whole-genome microarrays, we also examined crossovers and their associated gene conversion events as well as gene conversion events that were unassociated with crossovers in all four spores of tetrads obtained by sporulation of diploids at 14°C, 30°C, or 37°C. Although tetrads from cells sporulated at 30°C had slightly (20%) more crossovers than those derived from cells sporulated at the other two temperatures, spore viability was good at all three temperatures. Thus, despite temperature-induced variation in the genetic maps, yeast cells produce viable haploid products at a wide variety of sporulation temperatures. IMPORTANCE In the yeast Saccharomyces cerevisiae , recombination is usually studied in cells that undergo meiosis at 25°C or 30°C. In a genome-wide analysis, we showed that the locations of genomic regions with high and low levels of meiotic recombination (hot spots and cold spots, respectively) differed

  15. Break-induced replication and recombinational telomere elongation in yeast.

    PubMed

    McEachern, Michael J; Haber, James E

    2006-01-01

    When a telomere becomes unprotected or if only one end of a chromosomal double-strand break succeeds in recombining with a template sequence, DNA can be repaired by a recombination-dependent DNA replication process termed break-induced replication (BIR). In budding yeasts, there are two BIR pathways, one dependent on the Rad51 recombinase protein and one Rad51 independent; these two repair processes lead to different types of survivors in cells lacking the telomerase enzyme that is required for normal telomere maintenance. Recombination at telomeres is triggered by either excessive telomere shortening or disruptions in the function of telomere-binding proteins. Telomere elongation by BIR appears to often occur through a "roll and spread" mechanism. In this process, a telomeric circle produced by recombination at a dysfunctional telomere acts as a template for a rolling circle BIR event to form an elongated telomere. Additional BIR events can then copy the elongated sequence to all other telomeres.

  16. Rescue of Targeted Regions of Mammalian Chromosomes by in Vivo Recombination in Yeast

    PubMed Central

    Kouprina, Natalya; Kawamoto, Kensaku; Barrett, J. Carl; Larionov, Vladimir; Koi, Minoru

    1998-01-01

    In contrast to other animal cell lines, the chicken pre-B cell lymphoma line, DT40, exhibits a high level of homologous recombination, which can be exploited to generate site-specific alterations in defined target genes or regions. In addition, the ability to generate human/chicken monochromosomal hybrids in the DT40 cell line opens a way for specific targeting of human genes. Here we describe a new strategy for direct isolation of a human chromosomal region that is based on targeting of the chromosome with a vector containing a yeast selectable marker, centromere, and an ARS element. This procedure allows rescue of the targeted region by transfection of total genomic DNA into yeast spheroplasts. Selection for the yeast marker results in isolation of chromosome sequences in the form of large circular yeast artificial chromosomes (YACs) up to 170 kb in size containing the targeted region. These YACs are generated by homologous recombination in yeast between common repeated sequences in the targeted chromosomal fragment. Alternatively, the targeted region can be rescued as a linear YACs when a YAC fragmentation vector is included in the yeast transformation mixture. Because the entire isolation procedure of the chromosomal region, once a target insertion is obtained, can be accomplished in ∼1 week, the new method greatly expands the utility of the homologous recombinationproficient DT40 chicken cell system. PMID:9647640

  17. Synthesis of recombinant human parainfluenza virus 1 and 3 nucleocapsid proteins in yeast Saccharomyces cerevisiae.

    PubMed

    Juozapaitis, Mindaugas; Zvirbliene, Aurelija; Kucinskaite, Indre; Sezaite, Indre; Slibinskas, Rimantas; Coiras, Mayte; de Ory Manchon, Fernando; López-Huertas, María Rosa; Pérez-Breña, Pilar; Staniulis, Juozas; Narkeviciute, Irena; Sasnauskas, Kestutis

    2008-05-01

    Human parainfluenza virus types 1 and 3 (HPIV1 and HPIV3, respectively), members of the virus family Paramyxoviridae, are common causes of lower respiratory tract infections in infants, young children, the immunocompromised, the chronically ill, and the elderly. In order to synthesize recombinant HPIV1 and HPIV3 nucleocapsid proteins, the coding sequences were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of recombinant virus nucleocapsid proteins expression (20-24 mg l(-1) of yeast culture) was obtained. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. These structures contained host RNA, which was resistant to RNase treatment. The nucleocapsid proteins were stable in yeast and were easily purified by caesium chloride gradient ultracentrifugation. Therefore, this system proved to be simple, efficient and cost-effective, suitable for high-level production of parainfluenza virus nucleocapsids as nucleocapsid-like particles. When used as coating antigens in an indirect ELISA, the recombinant N proteins reacted with sera of patients infected with HPIV1 or 3. Serological assays to detect HPIV-specific antibodies could be designed on this basis.

  18. Mitochondrial Recombination Reveals Mito-Mito Epistasis in Yeast.

    PubMed

    Wolters, John F; Charron, Guillaume; Gaspary, Alec; Landry, Christian R; Fiumera, Anthony C; Fiumera, Heather L

    2018-05-01

    Genetic variation in mitochondrial DNA (mtDNA) provides adaptive potential although the underlying genetic architecture of fitness components within mtDNAs is not known. To dissect functional variation within mtDNAs, we first identified naturally occurring mtDNAs that conferred high or low fitness in Saccharomyces cerevisiae by comparing growth in strains containing identical nuclear genotypes but different mtDNAs. During respiratory growth under temperature and oxidative stress conditions, mitotype effects were largely independent of nuclear genotypes even in the presence of mito-nuclear interactions. Recombinant mtDNAs were generated to determine fitness components within high- and low-fitness mtDNAs. Based on phenotypic distributions of isogenic strains containing recombinant mtDNAs, we found that multiple loci contributed to mitotype fitness differences. These mitochondrial loci interacted in epistatic, nonadditive ways in certain environmental conditions. Mito-mito epistasis ( i.e. , nonadditive interactions between mitochondrial loci) influenced fitness in progeny from four different crosses, suggesting that mito-mito epistasis is a widespread phenomenon in yeast and other systems with recombining mtDNAs. Furthermore, we found that interruption of coadapted mito-mito interactions produced recombinant mtDNAs with lower fitness. Our results demonstrate that mito-mito epistasis results in functional variation through mitochondrial recombination in fungi, providing modes for adaptive evolution and the generation of mito-mito incompatibilities. Copyright © 2018 by the Genetics Society of America.

  19. Evaluation of a recombinant yeast cell estrogen screening assay.

    PubMed Central

    Coldham, N G; Dave, M; Sivapathasundaram, S; McDonnell, D P; Connor, C; Sauer, M J

    1997-01-01

    A wide range of chemicals with diverse structures derived from plant and environmental origins are reported to have hormonal activity. The potential for appreciable exposure of humans to such substances prompts the need to develop sensitive screening methods to quantitate and evaluate the risk to the public. Yeast cells transformed with plasmids encoding the human estrogen receptor and an estrogen responsive promoter linked to a reporter gene were evaluated for screening compounds for estrogenic activity. Relative sensitivity to estrogens was evaluated by reference to 17 beta-estradiol (E2) calibration curves derived using the recombinant yeast cells, MCF-7 human breast cancer cells, and a prepubertal mouse uterotrophic bioassay. The recombinant yeast cell bioassay (RCBA) was approximately two and five orders of magnitude more sensitive to E2 than MCF-7 cells and the uterotrophic assay, respectively. The estrogenic potency of 53 chemicals, including steroid hormones, synthetic estrogens, environmental pollutants, and phytoestrogens, was measured using the RCBA. Potency values produced with the RCBA relative to E2 (100) included estrone (9.6), diethylstilbestrol (74.3), tamoxifen (0.0047), alpha-zearalanol (1.3), equol (0.085), 4-nonylphenol (0.005), and butylbenzyl phathalate (0.0004), which were similar to literature values but generally higher than those produced by the uterotrophic assay. Exquisite sensitivity, absence of test compound biotransformation, ease of use, and the possibility of measuring antiestrogenic activity are important attributes that argue for the suitability of the RCBA in screening for potential xenoestrogens to evaluate risk to humans, wildlife, and the environment. Images Figure 1. Figure 2. Figure 3. Figure 4. PMID:9294720

  20. Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast

    PubMed Central

    Silva, J.V.J.; Arenhart, S.; Santos, H.F.; Almeida-Queiroz, S.R.; Silva, A.N.M.R.; Trevisol, I.M.; Bertani, G.R.; Gil, L.H.V.G.

    2014-01-01

    The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world’s first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development. PMID:25763067

  1. Correlation of Meiotic DSB Formation and Transcription Initiation Around Fission Yeast Recombination Hotspots.

    PubMed

    Yamada, Shintaro; Okamura, Mika; Oda, Arisa; Murakami, Hiroshi; Ohta, Kunihiro; Yamada, Takatomi

    2017-06-01

    Meiotic homologous recombination, a critical event for ensuring faithful chromosome segregation and creating genetic diversity, is initiated by programmed DNA double-strand breaks (DSBs) formed at recombination hotspots. Meiotic DSB formation is likely to be influenced by other DNA-templated processes including transcription, but how DSB formation and transcription interact with each other has not been understood well. In this study, we used fission yeast to investigate a possible interplay of these two events. A group of hotspots in fission yeast are associated with sequences similar to the cyclic AMP response element and activated by the ATF/CREB family transcription factor dimer Atf1-Pcr1. We first focused on one of those hotspots, ade6-3049 , and Atf1. Our results showed that multiple transcripts, shorter than the ade6 full-length messenger RNA, emanate from a region surrounding the ade6-3049 hotspot. Interestingly, we found that the previously known recombination-activation region of Atf1 is also a transactivation domain, whose deletion affected DSB formation and short transcript production at ade6-3049 These results point to a possibility that the two events may be related to each other at ade6-3049 In fact, comparison of published maps of meiotic transcripts and hotspots suggested that hotspots are very often located close to meiotically transcribed regions. These observations therefore propose that meiotic DSB formation in fission yeast may be connected to transcription of surrounding regions. Copyright © 2017 by the Genetics Society of America.

  2. Compartmentalization of the yeast meiotic nucleus revealed by analysis of ectopic recombination.

    PubMed

    Schlecht, Hélène B; Lichten, Michael; Goldman, Alastair S H

    2004-11-01

    As yeast cells enter meiosis, chromosomes move from a centromere-clustered (Rabl) to a telomere-clustered (bouquet) configuration and then to states of progressive homolog pairing where telomeres are more dispersed. It is uncertain at which stage of this process sequences commit to recombine with each other. Previous analyses using recombination between dispersed homologous sequences (ectopic recombination) support the view that, on average, homologs are aligned end to end by the time of commitment to recombination. We have undertaken further analyses incorporating new inserts, chromosome rearrangements, an alternate mode of recombination initiation, and mutants that disrupt nuclear structure or telomere metabolism. Our findings support previous conclusions and reveal that distance from the nearest telomere is an important parameter influencing recombination between dispersed sequences. In general, the farther dispersed sequences are from their nearest telomere, the less likely they are to engage in ectopic recombination. Neither the mode of initiating recombination nor the formation of the bouquet appears to affect this relationship. We suggest that aspects of telomere localization and behavior influence the organization and mobility of chromosomes along their entire length, during a critical period of meiosis I prophase that encompasses the homology search.

  3. Comparative Polygenic Analysis of Maximal Ethanol Accumulation Capacity and Tolerance to High Ethanol Levels of Cell Proliferation in Yeast

    PubMed Central

    Pais, Thiago M.; Foulquié-Moreno, María R.; Hubmann, Georg; Duitama, Jorge; Swinnen, Steve; Goovaerts, Annelies; Yang, Yudi; Dumortier, Françoise; Thevelein, Johan M.

    2013-01-01

    The yeast Saccharomyces cerevisiae is able to accumulate ≥17% ethanol (v/v) by fermentation in the absence of cell proliferation. The genetic basis of this unique capacity is unknown. Up to now, all research has focused on tolerance of yeast cell proliferation to high ethanol levels. Comparison of maximal ethanol accumulation capacity and ethanol tolerance of cell proliferation in 68 yeast strains showed a poor correlation, but higher ethanol tolerance of cell proliferation clearly increased the likelihood of superior maximal ethanol accumulation capacity. We have applied pooled-segregant whole-genome sequence analysis to identify the polygenic basis of these two complex traits using segregants from a cross of a haploid derivative of the sake strain CBS1585 and the lab strain BY. From a total of 301 segregants, 22 superior segregants accumulating ≥17% ethanol in small-scale fermentations and 32 superior segregants growing in the presence of 18% ethanol, were separately pooled and sequenced. Plotting SNP variant frequency against chromosomal position revealed eleven and eight Quantitative Trait Loci (QTLs) for the two traits, respectively, and showed that the genetic basis of the two traits is partially different. Fine-mapping and Reciprocal Hemizygosity Analysis identified ADE1, URA3, and KIN3, encoding a protein kinase involved in DNA damage repair, as specific causative genes for maximal ethanol accumulation capacity. These genes, as well as the previously identified MKT1 gene, were not linked in this genetic background to tolerance of cell proliferation to high ethanol levels. The superior KIN3 allele contained two SNPs, which are absent in all yeast strains sequenced up to now. This work provides the first insight in the genetic basis of maximal ethanol accumulation capacity in yeast and reveals for the first time the importance of DNA damage repair in yeast ethanol tolerance. PMID:23754966

  4. The yeast MSH1 gene is not involved in DNA repair or recombination during meiosis.

    PubMed

    Sia, Elaine A; Kirkpatrick, David T

    2005-02-03

    Six strong homologs of the bacterial MutS DNA mismatch repair (MMR) gene have been identified in the yeast Saccharomyces cerevisiae. With the exception of the MSH1 gene, the involvement of each homolog in DNA repair and recombination during meiosis has been determined previously. Five of the homologs have been demonstrated to act in meiotic DNA repair (MSH2, MSH3, MSH6 and MSH4) and/or meiotic recombination (MSH4 and MSH5). Unfortunately the loss of mitochondrial function that results from deletion of MSH1 disrupts meiotic progression, precluding an analysis of MSH1 function in meiotic DNA repair and recombination. However, the recent identification of two separation-of-function alleles of MSH1 that interfere with protein function but still maintain functional mitochondria allow the meiotic activities of MSH1 to be determined. We show that the G776D and F105A alleles of MSH1 exhibit no defects in meiotic recombination, repair base-base mismatches and large loop mismatches efficiently during meiosis, and have high levels of spore viability. These data indicate that the MSH1 protein, unlike other MutS homologs in yeast, plays no role in DNA repair or recombination during meiosis.

  5. Effects of Temperature on the Meiotic Recombination Landscape of the Yeast Saccharomyces cerevisiae

    PubMed Central

    Zhang, Ke; Wu, Xue-Chang

    2017-01-01

    ABSTRACT Although meiosis in warm-blooded organisms takes place in a narrow temperature range, meiosis in many organisms occurs over a wide variety of temperatures. We analyzed the properties of meiosis in the yeast Saccharomyces cerevisiae in cells sporulated at 14°C, 30°C, or 37°C. Using comparative-genomic-hybridization microarrays, we examined the distribution of Spo11-generated meiosis-specific double-stranded DNA breaks throughout the genome. Although there were between 300 and 400 regions of the genome with high levels of recombination (hot spots) observed at each temperature, only about 20% of these hot spots were found to have occurred independently of the temperature. In S. cerevisiae, regions near the telomeres and centromeres tend to have low levels of meiotic recombination. This tendency was observed in cells sporulated at 14°C and 30°C, but not at 37°C. Thus, the temperature of sporulation in yeast affects some global property of chromosome structure relevant to meiotic recombination. Using single-nucleotide polymorphism (SNP)-specific whole-genome microarrays, we also examined crossovers and their associated gene conversion events as well as gene conversion events that were unassociated with crossovers in all four spores of tetrads obtained by sporulation of diploids at 14°C, 30°C, or 37°C. Although tetrads from cells sporulated at 30°C had slightly (20%) more crossovers than those derived from cells sporulated at the other two temperatures, spore viability was good at all three temperatures. Thus, despite temperature-induced variation in the genetic maps, yeast cells produce viable haploid products at a wide variety of sporulation temperatures. PMID:29259092

  6. [Intragenic mitotic recombination induced by ultraviolet and gamma rays in radiosensitive mutants of Saccharomyces cerevisiae yeasts].

    PubMed

    Zakharov, I A; Kasinova, G V; Koval'tsova, S V

    1983-01-01

    The effect of UV- and gamma-irradiation on the survival and intragenic mitotic recombination (gene conversion) of 5 radiosensitive mutants was studied in comparison with the wild type. The level of spontaneous conversion was similar for RAD, rad2 and rad15, mutations xrs2 and xrs4 increasing and rad54 significantly decreasing it. The frequency of conversion induced by UV-light was greater in rad2, rad15 and xrs2 mutants and lower in xrs4, as compared to RAD. Gamma-irradiation caused induction of gene conversion with an equal frequency in RAD, rad2, rad15. Xrs2 and xrs4 mutations slightly decreased gamma-induced conversion. In rad54 mutant, UV-and gamma-induced conversion was practically absent. In the wild type yeast, a diploid strain is more resistant than a haploid, whereas in rad54 a diploid strain has the same or an increased sensitivity, as compared to a haploid strain (the "inverse ploidy effect"). This effect and also the block of induced mitotic recombination caused by rad54 indicate the presence in the yeast Saccharomyces cerevisiae of repair pathways of UV- and gamma-induced damages acting in diploid cells and realised by recombination. The data obtained as a result of many years' investigation of genetic effects in radiosensitive mutants of yeast are summarised and considered.

  7. Targeted mutagenesis of dengue virus type 2 replicon RNA by yeast in vivo recombination.

    PubMed

    Manzano, Mark; Padmanabhan, Radhakrishnan

    2014-01-01

    The use of cDNA infectious clones or subgenomic replicons is indispensable in studying flavivirus biology. Mutating nucleotides or amino acid residues gives important clues to their function in the viral life cycle. However, a major challenge to the establishment of a reverse genetics system for flaviviruses is the instability of their nucleotide sequences in Escherichia coli. Thus, direct cloning using conventional restriction enzyme-based procedures usually leads to unwanted rearrangements of the construct. In this chapter, we discuss a cloning strategy that bypasses traditional cloning procedures. We take advantage of the observations from previous studies that (1) unstable sequences in bacteria can be cloned in eukaryotic systems and (2) Saccharomyces cerevisiae has a well-studied genetics system to introduce sequences using homologous recombination. We describe a protocol to perform targeted mutagenesis in a subgenomic dengue virus 2 replicon. Our method makes use of homologous recombination in yeast using a linearized replicon and a PCR product containing the desired mutation. Constructs derived from this method can be propagated in E. coli with improved stability. Thus, yeast in vivo recombination provides an excellent strategy to genetically engineer flavivirus infectious clones or replicons because this system is compatible with inherently unstable sequences of flaviviruses and is not restricted by the limitations of traditional cloning procedures.

  8. UV-dependent production of 25-hydroxyvitamin D{sub 2} in the recombinant yeast cells expressing human CYP2R1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yasuda, Kaori; Endo, Mariko; Ikushiro, Shinichi

    Highlights: •We produce 25-hydroxyvitamin D in the recombinant yeast expressing human CYP2R1. •Vitamin D2 is produced in yeast from endogenous ergosterol with UV irradiation. •We produce 25-hydroxyvitamin D2 in the recombinant yeast without added substrate. -- Abstract: CYP2R1 is known to be a physiologically important vitamin D 25-hydroxylase. We have successfully expressed human CYP2R1 in Saccharomyces cerevisiae to reveal its enzymatic properties. In this study, we examined production of 25-hydroxylated vitamin D using whole recombinant yeast cells that expressed CYP2R1. When vitamin D{sub 3} or vitamin D{sub 2} was added to the cell suspension of CYP2R1-expressing yeast cells in amore » buffer containing glucose and β-cyclodextrin, the vitamins were converted into their 25-hydroxylated products. Next, we irradiated the cell suspension with UVB and incubated at 37 °C. Surprisingly, the 25-hydroxy vitamin D{sub 2} was produced without additional vitamin D{sub 2}. Endogenous ergosterol was likely converted into vitamin D{sub 2} by UV irradiation and thermal isomerization, and then the resulting vitamin D{sub 2} was converted to 25-hydroxyvitamin D{sub 2} by CYP2R1. This novel method for producing 25-hydroxyvitamin D{sub 2} without a substrate could be useful for practical purposes.« less

  9. The synthesis of recombinant membrane proteins in yeast for structural studies.

    PubMed

    Routledge, Sarah J; Mikaliunaite, Lina; Patel, Anjana; Clare, Michelle; Cartwright, Stephanie P; Bawa, Zharain; Wilks, Martin D B; Low, Floren; Hardy, David; Rothnie, Alice J; Bill, Roslyn M

    2016-02-15

    Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  10. High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography.

    PubMed

    Mizutani, Kimihiko

    2015-01-01

    Homologous recombination is a system for repairing the broken genomes of living organisms by connecting two DNA strands at their homologous sequences. Today, homologous recombination in yeast is used for plasmid construction as a substitute for traditional methods using restriction enzymes and ligases. This method has various advantages over the traditional method, including flexibility in the position of DNA insertion and ease of manipulation. Recently, the author of this review reported the construction of plasmids by homologous recombination in the methanol-utilizing yeast Pichia pastoris, which is known to be an excellent expression host for secretory proteins and membrane proteins. The method enabled high-throughput construction of expression systems of proteins using P. pastoris; the constructed expression systems were used to investigate the expression conditions of membrane proteins and to perform X-ray crystallography of secretory proteins. This review discusses the mechanisms and applications of homologous recombination, including the production of proteins for X-ray crystallography.

  11. Further Improvement of the Robust Recombinant Saccharomyces Yeast for the Conversion of Lignocellulosic Biomass to Ethanol

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ho, Nancy W. Y.; Adamec, Jiri; Mosier, Nathan, S.

    2011-04-09

    Since 1980, the PI’s laboratory at Purdue University has been at the forefront in developing recombinant Saccharomyces yeast for cellulosic ethanol production. Their innovation enabled them to successfully develop the recombinant Saccharomyces yeast strain 424A(LNH-ST) that has been validated by scientists in industry, universities, and National Laboratories. Strain 424A(LNH-ST) has also been used by a company to produce cellulosic ethanol since 2004. Nevertheless, this strain still needs improvement, particularly to achieve high ethanol titer when cellulosic biomass hydrolysates are used for ethanol production. In this project, we were able to carry out a total genetic overhaul of our yeast bymore » carrying out nine different tasks to improve our 424A(LNH-ST) strain. Through these tasks we enabled the yeast to co-ferment arabinose together with other four sugars generally present in all cellulosic biomass. Thus 424A(LNH-ST) can now ferment all five sugars, glucose, xylose, mannose, galactose and arabinose present in any cellulosic biomass. We also successfully used adaptation techniques and direct genetic improvements to develop improved 424A(LNH-ST) strains that are more resistant to acetic acid or ethanol. These are the most significant inhibitors of those commonly present in cellulosic hydrolysates that prevent 424A(LNH-ST) from producing high concentrations of cellulosic ethanol. The acetic acid resistant strain has 89% better xylose utilization in the presence of acetic acid and 25% better overall ethanol yield. The ethanol resistant strain has 250% better ethanol volumetric productivity. The three tasks for improving the main metabolic pathways have all been successfully completed but the impact of these improvements was less dramatic. This demonstrates our yeast already has effective metabolic systems for co-fermenting cellulosic sugars. However, our attempt to improve the yeast to transport xylose and arabinose more efficiently had only limited

  12. Further Improvement of the Robust Recombinant Saccharomyces Yeast for the Conversion of Lignocellulosic Biomass to Ethanol

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ho, Nancy, W. Y.; Adamec, Jiri; Mosier, Nathan, S.

    2011-04-07

    Since 1980, the PI's laboratory at Purdue University has been at the forefront in developing recombinant Saccharomyces yeast for cellulosic ethanol production. Their innovation enabled them to successfully develop the recombinant Saccharomyces yeast strain 424A(LNH-ST) that has been validated by scientists in industry, universities, and National Laboratories. Strain 424A(LNH-ST) has also been used by a company to produce cellulosic ethanol since 2004. Nevertheless, this strain still needs improvement, particularly to achieve high ethanol titer when cellulosic biomass hydrolysates are used for ethanol production. In this project, we were able to carry out a total genetic overhaul of our yeast bymore » carrying out nine different tasks to improve our 424A(LNH-ST) strain. Through these tasks we enabled the yeast to co-ferment arabinose together with other four sugars generally present in all cellulosic biomass. Thus 424A(LNH-ST) can now ferment all five sugars, glucose, xylose, mannose, galactose and arabinose present in any cellulosic biomass. We also successfully used adaptation techniques and direct genetic improvements to develop improved 424A(LNH-ST) strains that are more resistant to acetic acid or ethanol. These are the most significant inhibitors of those commonly present in cellulosic hydrolysates that prevent 424A(LNH-ST) from producing high concentrations of cellulosic ethanol. The acetic acid resistant strain has 89% better xylose utilization in the presence of acetic acid and 25% better overall ethanol yield. The ethanol resistant strain has 250% better ethanol volumetric productivity. The three tasks for improving the main metabolic pathways have all been successfully completed but the impact of these improvements was less dramatic. This demonstrates our yeast already has effective metabolic systems for co-fermenting cellulosic sugars. However, our attempt to improve the yeast to transport xylose and arabinose more efficiently had only limited success

  13. Combining yeast display and competitive FACS to select rare hapten-specific clones from recombinant antibody libraries

    DOE PAGES

    Sun, Yue; Ban, Bhupal; Bradbury, Andrew; ...

    2016-08-29

    The development of antibodies to low molecular weight haptens remains challenging due to both the low immunogenicity of many haptens and the cross-reactivity of the protein carriers used to generate the immune response. Recombinant antibodies and novel display technologies have greatly advanced antibody development; however, new techniques are still required to select rare hapten-specific antibodies from large recombinant libraries. In the present study, we used a combination of phage and yeast display to screen an immune antibody library (size, 4.4 × 10 6 ) against hapten markers for petroleum contamination (phenanthrene and methylphenanthrenes). Selection via phage display was used firstmore » to enrich the library between 20- and 100- fold for clones that bound to phenanthrene-protein conjugates. The enriched libraries were subsequently transferred to a yeast display system and a newly developed competitive FACS procedure was employed to select rare hapten-specific clones. Competitive FACS increased the frequency of hapten-specific scFvs in our yeast-displayed scFvs from 0.025 to 0.005% in the original library to between 13 and 35% in selected pools. The presence of hapten-specific scFvs was confirmed by competitive ELISA using periplasmic protein. Three distinct antibody clones that recognize phenanthrene and methylphenanthrenes were selected, and their distinctive binding properties were characterized. To our knowledge, these are first antibodies that can distinguish between methylated (petrogenic) versus unmethylated (pyrogenic) phenanthrenes; such antibodies will be useful in detecting the sources of environmental contamination. Furthermore, this selection method could be generally adopted in the selection of other hapten-specific recombinant antibodies.« less

  14. Combining yeast display and competitive FACS to select rare hapten-specific clones from recombinant antibody libraries

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sun, Yue; Ban, Bhupal; Bradbury, Andrew

    The development of antibodies to low molecular weight haptens remains challenging due to both the low immunogenicity of many haptens and the cross-reactivity of the protein carriers used to generate the immune response. Recombinant antibodies and novel display technologies have greatly advanced antibody development; however, new techniques are still required to select rare hapten-specific antibodies from large recombinant libraries. In the present study, we used a combination of phage and yeast display to screen an immune antibody library (size, 4.4 × 10 6 ) against hapten markers for petroleum contamination (phenanthrene and methylphenanthrenes). Selection via phage display was used firstmore » to enrich the library between 20- and 100- fold for clones that bound to phenanthrene-protein conjugates. The enriched libraries were subsequently transferred to a yeast display system and a newly developed competitive FACS procedure was employed to select rare hapten-specific clones. Competitive FACS increased the frequency of hapten-specific scFvs in our yeast-displayed scFvs from 0.025 to 0.005% in the original library to between 13 and 35% in selected pools. The presence of hapten-specific scFvs was confirmed by competitive ELISA using periplasmic protein. Three distinct antibody clones that recognize phenanthrene and methylphenanthrenes were selected, and their distinctive binding properties were characterized. To our knowledge, these are first antibodies that can distinguish between methylated (petrogenic) versus unmethylated (pyrogenic) phenanthrenes; such antibodies will be useful in detecting the sources of environmental contamination. Furthermore, this selection method could be generally adopted in the selection of other hapten-specific recombinant antibodies.« less

  15. Meiotic Recombination Initiated by a Double-Strand Break in Rad50Δ Yeast Cells Otherwise Unable to Initiate Meiotic Recombination

    PubMed Central

    Malkova, A.; Ross, L.; Dawson, D.; Hoekstra, M. F.; Haber, J. E.

    1996-01-01

    Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand breaks (DSBs). We have developed a system to compare the properties of meiotic DSBs with those created by the site-specific HO endonuclease. HO endonuclease was expressed under the control of the meiotic-specific SPO13 promoter, creating a DSB at a single site on one of yeast's 16 chromosomes. In Rad(+) strains the times of appearance of the HO-induced DSBs and of subsequent recombinants are coincident with those induced by normal meiotic DSBs. Physical monitoring of DNA showed that SPO13::HO induced gene conversions both in Rad(+) and in rad50Δ cells that cannot initiate normal meiotic DSBs. We find that the RAD50 gene is important, but not essential, for recombination even after a DSB has been created in a meiotic cell. In rad50Δ cells, some DSBs are not repaired until a broken chromosome has been packaged into a spore and is subsequently germinated. This suggests that a broken chromosome does not signal an arrest of progression through meiosis. The recombination defect in rad50Δ diploids is not, however, meiotic specific, as mitotic rad50 diploids, experiencing an HO-induced DSB, exhibit similar departures from wild-type recombination. PMID:8725223

  16. Vital Roles of the Second DNA-binding Site of Rad52 Protein in Yeast Homologous Recombination*

    PubMed Central

    Arai, Naoto; Kagawa, Wataru; Saito, Kengo; Shingu, Yoshinori; Mikawa, Tsutomu; Kurumizaka, Hitoshi; Shibata, Takehiko

    2011-01-01

    RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a “recombination mediator” to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing. PMID:21454474

  17. Immunological targeting of tumor cells undergoing an epithelial-mesenchymal transition via a recombinant brachyury-yeast vaccine

    PubMed Central

    Jales, Alessandra; Huang, Bruce; Fernando, Romaine I.; Hodge, James W.; Ardiani, Andressa; Apelian, David

    2013-01-01

    The embryonic T-box transcription factor brachyury is aberrantly expressed in a range of human tumors. Previous studies have demonstrated that brachyury is a driver of the epithelial-mesenchymal transition (EMT), a process associated with cancer progression. Brachyury expression in human tumor cells enhances tumor invasiveness in vitro and metastasis in vivo, and induces resistance to various conventional therapeutics including chemotherapy and radiation. These characteristics, and the selective expression of brachyury for a range of human tumor types vs. normal adult tissues, make brachyury an attractive tumor target. Due to its intracellular localization and the “undruggable” character of transcription factors, available options to target brachyury are currently limited. Here we report on the development and characterization of an immunological platform for the efficient targeting of brachyury-positive tumors consisting of a heat-killed, recombinant Saccharomyces cerevisiae (yeast)–brachyury vector-based vaccine (designated as GI-6301) that expresses the full-length human brachyury protein. We demonstrate that human dendritic cells treated with recombinant yeast-brachyury can activate and expand brachyury-specific CD4+ and CD8+ T cells in vitro that, in turn, can effectively lyse human tumor cells expressing the brachyury protein. Vaccination of mice with recombinant yeast-brachyury is also shown here to elicit brachyury-specific CD4+ and CD8+ T-cell responses, and to induce anti-tumor immunity in the absence of toxicity. Based on these results, a Phase I clinical trial of GI-6301 is currently ongoing in patients with advanced tumors; to our knowledge, this is the first vaccine platform aimed at targeting a driver of tumor EMT that has successfully reached the clinical stage. PMID:24125763

  18. Conditional genomic rearrangement by designed meiotic recombination using VDE (PI-SceI) in yeast.

    PubMed

    Fukuda, Tomoyuki; Ohya, Yoshikazu; Ohta, Kunihiro

    2007-10-01

    Meiotic recombination plays critical roles in the acquisition of genetic diversity and has been utilized for conventional breeding of livestock and crops. The frequency of meiotic recombination is normally low, and is extremely low in regions called "recombination cold domains". Here, we describe a new and highly efficient method to modulate yeast meiotic gene rearrangements using VDE (PI-SceI), an intein-encoded endonuclease that causes an efficient unidirectional meiotic gene conversion at its recognition sequence (VRS). We designed universal targeting vectors, by use of which the strain that inserts the VRS at a desired site is acquired. Meiotic induction of the strains provided unidirectional gene conversions and frequent genetic rearrangements of flanking genes with little impact on cell viability. This system thus opens the way for the designed modulation of meiotic gene rearrangements, regardless of recombinational activity of chromosomal domains. Finally, the VDE-VRS system enabled us to conduct meiosis-specific conditional knockout of genes where VDE-initiated gene conversion disrupts the target gene during meiosis, serving as a novel approach to examine the functions of genes during germination of resultant spores.

  19. [Distiller Yeasts Producing Antibacterial Peptides].

    PubMed

    Klyachko, E V; Morozkina, E V; Zaitchik, B Ts; Benevolensky, S V

    2015-01-01

    A new method of controlling lactic acid bacteria contamination was developed with the use of recombinant Saccharomyces cerevisiae strains producing antibacterial peptides. Genes encoding the antibacterial peptides pediocin and plantaricin with codons preferable for S. cerevisiae were synthesized, and a system was constructed for their secretory expression. Recombinant S. cerevisiae strains producing antibacterial peptides effectively inhibit the growth of Lactobacillus sakei, Pediacoccus pentasaceus, Pediacoccus acidilactici, etc. The application of distiller yeasts producing antibacterial peptides enhances the ethanol yield in cases of bacterial contamination. Recombinant yeasts producing the antibacterial peptides pediocin and plantaricin can successfully substitute the available industrial yeast strains upon ethanol production.

  20. A yeast-based genetic screening to identify human proteins that increase homologous recombination.

    PubMed

    Collavoli, Anita; Comelli, Laura; Rainaldi, Giuseppe; Galli, Alvaro

    2008-05-01

    To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.

  1. [The Engineering of a Yarrowia lipolytica Yeast Strain Capable of Homologous Recombination of the Mitochondrial Genome].

    PubMed

    Isakova, E P; Epova, E Yu; Sekova, V Yu; Trubnikova, E V; Kudykina, Yu K; Zylkova, M V; Guseva, M A; Deryabina, Yu I

    2015-01-01

    None of the studied eukaryotic species has a natural system for homologous recombination of the mitochondrial genome. We propose an integrated genetic construct pQ-SRUS, which allows introduction of the recA gene from Bacillus subtilis into the nuclear genome of an extremophilic yeast, Yarrowia lipolytica. The targeting of recombinant RecA to the yeast mitochondria is provided by leader sequences (5'-UTR and 3'-UTR) derived from the SOD2 gene mRNA, which exhibits affinity to the outer mitochondrial membrane and thus provides cotranslational transport of RecA to the inner space of the mitochondria. The Y. lipolytica strain bearing the pQ-SRUS construct has the unique ability to integrate DNA constructs into the mitochondrial genome. This fact was confirmed using a tester construct, pQ-NIHN, intended for the introduction of the EYFP gene into the translation initiation region of the Y. lipolytica ND1 mitochondrial gene. The Y. lipolytica strain bearing pQ-SRUS makes it possible to engineer recombinant producers based on Y. lipolytica bearing transgenes in the mitochondrial genome. They are promising for the construction of a genetic system for in vivo replication and modification of the human mitochondrial genome. These strains may be used as a tool for the treatment of human mitochondrial diseases (including genetically inherited ones).

  2. To Nick or Not to Nick: Comparison of I-SceI Single- and Double-Strand Break-Induced Recombination in Yeast and Human Cells

    PubMed Central

    Katz, Samantha S.; Gimble, Frederick S.; Storici, Francesca

    2014-01-01

    Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, or chromosomal rearrangements. By contrast, generation of a DNA single-strand break (SSB), or nick, can stimulate gene correction without the problems of DSB repair because the uncut DNA strand acts as a template to permit healing without alteration of genetic material. Here, we examine the ability of a nicking variant of the I-SceI endonuclease (K223I I-SceI) to stimulate gene targeting in yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK-293) cells. K223I I-SceI is proficient in both yeast and human cells and promotes gene correction up to 12-fold. We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick. We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells. These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy. PMID:24558436

  3. Acetylated Histone H3K9 is associated with meiotic recombination hotspots, and plays a role in recombination redundantly with other factors including the H3K4 methylase Set1 in fission yeast

    PubMed Central

    Yamada, Shintaro; Ohta, Kunihiro; Yamada, Takatomi

    2013-01-01

    Histone modifications are associated with meiotic recombination hotspots, discrete sites with augmented recombination frequency. For example, trimethylation of histone H3 lysine4 (H3K4me3) marks most hotspots in budding yeast and mouse. Modified histones are known to regulate meiotic recombination partly by promoting DNA double-strand break (DSB) formation at hotspots, but the role and precise landscape of involved modifications remain unclear. Here, we studied hotspot-associated modifications in fission yeast and found general features: acetylation of H3 lysine9 (H3K9ac) is elevated, and H3K4me3 is not significantly enriched. Mutating H3K9 to non-acetylatable alanine mildly reduced levels of the DSB-inducing protein Rec12 (the fission yeast homologue of Spo11) and DSB at hotspots, indicating that H3K9ac may be involved in DSB formation by enhancing the interaction between Rec12 and hotspots. In addition, we found that the lack of the H3K4 methyltransferase Set1 generally increased Rec12 binding to chromatin but partially reduced DSB formation at some loci, suggesting that Set1 is also involved in DSB formation. These results suggest that meiotic DSB formation is redundantly regulated by multiple chromatin-related factors including H3K9ac and Set1 in fission yeast. PMID:23382177

  4. A Cytoplasmic Dynein Heavy Chain Is Required for Oscillatory Nuclear Movement of Meiotic Prophase and Efficient Meiotic Recombination in Fission Yeast

    PubMed Central

    Yamamoto, Ayumu; West, Robert R.; McIntosh, J. Richard; Hiraoka, Yasushi

    1999-01-01

    Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination. PMID:10366596

  5. Small-angle neutron scattering study of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particle

    NASA Astrophysics Data System (ADS)

    Sato, M.; Ito, Y.; Kameyama, K.; Imai, M.; Ishikawa, N.; Takagi, T.

    1995-02-01

    The overall and internal structure of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particles was investigated by small-angle neutron scattering using the contrast variation method. The vaccine is a nearly spherical particle, and its contrast-matching point was determined to be at about 24% D 2O content, indicating that a large part of the vaccine particle is occupied by lipids and carbohydrates from the yeast. The Stuhrmann plot suggests that the surface antigens exist predominantly in the peripheral region of the particle, which is favorable to the induction of anti-virus antibodies.

  6. Distinct Domestication Trajectories in Top-Fermenting Beer Yeasts and Wine Yeasts.

    PubMed

    Gonçalves, Margarida; Pontes, Ana; Almeida, Pedro; Barbosa, Raquel; Serra, Marta; Libkind, Diego; Hutzler, Mathias; Gonçalves, Paula; Sampaio, José Paulo

    2016-10-24

    Beer is one of the oldest alcoholic beverages and is produced by the fermentation of sugars derived from starches present in cereal grains. Contrary to lager beers, made by bottom-fermenting strains of Saccharomyces pastorianus, a hybrid yeast, ale beers are closer to the ancient beer type and are fermented by S. cerevisiae, a top-fermenting yeast. Here, we use population genomics to investigate (1) the closest relatives of top-fermenting beer yeasts; (2) whether top-fermenting yeasts represent an independent domestication event separate from those already described; (3) whether single or multiple beer yeast domestication events can be inferred; and (4) whether top-fermenting yeasts represent non-recombinant or recombinant lineages. Our results revealed that top-fermenting beer yeasts are polyphyletic, with a main clade composed of at least three subgroups, dominantly represented by the German, British, and wheat beer strains. Other beer strains were phylogenetically close to sake, wine, or bread yeasts. We detected genetic signatures of beer yeast domestication by investigating genes previously linked to brewing and using genome-wide scans. We propose that the emergence of the main clade of beer yeasts is related with a domestication event distinct from the previously known cases of wine and sake yeast domestication. The nucleotide diversity of the main beer clade more than doubled that of wine yeasts, which might be a consequence of fundamental differences in the modes of beer and wine yeast domestication. The higher diversity of beer strains could be due to the more intense and different selection regimes associated to brewing. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Direct Cloning of Yeast Genes from an Ordered Set of Lambda Clones in Saccharomyces Cerevisiae by Recombination in Vivo

    PubMed Central

    Erickson, J. R.; Johnston, M.

    1993-01-01

    We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Esherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura(+) transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning. PMID:8514124

  8. Effects of cryoprotectants on the viability and activity of freeze dried recombinant yeasts as novel oral drug delivery systems assessed by an artificial digestive system.

    PubMed

    Blanquet, Stéphanie; Garrait, Ghislain; Beyssac, Erick; Perrier, Céline; Denis, Sylvain; Hébrard, Géraldine; Alric, Monique

    2005-09-01

    The aim of this study was to investigate, in a gastric-small intestinal system TIM-1, the effect of cryoprotectants on the survival of freeze-dried Saccharomyces cerevisiae expressing the heterologous P450 73A1 and their ability to convert trans-cinnamic acid into p-coumaric acid. Yeasts were lyophilized in suspensions of trehalose, maltose, lactose, or a milk proteins/trehalose mix. Freeze-dried or native yeasts and trans-cinnamic acid were introduced simultaneously into TIM-1 at the beginning of digestion. Yeast survival rate was evaluated by cell counting in the ileal effluents. P450 73A1 activity was followed by HPLC assay of p-coumaric acid. Freeze-dried yeasts showed high tolerance to digestive conditions. Nevertheless, their survival rate was lower than that of non-dried cells (around 80% whatever the protective agent vs. 96%). The ability of recombinant freeze-dried S. cerevisiae to perform a bioconversion reaction in the digestive tract was shown with all the protectants. The highest trans-cinnamic acid conversion rate (24 vs. 41% for native yeasts) was obtained with the milk proteins/trehalose mix. These results show that freeze-drying might be considered for the pharmaceutical formulation of new drug delivery systems based on orally administered recombinant yeasts and that TIM-1 could be a helpful tool for the pre-screening of oral dosage forms.

  9. Plasticity of laccase generated by homeologous recombination in yeast.

    PubMed

    Cusano, Angela M; Mekmouche, Yasmina; Meglecz, Emese; Tron, Thierry

    2009-10-01

    Laccase-encoding sequences sharing 65-71% identity were shuffledin vivo by homeologous recombination. Yeast efficiently repaired linearized plasmids containing clac1, clac2 or clac5 Trametes sp. C30 cDNAs using a clac3 PCR fragment. From transformants secreting active variants, three chimeric laccases (LAC131, LAC232 and LAC535), each resulting from double crossovers, were purified, and their apparent kinetic parameters were determined using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and syringaldazine (SGZ) as substrates. At acidic pH, the apparent kinetic parameters of the chimera were not distinguishable from each other or from those obtained for the LAC3 enzyme used as reference. On the other hand, the pH tolerance of the variants was visibly extended towards alkaline pH values. Compared to the parental LAC3, a 31-fold increase in apparent k(cat) was observed for LAC131 at pH 8. This factor is one of the highest ever observed for laccase in a single mutagenesis step.

  10. Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity.

    PubMed

    Chen, Mei-ling; Guo, Qin; Wang, Rui-zhi; Xu, Juan; Zhou, Chen-wei; Ruan, Hui; He, Guo-qing

    2011-07-01

    Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

  11. Methods and materials for the production of L-lactic acid in yeast

    DOEpatents

    Hause, Ben [Jordan, MN; Rajgarhia, Vineet [Minnetonka, MN; Suominen, Pirkko [Maple Grove, MN

    2009-05-19

    Recombinant yeast are provided having, in one aspect, multiple exogenous LDH genes integrated into the genome, while leaving native PDC genes intact. In a second aspect, recombinant yeast are provided having an exogenous LDH gene integrated into its genome at the locus of a native PDC gene, with deletion of the native PDC gene. The recombinant yeast are useful in fermentation process for producing lactic acid.

  12. Extensive Horizontal Transfer and Homologous Recombination Generate Highly Chimeric Mitochondrial Genomes in Yeast.

    PubMed

    Wu, Baojun; Buljic, Adnan; Hao, Weilong

    2015-10-01

    The frequency of horizontal gene transfer (HGT) in mitochondrial DNA varies substantially. In plants, HGT is relatively common, whereas in animals it appears to be quite rare. It is of considerable importance to understand mitochondrial HGT across the major groups of eukaryotes at a genome-wide level, but so far this has been well studied only in plants. In this study, we generated ten new mitochondrial genome sequences and analyzed 40 mitochondrial genomes from the Saccharomycetaceae to assess the magnitude and nature of mitochondrial HGT in yeasts. We provide evidence for extensive, homologous-recombination-mediated, mitochondrial-to-mitochondrial HGT occurring throughout yeast mitochondrial genomes, leading to genomes that are highly chimeric evolutionarily. This HGT has led to substantial intraspecific polymorphism in both sequence content and sequence divergence, which to our knowledge has not been previously documented in any mitochondrial genome. The unexpectedly high frequency of mitochondrial HGT in yeast may be driven by frequent mitochondrial fusion, relatively low mitochondrial substitution rates and pseudohyphal fusion to produce heterokaryons. These findings suggest that mitochondrial HGT may play an important role in genome evolution of a much broader spectrum of eukaryotes than previously appreciated and that there is a critical need to systematically study the frequency, extent, and importance of mitochondrial HGT across eukaryotes. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. History of genome editing in yeast.

    PubMed

    Fraczek, Marcin G; Naseeb, Samina; Delneri, Daniela

    2018-05-01

    For thousands of years humans have used the budding yeast Saccharomyces cerevisiae for the production of bread and alcohol; however, in the last 30-40 years our understanding of the yeast biology has dramatically increased, enabling us to modify its genome. Although S. cerevisiae has been the main focus of many research groups, other non-conventional yeasts have also been studied and exploited for biotechnological purposes. Our experiments and knowledge have evolved from recombination to high-throughput PCR-based transformations to highly accurate CRISPR methods in order to alter yeast traits for either research or industrial purposes. Since the release of the genome sequence of S. cerevisiae in 1996, the precise and targeted genome editing has increased significantly. In this 'Budding topic' we discuss the significant developments of genome editing in yeast, mainly focusing on Cre-loxP mediated recombination, delitto perfetto and CRISPR/Cas. © 2018 The Authors. Yeast published by John Wiley & Sons, Ltd.

  14. Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

    PubMed Central

    Rodríguez-López, Alexander; Alméciga-Díaz, Carlos J.; Sánchez, Jhonnathan; Moreno, Jefferson; Beltran, Laura; Díaz, Dennis; Pardo, Andrea; Ramírez, Aura María; Espejo-Mojica, Angela J.; Pimentel, Luisa; Barrera, Luis A.

    2016-01-01

    Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A. PMID:27378276

  15. Mus81 and Yen1 promote reciprocal exchange during mitotic recombination to maintain genome integrity in budding yeast

    PubMed Central

    Ho, Chu Kwen; Mazón, Gerard; Lam, Alicia F.; Symington, Lorraine S.

    2010-01-01

    Holliday junction (HJ) resolution is required for segregation of chromosomes and for formation of crossovers during homologous recombination. The identity of the resolvase(s) that functions in vivo has yet to be established, although several proteins able to cut HJs in vitro have been identified as candidates in yeasts and mammals. Using an assay to detect unselected products of mitotic recombination we found a significant decrease in crossovers in the Saccharomyces cerevisiae mus81Δ mutant. Yen1 serves a back-up function responsible for resolving intermediates in mus81Δ mutants, or when conversion tracts are short. In the absence of both Mus81 and Yen1 intermediates are not channeled exclusively to non-crossover recombinants, but instead are processed by Pol32-dependent break-induced replication (BIR). The channeling of recombination from reciprocal exchange to BIR results in greatly increased spontaneous loss of heterozygosity (LOH) and chromosome mis-segregation in the mus81Δ yen1Δ mutant, typical of the genomic instability found in tumor cells. PMID:21172663

  16. Finding trans-regulatory genes and protein complexes modulating meiotic recombination hotspots of human, mouse and yeast.

    PubMed

    Wu, Min; Kwoh, Chee-Keong; Li, Xiaoli; Zheng, Jie

    2014-09-11

    The regulatory mechanism of recombination is one of the most fundamental problems in genomics, with wide applications in genome wide association studies (GWAS), birth-defect diseases, molecular evolution, cancer research, etc. Recombination events cluster into short genomic regions called "recombination hotspots". Recently, a zinc finger protein PRDM9 was reported to regulate recombination hotspots in human and mouse genomes. In addition, a 13-mer motif contained in the binding sites of PRDM9 is found to be enriched in human hotspots. However, this 13-mer motif only covers a fraction of hotspots, indicating that PRDM9 is not the only regulator of recombination hotspots. Therefore, the challenge of discovering other regulators of recombination hotspots becomes significant. Furthermore, recombination is a complex process. Hence, multiple proteins acting as machinery, rather than individual proteins, are more likely to carry out this process in a precise and stable manner. Therefore, the extension of the prediction of individual trans-regulators to protein complexes is also highly desired. In this paper, we introduce a pipeline to identify genes and protein complexes associated with recombination hotspots. First, we prioritize proteins associated with hotspots based on their preference of binding to hotspots and coldspots. Second, using the above identified genes as seeds, we apply the Random Walk with Restart algorithm (RWR) to propagate their influences to other proteins in protein-protein interaction (PPI) networks. Hence, many proteins without DNA-binding information will also be assigned a score to implicate their roles in recombination hotspots. Third, we construct sub-PPI networks induced by top genes ranked by RWR for various species (e.g., yeast, human and mouse) and detect protein complexes in those sub-PPI networks. The GO term analysis show that our prioritizing methods and the RWR algorithm are capable of identifying novel genes associated with

  17. Co-fermentation using Recombinant Saccharomyces cerevisiae Yeast Strains Hyper-secreting Different Cellulases for the Production of Cellulosic Bioethanol.

    PubMed

    Lee, Cho-Ryong; Sung, Bong Hyun; Lim, Kwang-Mook; Kim, Mi-Jin; Sohn, Min Jeong; Bae, Jung-Hoon; Sohn, Jung-Hoon

    2017-06-30

    To realize the economical production of ethanol and other bio-based chemicals from lignocellulosic biomass by consolidated bioprocessing (CBP), various cellulases from different sources were tested to improve the level of cellulase secretion in the yeast Saccharomyces cerevisiae by screening an optimal translational fusion partner (TFP) as both a secretion signal and fusion partner. Among them, four indispensable cellulases for cellulose hydrolysis, including Chaetomium thermophilum cellobiohydrolase (CtCBH1), Chrysosporium lucknowense cellobiohydrolase (ClCBH2), Trichoderma reesei endoglucanase (TrEGL2), and Saccharomycopsis fibuligera β-glucosidase (SfBGL1), were identified to be highly secreted in active form in yeast. Despite variability in the enzyme levels produced, each recombinant yeast could secrete approximately 0.6-2.0 g/L of cellulases into the fermentation broth. The synergistic effect of the mixed culture of the four strains expressing the essential cellulases with the insoluble substrate Avicel and several types of cellulosic biomass was demonstrated to be effective. Co-fermentation of these yeast strains produced approximately 14 g/L ethanol from the pre-treated rice straw containing 35 g/L glucan with 3-fold higher productivity than that of wild type yeast using a reduced amount of commercial cellulases. This process will contribute to the cost-effective production of bioenergy such as bioethanol and biochemicals from cellulosic biomass.

  18. Fission yeast Tup1-like repressors repress chromatin remodeling at the fbp1+ promoter and the ade6-M26 recombination hotspot.

    PubMed Central

    Hirota, Kouji; Hoffman, Charles S; Shibata, Takehiko; Ohta, Kunihiro

    2003-01-01

    Chromatin remodeling plays crucial roles in the regulation of gene expression and recombination. Transcription of the fission yeast fbp1(+) gene and recombination at the meiotic recombination hotspot ade6-M26 (M26) are both regulated by cAMP responsive element (CRE)-like sequences and the CREB/ATF-type transcription factor Atf1*Pcr1. The Tup11 and Tup12 proteins, the fission yeast counterparts of the Saccharomyces cerevisiae Tup1 corepressor, are involved in glucose repression of the fbp1(+) transcription. We have analyzed roles of the Tup1-like corepressors in chromatin regulation around the fbp1(+) promoter and the M26 hotspot. We found that the chromatin structure around two regulatory elements for fbp1(+) was remodeled under derepressed conditions in concert with the robust activation of fbp1(+) transcription. Strains with tup11delta tup12delta double deletions grown in repressed conditions exhibited the chromatin state associated with wild-type cells grown in derepressed conditions. Interestingly, deletion of rst2(+), encoding a transcription factor controlled by the cAMP-dependent kinase, alleviated the tup11delta tup12delta defects in chromatin regulation but not in transcription repression. The chromatin at the M26 site in mitotic cultures of a tup11delta tup12delta mutant resembled that of wild-type meiotic cells. These observations suggest that these fission yeast Tup1-like corepressors repress chromatin remodeling at CRE-related sequences and that Rst2 antagonizes this function. PMID:14573465

  19. The high mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates in vivo.

    PubMed

    MacAlpine, D M; Perlman, P S; Butow, R A

    1998-06-09

    Abf2p is a high mobility group (HMG) protein found in yeast mitochondria that is required for the maintenance of wild-type (rho+) mtDNA in cells grown on fermentable carbon sources, and for efficient recombination of mtDNA markers in crosses. Here, we show by two-dimensional gel electrophoresis that Abf2p promotes or stabilizes Holliday recombination junction intermediates in rho+ mtDNA in vivo but does not influence the high levels of recombination intermediates readily detected in the mtDNA of petite mutants (rho-). mtDNA recombination junctions are not observed in rho+ mtDNA of wild-type cells but are elevated to detectable levels in cells with a null allele of the MGT1 gene (Deltamgt1), which codes for a mitochondrial cruciform-cutting endonuclease. The level of recombination intermediates in rho+ mtDNA of Deltamgt1 cells is decreased about 10-fold if those cells contain a null allele of the ABF2 gene. Overproduction of Abf2p by >/= 10-fold in wild-type rho+ cells, which leads to mtDNA instability, results in a dramatic increase in mtDNA recombination intermediates. Specific mutations in the two Abf2p HMG boxes required for DNA binding diminishes these responses. We conclude that Abf2p functions in the recombination of rho+ mtDNA.

  20. Mapping the yeast host cell response to recombinant membrane protein production: relieving the biological bottlenecks.

    PubMed

    Ashe, Mark P; Bill, Roslyn M

    2011-06-01

    Understanding the structures and functions of membrane proteins is an active area of research within bioscience. Membrane proteins are key players in essential cellular processes such as the uptake of nutrients, the export of waste products, and the way in which cells communicate with their environment. It is therefore not surprising that membrane proteins are targeted by over half of all prescription drugs. Since most membrane proteins are not abundant in their native membranes, it is necessary to produce them in recombinant host cells to enable further structural and functional studies. Unfortunately, achieving the required yields of functional recombinant membrane proteins is still a bottleneck in contemporary bioscience. This has highlighted the need for defined and rational optimization strategies based upon experimental observation rather than relying on trial and error. We have published a transcriptome and subsequent genetic analysis that has identified genes implicated in high-yielding yeast cells. These results have highlighted a role for alterations to a cell's protein synthetic capacity in the production of high yields of recombinant membrane protein: paradoxically, reduced protein synthesis favors higher yields. These results highlight a potential bottleneck at the protein folding or translocation stage of protein production. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Recombination Is Responsible for the Increased Recovery of Drug-Resistant Mutants with Hypermutated Genomes in Resting Yeast Diploids Expressing APOBEC Deaminases

    PubMed Central

    Lada, Artem G.; Stepchenkova, Elena I.; Zhuk, Anna S.; Kliver, Sergei F.; Rogozin, Igor B.; Polev, Dmitrii E.; Dhar, Alok; Pavlov, Youri I.

    2017-01-01

    DNA editing deaminases (APOBECs) are implicated in generation of mutations in somatic cells during tumorigenesis. APOBEC-dependent mutagenesis is thought to occur during transient exposure of unprotected single-stranded DNA. Mutations frequently occur in clusters (kataegis). We investigated mechanisms of mutant generation in growing and resting diploid yeast expressing APOBEC from sea lamprey, PmCDA1, whose kataegistic effect was previously shown to be associated with transcription. We have found that the frequency of canavanine-resistant mutants kept raising after growth cessation, while the profile of transcription remained unchanged. Surprisingly, the overall number of mutations in the genomes did not elevate in resting cells. Thus, mutations were accumulated during vigorous growth stage with both intense replication and transcription. We found that the elevated recovery of can1 mutant clones in non-growing cells is the result of loss of heterozygosity (LOH) leading to clusters of homozygous mutations in the chromosomal regions distal to the reporter gene. We confirmed that recombination frequency in resting cells was elevated by orders of magnitude, suggesting that cells were transiently committed to meiotic levels of recombination, a process referred to in yeast genetics as return-to-growth. In its extreme, on day 6 of starvation, a few mutant clones were haploid, likely resulting from completed meiosis. Distribution of mutations along chromosomes indicated that PmCDA1 was active during ongoing recombination events and sometimes produced characteristic kataegis near initial breakpoints. AID and APOBEC1 behaved similar to PmCDA1. We conclude that replication, transcription, and mitotic recombination contribute to the recovered APOBEC-induced mutations in resting diploids. The mechanism is relevant to the initial stages of oncogenic transformation in terminally differentiated cells, when recombination may lead to the LOH exposing recessive mutations induced by

  2. Recombination Is Responsible for the Increased Recovery of Drug-Resistant Mutants with Hypermutated Genomes in Resting Yeast Diploids Expressing APOBEC Deaminases.

    PubMed

    Lada, Artem G; Stepchenkova, Elena I; Zhuk, Anna S; Kliver, Sergei F; Rogozin, Igor B; Polev, Dmitrii E; Dhar, Alok; Pavlov, Youri I

    2017-01-01

    DNA editing deaminases (APOBECs) are implicated in generation of mutations in somatic cells during tumorigenesis. APOBEC-dependent mutagenesis is thought to occur during transient exposure of unprotected single-stranded DNA. Mutations frequently occur in clusters ( kataegis ). We investigated mechanisms of mutant generation in growing and resting diploid yeast expressing APOBEC from sea lamprey, PmCDA1, whose kataegistic effect was previously shown to be associated with transcription. We have found that the frequency of canavanine-resistant mutants kept raising after growth cessation, while the profile of transcription remained unchanged. Surprisingly, the overall number of mutations in the genomes did not elevate in resting cells. Thus, mutations were accumulated during vigorous growth stage with both intense replication and transcription. We found that the elevated recovery of can1 mutant clones in non-growing cells is the result of loss of heterozygosity (LOH) leading to clusters of homozygous mutations in the chromosomal regions distal to the reporter gene. We confirmed that recombination frequency in resting cells was elevated by orders of magnitude, suggesting that cells were transiently committed to meiotic levels of recombination, a process referred to in yeast genetics as return-to-growth. In its extreme, on day 6 of starvation, a few mutant clones were haploid, likely resulting from completed meiosis. Distribution of mutations along chromosomes indicated that PmCDA1 was active during ongoing recombination events and sometimes produced characteristic kataegis near initial breakpoints. AID and APOBEC1 behaved similar to PmCDA1. We conclude that replication, transcription, and mitotic recombination contribute to the recovered APOBEC-induced mutations in resting diploids. The mechanism is relevant to the initial stages of oncogenic transformation in terminally differentiated cells, when recombination may lead to the LOH exposing recessive mutations induced by

  3. Ultra-wide band electromagnetic radiation does not affect UV-induced recombination and mutagenesis in yeast.

    PubMed

    Pakhomova, O N; Belt, M L; Mathur, S P; Lee, J C; Akyel, Y

    1998-01-01

    Cell samples of the yeast Saccharomyces cerevisiae were exposed to 100 J/m2 of 254 nm ultraviolet (UV) radiation followed by a 30 min treatment with ultra-wide band (UWB) electromagnetic pulses. The UWB pulses (101-104 kV/m, 1.0 ns width, 165 ps rise time) were applied at the repetition rates of 0 Hz (sham), 16 Hz, or 600 Hz. The effect of exposures was evaluated from the colony-forming ability of the cells on complete and selective media and the number of aberrant colonies. The experiments established no effect of UWB exposure on the UV-induced reciprocal and non-reciprocal recombination, mutagenesis, or cell survival.

  4. Divergence, hybridization, and recombination in the mitochondrial genome of the human pathogenic yeast Cryptococcus gattii.

    PubMed

    Xu, Jianping; Yan, Zhun; Guo, Hong

    2009-06-01

    The inheritance of mitochondrial genes and genomes are uniparental in most sexual eukaryotes. This pattern of inheritance makes mitochondrial genomes in natural populations effectively clonal. Here, we examined the mitochondrial population genetics of the emerging human pathogenic fungus Cryptococcus gattii. The DNA sequences for five mitochondrial DNA fragments were obtained from each of 50 isolates belonging to two evolutionary divergent lineages, VGI and VGII. Our analyses revealed a greater sequence diversity within VGI than that within VGII, consistent with observations of the nuclear genes. The combined analyses of all five gene fragments indicated significant divergence between VGI and VGII. However, the five individual genealogies showed different relationships among the isolates, consistent with recent hybridization and mitochondrial gene transfer between the two lineages. Population genetic analyses of the multilocus data identified evidence for predominantly clonal mitochondrial population structures within both lineages. Interestingly, there were clear signatures of recombination among mitochondrial genes within the VGII lineage. Our analyses suggest historical mitochondrial genome divergence within C. gattii, but there is evidence for recent hybridization and recombination in the mitochondrial genome of this important human yeast pathogen.

  5. mlh3 mutations in baker’s yeast alter meiotic recombination outcomes by increasing noncrossover events genome-wide

    PubMed Central

    Al-Sweel, Najla; Raghavan, Vandana; Khondakar, Nabila; Manhart, Carol M.; Surtees, Jennifer A.

    2017-01-01

    Mlh1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how Mlh1-Mlh3 functions in both meiosis and MMR, we analyzed in baker’s yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each class were purified and characterized. Both Mlh1-mlh3-32 (MMR+, crossover-) and Mlh1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with Mlh1-Mlh3 in MMR, stimulated the endonuclease activity of Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for wild-type and MMR+ crossover-, MMR- crossover+, endonuclease defective and null mlh3 mutants in an S288c/YJM789 hybrid background. Compared to wild-type, all of the mlh3 mutants showed increases in the number of noncrossover events, consistent with recombination intermediates being resolved through alternative recombination pathways. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating Mlh1-Mlh3’s enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated. PMID:28827832

  6. Evaluation of a recombinant insect-derived amylase performance in simultaneous saccharification and fermentation process with industrial yeasts.

    PubMed

    Celińska, Ewelina; Borkowska, Monika; Białas, Wojciech

    2016-03-01

    Starch is the dominant feedstock consumed for the bioethanol production, accounting for 60 % of its global production. Considering the significant contribution of bioethanol to the global fuel market, any improvement in its major operating technologies is economically very attractive. It was estimated that up to 40 % of the final ethanol unit price is derived from the energy input required for the substrate pre-treatment. Application of raw starch hydrolyzing enzymes (RSHE), combined with operation of the process according to a simultaneous saccharification and fermentation (SSF) strategy, constitutes the most promising solutions to the current technologies limitations. In this study, we expressed the novel RSHE derived from an insect in Saccharomyces cerevisiae strain dedicated for the protein overexpression. Afterwards, the enzyme performance was assessed in SSF process conducted by industrial ethanologenic or thermotolerant yeast species. Comparison of the insect-derived RSHE preparation with commercially available amylolytic RSH preparation was conducted. Our results demonstrate that the recombinant alpha-amylase from rice weevil can be efficiently expressed and secreted with its native signal peptide in S. cerevisiae INVSc-pYES2-Amy1 expression system (accounting for nearly 72 % of the strain's secretome). Application of the recombinant enzyme-based preparation in SSF process secured sufficient amylolytic activity for the yeast cell propagation and ethanol formation from raw starch. (Oligo)saccharide profiles generated by the compared preparations differed with respect to homogeneity of the sugar mixtures. Concomitantly, as demonstrated by a kinetic model developed in this study, the kinetic parameters describing activity of the compared preparations were different.

  7. Construction of a laccase chimerical gene: recombinant protein characterization and gene expression via yeast surface display.

    PubMed

    Bleve, G; Lezzi, C; Spagnolo, S; Rampino, P; Perrotta, C; Mita, G; Grieco, Francesco

    2014-03-01

    The ERY4 laccase gene from Pleurotus eryngii was expressed in Saccharomyces cerevisiae and the recombinant laccase resulted to be not biologically active. This gene was thus modified to obtain chimerical enzymes derived from the substitution of N-, C- and both N- and C-terminal regions with the corresponding regions of Ery3 laccase, another laccase isoform of P. eryngii. The chimerical isoform named 4NC3, derived from the substitution of both N- and C-terminal regions, showed the best performances in terms of enzymatic activities, affinities for different substrates and stability at a broad range of temperatures and pHs. The chimerical 4NC3 laccase isoform was displayed on the cell surface of S. cerevisiae using the N-terminal fusion with either the Pir2 or the Flo1 S. cerevisiae proteins as anchor attachment sequence. Immunofluorescence microscopy and Western blot analyses confirmed the localization of 4NC3 on the yeast cell surface. The enzyme activity on specific laccase substrates revealed that 4NC3 laccase was immobilized in active form on the cell surface. To our knowledge, this is the first example of expression of a chimerical fungal laccase by yeast cell display.

  8. The construction of recombinant industrial yeasts free of bacterial sequences by directed gene replacement into a nonessential region of the genome.

    PubMed

    Xiao, W; Rank, G H

    1989-03-15

    The yeast SMR1 gene was used as a dominant resistance-selectable marker for industrial yeast transformation and for targeting integration of an economically important gene at the homologous ILV2 locus. A MEL1 gene, which codes for alpha-galactosidase, was inserted into a dispensable upstream region of SMR1 in vitro; different treatments of the plasmid (pWX813) prior to transformation resulted in 3' end, 5' end and replacement integrations that exhibited distinct integrant structures. One-step replacement within a nonessential region of the host genome generated a stable integration of MEL1 devoid of bacterial plasmid DNA. Using this method, we have constructed several alpha-galactosidase positive industrial Saccharomyces strains. Our study provides a general method for stable gene transfer in most industrial Saccharomyces yeasts, including those used in the baking, brewing (ale and lager), distilling, wine and sake industries, with solely nucleotide sequences of interest. The absence of bacterial DNA in the integrant structure facilitates the commercial application of recombinant DNA technology in the food and beverage industry.

  9. Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha

    PubMed Central

    2012-01-01

    Background Currently, the two most commonly used fibrinolytic agents in thrombolytic therapy are recombinant tissue plasminogen activator (rt-PA) and streptokinase (SK). Whereas SK has the advantage of substantially lower costs when compared to other agents, it is less effective than either rt-PA or related variants, has significant allergenic potential, lacks fibrin selectivity and causes transient hypotensive effects in high dosing schedules. Therefore, development of an alternative fibrinolytic agent having superior efficacy to SK, approaching that of rt-PA, together with a similar or enhanced safety profile and advantageous cost-benefit ratio, would be of substantial importance. Pre-clinical data suggest that the novel fibrinolytic recombinant staphylokinase (rSAK), or related rSAK variants, could be candidates for such development. However, since an efficient expression system for rSAK is still lacking, it has not yet been fully developed or evaluated for clinical purposes. This study’s goal was development of an efficient fermentation process for the production of a modified, non-glycosylated, biologically active rSAK, namely rSAK-2, using the well-established single cell yeast Hansenula polymorpha expression system. Results The development of an efficient large scale (80 L) Hansenula polymorpha fermentation process of short duration for rSAK-2 production is described. It evolved from an initial 1mL HTP methodology by successive scale-up over almost 5 orders of magnitude and improvement steps, including the optimization of critical process parameters (e.g. temperature, pH, feeding strategy, medium composition, etc.). Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif. Expression at high yields (≥ 1g rSAK-2/L cell culture broth) of biologically active rSAK-2 of expected molecular weight was achieved. Conclusion The optimized production process described for rSAK-2 in

  10. Industrial brewing yeast engineered for the production of primary flavor determinants in hopped beer.

    PubMed

    Denby, Charles M; Li, Rachel A; Vu, Van T; Costello, Zak; Lin, Weiyin; Chan, Leanne Jade G; Williams, Joseph; Donaldson, Bryan; Bamforth, Charles W; Petzold, Christopher J; Scheller, Henrik V; Martin, Hector Garcia; Keasling, Jay D

    2018-03-20

    Flowers of the hop plant provide both bitterness and "hoppy" flavor to beer. Hops are, however, both a water and energy intensive crop and vary considerably in essential oil content, making it challenging to achieve a consistent hoppy taste in beer. Here, we report that brewer's yeast can be engineered to biosynthesize aromatic monoterpene molecules that impart hoppy flavor to beer by incorporating recombinant DNA derived from yeast, mint, and basil. Whereas metabolic engineering of biosynthetic pathways is commonly enlisted to maximize product titers, tuning expression of pathway enzymes to affect target production levels of multiple commercially important metabolites without major collateral metabolic changes represents a unique challenge. By applying state-of-the-art engineering techniques and a framework to guide iterative improvement, strains are generated with target performance characteristics. Beers produced using these strains are perceived as hoppier than traditionally hopped beers by a sensory panel in a double-blind tasting.

