Alcoholic fermentation of d-xylose by yeasts. [Brettanomyces naardenensis; Candida shehatae; Candida tenuis; Pachysolen tannaphilus, Pichia segobiensis; Pichia stipitis

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

Toivola, A.; Yarrow, D.; van den Bosch, E.

1984-06-01

Type strains of 200 species of yeasts able to ferment glucose and grow on xylose were screened for fermentation of D-xylose. In most of the strains tested, ethanol production was negligible. Nineteen were found to produce between 0.1 and 1.0 g of ethanol per liter. Strains of the following species produce more than 1 g of ethanol per liter in the fermentation test with 2% xylose: Brettanomyces naardenensis, Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis. Subsequent screening of these yeasts for their capacity to ferment D-cellobiose revealed that only Candida tenuis CBS 4435 was a goodmore » fermenter of both xylose and cellobiose under the test conditions used.« less

Group X hybrid histidine kinase Chk1 is dispensable for stress adaptation, host-pathogen interactions and virulence in the opportunistic yeast Candida guilliermondii.

PubMed

Navarro-Arias, María J; Dementhon, Karine; Defosse, Tatiana A; Foureau, Emilien; Courdavault, Vincent; Clastre, Marc; Le Gal, Solène; Nevez, Gilles; Le Govic, Yohann; Bouchara, Jean-Philippe; Giglioli-Guivarc'h, Nathalie; Noël, Thierry; Mora-Montes, Hector M; Papon, Nicolas

2017-09-01

Hybrid histidine kinases (HHKs) progressively emerge as prominent sensing proteins in the fungal kingdom and as ideal targets for future therapeutics. The group X HHK is of major interest, since it was demonstrated to play an important role in stress adaptation, host-pathogen interactions and virulence in some yeast and mold models, and particularly Chk1, that corresponds to the sole group X HHK in Candida albicans. In the present work, we investigated the role of Chk1 in the low-virulence species Candida guilliermondii, in order to gain insight into putative conservation of the role of group X HHK in opportunistic yeasts. We demonstrated that disruption of the corresponding gene CHK1 does not influence growth, stress tolerance, drug susceptibility, protein glycosylation or cell wall composition in C. guilliermondii. In addition, we showed that loss of CHK1 does not affect C. guilliermondii ability to interact with macrophages and to stimulate cytokine production by human peripheral blood mononuclear cells. Finally, the C. guilliermondii chk1 null mutant was found to be as virulent as the wild-type strain in the experimental model Galleria mellonella. Taken together, our results demonstrate that group X HHK function is not conserved in Candida species. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Chemical composition and in vitro antimicrobial activity of the volatile oils from Gliomastix murorum and Pichia guilliermondii, two endophytic fungi in Paris polyphylla var. yunnanensis.

PubMed

Zhao, Jianglin; Shan, Tijiang; Huang, Yongfu; Liu, Xili; Gao, Xiwu; Wang, Mingan; Jiang, Weibo; Zhou, Ligang

2009-11-01

Volatile oils were obtained by hydro-distillation from Gliomastix murorum and Pichia guilliermondii, two endophytic fungi isolated from the traditional Chinese medicinal herb Paris polyphylla var. yunnanensis. The oils were analyzed for their chemical composition by gas chromatography-mass spectrometry (GC-MS). Palmitic acid (15.5%), (E)-9-octadecenoic acid (11.6%), 6-pentyl-5,6-dihydropyran-2-one (9.7%), and (7Z,10Z)-7,10- hexadecadienoic acid (8.3%) were the major compounds of the 40 identified components in G. murorum volatile oil. 1,1,3a,7-Tetramethyl-1a,2,3,3a,4,5,6,7b-octahydro-1H-cyclopropa[a]- naphthalene (25.9%), palmitic acid (15.5%), 1-methyl-2,4-di- (prop-1-en-2-yl)-1- vinylcyclohexane (7.9%), (E)-9-octadecenoic acid (7.3%), and (9E,12E)-ethyl-9,12-octadecadienoate (5.2%) were the major compounds of the 27 identified components in P. guilliermondii volatile oil. The in vitro antimicrobial activity of the volatile oils was also investigated to evaluate their efficacy against six bacteria and one phytopathogenic fungus. The minimum inhibitory concentration (MIC) values of the volatile oils against the test bacteria ranged from 0.20 mg/mL to 1.50 mg/mL. One of the most sensitive bacteria was Xanthomonas vesicatoria with an MIC of 0.20 mg/mL and 0.40 mg/mL for G. murorum and P. guilliermondii, respectively. The mean inhibitory concentration (IC50) of the volatile oils against spore germination of Magnaporthe oryzae was 0.84 mg/mL for G. murorum and 1.56 mg/mL for P. guilliermondii. These results indicated that the volatile oils from the endophytic fungi have strong antimicrobial activity and could be a potential source of antimicrobial ingredients.

Potential spoilage yeasts in winery environments: Characterization and proteomic analysis of Trigonopsis cantarellii.

PubMed

Portugal, Cauré; Pinto, Luís; Ribeiro, Miguel; Tenorio, Carmen; Igrejas, Gilberto; Ruiz-Larrea, Fernanda

2015-10-01

Wine microbiota is complex and includes a wide diversity of yeast species. Few of them are able to survive under the restrictive conditions of dry red wines. In our study we detected and identified seven yeast species of the order Saccharomycetales that can be considered potential spoilers of wines due to physiological traits such as acidogenic metabolism and off-odor generation: Arthroascus schoenii, Candida ishiwadae, Meyerozyma guilliermondii, Pichia holstii, Pichia manshurica, Trigonopsis cantarellii, and Trigonopsis variabilis. Based on the prevalence of T. cantarellii isolates in the wine samples of our study, we further characterized this species, determined molecular and phenotypic features, and performed a proteomic analysis to identify differentially expressed proteins at mid-exponential growth phase in the presence of ethanol in the culture broth. This yeast species is shown to be able to grow in the presence of ethanol by expressing heat shock proteins (Hsp70, Hsp71) and a DNA damage-related protein (Rad24), and to be able to confer spoilage characteristics on wine. Copyright © 2015 Elsevier B.V. All rights reserved.

Reliable differentiation of Meyerozyma guilliermondii from Meyerozyma caribbica by internal transcribed spacer restriction fingerprinting.