  11. Solving the influence maximization problem reveals regulatory organization of the yeast cell cycle.

    PubMed

    Gibbs, David L; Shmulevich, Ilya

    2017-06-01

    The Influence Maximization Problem (IMP) aims to discover the set of nodes with the greatest influence on network dynamics. The problem has previously been applied in epidemiology and social network analysis. Here, we demonstrate the application to cell cycle regulatory network analysis for Saccharomyces cerevisiae. Fundamentally, gene regulation is linked to the flow of information. Therefore, our implementation of the IMP was framed as an information theoretic problem using network diffusion. Utilizing more than 26,000 regulatory edges from YeastMine, gene expression dynamics were encoded as edge weights using time lagged transfer entropy, a method for quantifying information transfer between variables. By picking a set of source nodes, a diffusion process covers a portion of the network. The size of the network cover relates to the influence of the source nodes. The set of nodes that maximizes influence is the solution to the IMP. By solving the IMP over different numbers of source nodes, an influence ranking on genes was produced. The influence ranking was compared to other metrics of network centrality. Although the top genes from each centrality ranking contained well-known cell cycle regulators, there was little agreement and no clear winner. However, it was found that influential genes tend to directly regulate or sit upstream of genes ranked by other centrality measures. The influential nodes act as critical sources of information flow, potentially having a large impact on the state of the network. Biological events that affect influential nodes and thereby affect information flow could have a strong effect on network dynamics, potentially leading to disease. Code and data can be found at: https://github.com/gibbsdavidl/miergolf.

  12. Functional Heterologous Protein Expression by Genetically Engineered Probiotic Yeast Saccharomyces boulardii

    PubMed Central

    Hudson, Lauren E.; Fasken, Milo B.; McDermott, Courtney D.; McBride, Shonna M.; Kuiper, Emily G.; Guiliano, David B.; Corbett, Anita H.; Lamb, Tracey J.

    2014-01-01

    Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders. PMID:25391025

  13. Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.

    PubMed

    Hudson, Lauren E; Fasken, Milo B; McDermott, Courtney D; McBride, Shonna M; Kuiper, Emily G; Guiliano, David B; Corbett, Anita H; Lamb, Tracey J

    2014-01-01

    Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.

  14. Recombinant yeast as a functional tool for understanding bitterness and cucurbitacin biosynthesis in watermelon (Citrullus spp.).

    PubMed

    Davidovich-Rikanati, Rachel; Shalev, Lior; Baranes, Nadine; Meir, Ayala; Itkin, Maxim; Cohen, Shahar; Zimbler, Kobi; Portnoy, Vitaly; Ebizuka, Yutaka; Shibuya, Masaaki; Burger, Yosef; Katzir, Nurit; Schaffer, Arthur A; Lewinsohn, Efraim; Tadmor, Ya'akov

    2015-01-01

    Cucurbitacins are a group of bitter-tasting oxygenated tetracyclic triterpenes that are produced in the family Cucurbitaceae and other plant families. The natural roles of cucurbitacins in plants are probably related to defence against pathogens and pests. Cucurbitadienol, a triterpene synthesized from oxidosqualene, is the first committed precursor to cucurbitacins produced by a specialized oxidosqualene cyclase termed cucurbitadienol synthase. We explored cucurbitacin accumulation in watermelon in relation to bitterness. Our findings show that cucurbitacins are accumulated in bitter-tasting watermelon, Citrullus lanatus var. citroides, as well as in their wild ancestor, C. colocynthis, but not in non-bitter commercial cultivars of sweet watermelon (C. lanatus var. lanatus). Molecular analysis of genes expressed in the roots of several watermelon accessions led to the isolation of three sequences (CcCDS1, CcCDS2 and ClCDS1), all displaying high similarity to the pumpkin CpCPQ, encoding a protein previously shown to possess cucurbitadienol synthase activity. We utilized the Saccharomyces cerevisiae strain BY4743, heterozygous for lanosterol synthase, to probe for possible encoded cucurbitadienol synthase activity of the expressed watermelon sequences. Functional expression of the two sequences isolated from C. colocynthis (CcCDS1 and CcCDS2) in yeast revealed that only CcCDS2 possessed cucurbitadienol synthase activity, while CcCDS1 did not display cucurbitadienol synthase activity in recombinant yeast. ClCDS1 isolated from C. lanatus var. lanatus is almost identical to CcCDS1. Our results imply that CcCDS2 plays a role in imparting bitterness to watermelon. Yeast has been an excellent diagnostic tool to determine the first committed step of cucurbitacin biosynthesis in watermelon. Copyright © 2014 John Wiley & Sons, Ltd.

  15. Cloning and expression of Saccharomyces cerevisiae SUC2 gene in yeast platform and characterization of recombinant enzyme biochemical properties.

    PubMed

    Mohandesi, Nooshin; Siadat, Seyed Omid Ranaei; Haghbeen, Kamahldin; Hesampour, Ardeshir

    2016-12-01

    Invertase (EC.3.2.1.26) catalyzes the hydrolysis of sucrose to an equimolar mixture of D-glucose and D-fructose which is of interest for various industrial applications. In this research, Saccharomyces cerevisiae invertase gene (SUC2) was optimized based on Pichia pastoris codon preference. The synthetic gene was introduced into the methylotrophic yeast Pichia pastoris under the control of the inducible AOX1 promoter. High level of the extracellular recombinant invertase (R-inv) production was achieved via methanol induction for 4 days and purified by His-Tag affinity chromatography which appeared to be a mixture of glycosylated proteins with various sizes of 85-95 kDa on SDS-PAGE. Deglycosylation of the proteins by Endo-H resulted in the proteins with average molecular weight of 60 kDa. The purified recombinant invertase biochemical properties and kinetic parameters determined a pH and temperature optimum at 4.8 and 60 °C, respectively, which in comparison with native S. cerevisiae invertase, thermal stability of recombinant invertase is highly increased in different heating treatment experiments. The purification of recombinant invertase resulted in an enzyme with specific activity of 178.56 U/mg with 3.83-fold of purification and the kinetic constants for enzyme were Km value of 19 mM and Vmax value of 300 μmol min -1  mg -1 With kinetic efficiency (Kcat/Km) of 13.15 s -1  mmol -1 it can be concluded that recombinant P. pastoris invertase can be more effective for industrial quality criteria. We conclude that recombinant P. pastoris enzyme with broad pH stability, substrate specificity and proper thermal stability can fulfil a series of predefined industrial quality criteria to be used in food, pharmaceutical and bio ethanol production industries.

  16. Antigenic characterisation of yeast-expressed lyssavirus nucleoproteins.

    PubMed

    Kucinskaite, Indre; Juozapaitis, Mindaugas; Serva, Andrius; Zvirbliene, Aurelija; Johnson, Nicholas; Staniulis, Juozas; Fooks, Anthony R; Müller, Thomas; Sasnauskas, Kestutis; Ulrich, Rainer G

    2007-12-01

    In Europe, three genotypes of the genus Lyssavirus, family Rhabdoviridae, are present, classical rabies virus (RABV, genotype 1), European bat lyssavirus type 1 (EBLV-1, genotype 5) and European bat lyssavirus type 2 (EBLV-2, genotype 6). The entire authentic nucleoprotein (N protein) encoding sequences of RABV (challenge virus standard, CVS, strain), EBLV-1 and EBLV-2 were expressed in yeast Saccharomyces cerevisiae at high level. Purification of recombinant N proteins by caesium chloride gradient centrifugation resulted in yields between 14-17, 25-29 and 18-20 mg/l of induced yeast culture for RABV-CVS, EBLV-1 and EBLV-2, respectively. The purified N proteins were evaluated by negative staining electron microscopy, which revealed the formation of nucleocapsid-like structures. The antigenic conformation of the N proteins was investigated for their reactivity with monoclonal antibodies (mAbs) directed against different lyssaviruses. The reactivity pattern of each mAb was virtually identical between immunofluorescence assay with virus-infected cells, and ELISA and dot blot assay using the corresponding recombinant N proteins. These observations lead us to conclude that yeast-expressed lyssavirus N proteins share antigenic properties with naturally expressed virus protein. These recombinant proteins have the potential for use as components of serological assays for lyssaviruses.

  17. Production and characterization of recombinant protein preparations of Endonuclease G-homologs from yeast, C. elegans and humans.

    PubMed

    Kieper, Jana; Lauber, Christiane; Gimadutdinow, Oleg; Urbańska, Anna; Cymerman, Iwona; Ghosh, Mahua; Szczesny, Bartosz; Meiss, Gregor

    2010-09-01

    Nuc1p, CPS-6, EndoG and EXOG are evolutionary conserved mitochondrial nucleases from yeast, Caenorhabditis elegans and humans, respectively. These enzymes play an important role in programmed cell death as well as mitochondrial DNA-repair and recombination. Whereas a significant interest has been given to the cell biology of these proteins, in particular their recruitment during caspase-independent apoptosis, determination of their biochemical properties has lagged behind. In part, biochemical as well as structural analysis of mitochondrial nucleases has been hampered by the fact that upon cloning and overexpression in Escherichia coli these enzymes can exert considerable toxicity and tend to aggregate and form inclusion bodies. We have, therefore, established a uniform E. coli expression system allowing us to obtain these four evolutionary related nucleases in active form from the soluble as well as insoluble fractions of E. coli cell lysates. Using preparations of recombinant Nuc1p, CPS-6, EndoG and EXOG we have compared biochemical properties and the substrate specificities of these related nucleases on selected substrates in parallel. Whereas Nuc1p and EXOG in addition to their endonuclease activity exert 5'-3'-exonuclease activity, CPS-6 and EndoG predominantly are endonucleases. These findings allow speculating that the mechanisms of action of these related nucleases in cell death as well as DNA-repair and recombination differ according to their enzyme activities and substrate specificities. Copyright 2010 Elsevier Inc. All rights reserved.

  18. Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria*

    PubMed Central

    Gerhold, Joachim M.; Sedman, Tiina; Visacka, Katarina; Slezakova, Judita; Tomaska, Lubomir; Nosek, Jozef; Sedman, Juhan

    2014-01-01

    Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations. PMID:24951592

  19. Induction of homologous recombination in Saccharomyces cerevisiae.

    PubMed

    Simon, J R; Moore, P D

    1988-09-01

    We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, or the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.

  20. Genetic interactions between the chromosome axis-associated protein Hop1 and homologous recombination determinants in Schizosaccharomyces pombe.

    PubMed

    Brown, Simon David; Jarosinska, Olga Dorota; Lorenz, Alexander

    2018-03-17

    Hop1 is a component of the meiosis-specific chromosome axis and belongs to the evolutionarily conserved family of HORMA domain proteins. Hop1 and its orthologs in higher eukaryotes are a major factor in promoting double-strand DNA break formation and inter-homolog recombination. In budding yeast and mammals, they are also involved in a meiotic checkpoint kinase cascade monitoring the completion of double-strand DNA break repair. We used the fission yeast, Schizosaccharomyces pombe, which lacks a canonical synaptonemal complex to test whether Hop1 has a role beyond supporting the generation of double-strand DNA breaks and facilitating inter-homolog recombination events. We determined how mutants of homologous recombination factors genetically interact with hop1, studied the role(s) of the HORMA domain of Hop1, and characterized a bio-informatically predicted interactor of Hop1, Aho1 (SPAC688.03c). Our observations indicate that in fission yeast, Hop1 does require its HORMA domain to support wild-type levels of meiotic recombination and localization to meiotic chromatin. Furthermore, we show that hop1∆ only weakly interacts genetically with mutants of homologous recombination factors, and in fission yeast likely has no major role beyond break formation and promoting inter-homolog events. We speculate that after the evolutionary loss of the synaptonemal complex, Hop1 likely has become less important for modulating recombination outcome during meiosis in fission yeast, and that this led to a concurrent rewiring of genetic pathways controlling meiotic recombination.

  1. Annealing Vs. Invasion in Phage λ Recombination

    PubMed Central

    Stahl, M. M.; Thomason, L.; Poteete, A. R.; Tarkowski, T.; Kuzminov, A.; Stahl, F. W.

    1997-01-01

    Genetic recombination catalyzed by λ's Red pathway was studied in rec(+) and recA mutant bacteria by examining both intracellular λ DNA and mature progeny particles. Recombination of nonreplicating phage chromosomes was induced by double-strand breaks delivered at unique sites in vivo. In rec(+) cells, cutting only one chromosome gave nearly maximal stimulation of recombination; the recombinants formed contained relatively short hybrid regions, suggesting strand invasion. In contrast, in recA mutant cells, cutting the two parental chromosomes at non-allelic sites was required for maximal stimulation; the recombinants formed tended to be hybrid over the entire region between the two cuts, implying strand annealing. We conclude that, in the absence of RecA and the presence of non-allelic DNA ends, the Red pathway of λ catalyzes recombination primarily by annealing. PMID:9383045

  2. Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis.

    PubMed

    Jo, Hyun-Joo; Noh, Jin-Seok; Kong, Kwang-Hoon

    2013-08-01

    Brazzein is an intensely sweet-tasting protein with high water solubility, heat stability, and taste properties resembling those of carbohydrate sweeteners. In the present study, we describe the expression of the synthetic gene encoding brazzein, a sweet protein in the yeast Kluyveromyces lactis. The synthetic brazzein gene was designed based on the biased codons of the yeast, so as to optimize its expression, as well as on the extracellular secretion for expression in an active, soluble form. The synthesized brazzein gene was cloned into the secretion vector pKLAC2, which contains the yeast prepropeptide signal from the Saccharomycescerevisiae α-mating factor. The constructed plasmid pKLAC2-des-pE1M-brazzein was introduced into the yeast K. lactis GG799. The yeast transformants were cultured for high-yield secretion of the recombinant des-pE1M-brazzein in YPGal medium for 96 h at 30°C. The expressed recombinant des-pE1M-brazzein was purified by CM-Sepharose column chromatography and approximately 104 mg/L was obtained. The purity and conformational state of the recombinant des-pE1M-brazzein were confirmed using SDS-PAGE, HPLC, and circular dichroism. The identity of the recombinant protein was also confirmed by N-terminal amino acid analysis and taste testing. The purified recombinant des-pE1M-brazzein had an intrinsic sweetness in its minor form, approximately 2130 times sweeter than sucrose on a weight basis. These results demonstrate that the K. lactis expression system is useful for producing the recombinant brazzein in active form at a high yield with attributes useful in the food industry. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Functional conservation of the yeast and Arabidopsis RAD54-like genes.

    PubMed

    Klutstein, Michael; Shaked, Hezi; Sherman, Amir; Avivi-Ragolsky, Naomi; Shema, Efrat; Zenvirth, Drora; Levy, Avraham A; Simchen, Giora

    2008-04-01

    The Saccharomyces cerevisiae RAD54 gene has critical roles in DNA double-strand break repair, homologous recombination, and gene targeting. Previous results show that the yeast gene enhances gene targeting when expressed in Arabidopsis thaliana. In this work we address the trans-species compatibility of Rad54 functions. We show that overexpression of yeast RAD54 in Arabidopsis enhances DNA damage resistance severalfold. Thus, the yeast gene is active in the Arabidopsis homologous-recombination repair system. Moreover, we have identified an A. thaliana ortholog of yeast RAD54, named AtRAD54. This gene, with close sequence similarity to RAD54, complements methylmethane sulfonate (MMS) sensitivity but not UV sensitivity or gene targeting defects of rad54Delta mutant yeast cells. Overexpression of AtRAD54 in Arabidopsis leads to enhanced resistance to DNA damage. This gene's assignment as a RAD54 ortholog is further supported by the interaction of AtRad54 with AtRad51 and the interactions between alien proteins (i.e., yeast Rad54 with AtRAD51 and yeast Rad51 with AtRad54) in a yeast two-hybrid experiment. These interactions hint at the molecular nature of this interkingdom complementation, although the stronger effect of the yeast Rad54 in plants than AtRad54 in yeast might be explained by an ability of the Rad54 protein to act alone, independently of its interaction with Rad51.

  4. Construction of a lactose-assimilating strain of baker's yeast.

    PubMed

    Adam, A C; Prieto, J A; Rubio-Texeira, M; Polaina, J

    1999-09-30

    A recombinant strain of baker's yeast has been constructed which can assimilate lactose efficiently. This strain has been designed to allow its propagation in whey, the byproduct resulting from cheese-making. The ability to metabolize lactose is conferred by the functional expression of two genes from Kluyveromyces lactis, LAC12 and LAC4, which encode a lactose permease and a beta-galactosidase, respectively. To make the recombinant strain more acceptable for its use in bread-making, the genetic transformation of the host baker's yeast was carried out with linear fragments of DNA of defined sequence, carrying as the only heterologous material the coding regions of the two K. lactis genes. Growth of the new strain on cheese whey affected neither the quality of bread nor the yeast gassing power. The significance of the newly developed strain is two-fold: it affords a cheap alternative to the procedure generally used for the propagation of baker's yeast, and it offers a profitable use for cheese whey. Copyright 1999 John Wiley & Sons, Ltd.

  5. The cyc1-11 mutation in yeast reverts by recombination with a nonallelic gene: composite genes determining the iso-cytochromes c.

    PubMed Central

    Ernst, J F; Stewart, J W; Sherman, F

    1981-01-01

    DNA sequence analysis of a cloned fragment directly established that the cyc1-11 mutation of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae is a two-base-pair substitution that changes the CCA proline codon at amino acid position 76 to a UAA nonsense codon. Analysis of 11 revertant proteins and one cloned revertant gene showed that reversion of the cyc1-11 mutation can occur in three ways: a single base-pair substitution, which produces a serine replacement at position 76; recombination with the nonallelic CYC7 gene of iso-2-cytochrome c, which causes replacement of a segment in the cyc1-11 gene by the corresponding segment of the CYC7 gene; and either a two-base-pair substitution or recombination with the CYC7 gene, which causes the formation of the normal iso-1-cytochrome c sequence. These results demonstrate the occurrence of low frequencies of recombination between nonallelic genes having extensive but not complete homology. The formation of composite genes that share sequences from nonallelic genes may be an evolutionary mechanism for producing protein diversities and for maintaining identical sequences at different loci. Images PMID:6273865

  6. Applications of yeast surface display for protein engineering

    PubMed Central

    Cherf, Gerald M.; Cochran, Jennifer R.

    2015-01-01

    The method of displaying recombinant proteins on the surface of Saccharomyces cerevisiae via genetic fusion to an abundant cell wall protein, a technology known as yeast surface display, or simply, yeast display, has become a valuable protein engineering tool for a broad spectrum of biotechnology and biomedical applications. This review focuses on the use of yeast display for engineering protein affinity, stability, and enzymatic activity. Strategies and examples for each protein engineering goal are discussed. Additional applications of yeast display are also briefly presented, including protein epitope mapping, identification of protein-protein interactions, and uses of displayed proteins in industry and medicine. PMID:26060074

  7. Immobilisation increases yeast cells' resistance to dehydration-rehydration treatment.

    PubMed

    Borovikova, Diana; Rozenfelde, Linda; Pavlovska, Ilona; Rapoport, Alexander

    2014-08-20

    This study was performed with the goal of revealing if the dehydration procedure used in our new immobilisation method noticeably decreases the viability of yeast cells in immobilised preparations. Various yeasts were used in this research: Saccharomyces cerevisiae cells that were rather sensitive to dehydration and had been aerobically grown in an ethanol-containing medium, a recombinant strain of S. cerevisiae grown in aerobic conditions which were completely non-resistant to dehydration and an anaerobically grown bakers' yeast strain S. cerevisiae, as well as a fairly resistant Pichia pastoris strain. Experiments performed showed that immobilisation of all these strains essentially increased their resistance to a dehydration-rehydration treatment. The increase of cells' viability (compared with control cells dehydrated in similar conditions) was from 30 to 60%. It is concluded that a new immobilisation method, which includes a dehydration stage, does not lead to an essential loss of yeast cell viability. Correspondingly, there is no risk of losing the biotechnological activities of immobilised preparations. The possibility of producing dry, active yeast preparations is shown, for those strains that are very sensitive to dehydration and which can be used in biotechnology in an immobilised form. Finally, the immobilisation approach can be used for the development of efficient methods for the storage of recombinant yeast strains. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Replication intermediates of the linear mitochondrial DNA of Candida parapsilosis suggest a common recombination based mechanism for yeast mitochondria.

    PubMed

    Gerhold, Joachim M; Sedman, Tiina; Visacka, Katarina; Slezakova, Judita; Tomaska, Lubomir; Nosek, Jozef; Sedman, Juhan

    2014-08-15

    Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. A nuclear mutation defective in mitochondrial recombination in yeast.

    PubMed

    Ling, F; Makishima, F; Morishima, N; Shibata, T

    1995-08-15

    Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homologous recombination in the stable inheritance of mitochondrial DNA. For the detection of this class of mutants, we devised a novel procedure, 'mitochondrial crossing in haploid', which has enabled us to examine many mutant clones. Using this procedure, we examined mutants of Saccharomyces cerevisiae that showed an elevated UV induction of respiration-deficient mutations. We obtained a mutant that was defective in both the omega-intron homing and Endo.SceI-induced homologous gene conversion. We found that the mutant cells are temperature sensitive in the maintenance of mitochondrial DNA. A tetrad analysis indicated that elevated UV induction of respiration-deficient mutations, recombination deficiency and temperature sensitivity are all caused by a single nuclear mutation (mhr1) on chromosome XII. The pleiotropic characteristics of the mutant suggest an essential role for the MHR1 gene in DNA repair, recombination and the maintenance of DNA in mitochondria.

  10. Yeast-produced recombinant virus-like particles of coxsackievirus A6 elicited protective antibodies in mice.

    PubMed

    Zhou, Yu; Shen, Chaoyun; Zhang, Chao; Zhang, Wei; Wang, Lili; Lan, Ke; Liu, Qingwei; Huang, Zhong

    2016-08-01

    Coxsackievirus A6 (CA6) has recently emerged as the predominant pathogen of hand, foot and mouth disease (HFMD), causing significant morbidity in children and adults. The increasing prevalence of CA6 infection and its associated disease burden underscore the need for effective CA6 vaccines. However, CA6 grows poorly in cultured cells, making it difficult to develop inactivated whole-virus or live attenuated vaccines. Here we report the development of a recombinant virus-like particle (VLP) based CA6 vaccine. CA6 VLPs were produced in Pichia pastoris yeast transformed with a vector encoding both P1 and 3CD proteins of CA6. Immunization with CA6 VLPs elicited CA6-specific serum antibodies in mice. Passive transfer of anti-VLP antisera protected recipient mice against lethal CA6 challenge. Collectively, these results demonstrate that CA6 VLPs represent a viable CA6 vaccine candidate which warrants further preclinical and clinical development. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Intrinsic biocontainment: Multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes

    PubMed Central

    Cai, Yizhi; Agmon, Neta; Choi, Woo Jin; Ubide, Alba; Stracquadanio, Giovanni; Caravelli, Katrina; Hao, Haiping; Bader, Joel S.; Boeke, Jef D.

    2015-01-01

    Biocontainment may be required in a wide variety of situations such as work with pathogens, field release applications of engineered organisms, and protection of intellectual properties. Here, we describe the control of growth of the brewer’s yeast, Saccharomyces cerevisiae, using both transcriptional and recombinational “safeguard” control of essential gene function. Practical biocontainment strategies dependent on the presence of small molecules require them to be active at very low concentrations, rendering them inexpensive and difficult to detect. Histone genes were controlled by an inducible promoter and controlled by 30 nM estradiol. The stability of the engineered genes was separately regulated by the expression of a site-specific recombinase. The combined frequency of generating viable derivatives when both systems were active was below detection (<10−10), consistent with their orthogonal nature and the individual escape frequencies of <10−6. Evaluation of escaper mutants suggests strategies for reducing their emergence. Transcript profiling and growth test suggest high fitness of safeguarded strains, an important characteristic for wide acceptance. PMID:25624482

  12. A recombinant 63-kDa form of Bacillus anthracis protective antigen produced in the yeast Saccharomyces cerevisiae provides protection in rabbit and primate inhalational challenge models of anthrax infection.

    PubMed

    Hepler, Robert W; Kelly, Rosemarie; McNeely, Tessie B; Fan, Hongxia; Losada, Maria C; George, Hugh A; Woods, Andrea; Cope, Leslie D; Bansal, Alka; Cook, James C; Zang, Gina; Cohen, Steven L; Wei, Xiaorong; Keller, Paul M; Leffel, Elizabeth; Joyce, Joseph G; Pitt, Louise; Schultz, Loren D; Jansen, Kathrin U; Kurtz, Myra

    2006-03-06

    Infection by Bacillus anthracis is preventable by prophylactic vaccination with several naturally derived and recombinant vaccine preparations. Existing data suggests that protection is mediated by antibodies directed against the protective antigen (PA) component of the anthrax toxin complex. PA is an 83-kDa protein cleaved in vivo to yield a biologically active 63-kDa protein. In an effort to evaluate the potential of yeast as an expression system for the production of recombinant PA, and to determine if the yeast-purified rPA63 can protect from a lethal inhalational challenge, the sequence of the 63-kDa form of PA was codon-optimized and expressed in the yeast Saccharomyces cerevisiae. Highly purified rPA63 isolated from Saccharomyces under denaturing conditions demonstrated reduced biological activity in a macrophage-killing assay compared to non-denatured rPA83 purified from Escherichia coli. Rabbits and non-human primates (NHP) immunized with rPA63 and later challenged with a lethal dose of B. anthracis spores were generally protected from infection. These results indicate that epitopes present in the 63-kDa from of PA can protect rabbits and non-human primates from a lethal spore challenge, and further suggest that a fully functional rPA63 is not required in order to provide these epitopes.

  13. Advances in yeast genome engineering.

    PubMed

    David, Florian; Siewers, Verena

    2015-02-01

    Genome engineering based on homologous recombination has been applied to yeast for many years. However, the growing importance of yeast as a cell factory in metabolic engineering and chassis in synthetic biology demands methods for fast and efficient introduction of multiple targeted changes such as gene knockouts and introduction of multistep metabolic pathways. In this review, we summarize recent improvements of existing genome engineering methods, the development of novel techniques, for example for advanced genome redesign and evolution, and the importance of endonucleases as genome engineering tools. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  14. Candida utilis and Cyberlindnera (Pichia) jadinii: yeast relatives with expanding applications.

    PubMed

    Buerth, Christoph; Tielker, Denis; Ernst, Joachim F

    2016-08-01

    The yeast Candida utilis is used as a food additive and as a host for heterologous gene expression to produce various metabolites and proteins. Reliable protocols for intracellular production of recombinant proteins are available for C. utilis and have now been expanded to secrete proteins into the growth medium or to achieve surface display by linkage to a cell wall protein. A recombinant C. utilis strain was recently shown to induce oral tolerance in a mouse model of multiple sclerosis suggesting future applications in autoimmune therapy. Whole genome sequencing of C. utilis and its presumed parent Cyberlindnera (Pichia) jadinii demonstrated different ploidy but high sequence identity, consistent with identical recombinant technologies for both yeasts. C. jadinii was recently described as an antagonist to the important human fungal pathogen Candida albicans suggesting its use as a probiotic agent. The review summarizes the status of recombinant protein production in C. utilis, as well as current and future biotechnological and medical applications of C. utilis and C. jadinii.

  15. Fine-scale variation in meiotic recombination in Mimulus inferred from population shotgun sequencing

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hellsten, Uffe; Wright, Kevin M.; Jenkins, Jerry

    2013-11-13

    Meiotic recombination rates can vary widely across genomes, with hotspots of intense activity interspersed among cold regions. In yeast, hotspots tend to occur in promoter regions of genes, whereas in humans and mice hotspots are largely defined by binding sites of the PRDM9 protein. To investigate the detailed recombination pattern in a flowering plant we use shotgun resequencing of a wild population of the monkeyflower Mimulus guttatus to precisely locate over 400,000 boundaries of historic crossovers or gene conversion tracts. Their distribution defines some 13,000 hotspots of varying strengths, interspersed with cold regions of undetectably low recombination. Average recombination ratesmore » peak near starts of genes and fall off sharply, exhibiting polarity. Within genes, recombination tracts are more likely to terminate in exons than in introns. The general pattern is similar to that observed in yeast, as well as in PRDM9-knockout mice, suggesting that recombination initiation described here in Mimulus may reflect ancient and conserved eukaryotic mechanisms« less

  16. High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2.

    PubMed

    Abécassis, V; Pompon, D; Truan, G

    2000-10-15

    The design of a family shuffling strategy (CLERY: Combinatorial Libraries Enhanced by Recombination in Yeast) associating PCR-based and in vivo recombination and expression in yeast is described. This strategy was tested using human cytochrome P450 CYP1A1 and CYP1A2 as templates, which share 74% nucleotide sequence identity. Construction of highly shuffled libraries of mosaic structures and reduction of parental gene contamination were two major goals. Library characterization involved multiprobe hybridization on DNA macro-arrays. The statistical analysis of randomly selected clones revealed a high proportion of chimeric genes (86%) and a homogeneous representation of the parental contribution among the sequences (55.8 +/- 2.5% for parental sequence 1A2). A microtiter plate screening system was designed to achieve colorimetric detection of polycyclic hydrocarbon hydroxylation by transformed yeast cells. Full sequences of five randomly picked and five functionally selected clones were analyzed. Results confirmed the shuffling efficiency and allowed calculation of the average length of sequence exchange and mutation rates. The efficient and statistically representative generation of mosaic structures by this type of family shuffling in a yeast expression system constitutes a novel and promising tool for structure-function studies and tuning enzymatic activities of multicomponent eucaryote complexes involving non-soluble enzymes.

  17. Recombinant S. cerevisiae expressing Old Yellow Enzymes from non-conventional yeasts: an easy system for selective reduction of activated alkenes

    PubMed Central

    2014-01-01

    Background Old Yellow Enzymes (OYEs) are flavin-dependent enoate reductases (EC 1.6.99.1) that catalyze the stereoselective hydrogenation of electron-poor alkenes. Their ability to generate up to two stereocenters by the trans-hydrogenation of the C = C double bond is highly demanded in asymmetric synthesis. Isolated redox enzymes utilization require the addition of cofactors and systems for their regeneration. Microbial whole-cells may represent a valid alternative combining desired enzymatic activity and efficient cofactor regeneration. Considerable efforts were addressed at developing novel whole-cell OYE biocatalysts, based on recombinant Saccharomyces cerevisiae expressing OYE genes. Results Recombinant S. cerevisiae BY4741∆Oye2 strains, lacking endogenous OYE and expressing nine separate OYE genes from non-conventional yeasts, were used as whole-cell biocatalysts to reduce substrates with an electron-poor double bond activated by different electron-withdrawing groups. Ketoisophorone, α-methyl-trans-cinnamaldehyde, and trans-β-methyl-β-nitrostyrene were successfully reduced with high rates and selectivity. A series of four alkyl-substituted cyclohex-2-enones was tested to check the versatility and efficiency of the biocatalysts. Reduction of double bond occurred with high rates and enantioselectivity, except for 3,5,5-trimethyl-2-cyclohexenone. DFT (density functional theory) computational studies were performed to investigate whether the steric hindrance and/or the electronic properties of the substrates were crucial for reactivity. The three-dimensional structure of enoate reductases from Kluyveromyces lodderae and Candida castellii, predicted through comparative modeling, resulted similar to that of S. cerevisiae OYE2 and revealed the key role of Trp116 both in substrate specificity and stereocontrol. All the modeling studies indicate that steric hindrance was a major determinant in the enzyme reactivity. Conclusions The OYE biocatalysts, based on

  18. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  19. Strand invasion structures in the inverted repeat of Candida albicans mitochondrial DNA reveal a role for homologous recombination in replication.

    PubMed

    Gerhold, Joachim M; Aun, Anu; Sedman, Tiina; Jõers, Priit; Sedman, Juhan

    2010-09-24

    Molecular recombination and transcription are proposed mechanisms to initiate mitochondrial DNA (mtDNA) replication in yeast. We conducted a comprehensive analysis of mtDNA from the yeast Candida albicans. Two-dimensional agarose gel electrophoresis of mtDNA intermediates reveals no bubble structures diagnostic of specific replication origins, but rather supports recombination-driven replication initiation of mtDNA in yeast. Specific species of Y structures together with DNA copy number analyses of a C. albicans mutant strain provide evidence that a region in a mainly noncoding inverted repeat is predominantly involved in replication initiation via homologous recombination. Our further findings show that the C. albicans mtDNA forms a complex branched network that does not contain detectable amounts of circular molecules. We provide topological evidence for recombination-driven mtDNA replication initiation and introduce C. albicans as a suitable model organism to study wild-type mtDNA maintenance in yeast. Copyright © 2010 Elsevier Inc. All rights reserved.

  20. A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists

    PubMed Central

    Helsdingen, Richard J. R.; Hamers, Astrid R. M.; van Duursen, Majorie B. M.; Nielen, Michel W. F.; Hoogenboom, Ron L. A. P.

    2007-01-01

    Public concern about the presence of natural and anthropogenic compounds which affect human health by modulating normal endocrine functions is continuously growing. Fast and simple high-throughput screening methods for the detection of hormone activities are thus indispensable. During the last two decades, a panel of different in vitro assays has been developed, mainly for compounds with an estrogenic mode of action. Here we describe the development of an androgen transcription activation assay that is easy to use in routine screening. Recombinant yeast cells were constructed that express the human androgen receptor and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. Compared with other reporters, the yEGFP reporter protein is very convenient because it is directly measurable in intact living cells, i.e., cell wall disruption and the addition of a substrate are not needed. When yeast was exposed to 17β-testosterone, the concentration where half-maximal activation is reached (EC50) was 50 nM. The relative androgenic potencies, defined as the ratio between the EC50 of 17β-testosterone and the EC50 of the compound, of 5α-dihydrotestosterone, methyltrienolone, and 17β-boldenone are 2.3, 1.4, and 0.15 respectively. The results presented in this paper demonstrate that this new yeast androgen bioassay is fast, sensitive, and very specific and also suited to detect compounds that have an antiandrogenic mode of action. PMID:17849102

  1. Mitochondrial Recombination and Introgression during Speciation by Hybridization.

    PubMed

    Leducq, Jean-Baptiste; Henault, Mathieu; Charron, Guillaume; Nielly-Thibault, Lou; Terrat, Yves; Fiumera, Heather L; Shapiro, B Jesse; Landry, Christian R

    2017-08-01

    Genome recombination is a major source of genotypic diversity and contributes to adaptation and speciation following interspecies hybridization. The contribution of recombination in these processes has been thought to be largely limited to the nuclear genome because organelles are mostly uniparentally inherited in animals and plants, which prevents recombination. Unicellular eukaryotes such as budding yeasts do, however, transmit mitochondria biparentally, suggesting that during hybridization, both parents could provide alleles that contribute to mitochondrial functions such as respiration and metabolism in hybrid populations or hybrid species. We examined the dynamics of mitochondrial genome transmission and evolution during speciation by hybridization in the natural budding yeast Saccharomyces paradoxus. Using population-scale mitochondrial genome sequencing in two endemic North American incipient species SpB and SpC and their hybrid species SpC*, we found that both parental species contributed to the hybrid mitochondrial genome through recombination. We support our findings by showing that mitochondrial recombination between parental types is frequent in experimental crosses that recreate the early step of this speciation event. In these artificial hybrids, we observed that mitochondrial genome recombination enhances phenotypic variation among diploid hybrids, suggesting that it could play a role in the phenotypic differentiation of hybrid species. Like the nuclear genome, the mitochondrial genome can, therefore, also play a role in hybrid speciation. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Recombinational DSBs-intersected genes converge on specific disease- and adaptability-related pathways.

    PubMed

    Yang, Zhi-Kai; Luo, Hao; Zhang, Yanming; Wang, Baijing; Gao, Feng

    2018-05-03

    The budding yeast Saccharomyces cerevisiae is a model species powerful for studying the recombination of eukaryotes. Although many recombination studies have been performed for this species by experimental methods, the population genomic study based on bioinformatics analyses is urgently needed to greatly increase the range and accuracy of recombination detection. Here, we carry out the population genomic analysis of recombination in S. cerevisiae to reveal the potential rules between recombination and evolution in eukaryotes. By population genomic analysis, we discover significantly more and longer recombination events in clinical strains, which indicates that adverse environmental conditions create an obviously wider range of genetic combination in response to the selective pressure. Based on the analysis of recombinational DSBs-intersected genes (RDIGs), we find that RDIGs significantly converge on specific disease- and adaptability-related pathways, indicating that recombination plays a biologically key role in the repair of DSBs related to diseases and environmental adaptability, especially the human neurological disorders (NDs). By evolutionary analysis of RDIGs, we find that the RDIGs highly prevailing in populations of yeast tend to be more evolutionarily conserved, indicating the accurate repair of DSBs in these RDIGs is critical to ensure the eukaryotic survival or fitness. fgao@tju.edu.cn. Supplementary data are available at Bioinformatics online.

  3. RTEL1 maintains genomic stability by suppressing homologous recombination.

    PubMed

    Barber, Louise J; Youds, Jillian L; Ward, Jordan D; McIlwraith, Michael J; O'Neil, Nigel J; Petalcorin, Mark I R; Martin, Julie S; Collis, Spencer J; Cantor, Sharon B; Auclair, Melissa; Tissenbaum, Heidi; West, Stephen C; Rose, Ann M; Boulton, Simon J

    2008-10-17

    Homologous recombination (HR) is an important conserved process for DNA repair and ensures maintenance of genome integrity. Inappropriate HR causes gross chromosomal rearrangements and tumorigenesis in mammals. In yeast, the Srs2 helicase eliminates inappropriate recombination events, but the functional equivalent of Srs2 in higher eukaryotes has been elusive. Here, we identify C. elegans RTEL-1 as a functional analog of Srs2 and describe its vertebrate counterpart, RTEL1, which is required for genome stability and tumor avoidance. We find that rtel-1 mutant worms and RTEL1-depleted human cells share characteristic phenotypes with yeast srs2 mutants: lethality upon deletion of the sgs1/BLM homolog, hyperrecombination, and DNA damage sensitivity. In vitro, purified human RTEL1 antagonizes HR by promoting the disassembly of D loop recombination intermediates in a reaction dependent upon ATP hydrolysis. We propose that loss of HR control after deregulation of RTEL1 may be a critical event that drives genome instability and cancer.

  4. Characterization of N-linked oligosaccharides assembled on secretory recombinant glucose oxidase and cell wall mannoproteins from the methylotrophic yeast Hansenula polymorpha.

    PubMed

    Kim, Moo Woong; Rhee, Sang Ki; Kim, Jeong-Yoon; Shimma, Yoh-ichi; Chiba, Yasunori; Jigami, Yoshifumi; Kang, Hyun Ah

    2004-03-01

    Presently almost no information is available on the oligosaccharide structure of the glycoproteins secreted from the methylotrophic yeast Hansenula polymorpha, a promising host for the production of recombinant proteins. In this study, we analyze the size distribution and structure of N-linked oligosaccharides attached to the recombinant glycoprotein glucose oxidase (GOD) and the cell wall mannoproteins obtained from H. polymorpha. Oligosaccharide profiling showed that the major oligosaccharide species derived from the H. polymorpha-secreted recombinant GOD (rGOD) had core-type structures (Man(8-12)GlcNAc(2)). Analyses using anti-alpha 1,3-mannose antibody and exoglycosidases specific for alpha 1,2- or alpha 1,6-mannose linkages revealed that the mannose outer chains of N-glycans on the rGOD have very short alpha 1,6 extensions and are mainly elongated in alpha 1,2-linkages without a terminal alpha 1,3-linked mannose addition. The N-glycans released from the H. polymorpha mannoproteins were shown to contain mostly mannose in their outer chains, which displayed almost identical size distribution and structure to those of H. polymorpha-derived rGOD. These results strongly indicate that the outer chain processing of N-glycans by H. polymorpha significantly differs from that by Saccharomyces cerevisiae, thus generating much shorter mannose outer chains devoid of terminal alpha 1,3-linked mannoses.

  5. A new methodology to obtain wine yeast strains overproducing mannoproteins.

    PubMed

    Quirós, Manuel; Gonzalez-Ramos, Daniel; Tabera, Laura; Gonzalez, Ramon

    2010-04-30

    Yeast mannoproteins are highly glycosylated proteins that are covalently bound to the beta-1,3-glucan present in the yeast cell wall. Among their outstanding enological properties, yeast mannoproteins contribute to several aspects of wine quality by protecting against protein haze, reducing astringency, retaining aroma compounds and stimulating growth of lactic-acid bacteria. The development of a non-recombinant method to obtain enological yeast strains overproducing mannoproteins would therefore be very useful. Our previous experience on the genetic determinants of the release of these molecules by Saccharomyces cerevisiae has allowed us to propose a new methodology to isolate and characterize wine yeast that overproduce mannoproteins. The described methodology is based on the resistance of the killer 9 toxin produced by Williopsis saturnus, a feature linked to an altered biogenesis of the yeast cell wall. Copyright 2010 Elsevier B.V. All rights reserved.

  6. Cell recycle batch fermentation of high-solid lignocellulose using a recombinant cellulase-displaying yeast strain for high yield ethanol production in consolidated bioprocessing.

    PubMed

    Matano, Yuki; Hasunuma, Tomohisa; Kondo, Akihiko

    2013-05-01

    The aim of this study is to develop a scheme of cell recycle batch fermentation (CRBF) of high-solid lignocellulosic materials. Two-phase separation consisting of rough removal of lignocellulosic residues by low-speed centrifugation and solid-liquid separation enabled effective collection of Saccharomyces cerevisiae cells with decreased lignin and ash. Five consecutive batch fermentation of 200 g/L rice straw hydrothermally pretreated led to an average ethanol titer of 34.5 g/L. Moreover, the display of cellulases on the recombinant yeast cell surface increased ethanol titer to 42.2 g/L. After, five-cycle fermentation, only 3.3 g/L sugar was retained in the fermentation medium, because cellulase displayed on the cell surface hydrolyzed cellulose that was not hydrolyzed by commercial cellulases or free secreted cellulases. Fermentation ability of the recombinant strain was successfully kept during a five-cycle repeated batch fermentation with 86.3% of theoretical yield based on starting biomass. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Human XPA and XRCC1 DNA repair proteins expressed in yeast, Saccharomyces cerevisiae.

    PubMed

    Pushnova, E A; Ostanin, K; Thelen, M P

    2001-11-01

    Human XPA and XRCC1 DNA repair proteins have been expressed in a series of novel yeast episomal vectors. Expression of XPA cDNA resulted in synthesis of anti-XPA crossreacting polypeptides of 40 and 42 kDa, the status of the native protein found in human cells. Likewise, the majority of the recombinant XRCC1 found in the yeast intracellular fraction corresponded to the molecular mass of the full-length human protein. Recombinant XPA protein expressed as an NH(2)-terminal polyhistidine fusion could be affinity purified using Ni(2+) agarose. Copyright 2001 Academic Press.

  8. The yeast stands alone: the future of protein biologic production.

    PubMed

    Love, Kerry R; Dalvie, Neil C; Love, J Christopher

    2017-12-22

    Yeasts are promising alternative hosts for the manufacturing of recombinant protein therapeutics because they simply and efficiently meet needs for both platform and small-market drugs. Fast accumulation of biomass and low-cost media reduce the cost-of-goods when using yeast, which in turn can enable agile, small-volume manufacturing facilities. Small, tractable yeast genomes are amenable to rapid process development, facilitating strain and product quality by design. Specifically, Pichia pastoris is becoming a widely accepted yeast for biopharmaceutical manufacturing in much of the world owing to a clean secreted product and the rapidly expanding understanding of its cell biology as a host organism. We advocate for a near term partnership spanning industry and academia to promote open source, timely development of yeast hosts. Copyright © 2017. Published by Elsevier Ltd.

  9. Yeast as a model for Ras signalling.

    PubMed

    Tisi, Renata; Belotti, Fiorella; Martegani, Enzo

    2014-01-01

    For centuries yeast species have been popular hosts for classical biotechnology processes, such as baking, brewing, and wine making, and more recently for recombinant proteins production, thanks to the advantages of unicellular organisms (i.e., ease of genetic manipulation and rapid growth) together with the ability to perform eukaryotic posttranslational modifications. Moreover, yeast cells have been used for few decades as a tool for identifying the genes and pathways involved in basic cellular processes such as the cell cycle, aging, and stress response. In the budding yeast S. cerevisiae the Ras/cAMP/PKA pathway is directly involved in the regulation of metabolism, cell growth, stress resistance, and proliferation in response to the availability of nutrients and in the adaptation to glucose, controlling cytosolic cAMP levels and consequently the cAMP-dependent protein kinase (PKA) activity. Moreover, Ras signalling has been identified in several pathogenic yeasts as a key controller for virulence, due to its involvement in yeast morphogenesis. Nowadays, yeasts are still useful for Ras-like proteins investigation, both as model organisms and as a test tube to study variants of heterologous Ras-like proteins.

  10. Process strategies to maximize lipid accumulations of novel yeast in acid and base treated hydrolyzates

    USDA-ARS?s Scientific Manuscript database

    Oleaginous yeasts can accumulate up to 70% of cell biomass as lipids, predominantly as triacylglycerols. Yeast lipid fatty acid profiles have been reported to be similar to that of vegetable oils and consist primarily of oleic, palmitic, stearic, and linoleic acids. This capability provides the oppo...

  11. Regulation of Meiotic Recombination

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gregory p. Copenhaver

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diversemore » as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a

  12. Yeasts in sustainable bioethanol production: A review.

    PubMed

    Mohd Azhar, Siti Hajar; Abdulla, Rahmath; Jambo, Siti Azmah; Marbawi, Hartinie; Gansau, Jualang Azlan; Mohd Faik, Ainol Azifa; Rodrigues, Kenneth Francis

    2017-07-01

    Bioethanol has been identified as the mostly used biofuel worldwide since it significantly contributes to the reduction of crude oil consumption and environmental pollution. It can be produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal biomass through fermentation process by microorganisms. Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes employed in ethanol production due to its high ethanol productivity, high ethanol tolerance and ability of fermenting wide range of sugars. However, there are some challenges in yeast fermentation which inhibit ethanol production such as high temperature, high ethanol concentration and the ability to ferment pentose sugars. Various types of yeast strains have been used in fermentation for ethanol production including hybrid, recombinant and wild-type yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks must be converted to fermentable sugars before it can be fermented to ethanol. The common processes involves in ethanol production are pretreatment, hydrolysis and fermentation. Production of bioethanol during fermentation depends on several factors such as temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review highlights the different types of yeast strains, fermentation process, factors affecting bioethanol production and immobilization of yeasts for better bioethanol production.

  13. Recombinant Saccharomyces cerevisiae serves as novel carrier for oral DNA vaccines in Carassius auratus.

    PubMed

    Yan, Nana; Xu, Kun; Li, Xinyi; Liu, Yuwan; Bai, Yichun; Zhang, Xiaohan; Han, Baoquan; Chen, Zhilong; Zhang, Zhiying

    2015-12-01

    Oral delivery of DNA vaccines represents a promising vaccinating method for fish. Recombinant yeast has been proved to be a safe carrier for delivering antigen proteins and DNAs to some species in vivo. However, whether recombinant yeast can be used to deliver functional DNAs for vaccination to fish is still unknown. In this study, red crucian carp (Carassius auratus) was orally administrated with recombinant Saccharomyces cerevisiae harboring CMV-EGFP expression cassette. On day 5 post the first vaccination, EGFP expression in the hindgut was detected under fluorescence microscope. To further study whether the delivered gene could induce specific immune responses, the model antigen ovalbumin (OVA) was used as immunogen, and oral administrations were conducted with recombinant S. cerevisiae harboring pCMV-OVA mammalian gene expression cassette as gene delivery or pADH1-OVA yeast gene expression cassette as protein delivery. Each administration was performed with three different doses, and the OVA-specific serum antibody was detected in all the experimental groups by western blotting and enzyme-linked immunosorbent assay (ELISA). ELISA assay also revealed that pCMV-OVA group with lower dose (pCMV-OVA-L) and pADH1-OVA group with moderate dose (pADH1-OVA-M) triggered relatively stronger antibody response than the other two doses. Moreover, the antibody level induced by pCMV-OVA-L group was significantly higher than pADH1-OVA-M group at the same serum dilutions. All the results suggested that recombinant yeast can be used as a potential carrier for oral DNA vaccines and would help to develop more practical strategies to control infectious diseases in aquaculture. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. The impact of respiration and oxidative stress response on recombinant α-amylase production by Saccharomyces cerevisiae.

    PubMed

    Martínez, José L; Meza, Eugenio; Petranovic, Dina; Nielsen, Jens

    2016-12-01

    Studying protein production is important for fundamental research on cell biology and applied research for biotechnology. Yeast Saccharomyces cerevisiae is an attractive workhorse for production of recombinant proteins as it does not secrete many endogenous proteins and it is therefore easy to purify a secreted product. However, recombinant production at high rates represents a significant metabolic burden for the yeast cells, which results in oxidative stress and ultimately affects the protein production capacity. Here we describe a method to reduce the overall oxidative stress by overexpressing the endogenous HAP1 gene in a S. cerevisiae strain overproducing recombinant α-amylase. We demonstrate how Hap1p can activate a set of oxidative stress response genes and meanwhile contribute to increase the metabolic rate of the yeast strains, therefore mitigating the negative effect of the ROS accumulation associated to protein folding and hence increasing the production capacity during batch fermentations.

  15. Mitochondrial inheritance in budding yeasts: towards an integrated understanding.

    PubMed

    Solieri, Lisa

    2010-11-01

    Recent advances in yeast mitogenomics have significantly contributed to our understanding of the diversity of organization, structure and topology in the mitochondrial genome of budding yeasts. In parallel, new insights on mitochondrial DNA (mtDNA) inheritance in the model organism Saccharomyces cerevisiae highlighted an integrated scenario where recombination, replication and segregation of mtDNA are intricately linked to mitochondrial nucleoid (mt-nucleoid) structure and organelle sorting. In addition to this, recent discoveries of bifunctional roles of some mitochondrial proteins have interesting implications on mito-nuclear genome interactions and the relationship between mtDNA inheritance, yeast fitness and speciation. This review summarizes the current knowledge on yeast mitogenomics, mtDNA inheritance with regard to mt-nucleoid structure and organelle dynamics, and mito-nuclear genome interactions. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. SPAR1/RTEL1 maintains genomic stability by suppressing homologous recombination

    PubMed Central

    Barber, Louise J.; Youds, Jillian L.; Ward, Jordan D.; McIlwraith, Michael J.; O’Neil, Nigel J.; Petalcorin, Mark I.R.; Martin, Julie S.; Collis, Spencer J.; Cantor, Sharon B.; Auclair, Melissa; Tissenbaum, Heidi; West, Stephen C.; Rose, Ann M.; Boulton, Simon J.

    2013-01-01

    SUMMARY Inappropriate homologous recombination (HR) can cause gross chromosomal rearrangements that in mammalian cells may lead to tumorigenesis. In yeast, the Srs2 protein is an anti-recombinase that eliminates inappropriate recombination events, but the functional equivalent of Srs2 in higher eukaryotes has proven to be elusive. In this work, we identify C. elegans SPAR-1 as a functional analogue of Srs2 and describe its vertebrate counterpart, SPAR1/RTEL1, which is required for genome stability and tumour avoidance. We find that spar-1 mutant worms and SPAR1 knockdown human cells share characteristic phenotypes with yeast srs2 mutants, including inviability upon deletion of the sgs1/BLM homologue, hyper-recombination, and DNA damage sensitivity. In vitro, purified human SPAR1 antagonises HR by promoting the disassembly of D loop recombination intermediates in a reaction dependent upon ATP hydrolysis. We propose that loss of HR control following deregulation of SPAR1/RTEL1 may be a critical event that drives genome instability and cancer. PMID:18957201

  17. Statistical approaches to maximize recombinant protein expression in Escherichia coli: a general review.

    PubMed

    Papaneophytou, Christos P; Kontopidis, George

    2014-02-01

    The supply of many valuable proteins that have potential clinical or industrial use is often limited by their low natural availability. With the modern advances in genomics, proteomics and bioinformatics, the number of proteins being produced using recombinant techniques is exponentially increasing and seems to guarantee an unlimited supply of recombinant proteins. The demand of recombinant proteins has increased as more applications in several fields become a commercial reality. Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, producing soluble proteins in E. coli is still a major bottleneck for structural biology projects. One of the most challenging steps in any structural biology project is predicting which protein or protein fragment will express solubly and purify for crystallographic studies. The production of soluble and active proteins is influenced by several factors including expression host, fusion tag, induction temperature and time. Statistical designed experiments are gaining success in the production of recombinant protein because they provide information on variable interactions that escape the "one-factor-at-a-time" method. Here, we review the most important factors affecting the production of recombinant proteins in a soluble form. Moreover, we provide information about how the statistical design experiments can increase protein yield and purity as well as find conditions for crystal growth. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. [Clone, construct, expression and verification of lactoferricin B gene and several sequence mutations in yeast].

    PubMed

    Feng, Yong-qian; Zha, Xiao-jun; Zhai, Chao-yang

    2007-07-01

    To construct the eucaryotic recombinant plasmid of pYES2/LactoferricinB expressing in yeast of S. cerevisiae, of which the expressed protein antibacterial activity was verified in preliminary. By self-template PCR method, the gene of Lactoferricin B and its several sequence mutations were amplified with the parts of the pre-synthesized single chains. And then Lactoferricin B gene and its mutants were cloned into the vector of pYES2 to construct the recombined expression plasmid pYES2/Lactoferricin B etc. extracted and used to transform the yeast S. cerevisiae. The expressions of proteins were determined after induced by galactose. The expression proteins were collected and purified by hydronium-exchange column, and the bacterial inhibited test was applied to identify the protein antibacterial activities. The PCR amplifying and DNA sequencing tests indicated that the purpose plasmid contained the Lactoferricin B gene and several mutations. The induced target proteins were confirmed by SDS-PAGE electrophoresis and mass spectrum test. The protein antibacterial activities of mutations were verified in preliminary. The recombined plasmid pYES2/Lactoferricin B etc. are successfully constructed and induced to express in yeast cell of S. cerevisiae; the obtained recombined protein of Lactoferricin B provides a basis for further research work on the biological function and antibacterial activity.

  19. Mitotic Recombination and Genetic Changes in Saccharomyces cerevisiae during Wine Fermentation

    PubMed Central

    Puig, Sergi; Querol, Amparo; Barrio, Eladio; Pérez-Ortín, José E.

    2000-01-01

    Natural strains of Saccharomyces cerevisiae are prototrophic homothallic yeasts that sporulate poorly, are often heterozygous, and may be aneuploid. This genomic constitution may confer selective advantages in some environments. Different mechanisms of recombination, such as meiosis or mitotic rearrangement of chromosomes, have been proposed for wine strains. We studied the stability of the URA3 locus of a URA3/ura3 wine yeast in consecutive grape must fermentations. ura3/ura3 homozygotes were detected at a rate of 1 × 10−5 to 3 × 10−5 per generation, and mitotic rearrangements for chromosomes VIII and XII appeared after 30 mitotic divisions. We used the karyotype as a meiotic marker and determined that sporulation was not involved in this process. Thus, we propose a hypothesis for the genome changes in wine yeasts during vinification. This putative mechanism involves mitotic recombination between homologous sequences and does not necessarily imply meiosis. PMID:10788381

  20. Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination.

    PubMed

    Vashee, Sanjay; Stockwell, Timothy B; Alperovich, Nina; Denisova, Evgeniya A; Gibson, Daniel G; Cady, Kyle C; Miller, Kristofer; Kannan, Krishna; Malouli, Daniel; Crawford, Lindsey B; Voorhies, Alexander A; Bruening, Eric; Caposio, Patrizia; Früh, Klaus

    2017-01-01

    Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae . Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae . Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain

  1. Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination

    PubMed Central

    Vashee, Sanjay; Stockwell, Timothy B.; Alperovich, Nina; Denisova, Evgeniya A.; Gibson, Daniel G.; Cady, Kyle C.; Miller, Kristofer; Kannan, Krishna; Malouli, Daniel; Crawford, Lindsey B.; Voorhies, Alexander A.; Bruening, Eric; Caposio, Patrizia

    2017-01-01

    ABSTRACT Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to

  2. Role of RNase MRP in viral RNA degradation and RNA recombination.

    PubMed

    Jaag, Hannah M; Lu, Qiasheng; Schmitt, Mark E; Nagy, Peter D

    2011-01-01

    RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.

  3. Generation of Recombinant Porcine Parvovirus Virus-Like Particles in Saccharomyces cerevisiae and Development of Virus-Specific Monoclonal Antibodies

    PubMed Central

    Tamošiūnas, Paulius Lukas; Petraitytė-Burneikienė, Rasa; Lasickienė, Rita; Sereika, Vilimas; Lelešius, Raimundas; Žvirblienė, Aurelija; Sasnauskas, Kęstutis

    2014-01-01

    Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance. PMID:25045718

  4. Mitochondrial recombination increases with age in Podospora anserina.

    PubMed

    van Diepeningen, Anne D; Goedbloed, Daniël J; Slakhorst, S Marijke; Koopmanschap, A Bertha; Maas, Marc F P M; Hoekstra, Rolf F; Debets, Alfons J M

    2010-05-01

    With uniparental inheritance of mitochondria, there seems little reason for homologous recombination in mitochondria, but the machinery for mitochondrial recombination is quite well-conserved in many eukaryote species. In fungi and yeasts heteroplasmons may be formed when strains fuse and transfer of organelles takes place, making it possible to study mitochondrial recombination when introduced mitochondria contain different markers. A survey of wild-type isolates from a local population of the filamentous fungus Podospora anserina for the presence of seven optional mitochondrial introns indicated that mitochondrial recombination does take place in nature. Moreover the recombination frequency appeared to be correlated with age: the more rapidly ageing fraction of the population had a significantly lower linkage disequilibrium indicating more recombination. Direct confrontation experiments with heterokaryon incompatible strains with different mitochondrial markers at different (relative) age confirmed that mitochondrial recombination increases with age. We propose that with increasing mitochondrial damage over time, mitochondrial recombination - even within a homoplasmic population of mitochondria - is a mechanism that may restore mitochondrial function. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

  5. Heterologous mitochondrial DNA recombination in human cells.

    PubMed

    D'Aurelio, Marilena; Gajewski, Carl D; Lin, Michael T; Mauck, William M; Shao, Leon Z; Lenaz, Giorgio; Moraes, Carlos T; Manfredi, Giovanni

    2004-12-15

    Inter-molecular heterologous mitochondrial DNA (mtDNA) recombination is known to occur in yeast and plants. Nevertheless, its occurrence in human cells is still controversial. To address this issue we have fused two human cytoplasmic hybrid cell lines, each containing a distinct pathogenic mtDNA mutation and specific sets of genetic markers. In this hybrid model, we found direct evidence of recombination between these two mtDNA haplotypes. Recombinant mtDNA molecules in the hybrid cells were identified using three independent experimental approaches. First, recombinant molecules containing genetic markers from both parental alleles were demonstrated with restriction fragment length polymorphism of polymerase chain reaction products, by measuring the relative frequencies of each marker. Second, fragments of recombinant mtDNA were cloned and sequenced to identify the regions involved in the recombination events. Finally, recombinant molecules were demonstrated directly by Southern blot using appropriate combinations of polymorphic restriction sites and probes. This combined approach confirmed the existence of heterogeneous species of recombinant mtDNA molecules in the hybrid cells. These findings have important implications for mtDNA-related diseases, the interpretation of human evolution and population genetics and forensic analyses based on mtDNA genotyping.

  6. Optimization of glutathione production in batch and fed-batch cultures by the wild-type and recombinant strains of the methylotrophic yeast Hansenula polymorpha DL-1

    PubMed Central

    2011-01-01

    Background Tripeptide glutathione (gamma-glutamyl-L-cysteinyl-glycine) is the most abundant non-protein thiol that protects cells from metabolic and oxidative stresses and is widely used as medicine, food additives and in cosmetic industry. The methylotrophic yeast Hansenula polymorpha is regarded as a rich source of glutathione due to the role of this thiol in detoxifications of key intermediates of methanol metabolism. Cellular and extracellular glutathione production of H. polymorpha DL-1 in the wild type and recombinant strains which overexpress genes of glutathione biosynthesis (GSH2) and its precursor cysteine (MET4) was studied. Results Glutathione producing capacity of H. polymorpha DL-1 depending on parameters of cultivation (dissolved oxygen tension, pH, stirrer speed), carbon substrate (glucose, methanol) and type of overexpressed genes of glutathione and its precursor biosynthesis during batch and fed-batch fermentations were studied. Under optimized conditions of glucose fed-batch cultivation, the glutathione productivity of the engineered strains was increased from ~900 up to ~ 2300 mg of Total Intracellular Glutathione (TIG) or GSH+GSSGin, per liter of culture medium. Meantime, methanol fed-batch cultivation of one of the recombinant strains allowed achieving the extracellular glutathione productivity up to 250 mg of Total Extracellular Glutathione (TEG) or GSH+GSSGex, per liter of the culture medium. Conclusions H. polymorpha is an competitive glutathione producer as compared to other known yeast and bacteria strains (Saccharomyces cerevisiae, Candida utilis, Escherichia coli, Lactococcus lactis etc.) with good perspectives for further improvement especially for production of extracellular form of glutathione. PMID:21255454

  7. Maximizing detergent stability and functional expression of a GPCR by exhaustive recombination and evolution.

    PubMed

    Schlinkmann, Karola M; Hillenbrand, Matthias; Rittner, Alexander; Künz, Madeleine; Strohner, Ralf; Plückthun, Andreas

    2012-09-21

    To identify structural features in a G-protein-coupled receptor (GPCR) crucial for biosynthesis, stability in the membrane and stability in detergent micelles, we developed an evolutionary approach using expression in the inner membrane of Escherichia coli. From the analysis of 800,000 sequences of the rat neurotensin receptor 1, in which every amino acid had been varied to all 64 codons, we uncovered several "shift" positions, where the selected population focuses on a residue different from wild type. Here, we employed in vitro DNA recombination and a comprehensive synthetic binary library made by the Slonomics® technology, allowing us to uncover additive and synergistic effects in the structure that maximize both detergent stability and functional expression. We identified variants with >25,000 functional molecules per E. coli cell, a 50-fold increase over wild type, and observed strong coevolution of detergent stability. We arrived at receptor variants highly stable in short-chain detergents, much more so than those found by alanine scanning on the same receptor. These evolved GPCRs continue to be able to signal through the G-protein. We discuss the structural reasons for these improvements achieved through directed evolution. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

    PubMed

    Gray, Sean A; Weigel, Kris M; Ali, Ibne K M; Lakey, Annie A; Capalungan, Jeremy; Domingo, Gonzalo J; Cangelosi, Gerard A

    2012-01-01

    The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv) in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

  9. Heterologous Expression of the Carrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

    PubMed

    Ko, Eunhye; Kim, Minhye; Park, Yunho; Ahn, Yeh-Jin

    2017-08-01

    In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter-carrot Hsp17.7 gene-Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.

  10. Phylogenetics of Saccharomycetales, the ascomycete yeasts.

    PubMed

    Suh, Sung-Oui; Blackwell, Meredith; Kurtzman, Cletus P; Lachance, Marc-André

    2006-01-01

    Ascomycete yeasts (phylum Ascomycota: subphylum Saccharomycotina: class Saccharomycetes: order Saccharomycetales) comprise a monophyletic lineage with a single order of about 1000 known species. These yeasts live as saprobes, often in association with plants, animals and their interfaces. A few species account for most human mycotic infections, and fewer than 10 species are plant pathogens. Yeasts are responsible for important industrial and biotechnological processes, including baking, brewing and synthesis of recombinant proteins. Species such as Saccharomyces cerevisiae are model organisms in research, some of which led to a Nobel Prize. Yeasts usually reproduce asexually by budding, and their sexual states are not enclosed in a fruiting body. The group also is well defined by synapomorphies visible at the ultrastructural level. Yeast identification and classification changed dramatically with the availability of DNA sequencing. Species identification now benefits from a constantly updated sequence database and no longer relies on ambiguous growth tests. A phylogeny based on single gene analyses has shown the order to be remarkably divergent despite morphological similarities among members. The limits of many previously described genera are not supported by sequence comparisons, and multigene phylogenetic studies are under way to provide a stable circumscription of genera, families and orders. One recent multigene study has resolved species of the Saccharomycetaceae into genera that differ markedly from those defined by analysis of morphology and growth responses, and similar changes are likely to occur in other branches of the yeast tree as additional sequences become available.

  11. Carbon metabolism influenced for promoters and temperature used in the heterologous protein production using Pichia pastoris yeast.

    PubMed

    Zepeda, Andrea B; Pessoa, Adalberto; Farías, Jorge G

    2018-05-19

    Nowadays, it is necessary to search for different high-scale production strategies to produce recombinant proteins of economic interest. Only a few microorganisms are industrially relevant for recombinant protein production: methylotrophic yeasts are known to use methanol efficiently as the sole carbon and energy source. Pichia pastoris is a methylotrophic yeast characterized as being an economical, fast and effective system for heterologous protein expression. Many factors can affect both the product and the production, including the promoter, carbon source, pH, production volume, temperature, and many others; but to control all of them most of the time is difficult and this depends on the initial selection of each variable. Therefore, this review focuses on the selection of the best promoter in the recombination process, considering different inductors, and the temperature as a culture medium variable in methylotrophic Pichia pastoris yeast. The goal is to understand the effects associated with different factors that influence its cell metabolism and to reach the construction of an expression system that fulfills the requirements of the yeast, presenting an optimal growth and development in batch, fed-batch or continuous cultures, and at the same time improve its yield in heterologous protein production. Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  12. Generation of Tioman virus nucleocapsid-like particles in yeast Saccharomyces cerevisiae.

    PubMed

    Petraityte, Rasa; Tamosiunas, Paulius L; Juozapaitis, Mindaugas; Zvirbliene, Aurelija; Sasnauskas, Kestutis; Shiell, Brian; Russell, Gail; Bingham, John; Michalski, Wojtek P

    2009-10-01

    Tioman virus (TioV) was isolated from a number of pooled urine samples of Tioman Island flying foxes (Pteropus hypomelanus) during the search for the reservoir host of Nipah virus. Studies have established TioV as a new virus in the family Paramyxoviridae. This novel paramyxovirus is antigenically related to Menangle virus that was isolated in Australia in 1997 during disease outbreak in pigs. TioV causes mild disease in pigs and has a predilection for lymphoid tissues. Recent serosurvey showed that 1.8% of Tioman Islanders had neutralizing antibodies against TioV, indicating probable past infection. For the development of convenient serological tests for this virus, recombinant TioV nucleocapsid (N) protein was expressed in the yeast Saccharomyces cerevisiae. High yields of recombinant TioV N protein were obtained. Electron microscopy demonstrated that purified recombinant N protein self-assembled into nucleocapsid-like particles which were identical in density and morphology to authentic nucleocapsids from paramyxovirus-infected cells. Different size nucleocapsid-like particles were stable and readily purified by CsCl gradient ultracentrifugation. Polyclonal sera raised in rabbits after immunization with recombinant TioV N protein reacted reliably with TioV infected tissues in immunohistochemistry tests. It confirmed that the antigenic properties of yeast derived TioV N protein are identical to authentic viral protein.

  13. Nonselective enrichment for yeast adenine mutants by flow cytometry

    NASA Technical Reports Server (NTRS)

    Bruschi, C. V.; Chuba, P. J.

    1988-01-01

    The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

  14. Replication-Dependent Sister Chromatid Recombination in Rad1 Mutants of Saccharomyces Cerevisiae

    PubMed Central

    Kadyk, L. C.; Hartwell, L. H.

    1993-01-01

    Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation. In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G(1) or the G(2) phases of the cell cycle than was observed in RAD1/rad1-1 cells. Since sister chromatids are not present during G(1), this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication. The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G(2) phase, but only when they are present during DNA replication. We propose that there are two types of sister chromatid recombination in yeast. In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells. In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G(2). Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination. PMID:8454200

  15. CRISPR-directed mitotic recombination enables genetic mapping without crosses.

    PubMed

    Sadhu, Meru J; Bloom, Joshua S; Day, Laura; Kruglyak, Leonid

    2016-05-27

    Linkage and association studies have mapped thousands of genomic regions that contribute to phenotypic variation, but narrowing these regions to the underlying causal genes and variants has proven much more challenging. Resolution of genetic mapping is limited by the recombination rate. We developed a method that uses CRISPR (clustered, regularly interspaced, short palindromic repeats) to build mapping panels with targeted recombination events. We tested the method by generating a panel with recombination events spaced along a yeast chromosome arm, mapping trait variation, and then targeting a high density of recombination events to the region of interest. Using this approach, we fine-mapped manganese sensitivity to a single polymorphism in the transporter Pmr1. Targeting recombination events to regions of interest allows us to rapidly and systematically identify causal variants underlying trait differences. Copyright © 2016, American Association for the Advancement of Science.

  16. Recent advances in the production of recombinant subunit vaccines in Pichia pastoris

    PubMed Central

    Wang, Man; Jiang, Shuai; Wang, Yefu

    2016-01-01

    ABSTRACT Recombinant protein subunit vaccines are formulated using defined protein antigens that can be produced in heterologous expression systems. The methylotrophic yeast Pichia pastoris has become an important host system for the production of recombinant subunit vaccines. Although many basic elements of P. pastoris expression system are now well developed, there is still room for further optimization of protein production. Codon bias, gene dosage, endoplasmic reticulum protein folding and culture condition are important considerations for improved production of recombinant vaccine antigens. Here we comment on current advances in the application of P. pastoris for the synthesis of recombinant subunit vaccines. PMID:27246656

  17. Genetic relationship and biological status of the industrially important yeast Saccharomyces eubayanus Sampaio et al.

    PubMed

    Naumov, G I

    2017-03-01

    The genomes of the recently discovered yeast Saccharomyces eubayanus and traditional S. cerevisiae are known to be found in the yeast S. pastorianus (syn. S. carlsbergensis), which are essential for brewing. The cryotolerant yeast S. bayanus var. uvarum is of great importance for production of some wines. Based on ascospore viability and meiotic recombination of the control parental markers in hybrids, we have shown that there is no complete interspecies post-zygotic isolation between the yeasts S. eubayanus, S. bayanus var. bayanus and S. bayanus var. uvarum. The genetic data presented indicate that all of the three taxa belong to the same species.

  18. Engineering baker's yeast: room for improvement.

    PubMed

    Randez-Gil, F; Sanz, P; Prieto, J A

    1999-06-01

    Bread making is one of the oldest food-manufacturing processes. However, it is only in the past few years that recombinant-DNA technology has led to dramatic changes in formulation, ingredients or processing conditions. New strains of baker's yeast that produce CO2 more rapidly, are more resistant to stress or produce proteins or metabolites that can modify bread flavour, dough rheology or shelf-life are now emerging.

  19. Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor.

    PubMed

    Tran, Anh-Minh; Nguyen, Thanh-Thao; Nguyen, Cong-Thuan; Huynh-Thi, Xuan-Mai; Nguyen, Cao-Tri; Trinh, Minh-Thuong; Tran, Linh-Thuoc; Cartwright, Stephanie P; Bill, Roslyn M; Tran-Van, Hieu

    2017-04-04

    Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts. Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1. Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production.

  20. RNAi and heterochromatin repress centromeric meiotic recombination

    PubMed Central

    Ellermeier, Chad; Higuchi, Emily C.; Phadnis, Naina; Holm, Laerke; Geelhood, Jennifer L.; Thon, Genevieve; Smith, Gerald R.

    2010-01-01

    During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essential in most species for proper homologue segregation. Nevertheless, recombination is repressed specifically in and around the centromeres of chromosomes, apparently because rare centromeric (or pericentromeric) recombination events, when they do occur, can disrupt proper segregation and lead to genetic disabilities, including birth defects. The basis by which centromeric meiotic recombination is repressed has been largely unknown. We report here that, in fission yeast, RNAi functions and Clr4-Rik1 (histone H3 lysine 9 methyltransferase) are required for repression of centromeric recombination. Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis. PMID:20421495

  1. The Budding Yeast Nucleus

    PubMed Central

    Taddei, Angela; Schober, Heiko; Gasser, Susan M.

    2010-01-01

    The budding yeast nucleus, like those of other eukaryotic species, is highly organized with respect to both chromosomal sequences and enzymatic activities. At the nuclear periphery interactions of nuclear pores with chromatin, mRNA, and transport factors promote efficient gene expression, whereas centromeres, telomeres, and silent chromatin are clustered and anchored away from pores. Internal nuclear organization appears to be function-dependent, reflecting localized sites for tRNA transcription, rDNA transcription, ribosome assembly, and DNA repair. Recent advances have identified new proteins involved in the positioning of chromatin and have allowed testing of the functional role of higher-order chromatin organization. The unequal distribution of silent information regulatory factors and histone modifying enzymes, which arises in part from the juxtaposition of telomeric repeats, has been shown to influence chromatin-mediated transcriptional repression. Other localization events suppress unwanted recombination. These findings highlight the contribution budding yeast genetics and cytology have made to dissecting the functional role of nuclear structure. PMID:20554704

  2. Maximizing the concentrations of wheat grain fructans in bread by exploring strategies to prevent their yeast ( Saccharomyces cerevisiae )-mediated degradation.

    PubMed

    Verspreet, Joran; Hemdane, Sami; Dornez, Emmie; Cuyvers, Sven; Delcour, Jan A; Courtin, Christophe M

    2013-02-13

    The degradation of endogenous wheat grain fructans, oligosaccharides with possible health-promoting potential, during wheat whole meal bread making was investigated, and several strategies to prevent their degradation were evaluated. Up to 78.4 ± 5.2% of the fructans initially present in wheat whole meal were degraded during bread making by the action of yeast ( Saccharomyces cerevisiae ) invertase. The addition of sucrose to dough delayed fructan degradation but had no effect on final fructan concentrations. However, yeast growth conditions and yeast genotype did have a clear impact. A 3-fold reduction of fructan degradation could be achieved when the commercial bread yeast strain was replaced by yeast strains with lower sucrose degradation activity. Finally, fructan degradation during bread making could be prevented completely by the use of a yeast strain lacking invertase. These results show that the nutritional profile of bread can be enhanced through appropriate yeast technology.

  3. Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe.

    PubMed

    Sangesland, Maya; Atwood-Moore, Angela; Rai, Sudhir K; Levin, Henry L

    2016-01-01

    Transposition and homologous recombination assays are valuable genetic tools to measure the production and integration of cDNA from the long terminal repeat (LTR) retrotransposon Tf1 in the fission yeast (Schizosaccharomyces pombe). Here we describe two genetic assays, one that measures the transposition activity of Tf1 by monitoring the mobility of a drug resistance marked Tf1 element expressed from a multi-copy plasmid and another assay that measures homologous recombination between Tf1 cDNA and the expression plasmid. While the transposition assay measures insertion of full-length Tf1 cDNA mediated by the transposon integrase, the homologous recombination assay measures levels of cDNA present in the nucleus and is independent of integrase activity. Combined, these assays can be used to systematically screen large collections of strains to identify mutations that specifically inhibit the integration step in the retroelement life cycle. Such mutations can be identified because they reduce transposition activity but nevertheless have wild-type frequencies of homologous recombination. Qualitative assays of yeast patches on agar plates detect large defects in integration and recombination, while the quantitative approach provides a precise method of determining integration and recombination frequencies.

  4. Production of recombinant porcine colipase secreted by Pichia pastoris and its application to improve dietary fat digestion and growth of postweaning piglets.

    PubMed

    Liu, Fang-Chueh; Chen, Hsiao-Ling; Chong, Kowit-Yu; Hsu, A-Li; Chen, Chuan-Mu

    2008-01-01

    Recombinant porcine pancreatic colipase (pCoL) was produced in the methylotrophic yeast Pichia pastoris. A synthetic yeast secretion cassette was constructed with the constitutive promoter of the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene and the yeast alpha-mating factor signal peptide. The pCoL cDNA corresponding in the coding sequence, excluding a 16-amino acid segment of the native signal sequence, was cloned into the pGAPZalphaB vector and integrated into the genome of P. pastoris. Yeast transformants were cultured and bioactive pCoL protein was detected in the supernatant at a high-level of 126.8 mg/L after 3 days of culture. The transformed yeast containing the highest recombinant colipase level (pCoL yeast) and native yeast GS 115 not containing pCoL (non-pCoL yeast, as a control group) were separately cultured and the supernatants were adsorbed by dried skim milk. In an animal trial, two concentrations of colipase activity (0 vs. 5,000 U/kg in the diet) were blended with the pig corn-soybean basal diet and fed to weaned piglets for 4 weeks. The pCoL-administrated test group gained significantly more weight than piglets in the control group when measured at Day 15 (11.84 +/- 0.70 vs. 10.59 +/- 0.39 kg, P < 0.05), Day 22 (15.84 +/- 0.95 vs. 14.32 +/- 0.59 kg, P < 0.01), and Day 28 (20.19 +/- 1.47 vs. 18.54 +/- 0.92 kg, P < 0.01) after weaning. The blood triglyceride (TG) concentrations were significantly increased in the experimental test group that received recombinant colipase on the 28th day of postweaning when compared with that of the control group (32.50 vs. 16.37 mg/dL; P < 0.0001). These experimental data suggest that the use of recombinant porcine colipase as a dietary supplement provides an alternative approach for improving fat digestion and enhancing growth in postweaning piglets.

  5. Primers-4-Yeast: a comprehensive web tool for planning primers for Saccharomyces cerevisiae.

    PubMed

    Yofe, Ido; Schuldiner, Maya

    2014-02-01

    The budding yeast Saccharomyces cerevisiae is a key model organism of functional genomics, due to its ease and speed of genetic manipulations. In fact, in this yeast, the requirement for homologous sequences for recombination purposes is so small that 40 base pairs (bp) are sufficient. Hence, an enormous variety of genetic manipulations can be performed by simply planning primers with the correct homology, using a defined set of transformation plasmids. Although designing primers for yeast transformations and for the verification of their correct insertion is a common task in all yeast laboratories, primer planning is usually done manually and a tool that would enable easy, automated primer planning for the yeast research community is still lacking. Here we introduce Primers-4-Yeast, a web tool that allows primers to be designed in batches for S. cerevisiae gene-targeting transformations, and for the validation of correct insertions. This novel tool enables fast, automated, accurate primer planning for large sets of genes, introduces consistency in primer planning and is therefore suggested to serve as a standard in yeast research. Primers-4-Yeast is available at: http://www.weizmann.ac.il/Primers-4-Yeast Copyright © 2013 John Wiley & Sons, Ltd.

  6. How Hot Are Drosophila Hotspots? Examining Recombination Rate Variation and Associations with Nucleotide Diversity, Divergence, and Maternal Age in Drosophila pseudoobscura

    PubMed Central

    Manzano-Winkler, Brenda; McGaugh, Suzanne E.; Noor, Mohamed A. F.

    2013-01-01

    Fine scale meiotic recombination maps have uncovered a large amount of variation in crossover rate across the genomes of many species, and such variation in mammalian and yeast genomes is concentrated to <5kb regions of highly elevated recombination rates (10–100x the background rate) called “hotspots.” Drosophila exhibit substantial recombination rate heterogeneity across their genome, but evidence for these highly-localized hotspots is lacking. We assayed recombination across a 40Kb region of Drosophila pseudoobscura chromosome 2, with one 20kb interval assayed every 5Kb and the adjacent 20kb interval bisected into 10kb pieces. We found that recombination events across the 40kb stretch were relatively evenly distributed across each of the 5kb and 10kb intervals, rather than concentrated in a single 5kb region. This, in combination with other recent work, indicates that the recombination landscape of Drosophila may differ from the punctate recombination pattern observed in many mammals and yeast. Additionally, we found no correlation of average pairwise nucleotide diversity and divergence with recombination rate across the 20kb intervals, nor any effect of maternal age in weeks on recombination rate in our sample. PMID:23967224

  7. Optimal quality control of bakers' yeast fed-batch culture using population dynamics.

    PubMed

    Dairaku, K; Izumoto, E; Morikawa, H; Shioya, S; Takamatsu, T

    1982-12-01

    An optimal quality control policy for the overall specific growth rate of bakers' yeast, which maximizes the fermentative activity in the making of bread, was obtained by direct searching based on the mathematical model proposed previously. The mathematical model had described the age distribution of bakers' yeast which had an essential relationship to the ability of fermentation in the making of bread. The mathematical model is a simple aging model with two periods: Nonbudding and budding. Based on the result obtained by direct searching, the quality control of bakers' yeast fed-batch culture was performed and confirmed to be experimentally valid.

  8. Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1).

    PubMed

    Bragança, Caio Roberto Soares; Colombo, Lívia Tavares; Roberti, Alvaro Soares; Alvim, Mariana Caroline Tocantins; Cardoso, Silvia Almeida; Reis, Kledna Constancio Portes; de Paula, Sérgio Oliveira; da Silveira, Wendel Batista; Passos, Flavia Maria Lopes

    2015-02-01

    The yeast Kluyveromyces marxianus is a convenient host for industrial synthesis of biomolecules. However, despite its potential, there are few studies reporting the expression of heterologous proteins using this yeast. Here, we report expression of a dengue virus protein in K. marxianus for the first time. The dengue virus type 1 nonstructural protein 1 (NS1) was integrated into the K. marxianus UFV-3 genome at the LAC4 locus using an adapted integrative vector designed for high-level expression of recombinant protein in Kluyveromyces lactis. The NS1 gene sequence was codon-optimized to increase the level of protein expression in yeast. The synthetic gene was cloned in frame with K. lactis α-mating factor signal peptide, and the recombinant plasmid obtained was used to transform K. marxianus UFV-3 by electroporation. The transformed cells, selected in yeast extract peptone dextrose containing 200 μg mL(-1) Geneticin, were mitotically stable. Analysis of recombinant strains by RT-PCR and protein detection using blot analysis confirmed both transcription and expression of extracellular NS1 polypeptide. After induction with galactose, the NS1 protein was analyzed by sodium dodecyl sulfate-PAGE and immunogenic detection. Protein production was investigated under two conditions: with galactose and biotin pulses at 24-h intervals during 96 h of induction and without galactose and biotin supplementation. Protease activity was not detected in post-growth medium. Our results indicate that recombinant K. marxianus is a good host for the production of dengue virus NS1 protein, which has potential for diagnostic applications.

  9. Deciphering how LIP2 and POX2 promoters can optimally regulate recombinant protein production in the yeast Yarrowia lipolytica.

    PubMed

    Sassi, Hosni; Delvigne, Frank; Kar, Tambi; Nicaud, Jean-Marc; Coq, Anne-Marie Crutz-Le; Steels, Sebastien; Fickers, Patrick

    2016-09-20

    In recent years, the non-conventional model yeast species Yarrowia lipolytica has received much attention because it is a useful cell factory for producing recombinant proteins. In this species, expression vectors involving LIP2 and POX2 promoters have been developed and used successfully for protein production at yields similar to or even higher than those of other cell factories, such as Pichia pastoris. However, production processes involving these promoters can be difficult to manage, especially if carried out at large scales in fed-batch bioreactors, because they require hydrophobic inducers, such as oleic acid or methyl oleate. Thus, the challenge has become to reduce loads of hydrophobic substrates while simultaneously promoting recombinant protein production. One possible solution is to replace a portion of the inducer with a co-substrate that can serve as an alternative energy source. However, implementing such an approach would require detailed knowledge of how carbon sources impact promoter regulation, which is surprisingly still lacking for the LIP2 and POX2 promoters. This study's aim was thus to better characterize promoter regulation and cell metabolism in Y. lipolytica cultures grown in media supplemented with different carbon sources. pPOX2 induction could be detected when glucose or glycerol was used as sole carbon source, which meant these carbon source could not prevent promoter induction. In addition, when a mixture of glucose and oleic acid was used in complex medium, pPOX2 induction level was lower that that of pLIP2. In contrast, pLIP2 induction was absent when glucose was present in the culture medium, which meant that cell growth could occur without any recombinant gene expression. When a 40/60 mixture of glucose and oleic acid (w/w) was used, a tenfold increase in promoter induction, as compared to when an oleic-acid-only medium was observed. It was also clear that individual cells were adapting metabolically to use both glucose and oleic

  10. Generation of a Uracil Auxotroph Strain of the Probiotic Yeast Saccharomyces boulardii as a Host for the Recombinant Protein Production

    PubMed Central

    Hamedi, Hassan; Misaghi, Ali; Modarressi, Mohammad Hossein; Salehi, Taghi Zahraei; Khorasanizadeh, Dorsa; Khalaj, Vahid

    2013-01-01

    Background Saccharomyces boulardii (S. boulardii) is the best known probiotic yeast. The genetic engineering of this probiotic strain requires the availability of appropriate mutants to accept various gene constructs carrying different selection markers. As the auxotrophy selection markers are under focus, we have generated a ura3 auxotroph mutant of S. boulardii for use in further genetic manipulations. Methods Classical UV mutagenesis was used for the generation of auxotroph mutants. The mutants were selected in the presence of 5-FOA (5-Fluoroorotic acid), uracil and uridine. Uracil auxotrophy phenotype was confirmed by the ability of mutants to grow in the presence of uracil and the lack of growth in the absence of this compound. To test whether the uracil auxotrophy phenotype is due to the inactivation of URA3, the mutants were transformed with a plasmid carrying the gene. An in vitro assay was used for the analysis of acid and bile resistance capacity of these mutants. Results Three mutants were found to be ura3 auxotroph as they were able to grow only in the presence of uracil. When the URA3 gene was added, these mutants were able to grow normally in the absence of uracil. Further in vitro analysis showed that the acid and bile resistance capacity of one of these mutants is intact and similar to the wild type. Conclusion A uracil auxotroph mutant of the probiotic yeast, S. boulardii, was generated and characterized. This auxotroph strain may have potential applications in the production and delivery of the recombinant pharmacuetics into the intestinal lumen. PMID:23626874

  11. DNA recombination protein-dependent mechanism of homoplasmy and its proposed functions.

    PubMed

    Shibata, Takehiko; Ling, Feng

    2007-01-01

    Homoplasmy is a basic genetic state of mitochondria, in which all of the hundreds to thousands of mitochondrial (mt)DNA copies within a cell or an individual have the same nucleotide-sequence. It was recently found that "vegetative segregation" to generate homoplasmic cells is an active process under genetic control. In the yeast Saccharomyces cerevisiae, the Mhr1 protein which catalyzes a key reaction in mtDNA homologous recombination, plays a pivotal role in vegetative segregation. Conversely, within the nuclear genome, homologous DNA recombination causes genetic diversity. Considering these contradictory roles of this key reaction in DNA recombination, possible functions of homoplasmy are discussed.

  12. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast.

    PubMed

    Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L; Hallström, Björn M; Liu, Zihe; Petranovic, Dina; Uhlén, Mathias; Joensson, Haakan N; Andersson-Svahn, Helene; Nielsen, Jens

    2015-08-25

    There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.

  13. Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

    PubMed

    Khulape, S A; Maity, H K; Pathak, D C; Mohan, C Madhan; Dey, S

    2015-09-01

    The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine.

  14. Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast

    PubMed Central

    Li, Ping; Jin, Hui; Yu, Hong-Guo

    2014-01-01

    During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. PMID:25103240

  15. Fractionation of yeast extract by nanofiltration process to assess key compounds involved in CHO cell culture improvement.

    PubMed

    Mosser, Mathilde; Kapel, Romain; Chevalot, Isabelle; Olmos, Eric; Marc, Ivan; Marc, Annie; Oriol, Eric

    2015-01-01

    Yeast extract (YE) is known to greatly enhance mammalian cell culture performances, but its undefined composition decreases process reliability. Accordingly, in the present study, the nature of YE compounds involved in the improvement of recombinant CHO cell growth and IgG production was investigated. First, the benefits of YE were verified, revealing that it increased maximal concentrations of viable cells and IgG up to 73 and 60%, respectively compared to a reference culture. Then, the analyses of YE composition highlighted the presence of molecules such as amino acids, vitamins, salts, nucleobase, and glucose that were contained in reference medium, while others including peptides, trehalose, polysaccharides, and nucleic acids were not. Consequently, YE was fractionated by a nanofiltration process to deeper evaluate its effects on CHO cell cultures. The YE molecules already contained in reference medium were mainly isolated in the permeate fraction together with trehalose and short peptides, while other molecules were concentrated in the retentate. Permeate, which was free of macromolecules, exhibited a similar positive effect than raw YE on maximal concentrations. Additional studies on cell energetic metabolism underlined that dipeptides and tripeptides in permeate were used as an efficient source of nitrogenous substrates. © 2015 American Institute of Chemical Engineers.

  16. Comparison of melibiose utilizing baker's yeast strains produced by genetic engineering and classical breeding.

    PubMed

    Vincent, S F; Bell, P J; Bissinger, P; Nevalainen, K M

    1999-02-01

    Yeast strains currently used in the baking industry cannot fully utilize the trisaccharide raffinose found in beet molasses due to the absence of melibiase (alpha-galactosidase) activity. To overcome this deficiency, the MEL1 gene encoding melibiase enzyme was introduced into baker's yeast by both classical breeding and recombinant DNA technology. Both types of yeast strains were capable of vigorous fermentation in the presence of high levels of sucrose, making them suitable for the rapidly developing Asian markets where high levels of sugar are used in bread manufacture. Melibiase expression appeared to be dosage-dependent, with relatively low expression sufficient for complete melibiose utilization in a model fermentation system.

  17. Cryopreservative effects of the recombinant ice-binding protein from the arctic yeast Leucosporidium sp. on red blood cells.

    PubMed

    Lee, Sung Gu; Koh, Hye Yeon; Lee, Jun Hyuck; Kang, Sung-Ho; Kim, Hak Jun

    2012-06-01

    Antifreeze proteins (AFPs) have important functions in many freeze-tolerant organisms. The proteins non-colligatively lower the freezing point and functionally inhibit ice recrystallization in frozen solutions. In our previous studies, we found that the Arctic yeast Leucosporidium sp. produces an AFP (LeIBP), and that the protein could be successfully produced in Pichia expression system. The present study showed that recombinant LeIBP possesses the ability to reduce the damage induced to red blood cells (RBCs) by freeze thawing. In addition to 40 % glycerol, both 0.4 and 0.8 mg/ml LeIBPs significantly reduced freeze-thaw-induced hemolysis at either rapid- (45 °C) or slow-warming (22 °C) temperatures. Post-thaw cell counts of the cryopreserved RBCs were dramatically enhanced, in particular, in 0.8 mg/ml LeIBP. Interestingly, the cryopreserved cells in the presence of LeIBP showed preserved cell size distribution. These results indicate that the ability of LeIBP to inhibit ice recrystallization helps the RBCs avoid critically damaging electrolyte concentrations, which are known as solution effects. Considering all these data, LeIBP can be thought of as a key component in improving RBC cryopreservation efficiency.

  18. High-throughput measurement of recombination rates and genetic interference in Saccharomyces cerevisiae.

    PubMed

    Raffoux, Xavier; Bourge, Mickael; Dumas, Fabrice; Martin, Olivier C; Falque, Matthieu

    2018-06-01

    Allelic recombination owing to meiotic crossovers is a major driver of genome evolution, as well as a key player for the selection of high-performing genotypes in economically important species. Therefore, we developed a high-throughput and low-cost method to measure recombination rates and crossover patterning (including interference) in large populations of the budding yeast Saccharomyces cerevisiae. Recombination and interference were analysed by flow cytometry, which allows time-consuming steps such as tetrad microdissection or spore growth to be avoided. Moreover, our method can also be used to compare recombination in wild-type vs. mutant individuals or in different environmental conditions, even if the changes in recombination rates are small. Furthermore, meiotic mutants often present recombination and/or pairing defects affecting spore viability but our method does not involve growth steps and thus avoids filtering out non-viable spores. Copyright © 2018 John Wiley & Sons, Ltd.

  19. Mechanisms and Regulation of Mitotic Recombination in Saccharomyces cerevisiae

    PubMed Central

    Symington, Lorraine S.; Rothstein, Rodney; Lisby, Michael

    2014-01-01

    Homology-dependent exchange of genetic information between DNA molecules has a profound impact on the maintenance of genome integrity by facilitating error-free DNA repair, replication, and chromosome segregation during cell division as well as programmed cell developmental events. This chapter will focus on homologous mitotic recombination in budding yeast Saccharomyces cerevisiae. However, there is an important link between mitotic and meiotic recombination (covered in the forthcoming chapter by Hunter et al. 2015) and many of the functions are evolutionarily conserved. Here we will discuss several models that have been proposed to explain the mechanism of mitotic recombination, the genes and proteins involved in various pathways, the genetic and physical assays used to discover and study these genes, and the roles of many of these proteins inside the cell. PMID:25381364

  20. Metabolic reconstruction and flux analysis of industrial Pichia yeasts.

    PubMed

    Chung, Bevan Kai-Sheng; Lakshmanan, Meiyappan; Klement, Maximilian; Ching, Chi Bun; Lee, Dong-Yup

    2013-03-01

    Pichia yeasts have been recognized as important microbial cell factories in the biotechnological industry. Notably, the Pichia pastoris and Pichia stipitis species have attracted much research interest due to their unique cellular physiology and metabolic capability: P. pastoris has the ability to utilize methanol for cell growth and recombinant protein production, while P. stipitis is capable of assimilating xylose to produce ethanol under oxygen-limited conditions. To harness these characteristics for biotechnological applications, it is highly required to characterize their metabolic behavior. Recently, following the genome sequencing of these two Pichia species, genome-scale metabolic networks have been reconstructed to model the yeasts' metabolism from a systems perspective. To date, there are three genome-scale models available for each of P. pastoris and P. stipitis. In this mini-review, we provide an overview of the models, discuss certain limitations of previous studies, and propose potential future works that can be conducted to better understand and engineer Pichia yeasts for industrial applications.

  1. Simple method to detect triacylglycerol biosynthesis in a yeast-based recombinant system

    USDA-ARS?s Scientific Manuscript database

    Standard methods to quantify the activity of triacylglycerol (TAG) synthesizing enzymes DGAT and PDAT (TAG-SE) require a sensitive but rather arduous laboratory assay based on radio-labeled substrates. Here we describe two straightforward methods to detect TAG production in baker’s yeast Saccharomyc...

  2. The proteasomal Rpn11 metalloprotease suppresses tombusvirus RNA recombination and promotes viral replication via facilitating assembly of the viral replicase complex.

    PubMed

    Prasanth, K Reddisiva; Barajas, Daniel; Nagy, Peter D

    2015-03-01

    RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role

  3. The Proteasomal Rpn11 Metalloprotease Suppresses Tombusvirus RNA Recombination and Promotes Viral Replication via Facilitating Assembly of the Viral Replicase Complex

    PubMed Central

    Prasanth, K. Reddisiva; Barajas, Daniel

    2014-01-01

    ABSTRACT RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a “matchmaker” that brings the viral p92pol replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. IMPORTANCE RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA

  4. Integrated lipase production and in situ biodiesel synthesis in a recombinant Pichia pastoris yeast: an efficient dual biocatalytic system composed of cell free enzymes and whole cell catalysts

    PubMed Central

    2014-01-01

    Background Lipase-catalyzed biotransformation of acylglycerides or fatty acids into biodiesel via immobilized enzymes or whole cell catalysts has been considered as one of the most promising methods to produce renewable and environmentally friendly alternative liquid fuels, thus being extensively studied so far. In all previously pursued approaches, however, lipase enzymes are prepared in an independent process separated from enzymatic biodiesel production, which would unavoidably increase the cost and energy consumption during industrial manufacture of this cost-sensitive energy product. Therefore, there is an urgent need to develop novel cost-effective biocatalysts and biocatalytic processes with genuine industrial feasibility. Result Inspired by the consolidated bioprocessing of lignocellulose to generate bioethanol, an integrated process with coupled lipase production and in situ biodiesel synthesis in a recombinant P. pastoris yeast was developed in this study. The novel and efficient dual biocatalytic system based on Thermomyces lanuginosus lipase took advantage of both cell free enzymes and whole cell catalysts. The extracellular and intracellular lipases of growing yeast cells were simultaneously utilized to produce biodiesel from waste cooking oils in situ and in one pot. This integrated system effectively achieved 58% and 72% biodiesel yield via concurrent esterified-transesterified methanolysis and stepwise hydrolysis-esterification at 3:1 molar ratio between methanol and waste cooking oils, respectively. Further increasing the molar ratio of methanol to waste cooking oils to 6:1 led to an 87% biodiesel yield using the stepwise strategy. Both water tolerance and methanol tolerance of this novel system were found to be significantly improved compared to previous non-integrated biodiesel production processes using separately prepared immobilized enzymes or whole cell catalysts. Conclusion We have proposed a new concept of integrated biodiesel production

  5. Effect of chromosome tethering on nuclear organization in yeast.

    PubMed

    Avşaroğlu, Barış; Bronk, Gabriel; Gordon-Messer, Susannah; Ham, Jungoh; Bressan, Debra A; Haber, James E; Kondev, Jane

    2014-01-01

    Interphase chromosomes in Saccharomyces cerevisiae are tethered to the nuclear envelope at their telomeres and to the spindle pole body (SPB) at their centromeres. Using a polymer model of yeast chromosomes that includes these interactions, we show theoretically that telomere attachment to the nuclear envelope is a major determinant of gene positioning within the nucleus only for genes within 10 kb of the telomeres. We test this prediction by measuring the distance between the SPB and the silent mating locus (HML) on chromosome III in wild-type and mutant yeast strains that contain altered chromosome-tethering interactions. In wild-type yeast cells we find that disruption of the telomere tether does not dramatically change the position of HML with respect to the SPB, in agreement with theoretical predictions. Alternatively, using a mutant strain with a synthetic tether that localizes an HML-proximal site to the nuclear envelope, we find a significant change in the SPB-HML distance, again as predicted by theory. Our study quantifies the importance of tethering at telomeres on the organization of interphase chromosomes in yeast, which has been shown to play a significant role in determining chromosome function such as gene expression and recombination.

  6. Construction of a recombinant wine yeast strain expressing beta-(1,4)-endoglucanase and its use in microvinification processes.

    PubMed Central

    Pérez-González, J A; González, R; Querol, A; Sendra, J; Ramón, D

    1993-01-01

    A genetic transformation system for an industrial wine yeast strain is presented here. The system is based on the acquisition of cycloheximide resistance and is a direct adaptation of a previously published procedure for brewing yeasts (L. Del Pozo, D. Abarca, M. G. Claros, and A. Jiménez, Curr. Genet. 19:353-358, 1991). Transformants arose at an optimal frequency of 0.5 transformant per microgram of DNA, are stable in the absence of selective pressure, and produce wine in the same way as the untransformed industrial strain. By using this transformation protocol, a filamentous fungal beta-(1,4)-endoglucanase gene has been expressed in an industrial wine yeast under the control of the yeast actin gene promoter. Endoglucanolytic wine yeast secretes the fungal enzyme to the must, producing a wine with an increased fruity aroma. Images PMID:8215355

  7. A Molecular Portrait of De Novo Genes in Yeasts.

    PubMed

    Vakirlis, Nikolaos; Hebert, Alex S; Opulente, Dana A; Achaz, Guillaume; Hittinger, Chris Todd; Fischer, Gilles; Coon, Joshua J; Lafontaine, Ingrid

    2018-03-01

    New genes, with novel protein functions, can evolve "from scratch" out of intergenic sequences. These de novo genes can integrate the cell's genetic network and drive important phenotypic innovations. Therefore, identifying de novo genes and understanding how the transition from noncoding to coding occurs are key problems in evolutionary biology. However, identifying de novo genes is a difficult task, hampered by the presence of remote homologs, fast evolving sequences and erroneously annotated protein coding genes. To overcome these limitations, we developed a procedure that handles the usual pitfalls in de novo gene identification and predicted the emergence of 703 de novo gene candidates in 15 yeast species from 2 genera whose phylogeny spans at least 100 million years of evolution. We validated 85 candidates by proteomic data, providing new translation evidence for 25 of them through mass spectrometry experiments. We also unambiguously identified the mutations that enabled the transition from noncoding to coding for 30 Saccharomyces de novo genes. We established that de novo gene origination is a widespread phenomenon in yeasts, only a few being ultimately maintained by selection. We also found that de novo genes preferentially emerge next to divergent promoters in GC-rich intergenic regions where the probability of finding a fortuitous and transcribed ORF is the highest. Finally, we found a more than 3-fold enrichment of de novo genes at recombination hot spots, which are GC-rich and nucleosome-free regions, suggesting that meiotic recombination contributes to de novo gene emergence in yeasts.

  8. Oral administration of myostatin-specific recombinant Saccharomyces cerevisiae vaccine in rabbit.

    PubMed

    Liu, Zhongtian; Zhou, Gang; Ren, Chonghua; Xu, Kun; Yan, Qiang; Li, Xinyi; Zhang, Tingting; Zhang, Zhiying

    2016-04-29

    Yeast is considered as a simple and cost-effective host for protein expression, and our previous studies have proved that Saccharomyces cerevisiae can deliver recombinant protein and DNA into mouse dendritic cells and can further induce immune responses as novel vaccines. In order to know whether similar immune responses can be induced in rabbit by oral administration of such recombinant S. cerevisiae vaccine, we orally fed the rabbits with heat-inactivated myostatin-recombinant S. cerevisiae for 5 weeks, and then myostatin-specific antibody in serum was detected successfully by western blotting and ELISA assay. The rabbits treated with myostatin-recombinant S. cerevisiae vaccine grew faster and their muscles were much heavier than that of the control group. As a common experimental animal and a meat livestock with great economic value, rabbit was proved to be the second animal species that have been successfully orally immunized by recombinant S. cerevisiae vaccine after mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. The induction of mutation and recombination following UV irradiation during meiosis in Saccharomyces cerevisiae.

    PubMed

    Kelly, S L; Parry, J M

    1983-03-01

    Irradiation of yeast cultures with ultraviolet light at discrete stages during meiosis produces cyclic variations in sensitivity, i.e. cells are more sensitive to the lethal effects of UV light prior to entry into the meiotic DNA synthesis, and this corresponds to a peak of induction of point mutation. Cells become more resistant to both induced point mutation and lethality as they enter meiotic DNA synthesis, but become more sensitive again during spore formation. The induced level of intragenic recombination rises during the period of commitment to recombination to a level indistinguishable from the full meiotic level of spontaneous intragenic recombination. Induced reciprocal recombination remains above the spontaneous level up to the point of commitment to sporulation.

  10. Over expression of anti-MUC1 single-domain antibody fragments in the yeast Pichia pastoris.

    PubMed

    Rahbarizadeh, Fatemeh; Rasaee, Mohammad J; Forouzandeh, Mehdi; Allameh, Abdol-Amir

    2006-02-01

    The methylotrophic yeast Pichia pastoris has become a highly popular expression host system for the recombinant production of a wide variety of proteins, such as antibody fragments. Camelids produce functional antibodies devoid of light chains and constant heavy-chain domain (CH1). The antigen binding fragments of such heavy chain antibodies are therefore comprised in one single domain, the so-called VH of the camelid heavy chain antibody (VHH). To test the feasibility of expressing VHHs in the yeast, which on account of their small size and antigen recognition properties would have a major impact on antibody engineering strategies, we constructed two VHH genes encoding the single-domain antibody fragments with specificity for a cancer associated mucin, MUC1. The recombinant strains of the yeast P. pastoris were developed which secrete single-domain antibody fragment to the culture supernatant as a biologically active protein. Supplementation of medium with sorbitol (in pre-induction phase) and casamino acid or EDTA (in induction phase) provided ideal condition of increasing the yield of VHH production compared to culture condition devoid of above recipe. The secreted protein was purified following a 80% ammonium sulfate precipitation step, followed by a affinity chromatography column. The specific activity in enzyme-linked immunosorbant assay (ELISA) of the purified yeast VHH was higher than that of a bacterial periplasmic counterpart. These results reaffirm that the yeast P. pastoris is a suitable host for high level and correctly folded production of VHH antibody fragments with potential in vivo diagnostic and therapeutic applications. This is the first report of expression of VHH in P. pastoris.

  11. Construction of a self-cloning sake yeast that overexpresses alcohol acetyltransferase gene by a two-step gene replacement protocol.

    PubMed

    Hirosawa, I; Aritomi, K; Hoshida, H; Kashiwagi, S; Nishizawa, Y; Akada, R

    2004-07-01

    The commercial application of genetically modified industrial microorganisms has been problematic due to public concerns. We constructed a "self-cloning" sake yeast strain that overexpresses the ATF1 gene encoding alcohol acetyltransferase, to improve the flavor profile of Japanese sake. A constitutive yeast overexpression promoter, TDH3p, derived from the glyceraldehyde-3-phosphate dehydrogenase gene from sake yeast was fused to ATF1; and the 5' upstream non-coding sequence of ATF1 was further fused to TDH3p-ATF1. The fragment was placed on a binary vector, pGG119, containing a drug-resistance marker for transformation and a counter-selection marker for excision of unwanted DNA. The plasmid was integrated into the ATF1 locus of a sake yeast strain. This integration constructed tandem repeats of ATF1 and TDH3p-ATF1 sequences, between which the plasmid was inserted. Loss of the plasmid, which occurs through homologous recombination between either the TDH3p downstream ATF1 repeats or the TDH3p upstream repeat sequences, was selected by growing transformants on counter-selective medium. Recombination between the downstream repeats led to reversion to a wild type strain, but that between the upstream repeats resulted in a strain that possessed TDH3p-ATF1 without the extraneous DNA sequences. The self-cloning TDH3p-ATF1 yeast strain produced a higher amount of isoamyl acetate. This is the first expression-controlled self-cloning industrial yeast.

  12. Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors.

    PubMed

    Vega, Juan M; Yu, Weichang; Han, Fangpu; Kato, Akio; Peters, Eric M; Zhang, Zhanyuan J; Birchler, James A

    2008-04-01

    The Cre/loxP site-specific recombination system has been applied in various plant species including maize (Zea mays) for marker gene removal, gene targeting, and functional genomics. A BIBAC vector system was adapted for maize transformation with a large fragment of genetic material including a herbicide resistance marker gene, a 30 kb yeast genomic fragment as a marker for fluorescence in situ hybridization (FISH), and a 35S-lox-cre recombination cassette. Seventy-five transgenic lines were generated from Agrobacterium-mediated transformation of a maize Hi II line with multiple B chromosomes. Eighty-four inserts have been localized among all 10 A chromosome pairs by FISH using the yeast DNA probe together with a karyotyping cocktail. No inserts were found on the B chromosomes; thus a bias against the B chromosomes by the Agrobacterium-mediated transformation was revealed. The expression of a cre gene was confirmed in 68 of the 75 transgenic lines by a reporter construct for cre/lox mediated recombination. The placement of the cre/lox site-specific recombination system in many locations in the maize genome will be valuable materials for gene targeting and chromosome engineering.

  13. 'Yeast mail': a novel Saccharomyces application (NSA) to encrypt messages.

    PubMed

    Rosemeyer, Helmut; Paululat, Achim; Heinisch, Jürgen J

    2014-09-01

    The universal genetic code is used by all life forms to encode biological information. It can also be used to encrypt semantic messages and convey them within organisms without anyone but the sender and recipient knowing, i.e., as a means of steganography. Several theoretical, but comparatively few experimental, approaches have been dedicated to this subject, so far. Here, we describe an experimental system to stably integrate encrypted messages within the yeast genome using a polymerase chain reaction (PCR)-based, one-step homologous recombination system. Thus, DNA sequences encoding alphabetical and/or numerical information will be inherited by yeast propagation and can be sent in the form of dried yeast. Moreover, due to the availability of triple shuttle vectors, Saccharomyces cerevisiae can also be used as an intermediate construction device for transfer of information to either Drosophila or mammalian cells as steganographic containers. Besides its classical use in alcoholic fermentation and its modern use for heterologous gene expression, we here show that baker's yeast can thus be employed in a novel Saccharomyces application (NSA) as a simple steganographic container to hide and convey messages. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

  14. A role for recombination junctions in the segregation of mitochondrial DNA in yeast.

    PubMed

    Lockshon, D; Zweifel, S G; Freeman-Cook, L L; Lorimer, H E; Brewer, B J; Fangman, W L

    1995-06-16

    In S. cerevisiae, mitochondrial DNA (mtDNA) molecules, in spite of their high copy number, segregate as if there were a small number of heritable units. The rapid segregation of mitochondrial genomes can be analyzed using mtDNA deletion variants. These small, amplified genomes segregate preferentially from mixed zygotes relative to wild-type mtDNA. This segregation advantage is abolished by mutations in a gene, MGT1, that encodes a recombination junction-resolving enzyme. We show here that resolvase deficiency causes a larger proportion of molecules to be linked together by recombination junctions, resulting in the aggregation of mtDNA into a small number of cytological structures. This change in mtDNA structure can account for the increased mitotic loss of mtDNA and the altered pattern of mtDNA segregation from zygotes. We propose that the level of unresolved recombination junctions influences the number of heritable units of mtDNA.

  15. Unveiling the mystery of mitochondrial DNA replication in yeasts.

    PubMed

    Chen, Xin Jie; Clark-Walker, George Desmond

    2018-01-01

    Conventional DNA replication is initiated from specific origins and requires the synthesis of RNA primers for both the leading and lagging strands. In contrast, the replication of yeast mitochondrial DNA is origin-independent. The replication of the leading strand is likely primed by recombinational structures and proceeded by a rolling circle mechanism. The coexistent linear and circular DNA conformers facilitate the recombination-based initiation. The replication of the lagging strand is poorly understood. Re-evaluation of published data suggests that the rolling circle may also provide structures for the synthesis of the lagging-strand by mechanisms such as template switching. Thus, the coupling of recombination with rolling circle replication and possibly, template switching, may have been selected as an economic replication mode to accommodate the reductive evolution of mitochondria. Such a replication mode spares the need for conventional replicative components, including those required for origin recognition/remodelling, RNA primer synthesis and lagging-strand processing. Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  16. The organization of repeating units in mitochondrial DNA from yeast petite mutants.

    PubMed

    Bos, J L; Heyting, C; Van der Horst, G; Borst, P

    1980-04-01

    We have reinvestigated the linkage orientation of repeating units in mtDNAs of yeast ρ(-) petite mutants containing an inverted duplication. All five petite mtDNAs studied contain a continuous segment of wild-type mtDNA, part of which is duplicated and present in inverted form in the repeat. We show by restriction enzyme analysis that the non-duplicated segments between the inverted duplications are present in random orientation in all five petite mtDNAs. There is no segregation of sub-types with unique orientation. We attribute this to the high rate of intramolecular recombination between the inverted duplications. The results provide additional evidence for the high rate of recombination of yeast mtDNA even in haploid ρ(-) petite cells.We conclude that only two types of stable sequence organization exist in petite mtDNA: petites without an inverted duplication have repeats linked in straight head-to-tail arrangement (abcabc); petites with an inverted duplication have repeats in which the non-duplicated segments are present in random orientation.

  17. Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system.

    PubMed

    Yang, Fang; Lei, Yingying; Zhou, Meiling; Yao, Qili; Han, Yichao; Wu, Xiang; Zhong, Wanshun; Zhu, Chenghang; Xu, Weize; Tao, Ran; Chen, Xi; Lin, Da; Rahman, Khaista; Tyagi, Rohit; Habib, Zeshan; Xiao, Shaobo; Wang, Dang; Yu, Yang; Chen, Huanchun; Fu, Zhenfang; Cao, Gang

    2018-02-16

    Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based 'library vs library' Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome-lysosome fusion. This concept can also be applied to other systems to screen protein-RNA and protein-DNA interactions and delineate signaling landscape in cells.

  18. Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology

    PubMed Central

    Kouprina, Natalay; Larionov, Vladimir

    2016-01-01

    Transformation-associated recombination (TAR) cloning represents a unique tool for isolation and manipulation of large DNA molecules. The technique exploits a high level of homologous recombination in the yeast Sacharomyces cerevisiae. So far, TAR cloning is the only method available to selectively recover chromosomal segments up to 300 kb in length from complex and simple genomes. In addition, TAR cloning allows the assembly and cloning of entire microbe genomes up to several Mb as well as engineering of large metabolic pathways. In this review, we summarize applications of TAR cloning for functional/structural genomics and synthetic biology. PMID:27116033

  19. Effect of Chromosome Tethering on Nuclear Organization in Yeast

    PubMed Central

    Avşaroğlu, Barış; Bronk, Gabriel; Gordon-Messer, Susannah; Ham, Jungoh; Bressan, Debra A.; Haber, James E.; Kondev, Jane

    2014-01-01

    Interphase chromosomes in Saccharomyces cerevisiae are tethered to the nuclear envelope at their telomeres and to the spindle pole body (SPB) at their centromeres. Using a polymer model of yeast chromosomes that includes these interactions, we show theoretically that telomere attachment to the nuclear envelope is a major determinant of gene positioning within the nucleus only for genes within 10 kb of the telomeres. We test this prediction by measuring the distance between the SPB and the silent mating locus (HML) on chromosome III in wild–type and mutant yeast strains that contain altered chromosome-tethering interactions. In wild-type yeast cells we find that disruption of the telomere tether does not dramatically change the position of HML with respect to the SPB, in agreement with theoretical predictions. Alternatively, using a mutant strain with a synthetic tether that localizes an HML-proximal site to the nuclear envelope, we find a significant change in the SPB-HML distance, again as predicted by theory. Our study quantifies the importance of tethering at telomeres on the organization of interphase chromosomes in yeast, which has been shown to play a significant role in determining chromosome function such as gene expression and recombination. PMID:25020108

  20. Functional inclusion bodies produced in the yeast Pichia pastoris.

    PubMed

    Rueda, Fabián; Gasser, Brigitte; Sánchez-Chardi, Alejandro; Roldán, Mònica; Villegas, Sandra; Puxbaum, Verena; Ferrer-Miralles, Neus; Unzueta, Ugutz; Vázquez, Esther; Garcia-Fruitós, Elena; Mattanovich, Diethard; Villaverde, Antonio

    2016-10-01

    Bacterial inclusion bodies (IBs) are non-toxic protein aggregates commonly produced in recombinant bacteria. They are formed by a mixture of highly stable amyloid-like fibrils and releasable protein species with a significant extent of secondary structure, and are often functional. As nano structured materials, they are gaining biomedical interest because of the combination of submicron size, mechanical stability and biological activity, together with their ability to interact with mammalian cell membranes for subsequent cell penetration in absence of toxicity. Since essentially any protein species can be obtained as IBs, these entities, as well as related protein clusters (e.g., aggresomes), are being explored in biocatalysis and in biomedicine as mechanically stable sources of functional protein. One of the major bottlenecks for uses of IBs in biological interfaces is their potential contamination with endotoxins from producing bacteria. To overcome this hurdle, we have explored here the controlled production of functional IBs in the yeast Pichia pastoris (Komagataella spp.), an endotoxin-free host system for recombinant protein production, and determined the main physicochemical and biological traits of these materials. Quantitative and qualitative approaches clearly indicate the formation of IBs inside yeast, similar in morphology, size and biological activity to those produced in E. coli, that once purified, interact with mammalian cell membranes and penetrate cultured mammalian cells in absence of toxicity. Structurally and functionally similar from those produced in E. coli, the controlled production of IBs in P. pastoris demonstrates that yeasts can be used as convenient platforms for the biological fabrication of self-organizing protein materials in absence of potential endotoxin contamination and with additional advantages regarding, among others, post-translational modifications often required for protein functionality.

  1. Functional Genomics Using the Saccharomyces cerevisiae Yeast Deletion Collections.

    PubMed

    Nislow, Corey; Wong, Lai Hong; Lee, Amy Huei-Yi; Giaever, Guri

    2016-09-01

    Constructed by a consortium of 16 laboratories, the Saccharomyces genome-wide deletion collections have, for the past decade, provided a powerful, rapid, and inexpensive approach for functional profiling of the yeast genome. Loss-of-function deletion mutants were systematically created using a polymerase chain reaction (PCR)-based gene deletion strategy to generate a start-to-stop codon replacement of each open reading frame by homologous recombination. Each strain carries two molecular barcodes that serve as unique strain identifiers, enabling their growth to be analyzed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays or through the use of next-generation sequencing technologies. Functional profiling of the deletion collections, using either strain-by-strain or parallel assays, provides an unbiased approach to systematically survey the yeast genome. The Saccharomyces yeast deletion collections have proved immensely powerful in contributing to the understanding of gene function, including functional relationships between genes and genetic pathways in response to diverse genetic and environmental perturbations. © 2016 Cold Spring Harbor Laboratory Press.

  2. In vitro activity of monoclonal and recombinant yeast killer toxin-like antibodies against antibiotic-resistant gram-positive cocci.

    PubMed Central

    Conti, S.; Magliani, W.; Arseni, S.; Dieci, E.; Frazzi, R.; Salati, A.; Varaldo, P. E.; Polonelli, L.

    2000-01-01

    BACKGROUND: Monoclonal (mAbKT) and recombinant single-chain (scFvKT) anti-idiotypic antibodies were produced to represent the internal image of a yeast killer toxin (KT) characterized by a wide spectrum of antimicrobial activity, including gram-positive cocci. Pathogenic eukaryotic and prokaryotic microorganisms, such as Candida albicans, Pneumocystis carinii, and a multidrug-resistant strain of Mycobacterium tuberculosis, presenting specific, although yet undefined, KT-cell wall receptors (KTR), have proven to be killed in vitro by mAbKT and scFvKT. mAbKT and scFvKT exert a therapeutic effect in vivo in experimental models of candidiasis and pneumocystosis by mimicking the functional activity of protective antibodies naturally produced in humans against KTR of infecting microorganisms. The swelling tide of concern over increasing bacterial resistance to antibiotic drugs gives the impetus to develop new therapeutic compounds against microbial threat. Thus, the in vitro bactericidal activity of mAbKT and scFvKT against gram-positive, drug-resistant cocci of major epidemiological interest was investigated. MATERIALS AND METHODS: mAbKT and scFvKT generated by hybridoma and DNA recombinant technology from the spleen lymphocytes of mice immunized with a KT-neutralizing monoclonal antibody (mAb KT4) were used in a conventional colony forming unit (CFU) assay to determine, from a qualitative point of view, their bactericidal activity against Staphylococcus aureus, S. haemolyticus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae strains. These bacterial strains are characterized by different patterns of resistance to antibiotics, including methicillin, vancomycin, and penicillin. RESULTS: According to the experimental conditions adopted, no bacterial isolate proved to be resistant to the activity of mAbKT and scFvKT. CONCLUSIONS: scFvKT exerted a microbicidal activity against multidrug resistant bacteria, which may represent the basis for the drug modeling

  3. The kinetochore prevents centromere-proximal crossover recombination during meiosis

    PubMed Central

    Vincenten, Nadine; Kuhl, Lisa-Marie; Lam, Isabel; Oke, Ashwini; Kerr, Alastair RW; Hochwagen, Andreas; Fung, Jennifer; Keeney, Scott; Vader, Gerben; Marston, Adèle L

    2015-01-01

    During meiosis, crossover recombination is essential to link homologous chromosomes and drive faithful chromosome segregation. Crossover recombination is non-random across the genome, and centromere-proximal crossovers are associated with an increased risk of aneuploidy, including Trisomy 21 in humans. Here, we identify the conserved Ctf19/CCAN kinetochore sub-complex as a major factor that minimizes potentially deleterious centromere-proximal crossovers in budding yeast. We uncover multi-layered suppression of pericentromeric recombination by the Ctf19 complex, operating across distinct chromosomal distances. The Ctf19 complex prevents meiotic DNA break formation, the initiating event of recombination, proximal to the centromere. The Ctf19 complex independently drives the enrichment of cohesin throughout the broader pericentromere to suppress crossovers, but not DNA breaks. This non-canonical role of the kinetochore in defining a chromosome domain that is refractory to crossovers adds a new layer of functionality by which the kinetochore prevents the incidence of chromosome segregation errors that generate aneuploid gametes. DOI: http://dx.doi.org/10.7554/eLife.10850.001 PMID:26653857

  4. Evidence for a high mutation rate at rapidly evolving yeast centromeres.

    PubMed

    Bensasson, Douda

    2011-07-18

    Although their role in cell division is essential, centromeres evolve rapidly in animals, plants and yeasts. Unlike the complex centromeres of plants and aminals, the point centromeres of Saccharomcyes yeasts can be readily sequenced to distinguish amongst the possible explanations for fast centromere evolution. Using DNA sequences of all 16 centromeres from 34 strains of Saccharomyces cerevisiae and population genomic data from Saccharomyces paradoxus, I show that centromeres in both species evolve 3 times more rapidly even than selectively unconstrained DNA. Exceptionally high levels of polymorphism seen in multiple yeast populations suggest that rapid centromere evolution does not result from the repeated selective sweeps expected under meiotic drive. I further show that there is little evidence for crossing-over or gene conversion within centromeres, although there is clear evidence for recombination in their immediate vicinity. Finally I show that the mutation spectrum at centromeres is consistent with the pattern of spontaneous mutation elsewhere in the genome. These results indicate that rapid centromere evolution is a common phenomenon in yeast species. Furthermore, these results suggest that rapid centromere evolution does not result from the mutagenic effect of gene conversion, but from a generalised increase in the mutation rate, perhaps arising from the unusual chromatin structure at centromeres in yeast and other eukaryotes.

  5. Metabolic regulation and maximal reaction optimization in the central metabolism of a yeast cell

    NASA Astrophysics Data System (ADS)

    Kasbawati, Gunawan, A. Y.; Hertadi, R.; Sidarto, K. A.

    2015-03-01

    Regulation of fluxes in a metabolic system aims to enhance the production rates of biotechnologically important compounds. Regulation is held via modification the cellular activities of a metabolic system. In this study, we present a metabolic analysis of ethanol fermentation process of a yeast cell in terms of continuous culture scheme. The metabolic regulation is based on the kinetic formulation in combination with metabolic control analysis to indicate the key enzymes which can be modified to enhance ethanol production. The model is used to calculate the intracellular fluxes in the central metabolism of the yeast cell. Optimal control is then applied to the kinetic model to find the optimal regulation for the fermentation system. The sensitivity results show that there are external and internal control parameters which are adjusted in enhancing ethanol production. As an external control parameter, glucose supply should be chosen in appropriate way such that the optimal ethanol production can be achieved. For the internal control parameter, we find three enzymes as regulation targets namely acetaldehyde dehydrogenase, pyruvate decarboxylase, and alcohol dehydrogenase which reside in the acetaldehyde branch. Among the three enzymes, however, only acetaldehyde dehydrogenase has a significant effect to obtain optimal ethanol production efficiently.

  6. Increase of ethanol productivity by cell-recycle fermentation of flocculating yeast.

    PubMed

    Wang, F Z; Xie, T; Hui, M

    2011-01-01

    Using the recombinant flocculating Angel yeast F6, long-term repeated batch fermentation for ethanol production was performed and a high volumetric productivity resulted from half cells not washed and the optimum opportunity of residual glucose 20 g l(-1) of last medium. The obtained highest productivity was 2.07 g l-(1) h(-1), which was improved by 75.4% compared with that of 1.18 g l(-1) h(-1) in the first batch fermentation. The ethanol concentration reached 8.4% corresponding to the yield of 0.46 g g(-1). These results will contribute greatly to the industrial production of fuel ethanol using the commercial method with the flocculating yeast.

  7. Yeast aconitase binds and provides metabolically coupled protection to mitochondrial DNA.

    PubMed

    Chen, Xin Jie; Wang, Xiaowen; Butow, Ronald A

    2007-08-21

    Aconitase (Aco1p) is a multifunctional protein: It is an enzyme of the tricarboxylic acid cycle. In animal cells, Aco1p also is a cytosolic protein binding to mRNAs to regulate iron metabolism. In yeast, Aco1p was identified as a component of mtDNA nucleoids. Here we show that yeast Aco1p protects mtDNA from excessive accumulation of point mutations and ssDNA breaks and suppresses reductive recombination of mtDNA. Aconitase binds to both ds- and ssDNA, with a preference for GC-containing sequences. Therefore, mitochondria are opportunistic organelles that seize proteins, such as metabolic enzymes, for construction of the nucleoid, an mtDNA maintenance/segregation apparatus.

  8. Long G2 accumulates recombination intermediates and disturbs chromosome segregation at dysfunction telomere in Schizosaccharomyces pombe

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Habib, Ahmed G.K.; Masuda, Kenta; Yukawa, Masashi

    Protection of telomere (Pot1) is a single-stranded telomere binding protein which is essential for chromosome ends protection. Fission yeast Rqh1 is a member of RecQ helicases family which has essential roles in the maintenance of genomic stability and regulation of homologous recombination. Double mutant between fission yeast pot1Δ and rqh1 helicase dead (rqh1-hd) maintains telomere by homologous recombination. In pot1Δ rqh1-hd double mutant, recombination intermediates accumulate near telomere which disturb chromosome segregation and make cells sensitive to microtubule inhibitors thiabendazole (TBZ). Deletion of chk1{sup +} or mutation of its kinase domain shortens the G2 of pot1Δ rqh1-hd double mutant andmore » suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of that double mutant. In this study, we asked whether the long G2 is the reason for the TBZ sensitivity of pot1Δ rqh1-hd double mutant. We found that shortening the G2 of pot1Δ rqh1-hd double mutant by additional mutations of wee1 and mik1 or gain of function mutation of Cdc2 suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of pot1Δ rqh1-hd double mutant. Our results suggest that long G2 of pot1Δ rqh1-hd double mutant may allow time for the accumulation of recombination intermediates which disturb chromosome segregation and make cells sensitive to TBZ. - Ηighlights: • We show link between long G2 and accumulation of toxic recombination intermediates. • Accumulation of recombination intermediates at telomere results in TBZ sensitivity. • Activation of DNA damage checkpoint worsens cells' viability in presence of TBZ.« less

  9. Engineered yeast for enhanced CO2 mineralization†

    PubMed Central

    Barbero, Roberto; Carnelli, Lino; Simon, Anna; Kao, Albert; Monforte, Alessandra d’Arminio; Riccò, Moreno; Bianchi, Daniele; Belcher, Angela

    2014-01-01

    In this work, a biologically catalyzed CO2 mineralization process for the capture of CO2 from point sources was designed, constructed at a laboratory scale, and, using standard chemical process scale-up protocols, was modeled and evaluated at an industrial scale. A yeast display system in Saccharomyces cerevisae was used to screen several carbonic anhydrase isoforms and mineralization peptides for their impact on CO2 hydration, CaCO3 mineralization, and particle settling rate. Enhanced rates for each of these steps in the CaCO3 mineralization process were confirmed using quantitative techniques in lab-scale measurements. The effect of these enhanced rates on the CO2 capture cost in an industrial scale CO2 mineralization process using coal fly ash as the CaO source was evaluated. The model predicts a process using bCA2- yeast and fly ash is ~10% more cost effective per ton of CO2 captured than a process with no biological molecules, a savings not realized by wild-type yeast and high-temperature stable recombinant CA2 alone or in combination. The levelized cost of electricity for a power plant using this process was calculated and scenarios in which this process compares favorably to CO2 capture by MEA absorption process are presented. PMID:25289021

  10. Recombination at DNA replication fork barriers is not universal and is differentially regulated by Swi1.

    PubMed

    Pryce, David W; Ramayah, Soshila; Jaendling, Alessa; McFarlane, Ramsay J

    2009-03-24

    DNA replication stress has been implicated in the etiology of genetic diseases, including cancers. It has been proposed that genomic sites that inhibit or slow DNA replication fork progression possess recombination hotspot activity and can form potential fragile sites. Here we used the fission yeast, Schizosaccharomyces pombe, to demonstrate that hotspot activity is not a universal feature of replication fork barriers (RFBs), and we propose that most sites within the genome that form RFBs do not have recombination hotspot activity under nonstressed conditions. We further demonstrate that Swi1, the TIMELESS homologue, differentially controls the recombination potential of RFBs, switching between being a suppressor and an activator of recombination in a site-specific fashion.

  11. Recombination at DNA replication fork barriers is not universal and is differentially regulated by Swi1

    PubMed Central

    Pryce, David W.; Ramayah, Soshila; Jaendling, Alessa; McFarlane, Ramsay J.

    2009-01-01

    DNA replication stress has been implicated in the etiology of genetic diseases, including cancers. It has been proposed that genomic sites that inhibit or slow DNA replication fork progression possess recombination hotspot activity and can form potential fragile sites. Here we used the fission yeast, Schizosaccharomyces pombe, to demonstrate that hotspot activity is not a universal feature of replication fork barriers (RFBs), and we propose that most sites within the genome that form RFBs do not have recombination hotspot activity under nonstressed conditions. We further demonstrate that Swi1, the TIMELESS homologue, differentially controls the recombination potential of RFBs, switching between being a suppressor and an activator of recombination in a site-specific fashion. PMID:19273851

  12. A termite symbiotic mushroom maximizing sexual activity at growing tips of vegetative hyphae.

    PubMed

    Hsieh, Huei-Mei; Chung, Mei-Chu; Chen, Pao-Yang; Hsu, Fei-Man; Liao, Wen-Wei; Sung, Ai-Ning; Lin, Chun-Ru; Wang, Chung-Ju Rachel; Kao, Yu-Hsin; Fang, Mei-Jane; Lai, Chi-Yung; Huang, Chieh-Chen; Chou, Jyh-Ching; Chou, Wen-Neng; Chang, Bill Chia-Han; Ju, Yu-Ming

    2017-09-19

    Termitomyces mushrooms are mutualistically associated with fungus-growing termites, which are widely considered to cultivate a monogenotypic Termitomyces symbiont within a colony. Termitomyces cultures isolated directly from termite colonies are heterokaryotic, likely through mating between compatible homokaryons. After pairing homokaryons carrying different haplotypes at marker gene loci MIP and RCB from a Termitomyces fruiting body associated with Odontotermes formosanus, we observed nuclear fusion and division, which greatly resembled meiosis, during each hyphal cell division and conidial formation in the resulting heterokaryons. Surprisingly, nuclei in homokaryons also behaved similarly. To confirm if meiotic-like recombination occurred within mycelia, we constructed whole-genome sequencing libraries from mycelia of two homokaryons and a heterokaryon resulting from mating of the two homokaryons. Obtained reads were aligned to the reference genome of Termitomyces sp. J132 for haplotype reconstruction. After removal of the recombinant haplotypes shared between the heterokaryon and either homokaryons, we inferred that 5.04% of the haplotypes from the heterokaryon were the recombinants resulting from homologous recombination distributed genome-wide. With RNA transcripts of four meiosis-specific genes, including SPO11, DMC1, MSH4, and MLH1, detected from a mycelial sample by real-time quantitative PCR, the nuclear behavior in mycelia was reconfirmed meiotic-like. Unlike other basidiomycetes where sex is largely restricted to basidia, Termitomyces maximizes sexuality at somatic stage, resulting in an ever-changing genotype composed of a myriad of coexisting heterogeneous nuclei in a heterokaryon. Somatic meiotic-like recombination may endow Termitomyces with agility to cope with termite consumption by maximized genetic variability.

  13. Phenylbutyrate inhibits homologous recombination induced by camptothecin and methyl methanesulfonate.

    PubMed

    Kaiser, Gitte S; Germann, Susanne M; Westergaard, Tine; Lisby, Michael

    2011-08-01

    Homologous recombination is accompanied by extensive changes to chromatin organization at the site of DNA damage. Some of these changes are mediated through acetylation/deacetylation of histones. Here, we show that recombinational repair of DNA damage induced by the anti-cancer drug camptothecin (CPT) and the alkylating agent methyl methanesulfonate (MMS) is blocked by sodium phenylbutyrate (PBA) in the budding yeast Saccharomyces cerevisiae. In particular, PBA suppresses CPT- and MMS-induced genetic recombination as well as DNA double-strand break repair during mating-type interconversion. Treatment with PBA is accompanied by a dramatic reduction in histone H4 lysine 8 acetylation. Live cell imaging of homologous recombination proteins indicates that repair of CPT-induced DNA damage is redirected to a non-recombinogenic pathway in the presence of PBA without loss in cell viability. In contrast, the suppression of MMS-induced recombination by PBA is accompanied by a dramatic loss in cell viability. Taken together, our results demonstrate that PBA inhibits DNA damage-induced homologous recombination likely by mediating changes in chromatin acetylation. Moreover, the combination of PBA with genotoxic agents can lead to different cell fates depending on the type of DNA damage inflicted. 2011 Elsevier B.V. All rights reserved.

  14. Trends in recombinant protein use in animal production.

    PubMed

    Gifre, Laia; Arís, Anna; Bach, Àlex; Garcia-Fruitós, Elena

    2017-03-04

    Recombinant technologies have made possible the production of a broad catalogue of proteins of interest, including those used for animal production. The most widely studied proteins for the animal sector are those with an important role in reproduction, feed efficiency, and health. Nowadays, mammalian cells and fungi are the preferred choice for recombinant production of hormones for reproductive purposes and fibrolytic enzymes to enhance animal performance, respectively. However, the development of low-cost products is a priority, particularly in livestock. The study of cell factories such as yeast and bacteria has notably increased in the last decades to make the new developed reproductive hormones and fibrolytic enzymes a real alternative to the marketed ones. Important efforts have also been invested to developing new recombinant strategies for prevention and therapy, including passive immunization and modulation of the immune system. This offers the possibility to reduce the use of antibiotics by controlling physiological processes and improve the efficacy of preventing infections. Thus, nowadays different recombinant fibrolytic enzymes, hormones, and therapeutic molecules with optimized properties have been successfully produced through cost-effective processes using microbial cell factories. However, despite the important achievements for reducing protein production expenses, alternative strategies to further reduce these costs are still required. In this context, it is necessary to make a giant leap towards the use of novel strategies, such as nanotechnology, that combined with recombinant technology would make recombinant molecules affordable for animal industry.

  15. The CAF-1 and Hir Histone Chaperones Associate with Sites of Meiotic Double-Strand Breaks in Budding Yeast

    PubMed Central

    Brachet, Elsa; Béneut, Claire; Serrentino, Maria-Elisabetta; Borde, Valérie

    2015-01-01

    In the meiotic prophase, programmed DNA double-strand breaks (DSB) are introduced along chromosomes to promote homolog pairing and recombination. Although meiotic DSBs usually occur in nucleosome-depleted, accessible regions of chromatin, their repair by homologous recombination takes place in a nucleosomal environment. Nucleosomes may represent an obstacle for the recombination machinery and their timely eviction and reincorporation into chromatin may influence the outcome of recombination, for instance by stabilizing recombination intermediates. Here we show in budding yeast that nucleosomes flanking a meiotic DSB are transiently lost during recombination, and that specific histone H3 chaperones, CAF-1 and Hir, are mobilized at meiotic DSBs. However, the absence of these chaperones has no effect on meiotic recombination, suggesting that timely histone reincorporation following their eviction has no influence on the recombination outcome, or that redundant pathways are activated. This study is the first example of the involvement of histone H3 chaperones at naturally occurring, developmentally programmed DNA double-strand breaks. PMID:25938567

  16. Improved sugar co-utilisation by encapsulation of a recombinant Saccharomyces cerevisiae strain in alginate-chitosan capsules

    PubMed Central

    2014-01-01

    Background Two major hurdles for successful production of second-generation bioethanol are the presence of inhibitory compounds in lignocellulosic media, and the fact that Saccharomyces cerevisiae cannot naturally utilise pentoses. There are recombinant yeast strains that address both of these issues, but co-utilisation of glucose and xylose is still an issue that needs to be resolved. A non-recombinant way to increase yeast tolerance to hydrolysates is by encapsulation of the yeast. This can be explained by concentration gradients occuring in the cell pellet inside the capsule. In the current study, we hypothesised that encapsulation might also lead to improved simultaneous utilisation of hexoses and pentoses because of such sugar concentration gradients. Results In silico simulations of encapsulated yeast showed that the presence of concentration gradients of inhibitors can explain the improved inhibitor tolerance of encapsulated yeast. Simulations also showed pronounced concentration gradients of sugars, which resulted in simultaneous xylose and glucose consumption and a steady state xylose consumption rate up to 220-fold higher than that found in suspension culture. To validate the results experimentally, a xylose-utilising S. cerevisiae strain, CEN.PK XXX, was constructed and encapsulated in semi-permeable alginate-chitosan liquid core gel capsules. In defined media, encapsulation not only increased the tolerance of the yeast to inhibitors, but also promoted simultaneous utilisation of glucose and xylose. Encapsulation of the yeast resulted in consumption of at least 50% more xylose compared with suspended cells over 96-hour fermentations in medium containing both sugars. The higher consumption of xylose led to final ethanol titres that were approximately 15% higher. In an inhibitory dilute acid spruce hydrolysate, freely suspended yeast cells consumed the sugars in a sequential manner after a long lag phase, whereas no lag phase was observed for the

  17. Evidence for a high mutation rate at rapidly evolving yeast centromeres

    PubMed Central

    2011-01-01

    Background Although their role in cell division is essential, centromeres evolve rapidly in animals, plants and yeasts. Unlike the complex centromeres of plants and aminals, the point centromeres of Saccharomcyes yeasts can be readily sequenced to distinguish amongst the possible explanations for fast centromere evolution. Results Using DNA sequences of all 16 centromeres from 34 strains of Saccharomyces cerevisiae and population genomic data from Saccharomyces paradoxus, I show that centromeres in both species evolve 3 times more rapidly even than selectively unconstrained DNA. Exceptionally high levels of polymorphism seen in multiple yeast populations suggest that rapid centromere evolution does not result from the repeated selective sweeps expected under meiotic drive. I further show that there is little evidence for crossing-over or gene conversion within centromeres, although there is clear evidence for recombination in their immediate vicinity. Finally I show that the mutation spectrum at centromeres is consistent with the pattern of spontaneous mutation elsewhere in the genome. Conclusions These results indicate that rapid centromere evolution is a common phenomenon in yeast species. Furthermore, these results suggest that rapid centromere evolution does not result from the mutagenic effect of gene conversion, but from a generalised increase in the mutation rate, perhaps arising from the unusual chromatin structure at centromeres in yeast and other eukaryotes. PMID:21767380

  18. Modulation of the endogenous production of protoporphyrin IX in a yeast-based model organism

    NASA Astrophysics Data System (ADS)

    Joniová, Jaroslava; Gerelli, Emmanuel; Wagnières, Georges

    2017-02-01

    The main aim of this study was to assess conditions at which simple yeast-based model organism produces maximal levels of protoporphyrin IX (PpIX) after an exogenous administration of its precursor, 5-aminolevulinic acid (ALA), and the ferrous-ion chelator 2,2'-bipyridyl. We observed that the fluorescing porphyrin, produced after these administrations, was likely to be PpIX since fluorescence spectroscopy of the porphyrins produced endogenously in yeast cells resembles that of PpIX in DMSO and in vivo in the chick's chorioallantoic membrane model. Also, fluorescence lifetimes of these porphyrins are very similar to that of PpIX in vitro and in vivo. This suggests that PpIX is the main fluorescent compound produced by yeast in our conditions. We found that the conditions at which yeast produces the maximal PpIX were a synchronous administration of 5 μM ALA and 1 mM 2,2'-bipyridyl for yeast incubated in aqueous glucose and 1 mM 2,2'-bipyridyl in the presence of YPD medium. Such a simple model is of high interest to study basic mechanisms involved in the mitochondrial respiration since PpIX, which is produced in this organelle, can be used as an oxygen sensor, or to perform photodynamic therapy and photodiagnosis. Since the absorption and scattering coefficients of this model are much smaller than those of soft tissues over the visible part of the spectrum, a version of this model loaded with appropriated amounts of light absorbing and scattering particles could be designed as a phantom to mimic tumors containing PpIX, a useful tool to optimize certain cancer photodetection set-ups.