PubMed

Romi, Wahengbam; Keisam, Santosh; Ahmed, Giasuddin; Jeyaram, Kumaraswamy

2014-02-28

Meyerozyma guilliermondii (anamorph Candida guilliermondii) and Meyerozyma caribbica (anamorph Candida fermentati) are closely related species of the genetically heterogenous M. guilliermondii complex. Conventional phenotypic methods frequently misidentify the species within this complex and also with other species of the Saccharomycotina CTG clade. Even the long-established sequencing of large subunit (LSU) rRNA gene remains ambiguous. We also faced similar problem during identification of yeast isolates of M. guilliermondii complex from indigenous bamboo shoot fermentation in North East India. There is a need for development of reliable and accurate identification methods for these closely related species because of their increasing importance as emerging infectious yeasts and associated biotechnological attributes. We targeted the highly variable internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) and identified seven restriction enzymes through in silico analysis for differentiating M. guilliermondii from M. caribbica. Fifty five isolates of M. guilliermondii complex which could not be delineated into species-specific taxonomic ranks by API 20 C AUX and LSU rRNA gene D1/D2 sequencing were subjected to ITS-restriction fragment length polymorphism (ITS-RFLP) analysis. TaqI ITS-RFLP distinctly differentiated the isolates into M. guilliermondii (47 isolates) and M. caribbica (08 isolates) with reproducible species-specific patterns similar to the in silico prediction. The reliability of this method was validated by ITS1-5.8S-ITS2 sequencing, mitochondrial DNA RFLP and electrophoretic karyotyping. We herein described a reliable ITS-RFLP method for distinct differentiation of frequently misidentified M. guilliermondii from M. caribbica. Even though in silico analysis differentiated other closely related species of M. guilliermondii complex from the above two species, it is yet to be confirmed by in vitro analysis using reference strains. This method can be used

TdKT, a new killer toxin produced by Torulaspora delbrueckii effective against wine spoilage yeasts.

PubMed

Villalba, María Leticia; Susana Sáez, Julieta; Del Monaco, Silvana; Lopes, Christian Ariel; Sangorrín, Marcela Paula

2016-01-18

Microbiological spoilage is a major concern throughout the wine industry, and control tools are limited. This paper addresses the identification and partial characterization of a new killer toxin from Torulaspora delbrueckii with potential biocontrol activity of Brettanomyces bruxellensis, Pichia guilliermondii, Pichia manshurica and Pichia membranifaciens wine spoilage. A panel of 18 different wine strains of T. delbrueckii killer yeasts was analysed, and the strain T. delbrueckii NPCC 1033 (TdKT producer) showed a significant inhibitory effect on the growth of all different spoilage yeasts evaluated. The TdKT toxin was then subjected to a partial biochemical characterization. Its estimated molecular weight was N30 kDa and it showed glucanase and chitinase enzymatic activities. The killer activity was stable between pH 4.2 and 4.8 and inactivated at temperature above 40 °C. Pustulan and chitin — but not other cell wall polysaccharides — prevented sensitive yeast cells from being killed by TdKT, suggesting that those may be the first toxin targets in the cell wall. TdKT provoked an increase in necrosis cell death after 3 h treatment and apoptotic cell death after 24 h showing time dependence in its mechanisms of action. Killer toxin extracts were active at oenological conditions, confirming their potential use as a biocontrol tool in winemaking.

Single and combined effects of acetic acid, furfural, and sugars on the growth of the pentose-fermenting yeast Meyerozyma guilliermondii.

PubMed

Perna, Michelle Dos Santos Cordeiro; Bastos, Reinaldo Gaspar; Ceccato-Antonini, Sandra Regina

2018-02-01

The tolerance of the pentose-fermenting yeast Meyerozyma guilliermondii to the inhibitors released after the biomass hydrolysis, such as acetic acid and furfural, was surveyed. We first verified the effects of acetic acid and cell concentrations and initial pH on the growth of a M. guilliermondii strain in a semi-synthetic medium containing acetic acid as the sole carbon source. Second, the single and combined effects of furfural, acetic acid, and sugars (xylose, arabinose, and glucose) on the sugar uptake, cell growth, and ethanol production were also analysed. Growth inhibition occurred in concentrations higher than 10.5 g l -1 acetic acid and initial pH 3.5. The maximum specific growth rate (µ) was 0.023 h -1 and the saturation constant (ks) was 0.75 g l -1 acetic acid. Initial cell concentration also influenced µ. Acetic acid (initial concentration 5 g l -1 ) was co-consumed with sugars even in the presence of 20 mg l -1 furfural without inhibition to the yeast growth. The yeast grew and fermented sugars in a sugar-based medium with acetic acid and furfural in concentrations much higher than those usually found in hemicellulosic hydrolysates.

Pichia insulana sp. nov., a novel cactophilic yeast from the Caribbean

PubMed Central

Ganter, Philip F.; Cardinali, Gianluigi; Boundy-Mills, Kyria

2010-01-01

A novel species of ascomycetous yeast, Pichia insulana sp. nov., is described from necrotic tissue of columnar cacti on Caribbean islands. P. insulana is closely related to and phenotypically very similar to Pichia cactophila and Pichia pseudocactophila. There are few distinctions between these taxa besides spore type, host preference and locality. Sporogenous strains of P. insulana that produce asci with four hat-shaped spores have been found only on Curaçao, whereas there was no evidence of sporogenous P. cactophila from that island. In addition, sequences of the D1/D2 fragment of the large-subunit rDNA from 12 Curaçao strains showed consistent differences from the sequences of the type strains of P. cactophila and P. pseudocactophila. The type strain of P. insulana is TSU00-106.5T (=CBS 11169T =UCD-FST 09-160T). PMID:19661524

Transcriptomic responses of the biocontrol yeast Pichia anomala to aflatoxigenic Aspergillus flavus

USDA-ARS?s Scientific Manuscript database

Pichia anomala (Wickerhamomyces anomalus) WRL-076 is a biocontrol yeast which has been shown to inhibit growth and aflatoxin production of Aspergillus flavus. The molecular mechanism of biological control was further characterized by the temporal transcriptome response of P. anomala to A. flavus in...