  19. The combined use of the PLHC-1 cell line and the recombinant yeast assay to assess the environmental quality of estuarine and coastal sediments.

    PubMed

    Schnell, Sabine; Olivares, Alba; Piña, Benjamin; Echavarri-Erasun, Beatriz; Lacorte, Silvia; Porte, Cinta

    2013-12-15

    Sediment contamination poses a potential risk for both ecosystems and human health. Risk assessment is troublesome as sediments contain complex mixtures of toxicants, and traditional chemical analyses can neither provide information about potential hazards to organisms nor identify and measure all present contaminants. This work combines the use of the PLHC-1 cell line and the recombinant yeast assay (RYA) to assess the environmental quality of estuarine and coastal sediments. The application of multiple endpoints (cytotoxicity, generation of oxidative stress, presence of CYP1A inducing agents, micronucleus formation and estrogenicity) revealed that the organic extracts of those sediments affected by industrial activities or collected near harbours and untreated urban discharges showed significant cytotoxicity, micronuclei and CYP1A induction. The study highlights the usefulness of the applied bioassays to identify those sediments that could pose risk to aquatic organisms and that require further action to improve their environmental quality. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. VDE-initiated intein homing in Saccharomyces cerevisiae proceeds in a meiotic recombination-like manner.

    PubMed

    Fukuda, Tomoyuki; Nogami, Satoru; Ohya, Yoshikazu

    2003-07-01

    Inteins and group I introns found in prokaryotic and eukaryotic organisms occasionally behave as mobile genetic elements. During meiosis of the yeast Saccharomyces cerevisiae, the site-specific endonuclease encoded by VMA1 intein, VDE, triggers a single double-strand break (DSB) at an inteinless allele, leading to VMA1 intein homing. Besides the accumulating information on the in vitro activity of VDE, very little has been known about the molecular mechanism of intein homing in yeast nucleus. We developed an assay to detect the product of VMA1 intein homing in yeast genome. We analysed mutant phenotypes of RecA homologs, Rad51p and Dmc1p, and their interacting proteins, Rad54p and Tid1p, and found that they all play critical roles in intein inheritance. The absence of DSB end processing proteins, Sae2p and those in the Mre11-Rad50-Xrs2 complex, also causes partial reduction in homing efficiency. As with meiotic recombination, crossover events are frequently observed during intein homing. We also observed that the absence of premeiotic DNA replication caused by hydroxyurea (HU) or clb5delta clb6delta mutation reduces VDE-mediated DSBs. The repairing system working in intein homing shares molecular machinery with meiotic recombination induced by Spo11p. Moreover, like Spo11p-induced DNA cleavage, premeiotic DNA replication is a prerequisite for a VDE-induced DSB. VMA1 intein thus utilizes several host factors involved in meiotic and recombinational processes to spread its genetic information and guarantee its progeny through establishment of a parasitic relationship with the organism.

  1. Process for Assembly and Transformation into Saccharomyces cerevisiae of a Synthetic Yeast Artificial Chromosome Containing a Multigene Cassette to Express Enzymes That Enhance Xylose Utilization Designed for an Automated Platform.

    PubMed

    Hughes, Stephen R; Cox, Elby J; Bang, Sookie S; Pinkelman, Rebecca J; López-Núñez, Juan Carlos; Saha, Badal C; Qureshi, Nasib; Gibbons, William R; Fry, Michelle R; Moser, Bryan R; Bischoff, Kenneth M; Liu, Siqing; Sterner, David E; Butt, Tauseef R; Riedmuller, Steven B; Jones, Marjorie A; Riaño-Herrera, Néstor M

    2015-12-01

    A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains. © 2015 Society for Laboratory Automation and Screening.

  2. DNA recombination-initiation plays a role in the extremely biased inheritance of yeast [rho-] mitochondrial DNA that contains the replication origin ori5.

    PubMed

    Ling, Feng; Hori, Akiko; Shibata, Takehiko

    2007-02-01

    Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance of a small mitochondrial DNA (mtDNA) fragment that contains a replication origin (HS [rho(-)] mtDNA). Our previous studies showed that concatemers (linear head-to-tail multimers) are obligatory intermediates for mtDNA partitioning and are primarily formed by rolling-circle replication mediated by Mhr1, a protein required for homologous mtDNA recombination. In this study, we found that Mhr1 is required for the hypersuppressiveness of HS [ori5] [rho(-)] mtDNA harboring ori5, one of the replication origins of normal ([rho(+)]) mtDNA. In addition, we detected an Ntg1-stimulated double-strand break at the ori5 locus. Purified Ntg1, a base excision repair enzyme, introduced a double-stranded break by itself into HS [ori5] [rho(-)] mtDNA at ori5 isolated from yeast cells. Both hypersuppressiveness and concatemer formation of HS [ori5] [rho(-)] mtDNA are simultaneously suppressed by the ntg1 null mutation. These results support a model in which, like homologous recombination, rolling-circle HS [ori5] [rho(-)] mtDNA replication is initiated by double-stranded breakage in ori5, followed by Mhr1-mediated homologous pairing of the processed nascent DNA ends with circular mtDNA. The hypersuppressiveness of HS [ori5] [rho(-)] mtDNA depends on a replication advantage furnished by the higher density of ori5 sequences and on a segregation advantage furnished by the higher genome copy number on transmitted concatemers.

  3. Generation of henipavirus nucleocapsid proteins in yeast Saccharomyces cerevisiae.

    PubMed

    Juozapaitis, Mindaugas; Serva, Andrius; Zvirbliene, Aurelija; Slibinskas, Rimantas; Staniulis, Juozas; Sasnauskas, Kestutis; Shiell, Brian J; Wang, Lin-Fa; Michalski, Wojtek P

    2007-03-01

    Hendra and Nipah viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the family Paramyxoviridae. Currently these viruses are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genes encoding HeV and NiV nucleocapsid proteins were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of expression of these proteins (18-20 mg l(-1) of yeast culture) was obtained. Mass spectrometric analysis confirmed the primary structure of both proteins with 92% sequence coverage obtained using MS/MS analysis. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. The nucleocapsid proteins revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. HeV nucleocapsid protein was detected by sera derived from fruit bats, humans, horses infected with HeV, and NiV nucleocapsid protein was immunodetected with sera from, fruit bats, humans and pigs. The development of an efficient and cost-effective system for generation of henipavirus nucleocapsid proteins might help to improve reagents for diagnosis of viruses.

  4. Microbial factories for recombinant pharmaceuticals

    PubMed Central

    Ferrer-Miralles, Neus; Domingo-Espín, Joan; Corchero, José Luis; Vázquez, Esther; Villaverde, Antonio

    2009-01-01

    Most of the hosts used to produce the 151 recombinant pharmaceuticals so far approved for human use by the Food and Drug Administration (FDA) and/or by the European Medicines Agency (EMEA) are microbial cells, either bacteria or yeast. This fact indicates that despite the diverse bottlenecks and obstacles that microbial systems pose to the efficient production of functional mammalian proteins, namely lack or unconventional post-translational modifications, proteolytic instability, poor solubility and activation of cell stress responses, among others, they represent convenient and powerful tools for recombinant protein production. The entering into the market of a progressively increasing number of protein drugs produced in non-microbial systems has not impaired the development of products obtained in microbial cells, proving the robustness of the microbial set of cellular systems (so far Escherichia coli and Saccharomyces cerevisae) developed for protein drug production. We summarize here the nature, properties and applications of all those pharmaceuticals and the relevant features of the current and potential producing hosts, in a comparative way. PMID:19317892

  5. Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha.

    PubMed

    Tikhomirova, L P; Ikonomova, R N; Kuznetsova, E N

    1986-01-01

    For the transformation of the yeast Hansenula polymorpha we have constructed a set of hybrid plasmids carrying the LEU2 gene of Saccharomyces cerevisiae as a selective marker and fragments of mitochondrial DNA of Candida utilis and H. polymorpha or chromosomal DNA fragments of H. polymorpha as replicator sequences. The replication properties of chimeric plasmids in the yeast H. polymorpha were investigated. We showed that for plasmids propagated autonomously in this yeast the plasmid monomers could be detected in the transformants only during the immediate time after the transformation event. Further growth under selective conditions led to the selection of polymeric forms of plasmid DNA as it was clearly shown for transformants carrying cosmid pL2 with mtDNA fragment of C. utilis. Such transformants carrying polymerized plasmids showed a remarkably increased stability of the transformed phenotype. Cosmid pL2 was able to shuttle between Escherichia coli, S. cerevisiae and H. polymorpha, whereas plasmids with DNA fragments from H. polymorpha did not transform S. cerevisiae effectively.

  6. Oral vaccination through voluntary consumption of the convict grouper Epinephelus septemfasciatus with yeast producing the capsid protein of red-spotted grouper nervous necrosis virus.

    PubMed

    Cho, Seo Young; Kim, Hyoung Jin; Lan, Nguyen Thi; Han, Hyun-Ja; Lee, Deok-Chan; Hwang, Jee Youn; Kwon, Mun-Gyeong; Kang, Bo Kyu; Han, Sang Yoon; Moon, Hyoungjoon; Kang, Hyun Ah; Kim, Hong-Jin

    2017-05-01

    Nervous necrosis viruses (NNV) cause serious economic losses in marine fish cultivation. The red-spotted grouper NNV (RGNNV) is the most common species of NNV worldwide. There have been many efforts to develop prophylactic NNV vaccines, and various types of vaccine candidate have been suggested. However, most were designed as injectable vaccines, which are not suitable for large-scale vaccination and cause too much stress to the fish. Oral vaccination through voluntary feeding is an ideal way to provide protective immunity to fish. In the present study, recombinant Saccharomyces cerevisiae producing RGNNV capsid protein was used as oral vaccine. The recombinant yeast was prepared in freeze-dried form after disruption. Convict groupers were divided into three groups, control, and oral and parenteral vaccination groups, each consisting of 700 fishes. The control group received no treatment, the parenteral group received one intraperitoneal injection of RGNNV virus-like particles, and the oral vaccination group consumed feed containing the lysed recombinant yeast; voluntary intake was allowed four times at one-week intervals. Both vaccination groups produced serum RGNNV neutralizing antibody titers of >10 3 (log 2, 9.96), sustained for at least 95days post-immunization. In addition, in response to challenge with RGNNV both groups suffered significantly reduced mortality and had reduced brain RGNNV titers. These results indicate that recombinant yeast-based oral fish vaccines have great potential for large-scale vaccination. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Medicinally important secondary metabolites in recombinant microorganisms or plants: progress in alkaloid biosynthesis.

    PubMed

    Schäfer, Holger; Wink, Michael

    2009-12-01

    Plants produce a high diversity of natural products or secondary metabolites which are important for the communication of plants with other organisms. A prominent function is the protection against herbivores and/or microbial pathogens. Some natural products are also involved in defence against abiotic stress, e.g. UV-B exposure. Many of the secondary metabolites have interesting biological properties and quite a number are of medicinal importance. Because the production of the valuable natural products, such as the anticancer drugs paclitaxel, vinblastine or camptothecin in plants is a costly process, biotechnological alternatives to produce these alkaloids more economically become increasingly important. This review provides an overview of the state of art to produce alkaloids in recombinant microorganisms, such as bacteria or yeast. Some progress has been made in metabolic engineering usually employing a single recombinant alkaloid gene. More importantly, for benzylisoquinoline, monoterpene indole and diterpene alkaloids (taxanes) as well as some terpenoids and phenolics the proof of concept for production of complex alkaloids in recombinant Escherichia coli and yeast has already been achieved. In a long-term perspective, it will probably be possible to generate gene cassettes for complete pathways, which could then be used for production of valuable natural products in bioreactors or for metabolic engineering of crop plants. This will improve their resistance against herbivores and/or microbial pathogens.

  8. Asy2/Mer2: an evolutionarily conserved mediator of meiotic recombination, pairing, and global chromosome compaction.

    PubMed

    Tessé, Sophie; Bourbon, Henri-Marc; Debuchy, Robert; Budin, Karine; Dubois, Emeline; Liangran, Zhang; Antoine, Romain; Piolot, Tristan; Kleckner, Nancy; Zickler, Denise; Espagne, Eric

    2017-09-15

    Meiosis is the cellular program by which a diploid cell gives rise to haploid gametes for sexual reproduction. Meiotic progression depends on tight physical and functional coupling of recombination steps at the DNA level with specific organizational features of meiotic-prophase chromosomes. The present study reveals that every step of this coupling is mediated by a single molecule: Asy2/Mer2. We show that Mer2, identified so far only in budding and fission yeasts, is in fact evolutionarily conserved from fungi (Mer2/Rec15/Asy2/Bad42) to plants (PRD3/PAIR1) and mammals (IHO1). In yeasts, Mer2 mediates assembly of recombination-initiation complexes and double-strand breaks (DSBs). This role is conserved in the fungus Sordaria However, functional analysis of 13 mer2 mutants and successive localization of Mer2 to axis, synaptonemal complex (SC), and chromatin revealed, in addition, three further important functions. First, after DSB formation, Mer2 is required for pairing by mediating homolog spatial juxtaposition, with implications for crossover (CO) patterning/interference. Second, Mer2 participates in the transfer/maintenance and release of recombination complexes to/from the SC central region. Third, after completion of recombination, potentially dependent on SUMOylation, Mer2 mediates global chromosome compaction and post-recombination chiasma development. Thus, beyond its role as a recombinosome-axis/SC linker molecule, Mer2 has important functions in relation to basic chromosome structure. © 2017 Tessé et al.; Published by Cold Spring Harbor Laboratory Press.

  9. Heterologous expression of type I antifreeze peptide GS-5 in baker's yeast increases freeze tolerance and provides enhanced gas production in frozen dough.

    PubMed

    Panadero, Joaquin; Randez-Gil, Francisca; Prieto, Jose Antonio

    2005-12-28

    The demand for frozen-dough products has increased notably in the baking industry. Nowadays, no appropriate industrial baker's yeast with optimal gassing capacity in frozen dough is, however, available, and it is unlikely that classical breeding programs could provide significant improvements of this trait. Antifreeze proteins, found in diverse organisms, display the ability to inhibit the growth of ice, allowing them to survive at temperatures below 0 degrees C. In this study a recombinant antifreeze peptide GS-5 was expressed from the polar fish grubby sculpin (Myoxocephalus aenaeus) in laboratory and industrial baker's yeast strains of Saccharomyces cerevisiae. Production of the recombinant protein increased freezing tolerance in both strains tested. Furthermore, expression of the GS-5 encoding gene enhanced notably the gassing rate and total gas production in frozen and frozen sweet doughs. These effects are unlikely to be due to reduced osmotic damage during freezing/thawing, because recombinant cells showed growth behavior similar to that of the parent under hypermosmotic stress conditions.

  10. Yeast mitochondrial HMG proteins: DNA-binding properties of the most evolutionarily divergent component of mitochondrial nucleoids.

    PubMed

    Bakkaiova, Jana; Marini, Victoria; Willcox, Smaranda; Nosek, Jozef; Griffith, Jack D; Krejci, Lumir; Tomaska, Lubomir

    2015-12-08

    Yeast mtDNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids). The principal mediators of nucleoid formation are mitochondrial high-mobility group (HMG)-box containing (mtHMG) proteins. Although these proteins are some of the fastest evolving components of mt-nucleoids, it is not known whether the divergence of mtHMG proteins on the level of their amino acid sequences is accompanied by diversification of their biochemical properties. In the present study we performed a comparative biochemical analysis of yeast mtHMG proteins from Saccharomyces cerevisiae (ScAbf2p), Yarrowia lipolytica (YlMhb1p) and Candida parapsilosis (CpGcf1p). We found that all three proteins exhibit relatively weak binding to intact dsDNA. In fact, ScAbf2p and YlMhb1p bind quantitatively to this substrate only at very high protein to DNA ratios and CpGcf1p shows only negligible binding to dsDNA. In contrast, the proteins exhibit much higher preference for recombination intermediates such as Holliday junctions (HJ) and replication forks (RF). Therefore, we hypothesize that the roles of the yeast mtHMG proteins in maintenance and compaction of mtDNA in vivo are in large part mediated by their binding to recombination/replication intermediates. We also speculate that the distinct biochemical properties of CpGcf1p may represent one of the prerequisites for frequent evolutionary tinkering with the form of the mitochondrial genome in the CTG-clade of hemiascomycetous yeast species. © 2016 Authors.

  11. Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast.

    PubMed

    Li, Ping; Jin, Hui; Yu, Hong-Guo

    2014-10-01

    During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. © 2014 Li et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  12. Human recombinant lysosomal enzymes produced in microorganisms.

    PubMed

    Espejo-Mojica, Ángela J; Alméciga-Díaz, Carlos J; Rodríguez, Alexander; Mosquera, Ángela; Díaz, Dennis; Beltrán, Laura; Díaz, Sergio; Pimentel, Natalia; Moreno, Jefferson; Sánchez, Jhonnathan; Sánchez, Oscar F; Córdoba, Henry; Poutou-Piñales, Raúl A; Barrera, Luis A

    2015-01-01

    Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Search for protein partners of mitochondrial single-stranded DNA-binding protein Rim1p using a yeast two-hybrid system.

    PubMed

    Kucejová, B; Foury, F

    2003-01-01

    RIM1 is a nuclear gene of the yeast Saccharomyces cerevisiae coding for a protein with single-stranded DNA-binding activity that is essential for mitochondrial genome maintenance. No protein partners of Rim1p have been described so far in yeast. To better understand the role of this protein in mitochondrial DNA replication and recombination, a search for protein interactors by the yeast two-hybrid system was performed. This approach led to the identification of several candidates, including a putative transcription factor, Azf1p, and Mph1p, a protein with an RNA helicase domain which is known to influence the mutation rate of nuclear and mitochondrial genomes.

  14. Passive transfer of growth-inhibitory antibodies raised against yeast-expressed recombinant Plasmodium falciparum merozoite surface protein-1(19).

    PubMed

    Gozalo, A; Lucas, C; Cachay, M; Wellde, B T; Hall, T; Bell, B; Wood, J; Watts, D; Wooster, M; Lyon, J A; Moch, J K; Haynes, J D; Williams, J S; Holland, C; Watson, E; Kester, K E; Kaslow, D C; Ballou, W R

    1998-12-01

    Purified rabbit immunoglobulin raised against yeast-expressed recombinant FVO or 3D7 Plasmodium falciparum merozoite surface protein-1 (MSP-1) 19k-D C terminal fragment (MSP-1(19)) was transfused into malaria-naive Aotus nancymai monkeys that were immediately challenged with FVO asexual stage malaria parasites. Control monkeys received rabbit immunoglobulin raised against the sexual stage antigen Pfs25 or Aotus hyperimmune serum obtained from monkeys immunized by P. falciparum infection and drug cure. Passive transfer of rabbit anti-MSP-1(19) failed to protect against homologous or heterologous challenge and, when compared with negative controls, there were no differences in prepatent periods or time to treatment. Interestingly, rabbit anti-MSP-1(19), but not anti-Pfs25, immunoglobulin, and immune monkey serum prevented the development of antibodies directed against MSP-1(19) fragment by infected monkeys, indicating that the antibodies were reactive with native MSP-1(19) antigen in vivo. The prepatent period and time to treatment was greatly delayed in the two monkeys that received Aotus immune serum, both of which developed a chronic intermittent low level infection. In vitro parasite growth inhibition assays (GIAs) confirmed the presence of inhibitory activity (40% maximum inhibition) in concentrated anti-MSP-1(19) immunoglobulin (4.8 mg/ml), but the peak concentrations we achieved in vivo (1 mg/ml) were not inhibitory in vitro. Subinhibitory levels of anti-MSP-1(19) antibodies achieved by passive transfer were not protective against P. falciparum challenge.

  15. Bioconversion of Airborne Methylamine by Immobilized Recombinant Amine Oxidase from the Thermotolerant Yeast Hansenula polymorpha

    PubMed Central

    Sigawi, Sasi; Nitzan, Yeshayahu

    2014-01-01

    Aliphatic amines, including methylamine, are air-pollutants, due to their intensive use in industry and the natural degradation of proteins, amino acids, and other nitrogen-containing compounds in biological samples. It is necessary to develop systems for removal of methylamine from the air, since airborne methylamine has a negative effect on human health. The primary amine oxidase (primary amine : oxygen oxidoreductase (deaminating) or amine oxidase, AMO; EC 1.4.3.21), a copper-containing enzyme from the thermotolerant yeast Hansenula polymorpha which was overexpressed in baker's yeast Saccharomyces cerevisiae, was tested for its ability to oxidize airborne methylamine. A continuous fluidized bed bioreactor (CFBR) was designed to enable bioconversion of airborne methylamine by AMO immobilized in calcium alginate (CA) beads. The results demonstrated that the bioreactor with immobilized AMO eliminates nearly 97% of the airborne methylamine. However, the enzymatic activity of AMO causes formation of formaldehyde. A two-step bioconversion process was therefore proposed. In the first step, airborne methylamine was fed into a CFBR which contained immobilized AMO. In the second step, the gas flow was passed through another CFBR, with alcohol oxidase from the yeast H. polymorpha immobilized in CA, in order to decompose the formaldehyde formed in the first step. The proposed system provided almost total elimination of the airborne methylamine and the formaldehyde. PMID:24672387

  16. Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination.

    PubMed

    Mbantenkhu, MacMillan; Wang, Xiaowen; Nardozzi, Jonathan D; Wilkens, Stephan; Hoffman, Elizabeth; Patel, Anamika; Cosgrove, Michael S; Chen, Xin Jie

    2011-12-09

    Homologous recombination is a conserved molecular process that has primarily evolved for the repair of double-stranded DNA breaks and stalled replication forks. However, the recombination machinery in mitochondria is poorly understood. Here, we show that the yeast mitochondrial nucleoid protein, Mgm101, is related to the Rad52-type recombination proteins that are widespread in organisms from bacteriophage to humans. Mgm101 is required for repeat-mediated recombination and suppression of mtDNA fragmentation in vivo. It preferentially binds to single-stranded DNA and catalyzes the annealing of ssDNA precomplexed with the mitochondrial ssDNA-binding protein, Rim1. Transmission electron microscopy showed that Mgm101 forms large oligomeric rings of ∼14-fold symmetry and highly compressed helical filaments. Specific mutations affecting ring formation reduce protein stability in vitro. The data suggest that the ring structure may provide a scaffold for stabilization of Mgm101 by preventing the aggregation of the otherwise unstable monomeric conformation. Upon binding to ssDNA, Mgm101 is remobilized from the rings to form distinct nucleoprotein filaments. These studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity.

  17. Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory

    PubMed Central

    2010-01-01

    Background Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions. Results Here we demonstrate at least a doubling in productivity over wild-type strains for three recombinant membrane proteins and one recombinant soluble protein produced in TM6* cells. In all cases, this was attributed to the improved biomass properties of the strain. The yield profile across the growth curve was also more stable than in a wild-type strain, and was not further improved by lowering culture temperatures. This has the added benefit that improved yields can be attained rapidly at the yeast's optimal growth conditions. Importantly, improved productivity could not be reproduced in wild-type strains by culturing them under glucose fed-batch conditions: despite having achieved very similar biomass yields to those achieved by TM6* cultures, the total volumetric yields were not concomitantly increased. Furthermore, the productivity of TM6* was unaffected by growing cultures in the presence of ethanol. These findings support the unique properties of TM6* as a microbial cell factory. Conclusions The accumulation of biomass in yeast cell factories is not necessarily correlated with a proportional increase in the functional yield of the recombinant protein being produced. The respiratory S. cerevisiae strain reported here is therefore a useful addition to the matrix of production hosts currently available as its improved biomass properties do lead to increased

  18. Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory.

    PubMed

    Ferndahl, Cecilia; Bonander, Nicklas; Logez, Christel; Wagner, Renaud; Gustafsson, Lena; Larsson, Christer; Hedfalk, Kristina; Darby, Richard A J; Bill, Roslyn M

    2010-06-17

    Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions. Here we demonstrate at least a doubling in productivity over wild-type strains for three recombinant membrane proteins and one recombinant soluble protein produced in TM6* cells. In all cases, this was attributed to the improved biomass properties of the strain. The yield profile across the growth curve was also more stable than in a wild-type strain, and was not further improved by lowering culture temperatures. This has the added benefit that improved yields can be attained rapidly at the yeast's optimal growth conditions. Importantly, improved productivity could not be reproduced in wild-type strains by culturing them under glucose fed-batch conditions: despite having achieved very similar biomass yields to those achieved by TM6* cultures, the total volumetric yields were not concomitantly increased. Furthermore, the productivity of TM6* was unaffected by growing cultures in the presence of ethanol. These findings support the unique properties of TM6* as a microbial cell factory. The accumulation of biomass in yeast cell factories is not necessarily correlated with a proportional increase in the functional yield of the recombinant protein being produced. The respiratory S. cerevisiae strain reported here is therefore a useful addition to the matrix of production hosts currently available as its improved biomass properties do lead to increased volumetric yields without the need to

  19. Glucose-free fructose production from Jerusalem artichoke using a recombinant inulinase-secreting Saccharomyces cerevisiae strain.

    PubMed

    Yu, Jing; Jiang, Jiaxi; Ji, Wangming; Li, Yuyang; Liu, Jianping

    2011-01-01

    Using inulin (polyfructose) obtained from Jerusalen artichokes, we have produced fructose free of residual glucose using a recombinant inulinase-secreting strain of Saccharomyces cerevisiae in a one-step fermentation of Jerusalem artichoke tubers. For producing fructose from inulin, a recombinant inulinase-producing Saccharomyce cerevisiae strain was constructed with a deficiency in fructose uptake by disruption of two hexokinase genes hxk1 and hxk2. The inulinase gene introduced into S. cerevisiae was cloned from Kluyveromyces cicerisporus. Extracellular inulinase activity of the recombinant hxk-mutated S. cerevisiae strain reached 31 U ml(-1) after 96 h growth. When grown in a medium containing Jerusalem artichoke tubers as the sole component without any additives, the recombinant yeast accumulated fructose up to 9.2% (w/v) in the fermentation broth with only 0.1% (w/v) glucose left after 24 h.

  20. PGASO: A synthetic biology tool for engineering a cellulolytic yeast

    PubMed Central

    2012-01-01

    Background To achieve an economical cellulosic ethanol production, a host that can do both cellulosic saccharification and ethanol fermentation is desirable. However, to engineer a non-cellulolytic yeast to be such a host requires synthetic biology techniques to transform multiple enzyme genes into its genome. Results A technique, named Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO), that employs overlapping oligonucleotides for recombinatorial assembly of gene cassettes with individual promoters, was developed. PGASO was applied to engineer Kluyveromycesmarxianus KY3, which is a thermo- and toxin-tolerant yeast. We obtained a recombinant strain, called KR5, that is capable of simultaneously expressing exoglucanase and endoglucanase (both of Trichodermareesei), a beta-glucosidase (from a cow rumen fungus), a neomycin phosphotransferase, and a green fluorescent protein. High transformation efficiency and accuracy were achieved as ~63% of the transformants was confirmed to be correct. KR5 can utilize beta-glycan, cellobiose or CMC as the sole carbon source for growth and can directly convert cellobiose and beta-glycan to ethanol. Conclusions This study provides the first example of multi-gene assembly in a single step in a yeast species other than Saccharomyces cerevisiae. We successfully engineered a yeast host with a five-gene cassette assembly and the new host is capable of co-expressing three types of cellulase genes. Our study shows that PGASO is an efficient tool for simultaneous expression of multiple enzymes in the kefir yeast KY3 and that KY3 can serve as a host for developing synthetic biology tools. PMID:22839502

  1. Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae.

    PubMed

    Barajas, Daniel; Aponte-Ubillus, Juan Jose; Akeefe, Hassibullah; Cinek, Tomas; Peltier, Joseph; Gold, Daniel

    2017-01-01

    The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast S. cerevisiae would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors.

  2. Integrated approach for selecting efficient Saccharomyces cerevisiae for industrial lignocellulosic fermentations: Importance of yeast chassis linked to process conditions.

    PubMed

    Costa, Carlos E; Romaní, Aloia; Cunha, Joana T; Johansson, Björn; Domingues, Lucília

    2017-03-01

    In this work, four robust yeast chassis isolated from industrial environments were engineered with the same xylose metabolic pathway. The recombinant strains were physiologically characterized in synthetic xylose and xylose-glucose medium, on non-detoxified hemicellulosic hydrolysates of fast-growing hardwoods (Eucalyptus and Paulownia) and agricultural residues (corn cob and wheat straw) and on Eucalyptus hydrolysate at different temperatures. Results show that the co-consumption of xylose-glucose was dependent on the yeast background. Moreover, heterogeneous results were obtained among different hydrolysates and temperatures for each individual strain pointing to the importance of designing from the very beginning a tailor-made yeast considering the specific raw material and process. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. The N-terminal DNA-binding domain of Rad52 promotes RAD51-independent recombination in Saccharomyces cerevisiae.

    PubMed Central

    Tsukamoto, Mariko; Yamashita, Kentaro; Miyazaki, Toshiko; Shinohara, Miki; Shinohara, Akira

    2003-01-01

    In Saccharomyces cerevisiae, the Rad52 protein plays a role in both RAD51-dependent and RAD51-independent recombination pathways. We characterized a rad52 mutant, rad52-329, which lacks the C-terminal Rad51-interacting domain, and studied its role in RAD51-independent recombination. The rad52-329 mutant is completely defective in mating-type switching, but partially proficient in recombination between inverted repeats. We also analyzed the effect of the rad52-329 mutant on telomere recombination. Yeast cells lacking telomerase maintain telomere length by recombination. The rad52-329 mutant is deficient in RAD51-dependent telomere recombination, but is proficient in RAD51-independent telomere recombination. In addition, we examined the roles of other recombination genes in the telomere recombination. The RAD51-independent recombination in the rad52-329 mutant is promoted by a paralogue of Rad52, Rad59. All components of the Rad50-Mre11-Xrs2 complex are also important, but not essential, for RAD51-independent telomere recombination. Interestingly, RAD51 inhibits the RAD51-independent, RAD52-dependent telomere recombination. These findings indicate that Rad52 itself, and more precisely its N-terminal DNA-binding domain, promote an essential reaction in recombination in the absence of RAD51. PMID:14704160

  4. Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate

    PubMed Central

    Chen, Wen-Hsiang; Du, Lanying; Chag, Shivali M; Ma, Cuiqing; Tricoche, Nancy; Tao, Xinrong; Seid, Christopher A; Hudspeth, Elissa M; Lustigman, Sara; Tseng, Chien-Te K; Bottazzi, Maria Elena; Hotez, Peter J; Zhan, Bin; Jiang, Shibo

    2014-01-01

    Development of vaccines for preventing a future pandemic of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and for biodefense preparedness is urgently needed. Our previous studies have shown that a candidate SARS vaccine antigen consisting of the receptor-binding domain (RBD) of SARS-CoV spike protein can induce potent neutralizing antibody responses and protection against SARS-CoV challenge in vaccinated animals. To optimize expression conditions for scale-up production of the RBD vaccine candidate, we hypothesized that this could be potentially achieved by removing glycosylation sites in the RBD protein. In this study, we constructed two RBD protein variants: 1) RBD193-WT (193-aa, residues 318–510) and its deglycosylated forms (RBD193-N1, RBD193-N2, RBD193-N3); 2) RBD219-WT (219-aa, residues 318–536) and its deglycosylated forms (RBD219-N1, RBD219-N2, and RBD219-N3). All constructs were expressed as recombinant proteins in yeast. The purified recombinant proteins of these constructs were compared for their antigenicity, functionality and immunogenicity in mice using alum as the adjuvant. We found that RBD219-N1 exhibited high expression yield, and maintained its antigenicity and functionality. More importantly, RBD219-N1 induced significantly stronger RBD-specific antibody responses and a higher level of neutralizing antibodies in immunized mice than RBD193-WT, RBD193-N1, RBD193-N3, or RBD219-WT. These results suggest that RBD219-N1 could be selected as an optimal SARS vaccine candidate for further development. PMID:24355931

  5. Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate.

    PubMed

    Chen, Wen-Hsiang; Du, Lanying; Chag, Shivali M; Ma, Cuiqing; Tricoche, Nancy; Tao, Xinrong; Seid, Christopher A; Hudspeth, Elissa M; Lustigman, Sara; Tseng, Chien-Te K; Bottazzi, Maria Elena; Hotez, Peter J; Zhan, Bin; Jiang, Shibo

    2014-01-01

    Development of vaccines for preventing a future pandemic of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and for biodefense preparedness is urgently needed. Our previous studies have shown that a candidate SARS vaccine antigen consisting of the receptor-binding domain (RBD) of SARS-CoV spike protein can induce potent neutralizing antibody responses and protection against SARS-CoV challenge in vaccinated animals. To optimize expression conditions for scale-up production of the RBD vaccine candidate, we hypothesized that this could be potentially achieved by removing glycosylation sites in the RBD protein. In this study, we constructed two RBD protein variants: 1) RBD193-WT (193-aa, residues 318-510) and its deglycosylated forms (RBD193-N1, RBD193-N2, RBD193-N3); 2) RBD219-WT (219-aa, residues 318-536) and its deglycosylated forms (RBD219-N1, RBD219-N2, and RBD219-N3). All constructs were expressed as recombinant proteins in yeast. The purified recombinant proteins of these constructs were compared for their antigenicity, functionality and immunogenicity in mice using alum as the adjuvant. We found that RBD219-N1 exhibited high expression yield, and maintained its antigenicity and functionality. More importantly, RBD219-N1 induced significantly stronger RBD-specific antibody responses and a higher level of neutralizing antibodies in immunized mice than RBD193-WT, RBD193-N1, RBD193-N3, or RBD219-WT. These results suggest that RBD219-N1 could be selected as an optimal SARS vaccine candidate for further development.

  6. Asy2/Mer2: an evolutionarily conserved mediator of meiotic recombination, pairing, and global chromosome compaction

    PubMed Central

    Tessé, Sophie; Bourbon, Henri-Marc; Debuchy, Robert; Budin, Karine; Dubois, Emeline; Liangran, Zhang; Antoine, Romain; Piolot, Tristan; Kleckner, Nancy; Zickler, Denise; Espagne, Eric

    2017-01-01

    Meiosis is the cellular program by which a diploid cell gives rise to haploid gametes for sexual reproduction. Meiotic progression depends on tight physical and functional coupling of recombination steps at the DNA level with specific organizational features of meiotic-prophase chromosomes. The present study reveals that every step of this coupling is mediated by a single molecule: Asy2/Mer2. We show that Mer2, identified so far only in budding and fission yeasts, is in fact evolutionarily conserved from fungi (Mer2/Rec15/Asy2/Bad42) to plants (PRD3/PAIR1) and mammals (IHO1). In yeasts, Mer2 mediates assembly of recombination–initiation complexes and double-strand breaks (DSBs). This role is conserved in the fungus Sordaria. However, functional analysis of 13 mer2 mutants and successive localization of Mer2 to axis, synaptonemal complex (SC), and chromatin revealed, in addition, three further important functions. First, after DSB formation, Mer2 is required for pairing by mediating homolog spatial juxtaposition, with implications for crossover (CO) patterning/interference. Second, Mer2 participates in the transfer/maintenance and release of recombination complexes to/from the SC central region. Third, after completion of recombination, potentially dependent on SUMOylation, Mer2 mediates global chromosome compaction and post-recombination chiasma development. Thus, beyond its role as a recombinosome–axis/SC linker molecule, Mer2 has important functions in relation to basic chromosome structure. PMID:29021238

  7. Macrocyclic lactones differ in interaction with recombinant P-glycoprotein 9 of the parasitic nematode Cylicocylus elongatus and ketoconazole in a yeast growth assay.

    PubMed

    Kaschny, Maximiliane; Demeler, Janina; Janssen, I Jana I; Kuzmina, Tetiana A; Besognet, Bruno; Kanellos, Theo; Kerboeuf, Dominique; von Samson-Himmelstjerna, Georg; Krücken, Jürgen

    2015-04-01

    Macrocyclic lactones (MLs) are widely used parasiticides against nematodes and arthropods, but resistance is frequently observed in parasitic nematodes of horses and livestock. Reports claiming resistance or decreased susceptibility in human nematodes are increasing. Since no target site directed ML resistance mechanisms have been identified, non-specific mechanisms were frequently implicated in ML resistance, including P-glycoproteins (Pgps, designated ABCB1 in vertebrates). Nematode genomes encode many different Pgps (e.g. 10 in the sheep parasite Haemonchus contortus). ML transport was shown for mammalian Pgps, Pgps on nematode egg shells, and very recently for Pgp-2 of H. contortus. Here, Pgp-9 from the equine parasite Cylicocyclus elongatus (Cyathostominae) was expressed in a Saccharomyces cerevisiae strain lacking seven endogenous efflux transporters. Pgp was detected on these yeasts by flow cytometry and chemiluminescence using the monoclonal antibody UIC2, which is specific for the active Pgp conformation. In a growth assay, Pgp-9 increased resistance to the fungicides ketoconazole, actinomycin D, valinomycin and daunorubicin, but not to the anthelmintic fungicide thiabendazole. Since no fungicidal activity has been described for MLs, their interaction with Pgp-9 was investigated in an assay involving two drugs: Yeasts were incubated with the highest ketoconazole concentration not affecting growth plus increasing concentrations of MLs to determine competition between or modulation of transport of both drugs. Already equimolar concentrations of ivermectin and eprinomectin inhibited growth, and at fourfold higher ML concentrations growth was virtually abolished. Selamectin and doramectin did not increase susceptibility to ketoconazole at all, although doramectin has been shown previously to strongly interact with human and canine Pgp. An intermediate interaction was observed for moxidectin. This was substantiated by increased binding of UIC2 antibodies in the

  8. Macrocyclic Lactones Differ in Interaction with Recombinant P-Glycoprotein 9 of the Parasitic Nematode Cylicocylus elongatus and Ketoconazole in a Yeast Growth Assay

    PubMed Central

    Kaschny, Maximiliane; Demeler, Janina; Janssen, I. Jana I.; Kuzmina, Tetiana A.; Besognet, Bruno; Kanellos, Theo; Kerboeuf, Dominique; von Samson-Himmelstjerna, Georg; Krücken, Jürgen

    2015-01-01

    Macrocyclic lactones (MLs) are widely used parasiticides against nematodes and arthropods, but resistance is frequently observed in parasitic nematodes of horses and livestock. Reports claiming resistance or decreased susceptibility in human nematodes are increasing. Since no target site directed ML resistance mechanisms have been identified, non-specific mechanisms were frequently implicated in ML resistance, including P-glycoproteins (Pgps, designated ABCB1 in vertebrates). Nematode genomes encode many different Pgps (e.g. 10 in the sheep parasite Haemonchus contortus). ML transport was shown for mammalian Pgps, Pgps on nematode egg shells, and very recently for Pgp-2 of H. contortus. Here, Pgp-9 from the equine parasite Cylicocyclus elongatus (Cyathostominae) was expressed in a Saccharomyces cerevisiae strain lacking seven endogenous efflux transporters. Pgp was detected on these yeasts by flow cytometry and chemiluminescence using the monoclonal antibody UIC2, which is specific for the active Pgp conformation. In a growth assay, Pgp-9 increased resistance to the fungicides ketoconazole, actinomycin D, valinomycin and daunorubicin, but not to the anthelmintic fungicide thiabendazole. Since no fungicidal activity has been described for MLs, their interaction with Pgp-9 was investigated in an assay involving two drugs: Yeasts were incubated with the highest ketoconazole concentration not affecting growth plus increasing concentrations of MLs to determine competition between or modulation of transport of both drugs. Already equimolar concentrations of ivermectin and eprinomectin inhibited growth, and at fourfold higher ML concentrations growth was virtually abolished. Selamectin and doramectin did not increase susceptibility to ketoconazole at all, although doramectin has been shown previously to strongly interact with human and canine Pgp. An intermediate interaction was observed for moxidectin. This was substantiated by increased binding of UIC2 antibodies in the

  9. Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection.

    PubMed

    Tipper, Donald J; Szomolanyi-Tsuda, Eva

    2016-01-01

    Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes β-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%.

  10. [Improvement of thermal adaptability and fermentation of industrial ethanologenic yeast by genomic DNA mutagenesis-based genetic recombination].

    PubMed

    Liu, Xiuying; He, Xiuping; Lu, Ying; Zhang, Borun

    2011-07-01

    Ethanol is an attractive alternative to fossil fuels. Saccharomyces cerevisiae is the most important ethanol producer. However, in the process of industrial production of ethanol, both cell growth and fermentation of ethanologenic S. cerevisiae are dramatically affected by environmental stresses, such as thermal stress. In this study, we improved both the thermotolerance and fermentation performance of industrial ethanologenic S. cerevisiae by combined usage of chemical mutagenesis and genomic DNA mutagenesis-based genetic recombination method. The recombinant S. cerevisiae strain T44-2 could grow at 44 degrees C, 3 degrees C higher than that of the original strain CE6. The survival rate of T44-2 was 1.84 and 1.87-fold of that of CE6 when heat shock at 48 degrees C and 52 degrees C for 1 h respectively. At temperature higher than 37 degrees C, recombinant strain T44-2 always gave higher cell growth and ethanol production than those of strain CE6. Meanwhile, from 30 degrees C to 40 degrees C, recombinant strain T44-2 produces 91.2-83.8 g/L of ethanol from 200 g/L of glucose, which indicated that the recombinant strain T44-2 had both thermotolerance and broad thermal adaptability. The work offers a novel method, called genomic DNA mutagenesis-based genetic recombination, to improve the physiological functions of S. cerevisiae.

  11. Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis.

    PubMed

    Medhi, Darpan; Goldman, Alastair Sh; Lichten, Michael

    2016-11-18

    The budding yeast genome contains regions where meiotic recombination initiates more frequently than in others. This pattern parallels enrichment for the meiotic chromosome axis proteins Hop1 and Red1. These proteins are important for Spo11-catalyzed double strand break formation; their contribution to crossover recombination remains undefined. Using the sequence-specific VMA1 -derived endonuclease (VDE) to initiate recombination in meiosis, we show that chromosome structure influences the choice of proteins that resolve recombination intermediates to form crossovers. At a Hop1-enriched locus, most VDE-initiated crossovers, like most Spo11-initiated crossovers, required the meiosis-specific MutLγ resolvase. In contrast, at a locus with lower Hop1 occupancy, most VDE-initiated crossovers were MutLγ-independent. In pch2 mutants, the two loci displayed similar Hop1 occupancy levels, and VDE-induced crossovers were similarly MutLγ-dependent. We suggest that meiotic and mitotic recombination pathways coexist within meiotic cells, and that features of meiotic chromosome structure determine whether one or the other predominates in different regions.

  12. Cloning and expression of phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica PI12

    NASA Astrophysics Data System (ADS)

    Jaafar, Nardiah Rizwana; Bakar, Farah Diba Abu; Murad, Abdul Munir Abdul; Mahadi, Nor Muhammad

    2015-09-01

    The conversion of 3-phosphoglycerate to 2-phosphoglycerate during glycolysis and gluconeogenesis is catalyzed by phosphoglycerate mutase (PGM). Better understanding of metabolic reactions performed by this enzyme has been studied extensively in prokaryotes and eukaryotes. Here, we report a phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica. cDNA encoding for PGM from G. antarctica PI12, a psychrophilic yeast isolated from sea ice at Casey Station, Antarctica was amplified. The gene was then cloned into a cloning vector and sequenced, which verified its identity as the gene putatively encoding for PGM. The recombinant protein was expressed in Escherichia coli BL21 (DE3) as inclusion bodies and this was confirmed by SDS-PAGE and Western blot.

  13. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

    PubMed Central

    2012-01-01

    Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed

  14. Optimization of the Production Process and Characterization of the Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1), a SARS Vaccine Candidate.

    PubMed

    Chen, Wen-Hsiang; Chag, Shivali M; Poongavanam, Mohan V; Biter, Amadeo B; Ewere, Ebe A; Rezende, Wanderson; Seid, Christopher A; Hudspeth, Elissa M; Pollet, Jeroen; McAtee, C Patrick; Strych, Ulrich; Bottazzi, Maria Elena; Hotez, Peter J

    2017-08-01

    From 2002 to 2003, a global pandemic of severe acute respiratory syndrome (SARS) spread to 5 continents and caused 8000 respiratory infections and 800 deaths. To ameliorate the effects of future outbreaks as well as to prepare for biodefense, a process for the production of a recombinant protein vaccine candidate is under development. Previously, we reported the 5 L scale expression and purification of a promising recombinant SARS vaccine candidate, RBD219-N1, the 218-amino acid residue receptor-binding domain (RBD) of SARS coronavirus expressed in yeast-Pichia pastoris X-33. When adjuvanted with aluminum hydroxide, this protein elicited high neutralizing antibody titers and high RBD-specific antibody titers. However, the yield of RBD219-N1 (60 mg RBD219-N1 per liter of fermentation supernatant; 60 mg/L FS) still required improvement to reach our target of >100 mg/L FS. In this study, we optimized the 10 L scale production process and increased the fermentation yield 6- to 7-fold to 400 mg/L FS with purification recovery >50%. A panel of characterization tests indicated that the process is reproducible and that the purified, tag-free RBD219-N1 protein has high purity and a well-defined structure and is therefore a suitable candidate for production under current Good Manufacturing Practice and future phase-1 clinical trials. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Studying the replicative life span of yeast cells.

    PubMed

    Sinclair, David A

    2013-01-01

    The budding yeast Saccharomyces cerevisiae is a useful model for elucidating the pathways that control life span and the influence of environmental factors, such as calorie restriction (CR). For 75 years, CR has been studied for its ability to delay diseases of aging in mammals, from cancer to cardiovascular disease (McCay et al., Nutr Rev 33:241-243, 1975). In many other species, reducing calorie intake extends life span, including unicellular organisms (Jiang et al., FASEB J 14:2135-2137, 2000; Lin et al., Science 289:2126-2128, 2000), invertebrates (Rogina and Helfand, Proc Natl Acad Sci U S A 101:15998-16003, 2004), and rodents (Martín-Montalvo et al., Oncogene 30:505-520, 2011). Here we describe how to calorically restrict yeast cells, the methods used to determine the replicative life span (RLS) of budding yeast cells, how to selectively kill daughter cells using the mother enrichment program (MEP), how to measure recombination frequency at the rDNA locus, how to isolate large quantities of old cells, and how to analyze the circular forms of DNA known as extrachromosomal rDNA circles (ERCs), a cause of aging in S. cerevisiae (Petes, Cell 19:765-774, 1980; Sinclair and Guarente, Cell 91:1033-1042, 1997; Defossez et al., Mol Cell 3:447-455, 1999).

  16. [Genetic system for maintaining the mitochondrial human genome in yeast Yarrowia lipolytica].

    PubMed

    Isakova, E P; Deryabina, Yu I; Velyakova, A V; Biryukova, J K; Teplova, V V; Shevelev, A B

    2016-01-01

    For the first time, the possibility of maintaining an intact human mitochondrial genome in a heterologous system in the mitochondria of yeast Yarrowia lipolytica is shown. A method for introducing directional changes into the structure of the mitochondrial human genome replicating in Y. lipolytica by an artificially induced ability of yeast mitochondria for homologous recombination is proposed. A method of introducing and using phenotypic selection markers for the presence or absence of defects in genes tRNA-Lys and tRNA-Leu of the mitochondrial genome is developed. The proposed system can be used to correct harmful mutations of the human mitochondrial genome associated with mitochondrial diseases and for preparative amplification of intact mitochondrial DNA with an adjusted sequence in yeast cells. The applicability of the new system for the correction of mutations in the genes of Lys- and Leu-specific tRNAs of the human mitochondrial genome associated with serious and widespread human mitochondrial diseases such as myoclonic epilepsy with lactic acidosis (MELAS) and myoclonic epilepsy with ragged-red fibers (MERRF) is shown.

  17. Companion Protease Inhibitors for the In Situ Protection of Recombinant Proteins in Plants.

    PubMed

    Robert, Stéphanie; Jutras, Philippe V; Khalf, Moustafa; D'Aoust, Marc-André; Goulet, Marie-Claire; Sainsbury, Frank; Michaud, Dominique

    2016-01-01

    We previously described a procedure for the use of plant protease inhibitors as "companion" accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265-273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues. A procedure is first described involving inhibitors co-expressed along-and co-migrating-with the protein of interest in host plant cells. An alternative, single transgene scheme is then described involving translational fusions of the recombinant protein and companion inhibitor. These approaches may allow for a significant improvement of protein steady-state levels in leaves, comparable to yield improvements observed with protease-deficient strains of less complex protein expression hosts such as E. coli or yeasts.

  18. Definition of culture conditions for Arxula adeninivorans, a rational basis for studying heterologous gene expression in this dimorphic yeast.

    PubMed

    Stöckmann, Christoph; Palmen, Thomas G; Schroer, Kirsten; Kunze, Gotthard; Gellissen, Gerd; Büchs, Jochen

    2014-06-01

    The yeast Arxula adeninivorans is considered to be a promising producer of recombinant proteins. However, growth characteristics are poorly investigated and no industrial process has been established yet. Though of vital interest for strain screening and production processes, rationally defined culture conditions remain to be developed. A cultivation system was evolved based on targeted sampling and mathematical analysis of rationally designed small-scale cultivations in shake flasks. The oxygen and carbon dioxide transfer rates were analyzed as conclusive online parameters. Oxygen limitation extended cultivation and led to ethanol formation in cultures supplied with glucose. Cultures were inhibited at pH-values below 2.8. The phosphorus demand was determined as 1.55 g phosphorus per 100 g cell dry weight. Synthetic SYN6 medium with 20 g glucose l(-1) was optimized for cultivation in shake flasks by buffering at pH 6.4 with 140 mmol MES l(-1). Optimized SYN6 medium and operating conditions provided non-limited cultivations without by-product formation. A maximal specific growth rate of 0.32 h(-1) and short fermentations of 15 h were achieved. A pH optimum curve was derived from the oxygen transfer rates of differently buffered cultures, showing maximal growth between pH 2.8 and 6.5. Furthermore, it was shown that the applied medium and cultivation conditions were also suitable for non-limiting growth and product formation of a genetically modified A. adeninivorans strain expressing a heterologous phytase.

  19. Design and fabrication of dichroic mirrors for color separation and recombination of the Kr-Ar laser (white laser)

    NASA Astrophysics Data System (ADS)

    Park, Jungho; Park, Youngjun; Hwang, Young M.

    1997-10-01

    Cut-off filters reject all the radiation below and transmit all the above a certain wavelength and vice versa. In this paper, we will study the design and fabrication of a short wave pass or a long wave pass dichroic mirrors for color separation and recombination from the R.G.B. color beam source. In the laser display system, color separation and recombination is very important. We designed the coating layers so that the best performance may be obtained from a 45 degree incident s-polarized light. The following fabrication specification is satisfied in our color separation/recombination of the Kr-Ar laser source. The first dichroic mirror for the blue color separation, maximized on reflectance and transmittance as R > 99% in the blue regions (400 approximately 490 nm) and T > 90% in the green and red region (510 approximately 700 nm). The second dichroic mirror for the color recombination maximized the reflectance and transmittance as R > 99% in the range of 510 approximately 700 nm and T > 90% in the blue color region. In the third dichroic mirror for which it used the color separation and recombination of the green and red simultaneously, maximized the reflectance and transmittance as R > 99% in the green region (510 approximately 560 nm) and T > 90% in the red region. These fabricated mirrors were applied in our laser display projection system. We obtained an excellent result.

  20. The Spheroplast Lysis Assay for Yeast in Microtiter Plate Format

    PubMed Central

    Ovalle, Rafael; Spencer, Moyah; Thiwanont, Monthiwa; Lipke, Peter N.

    1999-01-01

    A yeast lysis assay in the microtiter plate format improved precision and throughput and led to an improved algorithm for estimating lag time. The assay reproducibly revealed differences of 10% or greater in the maximal lysis rate and 50% or greater in the lag time. Clonal differences were determined to be the major source of variation. Microtiter-based assays should be useful for screening for drug susceptibility and for analyzing mutant phenotypes. PMID:10427014

  1. A novel and robust recombinant Pichia pastoris yeast whole cell biocatalyst with intracellular overexpression of a Thermomyces lanuginosus lipase: preparation, characterization and application in biodiesel production.

    PubMed

    Yan, Jinyong; Zheng, Xianliang; Li, Shengying

    2014-01-01

    A novel and robust recombinant Pichia pastoris yeast whole cell catalyst (WCC) with functional intracellular expression of Thermomyces lanuginosus lipase (Tll) was constructed and characterized for biodiesel production from waste cooking oils. This permeabilized WCC was able to convert waste cooking oils to biodiesel with 82% yield within 84 h at 6% dosage whole cells. The WCC showed two fold catalytic activity of 0.73 U/mg DCW compared to its commercial counterpart Lipozyme TLIM (immobilized Tll). Short chain alcohol tolerance of this WCC was significantly improved compared to Lipozyme TLIM. This beneficial property enabled it to catalyze biodiesel production efficiently with one step addition of methanol. The reusability of this biocatalyst retained 78% activity after three batch cycles. This easily prepared and cost-effective WCC showed better catalytic performance than Lipozyme TLIM with respect to biodiesel yield and productivity, thus suggesting a promising cost-effective biocatalyst for biodiesel production. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Recombinant antibodies and their use in biosensors.

    PubMed

    Zeng, Xiangqun; Shen, Zhihong; Mernaugh, Ray

    2012-04-01

    Inexpensive, noninvasive immunoassays can be used to quickly detect disease in humans. Immunoassay sensitivity and specificity are decidedly dependent upon high-affinity, antigen-specific antibodies. Antibodies are produced biologically. As such, antibody quality and suitability for use in immunoassays cannot be readily determined or controlled by human intervention. However, the process through which high-quality antibodies can be obtained has been shortened and streamlined by use of genetic engineering and recombinant antibody techniques. Antibodies that traditionally take several months or more to produce when animals are used can now be developed in a few weeks as recombinant antibodies produced in bacteria, yeast, or other cell types. Typically most immunoassays use two or more antibodies or antibody fragments to detect antigens that are indicators of disease. However, a label-free biosensor, for example, a quartz-crystal microbalance (QCM) needs one antibody only. As such, the cost and time needed to design and develop an immunoassay can be substantially reduced if recombinant antibodies and biosensors are used rather than traditional antibody and assay (e.g. enzyme-linked immunosorbant assay, ELISA) methods. Unlike traditional antibodies, recombinant antibodies can be genetically engineered to self-assemble on biosensor surfaces, at high density, and correctly oriented to enhance antigen-binding activity and to increase assay sensitivity, specificity, and stability. Additionally, biosensor surface chemistry and physical and electronic properties can be modified to further increase immunoassay performance above and beyond that obtained by use of traditional methods. This review describes some of the techniques investigators have used to develop highly specific and sensitive, recombinant antibody-based biosensors for detection of antigens in simple or complex biological samples.

  3. The temporal response of recombination events to gamma radiation of meiotic cells in Sordaria brevicollis.

    PubMed

    Lewis, L A

    1982-01-01

    The temporal frequencies of different stages of prophase I were determined cytologically in Sordaria brevicollis (Olive and Fantini) as the basis for ascertaining the degree of synchrony in meiosis in this ascomycete. Croziers, karyogamy-zygotene and pachytene asci were shown to be in significant majorities at three distinct periods of the meiotic cycle. The response of recombination frequency to ionizing radiation was examined for the entire meiotic cycle. Three radiosensitive periods were determined. This response, which correlated temporally with each of the three peaks in ascal frequency, is interpreted as showing that the meiotic cycle of this organism is divided into periods of recombination commitment (radiation reduced frequencies) during the pre-meiotic S phase and recombination consummation (radiation induced frequencies) during zygotene and pachytene. The results are discussed in the context of the time at which recombination is consummated in eukaryotes such as yeast and Drosophila.

  4. Viral vectors for production of recombinant proteins in plants.

    PubMed

    Lico, Chiara; Chen, Qiang; Santi, Luca

    2008-08-01

    Global demand for recombinant proteins has steadily accelerated for the last 20 years. These recombinant proteins have a wide range of important applications, including vaccines and therapeutics for human and animal health, industrial enzymes, new materials and components of novel nano-particles for various applications. The majority of recombinant proteins are produced by traditional biological "factories," that is, predominantly mammalian and microbial cell cultures along with yeast and insect cells. However, these traditional technologies cannot satisfy the increasing market demand due to prohibitive capital investment requirements. During the last two decades, plants have been under intensive investigation to provide an alternative system for cost-effective, highly scalable, and safe production of recombinant proteins. Although the genetic engineering of plant viral vectors for heterologous gene expression can be dated back to the early 1980s, recent understanding of plant virology and technical progress in molecular biology have allowed for significant improvements and fine tuning of these vectors. These breakthroughs enable the flourishing of a variety of new viral-based expression systems and their wide application by academic and industry groups. In this review, we describe the principal plant viral-based production strategies and the latest plant viral expression systems, with a particular focus on the variety of proteins produced and their applications. We will summarize the recent progress in the downstream processing of plant materials for efficient extraction and purification of recombinant proteins. (c) 2008 Wiley-Liss, Inc.

  5. Variant forms of mitochondrial translation products in yeast: evidence for location of determinants on mitochondrial DNA.

    PubMed

    Douglas, M G; Butow, R A

    1976-04-01

    Products of mitochondrial protein synthesis in yeast have been labeled in vivo with 35SO42-. More than 20 polypeptide species fulfilling the criteria of mitochondrial translation products have been detected by analysis on sodium dodecyl sulfate-exponential polyacrylamide slab gels. A comparison of mitochondrial translation products in two wild-type strains has revealed variant forms of some polypeptide species which show genetic behavior consistent with the location of their structural genes on mtDNA. Our results demonstrate the feasibility of performing genetic analysis on putative gene products of mtDNA in wild-type yeast by direct examination of the segregation and recombination behavior of specific polypeptide species.

  6. An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species

    PubMed Central

    Sitepu, I.R.; Ignatia, L.; Franz, A. K.; Wong, D. M.; Faulina, S.A.; Tsui, M.; Kanti, A.; Boundy-Mills, K.

    2012-01-01

    A rapid and inexpensive method for estimating lipid content of yeasts is needed for screening large numbers of yeasts samples. Nile red is a fluorescent lipophilic dye used for detection and quantification of intracellular lipid droplets in various biological system including algae, yeasts and filamentous fungi. However, a published assay for yeast is affected by variable diffusion across the cell membrane, and variation in the time required to reach maximal fluorescence emission. In this study, parameters that may influence the emission were varied to determine optimal assay conditions. An improved assay with a high-throughput capability was developed that includes the addition of dimethyl sulfoxide (DMSO) solvent to improve cell permeability, elimination of the washing step, the reduction of Nile red concentration, kinetic readings rather than single time-point reading, and utilization of a black 96-well microplate. The improved method was validated by comparison to gravimetric determination of lipid content of a broad variety of ascomycete and basidiomycete yeast species. PMID:22985718

  7. Yeast-expressed recombinant As16 protects mice against Ascaris suum infection through induction of a Th2-skewed immune response

    PubMed Central

    Liu, Zhuyun; Keegan, Brian; Gazzinelli-Guimarães, Ana Clara; Fujiwara, Ricardo T.; Briggs, Neima; Jones, Kathryn M.; Strych, Ulrich; Beaumier, Coreen M.; Bottazzi, Maria Elena; Zhan, Bin

    2017-01-01

    Background Ascariasis remains the most common helminth infection in humans. As an alternative or complementary approach to global deworming, a pan-anthelminthic vaccine is under development targeting Ascaris, hookworm, and Trichuris infections. As16 and As14 have previously been described as two genetically related proteins from Ascaris suum that induced protective immunity in mice when formulated with cholera toxin B subunit (CTB) as an adjuvant, but the exact protective mechanism was not well understood. Methodology/Principal findings As16 and As14 were highly expressed as soluble recombinant proteins (rAs16 and rAs14) in Pichia pastoris. The yeast-expressed rAs16 was highly recognized by immune sera from mice infected with A. suum eggs and elicited 99.6% protection against A. suum re-infection. Mice immunized with rAs16 formulated with ISA720 displayed significant larva reduction (36.7%) and stunted larval development against A. suum eggs challenge. The protective immunity was associated with a predominant Th2-type response characterized by high titers of serological IgG1 (IgG1/IgG2a > 2000) and high levels of IL-4 and IL-5 produced by restimulated splenocytes. A similar level of protection was observed in mice immunized with rAs16 formulated with alum (Alhydrogel), known to induce mainly a Th2-type immune response, whereas mice immunized with rAs16 formulated with MPLA or AddaVax, both known to induce a Th1-type biased response, were not significantly protected against A. suum infection. The rAs14 protein was not recognized by A. suum infected mouse sera and mice immunized with rAs14 formulated with ISA720 did not show significant protection against challenge infection, possibly due to the protein’s inaccessibility to the host immune system or a Th1-type response was induced which would counter a protective Th2-type response. Conclusions/Significance Yeast-expressed rAs16 formulated with ISA720 or alum induced significant protection in mice against A. suum

  8. Cdc13 prevents telomere uncapping and Rad50-dependent homologous recombination

    PubMed Central

    Grandin, Nathalie; Damon, Christelle; Charbonneau, Michel

    2001-01-01

    Cdc13 performs an essential function in telomere end protection in budding yeast. Here, we analyze the consequences on telomere dynamics of cdc13-induced telomeric DNA damage in proliferating cells. Checkpoint-deficient cdc13-1 cells accumulated DNA damage and eventually senesced. However, these telomerase-proficient cells could survive by using homologous recombination but, contrary to telomerase-deficient cells, did so without prior telomere shortening. Strikingly, homologous recombination in cdc13-1 mec3, as well as in telomerase-deficient cdc13-1 cells, which were Rad52- and Rad50-dependent but Rad51-independent, exclusively amplified the TG1–3 repeats. This argues that not only short telomeres are substrates for type II recombination. The Cdc13-1 mutant protein harbored a defect in its association with Stn1 and Ten1 but also an additional, unknown, defect that could not be cured by expressing a Cdc13-1– Ten1–Stn1 fusion. We propose that Cdc13 prevents telomere uncapping and inhibits recombination between telomeric sequences through a pathway distinct from and complementary to that used by telomerase. PMID:11689452

  9. Trans-Lesion DNA Polymerases May Be Involved in Yeast Meiosis

    PubMed Central

    Arbel-Eden, Ayelet; Joseph-Strauss, Daphna; Masika, Hagit; Printzental, Oxana; Rachi, Eléanor; Simchen, Giora

    2013-01-01

    Trans-lesion DNA polymerases (TLSPs) enable bypass of DNA lesions during replication and are also induced under stress conditions. Being only weakly dependent on their template during replication, TLSPs introduce mutations into DNA. The low processivity of these enzymes ensures that they fall off their template after a few bases are synthesized and are then replaced by the more accurate replicative polymerase. We find that the three TLSPs of budding yeast Saccharomyces cerevisiae Rev1, PolZeta (Rev3 and Rev7), and Rad30 are induced during meiosis at a time when DNA double-strand breaks (DSBs) are formed and homologous chromosomes recombine. Strains deleted for one or any combination of the three TLSPs undergo normal meiosis. However, in the triple-deletion mutant, there is a reduction in both allelic and ectopic recombination. We suggest that trans-lesion polymerases are involved in the processing of meiotic double-strand breaks that lead to mutations. In support of this notion, we report significant yeast two-hybrid (Y2H) associations in meiosis-arrested cells between the TLSPs and DSB proteins Rev1-Spo11, Rev1-Mei4, and Rev7-Rec114, as well as between Rev1 and Rad30. We suggest that the involvement of TLSPs in processing of meiotic DSBs could be responsible for the considerably higher frequency of mutations reported during meiosis compared with that found in mitotically dividing cells, and therefore may contribute to faster evolutionary divergence than previously assumed. PMID:23550131

  10. Gateway Vectors for Efficient Artificial Gene Assembly In Vitro and Expression in Yeast Saccharomyces cerevisiae

    PubMed Central

    Giuraniuc, Claudiu V.; MacPherson, Murray; Saka, Yasushi

    2013-01-01

    Construction of synthetic genetic networks requires the assembly of DNA fragments encoding functional biological parts in a defined order. Yet this may become a time-consuming procedure. To address this technical bottleneck, we have created a series of Gateway shuttle vectors and an integration vector, which facilitate the assembly of artificial genes and their expression in the budding yeast Saccharomyces cerevisiae. Our method enables the rapid construction of an artificial gene from a promoter and an open reading frame (ORF) cassette by one-step recombination reaction in vitro. Furthermore, the plasmid thus created can readily be introduced into yeast cells to test the assembled gene’s functionality. As flexible regulatory components of a synthetic genetic network, we also created new versions of the tetracycline-regulated transactivators tTA and rtTA by fusing them to the auxin-inducible degron (AID). Using our gene assembly approach, we made yeast expression vectors of these engineered transactivators, AIDtTA and AIDrtTA and then tested their functions in yeast. We showed that these factors can be regulated by doxycycline and degraded rapidly after addition of auxin to the medium. Taken together, the method for combinatorial gene assembly described here is versatile and would be a valuable tool for yeast synthetic biology. PMID:23675537

  11. Yeast Based Sensors

    NASA Astrophysics Data System (ADS)

    Shimomura-Shimizu, Mifumi; Karube, Isao

    Since the first microbial cell sensor was studied by Karube et al. in 1977, many types of yeast based sensors have been developed as analytical tools. Yeasts are known as facultative anaerobes. Facultative anaerobes can survive in both aerobic and anaerobic conditions. The yeast based sensor consisted of a DO electrode and an immobilized omnivorous yeast. In yeast based sensor development, many kinds of yeast have been employed by applying their characteristics to adapt to the analyte. For example, Trichosporon cutaneum was used to estimate organic pollution in industrial wastewater. Yeast based sensors are suitable for online control of biochemical processes and for environmental monitoring. In this review, principles and applications of yeast based sensors are summarized.

  12. Construction of C35 gene bait recombinants and T47D cell cDNA library.

    PubMed

    Yin, Kun; Xu, Chao; Zhao, Gui-Hua; Liu, Ye; Xiao, Ting; Zhu, Song; Yan, Ge

    2017-11-20

    C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C35 1-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation. The three kinds of C35 genes were successfully amplified and inserted into pGBKT7 to construct bait recombinant plasmids pGBKT7-C351-348bp, pGBKT7-C351-153bp and pGBKT7-C35154-348bp, then transformed into Y187 yeast cells by the lithium acetate method. Auto-activation and toxicity of C35 baits were detected using nutritional deficient medium and X-α-Gal assays. The T47D cell ds cDNA was generated by SMART TM technology and the library was constructed using in vivo recombination-mediated cloning in the AH109 yeast strain using a pGADT7-Rec plasmid. The transformed Y187/pGBKT7-C351-348bp line was intensively inhibited while the truncated Y187/pGBKT7-C35 lines had no auto-activation and toxicity in yeast cells. The titer of established cDNA library was 2 × 10 7 pfu/mL with high transformation efficiency of 1.4 × 10 6 , and the insert size of ds cDNA was distributed homogeneously between 0.5-2.0 kb. Our research generated a T47D cell cDNA library with high titer, and the constructed two C35 "baits" contained a respective functional immunoreceptor tyrosine based activation motif (ITAM) and the conserved last four amino acids Cys-Ile-Leu-Val (CILV) motif, and therefore laid a foundation for screening the C35 interaction factors in a BC cell line.

  13. The potential of genetic engineering for improving brewing, wine-making and baking yeasts.

    PubMed

    Dequin, S

    2001-09-01

    The end of the twentieth century was marked by major advances in life technology, particularly in areas related to genetics and more recently genomics. Considerable progress was made in the development of genetically improved yeast strains for the wine, brewing and baking industries. In the last decade, recombinant DNA technology widened the possibilities for introducing new properties. The most remarkable advances, which are discussed in this Mini-Review, are improved process performance, off-flavor elimination, increased formation of by-products, improved hygienic properties or extension of substrate utilization. Although the introduction of this technology into traditional industries is currently limited by public perception, the number of potential applications of genetically modified industrial yeast is likely to increase in the coming years, as our knowledge derived from genomic analyses increases.

  14. Directed alteration of Saccharomyces cerevisiae mitochondrial DNA by biolistic transformation and homologous recombination.

    PubMed

    Bonnefoy, Nathalie; Fox, Thomas D

    2007-01-01

    Saccharomyces cerevisiae is currently the only species in which genetic transformation of mitochondria can be used to generate a wide variety of defined alterations in mitochondrial deoxyribonucleic acid (mtDNA). DNA sequences can be delivered into yeast mitochondria by microprojectile bombardment (biolistic transformation) and subsequently incorporated into mtDNA by the highly active homologous recombination machinery present in the organelle. Although transformation frequencies are relatively low, the availability of strong mitochondrial selectable markers for the yeast system, both natural and synthetic, makes the isolation of transformants routine. The strategies and procedures reviewed here allow the researcher to insert defined mutations into endogenous mitochondrial genes and to insert new genes into mtDNA. These methods provide powerful in vivo tools for the study of mitochondrial biology.

  15. Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    PubMed Central

    Medhi, Darpan; Goldman, Alastair SH; Lichten, Michael

    2016-01-01

    The budding yeast genome contains regions where meiotic recombination initiates more frequently than in others. This pattern parallels enrichment for the meiotic chromosome axis proteins Hop1 and Red1. These proteins are important for Spo11-catalyzed double strand break formation; their contribution to crossover recombination remains undefined. Using the sequence-specific VMA1-derived endonuclease (VDE) to initiate recombination in meiosis, we show that chromosome structure influences the choice of proteins that resolve recombination intermediates to form crossovers. At a Hop1-enriched locus, most VDE-initiated crossovers, like most Spo11-initiated crossovers, required the meiosis-specific MutLγ resolvase. In contrast, at a locus with lower Hop1 occupancy, most VDE-initiated crossovers were MutLγ-independent. In pch2 mutants, the two loci displayed similar Hop1 occupancy levels, and VDE-induced crossovers were similarly MutLγ-dependent. We suggest that meiotic and mitotic recombination pathways coexist within meiotic cells, and that features of meiotic chromosome structure determine whether one or the other predominates in different regions. DOI: http://dx.doi.org/10.7554/eLife.19669.001 PMID:27855779

  16. Molecular and physiological characteristics of a grape yeast strain containing atypical genetic material.

    PubMed

    Cappello, M S; Poltronieri, P; Blaiotta, G; Zacheo, G

    2010-11-15

    The knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae and Saccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from "Primitivo" grape (http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic material from a non-Saccharomyces species. The study was focused on the analysis of chromosomal and mitochondrial gene sequences (ITS and 26S rRNA, SSU and COXII, ACTIN-1 and TEF), 2D-PAGE mitochondrial proteins, and spore viability. The results allowed us to formulate the hypothesis that in the MSH199 isolate a DNA containing an rDNA sequence from Hanseniaspora vineae, a non-Saccharomyces yeast, was incorporated through homologous recombination in the grape environment where yeast species are propagated. Moreover, physiological characterization showed that the MSH199 isolate possesses high technological quality traits (fermentation performance) and glycerol production, resistance to ethanol, SO₂ and temperature) useful for industrial application. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. Extracellular Polysaccharides Produced by Yeasts and Yeast-Like Fungi

    NASA Astrophysics Data System (ADS)

    van Bogaert, Inge N. A.; de Maeseneire, Sofie L.; Vandamme, Erick J.

    Several yeasts and yeast-like fungi are known to produce extracellular polysaccharides. Most of these contain D-mannose, either alone or in combination with other sugars or phosphate. A large chemical and structural variability is found between yeast species and even among different strains. The types of polymers that are synthesized can be chemically characterized as mannans, glucans, phosphoman-nans, galactomannans, glucomannans and glucuronoxylomannans. Despite these differences, almost all of the yeast exopolysaccharides display some sort of biological activity. Some of them have already applications in chemistry, pharmacy, cosmetics or as probiotic. Furthermore, some yeast exopolysaccharides, such as pullulan, exhibit specific physico-chemical and rheological properties, making them useful in a wide range of technical applications. A survey is given here of the production, the characteristics and the application potential of currently well studied yeast extracellular polysaccharides.

  18. Yeast Infection (Vaginal)

    MedlinePlus

    Yeast infection (vaginal) Overview A vaginal yeast infection is a fungal infection that causes irritation, discharge and intense itchiness ... symptoms Causes The fungus candida causes a vaginal yeast infection. Your vagina naturally contains a balanced mix of yeast, including ...

  19. Yeast cell surface display for lipase whole cell catalyst and its applications

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Yun; Zhang, Rui; Lian, Zhongshuai

    The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chainmore » length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.« less

  20. Genotoxicity of diphenyl diselenide in bacteria and yeast.

    PubMed

    Rosa, Renato Moreira; Sulzbacher, Krisley; Picada, Jaqueline Nascimento; Roesler, Rafael; Saffi, Jenifer; Brendel, Martin; Henriques, João Antonio Pêgas

    2004-10-10

    Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds. This may increase the risk of human exposure to the chemical at the workplace. We have determined its mutagenic potential in the Salmonella/microsome assay and used the yeast Saccharomyces cerevisiae to assay for putative genotoxicity, recombinogenicity and to determine whether DNA damage produced by DPDS is repairable. Only in exponentially growing cultures was DPDS able to induce frameshift mutations in S. typhimurium and haploid yeast and to increase crossing over and gene conversion frequencies in diploid strains of S. cerevisiae. Thus, DPDS presents a behavior similar to that of an intercalating agent. Mutants defective in excision-resynthesis repair (rad3, rad1), in error-prone repair (rad6) and in recombinational repair (rad52) showed higher than WT-sensitivity to DPDS. It appears that this compound is capable of inducing single and/or double strand breaks in DNA. An epistatic interaction was shown between rad3-e5 and rad52-1 mutant alleles, indicating that excision-resynthesis and strand-break repair may possess common steps in the repair of DNA damage induced by DPDS. DPDS was able to enhance the mutagenesis induced by oxidative mutagens in bacteria. N-acetylcysteine, a glutathione biosynthesis precursor, prevented mutagenesis induced by DPDS in yeast. We have shown that DPDS is a weak mutagen which probably generates DNA strand breaks through both its intercalating action and pro-oxidant effect.

  1. Yeast derivatives and wheat germ in the adult diet modulates fecundity in a tephritid pest.

    PubMed

    Goane, L; Pereyra, P M; Castro, F; Ruiz, M J; Juárez, M L; Segura, D F; Vera, M T

    2018-05-22

    Anastrepha fraterculus (Wiedemann), a pest of great economic importance in South America, needs urgently to be controlled by environmentally friendly methods such as the sterile insect technique for which mass rearing of insects is required. Because oogenesis takes place during the adult stage, mass-rearing facilities should provide the females a diet that maximizes egg production at the lowest cost. Accordingly, we investigated the effect of artificial protein sources in the adult diet (yeast derivatives of different cost but with similar amino acids profiles, and the addition of wheat germ) on fecundity. Additionally, we evaluated different ratios of yeast derivatives or wheat germ on ovary maturation, fecundity, and fertility as well as their association with the nutrient content of females. Females fed hydrolyzed yeast and yeast extract attained the highest fecundity level, and those fed brewer's yeast the lowest. Reducing the amount of hydrolyzed yeast, an expensive protein source, in the diet negatively affected fecundity and ovary maturation. Increasing the amount of brewer's yeast, a low-cost protein source, did not favor fecundity. The addition of wheat germ in the adult diet improved fecundity regardless of the yeast derivate considered. Percentage of egg hatch was not affected by the diet. Nutrient content of A. fraterculus females varied according to the adult diet provided and mating status. Our findings provide novel baseline information to understand the role of nutrition on reproductive performance of A. fraterculus females and are discussed in the context of resource allocation. They also provide valuable advances in the search for cost-effective adult diets at fruit fly mass rearing facilities.

  2. Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae.

    PubMed

    Park, Yang-Nim; Masison, Daniel; Eisenberg, Evan; Greene, Lois E

    2011-09-01

    The yeast Saccharomyces cerevisiae has proved to be an excellent model organism to study the function of proteins. One of the many advantages of yeast is the many genetic tools available to manipulate gene expression, but there are still limitations. To complement the many methods used to control gene expression in yeast, we have established a conditional gene deletion system by using the FLP/FRT system on yeast vectors to conditionally delete specific yeast genes. Expression of Flp recombinase, which is under the control of the GAL1 promoter, was induced by galactose, which in turn excised FRT sites flanked genes. The efficacy of this system was examined using the FRT site-flanked genes HSP104, URA3 and GFP. The pre-excision frequency of this system, which might be caused by the basal activity of the GAL1 promoter or by spontaneous recombination between FRT sites, was detected ca. 2% under the non-selecting condition. After inducing expression of Flp recombinase, the deletion efficiency achieved ca. 96% of cells in a population within 9 h. After conditional deletion of the specific gene, protein degradation and cell division then diluted out protein that was expressed from this gene prior to its excision. Most importantly, the specific protein to be deleted could be expressed under its own promoter, so that endogenous levels of protein expression were maintained prior to excision by the Flp recombinase. Therefore, this system provides a useful tool for the conditional deletion of genes in yeast. Published in 2011 by John Wiley & Sons, Ltd.

  3. Mitochondrial fusion increases the mitochondrial DNA copy number in budding yeast.

    PubMed

    Hori, Akiko; Yoshida, Minoru; Ling, Feng

    2011-05-01

    Mitochondrial fusion plays an important role in mitochondrial DNA (mtDNA) maintenance, although the underlying mechanisms are unclear. In budding yeast, certain levels of reactive oxygen species (ROS) can promote recombination-mediated mtDNA replication, and mtDNA maintenance depends on the homologous DNA pairing protein Mhr1. Here, we show that the fusion of isolated yeast mitochondria, which can be monitored by the bimolecular fluorescence complementation-derived green fluorescent protein (GFP) fluorescence, increases the mtDNA copy number in a manner dependent on Mhr1. The fusion event, accompanied by the degradation of dissociated electron transport chain complex IV and transient reductions in the complex IV subunits by the inner membrane AAA proteases such as Yme1, increases ROS levels. Analysis of the initial stage of mitochondrial fusion in early log-phase cells produced similar results. Moreover, higher ROS levels in mitochondrial fusion-deficient mutant cells increased the amount of newly synthesized mtDNA, resulting in increases in the mtDNA copy number. In contrast, reducing ROS levels in yme1 null mutant cells significantly decreased the mtDNA copy number, leading to an increase in cells lacking mtDNA. Our results indicate that mitochondrial fusion induces mtDNA synthesis by facilitating ROS-triggered, recombination-mediated replication and thereby prevents the generation of mitochondria lacking DNA. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  4. Yeast surface displaying glucose oxidase as whole-cell biocatalyst: construction, characterization, and its electrochemical glucose sensing application.

    PubMed

    Wang, Hongwei; Lang, Qiaolin; Li, Liang; Liang, Bo; Tang, Xiangjiang; Kong, Lingrang; Mascini, Marco; Liu, Aihua

    2013-06-18

    The display of glucose oxidase (GOx) on yeast cell surface using a-agglutinin as an anchor motif was successfully developed. Both the immunochemical analysis and enzymatic assay showed that active GOx was efficiently expressed and translocated on the cell surface. Compared with conventional GOx, the yeast cell surface that displayed GOx (GOx-yeast) demonstrated excellent enzyme properties, such as good stability within a wide pH range (pH 3.5-11.5), good thermostability (retaining over 94.8% enzyme activity at 52 °C and 84.2% enzyme activity at 56 °C), and high d-glucose specificity. In addition, direct electrochemistry was achieved at a GOx-yeast/multiwalled-carbon-nanotube modified electrode, suggesting that the host cell of yeast did not have any adverse effect on the electrocatalytic property of the recombinant GOx. Thus, a novel electrochemical glucose biosensor based on this GOx-yeast was developed. The as-prepared biosensor was linear with the concentration of d-glucose within the range of 0.1-14 mM and a low detection limit of 0.05 mM (signal-to-noise ratio of S/N = 3). Moreover, the as-prepared biosensor is stable, specific, reproducible, simple, and cost-effective, which can be applicable for real sample detection. The proposed strategy to construct robust GOx-yeast may be applied to explore other oxidase-displaying-system-based whole-cell biocatalysts, which can find broad potential application in biosensors, bioenergy, and industrial catalysis.

  5. The impact of oxygen on the transcriptome of recombinant S. cerevisiae and P. pastoris - a comparative analysis.

    PubMed

    Baumann, Kristin; Dato, Laura; Graf, Alexandra B; Frascotti, Gianni; Dragosits, Martin; Porro, Danilo; Mattanovich, Diethard; Ferrer, Pau; Branduardi, Paola

    2011-05-09

    Saccharomyces cerevisiae and Pichia pastoris are two of the most relevant microbial eukaryotic platforms for the production of recombinant proteins. Their known genome sequences enabled several transcriptomic profiling studies under many different environmental conditions, thus mimicking not only perturbations and adaptations which occur in their natural surroundings, but also in industrial processes. Notably, the majority of such transcriptome analyses were performed using non-engineered strains.In this comparative study, the gene expression profiles of S. cerevisiae and P. pastoris, a Crabtree positive and Crabtree negative yeast, respectively, were analyzed for three different oxygenation conditions (normoxic, oxygen-limited and hypoxic) under recombinant protein producing conditions in chemostat cultivations. The major differences in the transcriptomes of S. cerevisiae and P. pastoris were observed between hypoxic and normoxic conditions, where the availability of oxygen strongly affected ergosterol biosynthesis, central carbon metabolism and stress responses, particularly the unfolded protein response. Steady state conditions under low oxygen set-points seemed to perturb the transcriptome of S. cerevisiae to a much lesser extent than the one of P. pastoris, reflecting the major tolerance of the baker's yeast towards oxygen limitation, and a higher fermentative capacity. Further important differences were related to Fab production, which was not significantly affected by oxygen availability in S. cerevisiae, while a clear productivity increase had been previously reported for hypoxically grown P. pastoris. The effect of three different levels of oxygen availability on the physiology of P. pastoris and S. cerevisiae revealed a very distinct remodelling of the transcriptional program, leading to novel insights into the different adaptive responses of Crabtree negative and positive yeasts to oxygen availability. Moreover, the application of such comparative

  6. High-resolution mapping, characterization, and optimization of autonomously replicating sequences in yeast

    PubMed Central

    Liachko, Ivan; Youngblood, Rachel A.; Keich, Uri; Dunham, Maitreya J.

    2013-01-01

    DNA replication origins are necessary for the duplication of genomes. In addition, plasmid-based expression systems require DNA replication origins to maintain plasmids efficiently. The yeast autonomously replicating sequence (ARS) assay has been a valuable tool in dissecting replication origin structure and function. However, the dearth of information on origins in diverse yeasts limits the availability of efficient replication origin modules to only a handful of species and restricts our understanding of origin function and evolution. To enable rapid study of origins, we have developed a sequencing-based suite of methods for comprehensively mapping and characterizing ARSs within a yeast genome. Our approach finely maps genomic inserts capable of supporting plasmid replication and uses massively parallel deep mutational scanning to define molecular determinants of ARS function with single-nucleotide resolution. In addition to providing unprecedented detail into origin structure, our data have allowed us to design short, synthetic DNA sequences that retain maximal ARS function. These methods can be readily applied to understand and modulate ARS function in diverse systems. PMID:23241746

  7. Separation by hydrophobic interaction chromatography and structural determination by mass spectrometry of mannosylated glycoforms of a recombinant transferrin-exendin-4 fusion protein from yeast.

    PubMed

    Zolodz, Melissa D; Herberg, John T; Narepekha, Halyna E; Raleigh, Emily; Farber, Matthew R; Dufield, Robert L; Boyle, Denis M

    2010-01-08

    Obtaining sufficient amounts of pure glycoprotein variants to characterize their structures is an important goal in both functional biology and the biotechnology industry. We have developed preparative HIC conditions that resolve glycoform variants on the basis of overall carbohydrate content for a recombinant transferrin-exendin-4 fusion protein. The fusion protein was expressed from the yeast Saccharomyces cerevisiae from high density fermentation and is post-translationally modified with mannose sugars through O-glycosidic linkages. Overall hydrophobic behavior appeared to be dominated by the N-terminal 39 amino acids from the exendin-4 and linker peptide sequences as compared to the less hydrophobic behavior of human transferrin alone. In addition, using LC techniques that measure total glycans released from the pure protein combined with new high resolution technologies using mass spectrometry, we have determined the locations and chain lengths of mannose residues on specific peptides derived from tryptic maps of the transferrin-exendin-4 protein. Though the protein is large (80,488kDa) and contains 78 possible serine and threonine residues as potential sites for sugar addition, mannosylation was observed on only two tryptic peptides located within the first 55 amino acids of the N-terminus. These glycopeptides were highly heterogeneous and contained between 1 and 10 mannose residues scattered among the various serine and threonine sites which were identified by electron transfer dissociation mass spectrometry. Glycan sequences from 1 to 6 linear mannose residues were detected, but mannose chain lengths of 3 or 4 were more common and formed 80% of the total oligosaccharides. This work introduces new technological capabilities for the purification and characterization of glycosylated variants of therapeutic recombinant proteins. Copyright 2009 Elsevier B.V. All rights reserved.

  8. Interplay between Synaptonemal Complex, Homologous Recombination, and Centromeres during Mammalian Meiosis

    PubMed Central

    Qiao, Huanyu; Chen, Jefferson K.; Reynolds, April; Höög, Christer; Paddy, Michael; Hunter, Neil

    2012-01-01

    The intimate synapsis of homologous chromosome pairs (homologs) by synaptonemal complexes (SCs) is an essential feature of meiosis. In many organisms, synapsis and homologous recombination are interdependent: recombination promotes SC formation and SCs are required for crossing-over. Moreover, several studies indicate that initiation of SC assembly occurs at sites where crossovers will subsequently form. However, recent analyses in budding yeast and fruit fly imply a special role for centromeres in the initiation of SC formation. In addition, in budding yeast, persistent SC–dependent centromere-association facilitates the disjunction of chromosomes that have failed to become connected by crossovers. Here, we examine the interplay between SCs, recombination, and centromeres in a mammal. In mouse spermatocytes, centromeres do not serve as SC initiation sites and are invariably the last regions to synapse. However, centromeres are refractory to de-synapsis during diplonema and remain associated by short SC fragments. Since SC–dependent centromere association is lost before diakinesis, a direct role in homolog segregation seems unlikely. However, post–SC disassembly, we find evidence of inter-centromeric connections that could play a more direct role in promoting homolog biorientation and disjunction. A second class of persistent SC fragments is shown to be crossover-dependent. Super-resolution structured-illumination microscopy (SIM) reveals that these structures initially connect separate homolog axes and progressively diminish as chiasmata form. Thus, DNA crossing-over (which occurs during pachynema) and axis remodeling appear to be temporally distinct aspects of chiasma formation. SIM analysis of the synapsis and crossover-defective mutant Sycp1−/− implies that SCs prevent unregulated fusion of homolog axes. We propose that SC fragments retained during diplonema stabilize nascent bivalents and help orchestrate local chromosome reorganization that promotes

  9. Prominent mitochondrial DNA recombination intermediates in human heart muscle.

    PubMed

    Kajander, O A; Karhunen, P J; Holt, I J; Jacobs, H T

    2001-11-01

    Recombination intermediates containing four-way (Holliday) junctions are generated during DNA repair and replication in many systems, including yeast mitochondrial DNA (mtDNA). In contrast, convincing evidence for recombination in mammalian mtDNA is lacking. We have used two-dimensional agarose-gel electrophoresis to analyse non-linear forms of mtDNA in human heart muscle. Replication intermediates from both the coupled and strand-asynchronous mtDNA replication pathways were detected. An additional class of non-linear molecules, with the electrophoretic properties of four-way junctions, was also prominent. These molecules were insensitive to topoisomerase I or RNase H, but were diminished by branch migration or RuvC treatment. Junctional molecules were detected in all regions of the mitochondrial genome, were found in myocardial DNA from young and old adults, but were present at lower levels in skeletal muscle and placenta. We suggest that they could represent intermediates of mtDNA repair, given their prevalence in the oxyradical-rich environment of heart muscle mitochondria.

  10. DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

    PubMed Central

    Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas S.; Lisby, Michael; Salanti, Ali; Fordyce, Sarah L.; Jespersen, Jakob S.; Carter, Richard; Deitsch, Kirk W.; Theander, Thor G.; Pedersen, Anders Gorm; Arnot, David E.

    2014-01-01

    Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens. PMID:24253306

  11. Suppression of genetic recombination in the pseudoautosomal region and at subtelomeres in mice with a hypomorphic Spo11 allele.

    PubMed

    Smagulova, Fatima; Brick, Kevin; Pu, Yongmei; Sengupta, Uttara; Camerini-Otero, R Daniel; Petukhova, Galina V

    2013-07-22

    Homologous recombination is the key process that generates genetic diversity and drives evolution. SPO11 protein triggers recombination by introducing DNA double stranded breaks at discreet areas of the genome called recombination hotspots. The hotspot locations are largely determined by the DNA binding specificity of the PRDM9 protein in human, mice and most other mammals. In budding yeast Saccharomyces cerevisae, which lacks a Prdm9 gene, meiotic breaks are formed opportunistically in the regions of accessible chromatin, primarily at gene promoters. The genome-wide distribution of hotspots in this organism can be altered by tethering Spo11 protein to Gal4 recognition sequences in the strain expressing Spo11 attached to the DNA binding domain of the Gal4 transcription factor. To establish whether similar re-targeting of meiotic breaks can be achieved in PRDM9-containing organisms we have generated a Gal4BD-Spo11 mouse that expresses SPO11 protein joined to the DNA binding domain of yeast Gal4. We have mapped the genome-wide distribution of the recombination initiation sites in the Gal4BD-Spo11 mice. More than two hundred of the hotspots in these mice were novel and were likely defined by Gal4BD, as the Gal4 consensus motif was clustered around the centers in these hotspots. Surprisingly, meiotic DNA breaks in the Gal4BD-Spo11 mice were significantly depleted near the ends of chromosomes. The effect is particularly striking at the pseudoautosomal region of the X and Y chromosomes - normally the hottest region in the genome. Our data suggest that specific, yet-unidentified factors influence the initiation of meiotic recombination at subtelomeric chromosomal regions.

  12. New Protocol Based on UHPLC-MS/MS for Quantitation of Metabolites in Xylose-Fermenting Yeasts

    NASA Astrophysics Data System (ADS)

    Campos, Christiane Gonçalves; Veras, Henrique César Teixeira; de Aquino Ribeiro, José Antônio; Costa, Patrícia Pinto Kalil Gonçalves; Araújo, Katiúscia Pereira; Rodrigues, Clenilson Martins; de Almeida, João Ricardo Moreira; Abdelnur, Patrícia Verardi

    2017-12-01

    Xylose fermentation is a bottleneck in second-generation ethanol production. As such, a comprehensive understanding of xylose metabolism in naturally xylose-fermenting yeasts is essential for prospection and construction of recombinant yeast strains. The objective of the current study was to establish a reliable metabolomics protocol for quantification of key metabolites of xylose catabolism pathways in yeast, and to apply this protocol to Spathaspora arborariae. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was used to quantify metabolites, and afterwards, sample preparation was optimized to examine yeast intracellular metabolites. S. arborariae was cultivated using xylose as a carbon source under aerobic and oxygen-limited conditions. Ion pair chromatography (IPC) and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) were shown to efficiently quantify 14 and 5 metabolites, respectively, in a more rapid chromatographic protocol than previously described. Thirteen and eleven metabolites were quantified in S. arborariae under aerobic and oxygen-limited conditions, respectively. This targeted metabolomics protocol is shown here to quantify a total of 19 metabolites, including sugars, phosphates, coenzymes, monosaccharides, and alcohols, from xylose catabolism pathways (glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle) in yeast. Furthermore, to our knowledge, this is the first time that intracellular metabolites have been quantified in S. arborariae after xylose consumption. The results indicated that fine control of oxygen levels during fermentation is necessary to optimize ethanol production by S. arborariae. The protocol presented here may be applied to other yeast species and could support yeast genetic engineering to improve second generation ethanol production. [Figure not available: see fulltext.

  13. Plastoglobules: a new address for targeting recombinant proteins in the chloroplast

    PubMed Central

    Vidi, Pierre-Alexandre; Kessler, Felix; Bréhélin, Claire

    2007-01-01

    Background The potential of transgenic plants for cost-effective production of pharmaceutical molecules is now becoming apparent. Plants have the advantage over established fermentation systems (bacterial, yeast or animal cell cultures) to circumvent the risk of pathogen contamination, to be amenable to large scaling up and to necessitate only established farming procedures. Chloroplasts have proven a useful cellular compartment for protein accumulation owing to their large size and number, as well as the possibility for organellar transformation. They therefore represent the targeting destination of choice for recombinant proteins in leaf crops such as tobacco. Extraction and purification of recombinant proteins from leaf material contribute to a large extent to the production costs. Developing new strategies facilitating these processes is therefore necessary. Results Here, we evaluated plastoglobule lipoprotein particles as a new subchloroplastic destination for recombinant proteins. The yellow fluorescent protein as a trackable cargo was targeted to plastoglobules when fused to plastoglobulin 34 (PGL34) as the carrier. Similar to adipocyte differentiation related protein (ADRP) in animal cells, most of the protein sequence of PGL34 was necessary for targeting to lipid bodies. The recombinant protein was efficiently enriched in plastoglobules isolated by simple flotation centrifugation. The viability of plants overproducing the recombinant protein was not affected, indicating that plastoglobule targeting did not significantly impair photosynthesis or sugar metabolism. Conclusion Our data identify plastoglobules as a new targeting destination for recombinant protein in leaf crops. The wide-spread presence of plastoglobules and plastoglobulins in crop species promises applications comparable to those of transgenic oilbody-oleosin technology in molecular farming. PMID:17214877

  14. Perchlorate Reduction by Yeast for Mars Exploration

    NASA Technical Reports Server (NTRS)

    Sharma, Alaisha

    2015-01-01

    Martian soil contains high levels (0.6 percentage by mass) of calcium perchlorate (Ca(ClO4)2), which readily dissociates into calcium and the perchlorate ion (ClO4-) in water. Even in trace amounts, perchlorates are toxic to humans and have been implicated in thyroid dysfunction. Devising methods to lessen perchlorate contamination is crucial to minimizing the health risks associated with human exploration and colonization of Mars. We designed a perchlorate reduction pathway, which sequentially reduces perchlorate to chloride (Cl-) and oxygen (O2), for implementation in the yeast Saccharomyces cerevisiae. Using genes obtained from perchlorate reducing bacteria Azospira oryzae and Dechloromonas aromatica, we plan to assemble this pathway directly within S. cerevisiae through recombinational cloning. A perchlorate reduction pathway would enable S. cerevisiae to lower perchlorate levels and produce oxygen, which may be harvested or used directly by S. cerevisiae for aerobic growth and compound synthesis. Moreover, using perchlorate as an external electron acceptor could improve the efficiency of redox-imbalanced production pathways in yeast. Although several perchlorate reducing bacteria have been identified and utilized in water treatment systems on Earth, the widespread use of S. cerevisiae as a synthetic biology platform justifies the development of a perchlorate reducing strain for implementation on Mars.

  15. The 2-micron plasmid as a nonselectable, stable, high copy number yeast vector

    NASA Technical Reports Server (NTRS)

    Ludwig, D. L.; Bruschi, C. V.

    1991-01-01

    The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.

  16. Synthesis of human parainfluenza virus 4 nucleocapsid-like particles in yeast and their use for detection of virus-specific antibodies in human serum.

    PubMed

    Bulavaitė, Aistė; Lasickienė, Rita; Tamošiūnas, Paulius Lukas; Simanavičius, Martynas; Sasnauskas, Kęstutis; Žvirblienė, Aurelija

    2017-04-01

    The aim of this study was to produce human parainfluenza virus type 4 (HPIV4) nucleocapsid (N) protein in yeast Saccharomyces cerevisiae expression system, to explore its structural and antigenic properties and to evaluate its applicability in serology. The use of an optimized gene encoding HPIV4 N protein amino acid (aa) sequence GenBank AGU90031.1 allowed high yield of recombinant N protein forming nucleocapsid-like particles (NLPs) in yeast. A substitution L332D disrupted self-assembly of NLPs, confirming the role of this position in the N proteins of Paramyxovirinae. Three monoclonal antibodies (MAbs) were generated against the NLP-forming HPIV4 N protein. They recognised HPIV4-infected cells, demonstrating the antigenic similarity between the recombinant and virus-derived N proteins. HPIV4 N protein was used as a coating antigen in an indirect IgG ELISA with serum specimens of 154 patients with respiratory tract infection. The same serum specimens were tested with previously generated N protein of a closely related HPIV2, another representative of genus Rubulavirus. Competitive ELISA was developed using related yeast-produced viral antigens to deplete the cross-reactive serum antibodies. In the ELISA either without or with competition using heterologous HPIV (2 or 4) N or mumps virus N proteins, the seroprevalence of HPIV4 N-specific IgG was, respectively, 46.8, 39.6 and 40.3% and the seroprevalence of HPIV2 N-specific IgG-47.4, 39.0 and 37.7%. In conclusion, yeast-produced HPIV4 N protein shares structural and antigenic properties of the native virus nucleocapsids. Yeast-produced HPIV4 and HPIV2 NLPs are prospective tools in serology.

  17. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  18. From mannan to bioethanol: cell surface co-display of β-mannanase and β-mannosidase on yeast Saccharomyces cerevisiae.

    PubMed

    Ishii, Jun; Okazaki, Fumiyoshi; Djohan, Apridah Cameliawati; Hara, Kiyotaka Y; Asai-Nakashima, Nanami; Teramura, Hiroshi; Andriani, Ade; Tominaga, Masahiro; Wakai, Satoshi; Kahar, Prihardi; Yopi; Prasetya, Bambang; Ogino, Chiaki; Kondo, Akihiko

    2016-01-01

    Mannans represent the largest hemicellulosic fraction in softwoods and also serve as carbohydrate stores in various plants. However, the utilization of mannans as sustainable resources has been less advanced in sustainable biofuel development. Based on a yeast cell surface-display technology that enables the immobilization of multiple enzymes on the yeast cell walls, we constructed a recombinant Saccharomyces cerevisiae strain that co-displays β-mannanase and β-mannosidase; this strain is expected to facilitate ethanol fermentation using mannan as a biomass source. Parental yeast S. cerevisiae assimilated mannose and glucose as monomeric sugars, producing ethanol from mannose. We constructed yeast strains that express tethered β-mannanase and β-mannosidase; co-display of the two enzymes on the cell surface was confirmed by immunofluorescence staining and enzyme activity assays. The constructed yeast cells successfully hydrolyzed 1,4-β-d-mannan and produced ethanol by assimilating the resulting mannose without external addition of enzymes. Furthermore, the constructed strain produced ethanol from 1,4-β-d-mannan continually during the third batch of repeated fermentation. Additionally, the constructed strain produced ethanol from ivory nut mannan; ethanol yield was improved by NaOH pretreatment of the substrate. We successfully displayed β-mannanase and β-mannosidase on the yeast cell surface. Our results clearly demonstrate the utility of the strain co-displaying β-mannanase and β-mannosidase for ethanol fermentation from mannan biomass. Thus, co-tethering β-mannanase and β-mannosidase on the yeast cell surface provides a powerful platform technology for yeast fermentation toward the production of bioethanol and other biochemicals from lignocellulosic materials containing mannan components.

  19. Saccharomyces interspecies hybrids as model organisms for studying yeast adaptation to stressful environments.

    PubMed

    Lopandic, Ksenija

    2018-01-01

    The strong development of molecular biology techniques and next-generation sequencing technologies in the last two decades has significantly improved our understanding of the evolutionary history of Saccharomyces yeasts. It has been shown that many strains isolated from man-made environments are not pure genetic lines, but contain genetic materials from different species that substantially increase their genome complexity. A number of strains have been described as interspecies hybrids, implying different yeast species that under specific circumstances exchange and recombine their genomes. Such fusing usually results in a wide variety of alterations at the genetic and chromosomal levels. The observed changes have suggested a high genome plasticity and a significant role of interspecies hybridization in the adaptation of yeasts to environmental stresses and industrial processes. There is a high probability that harsh wine and beer fermentation environments, from which the majority of interspecies hybrids have been isolated so far, influence their selection and stabilization as well as their genomic and phenotypic heterogeneity. The lessons we have learned about geno- and phenotype plasticity and the diversity of natural and commercial yeast hybrids have already had a strong impact on the development of artificial hybrids that can be successfully used in the fermentation-based food and beverage industry. The creation of artificial hybrids through the crossing of strains with desired attributes is a possibility to obtain a vast variety of new, but not genetically modified yeasts with a range of improved and beneficial traits. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  20. MTO1 mutations are associated with hypertrophic cardiomyopathy and lactic acidosis and cause respiratory chain deficiency in humans and yeast.

    PubMed

    Baruffini, Enrico; Dallabona, Cristina; Invernizzi, Federica; Yarham, John W; Melchionda, Laura; Blakely, Emma L; Lamantea, Eleonora; Donnini, Claudia; Santra, Saikat; Vijayaraghavan, Suresh; Roper, Helen P; Burlina, Alberto; Kopajtich, Robert; Walther, Anett; Strom, Tim M; Haack, Tobias B; Prokisch, Holger; Taylor, Robert W; Ferrero, Ileana; Zeviani, Massimo; Ghezzi, Daniele

    2013-11-01

    We report three families presenting with hypertrophic cardiomyopathy, lactic acidosis, and multiple defects of mitochondrial respiratory chain (MRC) activities. By direct sequencing of the candidate gene MTO1, encoding the mitochondrial-tRNA modifier 1, or whole exome sequencing analysis, we identified novel missense mutations. All MTO1 mutations were predicted to be deleterious on MTO1 function. Their pathogenic role was experimentally validated in a recombinant yeast model, by assessing oxidative growth, respiratory activity, mitochondrial protein synthesis, and complex IV activity. In one case, we also demonstrated that expression of wt MTO1 could rescue the respiratory defect in mutant fibroblasts. The severity of the yeast respiratory phenotypes partly correlated with the different clinical presentations observed in MTO1 mutant patients, although the clinical outcome was highly variable in patients with the same mutation and seemed also to depend on timely start of pharmacological treatment, centered on the control of lactic acidosis by dichloroacetate. Our results indicate that MTO1 mutations are commonly associated with a presentation of hypertrophic cardiomyopathy, lactic acidosis, and MRC deficiency, and that ad hoc recombinant yeast models represent a useful system to test the pathogenic potential of uncommon variants, and provide insight into their effects on the expression of a biochemical phenotype. © 2013 The Authors. *Human Mutation published by Wiley Periodicals, Inc.

  1. Identifying Cis-Regulatory Changes Involved in the Evolution of Aerobic Fermentation in Yeasts

    PubMed Central

    Lin, Zhenguo; Wang, Tzi-Yuan; Tsai, Bing-Shi; Wu, Fang-Ting; Yu, Fu-Jung; Tseng, Yu-Jung; Sung, Huang-Mo; Li, Wen-Hsiung

    2013-01-01

    Gene regulation change has long been recognized as an important mechanism for phenotypic evolution. We used the evolution of yeast aerobic fermentation as a model to explore how gene regulation has evolved and how this process has contributed to phenotypic evolution and adaptation. Most eukaryotes fully oxidize glucose to CO2 and H2O in mitochondria to maximize energy yield, whereas some yeasts, such as Saccharomyces cerevisiae and its relatives, predominantly ferment glucose into ethanol even in the presence of oxygen, a phenomenon known as aerobic fermentation. We examined the genome-wide gene expression levels among 12 different yeasts and found that a group of genes involved in the mitochondrial respiration process showed the largest reduction in gene expression level during the evolution of aerobic fermentation. Our analysis revealed that the downregulation of these genes was significantly associated with massive loss of binding motifs of Cbf1p in the fermentative yeasts. Our experimental assays confirmed the binding of Cbf1p to the predicted motif and the activator role of Cbf1p. In summary, our study laid a foundation to unravel the long-time mystery about the genetic basis of evolution of aerobic fermentation, providing new insights into understanding the role of cis-regulatory changes in phenotypic evolution. PMID:23650209

  2. Transcription in Yeast: Separation and Properties of Multiple RNA Polymerases

    PubMed Central

    Adman, Ray; Schultz, Loren D.; Hall, Benjamin D.

    1972-01-01

    Four peaks of DNA-directed RNA polymerase activity are resolved by salt gradient elution of a sonicated yeast cell extract on DEAE-Sephadex. The enzymes, which are named IA, IB, II, and III in order of elution, all appear to come from cell nuclei. Only enzyme II is sensitive to α-amanitin. All enzymes are more active with Mn++ than with Mg++ as divalent ion. Enzymes IB and II have salt optima in the range 0.05-0.10 M (NH4)2SO4, whereas enzyme III is maximally active at 0.20-0.25 M (NH4)2SO4. With optimal salt concentration and saturating DNA, the template preference ratio, activity on native calfthymus DNA divided by activity on denatured calf-thymus DNA, is 2.2 for IB, 0.4 for II, and 3.5 for III. None of the yeast polymerases was inhibited by rifamycin SV. Rifamycin AF/013 effectively inhibited polymerases IB, II, and III. PMID:4558656

  3. Drosophila Regulate Yeast Density and Increase Yeast Community Similarity in a Natural Substrate

    PubMed Central

    Stamps, Judy A.; Yang, Louie H.; Morales, Vanessa M.; Boundy-Mills, Kyria L.

    2012-01-01

    Drosophila melanogaster adults and larvae, but especially larvae, had profound effects on the densities and community structure of yeasts that developed in banana fruits. Pieces of fruit exposed to adult female flies previously fed fly-conditioned bananas developed higher yeast densities than pieces of the same fruits that were not exposed to flies, supporting previous suggestions that adult Drosophila vector yeasts to new substrates. However, larvae alone had dramatic effects on yeast density and species composition. When yeast densities were compared in pieces of the same fruits assigned to different treatments, fruits that developed low yeast densities in the absence of flies developed significantly higher yeast densities when exposed to larvae. Across all of the fruits, larvae regulated yeast densities within narrow limits, as compared to a much wider range of yeast densities that developed in pieces of the same fruits not exposed to flies. Larvae also affected yeast species composition, dramatically reducing species diversity across fruits, reducing variation in yeast communities from one fruit to the next (beta diversity), and encouraging the consistent development of a yeast community composed of three species of yeast (Candida californica, C. zemplinina, and Pichia kluvyeri), all of which were palatable to larvae. Larvae excreted viable cells of these three yeast species in their fecal pools, and discouraged the growth of filamentous fungi, processes which may have contributed to their effects on the yeast communities in banana fruits. These and other findings suggest that D. melanogaster adults and their larval offspring together engage in ‘niche construction’, facilitating a predictable microbial environment in the fruit substrates in which the larvae live and develop. PMID:22860093

  4. TOPBP1Dpb11 plays a conserved role in homologous recombination DNA repair through the coordinated recruitment of 53BP1Rad9

    PubMed Central

    Sims, Jennie Rae; Freire, Raimundo

    2017-01-01

    Genome maintenance and cancer suppression require homologous recombination (HR) DNA repair. In yeast and mammals, the scaffold protein TOPBP1Dpb11 has been implicated in HR, although its precise function and mechanism of action remain elusive. In this study, we show that yeast Dpb11 plays an antagonistic role in recombination control through regulated protein interactions. Dpb11 mediates opposing roles in DNA end resection by coordinating both the stabilization and exclusion of Rad9 from DNA lesions. The Mec1 kinase promotes the pro-resection function of Dpb11 by mediating its interaction with the Slx4 scaffold. Human TOPBP1Dpb11 engages in interactions with the anti-resection factor 53BP1 and the pro-resection factor BRCA1, suggesting that TOPBP1 also mediates opposing functions in HR control. Hyperstabilization of the 53BP1–TOPBP1 interaction enhances the recruitment of 53BP1 to nuclear foci in the S phase, resulting in impaired HR and the accumulation of chromosomal aberrations. Our results support a model in which TOPBP1Dpb11 plays a conserved role in mediating a phosphoregulated circuitry for the control of recombinational DNA repair. PMID:28228534

  5. 8-Methoxypsoralen photoinduced plasmid-chromosome recombination in Saccharomyces cerevisiae using a centromeric vector.

    PubMed Central

    Meira, L B; Henriques, J A; Magaña-Schwencke, N

    1995-01-01

    The characterization of a new system to study the induction of plasmid-chromosome recombination is described. Single-stranded and double-stranded centromeric vectors bearing 8-methoxypsoralen photoinduced lesions were used to transform a wild-type yeast strain bearing the leu2-3,112 marker. Using the SSCP methodology and DNA sequencing, it was demonstrated that repair of the lesions in plasmid DNA was mainly due to conversion of the chromosomal allele to the plasmid DNA. Images PMID:7784218

  6. Expression of LIP1 and LIP2 genes from Geotrichum species in Baker's yeast strains and their application to the bread-making process.

    PubMed

    Monfort, A; Blasco, A; Sanz, P; Prieto, J A

    1999-02-01

    Lipolytic baker's yeast strains able to produce extracellular active lipase have been constructed by transformation with plasmids containing the LIP1 and LIP2 genes from Geotrichum sp. under the control of the Saccharomyces cerevisiae actin promoter (pACT1). Lipase productivity differed between both constructs, YEpACT-LIP1-t and YEpACT-LIP2-t, being higher for the strain bearing the LIP2 gene in all culture media tested. This result appeared not to be the consequence of a defect in the transcription of the LIP1 gene as revealed by Northern blot analysis. Replacing the signal sequence of LIP1 by that of LIP2 in the YEpACT-LIP1-t plasmid enhanced significantly the secretion of lipase 1, but the levels of lipase activity were still lower than those found for the YEpACT-LIP2-t transformant. Recombinant lipase 2 protein produced by baker's yeast exhibited biochemical properties similar to those of the natural enzyme. Fermented dough prepared with YEpACT-LIP2-t-carrying cells rendered a bread with a higher loaf volume and a more uniform crumb structure than that prepared with control yeast. These effects were stronger by the addition in the bread dough formulas of a preferment enriched in recombinant lipase 2.

  7. Ergothioneine production using Methylobacterium species, yeast, and fungi.

    PubMed

    Fujitani, Yoshiko; Alamgir, Kabir Md; Tani, Akio

    2018-06-14

    Ergothioneine (EGT) is a sulfur-containing, anti-oxidative amino acid derived from histidine. EGT is synthesized in bacteria and fungi but not in animals and plants, and is now recognized as important for human health. Its cost-effective fermentative production has not been elucidated due to the lack of information for productive microorganisms. In this study, we doubled the gene copy for EGT synthesis and deleted the histidine ammonia-lyase gene in a potent EGT-producing methylotrophic bacterium Methylobacterium aquaticum strain 22A, and optimized its culture conditions, resulting in increased EGT production of 7.0 mg EGT/g dry cell weight and 100 μg EGT/5 mL/7 days. In addition, through screening we found EGT-producing eukaryotic strains of Aureobasidium pullulans and Rhodotorula mucilaginosa, which can produce 1.0 and 3.2 mg EGT/g dry cell weight, 70 and 120 μg EGT/5 mL/7 days, respectively. This study proposes practical uses of potent EGT-producing recombinant Methylobacterium species and non-recombinant yeast and fungal strains. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  8. [Engineering of a System for the Production of Mutant Human Alpha-Fetoprotein in the Methylotrophic Yeast Pichia pastoris].

    PubMed

    Morozkina, E V; Vavilova, E A; Zatsepin, S S; Klyachko, E V; Yagudin, T A; Chulkin, A M; Dudich, I V; Semenkova, L N; Churilova, I V; Benevolenskii, S V

    2016-01-01

    A system for the production of mutant recombinant human alpha-fetoprotein (rhAFPO) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS 115/pPICZ?A/rhAFP0, which produces unglycosylated rhAFPO and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.

  9. Cloning and characterization of an inulinase gene from the marine yeast Candida membranifaciens subsp. flavinogenie W14-3 and its expression in Saccharomyces sp. W0 for ethanol production.

    PubMed

    Zhang, Lin-Lin; Tan, Mei-Juan; Liu, Guang-Lei; Chi, Zhe; Wang, Guang-Yuan; Chi, Zhen-Ming

    2015-04-01

    The INU1 gene encoding an exo-inulinase from the marine-derived yeast Candida membranifaciens subsp. flavinogenie W14-3 was cloned and characterized. It had an open reading frame of 1,536 bp long encoding an inulinase. The coding region of it was not interrupted by any intron. The cloned gene encoded 512 amino acid residues of a protein with a putative signal peptide of 23 amino acids and a calculated molecular mass of 57.8 kDa. The protein sequence deduced from the inulinase gene contained the inulinase consensus sequences (WMNDPNGL), (RDP), ECP FS and Q. The protein also had six conserved putative N-glycosylation sites. The deduced inulinase from the yeast strain W14-3 was found to be closely related to that from Candida kutaonensis sp. nov. KRF1, Kluyveromyces marxianus, and Cryptococcus aureus G7a. The inulinase gene with its signal peptide encoding sequence was subcloned into the pMIRSC11 expression vector and expressed in Saccharomyces sp. W0. The recombinant yeast strain W14-3-INU-112 obtained could produce 16.8 U/ml of inulinase activity and 12.5 % (v/v) ethanol from 250 g/l of inulin within 168 h. The monosaccharides were detected after the hydrolysis of inulin with the crude inulinase (the yeast culture). All the results indicated that the cloned gene and the recombinant yeast strain W14-3-INU-112 had potential applications in biotechnology.

  10. Oral Administration of Recombinant Saccharomyces boulardii Expressing Ovalbumin-CPE Fusion Protein Induces Antibody Response in Mice

    PubMed Central

    Bagherpour, Ghasem; Ghasemi, Hosnie; Zand, Bahare; Zarei, Najmeh; Roohvand, Farzin; Ardakani, Esmat M.; Azizi, Mohammad; Khalaj, Vahid

    2018-01-01

    Saccharomyces boulardii, a subspecies of Saccharomyces cerevisiae, is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi®) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the ura3- S. boulardii. To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from Clostridium perfringens enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group (P < 0.001) compared to control groups (receiving wild type S. boulardii or PBS), and the fecal IgA titer was significantly higher in test group (P < 0.05) than control groups. In parallel, a recombinant S. boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii, as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins. PMID

  11. Oral Administration of Recombinant Saccharomyces boulardii Expressing Ovalbumin-CPE Fusion Protein Induces Antibody Response in Mice.

    PubMed

    Bagherpour, Ghasem; Ghasemi, Hosnie; Zand, Bahare; Zarei, Najmeh; Roohvand, Farzin; Ardakani, Esmat M; Azizi, Mohammad; Khalaj, Vahid

    2018-01-01

    Saccharomyces boulardii , a subspecies of Saccharomyces cerevisiae , is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi ® ) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the ura3 - S. boulardii . To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from Clostridium perfringens enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group ( P < 0.001) compared to control groups (receiving wild type S. boulardii or PBS), and the fecal IgA titer was significantly higher in test group ( P < 0.05) than control groups. In parallel, a recombinant S. boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii , as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins.

  12. Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

    PubMed Central

    Dälken, Benjamin; Jabulowsky, Robert A.; Oberoi, Pranav; Benhar, Itai; Wels, Winfried S.

    2010-01-01

    Background The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. Methods and Findings We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. Conclusions Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins. PMID:21203542

  13. Recombinant Expression of Tandem-HBc Virus-Like Particles (VLPs).

    PubMed

    Stephen, Sam L; Beales, Lucy; Peyret, Hadrien; Roe, Amy; Stonehouse, Nicola J; Rowlands, David J

    2018-01-01

    recombinant structures to assemble into regular isometric particles. Here, we describe the production of recombinant tandem-core particles in bacterial, yeast and plant expression systems.

  14. Improving the Organoleptic Properties of a Craft Mezcal Beverage by Increasing Fatty Acid Ethyl Ester Contents through ATF1 Expression in an Engineered Kluyveromyces marxianus UMPe-1 Yeast.

    PubMed

    Campos-García, Jesús; Vargas, Alejandra; Farías-Rosales, Lorena; Miranda, Ana L; Meza-Carmen, Víctor; Díaz-Pérez, Alma L

    2018-05-02

    Mezcal, a traditional beverage that originated in Mexico, is produced from species of the Agavaceae family. The esters associated with the yeasts utilized during fermentation are important for improving the organoleptic properties of the beverage. We improved the ester contents in a mezcal beverage by using the yeast Kluyveromyces marxianus, which was engineered with the ATF1 gene. ATF1 expression in the recombinant yeast significantly increased compared with that in the parental yeast, but its fermentative parameters were unchanged. Volatile-organic-compound-content analysis showed that esters had significantly increased in the mezcal produced with the engineered yeast. In a sensory-panel test, 48% of the panelists preferred the mezcal produced from the engineered yeast, 30% preferred the mezcal produced from the wild type, and 15 and 7% preferred the two mezcal types produced following the routine procedure. Correlation analysis showed that the fruitiness/sweetness description of the mezcal produced using the ATF1-engineered K. marxianus yeast correlated with the content of the esters, whose presence improved the organoleptic properties of the craft mezcal beverage.

  15. Pichia pastoris production of a prolyl 4-hydroxylase derived from Chondrosia reniformis sponge: A new biotechnological tool for the recombinant production of marine collagen.

    PubMed

    Pozzolini, Marina; Scarfì, Sonia; Mussino, Francesca; Salis, Annalisa; Damonte, Gianluca; Benatti, Umberto; Giovine, Marco

    2015-08-20

    Prolyl 4-hydroxylase (P4H) is a α2β2 tetramer catalyzing the post-translational hydroxylation of prolines in collagen. Its recombinant production is mainly pursued to realize biotechnological tools able to generate animal contaminant-free hydroxylated collagen. One promising candidate for biomedical applications is the collagen extracted from the marine sponge Chondrosia reniformis, because of its biocompatibility and because is devoid of the health risks associated with bovine and porcine collagens. Here we report on the production and selection, by enzymatic and biomolecular analyses, of a triple transformed Pichia pastoris strain expressing a stable P4H tetramer derived from C. reniformis sponge and a hydroxylated non fibrillar procollagen polypeptide from the same animal. The percentage of recombinant procollagen hydroxylated prolines inside the transformed yeast was of 36.3% analyzed by mass spectrometry indicating that the recombinant enzyme is active on its natural substrate inside the yeast cell host. Furthermore, the recombinant sponge P4H has the ability to hydroxylate its natural substrate in both X and Y positions in the Xaa-Yaa-Gly collagenous triplets. In conclusion this Pichia system seems ideal for high-level production of hydroxylated sponge- or marine-derived collagen polypeptides as well as of conotoxins or other marine proteins of high pharmacological interest needing this particular post-translational modification. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Prions in Yeast

    PubMed Central

    Liebman, Susan W.; Chernoff, Yury O.

    2012-01-01

    The concept of a prion as an infectious self-propagating protein isoform was initially proposed to explain certain mammalian diseases. It is now clear that yeast also has heritable elements transmitted via protein. Indeed, the “protein only” model of prion transmission was first proven using a yeast prion. Typically, known prions are ordered cross-β aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in understanding cellular control of prion propagation, prion structure, mechanisms of de novo prion formation, specificity of prion transmission, and the biological roles of prions. This review summarizes what has been learned from yeast prions. PMID:22879407

  17. Specific Modifications of Histone Tails, but Not DNA Methylation, Mirror the Temporal Variation of Mammalian Recombination Hotspots

    PubMed Central

    Zeng, Jia; Yi, Soojin V.

    2014-01-01

    Recombination clusters nonuniformly across mammalian genomes at discrete genomic loci referred to as recombination hotspots. Despite their ubiquitous presence, individual hotspots rapidly lose their activities, and the molecular and evolutionary mechanisms underlying such frequent hotspot turnovers (the so-called “recombination hotspot paradox”) remain unresolved. Even though some sequence motifs are significantly associated with hotspots, multiple lines of evidence indicate that factors other than underlying sequences, such as epigenetic modifications, may affect the evolution of recombination hotspots. Thus, identifying epigenetic factors that covary with recombination at fine-scale is a promising step for this important research area. It was previously reported that recombination rates correlate with indirect measures of DNA methylation in the human genome. Here, we analyze experimentally determined DNA methylation and histone modification of human sperms, and show that the correlation between DNA methylation and recombination in long-range windows does not hold with respect to the spatial and temporal variation of recombination at hotspots. On the other hand, two histone modifications (H3K4me3 and H3K27me3) overlap extensively with recombination hotspots. Similar trends were observed in mice. These results indicate that specific histone modifications rather than DNA methylation are associated with the rapid evolution of recombination hotspots. Furthermore, many human recombination hotspots occupy “bivalent” chromatin regions that harbor both active (H3K4me3) and repressive (H3K27me3) marks. This may explain why human recombination hotspots tend to occur in nongenic regions, in contrast to yeast and Arabidopsis hotspots that are characterized by generally active chromatins. Our results highlight the dynamic epigenetic underpinnings of recombination hotspot evolution. PMID:25326136

  18. Significant competitive advantage conferred by meiosis and syngamy in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Birdsell, J; Wills, C

    1996-01-01

    The presumed advantages of genetic recombinations are difficult to demonstrate directly. To investigate the effects of recombination and background heterozygosity on competitive ability, we have performed serial-transfer competition experiments between isogenic sexual and asexual strains of the yeast Saccharomyces cerevisiae. The members of these diploid pairs of strains differed only in being heterozygous (sexual) or homozygous (asexual) at the mating type or MAT locus. Competing pairs had either a completely homozygous or a heterozygous genetic background, the latter being heterozygous at many different loci throughout the genome. A round of meiotic recombination (automixis) conferred a large and statistically significant enhancement of competitive ability on sexual strains with a heterozygous genetic background. By contrast, in homozygous background competitions, meiosis decreased the sexual strains' initial relative competitive ability. In all cases, however, the sexual strains outcompeted their isogenic asexual counterparts, whether meiotic recombination had occurred or not. In some genetic backgrounds, this was due in part to an overdominance effect on competitive advantage of heterozygosity at the MAT locus. The advantage of the sexual strains also increased significantly during the course of the homozygous background competitions, particularly when meiosis had occurred. This latter effect either did not occur or was very weak in heterozygous background competitions. Overall, sexual strains with heterozygous genetic backgrounds had a significantly higher initial relative competitive ability than those with homozygous backgrounds. The advantage of mating type heterozygosity in this organism extends far beyond the ability to recombine meiotically. PMID:8570658

  19. Personalised Medicine: Genome Maintenance Lessons Learned from Studies in Yeast as a Model Organism.

    PubMed

    Abugable, Arwa A; Awwad, Dahlia A; Fleifel, Dalia; Ali, Mohamed M; El-Khamisy, Sherif; Elserafy, Menattallah

    2017-01-01

    Yeast research has been tremendously contributing to the understanding of a variety of molecular pathways due to the ease of its genetic manipulation, fast doubling time as well as being cost-effective. The understanding of these pathways did not only help scientists learn more about the cellular functions but also assisted in deciphering the genetic and cellular defects behind multiple diseases. Hence, yeast research not only opened the doors for transforming basic research into applied research, but also paved the roads for improving diagnosis and innovating personalized therapy of different diseases. In this chapter, we discuss how yeast research has contributed to understanding major genome maintenance pathways such as the S-phase checkpoint activation pathways, repair via homologous recombination and non-homologous end joining as well as topoisomerases-induced protein linked DNA breaks repair. Defects in these pathways lead to neurodegenerative diseases and cancer. Thus, the understanding of the exact genetic defects underlying these diseases allowed the development of personalized medicine, improving the diagnosis and treatment and overcoming the detriments of current conventional therapies such as the side effects, toxicity as well as drug resistance.

  20. Directed evolution for improved secretion of cancer-testis antigen NY-ESO-1 from yeast.

    PubMed

    Piatesi, Andrea; Howland, Shanshan W; Rakestraw, James A; Renner, Christoph; Robson, Neil; Cebon, Jonathan; Maraskovsky, Eugene; Ritter, Gerd; Old, Lloyd; Wittrup, K Dane

    2006-08-01

    NY-ESO-1 is a highly immunogenic tumor antigen and a promising vaccine candidate in cancer immunotherapy. Access to purified protein both for vaccine formulations and for monitoring antigen-specific immune responses is vital to vaccine development. Currently available recombinant Escherichia coli-derived NY-ESO-1 is isolated from inclusion bodies as a complex protein mixture and efforts to improve the purity of this antigen are required, especially for later-stage clinical trials. Using yeast cell surface display and fluorescence activated cell sorting techniques, we have engineered an NY-ESO-1 variant (NY-ESO-L5; C(75)A C(76)A C(78)A L(153)H) with a 100x improved display level on yeast compared to the wild-type protein. This mutant can be effectively produced as an Aga2p-fusion and purified in soluble form directly from the yeast cell wall. In the process, we have identified the epitope recognized by anti-NY-ESO-1 mAb E978 (79-87, GARGPESRL). The availability of an alternative expression host for this important antigen will help avoid artifactual false positive tests of patient immune response due to reaction against expression-host-specific contaminants.

  1. Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins.

    PubMed

    Irani, Zahra Azimzadeh; Kerkhoven, Eduard J; Shojaosadati, Seyed Abbas; Nielsen, Jens

    2016-05-01

    Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better prediction of protein yield, demonstrates the effect of the different types of N-glycosylation of protein yield, and can be used to predict potential targets for strain improvement. The model represents a step towards a more complete description of protein production in P. pastoris, which is required for using these models to understand and optimize protein production processes. © 2015 Wiley Periodicals, Inc.

  2. Reiterative Recombination for the in vivo assembly of libraries of multigene pathways.

    PubMed

    Wingler, Laura M; Cornish, Virginia W

    2011-09-13

    The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop "Reiterative Recombination," a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae, and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 10(4) biosynthetic pathways. We anticipate that our system's simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo.

  3. Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

    PubMed Central

    Wingler, Laura M.; Cornish, Virginia W.

    2011-01-01

    The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop “Reiterative Recombination,” a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae, and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 104 biosynthetic pathways. We anticipate that our system’s simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo. PMID:21876185

  4. Control of the yeast telomeric senescence survival pathways of recombination by the Mec1 and Mec3 DNA damage sensors and RPA

    PubMed Central

    Grandin, Nathalie; Charbonneau, Michel

    2007-01-01

    Saccharomyces cerevisiae telomerase-negative cells undergo homologous recombination on subtelomeric or TG1–3 telomeric sequences, thus allowing Type I or Type II post-senescence survival, respectively. Here, we find that the DNA damage sensors, Mec1, Mec3 and Rad24 control Type II recombination, while the Rad9 adaptor protein and the Rad53 and Chk1 effector kinases have no effect on survivor type selection. Therefore, the Mec1 and Mec3 checkpoint complexes control telomeric recombination independently of their roles in generating and amplifying the Mec1-Rad53-Chk1 kinase cascade. rfa1-t11 mutant cells, bearing a mutation in Replication Protein A (RPA) conferring a defect in recruiting Mec1-Ddc2, were also deficient in both types of telomeric recombination. Importantly, expression of an Rfa1-t11-Ddc2 hybrid fusion protein restored checkpoint-dependent arrest, but did not rescue defective telomeric recombination. Therefore, the Rfa1-t11-associated defect in telomeric recombination is not solely due to its failure to recruit Mec1. We have also isolated novel alleles of RFA1 that were deficient in Type I but not in Type II recombination and proficient in checkpoint control. Therefore, the checkpoint and recombination functions of RPA can be genetically separated, as can the RPA-mediated control of the two types of telomeric recombination. PMID:17202155

  5. Engineering tolerance to industrially relevant stress factors in yeast cell factories.

    PubMed

    Deparis, Quinten; Claes, Arne; Foulquié-Moreno, Maria R; Thevelein, Johan M

    2017-06-01

    The main focus in development of yeast cell factories has generally been on establishing optimal activity of heterologous pathways and further metabolic engineering of the host strain to maximize product yield and titer. Adequate stress tolerance of the host strain has turned out to be another major challenge for obtaining economically viable performance in industrial production. Although general robustness is a universal requirement for industrial microorganisms, production of novel compounds using artificial metabolic pathways presents additional challenges. Many of the bio-based compounds desirable for production by cell factories are highly toxic to the host cells in the titers required for economic viability. Artificial metabolic pathways also turn out to be much more sensitive to stress factors than endogenous pathways, likely because regulation of the latter has been optimized in evolution in myriads of environmental conditions. We discuss different environmental and metabolic stress factors with high relevance for industrial utilization of yeast cell factories and the experimental approaches used to engineer higher stress tolerance. Improving stress tolerance in a predictable manner in yeast cell factories should facilitate their widespread utilization in the bio-based economy and extend the range of products successfully produced in large scale in a sustainable and economically profitable way. © FEMS 2017.

  6. Engineering tolerance to industrially relevant stress factors in yeast cell factories

    PubMed Central

    Deparis, Quinten; Claes, Arne; Foulquié-Moreno, Maria R.

    2017-01-01

    Abstract The main focus in development of yeast cell factories has generally been on establishing optimal activity of heterologous pathways and further metabolic engineering of the host strain to maximize product yield and titer. Adequate stress tolerance of the host strain has turned out to be another major challenge for obtaining economically viable performance in industrial production. Although general robustness is a universal requirement for industrial microorganisms, production of novel compounds using artificial metabolic pathways presents additional challenges. Many of the bio-based compounds desirable for production by cell factories are highly toxic to the host cells in the titers required for economic viability. Artificial metabolic pathways also turn out to be much more sensitive to stress factors than endogenous pathways, likely because regulation of the latter has been optimized in evolution in myriads of environmental conditions. We discuss different environmental and metabolic stress factors with high relevance for industrial utilization of yeast cell factories and the experimental approaches used to engineer higher stress tolerance. Improving stress tolerance in a predictable manner in yeast cell factories should facilitate their widespread utilization in the bio-based economy and extend the range of products successfully produced in large scale in a sustainable and economically profitable way. PMID:28586408

  7. Recent advances in yeast cell-surface display technologies for waste biorefineries.

    PubMed

    Liu, Zhuo; Ho, Shih-Hsin; Hasunuma, Tomohisa; Chang, Jo-Shu; Ren, Nan-Qi; Kondo, Akihiko

    2016-09-01

    Waste biorefinery aims to maximize the output of value-added products from various artificial/agricultural wastes by using integrated bioprocesses. To make waste biorefinery economically feasible, it is thus necessary to develop a low-cost, environment-friendly technique to perform simultaneous biodegradation and bioconversion of waste materials. Cell-surface display engineering is a novel, cost-effective technique that can auto-immobilize proteins on the cell exterior of microorganisms, and has been applied for use with waste biofinery. Through tethering different enzymes (e.g., cellulase, lipase, and protease) or metal-binding peptides on cell surfaces, various yeast strains can effectively produce biofuels and biochemicals from sugar/protein-rich waste materials, catalyze waste oils into biodiesels, or retrieve heavy metals from wastewater. This review critically summarizes recent applications of yeast cell-surface display on various types of waste biorefineries, highlighting its potential and future challenges with regard to commercializing this technology. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Chloroplast NADPH-Dependent Thioredoxin Reductase from Chlorella vulgaris Alleviates Environmental Stresses in Yeast Together with 2-Cys Peroxiredoxin

    PubMed Central

    Machida, Takeshi; Ishibashi, Akiko; Kirino, Ai; Sato, Jun-ichi; Kawasaki, Shinji; Niimura, Youichi; Honjoh, Ken-ichi; Miyamoto, Takahisa

    2012-01-01

    Chloroplast NADPH-dependent thioredoxin reductase (NTRC) catalyzes the reduction of 2-Cys peroxiredoxin (2-Cys Prx) and, thus, probably functions as an antioxidant system. The functions of the enzyme in oxidative and salt stresses have been reported previously. We have previously identified and characterized NTRC in Chlorella vulgaris. In the present study, we isolated a full-length cDNA clone encoding 2-Cys Prx from C. vulgaris and investigated the involvement of Chlorella NTRC/2-Cys Prx system in several environmental stress tolerances by using yeast as a eukaryotic model. Deduced Chlorella 2-Cys Prx was homologous to those of chloroplast 2-Cys Prxs from plants, and two conserved cysteine residues were found in the deduced sequence. Enzyme assay showed that recombinant mature C. vulgaris NTRC (mCvNTRC) transferred electrons from NADPH to recombinant mature C. vulgaris 2-Cys Prx (mCvPrx), and mCvPrx decomposed hydrogen peroxide, tert-butyl hydroperoxide, and peroxynitrite by cooperating with mCvNTRC. Based on the results, the mCvNTRC/mCvPrx antioxidant system was identified in Chlorella. The antioxidant system genes were expressed in yeast separately or coordinately. Stress tolerances of yeast against freezing, heat, and menadione-induced oxidative stresses were significantly improved by expression of mCvNTRC, and the elevated tolerances were more significant when both mCvNTRC and mCvPrx were co-expressed. Our results reveal a novel feature of NTRC: it functions as an antioxidant system with 2-Cys Prx in freezing and heat stress tolerances. PMID:23029353

  9. Yeast for virus research

    PubMed Central

    Zhao, Richard Yuqi

    2017-01-01

    Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are two popular model organisms for virus research. They are natural hosts for viruses as they carry their own indigenous viruses. Both yeasts have been used for studies of plant, animal and human viruses. Many positive sense (+) RNA viruses and some DNA viruses replicate with various levels in yeasts, thus allowing study of those viral activities during viral life cycle. Yeasts are single cell eukaryotic organisms. Hence, many of the fundamental cellular functions such as cell cycle regulation or programed cell death are highly conserved from yeasts to higher eukaryotes. Therefore, they are particularly suited to study the impact of those viral activities on related cellular activities during virus-host interactions. Yeasts present many unique advantages in virus research over high eukaryotes. Yeast cells are easy to maintain in the laboratory with relative short doubling time. They are non-biohazardous, genetically amendable with small genomes that permit genome-wide analysis of virologic and cellular functions. In this review, similarities and differences of these two yeasts are described. Studies of virologic activities such as viral translation, viral replication and genome-wide study of virus-cell interactions in yeasts are highlighted. Impacts of viral proteins on basic cellular functions such as cell cycle regulation and programed cell death are discussed. Potential applications of using yeasts as hosts to carry out functional analysis of small viral genome and to develop high throughput drug screening platform for the discovery of antiviral drugs are presented. PMID:29082230

  10. Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle.

    PubMed

    Daigaku, Yasukazu; Mashiko, Satsuki; Mishiba, Keiichiro; Yamamura, Saburo; Ui, Ayako; Enomoto, Takemi; Yamamoto, Kazuo

    2006-08-30

    A CAN1/can1Delta heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.

  11. Characterization of single chain antibody targets through yeast two hybrid

    PubMed Central

    2010-01-01

    Background Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain. Results We describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region. Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed. Conclusions Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear

  12. L-arabinose fermenting yeast

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Min; Singh, Arjun; Suominen, Pirkko

    An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. A yeast strain engineered to metabolize arabinose through a novel pathway is also disclosed. Methods of producing ethanol include utilizing these modified yeast strains.

  13. L-arabinose fermenting yeast

    DOEpatents

    Zhang, Min; Singh, Arjun; Suominen, Pirkko; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric

    2014-09-23

    An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. A yeast strain engineered to metabolize arabinose through a novel pathway is also disclosed. Methods of producing ethanol include utilizing these modified yeast strains.

  14. Improvement of lipid production by the oleaginous yeast Rhodosporidium toruloides through UV mutagenesis.

    PubMed

    Yamada, Ryosuke; Kashihara, Tomomi; Ogino, Hiroyasu

    2017-05-01

    Oleaginous yeasts are considered a promising alternative lipid source for biodiesel fuel production. In this study, we attempted to improve the lipid productivity of the oleaginous yeast Rhodosporidium toruloides through UV irradiation mutagenesis and selection based on ethanol and H 2 O 2 tolerance or cerulenin, a fatty acid synthetase inhibitor. Glucose consumption, cell growth, and lipid production of mutants were evaluated. The transcription level of genes involved in lipid production was also evaluated in mutants. The ethanol and H 2 O 2 tolerant strain 8766 2-31M and the cerulenin resistant strain 8766 3-11C were generated by UV mutagenesis. The 8766 2-31M mutant showed a higher lipid production rate, and the 8766 3-11C mutant produced a larger amount of lipid and had a higher lipid production rate than the wild type strain. Transcriptional analysis revealed that, similar to the wild type strain, the ACL1 and GND1 genes were expressed at significantly low levels, whereas IDP1 and ME1 were highly expressed. In conclusion, lipid productivity in the oleaginous yeast R. toruloides was successfully improved via UV mutagenesis and selection. The study also identified target genes for improving lipid productivity through gene recombination.

  15. Prevention of Yeast Spoilage in Feed and Food by the Yeast Mycocin HMK

    PubMed Central

    Lowes, K. F.; Shearman, C. A.; Payne, J.; MacKenzie, D.; Archer, D. B.; Merry, R. J.; Gasson, M. J.