Biodegradation Mechanisms of Patulin in Candida guilliermondii: An iTRAQ-Based Proteomic Analysis

PubMed Central

Chen, Yong; Peng, Huai-Min; Wang, Xiao; Li, Bo-Qiang; Long, Man-Yuan; Tian, Shi-Ping

2017-01-01

Patulin, a potent mycotoxin, contaminates fruits and derived products worldwide, and is a serious health concern. Several yeast strains have shown the ability to effectively degrade patulin. However, the mechanisms of its biodegradation still remain unclear at this time. In the present study, biodegradation and involved mechanisms of patulin by an antagonistic yeast Candida guilliermondii were investigated. The results indicated that C. guilliermondii was capable of not only multiplying to a high population in medium containing patulin, but also effectively reducing patulin content in culture medium. Degradation of patulin by C. guilliermondii was dependent on the yeast cell viability, and mainly occurred inside cells. E-ascladiol was the main degradation product of patulin. An iTRAQ-based proteomic analysis revealed that the responses of C. guilliermondii to patulin were complex. A total of 30 differential proteins involved in 10 biological processes were identified, and more than two-thirds of the differential proteins were down-accumulated. Notably, a short-chain dehydrogenase (gi|190348612) was markedly induced by patulin at both the protein and mRNA levels. Our findings will provide a foundation to help enable the commercial development of an enzyme formulation for the detoxification of patulin in fruit-derived products. PMID:28208714

Rational selection of non-Saccharomyces wine yeasts for mixed starters based on ester formation and enological traits.

PubMed

Viana, Fernando; Gil, José V; Genovés, Salvador; Vallés, Salvador; Manzanares, Paloma

2008-09-01

Thirty-eight yeast strains belonging to the genera Candida, Hanseniaspora, Pichia, Torulaspora and Zygosaccharomyces were screened for ester formation on synthetic microbiological medium. The genera Hanseniaspora and Pichia stood out as the best acetate ester producers. Based on the ester profile Hanseniaspora guilliermondii 11027 and 11102, Hanseniaspora osmophila 1471 and Pichia membranifaciens 10113 and 10550 were selected for further characterization of enological traits. When growing on must H. osmophila 1471, which displayed a glucophilic nature and was able to consume more than 90% of initial must sugars, produced levels of acetic acid, medium chain fatty acids and ethyl acetate, within the ranges described for wine. On the other hand, it was found to be a strong producer of 2-phenylethyl acetate. Our data suggest that H. osmophila 1471 is a good candidate for mixed starters, although the possible interactions with S. cerevisiae deserve further research.

Screening wild yeast strains for alcohol fermentation from various fruits.

PubMed

Lee, Yeon-Ju; Choi, Yu-Ri; Lee, So-Young; Park, Jong-Tae; Shim, Jae-Hoon; Park, Kwan-Hwa; Kim, Jung-Wan

2011-03-01

Wild yeasts on the surface of various fruits including grapes were surveyed to obtain yeast strains suitable for fermenting a novel wine with higher alcohol content and supplemented with rice starch. We considered selected characteristics, such as tolerance to alcohol and osmotic pressure, capability of utilizing maltose, and starch hydrolysis. Among 637 putative yeast isolates, 115 strains exhibiting better growth in yeast-peptone-dextrose broth containing 30% dextrose, 7% alcohol, or 2% maltose were selected, as well as five α-amylase producers. Nucleotide sequence analysis of the 26S rDNA gene classified the strains into 13 species belonging to five genera; Pichia anomala was the most prevalent (41.7%), followed by Wickerhamomyces anomalus (19.2%), P. guilliermondii (15%), Candida spp. (5.8%), Kodamaea ohmeri (2.5%), and Metschnikowia spp. (2.5%). All of the α-amylase producers were Aureobasidium pullulans. Only one isolate (NK28) was identified as Saccharomyces cerevisiae. NK28 had all of the desired properties for the purpose of this study, except α-amylase production, and fermented alcohol better than commercial wine yeasts.

Genome sequence of the lignocellulose-bioconverting and xylose-fermenting yeast Pichia stipitis

Treesearch

Thomas W. Jeffries; Igor V. Grigroriev; Jane Grimwood; Jose M. Laplaza; Andrea Aerts; Asaf Salamov; Jeremy Schmutz; Erika Lindquist; Paramvir Dehal; Harris Shapiro; Yong-Su Jin; Volkmar Passoth; Paul M. Richardson

2007-01-01

Xylose is a major constituent of plant lignocellulose, and its fermentation is important for the bioconversion of plant biomass to fuels and chemicals. Pichia stipitis is a well-studied, native xylose-fermenting yeast. The mechanism and regulation of xylose metabolism in P. stipitis have been characterized and genes from P. stipitis have been used to engineer xylose...

Influence of growth conditions on adhesion of yeast Candida spp. and Pichia spp. to stainless steel surfaces.

PubMed

Tomičić, Ružica; Raspor, Peter

2017-08-01

An understanding of adhesion behavior of Candida and Pichia yeast under different environmental conditions is key to the development of effective preventive measures against biofilm-associated infection. Hence in this study we investigated the impact of growth medium and temperature on Candida and Pichia adherence using stainless steel (AISI 304) discs with different degrees of surface roughness (Ra = 25.20-961.9 nm), material typical for the food processing industry as well as medical devices. The adhesion of the yeast strains to stainless steel surfaces grown in Malt Extract broth (MEB) or YPD broth at three temperatures (7 °C, 37 °C, 43 °C for Candida strains and 7 °C, 27 °C, 32 °C for Pichia strains) was assessed by crystal violet staining. The results showed that the nutrient content of medium significantly influenced the quantity of adhered cells by the tested yeasts. Adhesion of C. albicans and C. glabrata on stainless steel surfaces were significantly higher in MEB, whereas for C. parapsilosis and C. krusei it was YPD broth. In the case with P. pijperi and P. membranifaciens, YPD broth was more effective in promoting adhesion than MEB. On the other hand, our data indicated that temperature is a very important factor which considerably affects the adhesion of these yeast. There was also significant difference in cell adhesion on all types of stainless steel surfaces for all tested yeast. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation, selection and evaluation of yeasts for use in fermentation of coffee beans by the wet process.