    2000-01-01

    The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts. PMID:10698773

  16. Non-homologous end joining-mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus.

    PubMed

    Hoshida, Hisashi; Murakami, Nobutada; Suzuki, Ayako; Tamura, Ryoko; Asakawa, Jun; Abdel-Banat, Babiker M A; Nonklang, Sanom; Nakamura, Mikiko; Akada, Rinji

    2014-01-01

    The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous end-joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR-amplified separately, mixed and directly used for the transformation. URA3(+) transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR-amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date. Copyright © 2013 John Wiley & Sons, Ltd.

  17. A non-canonical DNA structure enables homologous recombination in various genetic systems.

    PubMed

    Masuda, Tokiha; Ito, Yutaka; Terada, Tohru; Shibata, Takehiko; Mikawa, Tsutomu

    2009-10-30

    Homologous recombination, which is critical to genetic diversity, depends on homologous pairing (HP). HP is the switch from parental to recombinant base pairs, which requires expansion of inter-base pair spaces. This expansion unavoidably causes untwisting of the parental double-stranded DNA. RecA/Rad51-catalyzed ATP-dependent HP is extensively stimulated in vitro by negative supercoils, which compensates for untwisting. However, in vivo, double-stranded DNA is relaxed by bound proteins and thus is an unfavorable substrate for RecA/Rad51. In contrast, Mhr1, an ATP-independent HP protein required for yeast mitochondrial homologous recombination, catalyzes HP without the net untwisting of double-stranded DNA. Therefore, we questioned whether Mhr1 uses a novel strategy to promote HP. Here, we found that, like RecA, Mhr1 induced the extension of bound single-stranded DNA. In addition, this structure was induced by all evolutionarily and structurally distinct HP proteins so far tested, including bacterial RecO, viral RecT, and human Rad51. Thus, HP includes the common non-canonical DNA structure and uses a common core mechanism, independent of the species of HP proteins. We discuss the significance of multiple types of HP proteins.

  18. Specific modifications of histone tails, but not DNA methylation, mirror the temporal variation of mammalian recombination hotspots.

    PubMed

    Zeng, Jia; Yi, Soojin V

    2014-10-16

    Recombination clusters nonuniformly across mammalian genomes at discrete genomic loci referred to as recombination hotspots. Despite their ubiquitous presence, individual hotspots rapidly lose their activities, and the molecular and evolutionary mechanisms underlying such frequent hotspot turnovers (the so-called "recombination hotspot paradox") remain unresolved. Even though some sequence motifs are significantly associated with hotspots, multiple lines of evidence indicate that factors other than underlying sequences, such as epigenetic modifications, may affect the evolution of recombination hotspots. Thus, identifying epigenetic factors that covary with recombination at fine-scale is a promising step for this important research area. It was previously reported that recombination rates correlate with indirect measures of DNA methylation in the human genome. Here, we analyze experimentally determined DNA methylation and histone modification of human sperms, and show that the correlation between DNA methylation and recombination in long-range windows does not hold with respect to the spatial and temporal variation of recombination at hotspots. On the other hand, two histone modifications (H3K4me3 and H3K27me3) overlap extensively with recombination hotspots. Similar trends were observed in mice. These results indicate that specific histone modifications rather than DNA methylation are associated with the rapid evolution of recombination hotspots. Furthermore, many human recombination hotspots occupy "bivalent" chromatin regions that harbor both active (H3K4me3) and repressive (H3K27me3) marks. This may explain why human recombination hotspots tend to occur in nongenic regions, in contrast to yeast and Arabidopsis hotspots that are characterized by generally active chromatins. Our results highlight the dynamic epigenetic underpinnings of recombination hotspot evolution. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for

  19. Purification and functional characterisation of the pyruvate (monocarboxylate) carrier from baker's yeast mitochondria (Saccharomyces cerevisiae).

    PubMed

    Nałecz, M J; Nałecz, K A; Azzi, A

    1991-08-09

    Isolated yeast mitochondria were subjected to solubilization by Triton X-114 and the detergent extract was subsequently chromatrographed on dry hydroxyapatite. Purification of the yeast monocarboxylate (pyruvate) carrier was achieved by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate, as described previously for bovine heart mitochondria (Bolli, R., Nałecz K.A. and Azzi, A. (1989) J. Biol. Chem. 264 18024-18030). The final preparation contained two polypeptides of apparent molecular mass 26 and 50 kDa. The yeast carrier appeared to be less abundant, but more active, than the analogous protein from higher eukaryotes. The carrier was able to catalyse the pyruvate / pyruvate and pyruvate / acetoacetate exchange reactions, both reactions being sensitive to cyanocinnamate and its derivatives, to phenylpyruvate and to mersalyl and p-chloromercuribenzoate. In the pyruvate / acetoacetate exchange reaction (200 mM internal acetoacetate, enzymatic assay), the Km value for external pyruvate was found to be 0.8 mM and the Vmax 135 mumol/min per mg protein. Among other substrates of the yeast carrier, all transported with similar affinity and identical maximal velocity against acetoacetate, we identified 2-oxoisocaproate, 2-oxoisovalerate and 2-oxo-3-methylvalerate. Lactate was not translocated by this carrier with a measurable rate, neither were di- or tricarboxylates.

  20. Adaptive evolution of baker's yeast in a dough‐like environment enhances freeze and salinity tolerance

    PubMed Central

    Aguilera, Jaime; Andreu, Pasqual; Randez‐Gil, Francisca; Prieto, Jose Antonio

    2010-01-01

    Summary We used adaptive evolution to improve freeze tolerance of industrial baker's yeast. Our hypothesis was that adaptation to low temperature is accompanied by enhanced resistance of yeast to freezing. Based on this hypothesis, yeast was propagated in a flour‐free liquid dough model system, which contained sorbitol and NaCl, by successive batch refreshments maintained constantly at 12°C over at least 200 generations. Relative to the parental population, the maximal growth rate (µmax) under the restrictive conditions, increased gradually over the time course of the experiment. This increase was accompanied by enhanced freeze tolerance. However, these changes were not the consequence of genetic adaptation to low temperature, a fact that was confirmed by prolonged selection of yeast cells in YPD at 12°C. Instead, the experimental populations showed a progressive increase in NaCl tolerance. This phenotype was likely achieved at the expense of others traits, since evolved cells showed a ploidy reduction, a defect in the glucose derepression mechanism and a loss in their ability to utilize gluconeogenic carbon sources. We discuss the genetic flexibility of S. cerevisiae in terms of adaptation to the multiple constraints of the experimental design applied to drive adaptive evolution and the technologically advantageous phenotype of the evolved population. PMID:21255321

  1. Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation.

    PubMed

    De Tullio, Luisina; Kaniecki, Kyle; Kwon, Youngho; Crickard, J Brooks; Sung, Patrick; Greene, Eric C

    2017-10-17

    Srs2 is a super-family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single-molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA) bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (∼170 nt per second) in the 3'→5' direction along ssDNA saturated with replication protein A (RPA). We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA and, in doing so, promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  2. The phytopathogenic virulent effector protein RipI induces apoptosis in budding yeast Saccharomyces cerevisiae.

    PubMed

    Deng, Meng-Ying; Sun, Yun-Hao; Li, Pai; Fu, Bei; Shen, Dong; Lu, Yong-Jun

    2016-10-01

    Virulent protein toxins secreted by the bacterial pathogens can cause cytotoxicity by various molecular mechanisms to combat host cell defense. On the other hand, these proteins can also be used as probes to investigate the defense pathway of host innate immunity. Ralstonia solanacearum, one of the most virulent bacterial phytopathogens, translocates more than 70 effector proteins via type III secretion system during infection. Here, we characterized the cytotoxicity of effector RipI in budding yeast Saccharomyce scerevisiae, an alternative host model. We found that over-expression of RipI resulted in severe growth defect and arginine (R) 117 within the predicted integrase motif was required for inhibition of yeast growth. The phenotype of death manifested the hallmarks of apoptosis. Our data also revealed that RipI-induced apoptosis was independent of Yca1 and mitochondria-mediated apoptotic pathways because Δyca1 and Δaif1 were both sensitive to RipI as compared with the wild type. We further demonstrated that RipI was localized in the yeast nucleus and the N-terminal 1-174aa was required for the localization. High-throughput RNA sequencing analysis showed that upon RipI over-expression, 101 unigenes of yeast ribosome presented lower expression level, and 42 GO classes related to the nucleus or recombination were enriched with differential expression levels. Taken together, our data showed that a nuclear-targeting effector RipI triggers yeast apoptosis, potentially dependent on its integrase function. Our results also provided an alternative strategy to dissect the signaling pathway of cytotoxicity induced by the protein toxins. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Effect of wine yeast monoculture practice on the biodiversity of non-Saccharomyces yeasts.

    PubMed

    Ganga, M A; Martínez, C

    2004-01-01

    The objective of this work was to study the effect of the use of Saccharomyces cerevisiae monocultures over the biodiversity of non-Saccharomyces yeasts in wine-producing areas in Chile. Microvinifications were carried out with grape musts of two areas. In one of them, the fermentation is carried out mainly in a spontaneous manner, whereas in the other the musts are inoculated with commercial yeasts. The isolated yeasts were identified by the internal transcribed (ITS)/restriction fragment length polymorphism technique. In the industrial production area less variability of yeast genera was observed as compared with the traditional area, an observation that is greatest at the end of the fermentation. Furthermore, a study of the production of extracellular enzymes was done. The majority of the yeasts showed at least one of the activities assayed with the exception of beta-glycosidase. The results suggest that in the industrialized area the diversity of yeasts is less in the traditional area. Likewise, the potentiality of the non-Saccharomyces yeasts as enzyme producers with industrial interest has been confirmed. This study shows the negative effect of the use of monocultures over the biodiversity of yeasts in wine-producing regions.

  4. Assessing the complex architecture of polygenic traits in diverged yeast populations.

    PubMed

    Cubillos, Francisco A; Billi, Eleonora; Zörgö, Enikö; Parts, Leopold; Fargier, Patrick; Omholt, Stig; Blomberg, Anders; Warringer, Jonas; Louis, Edward J; Liti, Gianni

    2011-04-01

    Phenotypic variation arising from populations adapting to different niches has a complex underlying genetic architecture. A major challenge in modern biology is to identify the causative variants driving phenotypic variation. Recently, the baker's yeast, Saccharomyces cerevisiae has emerged as a powerful model for dissecting complex traits. However, past studies using a laboratory strain were unable to reveal the complete architecture of polygenic traits. Here, we present a linkage study using 576 recombinant strains obtained from crosses of isolates representative of the major lineages. The meiotic recombinational landscape appears largely conserved between populations; however, strain-specific hotspots were also detected. Quantitative measurements of growth in 23 distinct ecologically relevant environments show that our recombinant population recapitulates most of the standing phenotypic variation described in the species. Linkage analysis detected an average of 6.3 distinct QTLs for each condition tested in all crosses, explaining on average 39% of the phenotypic variation. The QTLs detected are not constrained to a small number of loci, and the majority are specific to a single cross-combination and to a specific environment. Moreover, crosses between strains of similar phenotypes generate greater variation in the offspring, suggesting the presence of many antagonistic alleles and epistatic interactions. We found that subtelomeric regions play a key role in defining individual quantitative variation, emphasizing the importance of the adaptive nature of these regions in natural populations. This set of recombinant strains is a powerful tool for investigating the complex architecture of polygenic traits. © 2011 Blackwell Publishing Ltd.

  5. Phenylalanine ammonia-lyase. Induction and purification from yeast and clearance in mammals.

    PubMed

    Fritz, R R; Hodgins, D S; Abell, C W

    1976-08-10

    Yeast phenylalanine ammonia-lyase (EC 4.3.1.5) catalyzes the deamination of L-phenylalanine to form trans-cinnamic acid and tyrosine to trans-coumaric acid. Maximal enzyme activity in Rhodotorula glutinis (2 units/g, wet weight, of yeast) was induced in late-log phase (12 to 14 hours) of growth in a culture medium containing 1.0% malt extract, 0.1% yeast extract, and 0.1% L-phenylalanine. A highly purified enzyme was obtained by fractionation with ammonium sulfate and sodium citrate followed by chromatography on DEAE-cellulose and Sephadex G-200. The active preparation yielded a major component on three different polyacrylamide gel electrophoretic systems. Antisera to phenylalanine ammonia-lyase was raised in rabbits and detected by double immunodiffusion. The antigen-antibody complex was enzymatically active in vitro. The biological half-life of the enzyme was approximately 21 hours in several mammalian species (mice without and with BW10232 adenocarcinoma and B16 melanoma, rats, and monkeys) after a single injection; however, upon repeated administration, phenylalanine ammonia-lyase had a much shorter biological half-life. The onset of rapid clearance occurred earlier in tumor-bearing than in nontumor-bearing mice indicating a direct or indirect influence by the tumor on the biological half-life of phenylalanine ammonia-lyase.

  6. New features of mitochondrial DNA replication system in yeast and man.

    PubMed

    Lecrenier, N; Foury, F

    2000-04-04

    In this review, we sum up the research carried out over two decades on mitochondrial DNA (mtDNA) replication, primarily by comparing this system in Saccharomyces cerevisiae and Homo sapiens. Brief incursions into systems of other organisms have also been achieved when they provide new information.S. cerevisiae and H. sapiens mitochondrial DNA (mtDNA) have been thought for a long time to share closely related architecture and replication mechanisms. However, recent studies suggest that mitochondrial genome of S. cerevisiae may be formed, at least partially, from linear multimeric molecules, while human mtDNA is circular. Although several proteins involved in the replication of these two genomes are very similar, divergences are also now increasingly evident. As an example, the recently cloned human mitochondrial DNA polymerase beta-subunit has no counterpart in yeast. Yet, yeast Abf2p and human mtTFA are probably not as closely functionally related as thought previously. Some mtDNA metabolism factors, like DNA ligases, were until recently largely uncharacterized, and have been found to be derived from alternative nuclear products. Many factors involved in the metabolism of mitochondrial DNA are linked through genetic or biochemical interconnections. These links are presented on a map. Finally, we discuss recent studies suggesting that the yeast mtDNA replication system diverges from that observed in man, and may involve recombination, possibly coupled to alternative replication mechanisms like rolling circle replication.

  7. Yeast cell differentiation: Lessons from pathogenic and non-pathogenic yeasts.

    PubMed

    Palková, Zdena; Váchová, Libuše

    2016-09-01

    Yeasts, historically considered to be single-cell organisms, are able to activate different differentiation processes. Individual yeast cells can change their life-styles by processes of phenotypic switching such as the switch from yeast-shaped cells to filamentous cells (pseudohyphae or true hyphae) and the transition among opaque, white and gray cell-types. Yeasts can also create organized multicellular structures such as colonies and biofilms, and the latter are often observed as contaminants on surfaces in industry and medical care and are formed during infections of the human body. Multicellular structures are formed mostly of stationary-phase or slow-growing cells that diversify into specific cell subpopulations that have unique metabolic properties and can fulfill specific tasks. In addition to the development of multiple protective mechanisms, processes of metabolic reprogramming that reflect a changed environment help differentiated individual cells and/or community cell constituents to survive harmful environmental attacks and/or to escape the host immune system. This review aims to provide an overview of differentiation processes so far identified in individual yeast cells as well as in multicellular communities of yeast pathogens of the Candida and Cryptococcus spp. and the Candida albicans close relative, Saccharomyces cerevisiae. Molecular mechanisms and extracellular signals potentially involved in differentiation processes are also briefly mentioned. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. L-arabinose fermenting yeast

    DOEpatents

    Zhang, Min; Singh, Arjun; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric; Suominen, Pirkko

    2010-12-07

    An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. Methods of producing ethanol include utilizing these modified yeast strains. ##STR00001##

  9. Phenomenology of maximal and near-maximal lepton mixing

    NASA Astrophysics Data System (ADS)

    Gonzalez-Garcia, M. C.; Peña-Garay, Carlos; Nir, Yosef; Smirnov, Alexei Yu.

    2001-01-01

    The possible existence of maximal or near-maximal lepton mixing constitutes an intriguing challenge for fundamental theories of flavor. We study the phenomenological consequences of maximal and near-maximal mixing of the electron neutrino with other (x=tau and/or muon) neutrinos. We describe the deviations from maximal mixing in terms of a parameter ɛ≡1-2 sin2 θex and quantify the present experimental status for \\|ɛ\\|<0.3. We show that both probabilities and observables depend on ɛ quadratically when effects are due to vacuum oscillations and they depend on ɛ linearly if matter effects dominate. The most important information on νe mixing comes from solar neutrino experiments. We find that the global analysis of solar neutrino data allows maximal mixing with confidence level better than 99% for 10-8 eV2<~Δm2<~2×10-7 eV2. In the mass ranges Δm2>~1.5×10-5 eV2 and 4×10-10 eV2<~Δm2<~2×10-7 eV2 the full interval \\|ɛ\\|<0.3 is allowed within ~4σ (99.995% CL) We suggest ways to measure ɛ in future experiments. The observable that is most sensitive to ɛ is the rate [NC]/[CC] in combination with the day-night asymmetry in the SNO detector. With theoretical and statistical uncertainties, the expected accuracy after 5 years is Δɛ~0.07. We also discuss the effects of maximal and near-maximal νe mixing in atmospheric neutrinos, supernova neutrinos, and neutrinoless double beta decay.

  10. DNA Sequence-Mediated, Evolutionarily Rapid Redistribution of Meiotic Recombination Hotspots

    PubMed Central

    Wahls, Wayne P.; Davidson, Mari K.

    2011-01-01

    Hotspots regulate the position and frequency of Spo11 (Rec12)-initiated meiotic recombination, but paradoxically they are suicidal and are somehow resurrected elsewhere in the genome. After the DNA sequence-dependent activation of hotspots was discovered in fission yeast, nearly two decades elapsed before the key realizations that (A) DNA site-dependent regulation is broadly conserved and (B) individual eukaryotes have multiple different DNA sequence motifs that activate hotspots. From our perspective, such findings provide a conceptually straightforward solution to the hotspot paradox and can explain other, seemingly complex features of meiotic recombination. We describe how a small number of single-base-pair substitutions can generate hotspots de novo and dramatically alter their distribution in the genome. This model also shows how equilibrium rate kinetics could maintain the presence of hotspots over evolutionary timescales, without strong selective pressures invoked previously, and explains why hotspots localize preferentially to intergenic regions and introns. The model is robust enough to account for all hotspots of humans and chimpanzees repositioned since their divergence from the latest common ancestor. PMID:22084420

  11. General introduction: recombinant protein production and purification of insoluble proteins.

    PubMed

    Ferrer-Miralles, Neus; Saccardo, Paolo; Corchero, José Luis; Xu, Zhikun; García-Fruitós, Elena

    2015-01-01

    Proteins are synthesized in heterologous systems because of the impossibility to obtain satisfactory yields from natural sources. The production of soluble and functional recombinant proteins is among the main goals in the biotechnological field. In this context, it is important to point out that under stress conditions, protein folding machinery is saturated and this promotes protein misfolding and, consequently, protein aggregation. Thus, the selection of the optimal expression organism and the most appropriate growth conditions to minimize the formation of insoluble proteins should be done according to the protein characteristics and downstream requirements. Escherichia coli is the most popular recombinant protein expression system despite the great development achieved so far by eukaryotic expression systems. Besides, other prokaryotic expression systems, such as lactic acid bacteria and psychrophilic bacteria, are gaining interest in this field. However, it is worth mentioning that prokaryotic expression system poses, in many cases, severe restrictions for a successful heterologous protein production. Thus, eukaryotic systems such as mammalian cells, insect cells, yeast, filamentous fungus, and microalgae are an interesting alternative for the production of these difficult-to-express proteins.

  12. Crystallization and preliminary crystallographic study of the yeast Malassezia sympodialis allergen Mala s 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vilhelmsson, Monica, E-mail: monica.vilhelmsson@medks.ki.se; Center for Infectious Medicine, Department of Medicine, Karolinska University Hospital, Huddinge, Stockholm; Hallberg, B. Martin

    2006-02-01

    Crystals of the M. sympodialis allergen Mala s 1 have been obtained using the hanging-drop vapour-diffusion method. A diffraction data set has been collected from native crystals to 1.35 Å resolution. The opportunistic yeast Malassezia sympodialis can act as an allergen and elicit specific IgE- and T-cell reactivity in patients with atopic eczema. The first identified major allergen from M. sympodialis, Mala s 1, is present on the cell surface of the yeast. Recombinant Mala s 1 was expressed in Escherichia coli, purified and refolded in a soluble form. Crystals of Mala s 1 were obtained in 25% PEG 8K,more » 0.2 M (NH{sub 4}){sub 2}SO{sub 4}. Crystals belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 44.4, b = 163.7, c = 50.6 Å, and diffract to 1.35 Å resolution.« less

  13. Nitrile Metabolizing Yeasts

    NASA Astrophysics Data System (ADS)

    Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

    Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing

  14. Pro-region engineering for improved yeast display and secretion of brain derived neurotrophic factor.

    PubMed

    Burns, Michael L; Malott, Thomas M; Metcalf, Kevin J; Puguh, Arthya; Chan, Jonah R; Shusta, Eric V

    2016-03-01

    Brain derived neurotrophic factor (BDNF) is a promising therapeutic candidate for a variety of neurological diseases. However, it is difficult to produce as a recombinant protein. In its native mammalian context, BDNF is first produced as a pro-protein with subsequent proteolytic removal of the pro-region to yield mature BDNF protein. Therefore, in an attempt to improve yeast as a host for heterologous BDNF production, the BDNF pro-region was first evaluated for its effects on BDNF surface display and secretion. Addition of the wild-type pro-region to yeast BDNF production constructs improved BDNF folding both as a surface-displayed and secreted protein in terms of binding its natural receptors TrkB and p75, but titers remained low. Looking to further enhance the chaperone-like functions provided by the pro-region, two rounds of directed evolution were performed, yielding mutated pro-regions that further improved the display and secretion properties of BDNF. Subsequent optimization of the protease recognition site was used to control whether the produced protein was in pro- or mature BDNF forms. Taken together, we have demonstrated an effective strategy for improving BDNF compatibility with yeast protein engineering and secretion platforms. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Genome dynamics and evolution in yeasts: A long-term yeast-bacteria competition experiment

    PubMed Central

    Katz, Michael; Knecht, Wolfgang; Compagno, Concetta; Piškur, Jure

    2018-01-01

    There is an enormous genetic diversity evident in modern yeasts, but our understanding of the ecological basis of such diversifications in nature remains at best fragmented so far. Here we report a long-term experiment mimicking a primordial competitive environment, in which yeast and bacteria co-exist and compete against each other. Eighteen yeasts covering a wide phylogenetic background spanning approximately 250 million years of evolutionary history were used to establish independent evolution lines for at most 130 passages. Our collection of hundreds of modified strains generated through such a rare two-species cross-kingdom competition experiment re-created the appearance of large-scale genomic rearrangements and altered phenotypes important in the diversification history of yeasts. At the same time, the methodology employed in this evolutionary study would also be a non-gene-technological method of reprogramming yeast genomes and then selecting yeast strains with desired traits. Cross-kingdom competition may therefore be a method of significant value to generate industrially useful yeast strains with new metabolic traits. PMID:29624585

  16. Destabilization and recovery of a yeast prion after mild heat shock

    PubMed Central

    Newnam, Gary P.; Birchmore, Jennifer L.; Chernoff, Yury O.

    2011-01-01

    Yeast prion [PSI+] is a self-perpetuating amyloid of the translational termination factor Sup35. Although [PSI+] propagation is modulated by heat shock proteins (Hsps), high temperature was previously reported to have little or no effect on [PSI+]. Our results show that short-term exposure of exponentially growing yeast culture to mild heat shock, followed by immediate resumption of growth, leads to [PSI+] destabilization, sometimes persisting for several cell divisions after heat shock. Prion loss occurring in the first division after heat shock is preferentially detected in a daughter cell, indicating the impairment of prion segregation that results in asymmetric prion distribution between a mother cell and a bud. Longer heat shock or prolonged incubation in the absence of nutrients after heat shock lead to [PSI+] recovery. Both prion destabilization and recovery during heat shock depend on protein synthesis. Maximal prion destabilization coincides with maximal imbalance between Hsp104 and other Hsps such as Hsp70-Ssa. Deletions of individual SSA genes increase prion destabilization and/or counteract recovery. Dynamics of prion aggregation during destabilization and recovery is consistent with the notion that efficient prion fragmentation and segregation require a proper balance between Hsp104 and other (e. g. Hsp70-Ssa) chaperones. In contrast to heat shock, [PSI+] destabilization by osmotic stressors does not always depend on cell proliferation and/or protein synthesis, indicating that different stresses may impact the prion via different mechanisms. Our data demonstrate that heat stress causes asymmetric prion distribution in a cell division, and confirm that effects of Hsps on prions are physiologically relevant. PMID:21392508

  17. Effect of carbon source on the accumulation of cytochrome P-450 in the yeast Saccharomyces cerevisiae.

    PubMed

    Kärenlampi, S O; Marin, E; Hänninen, O O

    1981-02-15

    The appearance of cytochrome P-450 in the yeast Saccharomyces cerevisiae depended on the substrate supporting growth. Cytochrome P-450 was apparent in yeast cells grown on a strongly fermentable sugar such as D-glucose, D-fructose or sucrose. When yeast was grown on D-galactose, D-mannose or maltose, where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. The cytochrome P-450 concentration was maximal at the beginning of the stationary phase of the culture. Thereafter the concentration decreased, reaching zero at a late-stationary phase. When the yeast was grown on a medium that contained lactose or pentoses (L-arabinose, L-rhamnose, D-ribose and D-xylose), cytochrome P-450 did not occur. When a non-fermentable energy source (glycerol, lactate or ethanol) was used, no cytochrome P-450 was detectable. Transfer of cells from D-glucose medium to ethanol medium caused a slow disappearance of cytochrome P-450, although the amount of the haemoprotein still continued to increase in the control cultures. Cytochrome P-450 appeared thus to accumulate in conditions where the rate of growth was fast and fermentation occurred. Occurrence of this haemoprotein is not necessarily linked, however, with the repression of mitochondrial haemoprotein synthesis.

  18. Bioprocess and downstream optimization of recombinant human growth hormone in Pichia pastoris

    PubMed Central

    Azadi, Saeed; Sadjady, Seyed Kazem; Mortazavi, Seyed Alireza; Naghdi, Nasser; Mahboubi, Arash; Solaimanian, Roya

    2018-01-01

    The methylotrophic yeast Pichia pastoris is a well-established expression host, which is often used in the production of protein pharmaceuticals. This work aimed to evaluate the effect of various concentrations of ascorbic acid in mixed feeding strategy with sorbitol/methanol on productivity of recombinant human growth hormone (r-hGH). The relevant concentration of ascorbic acid (5, 10, or 20 mmol) and 50 g/L sorbitol were added in batch-wise mode to the medium at the beginning of induction phase. The rate of methanol addition was increased stepwise during the first 12 h of production and then kept constant. Total protein and r-hGH concentrations were analyzed and the results compared with sorbitol/methanol feeding using one-way analysis of variance. Moreover, an effective clarification process using activated carbon was developed to remove process contaminants like pigments and endotoxins. Finally, a three-step chromatographic process was applied to purify the product. According to the obtained results, addition of 10 mmol ascorbic acid to sorbitol/methanol co-feeding could significantly increase cell biomass (1.7 fold), total protein (1.14 fold), and r-hGH concentration (1.43 fold). One percent activated carbon could significantly decrease pigments and endotoxins without any significant changes in r-hGH assay. The result of the study concluded that ascorbic acid in combination with sorbitol could effectively enhance the productivity of r-hGH. This study also demonstrated that activated carbon clarification is a simple method for efficient removal of endotoxin and pigment in production of recombinant protein in the yeast expression system. PMID:29853932

  19. Evolutionarily diverse determinants of meiotic DNA break and recombination landscapes across the genome

    PubMed Central

    Fowler, Kyle R.; Sasaki, Mariko; Milman, Neta

    2014-01-01

    Fission yeast Rec12 (Spo11 homolog) initiates meiotic recombination by forming developmentally programmed DNA double-strand breaks (DSBs). DSB distributions influence patterns of heredity and genome evolution, but the basis of the highly nonrandom choice of Rec12 cleavage sites is poorly understood, largely because available maps are of relatively low resolution and sensitivity. Here, we determined DSBs genome-wide at near-nucleotide resolution by sequencing the oligonucleotides attached to Rec12 following DNA cleavage. The single oligonucleotide size class allowed us to deeply sample all break events. We find strong evidence across the genome for differential DSB repair accounting for crossover invariance (constant cM/kb in spite of DSB hotspots). Surprisingly, about half of all crossovers occur in regions where DSBs occur at low frequency and are widely dispersed in location from cell to cell. These previously undetected, low-level DSBs thus play an outsized and crucial role in meiosis. We further find that the influence of underlying nucleotide sequence and chromosomal architecture differs in multiple ways from that in budding yeast. DSBs are not strongly restricted to nucleosome-depleted regions, as they are in budding yeast, but are nevertheless spatially influenced by chromatin structure. Our analyses demonstrate that evolutionarily fluid factors contribute to crossover initiation and regulation. PMID:25024163

  20. Ntg1p, the base excision repair protein, generates mutagenic intermediates in yeast mitochondrial DNA.

    PubMed

    Phadnis, Naina; Mehta, Reema; Meednu, Nida; Sia, Elaine A

    2006-07-13

    Mitochondrial DNA is predicted to be highly prone to oxidative damage due to its proximity to free radicals generated by oxidative phosphorylation. Base excision repair (BER) is the primary repair pathway responsible for repairing oxidative damage in nuclear and mitochondrial genomes. In yeast mitochondria, three N-glycosylases have been identified so far, Ntg1p, Ogg1p and Ung1p. Ntg1p, a broad specificity N-glycosylase, takes part in catalyzing the first step of BER that involves the removal of the damaged base. In this study, we examined the role of Ntg1p in maintaining yeast mitochondrial genome integrity. Using genetic reporters and assays to assess mitochondrial mutations, we found that loss of Ntg1p suppresses mitochondrial point mutation rates, frameshifts and recombination rates. We also observed a suppression of respiration loss in the ntg1-Delta cells in response to ultraviolet light exposure implying an overlap between BER and UV-induced damage in the yeast mitochondrial compartment. Over-expression of the BER AP endonuclease, Apn1p, did not significantly affect the mitochondrial mutation rate in the presence of Ntg1p, whereas Apn1p over-expression in an ntg1-Delta background increased the frequency of mitochondrial mutations. In addition, loss of Apn1p also suppressed mitochondrial point mutations. Our work suggests that both Ntg1p and Apn1p generate mutagenic intermediates in the yeast mitochondrial genome.

  1. Conversion of Deletions during Recombination in Pneumococcal Transformation

    PubMed Central

    Lefevre, J. C.; Mostachfi, P.; Gasc, A. M.; Guillot, E.; Pasta, F.; Sicard, M.

    1989-01-01

    Genetic analysis of 16 deletions obtained in the amiA locus of pneumococcus is described. When present on donor DNA, all deletions increased drastically the frequency of wild-type recombinants in two-point crosses. This effect was maximal for deletions longer than 200 bases. It was reduced for heterologies shorter than 76 bases and did not exist for very short deletions. In three-point crosses in which the deletion was localized between two point mutations, we demonstrated that this excess of wild-type recombinants was the result of a genetic conversion. This conversion extended over several scores of bases outside the deletion. Conversion takes place during the heteroduplex stage of recombination. Therefore, in pneumococcal transformation, long heterologies participated in this heteroduplex configuration. As this conversion did not require an active DNA polymerase A gene it is proposed that the mechanism of conversion is not a DNA repair synthesis but involves breakage and ligation between DNA molecules. Conversion of deletions did not require the Hex system of correction of mismatched bases. It differs also from localized conversion. It appears that it is a process that evolved to correct errors of replication which lead to long heterologies and which are not eliminated by other systems. PMID:2599365

  2. Largely enhanced bioethanol production through the combined use of lignin-modified sugarcane and xylose fermenting yeast strain.

    PubMed

    Ko, Ja Kyong; Jung, Je Hyeong; Altpeter, Fredy; Kannan, Baskaran; Kim, Ha Eun; Kim, Kyoung Heon; Alper, Hal S; Um, Youngsoon; Lee, Sun-Mi

    2018-05-01

    The recalcitrant structure of lignocellulosic biomass is a major barrier in efficient biomass-to-ethanol bioconversion processes. The combination of feedstock engineering via modification in the lignin synthesis pathway of sugarcane and co-fermentation of xylose and glucose with a recombinant xylose utilizing yeast strain produced 148% more ethanol compared to that of the wild type biomass and control strain. The lignin reduced biomass led to a substantially increased release of fermentable sugars (glucose and xylose). The engineered yeast strain efficiently co-utilized glucose and xylose for fermentation, elevating ethanol yields. In this study, it was experimentally demonstrated that the combined efforts of engineering both feedstock and microorganisms largely enhances the bioconversion of lignocellulosic feedstock to bioethanol. This strategy will significantly improve the economic feasibility of lignocellulosic biofuels production. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. New yeasts-new brews: modern approaches to brewing yeast design and development.

    PubMed

    Gibson, B; Geertman, J-M A; Hittinger, C T; Krogerus, K; Libkind, D; Louis, E J; Magalhães, F; Sampaio, J P

    2017-06-01

    The brewing industry is experiencing a period of change and experimentation largely driven by customer demand for product diversity. This has coincided with a greater appreciation of the role of yeast in determining the character of beer and the widespread availability of powerful tools for yeast research. Genome analysis in particular has helped clarify the processes leading to domestication of brewing yeast and has identified domestication signatures that may be exploited for further yeast development. The functional properties of non-conventional yeast (both Saccharomyces and non-Saccharomyces) are being assessed with a view to creating beers with new flavours as well as producing flavoursome non-alcoholic beers. The discovery of the psychrotolerant S. eubayanus has stimulated research on de novo S. cerevisiae × S. eubayanus hybrids for low-temperature lager brewing and has led to renewed interest in the functional importance of hybrid organisms and the mechanisms that determine hybrid genome function and stability. The greater diversity of yeast that can be applied in brewing, along with an improved understanding of yeasts' evolutionary history and biology, is expected to have a significant and direct impact on the brewing industry, with potential for improved brewing efficiency, product diversity and, above all, customer satisfaction. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Induction of homologous recombination following in utero exposure to DNA-damaging agents.

    PubMed

    Karia, Bijal; Martinez, Jo Ann; Bishop, Alexander J R

    2013-11-01

    Much of our understanding of homologous recombination, as well as the development of the working models for these processes, has been derived from extensive work in model organisms, such as yeast and fruit flies, and mammalian systems by studying the repair of induced double strand breaks or repair following exposure to genotoxic agents in vitro. We therefore set out to expand this in vitro work to ask whether DNA-damaging agents with varying modes of action could induce somatic change in an in vivo mouse model of homologous recombination. We exposed pregnant dams to DNA-damaging agents, conferring a variety of lesions at a specific time in embryo development. To monitor homologous recombination frequency, we used the well-established retinal pigment epithelium pink-eyed unstable assay. Homologous recombination resulting in the deletion of a duplicated 70 kb fragment in the coding region of the Oca2 gene renders this gene functional and can be visualized as a pigmented eyespot in the retinal pigment epithelium. We observed an increased frequency of pigmented eyespots in resultant litters following exposure to cisplatin, methyl methanesulfonate, ethyl methanesulfonate, 3-aminobenzamide, bleomycin, and etoposide with a contrasting decrease in the frequency of detectable reversion events following camptothecin and hydroxyurea exposure. The somatic genomic rearrangements that result from such a wide variety of differently acting damaging agents implies long-term potential effects from even short-term in utero exposures. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Estrogenic activity of phenolic additives determined by an in vitro yeast bioassay.

    PubMed Central

    Miller, D; Wheals, B B; Beresford, N; Sumpter, J P

    2001-01-01

    We used a recombinant yeast estrogen assay to assess the activity of 73 phenolic additives that are used as sunscreens, preservatives, disinfectants, antioxidants, flavorings, or for perfumery. Thirty-two of these compounds displayed activity: 22 with potencies relative to 17beta-estradiol, ranging from 1/3,000 to < 1/3,000,000, and 10 compounds with an impaired response that could not be directly compared with 17beta-estradiol. Forty-one compounds were inactive. The major criteria for activity appear to be the presence of an unhindered phenolic OH group in a para position and a molecular weight of 140-250 Da. PMID:11266322

  6. Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae

    PubMed Central

    Salusjärvi, Laura; Kankainen, Matti; Soliymani, Rabah; Pitkänen, Juha-Pekka; Penttilä, Merja; Ruohonen, Laura

    2008-01-01

    Background Considerable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose. Results Genes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators HAP4, CAT8 and SIP1-2 and 4. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor RGT1 and its target genes HXT2-3, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells. Conclusion The results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant S. cerevisiae strains

  7. Low-frequency chimeric yeast artificial chromosome libraries from flow-sorted human chromosomes 16 and 21.

    PubMed Central

    McCormick, M K; Campbell, E; Deaven, L; Moyzis, R

    1993-01-01

    Construction of chromosome-specific yeast artificial chromosome (YAC) libraries from sorted chromosomes was undertaken (i) to eliminate drawbacks associated with first-generation total genomic YAC libraries, such as the high frequency of chimeric YACs, and (ii) to provide an alternative method for generating chromosome-specific YAC libraries in addition to isolating such collections from a total genomic library. Chromosome-specific YAC libraries highly enriched for human chromosomes 16 and 21 were constructed. By maximizing the percentage of fragments with two ligatable ends and performing yeast transformations with less than saturating amounts of DNA in the presence of carrier DNA, YAC libraries with a low percentage of chimeric clones were obtained. The smaller number of YAC clones in these chromosome-specific libraries reduces the effort involved in PCR-based screening and allows hybridization methods to be a manageable screening approach. Images PMID:8430075

  8. Vaginal yeast infection

    MedlinePlus

    Yeast infection - vagina; Vaginal candidiasis; Monilial vaginitis ... Most women have a vaginal yeast infection at some time. Candida albicans is a common type of fungus. It is often found in small amounts in the ...

  9. Molecular analysis of recombination in a family with Duchenne muscular dystrophy and a large pericentric X chromosome inversion.

    PubMed Central

    Shashi, V.; Golden, W. L.; Allinson, P. S.; Blanton, S. H.; von Kap-Herr, C.; Kelly, T. E.

    1996-01-01

    It has been demonstrated in animal studies that, in animals heterozygous for pericentric chromosomal inversions, loop formation is greatly reduced during meiosis. This results in absence of recombination within the inverted segment, with recombination seen only outside the inversion. A recent study in yeast has shown that telomeres, rather than centromeres, lead in chromosome movement just prior to meiosis and may be involved in promoting recombination. We studied by cytogenetic analysis and DNA polymorphisms the nature of meiotic recombination in a three-generation family with a large pericentric X chromosome inversion, inv(X)(p21.1q26), in which Duchenne muscular dystrophy (DMD) was cosegregating with the inversion. On DNA analysis there was no evidence of meiotic recombination between the inverted and normal X chromosomes in the inverted segment. Recombination was seen at the telomeric regions, Xp22 and Xq27-28. No deletion or point mutation was found on analysis of the DMD gene. On the basis of the FISH results, we believe that the X inversion is the mutation responsible for DMD in this family. Our results indicate that (1) pericentric X chromosome inversions result in reduction of recombination between the normal and inverted X chromosomes; (2) meiotic X chromosome pairing in these individuals is likely initiated at the telomeres; and (3) in this family DMD is caused by the pericentric inversion. Images Figure 2 Figure 5 Figure 6 Figure 7 PMID:8651300

  10. Schizosaccharomyces japonicus: the fission yeast is a fusion of yeast and hyphae.

    PubMed

    Niki, Hironori

    2014-03-01

    The clade of Schizosaccharomyces includes 4 species: S. pombe, S. octosporus, S. cryophilus, and S. japonicus. Although all 4 species exhibit unicellular growth with a binary fission mode of cell division, S. japonicus alone is dimorphic yeast, which can transit from unicellular yeast to long filamentous hyphae. Recently it was found that the hyphal cells response to light and then synchronously activate cytokinesis of hyphae. In addition to hyphal growth, S. japonicas has many properties that aren't shared with other fission yeast. Mitosis of S. japonicas is referred to as semi-open mitosis because dynamics of nuclear membrane is an intermediate mode between open mitosis and closed mitosis. Novel genetic tools and the whole genomic sequencing of S. japonicas now provide us with an opportunity for revealing unique characters of the dimorphic yeast. © 2013 The Author. Yeast Published by John Wiley & Sons Ltd.

  11. Bioethanol production performance of five recombinant strains of laboratory and industrial xylose-fermenting Saccharomyces cerevisiae.

    PubMed

    Matsushika, Akinori; Inoue, Hiroyuki; Murakami, Katsuji; Takimura, Osamu; Sawayama, Shigeki

    2009-04-01

    In this study, five recombinant Saccharomyces cerevisiae strains were compared for their xylose-fermenting ability. The most efficient xylose-to-ethanol fermentation was found by using the industrial strain MA-R4, in which the genes for xylose reductase and xylitol dehydrogenase from Pichia stipitis along with an endogenous xylulokinase gene were expressed by chromosomal integration of the flocculent yeast strain IR-2. The MA-R4 strain rapidly converted xylose to ethanol with a low xylitol yield. Furthermore, the MA-R4 strain had the highest ethanol production when fermenting not only a mixture of glucose and xylose, but also mixed sugars in the detoxified hydrolysate of wood chips. These results collectively suggest that MA-R4 may be a suitable recombinant strain for further study into large-scale ethanol production from mixed sugars present in lignocellulosic hydrolysates.

  12. Ndj1, a telomere-associated protein, regulates centrosome separation in budding yeast meiosis

    PubMed Central

    Li, Ping; Shao, Yize; Jin, Hui

    2015-01-01

    Yeast centrosomes (called spindle pole bodies [SPBs]) remain cohesive for hours during meiotic G2 when recombination takes place. In contrast, SPBs separate within minutes after duplication in vegetative cells. We report here that Ndj1, a previously known meiosis-specific telomere-associated protein, is required for protecting SPB cohesion. Ndj1 localizes to the SPB but dissociates from it ∼16 min before SPB separation. Without Ndj1, meiotic SPBs lost cohesion prematurely, whereas overproduction of Ndj1 delayed SPB separation. When produced ectopically in vegetative cells, Ndj1 caused SPB separation defects and cell lethality. Localization of Ndj1 to the SPB depended on the SUN domain protein Mps3, and removal of the N terminus of Mps3 allowed SPB separation and suppressed the lethality of NDJ1-expressing vegetative cells. Finally, we show that Ndj1 forms oligomeric complexes with Mps3, and that the Polo-like kinase Cdc5 regulates Ndj1 protein stability and SPB separation. These findings reveal the underlying mechanism that coordinates yeast centrosome dynamics with meiotic telomere movement and cell cycle progression. PMID:25897084

  13. Direct conversion of starch to ethanol using recombınant Saccharomyces cerevisiae containing glucoamylase gene

    NASA Astrophysics Data System (ADS)

    Purkan, P.; Baktir, A.; Puspaningsih, N. N. T.; Ni'mah, M.

    2017-09-01

    Saccharomyces cerevisiae is known for its high fermentative capacity, high ethanol yield and its high ethanol tolerance. The yeast is inability converting starch (relatively inexpensive substrate) into biofuel ethanol. Insertion of glucoamylase gene in yeast cell of Saccharomyces cerevisiae had been done to increase the yeast function in ethanol fermentation from starch. Transformation of yeast of S. cerevisiae with recombinant plasmid yEP-GLO1 carrying gene encoding glucoamylase (GLO1) produced the recombinant yeast which enable to degrade starch. Optimizing of bioconversion process of starch into ethanol by the yeast of recombinant Saccharomyces cerevisiae [yEP-GLO1] had been also done. Starch concentration which could be digested by recombinant yeast of S. cerevisiae [yEP-GLO1] was 10% (w/v). Bioconversion of starch having concentration 10% (b/v) using recombinant yeast of S. cerevisiae BY5207 [yEP-GLO1] could result ethanol as 20% (v/v) to alcoholmeter and 19,5% (v/v) to gas of chromatography. Otherwise, using recombinant yeast S. cerevisiae S. cerevisiae AS3324 [yEP-GLO1] resulted ethanol as 17% (v/v) to alcoholmeter and 17,5% (v/v) to gas of chromatography. The highest ethanol in starch bioconversion using both recombinant yeasts BY5207 and AS3324 could be resulted on 144 hours of fermentation time as well as in pH 5.

  14. Marine yeast isolation and industrial application.

    PubMed

    Zaky, Abdelrahman Saleh; Tucker, Gregory A; Daw, Zakaria Yehia; Du, Chenyu

    2014-09-01

    Over the last century, terrestrial yeasts have been widely used in various industries, such as baking, brewing, wine, bioethanol and pharmaceutical protein production. However, only little attention has been given to marine yeasts. Recent research showed that marine yeasts have several unique and promising features over the terrestrial yeasts, for example higher osmosis tolerance, higher special chemical productivity and production of industrial enzymes. These indicate that marine yeasts have great potential to be applied in various industries. This review gathers the most recent techniques used for marine yeast isolation as well as the latest applications of marine yeast in bioethanol, pharmaceutical and enzyme production fields. © 2014 The Authors FEMS Yeast Research published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

  15. Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization.

    PubMed

    Schwarzhans, Jan-Philipp; Wibberg, Daniel; Winkler, Anika; Luttermann, Tobias; Kalinowski, Jörn; Friehs, Karl

    2016-05-20

    The classic AOX1 replacement approach is still one of the most often used techniques for expression of recombinant proteins in the methylotrophic yeast Pichia pastoris. Although this approach is largely successful, it frequently delivers clones with unpredicted production characteristics and a work-intense screening process is required to find the strain with desired productivity. In this project 845 P. pastoris clones, transformed with a GFP expression cassette, were analyzed for their methanol-utilization (Mut)-phenotypes, GFP gene expression levels and gene copy numbers. Several groups of strains with irregular features were identified. Such features include GFP expression that is markedly higher or lower than expected based on gene copy number as well as strains that grew under selective conditions but where the GFP gene cassette and its expression could not be detected. From these classes of strains 31 characteristic clones were selected and their genomes sequenced. By correlating the assembled genome data with the experimental phenotypes novel insights were obtained. These comprise a clear connection between productivity and cassette-to-cassette orientation in the genome, the occurrence of false-positive clones due to a secondary recombination event, and lower total productivity due to the presence of untransformed cells within the isolates were discovered. To cope with some of these problems, the original vector was optimized by replacing the AOX1 terminator, preventing the occurrence of false-positive clones due to the secondary recombination event. Standard methods for transformation of P. pastoris led to a multitude of unintended and sometimes detrimental integration events, lowering total productivity. By documenting the connections between productivity and integration event we obtained a deeper understanding of the genetics of mutation in P. pastoris. These findings and the derived improved mutagenesis and transformation procedures and tools will help

  16. Cellulases for biomass degradation: comparing recombinant cellulase expression platforms.

    PubMed

    Garvey, Megan; Klose, Holger; Fischer, Rainer; Lambertz, Camilla; Commandeur, Ulrich

    2013-10-01

    Improvement of cellulase expression has the potential to change the nature of the biofuel industry. Increasing the economic feasibility of cellulase systems would significantly broaden the range of practicable biomass conversion, lowering the environmental impact of our civilisations' fuel needs. Cellulases are derived from certain fungi and bacteria, which are often difficult to culture on an industrial scale. Accordingly, methods to recombinantly express important cellulases and other glycosyl hydrolase (GH) enzymes are under serious investigation. Herein, we examine the latest developments in bacterial, yeast, plant, and fungal expression systems. We discuss current strategies for producing cellulases, and evaluate the benefits and drawbacks in yield, stability, and activity of enzymes from each system, and the overall progress in the field. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Recombinant human leptin in women with hypothalamic amenorrhea.

    PubMed

    Welt, Corrine K; Chan, Jean L; Bullen, John; Murphy, Robyn; Smith, Patricia; DePaoli, Alex M; Karalis, Aspasia; Mantzoros, Christos S

    2004-09-02

    Disruptions in hypothalamic-gonadal and other endocrine axes due to energy deficits are associated with low levels of the adipocyte-secreted hormone leptin and may result in hypothalamic amenorrhea. We hypothesized that exogenous recombinant leptin replacement would improve reproductive and neuroendocrine function in women with hypothalamic amenorrhea. Eight women with hypothalamic amenorrhea due to strenuous exercise or low weight were studied for one month before receiving recombinant human leptin and then while receiving treatment for up to three months. Six control subjects with hypothalamic amenorrhea received no treatment and were studied for a mean (+/-SD) of 8.5+/-8.1 months. Luteinizing hormone (LH) pulsatility, body weight, ovarian variables, and hormone levels did not change significantly over time in the controls and during a one-month control period before recombinant leptin therapy in the treated subjects. In contrast, recombinant leptin treatment increased mean LH levels and LH pulse frequency after two weeks and increased maximal follicular diameter, the number of dominant follicles, ovarian volume, and estradiol levels over a period of three months. Three patients had an ovulatory menstrual cycle (P<0.05 for the comparison with an expected rate of spontaneous ovulation of 10 percent); two others had preovulatory follicular development and withdrawal bleeding during treatment (P<0.05). Recombinant leptin significantly increased levels of free triiodothyronine, free thyroxine, insulin-like growth factor 1, insulin-like growth factor-binding protein 3, bone alkaline phosphatase, and osteocalcin but not cortisol, corticotropin, or urinary N-telopeptide. Leptin administration for the relative leptin deficiency in women with hypothalamic amenorrhea appears to improve reproductive, thyroid, and growth hormone axes and markers of bone formation, suggesting that leptin, a peripheral signal reflecting the adequacy of energy stores, is required for normal

  18. Maximal Oxygen Intake and Maximal Work Performance of Active College Women.

    ERIC Educational Resources Information Center

    Higgs, Susanne L.

    Maximal oxygen intake and associated physiological variables were measured during strenuous exercise on women subjects (N=20 physical education majors). Following assessment of maximal oxygen intake, all subjects underwent a performance test at the work level which had elicited their maximal oxygen intake. Mean maximal oxygen intake was 41.32…

  19. Yeast ecology of Kombucha fermentation.

    PubMed

    Teoh, Ai Leng; Heard, Gillian; Cox, Julian

    2004-09-01

    Kombucha is a traditional fermentation of sweetened tea, involving a symbiosis of yeast species and acetic acid bacteria. Despite reports of different yeast species being associated with the fermentation, little is known of the quantitative ecology of yeasts in Kombucha. Using oxytetracycline-supplemented malt extract agar, yeasts were isolated from four commercially available Kombucha products and identified using conventional biochemical and physiological tests. During the fermentation of each of the four products, yeasts were enumerated from both the cellulosic pellicle and liquor of the Kombucha. The number and diversity of species varied between products, but included Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii and Zygosaccharomyces bailii. While these yeast species are known to occur in Kombucha, the enumeration of each species present throughout fermentation of each of the four Kombucha cultures demonstrated for the first time the dynamic nature of the yeast ecology. Kombucha fermentation is, in general, initiated by osmotolerant species, succeeded and ultimately dominated by acid-tolerant species.

  20. Efficient Production of γ-GABA Using Recombinant E. coli Expressing Glutamate Decarboxylase (GAD) Derived from Eukaryote Saccharomyces cerevisiae.

    PubMed

    Xiong, Qiang; Xu, Zheng; Xu, Lu; Yao, Zhong; Li, Sha; Xu, Hong

    2017-12-01

    γ-Aminobutyric acid (γ-GABA) is a non-proteinogenic amino acid, which acts as a major regulator in the central nervous system. Glutamate decarboxylase (namely GAD, EC 4.1.1.15) is known to be an ideal enzyme for γ-GABA production using L-glutamic acid as substrate. In this study, we cloned and expressed GAD gene from eukaryote Saccharomyces cerevisiae (ScGAD) in E. coli BL21(DE3). This enzyme was further purified and its optimal reaction temperature and pH were 37 °C and pH 4.2, respectively. The cofactor of ScGAD was verified to be either pyridoxal 5'-phosphate (PLP) or pyridoxal hydrochloride. The optimal concentration of either cofactor was 50 mg/L. The optimal medium for E. coli-ScGAD cultivation and expression were 10 g/L lactose, 5 g/L glycerol, 20 g/L yeast extract, and 10 g/L sodium chloride, resulting in an activity of 55 U/mL medium, three times higher than that of using Luria-Bertani (LB) medium. The maximal concentration of γ-GABA was 245 g/L whereas L-glutamic acid was near completely converted. These findings provided us a good example for bio-production of γ-GABA using recombinant E. coli expressing a GAD enzyme derived from eukaryote.

  1. Marine yeast isolation and industrial application

    PubMed Central

    Zaky, Abdelrahman Saleh; Tucker, Gregory A; Daw, Zakaria Yehia; Du, Chenyu

    2014-01-01

    Over the last century, terrestrial yeasts have been widely used in various industries, such as baking, brewing, wine, bioethanol and pharmaceutical protein production. However, only little attention has been given to marine yeasts. Recent research showed that marine yeasts have several unique and promising features over the terrestrial yeasts, for example higher osmosis tolerance, higher special chemical productivity and production of industrial enzymes. These indicate that marine yeasts have great potential to be applied in various industries. This review gathers the most recent techniques used for marine yeast isolation as well as the latest applications of marine yeast in bioethanol, pharmaceutical and enzyme production fields. PMID:24738708

  2. Maintenance and integrity of the mitochondrial genome: a plethora of nuclear genes in the budding yeast.

    PubMed

    Contamine, V; Picard, M

    2000-06-01

    Instability of the mitochondrial genome (mtDNA) is a general problem from yeasts to humans. However, its genetic control is not well documented except in the yeast Saccharomyces cerevisiae. From the discovery, 50 years ago, of the petite mutants by Ephrussi and his coworkers, it has been shown that more than 100 nuclear genes directly or indirectly influence the fate of the rho(+) mtDNA. It is not surprising that mutations in genes involved in mtDNA metabolism (replication, repair, and recombination) can cause a complete loss of mtDNA (rho(0) petites) and/or lead to truncated forms (rho(-)) of this genome. However, most loss-of-function mutations which increase yeast mtDNA instability act indirectly: they lie in genes controlling functions as diverse as mitochondrial translation, ATP synthase, iron homeostasis, fatty acid metabolism, mitochondrial morphology, and so on. In a few cases it has been shown that gene overexpression increases the levels of petite mutants. Mutations in other genes are lethal in the absence of a functional mtDNA and thus convert this petite-positive yeast into a petite-negative form: petite cells cannot be recovered in these genetic contexts. Most of the data are explained if one assumes that the maintenance of the rho(+) genome depends on a centromere-like structure dispensable for the maintenance of rho(-) mtDNA and/or the function of mitochondrially encoded ATP synthase subunits, especially ATP6. In fact, the real challenge for the next 50 years will be to assemble the pieces of this puzzle by using yeast and to use complementary models, especially in strict aerobes.

  3. Recombinant protein expression of Moringa oleifera lectin in methylotrophic yeast as active coagulant for sustainable high turbid water treatment.

    PubMed

    Abd Wahid, Muhamad Azhar; Megat Mohd Noor, Megat Johari; Goto, Masafumi; Sugiura, Norio; Othman, Nor'azizi; Zakaria, Zuriati; Ahmad Mohammed, Thamer; Jusoh, Ahmad; Hara, Hirofumi

    2017-08-01

    The natural coagulant Moringa oleifera lectin (MoL) as cationic protein is a promising candidate in coagulation process of water treatment plant. Introducing the gene encoding MoL into a host, Pichia pastoris, to secrete soluble recombinant protein is assessed in this study. Initial screening using PCR confirmed the insertion of MoL gene, and SDS-PAGE analysis detected the MoL protein at 8 kDa. Cultured optimization showed the highest MoL protein at 520 mg/L was observed at 28 °C for 144 h of culturing by induction in 1% methanol. Approximately, 0.40 mg/mL of recombinant MoL protein showed 95 ± 2% turbidity removal of 1% kaolin suspension. In 0.1% kaolin suspension, the concentration of MoL at 10 μg/mL exhibits the highest turbidity reduction at 68 ± 1%. Thus, recombinant MoL protein from P. pastoris is an effective coagulant for water treatment.

  4. Development and evaluation of yeast-based GFP and luciferase reporter assays for chemical-induced genotoxicity and oxidative damage.

    PubMed

    Suzuki, Hajime; Sakabe, Takahiro; Hirose, Yuu; Eki, Toshihiko

    2017-01-01

    We aimed to develop the bioassays for genotixicity and/or oxidative damage using the recombinant yeast. A genotoxicity assay was developed using recombinant Saccharomyces cerevisiae strain BY4741 with a green fluorescent protein (GFP) reporter plasmid, driven by the DNA damage-responsive RNR3 promoter. Enhanced fluorescence induction was observed in DNA repair-deficient strains treated with methyl methanesulfonate, but not with hydrogen peroxide. A GFP reporter yeast strain driven by the oxidative stress-responsive TRX2 promoter was newly developed to assess oxidative damage, but fluorescence was poorly induced by oxidants. In place of GFP, yeast strains with luciferase gene reporter plasmids (luc2 and luc2CP, encoding stable and unstable luciferase, respectively) were prepared. Transient induction of luciferase activity was clearly detected only in a TRX2 promoter-driven luc2CP reporter strain within 90 min of oxidant exposure. However, luciferase was strongly induced by hydroxyurea in the RNR3 promoter-driven luc2 and GFP reporter strains over 8 h after the exposure, suggesting that the RNR3 promoter is continuously upregulated by DNA damage, whereas the TRX2 promoter is transiently activated by oxidative agents. Luciferase activity levels were also increased in a TRX2-promoter-driven luc2CP reporter strain treated with tert-butyl hydroperoxide and menadione and weakly induced with diamide and diethyl maleate. Weakly enhanced luciferase activity induction was detected in the sod1Δ, sod2Δ, and rad27Δ strains treated with hydrogen peroxide compared with that in the wild-type strain. In conclusion, tests using GFP and stable luciferase reporters are useful for genotoxicity, and oxidative damage can be clearly detected by assay with an unstable luciferase reporter.

  5. Destabilization and recovery of a yeast prion after mild heat shock.

    PubMed

    Newnam, Gary P; Birchmore, Jennifer L; Chernoff, Yury O

    2011-05-06

    Yeast prion [PSI(+)] is a self-perpetuating amyloid of the translational termination factor Sup35. Although [PSI(+)] propagation is modulated by heat shock proteins (Hsps), high temperature was previously reported to have little or no effect on [PSI(+)]. Our results show that short-term exposure of exponentially growing yeast culture to mild heat shock, followed by immediate resumption of growth, leads to [PSI(+)] destabilization, sometimes persisting for several cell divisions after heat shock. Prion loss occurring in the first division after heat shock is preferentially detected in a daughter cell, indicating the impairment of prion segregation that results in asymmetric prion distribution between a mother cell and a bud. Longer heat shock or prolonged incubation in the absence of nutrients after heat shock led to [PSI(+)] recovery. Both prion destabilization and recovery during heat shock depend on protein synthesis. Maximal prion destabilization coincides with maximal imbalance between Hsp104 and other Hsps such as Hsp70-Ssa. Deletions of individual SSA genes increase prion destabilization and/or counteract recovery. The dynamics of prion aggregation during destabilization and recovery are consistent with the notion that efficient prion fragmentation and segregation require a proper balance between Hsp104 and other (e.g., Hsp70-Ssa) chaperones. In contrast to heat shock, [PSI(+)] destabilization by osmotic stressors does not always depend on cell proliferation and/or protein synthesis, indicating that different stresses may impact the prion via different mechanisms. Our data demonstrate that heat stress causes asymmetric prion distribution in a cell division and confirm that the effects of Hsps on prions are physiologically relevant. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Fermentative production of l-galactonate by using recombinant Saccharomyces cerevisiae containing the endogenous galacturonate reductase gene from Cryptococcus diffluens.

    PubMed

    Matsubara, Takeo; Hamada, Shohei; Wakabayashi, Ayaka; Kishida, Masao

    2016-11-01

    The GAR1 gene, encoding d-galacturonate reductase in Cryptococcus diffluens, was isolated, and the GAR1-expression plasmid was constructed by insertion of GAR1 downstream of the yeast constitutive promoter in the yeast-integrating vector. Recombinant Saccharomyces cerevisiae expressing C. diffluensd-galacturonate reductase from a genome integrated copy of the gene was cultured for use the conversion of d-galacturonic acid to l-galactonic acid. The optimum conditions for l-galactonic acid production were determined in terms of the initial concentration of d-galacturonic acid, fermentation pH, and mixed sugars. The following conditions yielded high efficiency in the conversion of d-galacturonic acid to l-galactonic acid in large-scale cultures: 0.1% initial d-galacturonic acid concentration, pH 3.5, and glucose as additional sugar. The aerobic condition was necessary for the conversion of d-galacturonic acid. Subculture of that recombinant was not showing to decrease of the d-galacturonic acid conversion rate even though it was repeated in ten generations. Culturing in scale-up, the conversion rate of d-galacturonic acid to l-galactonic acid was increased. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  7. Factors beyond Enolase 2 and Mitochondrial Lysyl-tRNA Synthetase Precursor Are Required for tRNA Import into Yeast Mitochondria.

    PubMed

    Baleva, M V; Meyer, M; Entelis, N; Tarassov, I; Kamenski, P; Masquida, B

    2017-11-01

    In yeast, the import of tRNA Lys with CUU anticodon (tRK1) relies on a complex mechanism where interaction with enolase 2 (Eno2p) dictates a deep conformational change of the tRNA. This event is believed to mask the tRNA from the cytosolic translational machinery to re-direct it towards the mitochondria. Once near the mitochondrial outer membrane, the precursor of the mitochondrial lysyl-tRNA synthetase (preMsk1p) takes over enolase to carry the tRNA within the mitochondrial matrix, where it is supposed to participate in translation following correct refolding. Biochemical data presented in this report focus on the role of enolase. They show that despite the inability of Eno2p alone to form a complex with tRK1, mitochondrial import can be recapitulated in vitro using fractions of yeast extracts sharing either recombinant or endogenous yeast Eno2p as one of the main components. Taken together, our data suggest the existence of a protein complex containing Eno2p that is involved in RNA mitochondrial import.

  8. Meiotic recombination protein Rec12: functional conservation, crossover homeostasis and early crossover/non-crossover decision

    PubMed Central

    Kan, Fengling; Davidson, Mari K.; Wahls, Wayne P.

    2011-01-01

    In fission yeast and other eukaryotes, Rec12 (Spo11) is thought to catalyze the formation of dsDNA breaks (DSBs) that initiate homologous recombination in meiosis. Rec12 is orthologous to the catalytic subunit of topoisomerase VI (Top6A). Guided by the crystal structure of Top6A, we engineered the rec12 locus to encode Rec12 proteins each with a single amino acid substitution in a conserved residue. Of 21 substitutions, 10 significantly reduced or abolished meiotic DSBs, gene conversion, crossover recombination and the faithful segregation of chromosomes. Critical residues map within the metal ion-binding pocket toprim (E179A, D229A, D231A), catalytic region 5Y-CAP (R94A, D95A, Y98F) and the DNA-binding interface (K201A, G202E, R209A, K242A). A subset of substitutions reduced DSBs but maintained crossovers, demonstrating crossover homeostasis. Furthermore, a strong separation of function mutation (R304A) suggests that the crossover/non-crossover decision is established early by a protein–protein interaction surface of Rec12. Fission yeast has multiple crossovers per bivalent, and chromosome segregation was robust above a threshold of about one crossover per bivalent, below which non-disjunction occurred. These results support structural and functional conservation among Rec12/Spo11/Top6A family members for the catalysis of DSBs, and they reveal how Rec12 regulates other features of meiotic chromosome dynamics. PMID:21030440

  9. Towards the design of an optimal strategy for the production of ergosterol from Saccharomyces cerevisiae yeasts.

    PubMed

    Náhlík, Jan; Hrnčiřík, Pavel; Mareš, Jan; Rychtera, Mojmír; Kent, Christopher A

    2017-05-01

    The total yield of ergosterol produced by the fermentation of the yeast Saccharomyces cerevisiae depends on the final amount of yeast biomass and the ergosterol content in the cells. At the same time ergosterol purity-defined as percentage of ergosterol in the total sterols in the yeast-is equally important for efficient downstream processing. This study investigated the development of both the ergosterol content and ergosterol purity in different physiological (metabolic) states of the microorganism S. cerevisiae with the aim of reaching maximal ergosterol productivity. To expose the yeast culture to different physiological states during fermentation an on-line inference of the current physiological state of the culture was used. The results achieved made it possible to design a new production strategy, which consists of two preferable metabolic states, oxidative-fermentative growth on glucose followed by oxidative growth on glucose and ethanol simultaneously. Experimental application of this strategy achieved a value of the total efficiency of ergosterol production (defined as product of ergosterol yield coefficient and volumetric productivity), 103.84 × 10 -6 g L -1 h -1 , more than three times higher than with standard baker's yeast fed-batch cultivations, which attained in average 32.14 × 10 -6 g L -1 h -1 . At the same time the final content of ergosterol in dry biomass was 2.43%, with a purity 86%. These results make the product obtained by the proposed control strategy suitable for effective down-stream processing. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:838-848, 2017. © 2017 American Institute of Chemical Engineers.

  10. Sequence requirement of the ade6-4095 meiotic recombination hotspot in Schizosaccharomyces pombe.

    PubMed

    Foulis, Steven J; Fowler, Kyle R; Steiner, Walter W

    2018-02-01

    Homologous recombination occurs at a greatly elevated frequency in meiosis compared to mitosis and is initiated by programmed double-strand DNA breaks (DSBs). DSBs do not occur at uniform frequency throughout the genome in most organisms, but occur preferentially at a limited number of sites referred to as hotspots. The location of hotspots have been determined at nucleotide-level resolution in both the budding and fission yeasts, and while several patterns have emerged regarding preferred locations for DSB hotspots, it remains unclear why particular sites experience DSBs at much higher frequency than other sites with seemingly similar properties. Short sequence motifs, which are often sites for binding of transcription factors, are known to be responsible for a number of hotspots. In this study we identified the minimum sequence required for activity of one of such motif identified in a screen of random sequences capable of producing recombination hotspots. The experimentally determined sequence, GGTCTRGACC, closely matches the previously inferred sequence. Full hotspot activity requires an effective sequence length of 9.5 bp, whereas moderate activity requires an effective sequence length of approximately 8.2 bp and shows significant association with DSB hotspots. In combination with our previous work, this result is consistent with a large number of different sequence motifs capable of producing recombination hotspots, and supports a model in which hotspots can be rapidly regenerated by mutation as they are lost through recombination.

  11. Wine yeasts for the future.

    PubMed

    Fleet, Graham H

    2008-11-01

    International competition within the wine market, consumer demands for newer styles of wines and increasing concerns about the environmental sustainability of wine production are providing new challenges for innovation in wine fermentation. Within the total production chain, the alcoholic fermentation of grape juice by yeasts is a key process where winemakers can creatively engineer wine character and value through better yeast management and, thereby, strategically tailor wines to a changing market. This review considers the importance of yeast ecology and yeast metabolic reactions in determining wine quality, and then discusses new directions for exploiting yeasts in wine fermentation. It covers criteria for selecting and developing new commercial strains, the possibilities of using yeasts other than those in the genus of Saccharomyces, the prospects for mixed culture fermentations and explores the possibilities for high cell density, continuous fermentations.

  12. Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells.

    PubMed

    Hong, Y K; Kim, D H; Beletskii, A; Lee, C; Memili, E; Strauss, W M

    2001-04-01

    Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells. Copyright 2001 Academic Press.

  13. Discussion of teleomorphic and anamorphic Ascomycetous yeasts and yeast-like taxa

    USDA-ARS?s Scientific Manuscript database

    The relationship of ascomycetous yeasts with other members of the ascomycete fungi (Ascomycota) has been controversial for over 100 years. Because yeasts are morphologically simple, it was proposed that they represent primitive forms of ascomycetes (e.g., Guilliermond 1912). Alternatively, the ide...

  14. An avian influenza H5N1 virus vaccine candidate based on the extracellular domain produced in yeast system as subviral particles protects chickens from lethal challenge.

    PubMed

    Pietrzak, Maria; Macioła, Agnieszka; Zdanowski, Konrad; Protas-Klukowska, Anna Maria; Olszewska, Monika; Śmietanka, Krzysztof; Minta, Zenon; Szewczyk, Bogusław; Kopera, Edyta

    2016-09-01

    Highly pathogenic avian influenza is an on-going problem in poultry and a potential human pandemic threat. Pandemics occur suddenly and vaccine production must be fast and effective to be of value in controlling the spread of the virus. In this study we evaluated the potential of a recombinant protein from the extracellular domain of an H5 hemagglutinin protein produced in a yeast expression system to act as an effective vaccine. Protein production was efficient, with up to 200 mg purified from 1 L of culture medium. We showed that the deletion of the multibasic cleavage site from the protein improves oligomerization and, consequentially, its immunogenicity. We also showed that immunization with this deleted protein protected chickens from challenge with a highly pathogenic avian influenza H5N1 virus. Our results suggest that this recombinant protein produced in yeast may be an effective vaccine against H5N1 virus in poultry. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Recombinant sterol esterase from Ophiostoma piceae: an improved biocatalyst expressed in Pichia pastoris.