PubMed

de Melo Pereira, Gilberto Vinícius; Soccol, Vanete Thomaz; Pandey, Ashok; Medeiros, Adriane Bianchi Pedroni; Andrade Lara, João Marcos Rodrigues; Gollo, André Luiz; Soccol, Carlos Ricardo

2014-10-01

During wet processing of coffee, the ripe cherries are pulped, then fermented and dried. This study reports an experimental approach for target identification and selection of indigenous coffee yeasts and their potential use as starter cultures during the fermentation step of wet processing. A total of 144 yeast isolates originating from spontaneously fermenting coffee beans were identified by molecular approaches and screened for their capacity to grow under coffee-associated stress conditions. According to ITS-rRNA gene sequencing, Pichia fermentans and Pichia kluyveri were the most frequent isolates, followed by Candida Candida glabrata, quercitrusa, Saccharomyces sp., Pichia guilliermondii, Pichia caribbica and Hanseniaspora opuntiae. Nine stress-tolerant yeast strains were evaluated for their ability to produce aromatic compounds in a coffee pulp simulation medium and for their pectinolytic activity. P. fermentans YC5.2 produced the highest concentrations of flavor-active ester compounds (viz., ethyl acetate and isoamyl acetate), while Saccharomyces sp. YC9.15 was the best pectinase-producing strain. The potential impact of these selected yeast strains to promote flavor development in coffee beverages was investigated for inoculating coffee beans during wet fermentation trials at laboratory scale. Inoculation of a single culture of P. fermentans YC5.2 and co-culture of P. fermentans YC5.2 and Saccharomyces sp. YC9.15 enhanced significantly the formation of volatile aroma compounds during the fermentation process compared to un-inoculated control. The sensory analysis indicated that the flavor of coffee beverages was influenced by the starter cultures, being rated as having the higher sensory scores for fruity, buttery and fermented aroma. This demonstrates a complementary role of yeasts associated with coffee quality through the synthesis of yeast-specific volatile constituents. The yeast strains P. fermentans YC5.2 and Saccharomyces sp. YC9.15 have a great

[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.

Effect of carbon sources on the growth and ethanol production of native yeast Pichia kudriavzevii ITV-S42 isolated from sweet sorghum juice.

PubMed

Díaz-Nava, L E; Montes-Garcia, N; Domínguez, J M; Aguilar-Uscanga, M G

2017-07-01

The importance of non-Saccharomyces yeast species in fermentation processes is widely acknowledged. Within this group, Pichia kudriavzevii ITV-S42 yeast strain shows particularly desirable characteristics for ethanol production. Despite this fact, a thorough study of the metabolic and kinetic characteristics of this strain is currently unavailable. The aim of this work is to study the nutritional requirements of Pichia kudriavzevii ITV-S42 strain and the effect of different carbon sources on the growth and ethanol production. Results showed that glucose and fructose were both assimilated and fermented, achieving biomass and ethanol yields of 0.37 and 0.32 gg -1 , respectively. Glycerol was assimilated but not fermented; achieving a biomass yield of 0.88 gg -1 . Xylose and sucrose were not metabolized by the yeast strain. Finally, the use of a culture medium enriched with salts and yeast extract favored glucose consumption both for growth and ethanol production, improving ethanol tolerance reported for this genre (35 g L -1 ) to 90 g L -1 maximum ethanol concentration (over 100%). Furthermore Pichia kudriavzevii ITV-S42 maintained its fermentative capacity up to 200 g L -1 initial glucose, demonstrating that this yeast is osmotolerant.

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.

Cloning and sequence analysis of the invertase gene INV 1 from the yeast Pichia anomala.

PubMed

Pérez, J A; Rodríguez, J; Rodríguez, L; Ruiz, T

1996-02-01

A genomic library from the yeast Pichia anomala has been constructed and employed to clone the gene encoding the sucrose-hydrolysing enzyme invertase by complementation of a sucrose non-fermenting mutant of Saccharomyces cerevisiae. The cloned gene, INV1, was sequenced and found to encode a polypeptide of 550 amino acids which contained a 22 amino-acid signal sequence and ten potential glycosylation sites. The amino-acid sequence shows significant identity with other yeast invertases and also with Kluyveromyces marxianus inulinase, a yeast beta-fructofuranosidase which has a different substrate specificity. The nucleotide sequences of the 5' and 3' non-coding regions were found to contain several consensus motifs probably involved in the initiation and termination of gene transcription.

Genetic and phenotypic diversity of autochthonous cider yeasts in a cellar from Asturias.

PubMed

Pando Bedriñana, R; Querol Simón, A; Suárez Valles, B

2010-06-01

This paper analyses yeast diversity and dynamics during the production of Asturian cider. Yeasts were isolated from apple juice and at different stages of fermentation in a cellar in Villaviciosa during two Asturian cider-apple harvests. The species identified by ITS-RFLP corresponded to Hanseniaspora valbyensis, Hanseniaspora uvarum, Metschnikowia pulcherrima, Pichia guilliermondii, Candida parapsilosis, Saccharomyces cerevisiae and Saccharomyces bayanus/Saccharomyces pastorianus/Saccharomyces kudriavzevii/Saccharomyces mikatae. The species C. parapsilosis is reported here for the first time in cider. The analysis of Saccharomyces mtDNA patterns showed great diversity, sequential substitution and the presence of a small number of yeast patterns (up to 8), present in both harvests. Killer (patterns nos. 22' and 47), sensitive (patterns nos. 12, 15, 33 and 61) and neutral phenotypes were found among the S. cerevisiae isolates. The detection of beta-glucosidase activity, with arbutin as the sole carbon source, allowed two S. cerevisiae strains (patterns nos. 3' and 19') to be differentiated by means of this enzymatic activity. Yeast strains producing the killer toxin or with beta-glucosidase activity are reported for the first time in autochthonous cider yeasts. 2009 Elsevier Ltd. All rights reserved.

GC-Rich DNA Elements Enable Replication Origin Activity in the Methylotrophic Yeast Pichia pastoris

PubMed Central

Liachko, Ivan; Youngblood, Rachel A.; Tsui, Kyle; Bubb, Kerry L.; Queitsch, Christine; Raghuraman, M. K.; Nislow, Corey; Brewer, Bonita J.; Dunham, Maitreya J.

2014-01-01

The well-studied DNA replication origins of the model budding and fission yeasts are A/T-rich elements. However, unlike their yeast counterparts, both plant and metazoan origins are G/C-rich and are associated with transcription start sites. Here we show that an industrially important methylotrophic budding yeast, Pichia pastoris, simultaneously employs at least two types of replication origins—a G/C-rich type associated with transcription start sites and an A/T-rich type more reminiscent of typical budding and fission yeast origins. We used a suite of massively parallel sequencing tools to map and dissect P. pastoris origins comprehensively, to measure their replication dynamics, and to assay the global positioning of nucleosomes across the genome. Our results suggest that some functional overlap exists between promoter sequences and G/C-rich replication origins in P. pastoris and imply an evolutionary bifurcation of the modes of replication initiation. PMID:24603708

GC-rich DNA elements enable replication origin activity in the methylotrophic yeast Pichia pastoris.

PubMed

Liachko, Ivan; Youngblood, Rachel A; Tsui, Kyle; Bubb, Kerry L; Queitsch, Christine; Raghuraman, M K; Nislow, Corey; Brewer, Bonita J; Dunham, Maitreya J

2014-03-01

The well-studied DNA replication origins of the model budding and fission yeasts are A/T-rich elements. However, unlike their yeast counterparts, both plant and metazoan origins are G/C-rich and are associated with transcription start sites. Here we show that an industrially important methylotrophic budding yeast, Pichia pastoris, simultaneously employs at least two types of replication origins--a G/C-rich type associated with transcription start sites and an A/T-rich type more reminiscent of typical budding and fission yeast origins. We used a suite of massively parallel sequencing tools to map and dissect P. pastoris origins comprehensively, to measure their replication dynamics, and to assay the global positioning of nucleosomes across the genome. Our results suggest that some functional overlap exists between promoter sequences and G/C-rich replication origins in P. pastoris and imply an evolutionary bifurcation of the modes of replication initiation.

Intracellular trehalose and sorbitol synergistically promoting cell viability of a biocontrol yeast Pichia anomala for aflatoxin reduction

USDA-ARS?s Scientific Manuscript database

Pichia anomala WRL-076 was discovered by a visual screening bioassay for its antagonism against Aspergillus flavus. The yeast was shown to significantly inhibit aflatoxin production and the growth of A. flavus. P. anomala is a potential biocontrol agent for reduction of mycotoxin in the food chain...

Yeasts are essential for cocoa bean fermentation.

PubMed

Ho, Van Thi Thuy; Zhao, Jian; Fleet, Graham

2014-03-17

Cocoa beans (Theobroma cacao) are the major raw material for chocolate production and fermentation of the beans is essential for the development of chocolate flavor precursors. In this study, a novel approach was used to determine the role of yeasts in cocoa fermentation and their contribution to chocolate quality. Cocoa bean fermentations were conducted with the addition of 200ppm Natamycin to inhibit the growth of yeasts, and the resultant microbial ecology and metabolism, bean chemistry and chocolate quality were compared with those of normal (control) fermentations. The yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii and Kluyveromyces marxianus, the lactic acid bacteria Lactobacillus plantarum and Lactobacillus fermentum and the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateurii were the major species found in the control fermentation. In fermentations with the presence of Natamycin, the same bacterial species grew but yeast growth was inhibited. Physical and chemical analyses showed that beans fermented without yeasts had increased shell content, lower production of ethanol, higher alcohols and esters throughout fermentation and lesser presence of pyrazines in the roasted product. Quality tests revealed that beans fermented without yeasts were purplish-violet in color and not fully brown, and chocolate prepared from these beans tasted more acid and lacked characteristic chocolate flavor. Beans fermented with yeast growth were fully brown in color and gave chocolate with typical characters which were clearly preferred by sensory panels. Our findings demonstrate that yeast growth and activity were essential for cocoa bean fermentation and the development of chocolate characteristics. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

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.

Yeast succession in the Amazon fruit Parahancornia amapa as resource partitioning among Drosophila spp.

PubMed Central

Morais, P B; Martins, M B; Klaczko, L B; Mendonça-Hagler, L C; Hagler, A N

1995-01-01

The succession of yeasts colonizing the fallen ripe amapa fruit, from Parahancornia amapa, was examined. The occupation of the substrate depended on both the competitive interactions of yeast species, such as the production of killer toxins, and the selective dispersion by the drosophilid guild of the amapa fruit. The yeast community associated with this Amazon fruit differed from those isolated from other fruits in the same forest. The physiological profile of these yeasts was mostly restricted to the assimilation of a few simple carbon sources, mainly L-sorbose, D-glycerol, DL-lactate, cellobiose, and salicin. Common fruit-associated yeasts of the genera Kloeckera and Hanseniaspora, Candida guilliermondii, and Candida krusei colonized fruits during the first three days after the fruit fell. These yeasts were dispersed and served as food for the invader Drosophila malerkotliana. The resident flies of the Drosophila willistoni group fed selectively on patches of yeasts colonizing fruits 3 to 10 days after the fruit fell. The killer toxin-producing yeasts Pichia kluyveri var. kluyveri and Candida fructus were probably involved in the exclusion of some species during the intermediate stages of fruit deterioration. An increase in pH, inhibiting toxin activity and the depletion of simple sugars, may have promoted an increase in yeast diversity in the later stages of decomposition. The yeast succession provided a patchy environment for the drosophilids sharing this ephemeral substrate. PMID:8534092

Molecular identification and physiological characterization of yeasts, lactic acid bacteria and acetic acid bacteria isolated from heap and box cocoa bean fermentations in West Africa.

PubMed

Visintin, Simonetta; Alessandria, Valentina; Valente, Antonio; Dolci, Paola; Cocolin, Luca

2016-01-04

Yeast, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) populations, isolated from cocoa bean heap and box fermentations in West Africa, have been investigated. The fermentation dynamicswere determined by viable counts, and 106 yeasts, 105 LAB and 82 AAB isolateswere identified by means of rep-PCR grouping and sequencing of the rRNA genes. During the box fermentations, the most abundant species were Saccharomyces cerevisiae, Candida ethanolica, Lactobacillus fermentum, Lactobacillus plantarum, Acetobacter pasteurianus and Acetobacter syzygii, while S. cerevisiae, Schizosaccharomyces pombe, Hanseniaspora guilliermondii, Pichia manshurica, C. ethanolica, Hanseniaspora uvarum, Lb. fermentum, Lb. plantarum, A. pasteurianus and Acetobacter lovaniensis were identified in the heap fermentations. Furthermore, the most abundant species were molecularly characterized by analyzing the rep-PCR profiles. Strains grouped according to the type of fermentations and their progression during the transformation process were also highlighted. The yeast, LAB and AAB isolates were physiologically characterized to determine their ability to grow at different temperatures, as well as at different pH, and ethanol concentrations, tolerance to osmotic stress, and lactic acid and acetic acid inhibition. Temperatures of 45 °C, a pH of 2.5 to 3.5, 12% (v/v) ethanol and high concentrations of lactic and acetic acid have a significant influence on the growth of yeasts, LAB and AAB. Finally, the yeastswere screened for enzymatic activity, and the S. cerevisiae, H. guilliermondii, H. uvarumand C. ethanolica species were shown to possess several enzymes that may impact the quality of the final product.