    PubMed

    Cedillo, Víctor Barba; Plou, Francisco J; Martínez, María Jesús

    2012-06-07

    The ascomycete Ophiostoma piceae produces a sterol esterase (OPE) with high affinity towards p-nitrophenol, glycerol and sterol esters. Its hydrolytic activity on natural mixtures of triglycerides and sterol esters has been proposed for pitch biocontrol in paper industry since these compounds produce important economic losses during paper pulp manufacture. Recently, this enzyme has been heterologously expressed in the methylotrophic yeast Pichia pastoris, and the hydrolytic activity of the recombinant protein (OPE*) studied. After the initial screening of different clones expressing the enzyme, only one was selected for showing the highest production rate. Different culture conditions were tested to improve the expression of the recombinant enzyme. Complex media were better than minimal media for production, but in any case the levels of enzymatic activity were higher (7-fold in the best case) than those obtained from O. piceae. The purified enzyme had a molecular mass of 76 kDa, higher than that reported for the native enzyme under SDS-PAGE (60 kDa). Steady-state kinetic characterization of the recombinant protein showed improved catalytic efficiency for this enzyme as compared to the native one, for all the assayed substrates (p-nitrophenol, glycerol, and cholesterol esters). Different causes for this were studied, as the increased glycosylation degree of the recombinant enzyme, their secondary structures or the oxidation of methionine residues. However, none of these could explain the improvements found in the recombinant protein. N-terminal sequencing of OPE* showed that two populations of this enzyme were expressed, having either 6 or 8 amino acid residues more than the native one. This fact affected the aggregation behaviour of the recombinant protein, as was corroborated by analytical ultracentrifugation, thus improving the catalytic efficiency of this enzyme. P. pastoris resulted to be an optimum biofactory for the heterologous production of recombinant

  16. Yeasts and yeast-like organisms associated with fruits and blossoms of different fruit trees.

    PubMed

    Vadkertiová, Renáta; Molnárová, Jana; Vránová, Dana; Sláviková, Elena

    2012-12-01

    Yeasts are common inhabitants of the phyllosphere, but our knowledge of their diversity in various plant organs is still limited. This study focused on the diversity of yeasts and yeast-like organisms associated with matured fruits and fully open blossoms of apple, plum, and pear trees, during 2 consecutive years at 3 localities in southwest Slovakia. The occurrence of yeasts and yeast-like organisms in fruit samples was 2½ times higher and the yeast community more diverse than that in blossom samples. Only 2 species (Aureobasidium pullulans and Metschnikowia pulcherrima) occurred regularly in the blossom samples, whereas Galactomyces candidus, Hanseniaspora guilliermondii, Hanseniaspora uvarum, M. pulcherrima, Pichia kluyveri, Pichia kudriavzevii, and Saccharomyces cerevisiae were the most frequently isolated species from the fruit samples. The ratio of the number of samples where only individual species were present to the number of samples where 2 or more species were found (consortium) was counted. The occurrence of individual species in comparison with consortia was much higher in blossom samples than in fruit samples. In the latter, consortia predominated. Aureobasidium pullulans, M. pulcherrima, and S. cerevisiae, isolated from both the fruits and blossoms, can be considered as resident yeast species of various fruit tree species cultivated in southwest Slovakia localities.

  17. A robust gene-stacking method utilizing yeast assembly for plant synthetic biology

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shih, Patrick M.; Vuu, Khanh; Mansoori, Nasim

    The advent and growth of synthetic biology has demonstrated its potential as a promising avenue of research to address many societal needs. But, plant synthetic biology efforts have been hampered by a dearth of DNA part libraries, versatile transformation vectors and efficient assembly strategies. We describe a versatile system (named jStack) utilizing yeast homologous recombination to efficiently assemble DNA into plant transformation vectors. We also demonstrate how this method can facilitate pathway engineering of molecules of pharmaceutical interest, production of potential biofuels and shuffling of disease-resistance traits between crop species. Our approach provides a powerful alternative to conventional strategies formore » stacking genes and traits to address many impending environmental and agricultural challenges.« less

  18. A robust gene-stacking method utilizing yeast assembly for plant synthetic biology

    DOE PAGES

    Shih, Patrick M.; Vuu, Khanh; Mansoori, Nasim; ...

    2016-10-26

    The advent and growth of synthetic biology has demonstrated its potential as a promising avenue of research to address many societal needs. But, plant synthetic biology efforts have been hampered by a dearth of DNA part libraries, versatile transformation vectors and efficient assembly strategies. We describe a versatile system (named jStack) utilizing yeast homologous recombination to efficiently assemble DNA into plant transformation vectors. We also demonstrate how this method can facilitate pathway engineering of molecules of pharmaceutical interest, production of potential biofuels and shuffling of disease-resistance traits between crop species. Our approach provides a powerful alternative to conventional strategies formore » stacking genes and traits to address many impending environmental and agricultural challenges.« less

  19. Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization.

    PubMed

    La Ferla, Marco; Mercatanti, Alberto; Rocchi, Giulia; Lodovichi, Samuele; Cervelli, Tiziana; Pignata, Luca; Caligo, Maria Adelaide; Galli, Alvaro

    2015-04-01

    The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the deubiquitination enzyme gene OTU1, the nuclear pore protein POM152 and the SNT1 that encodes for the Set3C subunit of the histone deacetylase complex. In these strains the PARP-1 level was roughly the same as in the wild type. PARP-1 localized in the nucleus more in the snt1Δ than in the wild type strain; after UV radiation, PARP-1 localized in the nucleus more in hho1 and pom152 deletion strains than in the wild type indicating that these functions may have a role on regulating PARP-1 level and activity in the nucleus. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Rad52/Rad59-dependent Recombination as a Means to Rectify Faulty Okazaki Fragment Processing*

    PubMed Central

    Lee, Miju; Lee, Chul-Hwan; Demin, Annie Albert; Munashingha, Palinda Ruvan; Amangyeld, Tamir; Kwon, Buki; Formosa, Tim; Seo, Yeon-Soo

    2014-01-01

    The correct removal of 5′-flap structures by Rad27 and Dna2 during Okazaki fragment maturation is crucial for the stable maintenance of genetic materials and cell viability. In this study, we identified RAD52, a key recombination protein, as a multicopy suppressor of dna2-K1080E, a lethal helicase-negative mutant allele of DNA2 in yeasts. In contrast, the overexpression of Rad51, which works conjointly with Rad52 in canonical homologous recombination, failed to suppress the growth defect of the dna2-K1080E mutation, indicating that Rad52 plays a unique and distinct role in Okazaki fragment metabolism. We found that the recombination-defective Rad52-QDDD/AAAA mutant did not rescue dna2-K1080E, suggesting that Rad52-mediated recombination is important for suppression. The Rad52-mediated enzymatic stimulation of Dna2 or Rad27 is not a direct cause of suppression observed in vivo, as both Rad52 and Rad52-QDDD/AAAA proteins stimulated the endonuclease activities of both Dna2 and Rad27 to a similar extent. The recombination mediator activity of Rad52 was dispensable for the suppression, whereas both the DNA annealing activity and its ability to interact with Rad59 were essential. In addition, we found that several cohesion establishment factors, including Rsc2 and Elg1, were required for the Rad52-dependent suppression of dna2-K1080E. Our findings suggest a novel Rad52/Rad59-dependent, but Rad51-independent recombination pathway that could ultimately lead to the removal of faulty flaps in conjunction with cohesion establishment factors. PMID:24711454

  1. Forces in yeast flocculation

    NASA Astrophysics Data System (ADS)

    El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Vincent, Stéphane P.; Abellán Flos, Marta; Hols, Pascal; Lipke, Peter N.; Dufrêne, Yves F.

    2015-01-01

    In the baker's yeast Saccharomyces cerevisiae, cell-cell adhesion (``flocculation'') is conferred by a family of lectin-like proteins known as the flocculin (Flo) proteins. Knowledge of the adhesive and mechanical properties of flocculins is important for understanding the mechanisms of yeast adhesion, and may help controlling yeast behaviour in biotechnology. We use single-molecule and single-cell atomic force microscopy (AFM) to explore the nanoscale forces engaged in yeast flocculation, focusing on the role of Flo1 as a prototype of flocculins. Using AFM tips labelled with mannose, we detect single flocculins on Flo1-expressing cells, showing they are widely exposed on the cell surface. When subjected to force, individual Flo1 proteins display two distinct force responses, i.e. weak lectin binding forces and strong unfolding forces reflecting the force-induced extension of hydrophobic tandem repeats. We demonstrate that cell-cell adhesion bonds also involve multiple weak lectin interactions together with strong unfolding forces, both associated with Flo1 molecules. Single-molecule and single-cell data correlate with microscale cell adhesion behaviour, suggesting strongly that Flo1 mechanics is critical for yeast flocculation. These results favour a model in which not only weak lectin-sugar interactions are involved in yeast flocculation but also strong hydrophobic interactions resulting from protein unfolding.

  2. The yeast actin cytoskeleton.

    PubMed

    Mishra, Mithilesh; Huang, Junqi; Balasubramanian, Mohan K

    2014-03-01

    The actin cytoskeleton is a complex network of dynamic polymers, which plays an important role in various fundamental cellular processes, including maintenance of cell shape, polarity, cell division, cell migration, endocytosis, vesicular trafficking, and mechanosensation. Precise spatiotemporal assembly and disassembly of actin structures is regulated by the coordinated activity of about 100 highly conserved accessory proteins, which nucleate, elongate, cross-link, and sever actin filaments. Both in vivo studies in a wide range of organisms from yeast to metazoans and in vitro studies of purified proteins have helped shape the current understanding of actin dynamics and function. Molecular genetics, genome-wide functional analysis, sophisticated real-time imaging, and ultrastructural studies in concert with biochemical analysis have made yeast an attractive model to understand the actin cytoskeleton, its molecular dynamics, and physiological function. Studies of the yeast actin cytoskeleton have contributed substantially in defining the universal mechanism regulating actin assembly and disassembly in eukaryotes. Here, we review some of the important insights generated by the study of actin cytoskeleton in two important yeast models the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  3. Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris.

    PubMed

    Kuddus, Md Ruhul; Rumi, Farhana; Tsutsumi, Motosuke; Takahashi, Rika; Yamano, Megumi; Kamiya, Masakatsu; Kikukawa, Takashi; Demura, Makoto; Aizawa, Tomoyasu

    2016-06-01

    Snakin-1 (SN-1) is a small cysteine-rich plant antimicrobial peptide with broad spectrum antimicrobial activity which was isolated from potato (Solanum tuberosum). Here, we carried out the expression of a recombinant SN-1 in the methylotrophic yeast Pichia pastoris, along with its purification and characterization. A DNA fragment encoding the mature SN-1 was cloned into pPIC9 vector and introduced into P. pastoris. A large amount of pure recombinant SN-1 (approximately 40 mg/1L culture) was obtained from a fed-batch fermentation culture after purification with a cation exchange column followed by RP-HPLC. The identity of the recombinant SN-1 was verified by MALDI-TOF MS, CD and (1)H NMR experiments. All these data strongly indicated that the recombinant SN-1 peptide had a folding with six disulfide bonds that was identical to the native SN-1. Our findings showed that SN-1 exhibited strong antimicrobial activity against test microorganisms and produced very weak hemolysis of mammalian erythrocytes. The mechanism of its antimicrobial action against Escherichia coli was investigated by both outer membrane permeability assay and cytoplasmic membrane depolarization assay. These assays demonstrated that SN-1 is a membrane-active antimicrobial peptide which can disrupt both outer and cytoplasmic membrane integrity. This is the first report on the recombinant expression and purification of a fully active SN-1 in P. pastoris. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. [Thermoresistance in Saccharomyces cerevisiae yeasts].

    PubMed

    Kaliuzhin, V A

    2011-01-01

    Under natural conditions, yeast Saccharomyces cerevisiae reproduce, as a rule, on the surface of solid or liquid medium. Thus, life cycle of yeast populations is substantially influenced by diurnal changes in ambient temperature. The pattern in the response of unrestricted yeast S. cerevisiae culture to changes in the temperature of cultivation is revealed experimentally. Yeast population, in the absence of environmental constraints on the functioning of cell chemosmotic bioenergetic system, demonstrates the ability of thermoresistance when the temperature of cultivation switches from the range of 12-36 degrees C to 37.5-40 degrees C. During the transient period that is associated with the temperature switching and lasts from 1 to 4 turnover cycles, yeast reproduction rate remains 1.5-2 times higher than under stationary conditions. This is due to evolutionary acquired adaptive activity of cell chemosmotic system. After the adaptive resources exhausting, yeast thermoresistance fully recovers at the temperature range of 12-36 degrees C within one generation time under conditions of both restricted and unrestricted nourishment. Adaptive significance of such thermoresistance seems obvious enough--it allows maintaining high reproduction rate in yeast when ambient temperature is reaching a brief maximum shortly after noon.

  5. Interaction Between Yeasts and Zinc

    NASA Astrophysics Data System (ADS)

    Nicola, Raffaele De; Walker, Graeme

    Zinc is an essential trace element in biological systems. For example, it acts as a cellular membrane stabiliser, plays a critical role in gene expression and genome modification and activates nearly 300 enzymes, including alcohol dehydrogenase. The present chapter will be focused on the influence of zinc on cell physiology of industrial yeast strains of Saccharomyces cerevisiae, with special regard to the uptake and subsequent utilisation of this metal. Zinc uptake by yeast is metabolism-dependent, with most of the available zinc translocated very quickly into the vacuole. At cell division, zinc is distributed from mother to daughter cells and this effectively lowers the individual cellular zinc concentration, which may become zinc depleted at the onset of the fermentation. Zinc influences yeast fermentative performance and examples will be provided relating to brewing and wine fermentations. Industrial yeasts are subjected to several stresses that may impair fermentation performance. Such stresses may also impact on yeast cell zinc homeostasis. This chapter will discuss the practical implications for the correct management of zinc bioavailability for yeast-based biotechnologies aimed at improving yeast growth, viability, fermentation performance and resistance to environmental stresses

  6. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Dried yeasts. 172.896 Section 172.896 Food and... PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis) and dried torula yeast (Candida utilis...

  7. Refined protocols of tamoxifen injection for inducible DNA recombination in mouse astroglia.

    PubMed

    Jahn, Hannah M; Kasakow, Carmen V; Helfer, Andreas; Michely, Julian; Verkhratsky, Alexei; Maurer, Hans H; Scheller, Anja; Kirchhoff, Frank

    2018-04-12

    Inducible DNA recombination of floxed alleles in vivo by liver metabolites of tamoxifen (TAM) is an important tool to study gene functions. Here, we describe protocols for optimal DNA recombination in astrocytes, based on the GLAST-Cre ERT2 /loxP system. In addition, we demonstrate that quantification of genomic recombination allows to determine the proportion of cell types in various brain regions. We analyzed the presence and clearance of TAM and its metabolites (N-desmethyl-tamoxifen, 4-hydroxytamoxifen and endoxifen) in brain and serum of mice by liquid chromatographic-high resolution-tandem mass spectrometry (LC-HR-MS/MS) and assessed optimal injection protocols by quantitative RT-PCR of several floxed target genes (p2ry1, gria1, gabbr1 and Rosa26-tdTomato locus). Maximal recombination could be achieved in cortex and cerebellum by single daily injections for five and three consecutive days, respectively. Furthermore, quantifying the loss of floxed alleles predicted the percentage of GLAST-positive cells (astroglia) per brain region. We found that astrocytes contributed 20 to 30% of the total cell number in cortex, hippocampus, brainstem and optic nerve, while in the cerebellum Bergmann glia, velate astrocytes and white matter astrocytes accounted only for 8% of all cells.

  8. Characterization of maltotriose transporters from the Saccharomyces eubayanus subgenome of the hybrid Saccharomyces pastorianus lager brewing yeast strain Weihenstephan 34/70.

    PubMed

    Cousseau, F E M; Alves, S L; Trichez, D; Stambuk, B U

    2013-01-01

    The genome from the Saccharomyces pastorianus industrial lager brewing strain Weihenstephan 34/70, a natural Saccharomyces cerevisiae/Saccharomyces eubayanus hybrid, indicated the presence of two different maltotriose transporter genes: a new gene in the S. eubayanus subgenome with 81% of homology to the AGT1 permease from S. cerevisiae, and an amplification of the S. eubayanus MTY1 maltotriose permease previously identified in S. pastorianus yeasts. To characterize these S. eubayanus transporter genes, we used a S. cerevisiae strain deleted in the AGT1 permease and introduced the desired permease gene(s) into this locus through homologous recombination. Our results indicate that both the MTY1 and AGT1 genes from the S. eubayanus subgenome encode functional maltotriose transporters that allow fermentation of this sugar by yeast cells, despite their apparent differences in the kinetics of maltotriose-H(+) symport activity. The presence of two maltotriose transporters in the S. eubayanus subgenome not only highlights the importance of sugar transport for efficient maltotriose utilization by industrial yeasts, but these new genes can be used in breeding and/or selection programs aimed at increasing yeast fitness for the efficient fermentation of brewer's wort. © 2012 The Society for Applied Microbiology.

  9. Segregation and transmission of mitochondrial markers in fusion products of the asporogenous yeast Torulopsis glabrata.

    PubMed

    Sriprakash, K S; Batum, C

    1981-09-01

    Using a protoplast fusion technique we have been able to locate to the mitochondrial genome of the asporogenous yeast Torulopsis glabrata mutations conferring resistance to oligomycin, antimycin and diuron. When two strains differing in the size of their mtDNAs were fused the mitochondrial markers from the parent with the larger mtDNA (71-91) were transmitted predominantly among the fusion products. Both genetical and physical evidence support the occurrence of recombination in T. glabrata mitochondrial genome. Segregation of the mitochondrial genome appears to take place before the separation of the first bud from the fusion product.

  10. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Dried yeasts. 172.896 Section 172.896 Food and... Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis) and dried torula yeast (Candida utilis) may be safely used in food provided the total folic acid...

  11. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Dried yeasts. 172.896 Section 172.896 Food and... Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis) and dried torula yeast (Candida utilis) may be safely used in food provided the total folic acid...

  12. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Dried yeasts. 172.896 Section 172.896 Food and... Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis) and dried torula yeast (Candida utilis) may be safely used in food provided the total folic acid...

  13. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Dried yeasts. 172.896 Section 172.896 Food and... Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis) and dried torula yeast (Candida utilis) may be safely used in food provided the total folic acid...

  14. Components of a Fanconi-like pathway control Pso2-independent DNA interstrand crosslink repair in yeast.

    PubMed

    Ward, Thomas A; Dudášová, Zuzana; Sarkar, Sovan; Bhide, Mangesh R; Vlasáková, Danuša; Chovanec, Miroslav; McHugh, Peter J

    2012-01-01

    Fanconi anemia (FA) is a devastating genetic disease, associated with genomic instability and defects in DNA interstrand cross-link (ICL) repair. The FA repair pathway is not thought to be conserved in budding yeast, and although the yeast Mph1 helicase is a putative homolog of human FANCM, yeast cells disrupted for MPH1 are not sensitive to ICLs. Here, we reveal a key role for Mph1 in ICL repair when the Pso2 exonuclease is inactivated. We find that the yeast FANCM ortholog Mph1 physically and functionally interacts with Mgm101, a protein previously implicated in mitochondrial DNA repair, and the MutSα mismatch repair factor (Msh2-Msh6). Co-disruption of MPH1, MGM101, MSH6, or MSH2 with PSO2 produces a lesion-specific increase in ICL sensitivity, the elevation of ICL-induced chromosomal rearrangements, and persistence of ICL-associated DNA double-strand breaks. We find that Mph1-Mgm101-MutSα directs the ICL-induced recruitment of Exo1 to chromatin, and we propose that Exo1 is an alternative 5'-3' exonuclease utilised for ICL repair in the absence of Pso2. Moreover, ICL-induced Rad51 chromatin loading is delayed when both Pso2 and components of the Mph1-Mgm101-MutSα and Exo1 pathway are inactivated, demonstrating that the homologous recombination stages of ICL repair are inhibited. Finally, the FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are also components of the genetic pathway controlled by Mph1-Mgm101-MutSα. Together this suggests that a prototypical FA-related ICL repair pathway operates in budding yeast, which acts redundantly with the pathway controlled by Pso2, and is required for the targeting of Exo1 to chromatin to execute ICL repair.

  15. Real-time measurement of the intracellular pH of yeast cells during glucose metabolism using ratiometric fluorescent nanosensors.

    PubMed

    Elsutohy, Mohamed M; Chauhan, Veeren M; Markus, Robert; Kyyaly, Mohammed Aref; Tendler, Saul J B; Aylott, Jonathan W

    2017-05-11

    Intracellular pH is a key parameter that influences many biochemical and metabolic pathways that can also be used as an indirect marker to monitor metabolic and intracellular processes. Herein, we utilise ratiometric fluorescent pH-sensitive nanosensors with an extended dynamic pH range to measure the intracellular pH of yeast (Saccharomyces cerevisiae) during glucose metabolism in real-time. Ratiometric fluorescent pH-sensitive nanosensors consisting of a polyacrylamide nanoparticle matrix covalently linked to two pH-sensitive fluorophores, Oregon green (OG) and 5(6)carboxyfluorescein (FAM), and a reference pH-insensitive fluorophore, 5(6)carboxytetramethylrhodamine (TAMRA), were synthesised. Nanosensors were functionalised with acrylamidopropyltrimethyl ammonium hydrochloride (ACTA) to confer a positive charge to the nanoparticle surfaces that facilitated nanosensor delivery to yeast cells, negating the need to use stress inducing techniques. The results showed that under glucose-starved conditions the intracellular pH of yeast population (n ≈ 200) was 4.67 ± 0.15. Upon addition of d-(+)-glucose (10 mM), this pH value decreased to pH 3.86 ± 0.13 over a period of 10 minutes followed by a gradual rise to a maximal pH of 5.21 ± 0.26, 25 minutes after glucose addition. 45 minutes after the addition of glucose, the intracellular pH of yeast cells returned to that of the glucose starved conditions. This study advances our understanding of the interplay between glucose metabolism and pH regulation in yeast cells, and indicates that the intracellular pH homestasis in yeast is highly regulated and demonstrates the utility of nanosensors for real-time intracellular pH measurements.

  16. Yeast flocculation: New story in fuel ethanol production.

    PubMed

    Zhao, X Q; Bai, F W

    2009-01-01

    Yeast flocculation has been used in the brewing industry to facilitate biomass recovery for a long time, and thus its mechanism of yeast flocculation has been intensively studied. However, the application of flocculating yeast in ethanol production garnered attention mainly in the 1980s and 1990s. In this article, updated research progress in the molecular mechanism of yeast flocculation and the impact of environmental conditions on yeast flocculation are reviewed. Construction of flocculating yeast strains by genetic approach and utilization of yeast flocculation for ethanol production from various feedstocks were presented. The concept of self-immobilized yeast cells through their flocculation is revisited through a case study of continuous ethanol fermentation with the flocculating yeast SPSC01, and their technical and economic advantages are highlighted by comparing with yeast cells immobilized with supporting materials and regular free yeast cells as well. Taking the flocculating yeast SPSC01 as an example, the ethanol tolerance of the flocculating yeast was also discussed.

  17. Brewing characteristics of piezosensitive sake yeasts

    NASA Astrophysics Data System (ADS)

    Nomura, Kazuki; Hoshino, Hirofumi; Igoshi, Kazuaki; Onozuka, Haruka; Tanaka, Erika; Hayashi, Mayumi; Yamazaki, Harutake; Takaku, Hiroaki; Iguchi, Akinori; Shigematsu, Toru

    2018-04-01

    Application of high hydrostatic pressure (HHP) treatment to food processing is expected as a non-thermal fermentation regulation technology that supresses over fermentation. However, the yeast Saccharomyces cerevisiae used for Japanese rice wine (sake) brewing shows high tolerance to HHP. Therefore, we aimed to generate pressure-sensitive (piezosensitive) sake yeast strains by mating sake with piezosensitive yeast strains to establish an HHP fermentation regulation technology and extend the shelf life of fermented foods. The results of phenotypic analyses showed that the generated yeast strains were piezosensitive and exhibited similar fermentation ability compared with the original sake yeast strain. In addition, primary properties of sake brewed using these strains, such as ethanol concentration, sake meter value and sake flavor compounds, were almost equivalent to those obtained using the sake yeast strain. These results suggest that the piezosensitive strains exhibit brewing characteristics essentially equivalent to those of the sake yeast strain.

  18. [Control levels of Sin3 histone deacetylase for spontaneous and UV-induced mutagenesis in yeasts Saccharomyces cerevisiae].

    PubMed

    Lebovka, I Iu; Kozhina, T N; Fedorova, I V; Peshekhonov, V T; Evstiukhina, T A; Chernenkov, A Iu; Korolev, V G

    2014-01-01

    SIN3 gene product operates as a repressor for a huge amount of genes in Saccharomyces cerevisiae. Sin3 protein with a mass of about 175 kDa is a member of the RPD3 protein complex with an assessed mass of greater than 2 million Da. It was previously shownthat RPD3 gene mutations influence recombination and repair processes in S. cerevisiae yeasts. We studied the impacts of the sin3 mutation on UV-light sensitivity and UV-induced mutagenesis in budding yeast cells. The deletion ofthe SIN3 gene causes weak UV-sensitivity of mutant budding cells as compared to the wild-type strain. These results show that the sin3 mutation decreases both spontaneous and UV-induced levels of levels. This fact is hypothetically related to themalfunction of ribonucleotide reductase activity regulation, which leads to a decrease in the dNTP pool and the inaccurate error-prone damage bypass postreplication repair pathway, which in turn provokes a reduction in the incidence of mutations.

  19. 21 CFR 172.898 - Bakers yeast glycan.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Bakers yeast glycan. 172.898 Section 172.898 Food... Bakers yeast glycan. Bakers yeast glycan may be safely used in food in accordance with the following conditions: (a) Bakers yeast glycan is the comminuted, washed, pasteurized, and dried cell walls of the yeast...

  20. Mitochondrial and cytoplasmic isoleucyl-, glutamyl- and arginyl-tRNA synthetases of yeast are encoded by separate genes.

    PubMed

    Tzagoloff, A; Shtanko, A

    1995-06-01

    Three complementation groups of a pet mutant collection have been found to be composed of respiratory-deficient deficient mutants with lesions in mitochondrial protein synthesis. Recombinant plasmids capable of restoring respiration were cloned by transformation of representatives of each complementation group with a yeast genomic library. The plasmids were used to characterize the complementing genes and to institute disruption of the chromosomal copies of each gene in respiratory-proficient yeast. The sequences of the cloned genes indicate that they code for isoleucyl-, arginyl- and glutamyl-tRNA synthetases. The properties of the mutants used to obtain the genes and of strains with the disrupted genes indicate that all three aminoacyl-tRNA synthetases function exclusively in mitochondrial proteins synthesis. The ISM1 gene for mitochondrial isoleucyl-tRNA synthetase has been localized to chromosome XVI next to UME5. The MSR1 gene for the arginyl-tRNA synthetase was previously located on yeast chromosome VIII. The third gene MSE1 for the mitochondrial glutamyl-tRNA synthetase has not been localized. The identification of three new genes coding for mitochondrial-specific aminoacyl-tRNA synthetases indicates that in Saccharomyces cerevisiae at least 11 members of this protein family are encoded by genes distinct from those coding for the homologous cytoplasmic enzymes.

  1. High temporal-resolution view of transcription and chromatin states across distinct metabolic states in budding yeast

    PubMed Central

    Kuang, Zheng; Cai, Ling; Zhang, Xuekui; Ji, Hongkai; Tu, Benjamin P.; Boeke, Jef D.

    2014-01-01

    Under continuous, glucose-limited conditions, budding yeast exhibit robust metabolic cycles associated with major oscillations of gene expression. How such fluctuations are linked to changes in chromatin status is not well understood. Here we examine the correlated genome-wide transcription and chromatin states across the yeast metabolic cycle at unprecedented temporal resolution, revealing a “just-in-time supply chain” by which components from specific cellular processes such as ribosome biogenesis become available in a highly coordinated manner. We identify distinct chromatin and splicing patterns associated with different gene categories and determine the relative timing of chromatin modifications to maximal transcription. There is unexpected variation in the chromatin modification and expression relationship, with histone acetylation peaks occurring with varying timing and “sharpness” relative to RNA expression both within and between cycle phases. Chromatin modifier occupancy reveals subtly distinct spatial and temporal patterns compared to the modifications themselves. PMID:25173176

  2. Improving the performance of industrial ethanol-producing yeast by expressing the aspartyl protease on the cell surface.

    PubMed

    Guo, Zhong-peng; Zhang, Liang; Ding, Zhong-yang; Wang, Zheng-Xiang; Shi, Gui-Yang

    2010-12-01

    The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell-surface anchored expressed in industrial ethanol-producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild-type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98-99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild-type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. Copyright © 2010 John Wiley & Sons, Ltd.

  3. Opportunistic Pathogenic Yeasts

    NASA Astrophysics Data System (ADS)

    Banerjee, Uma

    Advances in medical research, made during the last few decades, have improved the prophylactic, diagnostic and therapeutic capabilities for variety of infections/diseases. However, many of the prophylactic and therapeutic procedures have been seen in many instances to exact a price of host-vulnerability to an expanding group of opportunistic pathogens and yeasts are one of the important members in it. Fortunately amongst the vast majority of yeasts present in nature only few are considered to have the capability to cause infections when certain opportunities predisposes and these are termed as ‘opportunistic pathogenic yeasts.’ However, the term ‘pathogenic’ is quite tricky, as it depends of various factors of the host, the ‘bug’ and the environment to manifest the clinical infection. The borderline is expanding. In the present century with unprecedented increase in number of immune-compromised host in various disciplines of health care settings, where any yeast, which has the capability to grow at 37 ° C (normal body temperature of human), can be pathogenic and cause infection in particular situation

  4. Hsl7 is a substrate-specific type II protein arginine methyltransferase in yeast

    PubMed Central

    Sayegh, Joyce; Clarke, Steven G.

    2008-01-01

    The Saccharomyces cerevisiae protein Hsl7 is a regulator of the Swe1 protein kinase in cell cycle checkpoint control. Hsl7 has been previously described as a type III protein arginine methyltransferase, catalyzing the formation of ω-monomethylarginine residues on non-physiological substrates. However, we show here that Hsl7 can also display type II activity, generating symmetric dimethylarginine residues on calf thymus histone H2A. Symmetric dimethylation is only observed when enzyme and the methyl-accepting substrate were incubated for extended times. We confirmed the Hsl7-dependent formation of symmetric dimethylarginine by amino acid analysis and thin layer chromatography with wild type and mutant recombinant enzymes expressed from both bacteria and yeast. This result is significant because no type II activity has been previously demonstrated in S. cerevisiae. We also show that Hsl7 has little or no activity on GST-GAR, a commonly used substrate for protein arginine methyltransferases, and only minimal activity on myelin basic protein. This enzyme thus may only recognize only a small subset of potential substrate proteins in yeast, in contrast to the situation with Rmt1, the major type I methyltransferase. PMID:18515076

  5. The CCR4-NOT Complex Is Implicated in the Viability of Aneuploid Yeasts

    PubMed Central

    Tange, Yoshie; Kurabayashi, Atsushi; Goto, Bunshiro; Hoe, Kwang-Lae; Kim, Dong-Uk; Park, Han-Oh; Hayles, Jacqueline; Chikashige, Yuji; Tsutumi, Chihiro; Hiraoka, Yasushi; Yamao, Fumiaki; Nurse, Paul; Niwa, Osami

    2012-01-01

    To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability. PMID:22737087

  6. Ndj1, a telomere-associated protein, regulates centrosome separation in budding yeast meiosis.

    PubMed

    Li, Ping; Shao, Yize; Jin, Hui; Yu, Hong-Guo

    2015-04-27

    Yeast centrosomes (called spindle pole bodies [SPBs]) remain cohesive for hours during meiotic G2 when recombination takes place. In contrast, SPBs separate within minutes after duplication in vegetative cells. We report here that Ndj1, a previously known meiosis-specific telomere-associated protein, is required for protecting SPB cohesion. Ndj1 localizes to the SPB but dissociates from it ∼16 min before SPB separation. Without Ndj1, meiotic SPBs lost cohesion prematurely, whereas overproduction of Ndj1 delayed SPB separation. When produced ectopically in vegetative cells, Ndj1 caused SPB separation defects and cell lethality. Localization of Ndj1 to the SPB depended on the SUN domain protein Mps3, and removal of the N terminus of Mps3 allowed SPB separation and suppressed the lethality of NDJ1-expressing vegetative cells. Finally, we show that Ndj1 forms oligomeric complexes with Mps3, and that the Polo-like kinase Cdc5 regulates Ndj1 protein stability and SPB separation. These findings reveal the underlying mechanism that coordinates yeast centrosome dynamics with meiotic telomere movement and cell cycle progression. © 2015 Li et al.

  7. HSF-1, HIF-1 and HSP90 expression on recombinant Pichia pastoris under fed-batch fermentation.

    PubMed

    Zepeda, Andrea B; Figueroa, Carolina A; Abdalla, Dulcineia S P; Maranhão, Andrea Q; Ulloa, Patricio H; Pessoa, Adalberto; Farías, Jorge G

    2014-01-01

    Pichia pastoris is a methylotrophic yeast used as an efficient expression system for heterologous protein production as compared to other expression systems. Considering that every cell must respond to environmental changes to survive and differentiate, determination of endogenous protein related to heat stress responses and hypoxia, it would necessary to establish the temperature and methanol concentration conditions for optimal growth. The aim of this study is characterize the culture conditions through the putative biomarkers in different conditions of temperature and methanol concentration. Three yeast cultures were performed: 3X = 3% methanol -10 °C, 4X = 3% methanol -30 °C, and 5X = 1% methanol -10 °C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. The western blot results of HIF-1α and HSP-90 did not indicate statistically significant in the culture conditions studied. Respect to biomarkers location, HIF-1α and HSP-90 presented differences between cultures. In conclusion, the results suggest the cultures in a hypoxic condition produce a high density and yeast cells smaller. Beside the high density would not necessary related with a high production of recombinant proteins in modified-genetically P. pastoris.

  8. Yeast Droplets

    NASA Astrophysics Data System (ADS)

    Nguyen, Baochi; Upadhyaya, Arpita; van Oudenaarden, Alexander; Brenner, Michael

    2002-11-01

    It is well known that the Young's law and surface tension govern the shape of liquid droplets on solid surfaces. Here we address through experiments and theory the shape of growing aggregates of yeast on agar substrates, and assess whether these ideas still hold. Experiments are carried out on Baker's yeast, with different levels of expressions of an adhesive protein governing cell-cell and cell-substrate adhesion. Changing either the agar concentration or the expression of this protein modifies the local contact angle of a yeast droplet. When the colony is small, the shape is a spherical cap with the contact angle obeying Young's law. However, above a critical volume this structure is unstable, and the droplet becomes nonspherical. We present a theoretical model where this instability is caused by bulk elastic effects. The model predicts that the transition depends on both volume and contact angle, in a manner quantitatively consistent with our experiments.

  9. Not your ordinary yeast: non-Saccharomyces yeasts in wine production uncovered.

    PubMed

    Jolly, Neil P; Varela, Cristian; Pretorius, Isak S

    2014-03-01

    Saccharomyces cerevisiae and grape juice are 'natural companions' and make a happy wine marriage. However, this relationship can be enriched by allowing 'wild' non-Saccharomyces yeast to participate in a sequential manner in the early phases of grape must fermentation. However, such a triangular relationship is complex and can only be taken to 'the next level' if there are no spoilage yeast present and if the 'wine yeast' - S. cerevisiae - is able to exert its dominance in time to successfully complete the alcoholic fermentation. Winemakers apply various 'matchmaking' strategies (e.g. cellar hygiene, pH, SO2 , temperature and nutrient management) to keep 'spoilers' (e.g. Dekkera bruxellensis) at bay, and allow 'compatible' wild yeast (e.g. Torulaspora delbrueckii, Pichia kluyveri, Lachancea thermotolerans and Candida/Metschnikowia pulcherrima) to harmonize with potent S. cerevisiae wine yeast and bring the best out in wine. Mismatching can lead to a 'two is company, three is a crowd' scenario. More than 40 of the 1500 known yeast species have been isolated from grape must. In this article, we review the specific flavour-active characteristics of those non-Saccharomyces species that might play a positive role in both spontaneous and inoculated wine ferments. We seek to present 'single-species' and 'multi-species' ferments in a new light and a new context, and we raise important questions about the direction of mixed-fermentation research to address market trends regarding so-called 'natural' wines. This review also highlights that, despite the fact that most frontier research and technological developments are often focussed primarily on S. cerevisiae, non-Saccharomyces research can benefit from the techniques and knowledge developed by research on the former. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  10. Photoionization and Recombination

    NASA Technical Reports Server (NTRS)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  11. Report on LEReC Recombination Monitor APEX Study on June 15th 2016

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Drees, A.; Bruno, D.; Curcio, T.

    2016-12-21

    During the prospective Low Energy RHIC electron Cooling (LEReC) operation, the electron beam will overlap and interact with the low energy ion beam to provide transverse cooling. Cooling is needed to facilitate reaching the BES-2 (Beam Energy Scan 2) program goals of an average store luminosity of 5 × 10 24 cm -2 s -1 at 3.85 GeV/n and 17.3 × 10 24 cm -2 s -1 at 9.1 GeV/n. The RHIC phase of BES-2 is currently planned for the RHIC runs in 2019 and 2020. Effective cooling will depend on the accuracy of velocity matching between the two beams.more » Another process, the rate of ion-electron recombination, is also maximized when the velocities are matched, but the exact matching requirement is less stringent. Therefore, as suggested by one of us, detecting and maximizing recombination signals should be helpful in finding the narrow velocity matching window conducive to cooling. When 197Au 79+ RHIC ions pick up an electron from the LEREeC electron beam they are converted into 197Au 78+ ions with nearly the same momentum while having about 1.3% higher magnetic rigidity than the original 197Au 79+ particles. The detection of the recombined ions can be done by driving the 197Au 78+ beam into the beam pipe wall, creating showers of secondary particles which then can be detected outside the cryostat by using appropriately positioned detectors. For the purpose of forcing losses of the expected off-momentum particles a dedicated lattice with large horizontal dispersion in one arc was proposed and designed.« less

  12. Protective immunity against tick infestation in cattle vaccinated with recombinant trypsin inhibitor of Rhipicephalus microplus.

    PubMed

    Andreotti, Renato; Cunha, Rodrigo Casquero; Soares, Mariana Aparecida; Guerrero, Felix D; Leite, Fábio P Leivas; de León, Adalberto A Pérez

    2012-10-19

    The cattle tick, Rhipicephalus microplus, is regarded as the most economically important ectoparasite of livestock globally. Control is achieved primarily through the use of acaricides. This approach is hampered by the development of resistance to commercial acaricides among cattle tick populations. Vaccination against R. microplus infestation is another technology that can be integrated for effective cattle tick control. Proteins belonging to the Kunitz-BPTI family are abundant in cattle tick salivary glands, midgut, and ovaries. These organs are attractive targets for the development of a novel cattle tick vaccine. Efficacy assessment against cattle tick infestation in bovines using a vaccine containing the recombinant form of a member of the Kunitz family from R. microplus produced in a yeast expression system is reported for the first time here. The yeast Pichia pastoris was bioengineered to produce the recombinant version of a trypsin inhibitor that is expressed in cattle tick larvae (rRmLTI). Immunization with rRmLTI afforded 32% efficacy against R. microplus. The estimated molecular weight of rRmLTI was 46 kDa. Structural homology to the native form of the larval trypsin inhibitor was documented by recognition of rRmLTI in Western-blots using polyclonal antibodies from mice immunized with cattle tick larval extract or rRmLTI. Bioinformatics analysis of the partial nucleotide and deduced amino acid sequences indicated that the rRmLTI closely resembles BmTI-6, which is a three-headed Kunitz protein present in cattle tick ovary and fat tissue. Published by Elsevier Ltd.

  13. Lager Yeast Comes of Age

    PubMed Central

    2014-01-01

    Alcoholic fermentations have accompanied human civilizations throughout our history. Lager yeasts have a several-century-long tradition of providing fresh beer with clean taste. The yeast strains used for lager beer fermentation have long been recognized as hybrids between two Saccharomyces species. We summarize the initial findings on this hybrid nature, the genomics/transcriptomics of lager yeasts, and established targets of strain improvements. Next-generation sequencing has provided fast access to yeast genomes. Its use in population genomics has uncovered many more hybridization events within Saccharomyces species, so that lager yeast hybrids are no longer the exception from the rule. These findings have led us to propose network evolution within Saccharomyces species. This “web of life” recognizes the ability of closely related species to exchange DNA and thus drain from a combined gene pool rather than be limited to a gene pool restricted by speciation. Within the domesticated lager yeasts, two groups, the Saaz and Frohberg groups, can be distinguished based on fermentation characteristics. Recent evidence suggests that these groups share an evolutionary history. We thus propose to refer to the Saaz group as Saccharomyces carlsbergensis and to the Frohberg group as Saccharomyces pastorianus based on their distinct genomes. New insight into the hybrid nature of lager yeast will provide novel directions for future strain improvement. PMID:25084862

  14. Expression of recombinant sea urchin cellulase SnEG54 using mammalian cell lines.

    PubMed

    Okumura, Fumihiko; Kameda, Hiroyuki; Ojima, Takao; Hatakeyama, Shigetsugu

    2010-05-07

    We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  15. Yeast killer systems.

    PubMed Central

    Magliani, W; Conti, S; Gerloni, M; Bertolotti, D; Polonelli, L

    1997-01-01

    The killer phenomenon in yeasts has been revealed to be a multicentric model for molecular biologists, virologists, phytopathologists, epidemiologists, industrial and medical microbiologists, mycologists, and pharmacologists. The surprisingly widespread occurrence of the killer phenomenon among taxonomically unrelated microorganisms, including prokaryotic and eukaryotic pathogens, has engendered a new interest in its biological significance as well as its theoretical and practical applications. The search for therapeutic opportunities by using yeast killer systems has conceptually opened new avenues for the prevention and control of life-threatening fungal diseases through the idiotypic network that is apparently exploited by the immune system in the course of natural infections. In this review, the biology, ecology, epidemiology, therapeutics, serology, and idiotypy of yeast killer systems are discussed. PMID:9227858

  16. Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.

    PubMed

    Keniya, Mikhail V; Holmes, Ann R; Niimi, Masakazu; Lamping, Erwin; Gillet, Jean-Pierre; Gottesman, Michael M; Cannon, Richard D

    2014-10-06

    ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-β mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.

  17. Multisite-specific tRNA:m5C-methyltransferase (Trm4) in yeast Saccharomyces cerevisiae: identification of the gene and substrate specificity of the enzyme.

    PubMed Central

    Motorin, Y; Grosjean, H

    1999-01-01

    Several genes encoding putative RNA:5-methylcytidine-transferases (m5C-transferases) from different organisms, including yeast, have been identified by sequence homology with the recently identified 16S rRNA:m5C967-methyltransferase (gene SUN) from Escherichia coli. One of the yeast ORFs (YBL024w) was amplified by PCR, inserted in the expression vector pET28b, and the corresponding protein was hyperexpressed in E. coli BL21 (DE3). The resulting N-terminally His6-tagged recombinant Ybl024p was purified to apparent homogeneity by one-step affinity chromatography on Ni2+-NTA-agarose column. The activity and substrate specificity of the purified Ybl024p were tested in vitro using T7 transcripts of different yeast tRNAs as substrates and S-adenosyl-L-methionine as a donor of the methyl groups. The results indicate that yeast ORF YBL024w encodes S-adenosyl-L-methionine-dependent tRNA: m5C-methyltransferase that is capable of methylating cytosine to m5C at several positions in different yeast tRNAs and pre-tRNAs containing intron. Modification of tRNA occurs at all four positions (34, 40, 48, and 49) at which m5C has been found in yeast tRNAs sequenced so far. Disruption of the ORF YBL024w leads to the complete absence of m5C in total yeast tRNA. Moreover no tRNA:m5C-methyltransferase activity towards all potential m5C methylation sites was detected in the extract of the disrupted yeast strain. These results demonstrate that the protein product of a single gene is responsible for complete m5C methylation of yeast tRNA. Because this newly characterized multisite-specific modification enzyme Ybl024p is the fourth tRNA-specific methyltransferase identified in yeast, we suggest designating it as TRM4, the gene corresponding to ORF YBL024w. PMID:10445884

  18. Yeasts as distinct life forms of fungi

    USDA-ARS?s Scientific Manuscript database

    This review describes all presently recognized genera of the Ascomycete yeasts (Saccharomycotina, budding yeasts, and the Taphrinomycotina, fission yeasts and related) as well as all currently recognized genera of the Basidiomycete yeasts. This update will be the lead chapter for a book entitled “Ye...

  19. Study of amyloids using yeast

    PubMed Central

    Wickner, Reed B.; Kryndushkin, Dmitry; Shewmaker, Frank; McGlinchey, Ryan; Edskes, Herman K.

    2012-01-01

    Summary Saccharomyces cerevisiae has been a useful model organism in such fields as the cell cycle, regulation of transcription, protein trafficking and cell biology, primarily because of its ease of genetic manipulation. This is no less so in the area of amyloid studies. The endogenous yeast amyloids described to date include prions, infectious proteins (Table 1), and some cell wall proteins (1). and amyloids of humans and a fungal prion have also been studied using the yeast system. Accordingly, the emphasis of this chapter will be on genetic, biochemical, cell biological and physical methods particularly useful in the study of yeast prions and other amyloids studied in yeast. We limit our description of these methods to those aspects which have been most useful in studying yeast prions, citing more detailed expositions in the literature. Volumes on yeast genetics methods (2–4), and on amyloids and prions (5, 6) are useful, and Masison has edited a volume of Methods on “Identification, analysis and characterization of fungal prions” which covers some of this territory (7). We also outline some useful physical methods, pointing the reader to more extensive and authoratative descriptions. PMID:22528100

  20. Discovery of a diaminoquinoxaline benzenesulfonamide antagonist of HIV-1 Nef function using a yeast-based phenotypic screen

    PubMed Central

    2013-01-01

    Background HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex. Results Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades. Conclusions Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I. PMID:24229420

  1. Evolutionary History of Ascomyceteous Yeasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Haridas, Sajeet; Riley, Robert; Salamov, Asaf

    2014-06-06

    Yeasts are important for many industrial and biotechnological processes and show remarkable diversity despite morphological similarities. We have sequenced the genomes of 16 ascomycete yeasts of taxonomic and industrial importance including members of Saccharomycotina and Taphrinomycotina. A comparison of these with several other previously published yeast genomes have added increased confidence to the phylogenetic positions of previously poorly placed species including Saitoella complicata, Babjeviella inositovora and Metschnikowia bicuspidata. Phylogenetic analysis also showed that yeasts with alternative nuclear codon usage where CUG encodes serine instead of leucine are monophyletic within the Saccharomycotina. Most of the yeasts have compact genomes with amore » large fraction of single exon genes with Lipomyces starkeyi and the previously published Pneumocystis jirovecii being notable exceptions. Intron analysis suggests that early diverging species have more introns. We also observed a large number of unclassified lineage specific non-simple repeats in these genomes.« less

  2. Eighteen new oleaginous yeast species.

    PubMed

    Garay, Luis A; Sitepu, Irnayuli R; Cajka, Tomas; Chandra, Idelia; Shi, Sandy; Lin, Ting; German, J Bruce; Fiehn, Oliver; Boundy-Mills, Kyria L

    2016-07-01

    Of 1600 known species of yeasts, about 70 are known to be oleaginous, defined as being able to accumulate over 20 % intracellular lipids. These yeasts have value for fundamental and applied research. A survey of yeasts from the Phaff Yeast Culture Collection, University of California Davis was performed to identify additional oleaginous species within the Basidiomycota phylum. Fifty-nine strains belonging to 34 species were grown in lipid inducing media, and total cell mass, lipid yield and triacylglycerol profiles were determined. Thirty-two species accumulated at least 20 % lipid and 25 species accumulated over 40 % lipid by dry weight. Eighteen of these species were not previously reported to be oleaginous. Triacylglycerol profiles were suitable for biodiesel production. These results greatly expand the number of known oleaginous yeast species, and reveal the wealth of natural diversity of triacylglycerol profiles within wild-type oleaginous Basidiomycetes.

  3. Engineering of the glycerol decomposition pathway and cofactor regulation in an industrial yeast improves ethanol production.

    PubMed

    Zhang, Liang; Tang, Yan; Guo, Zhongpeng; Shi, Guiyang

    2013-10-01

    Glycerol is a major by-product of industrial ethanol production and its formation consumes up to 4 % of the sugar substrate. This study modified the glycerol decomposition pathway of an industrial strain of Saccharomyces cerevisiae to optimize the consumption of substrate and yield of ethanol. This study is the first to couple glycerol degradation with ethanol formation, to the best of our knowledge. The recombinant strain overexpressing GCY1 and DAK1, encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, in glycerol degradation pathway, exhibited a moderate increase in ethanol yield (2.9 %) and decrease in glycerol yield (24.9 %) compared to the wild type with the initial glucose concentration of 15 % under anaerobic conditions. However, when the mhpF gene, encoding acetylating NAD⁺-dependent acetaldehyde dehydrogenase from Escherichia coli, was co-expressed in the aforementioned recombinant strain, a further increase in ethanol yield by 5.5 % and decrease in glycerol yield by 48 % were observed for the resultant recombinant strain GDMS1 when acetic acid was added into the medium prior to inoculation compared to the wild type. The process outlined in this study which enhances glycerol consumption and cofactor regulation in an industrial yeast is a promising metabolic engineering strategy to increase ethanol production by reducing the formation of glycerol.

  4. Dmc1 of Schizosaccharomyces pombe plays a role in meiotic recombination.

    PubMed

    Fukushima, K; Tanaka, Y; Nabeshima, K; Yoneki, T; Tougan, T; Tanaka, S; Nojima, H

    2000-07-15

    We report here a Schizosaccharomyces pombe gene (dmc1(+)) that resembles budding yeast DMC1 in the region immediately upstream of the rad24(+) gene. We showed by northern and Southern blot analysis that dmc1(+) and rad24(+) are co-transcribed as a bicistronic mRNA of 2.8 kb with meiotic specificity, whereas rad24(+) itself is constitutively transcribed as a 1.0-kb mRNA species during meiosis. Induction of the bicistronic transcript is under the control of a meiosis-specific transcription factor, Ste11. Disruption of both dmc1(+) and rad24(+) had no effect on mitosis or spore formation, and dmc1Delta cells displayed no change in sensitivity to UV or gamma irradiation relative to the wild type. Tetrad analysis indicated that Dmc1 is involved in meiotic recombination. Analysis of gene conversion frequencies using single and double mutants of dmc1 and rhp51 indicated that both Dmc1 and Rhp51 function in meiotic gene conversion. These observations, together with a high level of sequence identity, indicate that the dmc1(+) gene of S. POMBE: is a structural homolog of budding yeast DMC1, sharing both similar and distinct functions in meiosis.

  5. Production of a recombinant phospholipase A2 in Escherichia coli using resonant acoustic mixing that improves oxygen transfer in shake flasks.

    PubMed

    Valdez-Cruz, Norma A; Reynoso-Cereceda, Greta I; Pérez-Rodriguez, Saumel; Restrepo-Pineda, Sara; González-Santana, Jesus; Olvera, Alejandro; Zavala, Guadalupe; Alagón, Alejandro; Trujillo-Roldán, Mauricio A

    2017-07-25

    Shake flasks are widely used during the development of bioprocesses for recombinant proteins. Cultures of recombinant Escherichia coli with orbital mixing (OM) have an oxygen limitation negatively affecting biomass growth and recombinant-protein production. With the aim to improve mixing and aeration in shake flask cultures, we analyzed cultures subjected to OM and the novel resonant acoustic mixing (RAM) by applying acoustic energy to E. coli BL21-Gold (DE3): a producer of recombinant phospholipase A2 (rPLA2) from Micrurus laticollaris snake venom. Comparing OM with RAM (200 rpm vs. 7.5g) at the same initial volumetric oxygen transfer coefficient (k L a ≈ 80 h -1 ) ~69% less biomass was obtained with OM compared with RAM. We analyzed two more conditions increasing agitation until maximal speed (12.5 and 20g), and ~1.6- and ~1.4-fold greater biomass was obtained as compared with cultures at 7.5g. Moreover, the specific growth rate was statistically similar in all cultures carried out in RAM, but ~1.5-fold higher than that in cultures carried out under OM. Almost half of the glucose was consumed in OM, whereas between 80 and 100% of the glucose was consumed in RAM cultures, doubling biomass per glucose yields. Differential organic acid production was observed, but acetate production was prevented at the maximal RAM (20g). The amount of rPLA2 in both, OM and RAM cultures, represented 38 ± 5% of the insoluble protein. A smaller proportion of α-helices and β-sheet of purified inclusion bodies (IBs) were appreciated by ATR-FTIR from cultures carried out under OM, than those from RAM. At maximal agitation by RAM, internal E. coli localization patterns of protein aggregation changed, as well as, IBs proteolytic degradation, in conjunction with the formation of small external vesicles, although these changes did not significantly affect the cell survival response. In moderate-cell-density recombinant E. coli BL21-Gold (DE3) cultures, the agitation increases in

  6. Oral yeast colonization throughout pregnancy

    PubMed Central

    Rio, Rute; Simões-Silva, Liliana; Garro, Sofia; Silva, Mário-Jorge; Azevedo, Álvaro

    2017-01-01

    Background Recent studies suggest that placenta may harbour a unique microbiome that may have origin in maternal oral microbiome. Although the major physiological and hormonal adjustments observed in pregnant women lead to biochemical and microbiological modifications of the oral environment, very few studies evaluated the changes suffered by the oral microbiota throughout pregnancy. So, the aim of our study was to evaluate oral yeast colonization throughout pregnancy and to compare it with non-pregnant women. Material and Methods The oral yeast colonization was assessed in saliva of 30 pregnant and non-pregnant women longitudinally over a 6-months period. Demographic information was collected, a non-invasive intra-oral examination was performed and saliva flow and pH were determined. Results Pregnant and non-pregnant groups were similar regarding age and level of education. Saliva flow rate did not differ, but saliva pH was lower in pregnant than in non-pregnant women. Oral yeast prevalence was higher in pregnant than in non-pregnant women, either in the first or in the third trimester, but did not attain statistical significance. In individuals colonized with yeast, the total yeast quantification (Log10CFU/mL) increase from the 1st to the 3rd trimester in pregnant women, but not in non-pregnant women. Conclusions Pregnancy may favour oral yeast growth that may be associated with an acidic oral environment. Key words:Oral yeast, fungi, pregnancy, saliva pH. PMID:28160578

  7. Biomedical applications of yeast- a patent view, part one: yeasts as workhorses for the production of therapeutics and vaccines.

    PubMed

    Roohvand, Farzin; Shokri, Mehdi; Abdollahpour-Alitappeh, Meghdad; Ehsani, Parastoo

    2017-08-01

    Yeasts, as Eukaryotes, offer unique features for ease of growth and genetic manipulation possibilities, making it an exceptional microbial host. Areas covered: This review provides general and patent-oriented insights into production of biopharmaceuticals by yeasts. Patents, wherever possible, were correlated to the original or review articles. The review describes applications of major GRAS (generally regarded as safe) yeasts for the production of therapeutic proteins and subunit vaccines; additionally, immunomodulatory properties of yeast cell wall components were reviewed for use of whole yeast cells as a new vaccine platform. The second part of the review will discuss yeast- humanization strategies and innovative applications. Expert opinion: Biomedical applications of yeasts were initiated by utilization of Saccharomyces cerevisiae, for production of leavened (fermented) products, and advanced to serve to produce biopharmaceuticals. Higher biomass production and expression/secretion yields, more similarity of glycosylation patterns to mammals and possibility of host-improvement strategies through application of synthetic biology might enhance selection of Pichia pastoris (instead of S. cerevisiae) as a host for production of biopharmaceutical in future. Immunomodulatory properties of yeast cell wall β-glucans and possibility of intracellular expression of heterologous pathogen/tumor antigens in yeast cells have expanded their application as a new platform, 'Whole Yeast Vaccines'.

  8. Identification of Candidate Genes Involved in the Salt Tolerance of Date Palm (Phoenix dactylifera L.) Based on a Yeast Functional Bioassay.

    PubMed

    Patankar, Himanshu V; Al-Harrasi, Ibtisam; Al-Yahyai, Rashid; Yaish, Mahmoud W

    2018-06-01

    Although date palm is a relatively salt-tolerant plant, the molecular basis of this tolerance is complex and poorly understood. Therefore, this study aimed to identify the genes involved in salinity tolerance using a basic yeast functional bioassay. To achieve this, a date palm cDNA library was overexpressed in Saccharomyces cerevisiae cells. The expression levels of selected genes that make yeast cells tolerant to salt were subsequently validated in the leaf and root tissues of date palm seedlings using a quantitative PCR method. About 6000 yeast transformant cells were replica printed and screened on a synthetic minimal medium containing 1.0 M of NaCl. The screening results showed the presence of 62 salt-tolerant transformant colonies. Sequence analysis of the recombinant yeast plasmids revealed the presence of a group of genes with potential salt-tolerance functions, such as aquaporins (PIP), serine/threonine protein kinases (STKs), ethylene-responsive transcription factor 1 (ERF1), and peroxidases (PRX). The expression pattern of the selected genes endorsed the hypothesis that these genes may be involved in salinity tolerance, as they showed a significant (p < 0.05) overexpression trend in both the leaf and root tissues in response to salinity. The genes identified in this project are suitable candidates for the further functional characterization of date palms.

  9. Nutrient supplements boost yeast transformation efficiency

    PubMed Central

    Yu, Sheng-Chun; Dawson, Alexander; Henderson, Alyssa C.; Lockyer, Eloise J.; Read, Emily; Sritharan, Gayathri; Ryan, Marjah; Sgroi, Mara; Ngou, Pok M.; Woodruff, Rosie; Zhang, Ruifeng; Ren Teen Chia, Travis; Liu, Yu; Xiang, Yiyu; Spanu, Pietro D.

    2016-01-01

    Efficiency of yeast transformation is determined by the rate of yeast endocytosis. The aim of this study was to investigate the effect of introducing amino acids and other nutrients (inositol, adenine, or p-aminobenzoic acid) in the transformation medium to develop a highly efficient yeast transformation protocol. The target of rapamycin complex 1 (TORC1) kinase signalling complex influences the rate of yeast endocytosis. TORC signaling is induced by amino acids in the media. Here, we found that increasing the concentration of amino acids and other nutrients in the growth media lead to an increase yeast transformation efficiency up to 107 CFU per μg plasmid DNA and per 108 cells with a 13.8 kb plasmid DNA. This is over 130 times that of current published methods. This improvement may facilitate more efficient experimentation in which transformation efficiency is critical, such as yeast two-hybrid screening. PMID:27760994

  10. Assessment of dioxin-like activity in ambient air particulate matter using recombinant yeast assays

    NASA Astrophysics Data System (ADS)

    Olivares, Alba; van Drooge, Barend L.; Pérez Ballesta, Pascual; Grimalt, Joan O.; Piña, Benjamin

    2011-01-01

    Ectopic activation of the aryl hydrocarbon receptor (AhR), also known as dioxin-like activity, is a major component of the toxicity associated with polycyclic aromatic hydrocarbons (PAH). Filtration of ambient air particulate matter through PM 10 filters followed by chemical determination of PAH concentrations and a yeast-based bioassay (RYA) were combined to evaluate and characterize dioxin-like activity in ambient air. Samples were collected in a semirural area of Northern Italy between September 2008 and February 2009. Total PAH contents ranged between 0.3 ng m -3 and 34 ng m -3 and were in correlation with seasonal variations of meteorological conditions and combustion processes. Dioxin-like activity values in air samples showed an excellent correlation (0.71 < R2 < 0.86) with the observed PAH concentrations and the predicted toxicity equivalents for PAH. This RYA-bioassay reported in the present study provides a simple and low-cost routine control for toxic PAH emissions, even at background air concentration levels.

  11. Recombinant antigens expressed in Pichia pastoris for the diagnosis of sleeping sickness caused by Trypanosoma brucei gambiense.

    PubMed

    Rogé, Stijn; Van Nieuwenhove, Liesbeth; Meul, Magali; Heykers, Annick; Brouwer de Koning, Annette; Bebronne, Nicolas; Guisez, Yves; Büscher, Philippe

    2014-07-01

    Screening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents. We have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture. The diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.

  12. An alkaline thermostable recombinant Humicola grisea var. thermoidea cellobiohydrolase presents bifunctional (endo/exoglucanase) activity on cellulosic substrates.

    PubMed

    Oliveira, G S; Ulhoa, C J; Silveira, M H L; Andreaus, J; Silva-Pereira, I; Poças-Fonseca, M J; Faria, F P

    2013-01-01

    Humicola grisea var. thermoidea is a deuteromycete which secretes a large spectrum of hydrolytic enzymes when grown on lignocellulosic residues. This study focused on the heterologous expression and recombinant enzyme analysis of the major secreted cellulase when the fungus is grown on sugarcane bagasse as the sole carbon source. Cellobiohydrolase 1.2 (CBH 1.2) cDNA was cloned in Pichia pastoris under control of the AOX1 promoter. Recombinant protein (rCBH1.2) was efficiently produced and secreted as a functional enzyme, presenting a molecular mass of 47 kDa. Maximum enzyme production was achieved at 96 h, in culture medium supplemented with 1.34 % urea and 1 % yeast extract and upon induction with 1 % methanol. Recombinant enzyme exhibited optimum activity at 60 °C and pH 8, and presented a remarkable thermostability, particularly at alkaline pH. Activity was evaluated on different cellulosic substrates (carboxymethyl cellulose, filter paper, microcrystalline cellulose and 4-para-nitrophenyl β-D-glucopyranoside). Interestingly, rCBH1.2 presented both exoglucanase and endoglucanase activities and mechanical agitation increased substrate hydrolysis. Results indicate that rCBH1.2 is a potential biocatalyst for applications in the textile industry or detergent formulation.

  13. Recombineering: A Homologous Recombination-Based Method of Genetic Engineering

    PubMed Central

    Sharan, Shyam K.; Thomason, Lynn C.; Kuznetsov, Sergey G.; Court, Donald L.

    2009-01-01

    Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in E. coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-strand linear DNA substrates, so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene tags can be inserted, and regions of bacterial artificial chromosomes (BACs) or the E. coli genome can be subcloned by gene retrieval using recombineering. Most of these constructs can be made within about a week's time. PMID:19180090

  14. Structural and functional characterization of the CAP domain of pathogen-related yeast 1 (Pry1) protein

    NASA Astrophysics Data System (ADS)

    Darwiche, Rabih; Kelleher, Alan; Hudspeth, Elissa M.; Schneiter, Roger; Asojo, Oluwatoyin A.

    2016-06-01

    The production, crystal structure, and functional characterization of the C-terminal cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domain of pathogen-related yeast protein-1 (Pry1) from Saccharomyces cerevisiae is presented. The CAP domain of Pry1 (Pry1CAP) is functional in vivo as its expression restores cholesterol export to yeast mutants lacking endogenous Pry1 and Pry2. Recombinant Pry1CAP forms dimers in solution, is sufficient for in vitro cholesterol binding, and has comparable binding properties as full-length Pry1. Two crystal structures of Pry1CAP are reported, one with Mg2+ coordinated to the conserved CAP tetrad (His208, Glu215, Glu233 and His250) in spacegroup I41 and the other without divalent cations in spacegroup P6122. The latter structure contains four 1,4-dioxane molecules from the crystallization solution, one of which sits in the cholesterol binding site. Both structures reveal that the divalent cation and cholesterol binding sites are connected upon dimerization, providing a structural basis for the observed Mg2+-dependent sterol binding by Pry1.

  15. Purification and characterization of VDE, a site-specific endonuclease from the yeast Saccharomyces cerevisiae.

    PubMed

    Gimble, F S; Thorner, J

    1993-10-15

    The 119-kDa primary translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes a self-catalyzed rearrangement ("protein splicing") that excises an internal 50-kDa segment of the polypeptide and joins the amino-terminal and carboxyl-terminal segments to generate the 69-kDa subunit of the vacuolar membrane-associated H(+)-ATPase. We have shown previously that the internal segment is a site-specific endonuclease (Gimble, F. S., and Thorner, J. (1992) Nature 357, 301-306). Here we describe methods for the high level expression and purification to near homogeneity of both the authentic VMA1-derived endonuclease (or VDE) from yeast (yield 18%) and a recombinant form of VDE made in bacteria (yield 29%). Detailed characterization of these preparations demonstrated that the yeast-derived and bacterially produced enzymes were indistinguishable, as judged by: (a) behavior during purification; (b) apparent native molecular mass (50 kDa); (c) immunological reactivity; and (d) catalytic properties (specific activity; cleavage site recognition; and optima for pH, temperature, divalent cation and ionic strength). The minimal site required for VDE cleavage was delimited to a 30-base pair sequence within its specific substrate (the VMA1 delta vde allele).

  16. Virgin olive oil yeasts: A review.

    PubMed

    Ciafardini, Gino; Zullo, Biagi Angelo

    2018-04-01

    This review summarizes current knowledge on virgin olive oil yeasts. Newly produced olive oil contains solid particles and micro drops of vegetation water in which yeasts reproduce to become the typical microbiota of olive oil. To date, about seventeen yeast species have been isolated from different types of olive oils and their by-products, of which six species have been identified as new species. Certain yeast species contribute greatly to improving the sensorial characteristics of the newly produced olive oil, whereas other species are considered harmful as they can damage the oil quality through the production of unpleasant flavors and triacylglycerol hydrolysis. Studies carried out in certain yeast strains have demonstrated the presence of defects in olive oil treated with Candida adriatica, Nakazawaea wickerhamii and Candida diddensiae specific strains, while other olive oil samples treated with other Candida diddensiae strains were defect-free after four months of storage and categorized as extra virgin. A new acetic acid producing yeast species, namely, Brettanomyces acidodurans sp. nov., which was recently isolated from olive oil, could be implicated in the wine-vinegary defect of the product. Other aspects related to the activity of the lipase-producing yeasts and the survival of the yeast species in the flavored olive oils are also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. The yeast Holliday junction resolvase, CCE1, can restore wild-type mitochondrial DNA to human cells carrying rearranged mitochondrial DNA.

    PubMed

    Sembongi, Hiroshi; Di Re, Miriam; Bokori-Brown, Monika; Holt, Ian J

    2007-10-01

    Rearrangements of mitochondrial DNA (mtDNA) are a well-recognized cause of human disease; deletions are more frequent, but duplications are more readily transmitted to offspring. In theory, partial duplications of mtDNA can be resolved to partially deleted and wild-type (WT) molecules, via homologous recombination. Therefore, the yeast CCE1 gene, encoding a Holliday junction resolvase, was introduced into cells carrying partially duplicated or partially triplicated mtDNA. Some cell lines carrying the CCE1 gene had substantial amounts of WT mtDNA suggesting that the enzyme can mediate intramolecular recombination in human mitochondria. However, high levels of expression of CCE1 frequently led to mtDNA loss, and so it is necessary to strictly regulate the expression of CCE1 in human cells to ensure the selection and maintenance of WT mtDNA.

  18. 21 CFR 184.1983 - Bakers yeast extract.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Bakers yeast extract. 184.1983 Section 184.1983... GRAS § 184.1983 Bakers yeast extract. (a) Bakers yeast extract is the food ingredient resulting from concentration of the solubles of mechanically ruptured cells of a selected strain of yeast, Saccharomyces...

  19. 21 CFR 184.1983 - Bakers yeast extract.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Bakers yeast extract. 184.1983 Section 184.1983... Listing of Specific Substances Affirmed as GRAS § 184.1983 Bakers yeast extract. (a) Bakers yeast extract... a selected strain of yeast, Saccharomyces cerevisiae. It may be concentrated or dried. (b) The...

  20. 21 CFR 184.1983 - Bakers yeast extract.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Bakers yeast extract. 184.1983 Section 184.1983... Listing of Specific Substances Affirmed as GRAS § 184.1983 Bakers yeast extract. (a) Bakers yeast extract... a selected strain of yeast, Saccharomyces cerevisiae. It may be concentrated or dried. (b) The...