Disruption of the cytochrome c gene in xylose-utilizing yeast Pichia stipitis leads to higher ethanol production

Treesearch

Nian-Qing Shi; Brian Davis; Fred Sherman; Jose Cruz; Thomas W. Jeffries

1999-01-01

The xylose-utilizing yeast, Pichia stipitis, has a complex respiratory system that contains cytochrome and non-cytochrome alternative electron transport chains in its mitochondria. To gain primary insights into the alternative respiratory pathway, a cytochrome c gene (PsCYC1, Accession No. AF030426) was cloned from wild-type P. stipitis CBS 6054 by cross-hybridization...

Pseudooutbreak of Candida guilliermondii fungemia in a neonatal intensive care unit.

PubMed

Yagupsky, P; Dagan, R; Chipman, M; Goldschmied-Reouven, A; Zmora, E; Karplus, M

1991-12-01

During a 3-week period multiple blood cultures obtained from 14 Neonatal Intensive Care Unit infants and 3 Newborn Unit babies grew Candida guilliermondii, a yeast rarely associated with infections in humans. At the time of detection of positive cultures, most infants had been hospitalized for days or weeks for serious perinatal conditions and treated with antibiotics and intravenous hyperalimentation. Two critically ill premature infants from whom the yeast was isolated were given amphotericin B. In 7 other infants, however, yeasts were recovered on the day of birth, raising the question of pseudofungemia. Exhaustive interrogation on the blood culture practices revealed that when drawing blood for a culture from small infants, "butterfly" needles were often flushed with a diluted heparin solution to prevent blood clotting. Culture of a single lot of diluted heparin vials, prepared at the hospital pharmacy and distributed to the Neonatal Intensive Care Unit and Newborn Unit shortly before the onset of the epidemic, grew between 10,000 and 15,000 colony-forming units of Candida guilliermondii/ml. Removal of contaminated heparin vials and discontinuation of heparinization of needles used for blood cultures resulted in cessation of the epidemic. The present outbreak illustrates the difficulties in recognizing pseudoinfections in sick premature infants and the importance of intensive investigation and intervention during such an outbreak.

A Candida guilliermondii lysine hyperproducer capable of elevated citric acid production.

PubMed

West, Thomas P

2016-05-01

A mutant of the yeast Candida guilliermondii ATCC 9058 exhibiting elevated citric acid production was isolated based upon its ability to overproduce lysine. This method involved the use of a solid medium containing a combination of lysine analogues to identify a mutant that produced a several-fold higher lysine level compared to its parent strain using glucose or glycerol as a carbon source. The mutant strain was also capable of producing more than a fivefold higher citric acid level on glycerol as a carbon source compared to its parent strain. It was concluded that the screening of yeast lysine hyperproducer strains could provide a rapid approach to isolate yeast citric acid hyperproducer strains.

Isolation and characterization of yeasts capable of efficient utilization of hemicellulosic hydrolyzate as the carbon source.

PubMed

Cassa-Barbosa, L A; Procópio, R E L; Matos, I T S R; Filho, S A

2015-09-28

Few yeasts have shown the potential to efficiently utilize hemicellulosic hydrolyzate as the carbon source. In this study, microorganisms isolated from the Manaus region in Amazonas, Brazil, were characterized based on their utilization of the pentoses, xylose, and arabinose. The yeasts that showed a potential to assimilate these sugars were selected for the better utilization of lignocellulosic biomass. Two hundred and thirty seven colonies of unicellular microorganisms grown on hemicellulosic hydrolyzate, xylose, arabinose, and yeast nitrogen base selective medium were analyzed. Of these, 231 colonies were subjected to sugar assimilation tests. One hundred and twenty five of these were shown to utilize hydrolyzed hemicellulose, xylose, or arabinose as the carbon source for growth. The colonies that showed the best growth (N = 57) were selected, and their internal transcribed spacer-5.8S rDNA was sequenced. The sequenced strains formed four distinct groups in the phylogenetic tree, and showed a high percentage of similarity with Meyerozyma caribbica, Meyerozyma guilliermondii, Trichosporon mycotoxinivorans, Trichosporon loubieri, Pichia kudriavzevii, Candida lignohabitans, and Candida ethanolica. The discovery of these xylose-fermenting yeasts could attract widespread interest, as these can be used in the cost-effective production of liquid fuel from lignocellulosic materials.

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.

In vivo assessing of nanometric changes on the surface of whole tomatoes that have been inoculated with Candida guilliermondii yeast.

PubMed

Infante, Esperanza Del Pilar

2014-08-01

The cuticle of plants that covers the epidermis of cells, an interface between the fruit and the environment, has an important role to play in fruit quality because it prevents water loss and mechanical damage while simultaneously forming a barrier as it prevents phytopathogens from entering the fruit. All these factors give rise to flaws in the appearance of the fruit, thus contributing to marketing problems in the form of financial loss. In the search for solutions to some of these problems, certain biocontrolling yeasts have been introduced in the last few years. In the study described here, the changes observed on the surface of the whole tomato were evaluated in vivo during the first 72 h after inoculation by spraying Candida guilliermondii yeast onto the fruit's surface. The measurements were taken on a nanometric scale using atomic force microscopy; images were created in both contact and tapping modes. The results showed diminished roughness of the surface, which could contribute to reduced phytopathogen adherence due to the thinner contact area. These results furthermore showed that a yeast biofilm was formed on the fruit which probably helps to improve water retention inside the fruit. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Utilization of xylan by yeasts and its conversion to ethanol by Pichia stipitis strains. [Cryptococcus; Pichia stipitis; Candida shehatae

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

Lee, H.; Biely, P.; Latta, R.K.

Yeasts able to grow on D-xylose were screened for the ability to hydrolyze xylan. Xylanase activity was found to be rare; a total of only 19 of more than 250 strains yielded a positive test result. The activity was localized largely in the genus Cryptococcus and in Pichia stipitis and its anamorph Candida shehatae. The ability to hydrolyze xylan was generally uncoupled from that to hydrolyze cellulose; only three of the xylan-positive strains also yielded a positive test for cellulolytic activity. Of the 19 xylanolytic strains. 2. P. stipitis CBS 5773 and CBS 5775, converted xylan into ethanol, with aboutmore » 60% of a theoretical yield computed on the basis of the amount of D-xylose present originally that could be released by acid hydrolysis.« less

Selection of yeasts with multifunctional features for application as starters in natural black table olive processing.

PubMed

Bonatsou, S; Benítez, A; Rodríguez-Gómez, F; Panagou, E Z; Arroyo-López, F N

2015-04-01

Yeasts are unicellular eukaryotic microorganisms with a great importance in the elaboration on many foods and beverages. In the last years, researches have focused their attention to determine the favourable effects that these microorganisms could provide to table olive processing. In this context, the present study assesses, at laboratory scale, the potential technological (resistance to salt, lipase, esterase and β-glucosidase activities) and probiotic (phytase activity, survival to gastric and pancreatic digestions) features of 12 yeast strains originally isolated from Greek natural black table olive fermentations. The multivariate classification analysis carried out with all information obtained (a total of 336 quantitative input data), revealed that the most promising strains (clearly discriminated from the rest of isolates) were Pichia guilliermondii Y16 (which showed overall the highest resistance to salt and simulated digestions) and Wickerhamomyces anomalus Y18 (with the overall highest technological enzymatic activities), while the rest of strains were grouped together in two clearly differentiated clusters. Thus, this work opens the possibility for the evaluation of these two selected yeasts as multifunctional starters, alone or in combination with lactic acid bacteria, in real table olive fermentations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dynamic genome-scale metabolic modeling of the yeast Pichia pastoris.

PubMed

Saitua, Francisco; Torres, Paulina; Pérez-Correa, José Ricardo; Agosin, Eduardo

2017-02-21

Pichia pastoris shows physiological advantages in producing recombinant proteins, compared to other commonly used cell factories. This yeast is mostly grown in dynamic cultivation systems, where the cell's environment is continuously changing and many variables influence process productivity. In this context, a model capable of explaining and predicting cell behavior for the rational design of bioprocesses is highly desirable. Currently, there are five genome-scale metabolic reconstructions of P. pastoris which have been used to predict extracellular cell behavior in stationary conditions. In this work, we assembled a dynamic genome-scale metabolic model for glucose-limited, aerobic cultivations of Pichia pastoris. Starting from an initial model structure for batch and fed-batch cultures, we performed pre/post regression diagnostics to ensure that model parameters were identifiable, significant and sensitive. Once identified, the non-relevant ones were iteratively fixed until a priori robust modeling structures were found for each type of cultivation. Next, the robustness of these reduced structures was confirmed by calibrating the model with new datasets, where no sensitivity, identifiability or significance problems appeared in their parameters. Afterwards, the model was validated for the prediction of batch and fed-batch dynamics in the studied conditions. Lastly, the model was employed as a case study to analyze the metabolic flux distribution of a fed-batch culture and to unravel genetic and process engineering strategies to improve the production of recombinant Human Serum Albumin (HSA). Simulation of single knock-outs indicated that deviation of carbon towards cysteine and tryptophan formation improves HSA production. The deletion of methylene tetrahydrofolate dehydrogenase could increase the HSA volumetric productivity by 630%. Moreover, given specific bioprocess limitations and strain characteristics, the model suggests that implementation of a decreasing

Saccharomyces cerevisiae and non-Saccharomyces yeasts in grape varieties of the São Francisco Valley

PubMed Central

de Ponzzes-Gomes, Camila M.P.B.S.; de Mélo, Dângelly L.F.M.; Santana, Caroline A.; Pereira, Giuliano E.; Mendonça, Michelle O.C.; Gomes, Fátima C.O.; Oliveira, Evelyn S.; Barbosa, Antonio M.; Trindade, Rita C.; Rosa, Carlos A.

2014-01-01

The aims of this work was to characterise indigenous Saccharomyces cerevisiae strains in the naturally fermented juice of grape varieties Cabernet Sauvignon, Grenache, Tempranillo, Sauvignon Blanc and Verdejo used in the São Francisco River Valley, northeastern Brazil. In this study, 155 S. cerevisiae and 60 non-Saccharomyces yeasts were isolated and identified using physiological tests and sequencing of the D1/D2 domains of the large subunit of the rRNA gene. Among the non-Saccharomyces species, Rhodotorula mucilaginosa was the most common species, followed by Pichia kudriavzevii, Candida parapsilosis, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Kloeckera apis, P. manshurica, C. orthopsilosis and C. zemplinina. The population counts of these yeasts ranged among 1.0 to 19 × 105 cfu/mL. A total of 155 isolates of S. cerevisiae were compared by mitochondrial DNA restriction analysis, and five molecular mitochondrial DNA restriction profiles were detected. Indigenous strains of S. cerevisiae isolated from grapes of the São Francisco Valley can be further tested as potential starters for wine production. PMID:25242923

Saccharomyces cerevisiae and non-Saccharomyces yeasts in grape varieties of the São Francisco Valley.