  1. Biosentinel: Improving Desiccation Tolerance of Yeast Biosensors for Deep-Space Missions

    NASA Technical Reports Server (NTRS)

    Dalal, Sawan; Santa Maria, Sergio R.; Liddell, Lauren; Bhattacharya, Sharmila

    2017-01-01

    BioSentinel is one of 13 secondary payloads to be deployed on Exploration Mission 1 (EM-1) in 2019. We will use the budding yeast Saccharomyces cerevisiae as a biosensor to determine how deep-space radiation affects living organisms and to potentially quantify radiation levels through radiation damage analysis. Radiation can damage DNA through double strand breaks (DSBs), which can normally be repaired by homologous recombination. Two yeast strains will be air-dried and stored in microfluidic cards within the payload: a wild-type control strain and a radiation sensitive rad51 mutant that is deficient in DSB repairs. Throughout the mission, the microfluidic cards will be rehydrated with growth medium and an indicator dye. Growth rates of each strain will be measured through LED detection of the reduction of the indicator dye, which correlates with DNA repair and the amount of radiation damage accumulated. Results from BioSentinel will be compared to analog experiments on the ISS and on Earth. It is well known that desiccation can damage yeast cells and decrease viability over time. We performed a screen for desiccation-tolerant rad51 strains. We selected 20 re-isolates of rad51 and ran a weekly screen for desiccation-tolerant mutants for five weeks. Our data shows that viability decreases over time, confirming previous research findings. Isolates L2, L5 and L14 indicate desiccation tolerance and are candidates for whole-genome sequencing. More time is needed to determine whether a specific strain is truly desiccation tolerant. Furthermore, we conducted an intracellular trehalose assay to test how intracellular trehalose concentrations affect or protect the mutant strains against desiccation stress. S. cerevisiae cell and reagent concentrations from a previously established intracellular trehalose protocol did not yield significant absorbance measurements, so we tested varying cell and reagent concentrations and determined proper concentrations for successful

  2. Vaccine potential of recombinant cathepsin B against Fasciola gigantica.

    PubMed

    Chantree, Pathanin; Phatsara, Manussabhorn; Meemon, Krai; Chaichanasak, Pannigan; Changklungmoa, Narin; Kueakhai, Pornanan; Lorsuwannarat, Natcha; Sangpairoj, Kant; Songkoomkrong, Sineenart; Wanichanon, Chaitip; Itagaki, Tadashi; Sobhon, Prasert

    2013-09-01

    In Fasciola gigantica, cathepsin Bs, especially cathepsin B2 and B3 are expressed in early juvenile stages, and are proposed to mediate the invasion of host tissues. Thus they are thought to be the target vaccine candidates that can block the invasion and migration of the juvenile parasite. To evaluate their vaccine potential, the recombinant cathepsin B2 (rFgCatB2) and cathepsin B3 (rFgCatB3) were expressed in yeast, Pichia pastoris, and used to immunize mice in combination with Freund's adjuvant to evaluate the protection against the infection by F. gigantica metacercariae, and the induction of immune responses. Mice immunized with both recombinant proteins exhibited high percent of parasite reduction at 60% for rFgCatB2 and 66% for rFgCatB3. Immunization by both antigens induced continuously increasing levels of IgG1 and IgG2a with a higher level of IgG1 isotype, indicating the mixed Th1/Th2 responses with Th2 predominating. When examined individually, the higher levels of IgG1 and IgG2a were correlated with the lower numbers of worm recoveries. Thus, both cathepsin B2 and cathepsin B3 are plausible vaccine candidates whose potential should be further tested in large economic animals. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. The homologous recombination machinery modulates the formation of RNA–DNA hybrids and associated chromosome instability

    PubMed Central

    Wahba, Lamia; Gore, Steven K; Koshland, Douglas

    2013-01-01

    Genome instability in yeast and mammals is caused by RNA–DNA hybrids that form as a result of defects in different aspects of RNA biogenesis. We report that in yeast mutants defective for transcription repression and RNA degradation, hybrid formation requires Rad51p and Rad52p. These proteins normally promote DNA–DNA strand exchange in homologous recombination. We suggest they also directly promote the DNA–RNA strand exchange necessary for hybrid formation since we observed accumulation of Rad51p at a model hybrid-forming locus. Furthermore, we provide evidence that Rad51p mediates hybridization of transcripts to homologous chromosomal loci distinct from their site of synthesis. This hybrid formation in trans amplifies the genome-destabilizing potential of RNA and broadens the exclusive co-transcriptional models that pervade the field. The deleterious hybrid-forming activity of Rad51p is counteracted by Srs2p, a known Rad51p antagonist. Thus Srs2p serves as a novel anti-hybrid mechanism in vivo. DOI: http://dx.doi.org/10.7554/eLife.00505.001 PMID:23795288

  4. Inventions on baker's yeast strains and specialty ingredients.

    PubMed

    Gélinas, Pierre

    2009-06-01

    Baker's yeast is one of the oldest food microbial starters. Between 1927 and 2008, 165 inventions on more than 337 baker's yeast strains were patented. The first generation of patented yeast strains claimed improved biomass yield at the yeast plant, higher gassing power in dough or better survival to drying to prepare active dry baker's yeast. Especially between 1980 and 1995, a major interest was given to strains for multiple bakery applications such as dough with variable sugar content and stored at refrigeration (cold) or freezing temperatures. During the same period, genetically engineered yeast strains became very popular but did not find applications in the baking industry. Since year 2000, patented baker's yeast strains claimed aroma, anti-moulding or nutritive properties to better meet the needs of the baking industry. In addition to patents on yeast strains, 47 patents were issued on baker's yeast specialty ingredients for niche markets. This review shows that patents on baker's yeast with improved characteristics such as aromatic or nutritive properties have regularly been issued since the 1920's. Overall, it also confirms recent interest for a very wide range of tailored-made yeast-based ingredients for bakery applications.

  5. Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability

    NASA Astrophysics Data System (ADS)

    Jiang, Fei; Kongsaeree, Puapong; Schilke, Karl; Lajoie, Curtis; Kelly, Christine

    The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris ctMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4-7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.

  6. 21 CFR 172.898 - Bakers yeast glycan.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Bakers yeast glycan. 172.898 Section 172.898 Food... Multipurpose Additives § 172.898 Bakers yeast glycan. Bakers yeast glycan may be safely used in food in accordance with the following conditions: (a) Bakers yeast glycan is the comminuted, washed, pasteurized, and...

  7. 21 CFR 172.898 - Bakers yeast glycan.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Bakers yeast glycan. 172.898 Section 172.898 Food... Multipurpose Additives § 172.898 Bakers yeast glycan. Bakers yeast glycan may be safely used in food in accordance with the following conditions: (a) Bakers yeast glycan is the comminuted, washed, pasteurized, and...

  8. Whole-Genome Sequencing of Sake Yeast Saccharomyces cerevisiae Kyokai no. 7

    PubMed Central

    Akao, Takeshi; Yashiro, Isao; Hosoyama, Akira; Kitagaki, Hiroshi; Horikawa, Hiroshi; Watanabe, Daisuke; Akada, Rinji; Ando, Yoshinori; Harashima, Satoshi; Inoue, Toyohisa; Inoue, Yoshiharu; Kajiwara, Susumu; Kitamoto, Katsuhiko; Kitamoto, Noriyuki; Kobayashi, Osamu; Kuhara, Satoru; Masubuchi, Takashi; Mizoguchi, Haruhiko; Nakao, Yoshihiro; Nakazato, Atsumi; Namise, Masahiro; Oba, Takahiro; Ogata, Tomoo; Ohta, Akinori; Sato, Masahide; Shibasaki, Seiji; Takatsume, Yoshifumi; Tanimoto, Shota; Tsuboi, Hirokazu; Nishimura, Akira; Yoda, Koji; Ishikawa, Takeaki; Iwashita, Kazuhiro; Fujita, Nobuyuki; Shimoi, Hitoshi

    2011-01-01

    The term ‘sake yeast’ is generally used to indicate the Saccharomyces cerevisiae strains that possess characteristics distinct from others including the laboratory strain S288C and are well suited for sake brewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sake yeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeated losses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the two strains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNA-mediated recombination of Ty elements. The present study advances our understanding of the functional and evolutionary genomics of the sake yeast. PMID:21900213

  9. Schizosaccharomyces pombe Retrotransposon Tf2 Mobilizes Primarily through Homologous cDNA Recombination

    PubMed Central

    Hoff, Eleanor F.; Levin, Henry L.; Boeke, Jef D.

    1998-01-01

    The Tf2 retrotransposon, found in the fission yeast Schizosaccharomyces pombe, is nearly identical to its sister element, Tf1, in its reverse transcriptase-RNase H and integrase domains but is very divergent in the gag domain, the protease, the 5′ untranslated region, and the U3 domain of the long terminal repeats. It has now been demonstrated that a neo-marked copy of Tf2 overexpressed from a heterologous promoter can mobilize into the S. pombe genome and produce true transposition events. However, the Tf2-neo mobilization frequency is 10- to 20-fold lower than that of Tf1-neo, and 70% of the Tf2-neo events are homologous recombination events generated independently of a functional Tf2 integrase. Thus, the Tf2 element is primarily dependent on homologous recombination with preexisting copies of Tf2 for its propagation. Finally, production of Tf2-neo proteins and cDNA was also analyzed; surprisingly, Tf2 was found to produce its reverse transcriptase as a single species in which it is fused to protease, unlike all other retroviruses and retrotransposons. PMID:9774697

  10. The wine and beer yeast Dekkera bruxellensis

    PubMed Central

    Schifferdecker, Anna Judith; Dashko, Sofia; Ishchuk, Olena P; Piškur, Jure

    2014-01-01

    Recently, the non-conventional yeast Dekkera bruxellensis has been gaining more and more attention in the food industry and academic research. This yeast species is a distant relative of Saccharomyces cerevisiae and is especially known for two important characteristics: on the one hand, it is considered to be one of the main spoilage organisms in the wine and bioethanol industry; on the other hand, it is 'indispensable' as a contributor to the flavour profile of Belgium lambic and gueuze beers. Additionally, it adds to the characteristic aromatic properties of some red wines. Recently this yeast has also become a model for the study of yeast evolution. In this review we focus on the recently developed molecular and genetic tools, such as complete genome sequencing and transformation, to study and manipulate this yeast. We also focus on the areas that are particularly well explored in this yeast, such as the synthesis of off-flavours, yeast detection methods, carbon metabolism and evolutionary history. © 2014 The Authors. Yeast published by John Wiley & Sons, Ltd. PMID:24932634

  11. The wine and beer yeast Dekkera bruxellensis.

    PubMed

    Schifferdecker, Anna Judith; Dashko, Sofia; Ishchuk, Olena P; Piškur, Jure

    2014-09-01

    Recently, the non-conventional yeast Dekkera bruxellensis has been gaining more and more attention in the food industry and academic research. This yeast species is a distant relative of Saccharomyces cerevisiae and is especially known for two important characteristics: on the one hand, it is considered to be one of the main spoilage organisms in the wine and bioethanol industry; on the other hand, it is 'indispensable' as a contributor to the flavour profile of Belgium lambic and gueuze beers. Additionally, it adds to the characteristic aromatic properties of some red wines. Recently this yeast has also become a model for the study of yeast evolution. In this review we focus on the recently developed molecular and genetic tools, such as complete genome sequencing and transformation, to study and manipulate this yeast. We also focus on the areas that are particularly well explored in this yeast, such as the synthesis of off-flavours, yeast detection methods, carbon metabolism and evolutionary history. © 2014 The Authors. Yeast published by John Wiley & Sons, Ltd.

  12. Inducible DNA-repair systems in yeast: competition for lesions.

    PubMed

    Mitchel, R E; Morrison, D P

    1987-03-01

    DNA lesions may be recognized and repaired by more than one DNA-repair process. If two repair systems with different error frequencies have overlapping lesion specificity and one or both is inducible, the resulting variable competition for the lesions can change the biological consequences of these lesions. This concept was demonstrated by observing mutation in yeast cells (Saccharomyces cerevisiae) exposed to combinations of mutagens under conditions which influenced the induction of error-free recombinational repair or error-prone repair. Total mutation frequency was reduced in a manner proportional to the dose of 60Co-gamma- or 254 nm UV radiation delivered prior to or subsequent to an MNNG exposure. Suppression was greater per unit radiation dose in cells gamma-irradiated in O2 as compared to N2. A rad3 (excision-repair) mutant gave results similar to wild-type but mutation in a rad52 (rec-) mutant exposed to MNNG was not suppressed by radiation. Protein-synthesis inhibition with heat shock or cycloheximide indicated that it was the mutation due to MNNG and not that due to radiation which had changed. These results indicate that MNNG lesions are recognized by both the recombinational repair system and the inducible error-prone system, but that gamma-radiation induction of error-free recombinational repair resulted in increased competition for the lesions, thereby reducing mutation. Similarly, gamma-radiation exposure resulted in a radiation dose-dependent reduction in mutation due to MNU, EMS, ENU and 8-MOP + UVA, but no reduction in mutation due to MMS. These results suggest that the number of mutational MMS lesions recognizable by the recombinational repair system must be very small relative to those produced by the other agents. MNNG induction of the inducible error-prone systems however, did not alter mutation frequencies due to ENU or MMS exposure but, in contrast to radiation, increased the mutagenic effectiveness of EMS. These experiments demonstrate

  13. Biotechnology of non-Saccharomyces yeasts-the basidiomycetes.

    PubMed

    Johnson, Eric A

    2013-09-01

    Yeasts are the major producer of biotechnology products worldwide, exceeding production in capacity and economic revenues of other groups of industrial microorganisms. Yeasts have wide-ranging fundamental and industrial importance in scientific, food, medical, and agricultural disciplines (Fig. 1). Saccharomyces is the most important genus of yeast from fundamental and applied perspectives and has been expansively studied. Non-Saccharomyces yeasts (non-conventional yeasts) including members of the Ascomycetes and Basidiomycetes also have substantial current utility and potential applicability in biotechnology. In an earlier mini-review, "Biotechnology of non-Saccharomyces yeasts-the ascomycetes" (Johnson Appl Microb Biotechnol 97: 503-517, 2013), the extensive biotechnological utility and potential of ascomycetous yeasts are described. Ascomycetous yeasts are particularly important in food and ethanol formation, production of single-cell protein, feeds and fodder, heterologous production of proteins and enzymes, and as model and fundamental organisms for the delineation of genes and their function in mammalian and human metabolism and disease processes. In contrast, the roles of basidiomycetous yeasts in biotechnology have mainly been evaluated only in the past few decades and compared to the ascomycetous yeasts and currently have limited industrial utility. From a biotechnology perspective, the basidiomycetous yeasts are known mainly for the production of enzymes used in pharmaceutical and chemical synthesis, for production of certain classes of primary and secondary metabolites such as terpenoids and carotenoids, for aerobic catabolism of complex carbon sources, and for bioremediation of environmental pollutants and xenotoxicants. Notwithstanding, the basidiomycetous yeasts appear to have considerable potential in biotechnology owing to their catabolic utilities, formation of enzymes acting on recalcitrant substrates, and through the production of unique primary

  14. Between science and industry-applied yeast research.

    PubMed

    Korhola, Matti

    2018-03-01

    I was fortunate to enter yeast research at the Alko Research Laboratories with a strong tradition in yeast biochemistry and physiology studies. At the same time in the 1980s there was a fundamental or paradigm change in molecular biology research with discoveries in DNA sequencing and other analytical and physical techniques for studying macromolecules and cells. Since that time biotechnological research has expanded the traditional fermentation industries to efficient production of industrial and other enzymes and specialty chemicals. Our efforts were directed towards improving the industrial production organisms: minerals enriched yeasts (Se, Cr, Zn) and high glutathione content yeast, baker´s, distiller´s, sour dough and wine yeasts, and the fungal Trichoderma reesei platform for enzyme production. I am grateful for the trust of my colleagues in several leadership positions at the Alko Research Laboratories, Yeast Industry Platform and at the international yeast community.

  15. Production of recombinant streptokinase from Streptococcus pyogenes isolate and its potential for thrombolytic therapy.

    PubMed

    Assiri, Abdullah S; El-Gamal, Basiouny A; Hafez, Elsayed E; Haidara, Mohamed A

    2014-12-01

    To produce an effective recombinant streptokinase (rSK) from pathogenic Streptococcus pyogenes isolate in yeast, and evaluate its potential for thrombolytic therapy. This study was conducted from November 2012 to December 2013 at King Khalid University, Abha, Kingdom of Saudi Arabia (KSA). Throat swabs collected from 45 pharyngitis patients in Asser Central Hospital, Abha, KSA were used to isolate Streptococcus pyogenes. The bacterial DNA was used for amplification of the streptokinase gene (1200 bp). The gene was cloned and in vitro transcribed in an eukaryotic expression vector that was transformed into yeast Pichia pastoris SMD1168, and the rSK protein was purified and tested for its thrombolytic activity. The Streptococcus pyogenes strain was isolated and its DNA nucleotide sequence revealed similarity to other Streptococcus pyogenes in the Gene bank. Sequencing of the amplified gene based on DNA nucleotide sequence revealed a SK gene closely related to other SK genes in the Gene bank. However, based on deduced amino acids sequence, the gene formed a separate cluster different from clusters formed by other examined genes, suggesting a new bacterial isolate and accordingly a new gene. The purified protein showed 82% clot lysis compared to a commercial SK (81%) at an enzyme concentration of 2000 U/ml. The present yeast rSK showed similar thrombolytic activity in vitro as that of a commercial SK, suggesting its potential for thrombolytic therapy and large scale production. 

  16. CRISPR/Cas9 System as a Valuable Genome Editing Tool for Wine Yeasts with Application to Decrease Urea Production

    PubMed Central

    Vigentini, Ileana; Gebbia, Marinella; Belotti, Alessandra; Foschino, Roberto; Roth, Frederick P.

    2017-01-01

    An extensive repertoire of molecular tools is available for genetic analysis in laboratory strains of S. cerevisiae. Although this has widely contributed to the interpretation of gene functionality within haploid laboratory isolates, the genetics of metabolism in commercially-relevant polyploid yeast strains is still poorly understood. Genetic engineering in industrial yeasts is undergoing major changes due to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) engineering approaches. Here we apply the CRISPR/Cas9 system to two commercial “starter” strains of S. cerevisiae (EC1118, AWRI796), eliminating the CAN1 arginine permease pathway to generate strains with reduced urea production (18.5 and 35.5% for EC1118 and AWRI796, respectively). In a wine-model environment based on two grape musts obtained from Chardonnay and Cabernet Sauvignon cultivars, both S. cerevisiae starter strains and CAN1 mutants completed the must fermentation in 8–12 days. However, recombinant strains carrying the can1 mutation failed to produce urea, suggesting that the genetic modification successfully impaired the arginine metabolism. In conclusion, the reduction of urea production in a wine-model environment confirms that the CRISPR/Cas9 system has been successfully established in S. cerevisiae wine yeasts. PMID:29163459

  17. Din7 and Mhr1 expression levels regulate double-strand-break–induced replication and recombination of mtDNA at ori5 in yeast

    PubMed Central

    Ling, Feng; Hori, Akiko; Yoshitani, Ayako; Niu, Rong; Yoshida, Minoru; Shibata, Takehiko

    2013-01-01

    The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5′-exodeoxyribonuclease activity. Using a small ρ− mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ− cells and increased deletion mutagenesis at the ori5 region in ρ+ cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5. PMID:23598996

  18. Din7 and Mhr1 expression levels regulate double-strand-break-induced replication and recombination of mtDNA at ori5 in yeast.

    PubMed

    Ling, Feng; Hori, Akiko; Yoshitani, Ayako; Niu, Rong; Yoshida, Minoru; Shibata, Takehiko

    2013-06-01

    The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5'-exodeoxyribonuclease activity. Using a small ρ(-) mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ(-) cells and increased deletion mutagenesis at the ori5 region in ρ(+) cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.

  19. Yeast exonuclease 5 is essential for mitochondrial genome maintenance.

    PubMed

    Burgers, Peter M; Stith, Carrie M; Yoder, Bonita L; Sparks, Justin L

    2010-03-01

    Yeast exonuclease 5 is encoded by the YBR163w (DEM1) gene, and this gene has been renamed EXO5. It is distantly related to the Escherichia coli RecB exonuclease class. Exo5 is localized to the mitochondria, and EXO5 deletions or nuclease-defective EXO5 mutants invariably yield petites, amplifying either the ori3 or ori5 region of the mitochondrial genome. These petites remain unstable and undergo continuous rearrangement. The mitochondrial phenotype of exo5Delta strains suggests an essential role for the enzyme in DNA replication and recombination. No nuclear phenotype associated with EXO5 deletions has been detected. Exo5 is a monomeric 5' exonuclease that releases dinucleotides as products. It is specific for single-stranded DNA and does not hydrolyze RNA. However, Exo5 has the capacity to slide across 5' double-stranded DNA or 5' RNA sequences and resumes cutting two nucleotides downstream of the double-stranded-to-single-stranded junction or RNA-to-DNA junction, respectively.

  20. Yeast-based biosensors: design and applications.

    PubMed

    Adeniran, Adebola; Sherer, Michael; Tyo, Keith E J

    2015-02-01

    Yeast-based biosensing (YBB) is an exciting research area, as many studies have demonstrated the use of yeasts to accurately detect specific molecules. Biosensors incorporating various yeasts have been reported to detect an incredibly large range of molecules including but not limited to odorants, metals, intracellular metabolites, carcinogens, lactate, alcohols, and sugars. We review the detection strategies available for different types of analytes, as well as the wide range of output methods that have been incorporated with yeast biosensors. We group biosensors into two categories: those that are dependent upon transcription of a gene to report the detection of a desired molecule and those that are independent of this reporting mechanism. Transcription-dependent biosensors frequently depend on heterologous expression of sensing elements from non-yeast organisms, a strategy that has greatly expanded the range of molecules available for detection by YBBs. Transcription-independent biosensors circumvent the problem of sensing difficult-to-detect analytes by instead relying on yeast metabolism to generate easily detected molecules when the analyte is present. The use of yeast as the sensing element in biosensors has proven to be successful and continues to hold great promise for a variety of applications. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  1. Evidence that meiotic pairing starts at the telomeres: Molecular analysis of recombination in a family with a pericentric X chromosome inversion

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shashi, V.; Allinson, P.S.; Golden, W.L.

    1994-09-01

    Recent studies in yeast have shown that telomeres rather than centromeres lead in chromosome movement just prior to meiosis and may have a role in recombination. Cytological studies of meiosis in Drosophila and mice have shown that in pericentric inversion heterozygotes there is lack of loop formation, with recobmination seen only outside the inversion. In a family with Duchenne muscular dystrophy (DMD) we recognized that only affected males and carrier females had a pericentric X chromosome inversion (inv X(p11.4;q26)). Since the short arm inversion breakpoint was proximal to the DMD locus, it could not be implicated in the mutational eventmore » causing DMD. There was no history of infertility, recurrent miscarriages or liveborn unbalanced females to suggest there was recombination within the inversion. We studied 22 members over three generations to understand the pattern of meiotic recombination between the normal and the inverted X chromosome. In total, 17 meioses involving the inverted X chromosome in females were studied by cytogenetic analysis and 16 CA repeat polymorphisms along the length of the X chromosome. Results: (a) There was complete concordance between the segregation of the DMD mutation and the inverted X chromosome. (b) On DNA analysis, there was complete absence of recombination within the inverted segment. We also found no recombination at the DMD locus. Recombination was seen only at Xp22 and Xq27-28. (c) Recombination was seen in the same individual at both Xp22 and Xq27-28 without recombination otherwise. Conclusions: (1) Pericentric X inversions reduce the genetic map length of the chromosome, with the physical map length being normal. (2) Meiotic X chromosome pairing in this family is initiated at the telomeres. (3) Following telomeric pairing in pericentric X chromosome inversions, there is inhibition of recombination within the inversion and adjacent regions.« less

  2. Synthetic signal sequences that enable efficient secretory protein production in the yeast Kluyveromyces marxianus.

    PubMed

    Yarimizu, Tohru; Nakamura, Mikiko; Hoshida, Hisashi; Akada, Rinji

    2015-02-14

    Targeting of cellular proteins to the extracellular environment is directed by a secretory signal sequence located at the N-terminus of a secretory protein. These signal sequences usually contain an N-terminal basic amino acid followed by a stretch containing hydrophobic residues, although no consensus signal sequence has been identified. In this study, simple modeling of signal sequences was attempted using Gaussia princeps secretory luciferase (GLuc) in the yeast Kluyveromyces marxianus, which allowed comprehensive recombinant gene construction to substitute synthetic signal sequences. Mutational analysis of the GLuc signal sequence revealed that the GLuc hydrophobic peptide length was lower limit for effective secretion and that the N-terminal basic residue was indispensable. Deletion of the 16th Glu caused enhanced levels of secreted protein, suggesting that this hydrophilic residue defined the boundary of a hydrophobic peptide stretch. Consequently, we redesigned this domain as a repeat of a single hydrophobic amino acid between the N-terminal Lys and C-terminal Glu. Stretches consisting of Phe, Leu, Ile, or Met were effective for secretion but the number of residues affected secretory activity. A stretch containing sixteen consecutive methionine residues (M16) showed the highest activity; the M16 sequence was therefore utilized for the secretory production of human leukemia inhibitory factor protein in yeast, resulting in enhanced secreted protein yield. We present a new concept for the provision of secretory signal sequence ability in the yeast K. marxianus, determined by the number of residues of a single hydrophobic residue located between N-terminal basic and C-terminal acidic amino acid boundaries.

  3. HSF-1, HIF-1and HSP90 expression on recombinant Pichia pastoris under fed-batch fermentation

    PubMed Central

    Zepeda, Andrea B.; Figueroa, Carolina A.; Abdalla, Dulcineia S.P.; Maranhão, Andrea Q.; Ulloa, Patricio H.; Pessoa, Adalberto; Farías, Jorge G.

    2014-01-01

    Pichia pastoris is a methylotrophic yeast used as an efficient expression system for heterologous protein production as compared to other expression systems. Considering that every cell must respond to environmental changes to survive and differentiate, determination of endogenous protein related to heat stress responses and hypoxia, it would necessary to establish the temperature and methanol concentration conditions for optimal growth. The aim of this study is characterize the culture conditions through the putative biomarkers in different conditions of temperature and methanol concentration. Three yeast cultures were performed: 3X = 3% methanol −10 °C, 4X = 3% methanol −30 °C, and 5X = 1% methanol −10 °C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. The western blot results of HIF-1α and HSP-90 did not indicate statistically significant in the culture conditions studied. Respect to biomarkers location, HIF-1α and HSP-90 presented differences between cultures. In conclusion, the results suggest the cultures in a hypoxic condition produce a high density and yeast cells smaller. Beside the high density would not necessary related with a high production of recombinant proteins in modified-genetically P. pastoris. PMID:25242931

  4. Oral yeast colonization throughout pregnancy.

    PubMed

    Rio, R; Simões-Silva, L; Garro, S; Silva, M-J; Azevedo, Á; Sampaio-Maia, B

    2017-03-01

    Recent studies suggest that placenta may harbour a unique microbiome that may have origin in maternal oral microbiome. Although the major physiological and hormonal adjustments observed in pregnant women lead to biochemical and microbiological modifications of the oral environment, very few studies evaluated the changes suffered by the oral microbiota throughout pregnancy. So, the aim of our study was to evaluate oral yeast colonization throughout pregnancy and to compare it with non-pregnant women. The oral yeast colonization was assessed in saliva of 30 pregnant and non-pregnant women longitudinally over a 6-months period. Demographic information was collected, a non-invasive intra-oral examination was performed and saliva flow and pH were determined. Pregnant and non-pregnant groups were similar regarding age and level of education. Saliva flow rate did not differ, but saliva pH was lower in pregnant than in non-pregnant women. Oral yeast prevalence was higher in pregnant than in non-pregnant women, either in the first or in the third trimester, but did not attain statistical significance. In individuals colonized with yeast, the total yeast quantification (Log10CFU/mL) increase from the 1st to the 3rd trimester in pregnant women, but not in non-pregnant women. Pregnancy may favour oral yeast growth that may be associated with an acidic oral environment.

  5. Biotechnological Applications of Dimorphic Yeasts

    NASA Astrophysics Data System (ADS)

    Doiphode, N.; Joshi, C.; Ghormade, V.; Deshpande, M. V.

    The dimorphic yeasts have the equilibrium between spherical growth (budding) and polarized (hyphal or pseudohyphal tip elongation) which can be triggered by change in the environmental conditions. The reversible growth phenomenon has made dimorphic yeasts as an useful model to understand fungal evolution and fungal differentiation, in general. In nature dimorphism is clearly evident in plant and animal fungal pathogens, which survive and most importantly proliferate in the respective hosts. However, number of organisms with no known pathogenic behaviour also show such a transition, which can be exploited for the technological applications due to their different biochemical make up under different morphologies. For instance, chitin and chitosan production using dimorphic Saccharomyces, Mucor, Rhizopus and Benjaminiella, oil degradation and biotransformation with yeast-form of Yarrowia species, bioremediation of organic pollutants, exopolysac-charide production by yeast-phase of Aureobasidium pullulans, to name a few. Myrothecium verrucaria can be used for seed dressing in its yeast form and it produces a mycolytic enzyme complex in its hyphal-form for the biocontrol of fungal pathogens, while Beauveria bassiana and other entomopathogens kill the insect pest by producing yeast- like cells in the insect body. The form-specific expression of protease, chitinase, lipase, ornithine decarboxylase, glutamate dehydrogenases, etc. make Benjaminiella poitrasii, Basidiobolus sp., and Mucor rouxii strains important in bioremediation, nanobiotechnology, fungal evolution and other areas.

  6. Recombination-dependent mtDNA partitioning: in vivo role of Mhr1p to promote pairing of homologous DNA.

    PubMed

    Ling, Feng; Shibata, Takehiko

    2002-09-02

    Yeast mhr1-1 was isolated as a defective mutation in mitochondrial DNA (mtDNA) recombination. About half of mhr1-1 cells lose mtDNA during growth at a higher temperature. Here, we show that mhr1-1 exhibits a defect in the partitioning of nascent mtDNA into buds and is a base-substitution mutation in MHR1 encoding a mitochondrial matrix protein. We found that the Mhr1 protein (Mhr1p) has activity to pair single-stranded DNA and homologous double-stranded DNA to form heteroduplex joints in vitro, and that mhr1-1 causes the loss of this activity, indicating its role in homologous mtDNA recombination. While the majority of the mtDNA in the mother cells consists of head-to-tail concatemers, more than half of the mtDNA in the buds exists as genome-sized monomers. The mhr1-1 deltacce1 double mutant cells do not maintain any mtDNA, indicating the strict dependence of mtDNA maintenance on recombination functions. These results suggest a mechanism for mtDNA inheritance similar to that operating in the replication and packaging of phage DNA.

  7. Yeasts of the soil – obscure but precious

    PubMed Central

    2018-01-01

    Abstract Pioneering studies performed in the nineteenth century demonstrated that yeasts are present in below‐ground sources. Soils were regarded more as a reservoir for yeasts that reside in habitats above it. Later studies showed that yeast communities in soils are taxonomically diverse and different from those above‐ground. Soil yeasts possess extraordinary adaptations that allow them to survive in a wide range of environmental conditions. A few species are promising sources of yeast oils and have been used in agriculture as potential antagonists of soil‐borne plant pathogens or as plant growth promoters. Yeasts have been studied mainly in managed soils such as vineyards, orchards and agricultural fields, and to a lesser extent under forests and grasslands. Our knowledge of soil yeasts is further biased towards temperate and boreal forests, whereas data from Africa, the Americas and Asia are scarce. Although soil yeast communities are often species‐poor in a single sample, they are more diverse on the biotope level. Soil yeasts display pronounced endemism along with a surprisingly high proportion of currently unidentified species. However, like other soil inhabitants, yeasts are threatened by habitat alterations owing to anthropogenic activities such as agriculture, deforestation and urbanization. In view of the rapid decline of many natural habitats, the study of soil yeasts in undisturbed or low‐managed biotopes is extremely valuable. The purpose of this review is to encourage researchers, both biologists and soil scientists, to include soil yeasts in future studies. PMID:29365211

  8. Genetic Evidence for a Silent SUC Gene in Yeast

    PubMed Central

    Carlson, Marian; Osmond, Barbara C.; Botstein, David

    1981-01-01

    The SUC genes (SUC1–SUC7) of Saccharomyces are a family of genes that are dispersed in the yeast genome. A SUC+ allele at any locus confers the ability to produce the enzyme invertase and, thus, to ferment sucrose. Most yeast strains do not carry SUC+ alleles at all possible SUC loci. We have investigated the naturally occurring negative (suc0) alleles present at SUC loci with the aim of distinguishing between two possible models for the structure of suc0 alleles: (1) suc0 alleles correspond to a simple absence of SUC genetic information; (2) suc0 alleles are "silent" SUC genes that either produce a defective product or are not expressed. To facilitate these studies, sucrose-nonfermenting strains were constructed that are congenic to S. cerevisiae strain S288C (SUC2+), but carry at the SUC2 locus the naturally occurring negative allele, suc20, of strain FL100 (Lacroute 1968). These strains were used to study the genetic properties of the suc20 allele of FL100 and the suc0 alleles (suc10, suc30, etc.) of S288C. The suc20 allele was shown to revert to an active Suc+ state and to provide functional information at three points in the SUC2 gene in recombination experiments; this suc20 gene thus appears to be a "silent" gene. Similar tests for silent SUC genes in S288C (corresponding to loci other than SUC2) failed to reveal any additional silent genes. PMID:7040164

  9. Electron transport chain in a thermotolerant yeast.

    PubMed

    Mejía-Barajas, Jorge A; Martínez-Mora, José A; Salgado-Garciglia, Rafael; Noriega-Cisneros, Ruth; Ortiz-Avila, Omar; Cortés-Rojo, Christian; Saavedra-Molina, Alfredo

    2017-04-01

    Yeasts capable of growing and surviving at high temperatures are regarded as thermotolerant. For appropriate functioning of cellular processes and cell survival, the maintenance of an optimal redox state is critical of reducing and oxidizing species. We studied mitochondrial functions of the thermotolerant Kluyveromyces marxianus SLP1 and the mesophilic OFF1 yeasts, through the evaluation of its mitochondrial membrane potential (ΔΨ m ), ATPase activity, electron transport chain (ETC) activities, alternative oxidase activity, lipid peroxidation. Mitochondrial membrane potential and the cytoplasmic free Ca 2+ ions (Ca 2+ cyt) increased in the SLP1 yeast when exposed to high temperature, compared with the mesophilic yeast OFF1. ATPase activity in the mesophilic yeast diminished 80% when exposed to 40° while the thermotolerant SLP1 showed no change, despite an increase in the mitochondrial lipid peroxidation. The SLP1 thermotolerant yeast exposed to high temperature showed a diminution of 33% of the oxygen consumption in state 4. The uncoupled state 3 of oxygen consumption did not change in the mesophilic yeast when it had an increase of temperature, whereas in the thermotolerant SLP1 yeast resulted in an increase of 2.5 times when yeast were grown at 30 o , while a decrease of 51% was observed when it was exposed to high temperature. The activities of the ETC complexes were diminished in the SLP1 when exposed to high temperature, but also it was distinguished an alternative oxidase activity. Our results suggest that the mitochondria state, particularly ETC state, is an important characteristic of the thermotolerance of the SLP1 yeast strain.

  10. Yeasts in floral nectar: a quantitative survey

    PubMed Central

    Herrera, Carlos M.; de Vega, Clara; Canto, Azucena; Pozo, María I.

    2009-01-01

    Background and Aims One peculiarity of floral nectar that remains relatively unexplored from an ecological perspective is its role as a natural habitat for micro-organisms. This study assesses the frequency of occurrence and abundance of yeast cells in floral nectar of insect-pollinated plants from three contrasting plant communities on two continents. Possible correlations between interspecific differences in yeast incidence and pollinator composition are also explored. Methods The study was conducted at three widely separated areas, two in the Iberian Peninsula (Spain) and one in the Yucatán Peninsula (Mexico). Floral nectar samples from 130 species (37–63 species per region) in 44 families were examined microscopically for the presence of yeast cells. For one of the Spanish sites, the relationship across species between incidence of yeasts in nectar and the proportion of flowers visited by each of five major pollinator categories was also investigated. Key Results Yeasts occurred regularly in the floral nectar of many species, where they sometimes reached extraordinary densities (up to 4 × 105 cells mm−3). Depending on the region, between 32 and 44 % of all nectar samples contained yeasts. Yeast cell densities in the order of 104 cells mm−3 were commonplace, and densities >105 cells mm−3 were not rare. About one-fifth of species at each site had mean yeast cell densities >104 cells mm−3. Across species, yeast frequency and abundance were directly correlated with the proportion of floral visits by bumble-bees, and inversely with the proportion of visits by solitary bees. Conclusions Incorporating nectar yeasts into the scenario of plant–pollinator interactions opens up a number of intriguing avenues for research. In addition, with yeasts being as ubiquitous and abundant in floral nectars as revealed by this study, and given their astounding metabolic versatility, studies focusing on nectar chemical features should carefully control for the presence

  11. Molecular mechanism of vde-initiated intein homing in yeast nuclear genome.

    PubMed

    Fukuda, Tomoyuki; Nagai, Yuri; Ohya, Yoshikazu

    2004-01-01

    In Saccharomyces cerevisiae, VMAI intein encodes a homing endonuclease termed VDE which is produced by an autocatalytic protein splicing reaction. VDE introduces a DSB at its recognition sequence on intein-minus allele, resulting in the lateral transfer of VMAI intein. In this review, we summarize a decade of in vitro study on VDE and describe our recent study on the in vivo behavior of both VDE and host proteins involved in intein mobility. Meiotic DSBs caused by VDE are repaired in the similar pathway to that working in meiotic recombination induced by Spollp-mediated DSBs. Meiosis-specific DNA cleavage and homing is shown to be guaranteed by the two distinct mechanisms, the subcellular localization of VDE and a requirement of premeiotic DNA replication. Based on these lines of evidence, we present the whole picture of molecular mechanism of VDEinitiated homing in yeast cells.

  12. Roles of exonucleases and translesion synthesis DNA polymerases during mitotic gap repair in yeast

    PubMed Central

    Guo, Xiaoge; Jinks-Robertson, Sue

    2013-01-01

    Transformation-based gap-repair assays have long been used to model the repair of mitotic double-strand breaks (DSBs) by homologous recombination in yeast. In the current study, we examine genetic requirements of two key processes involved in DSB repair: (1) the processive 5′-end resection that is required to efficiently engage a repair template and (2) the filling of resected ends by DNA polymerases. The specific gap-repair assay used allows repair events resolved as crossover versus noncrossover products to be distinguished, as well as the extent of heteroduplex DNA formed during recombination to be measured. To examine end resection, the efficiency and outcome of gap repair were monitored in the absence of the Exo1 exonuclease and the Sgs1 helicase. We found that either Exo1 or Sgs1 presence is sufficient to inhibit gap-repair efficiency over 10-fold, consistent with resection-mediated destruction of the introduced plasmid. In terms of DNA polymerase requirements for gap repair, we focused specifically on potential roles of the Pol ζ and Pol η translesion synthesis DNA polymerases. We found that both Pol ζ and Pol η are necessary for efficient gap repair and that each functions independently of the other. These polymerases may be either in the initiation of DNA synthesis from the an invading end, or in a gap-filling process that is required to complete recombination. PMID:24210827

  13. Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield

    PubMed Central

    2011-01-01

    Background Pichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast. Results Addition of defined concentrations of Antifoam A (Sigma), Antifoam C (Sigma), J673A (Struktol), P2000 (Fluka) or SB2121 (Struktol) to shake-flask cultures of P. pastoris increased the total amount of recombinant GFP in the culture medium (the total yield) and in the case of P2000, SB2121 and J673A almost doubled it. When normalized to the culture density, the GFP specific yield (μg OD595-1) was only increased for Antifoam A, Antifoam C and J673A. Whilst none of the antifoams affected the growth rate of the cells, addition of P2000 or SB2121 was found to increase culture density. There was no correlation between total yield, specific yield or specific growth rate and the volumetric oxygen mass transfer coefficient (kLa) in the presence of antifoam. Moreover, the antifoams did not affect the dissolved oxygen concentration of the cultures. A comparison of the amount of GFP retained in the cell by flow cytometry with that in the culture medium by fluorimetry suggested that addition of Antifoam A, Antifoam C or J673A increased the specific yield of GFP by increasing the proportion secreted into the medium

  14. Interactions between Drosophila and its natural yeast symbionts—Is Saccharomyces cerevisiae a good model for studying the fly-yeast relationship?

    PubMed Central

    Hoang, Don; Kopp, Artyom

    2015-01-01

    Yeasts play an important role in the biology of the fruit fly, Drosophila melanogaster. In addition to being a valuable source of nutrition, yeasts affect D. melanogaster behavior and interact with the host immune system. Most experiments investigating the role of yeasts in D. melanogaster biology use the baker’s yeast, Saccharomyces cerevisiae. However, S. cerevisiae is rarely found with natural populations of D. melanogaster or other Drosophila species. Moreover, the strain of S. cerevisiae used most often in D. melanogaster experiments is a commercially and industrially important strain that, to the best of our knowledge, was not isolated from flies. Since disrupting natural host–microbe interactions can have profound effects on host biology, the results from D. melanogaster–S. cerevisiae laboratory experiments may not be fully representative of host–microbe interactions in nature. In this study, we explore the D. melanogaster-yeast relationship using five different strains of yeast that were isolated from wild Drosophila populations. Ingested live yeasts have variable persistence in the D. melanogaster gastrointestinal tract. For example, Hanseniaspora occidentalis persists relative to S. cerevisiae, while Brettanomyces naardenensis is removed. Despite these differences in persistence relative to S. cerevisiae, we find that all yeasts decrease in total abundance over time. Reactive oxygen species (ROS) are an important component of the D. melanogaster anti-microbial response and can inhibit S. cerevisiae growth in the intestine. To determine if sensitivity to ROS explains the differences in yeast persistence, we measured yeast growth in the presence and absence of hydrogen peroxide. We find that B. naardenesis is completely inhibited by hydrogen peroxide, while H. occidentalis is not, which is consistent with yeast sensitivity to ROS affecting persistence within the D. melanogaster gastrointestinal tract. We also compared the feeding preference of D

  15. Interactions between Drosophila and its natural yeast symbionts-Is Saccharomyces cerevisiae a good model for studying the fly-yeast relationship?

    PubMed

    Hoang, Don; Kopp, Artyom; Chandler, James Angus

    2015-01-01

    Yeasts play an important role in the biology of the fruit fly, Drosophila melanogaster. In addition to being a valuable source of nutrition, yeasts affect D. melanogaster behavior and interact with the host immune system. Most experiments investigating the role of yeasts in D. melanogaster biology use the baker's yeast, Saccharomyces cerevisiae. However, S. cerevisiae is rarely found with natural populations of D. melanogaster or other Drosophila species. Moreover, the strain of S. cerevisiae used most often in D. melanogaster experiments is a commercially and industrially important strain that, to the best of our knowledge, was not isolated from flies. Since disrupting natural host-microbe interactions can have profound effects on host biology, the results from D. melanogaster-S. cerevisiae laboratory experiments may not be fully representative of host-microbe interactions in nature. In this study, we explore the D. melanogaster-yeast relationship using five different strains of yeast that were isolated from wild Drosophila populations. Ingested live yeasts have variable persistence in the D. melanogaster gastrointestinal tract. For example, Hanseniaspora occidentalis persists relative to S. cerevisiae, while Brettanomyces naardenensis is removed. Despite these differences in persistence relative to S. cerevisiae, we find that all yeasts decrease in total abundance over time. Reactive oxygen species (ROS) are an important component of the D. melanogaster anti-microbial response and can inhibit S. cerevisiae growth in the intestine. To determine if sensitivity to ROS explains the differences in yeast persistence, we measured yeast growth in the presence and absence of hydrogen peroxide. We find that B. naardenesis is completely inhibited by hydrogen peroxide, while H. occidentalis is not, which is consistent with yeast sensitivity to ROS affecting persistence within the D. melanogaster gastrointestinal tract. We also compared the feeding preference of D

  16. A Delicate Balance Between Repair and Replication Factors Regulates Recombination Between Divergent DNA Sequences in Saccharomyces cerevisiae

    PubMed Central

    Chakraborty, Ujani; George, Carolyn M.; Lyndaker, Amy M.; Alani, Eric

    2016-01-01

    Single-strand annealing (SSA) is an important homologous recombination mechanism that repairs DNA double strand breaks (DSBs) occurring between closely spaced repeat sequences. During SSA, the DSB is acted upon by exonucleases to reveal complementary sequences that anneal and are then repaired through tail clipping, DNA synthesis, and ligation steps. In baker’s yeast, the Msh DNA mismatch recognition complex and the Sgs1 helicase act to suppress SSA between divergent sequences by binding to mismatches present in heteroduplex DNA intermediates and triggering a DNA unwinding mechanism known as heteroduplex rejection. Using baker’s yeast as a model, we have identified new factors and regulatory steps in heteroduplex rejection during SSA. First we showed that Top3-Rmi1, a topoisomerase complex that interacts with Sgs1, is required for heteroduplex rejection. Second, we found that the replication processivity clamp proliferating cell nuclear antigen (PCNA) is dispensable for heteroduplex rejection, but is important for repairing mismatches formed during SSA. Third, we showed that modest overexpression of Msh6 results in a significant increase in heteroduplex rejection; this increase is due to a compromise in Msh2-Msh3 function required for the clipping of 3′ tails. Thus 3′ tail clipping during SSA is a critical regulatory step in the repair vs. rejection decision; rejection is favored before the 3′ tails are clipped. Unexpectedly, Msh6 overexpression, through interactions with PCNA, disrupted heteroduplex rejection between divergent sequences in another recombination substrate. These observations illustrate the delicate balance that exists between repair and replication factors to optimize genome stability. PMID:26680658

  17. Evaluation of Automated Yeast Identification System

    NASA Technical Reports Server (NTRS)

    McGinnis, M. R.

    1996-01-01

    One hundred and nine teleomorphic and anamorphic yeast isolates representing approximately 30 taxa were used to evaluate the accuracy of the Biolog yeast identification system. Isolates derived from nomenclatural types, environmental, and clinica isolates of known identity were tested in the Biolog system. Of the isolates tested, 81 were in the Biolog database. The system correctly identified 40, incorrectly identified 29, and was unable to identify 12. Of the 28 isolates not in the database, 18 were given names, whereas 10 were not. The Biolog yeast identification system is inadequate for the identification of yeasts originating from the environment during space program activities.

  18. Evidence for human meiotic recombination interference obtained through construction of a short tandem repeat-polymorphism linkage map of chromosome 19

    PubMed Central

    Weber, James L.; Wang, Zhenyuan; Hansen, Kevin; Stephenson, Matt; Kappel, Clarisse; Salzman, Sherry; Wilkie, Patricia J.; Keats, Bronya; Dracopoli, Nicholas C.; Brandriff, Brigitte F.; Olsen, Anne S.

    1993-01-01

    An improved linkage map for human chromosome 19 containing 35 short tandem repeat polymorphisms (STRPs) and one VNTR (D19S20) was constructed. The map included 12 new (GATA)n tetranucleotide STRPs. Although total lengths of the male (114 cM) and female (128 cM) maps were similar, at both ends of the chromosome male recombination exceeded female recombination, while in the interior portion of the map female recombination was in excess. Cosmid clones containing the STRP sequences were identified and were positioned along the chromosome by fluorescent in situ hybridization. Four rounds of careful checking and removal of genotyping errors allowed biologically relevant conclusions to be made concerning the numbers and distributions of recombination events on chromosome 19. The average numbers of recombinations per chromosome matched closely the lengths of the genetic maps computed by using the program CRIMAP. Significant numbers of chromosomes with zero, one, two, or three recombinations were detected as products of both female and male meioses. On the basis of the total number of observed pairs of recombination events in which only a single informative marker was situated between the two recombinations, a maximal estimate for the rate of meiotic STRP “gene” conversion without recombination was calculated as 3 × 10−4/meiosis. For distances up to 30 cM between recombinations, many fewer chromosomes which had undergone exactly two recombinations were observed than were expected on the basis of the assumption of independent recombination locations. This strong new evidence for human meiotic interference will help to improve the accuracy of interpretation of clinical DNA test results involving polymorphisms flanking a genetic abnormality. PMID:8213834

  19. Implications of evolutionary engineering for growth and recombinant protein production in methanol-based growth media in the yeast Pichia pastoris.

    PubMed

    Moser, Josef W; Prielhofer, Roland; Gerner, Samuel M; Graf, Alexandra B; Wilson, Iain B H; Mattanovich, Diethard; Dragosits, Martin

    2017-03-17

    Pichia pastoris is a widely used eukaryotic expression host for recombinant protein production. Adaptive laboratory evolution (ALE) has been applied in a wide range of studies in order to improve strains for biotechnological purposes. In this context, the impact of long-term carbon source adaptation in P. pastoris has not been addressed so far. Thus, we performed a pilot experiment in order to analyze the applicability and potential benefits of ALE towards improved growth and recombinant protein production in P. pastoris. Adaptation towards growth on methanol was performed in replicate cultures in rich and minimal growth medium for 250 generations. Increased growth rates on these growth media were observed at the population and single clone level. Evolved populations showed various degrees of growth advantages and trade-offs in non-evolutionary growth conditions. Genome resequencing revealed a wide variety of potential genetic targets associated with improved growth performance on methanol-based growth media. Alcohol oxidase represented a mutational hotspot since four out of seven evolved P. pastoris clones harbored mutations in this gene, resulting in decreased Aox activity, despite increased growth rates. Selected clones displayed strain-dependent variations for AOX-promoter based recombinant protein expression yield. One particularly interesting clone showed increased product titers ranging from a 2.5-fold increase in shake flask batch culture to a 1.8-fold increase during fed batch cultivation. Our data indicate a complex correlation of carbon source, growth context and recombinant protein production. While similar experiments have already shown their potential in other biotechnological areas where microbes were evolutionary engineered for improved stress resistance and growth, the current dataset encourages the analysis of the potential of ALE for improved protein production in P. pastoris on a broader scale.

  20. Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators.

    PubMed Central

    Shimomura, O; Inouye, S; Musicki, B; Kishi, Y

    1990-01-01

    Properties of a recombinant aequorin were investigated in comparison with those of natural aequorin. In chromatographic behaviour the recombinant aequorin did not match any of ten isoaequorins tested, although it was very similar to aequorin J. Its sensitivity to Ca2+ was found to be higher than that of any isoaequorin except aequorin D. The recombinant aequorin exhibited no toxicity when tested in various kinds of cells, even where samples of natural aequorin had been found to be toxic. Properties of four recombinant semi-synthetic aequorins (fch-, hcp-, e- and n-types), prepared from the recombinant apo-aequorin and synthetic analogues of coelenterazine, were approximately parallel with those of corresponding semi-synthetic aequorins prepared from natural apo-aequorin. Both recombinant e-aequorin and natural e-aequorin J luminesced with high values of the luminescence intensity ratio I400/I465, although the ratios were not pCa-dependent. The recombinant aequorin and recombinant semi-synthetic aequorins are highly suited for monitoring cellular Ca2+. PMID:2400391

  1. Genomics and the making of yeast biodiversity.

    PubMed

    Hittinger, Chris Todd; Rokas, Antonis; Bai, Feng-Yan; Boekhout, Teun; Gonçalves, Paula; Jeffries, Thomas W; Kominek, Jacek; Lachance, Marc-André; Libkind, Diego; Rosa, Carlos A; Sampaio, José Paulo; Kurtzman, Cletus P

    2015-12-01

    Yeasts are unicellular fungi that do not form fruiting bodies. Although the yeast lifestyle has evolved multiple times, most known species belong to the subphylum Saccharomycotina (syn. Hemiascomycota, hereafter yeasts). This diverse group includes the premier eukaryotic model system, Saccharomyces cerevisiae; the common human commensal and opportunistic pathogen, Candida albicans; and over 1000 other known species (with more continuing to be discovered). Yeasts are found in every biome and continent and are more genetically diverse than angiosperms or chordates. Ease of culture, simple life cycles, and small genomes (∼10-20Mbp) have made yeasts exceptional models for molecular genetics, biotechnology, and evolutionary genomics. Here we discuss recent developments in understanding the genomic underpinnings of the making of yeast biodiversity, comparing and contrasting natural and human-associated evolutionary processes. Only a tiny fraction of yeast biodiversity and metabolic capabilities has been tapped by industry and science. Expanding the taxonomic breadth of deep genomic investigations will further illuminate how genome function evolves to encode their diverse metabolisms and ecologies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Accelerating Yeast Prion Biology using Droplet Microfluidics

    NASA Astrophysics Data System (ADS)

    Ung, Lloyd; Rotem, Assaf; Jarosz, Daniel; Datta, Manoshi; Lindquist, Susan; Weitz, David

    2012-02-01

    Prions are infectious proteins in a misfolded form, that can induce normal proteins to take the misfolded state. Yeast prions are relevant, as a model of human prion diseases, and interesting from an evolutionary standpoint. Prions may also be a form of epigenetic inheritance, which allow yeast to adapt to stressful conditions at rates exceeding those of random mutations and propagate that adaptation to their offspring. Encapsulation of yeast in droplet microfluidic devices enables high-throughput measurements with single cell resolution, which would not be feasible using bulk methods. Millions of populations of yeast can be screened to obtain reliable measurements of prion induction and loss rates. The population dynamics of clonal yeast, when a fraction of the cells are prion expressing, can be elucidated. Furthermore, the mechanism by which certain strains of bacteria induce yeast to express prions in the wild can be deduced. Integrating the disparate fields of prion biology and droplet microfluidics reveals a more complete picture of how prions may be more than just diseases and play a functional role in yeast.

  3. [Groups and sources of yeasts in house dust].

    PubMed

    Glushakova, A M; Zheltikova, T M; Chernov, I Iu

    2004-01-01

    House dust contains bacteria, mycelial fungi, microarthropods, and yeasts. The house dust samples collected in 25 apartments in Moscow and the Moscow region were found to contain yeasts belonging to the genera Candida, Cryptococcus, Debaryomyces, Rhodotorula, Sporobolomyces, and Trichosporon. The most frequently encountered microorganisms were typical epiphytic yeasts, such as Cryptococcus diffluens and Rhodotorula mucilaginosa, which are capable of long-term preservation in an inactive state. The direct source of epiphytic yeasts occurring in the house dust might be the indoor plants, which were contaminated with these yeasts, albeit to a lesser degree than outdoor plants. Along with the typical epiphytic yeasts, the house dust contained the opportunistic yeast pathogens Candida catenulata, C. guillermondii, C. haemulonii, C. rugosa, and C. tropicalis, which are known as the causal agents of candidiasis. We failed to reveal any correlation between the abundance of particular yeast species in the house dust, residential characteristics, and the atopic dermatitis of the inhabitants.

  4. High-temperature ethanol production using thermotolerant yeast newly isolated from Greater Mekong Subregion.

    PubMed

    Techaparin, Atiya; Thanonkeo, Pornthap; Klanrit, Preekamol

    The application of high-potential thermotolerant yeasts is a key factor for successful ethanol production at high temperatures. Two hundred and thirty-four yeast isolates from Greater Mekong Subregion (GMS) countries, i.e., Thailand, The Lao People's Democratic Republic (Lao PDR) and Vietnam were obtained. Five thermotolerant yeasts, designated Saccharomyces cerevisiae KKU-VN8, KKU-VN20, and KKU-VN27, Pichia kudriavzevii KKU-TH33 and P. kudriavzevii KKU-TH43, demonstrated high temperature and ethanol tolerance levels up to 45°C and 13% (v/v), respectively. All five strains produced higher ethanol concentrations and exhibited greater productivities and yields than the industrial strain S. cerevisiae TISTR5606 during high-temperature fermentation at 40°C and 43°C. S. cerevisiae KKU-VN8 demonstrated the best performance for ethanol production from glucose at 37°C with an ethanol concentration of 72.69g/L, a productivity of 1.59g/L/h and a theoretical ethanol yield of 86.27%. The optimal conditions for ethanol production of S. cerevisiae KKU-VN8 from sweet sorghum juice (SSJ) at 40°C were achieved using the Box-Behnken experimental design (BBD). The maximal ethanol concentration obtained during fermentation was 89.32g/L, with a productivity of 2.48g/L/h and a theoretical ethanol yield of 96.32%. Thus, the newly isolated thermotolerant S. cerevisiae KKU-VN8 exhibits a great potential for commercial-scale ethanol production in the future. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  5. Differential effects of the mismatch repair genes MSH2 and MSH3 on homeologous recombination in Saccharomyces cerevisiae.

    PubMed

    Selva, E M; Maderazo, A B; Lahue, R S

    1997-12-01

    The products of the yeast mismatch repair genes MSH2 and MSH3 participate in the inhibition of genetic recombination between homeologous (divergent) DNA sequences. In strains deficient for these genes, homeologous recombination rates between repeated elements are elevated due to the loss of this inhibition. In this study, the effects of these mutations were further analyzed by quantitation of mitotic homeologous recombinants as crossovers, gene conversions or exceptional events in wild-type, msh2, msh3 and msh2 msh3 mutant strains. When homeologous sequences were present as a direct repeat in one orientation, crossovers and gene conversions were elevated in msh2, msh3 and msh2 msh3 strains. The increases were greater in the msh2 msh3 double mutant than in either single mutant. When the order of the homeologous sequences was reversed, the msh2 mutation again yielded increased rates of crossovers and gene conversions. However, in an msh3 strain, gene conversions occurred at higher levels but interchromosomal crossovers were not increased and intrachromosomal crossovers were reduced relative to wild type. The msh2 msh3 double mutant behaved like the msh2 single mutant in this orientation. Control strains harboring homologous duplications were largely but not entirely unaffected in mutant strains, suggesting specificity for the mismatched intermediates of homeologous recombination. In all strains, very few (< 10%) recombinants could be attributed to exceptional events. These results suggest that MSH2 and MSH3 can function differentially to control homeologous exchanges.

  6. Accumulation of linear mitochondrial DNA fragments in the nucleus shortens the chronological life span of yeast.

    PubMed

    Cheng, Xin; Ivessa, Andreas S

    2012-10-01

    Translocation of mitochondrial DNA (mtDNA) fragments to the nucleus and insertion of those fragments into nuclear DNA has been observed in several organisms ranging from yeast to plants and mammals. Disruption of specific nuclear genes by de novo insertions of mtDNA fragments has even been linked to the initiation of several human diseases. Recently, we demonstrated that baker's yeast strains with high rates of mtDNA fragments migrating to the nucleus (yme1-1 mutant) exhibit short chronological life spans (CLS). The yeast CLS is determined by the survival of non-dividing cell populations. Here, we show that lack of the non-homologous-end-joining enzyme DNA ligase IV (DNL4) can rescue the short CLS of the yme1-1 mutant. In fission yeast, DNA ligase IV has been shown to be required for the capture of mtDNA fragments during the repair of double-stranded DNA breaks in nuclear DNA. In further analyses using pulse field gel and 2D gel electrophoresis we demonstrate that linear mtDNA fragments with likely nuclear localization accumulate in the yme1-1 mutant. The accumulation of the linear mtDNA fragments in the yme1-1 mutant is suppressed when Dnl4 is absent. We propose that the linear nuclear mtDNA fragments accelerate the aging process in the yme1-1 mutant cells by possibly affecting nuclear processes including DNA replication, recombination, and repair as well as transcription of nuclear genes. We speculate further that Dnl4 protein has besides its function as a ligase also a role in DNA protection. Dnl4 protein may stabilize the linear mtDNA fragments in the nucleus by binding to their physical ends. In the absence of Dnl4 protein the linear fragments are therefore unprotected and possibly degraded by nuclear nucleases. Copyright © 2012 Elsevier GmbH. All rights reserved.

  7. Genomics and the making of yeast biodiversity

    USDA-ARS?s Scientific Manuscript database

    Yeasts are unicellular fungi that do not form fruiting bodies. Although the yeast lifestyle has evolved multiple times, most known species belong to the subphylum Saccharomycotina (syn. Hemiascomycota, hereafter yeasts). This diverse group includes the premier eukaryotic model system, Saccharomyces ...

  8. Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

    PubMed

    Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

    2014-01-01

    The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

  9. Protein glycosylation in diverse cell systems: implications for modification and analysis of recombinant proteins.

    PubMed

    Brooks, Susan A

    2006-06-01

    A major challenge for the biotechnology industry is to engineer the glycosylation pathways of expression systems to synthesize recombinant proteins with human glycosylation. Inappropriate glycosylation can result in reduced activity, limited half-life in circulation and unwanted immunogenicity. In this review, the complexities of glycosylation in human cells are explained and compared with glycosylation in bacteria, yeasts, fungi, insects, plants and nonhuman mammalian species. Key advances in the engineering of the glycosylation of expression systems are highlighted. Advances in the challenging and technically complex field of glycan analysis are also described. The emergence of a new generation of expression systems with sophisticated engineering for humanized glycosylation of glycoproteins appears to be on the horizon.

  10. 21 CFR 172.590 - Yeast-malt sprout extract.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Yeast-malt sprout extract. 172.590 Section 172.590... Substances § 172.590 Yeast-malt sprout extract. Yeast-malt sprout extract, as described in this section, may... produced by partial hydrolysis of yeast extract (derived from Saccharomyces cereviseae, Saccharomyces...

  11. Recombination Can Cause Telomere Elongations as Well as Truncations Deep within Telomeres in Wild-Type Kluyveromyces lactis Cells ▿

    PubMed Central

    Bechard, Laura H.; Jamieson, Nathan; McEachern, Michael J.

    2011-01-01

    In this study, we examined the role of recombination at the telomeres of the yeast Kluyveromyces lactis. We demonstrated that an abnormally long and mutationally tagged telomere was subject to high rates of telomere rapid deletion (TRD) that preferentially truncated the telomere to near-wild-type size. Unlike the case in Saccharomyces cerevisiae, however, there was not a great increase in TRD in meiosis. About half of mitotic TRD events were associated with deep turnover of telomeric repeats, suggesting that telomeres were often cleaved to well below normal length prior to being reextended by telomerase. Despite its high rate of TRD, the long telomere showed no increase in the rate of subtelomeric gene conversion, a highly sensitive test of telomere dysfunction. We also showed that the long telomere was subject to appreciable rates of becoming elongated substantially further through a recombinational mechanism that added additional tagged repeats. Finally, we showed that the deep turnover that occurs within normal-length telomeres was diminished in the absence of RAD52. Taken together, our results suggest that homologous recombination is a significant process acting on both abnormally long and normally sized telomeres in K. lactis. PMID:21148753

  12. Yeasts Diversity in Fermented Foods and Beverages

    NASA Astrophysics Data System (ADS)

    Tamang, Jyoti Prakash; Fleet, Graham H.

    People across the world have learnt to culture and use the essential microorganisms for production of fermented foods and alcoholic beverages. A fermented food is produced either spontaneously or by adding mixed/pure starter culture(s). Yeasts are among the essential functional microorganisms encountered in many fermented foods, and are commercially used in production of baker's yeast, breads, wine, beer, cheese, etc. In Asia, moulds are predominant followed by amylolytic and alcohol-producing yeasts in the fermentation processes, whereas in Africa, Europe, Australia and America, fermented products are prepared exclusively using bacteria or bacteria-yeasts mixed cultures. This chapter would focus on the varieties of fermented foods and alcoholic beverages produced by yeasts, their microbiology and role in food fermentation, widely used commercial starters (pilot production, molecular aspects), production technology of some common commercial fermented foods and alcoholic beverages, toxicity and food safety using yeasts cultures and socio-economy

  13. The yeast Fun30 and human SMARCAD1 chromatin remodelers promote DNA end resection

    PubMed Central

    Costelloe, Thomas; Louge, Raphaël; Tomimatsu, Nozomi; Mukherjee, Bipasha; Martini, Emmanuelle; Khadaroo, Basheer; Dubois, Kenny; Wiegant, Wouter W.; Thierry, Agnès; Burma, Sandeep; van Attikum, Haico; Llorente, Bertrand

    2012-01-01

    Several homology-dependent pathways can repair potentially lethal DNA double-strand breaks (DSBs). The first step common to all homologous recombination reactions is the 5′-3′ degradation of DSB ends that yields 3′ single-stranded DNA (ssDNA) required for loading of checkpoint and recombination proteins. The Mre11-Rad50-Xrs2/NBS1 complex and Sae2/CtIP initiate end resection while long-range resection depends on the exonuclease Exo1 or the helicase-topoisomerase complex Sgs1-Top3-Rmi1 with the endonuclease Dna21-6. DSBs occur in the context of chromatin, but how the resection machinery navigates through nucleosomal DNA is a process that is not well understood7. Here, we show that the yeast S. cerevisiae Fun30 protein and its human counterpart SMARCAD18, two poorly characterized ATP-dependent chromatin remodelers of the Snf2 ATPase family, are novel factors that are directly involved in the DSB response. Fun30 physically associates with DSB ends and directly promotes both Exo1- and Sgs1-dependent end resection through a mechanism involving its ATPase activity. The function of Fun30 in resection facilitates repair of camptothecin (CPT)-induced DNA lesions, and it becomes dispensable when Exo1 is ectopically overexpressed. Interestingly, SMARCAD1 is also recruited to DSBs and the kinetics of recruitment is similar to that of Exo1. Loss of SMARCAD1 impairs end resection, recombinational DNA repair and renders cells hypersensitive to DNA damage resulting from CPT or PARP inhibitor treatments. These findings unveil an evolutionarily conserved role for the Fun30 and SMARCAD1 chromatin remodelers in controlling end resection, homologous recombination and genome stability in the context of chromatin. PMID:22960744

  14. Genetics of Yeasts

    NASA Astrophysics Data System (ADS)

    Querol, Amparo; Fernández-Espinar, M. Teresa; Belloch, Carmela

    The use of yeasts in biotechnology processes dates back to ancient days. Before 7000 BC, beer was produced in Sumeria. Wine was made in Assyria in 3500 BC, and ancient Rome had over 250 bakeries, which were making leavened bread by 100 BC. And milk has been made into Kefyr and Koumiss in Asia for many centuries (Demain, Phaff, & Kurtzman, 1999). However, the importance of yeast in the food and beverage industries was only realized about 1860, when their role in food manufacturing became evident.

  15. 21 CFR 172.590 - Yeast-malt sprout extract.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Yeast-malt sprout extract. 172.590 Section 172.590... CONSUMPTION Flavoring Agents and Related Substances § 172.590 Yeast-malt sprout extract. Yeast-malt sprout... prescribed conditions: (a) The additive is produced by partial hydrolysis of yeast extract (derived from...

  16. 21 CFR 172.590 - Yeast-malt sprout extract.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Yeast-malt sprout extract. 172.590 Section 172.590... CONSUMPTION Flavoring Agents and Related Substances § 172.590 Yeast-malt sprout extract. Yeast-malt sprout... prescribed conditions: (a) The additive is produced by partial hydrolysis of yeast extract (derived from...

  17. 21 CFR 172.590 - Yeast-malt sprout extract.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Yeast-malt sprout extract. 172.590 Section 172.590... CONSUMPTION Flavoring Agents and Related Substances § 172.590 Yeast-malt sprout extract. Yeast-malt sprout... prescribed conditions: (a) The additive is produced by partial hydrolysis of yeast extract (derived from...

  18. 21 CFR 172.590 - Yeast-malt sprout extract.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Yeast-malt sprout extract. 172.590 Section 172.590... CONSUMPTION Flavoring Agents and Related Substances § 172.590 Yeast-malt sprout extract. Yeast-malt sprout... prescribed conditions: (a) The additive is produced by partial hydrolysis of yeast extract (derived from...

  19. Characteristics of alpha-glucosidase production from recombinant Aspergillus oryzae by membrane-surface liquid culture in comparison with various cultivation methods.

    PubMed

    Morita, Masakazu; Shimamura, Hiroko; Ishida, Natsuko; Imamura, Koreyoshi; Sakiyama, Takaharu; Nakanishi, Kazuhiro

    2004-01-01

    alpha-Glucosidase was produced using recombinant Aspergillus oryzae by membrane-surface liquid culture (MSLC), a method previously developed by the authors and the results compared with other methods, including shaking flask culture (SFC), agar-plate culture (APC), culture on urethane sponge supports (USC), and liquid surface culture (LSC) to determine possible reasons for the advantageous features of MSLC. When yeast extract was used as a nitrogen source, the amount of enzyme produced by MSLC was 5 or more times higher than those for SFC and LSC, but similar to that using APC. Enzyme production in USC was slightly lower than in MSLC and APC. Cell growth was similar irrespective of the cultivation method used. When NaNO3, a typical inorganic nitrogen source was used, enzyme production in all the cultures was lower than that using yeast extract. However, even using NaNO3, the amount of the enzyme produced by MSLC was 8 to 20 times higher than those by SFC, APC, USC, and LSC. Although cell growth using NaNO3 was similar to that for yeast extract in MSLC, it was markedly decreased in SFC, APC, and LSC. The reason for the difference in enzyme productivity for various cultivation methods using yeast extract and NaNO3 as a nitrogen source is discussed, on the basis of the experimental findings. The role of the oxygen transfer effect and gene expression levels in enzyme production were also examined.

  20. Dominant genetics using a yeast genomic library under the control of a strong inducible promoter.

    PubMed

    Ramer, S W; Elledge, S J; Davis, R W

    1992-12-01

    In Saccharomyces cerevisiae, numerous genes have been identified by selection from high-copy-number libraries based on "multicopy suppression" or other phenotypic consequences of overexpression. Although fruitful, this approach suffers from two major drawbacks. First, high copy number alone may not permit high-level expression of tightly regulated genes. Conversely, other genes expressed in proportion to dosage cannot be identified if their products are toxic at elevated levels. This work reports construction of a genomic DNA expression library for S. cerevisiae that circumvents both limitations by fusing randomly sheared genomic DNA to the strong, inducible yeast GAL1 promoter, which can be regulated by carbon source. The library obtained contains 5 x 10(7) independent recombinants, representing a breakpoint at every base in the yeast genome. This library was used to examine aberrant gene expression in S. cerevisiae. A screen for dominant activators of yeast mating response identified eight genes that activate the pathway in the absence of exogenous mating pheromone, including one previously unidentified gene. One activator was a truncated STE11 gene lacking approximately 1000 base pairs of amino-terminal coding sequence. In two different clones, the same GAL1 promoter-proximal ATG is in-frame with the coding sequence of STE11, suggesting that internal initiation of translation there results in production of a biologically active, truncated STE11 protein. Thus this library allows isolation based on dominant phenotypes of genes that might have been difficult or impossible to isolate from high-copy-number libraries.