PubMed

de Ponzzes-Gomes, Camila M P B S; de Mélo, Dângelly L F M; Santana, Caroline A; Pereira, Giuliano E; Mendonça, Michelle O C; Gomes, Fátima C O; Oliveira, Evelyn S; Barbosa, Antonio M; Trindade, Rita C; Rosa, Carlos A

2014-01-01

The aims of this work was to characterise indigenous Saccharomyces cerevisiae strains in the naturally fermented juice of grape varieties Cabernet Sauvignon, Grenache, Tempranillo, Sauvignon Blanc and Verdejo used in the São Francisco River Valley, northeastern Brazil. In this study, 155 S. cerevisiae and 60 non-Saccharomyces yeasts were isolated and identified using physiological tests and sequencing of the D1/D2 domains of the large subunit of the rRNA gene. Among the non-Saccharomyces species, Rhodotorula mucilaginosa was the most common species, followed by Pichia kudriavzevii, Candida parapsilosis, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Kloeckera apis, P. manshurica, C. orthopsilosis and C. zemplinina. The population counts of these yeasts ranged among 1.0 to 19 × 10(5) cfu/mL. A total of 155 isolates of S. cerevisiae were compared by mitochondrial DNA restriction analysis, and five molecular mitochondrial DNA restriction profiles were detected. Indigenous strains of S. cerevisiae isolated from grapes of the São Francisco Valley can be further tested as potential starters for wine production.

Effects of indigenous yeasts on physicochemical and microbial properties of Korean soy sauce prepared by low-salt fermentation.

PubMed

Song, Young-Ran; Jeong, Do-Youn; Baik, Sang-Ho

2015-10-01

This study deals with understanding the effects of salt reduction on both the physicochemical and microbiological properties of soy sauce fermentation and also the application of indigenous yeast starters to compensate for undesirable changes occurring in salt-reduced processes. Fermentation was tested in situ at a Korean commercial soy sauce processing unit. Salt reduction resulted in higher acidity as well as lower pH and contents of residual sugar and ethanol. Moreover, undesired flavor characteristics, due to a lack of distinctive compounds, was observed. In addition, putrefactive Staphylococcus and Enterococcus spp. were present only during salt-reduced fermentation. To control these adverse effects, a single or mixed culture of two indigenous yeasts, Torulaspora delbrueckii and Pichia guilliermondii, producing high ethanol and 3-methyl-1-butanol, respectively, were tested. Overall, all types of yeast applications inhibited undesirable bacterial growth despite salt reduction. Of the starter cultures tested, the mixed culture resulted in a balance of more complex and richer flavors with an identical flavor profile pattern to that obtained from high salt soy sauce. Hence, this strategy using functional yeast cultures offers a technological option to manufacture salt-reduced soy sauce while preserving its typical sensory characteristics without affecting safety. Copyright © 2015 Elsevier Ltd. All rights reserved.

Two Pathways of Sphingolipid Biosynthesis Are Separated in the Yeast Pichia pastoris*

PubMed Central

Ternes, Philipp; Wobbe, Tobias; Schwarz, Marnie; Albrecht, Sandra; Feussner, Kirstin; Riezman, Isabelle; Cregg, James M.; Heinz, Ernst; Riezman, Howard; Feussner, Ivo; Warnecke, Dirk

2011-01-01

Although the yeast Saccharomyces cerevisiae has only one sphingolipid class with a head group based on phosphoinositol, the yeast Pichia pastoris as well as many other fungi have a second class, glucosylceramide, which has a glucose head group. These two sphingolipid classes are in addition distinguished by a characteristic structure of their ceramide backbones. Here, we investigate the mechanisms controlling substrate entry into the glucosylceramide branch of the pathway. By a combination of enzymatic in vitro studies and lipid analysis of genetically engineered yeast strains, we show that the ceramide synthase Bar1p occupies a key branching point in sphingolipid biosynthesis in P. pastoris. By preferring dihydroxy sphingoid bases and C16/C18 acyl-coenzyme A as substrates, Bar1p produces a structurally well defined group of ceramide species, which is the exclusive precursor for glucosylceramide biosynthesis. Correlating with the absence of glucosylceramide in this yeast, a gene encoding Bar1p is missing in S. cerevisiae. We could not successfully investigate the second ceramide synthase in P. pastoris that is orthologous to S. cerevisiae Lag1p/Lac1p. By analyzing the ceramide and glucosylceramide species in a collection of P. pastoris knock-out strains in which individual genes encoding enzymes involved in glucosylceramide biosynthesis were systematically deleted, we show that the ceramide species produced by Bar1p have to be modified by two additional enzymes, sphingolipid Δ4-desaturase and fatty acid α-hydroxylase, before the final addition of the glucose head group by the glucosylceramide synthase. Together, this set of four enzymes specifically defines the pathway leading to glucosylceramide biosynthesis. PMID:21303904

Secretion of non-cell-bound phytase by the yeast Pichia kudriavzevii TY13.

PubMed

Hellström, A; Qvirist, L; Svanberg, U; Veide Vilg, J; Andlid, T

2015-05-01

Mineral deficiencies cause several health problems in the world, especially for populations consuming cereal-based diets rich in the anti-nutrient phytate. Our aim was to characterize the phytate-degrading capacity of the yeast Pichia kudriavzevii TY13 and its secretion of phytase. The phytase activity in cell-free supernatants from cultures with 100% intact cells was 35-190 mU ml(-1) depending on the media. The Km was 0.28 mmol l(-1) and the specific phytase activity 0.32 U mg(-1) total protein. The phytase activity and secretion of extracellular non-cell-bound phytase was affected by the medium phosphate concentrations. Further, addition of yeast extract had a clearly inducing effect, resulting in over 60% of the cultures total phytase activity as non-cell-bound. Our study reveals that it is possible to achieve high extracellular phytase activity from the yeast P. kudriavzevii TY13 by proper composition of the growth medium. TY13 could be a promising future starter culture for fermented foods with improved mineral bioavailability. Using strains that secrete phytase to the food matrix may significantly improve the phytate degradation by facilitating the enzyme-to-substrate interaction. The secreted non-cell-bound phytase activities by TY13 could further be advantageous in industrial production of phytase. © 2015 The Society for Applied Microbiology.

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris.

PubMed

Grillitsch, Karlheinz; Tarazona, Pablo; Klug, Lisa; Wriessnegger, Tamara; Zellnig, Günther; Leitner, Erich; Feussner, Ivo; Daum, Günther

2014-07-01

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production. Copyright © 2014 Elsevier B.V. All rights reserved.

[Posttraumatic primary cutaneous aspergillosis with Candida guilliermondii infection in a healthy host].

PubMed

Türkşen, Zeynep; Yağcı, Server; Karadağ, Ayşe Serap; Tezer, Ayla; Taner, Omer Faruk; Tekin, Fatih; Arıkan Akdağlı, Sevtap

2010-10-01

Opportunistic fungal infections are usually seen in immunocompromised patients. While Candida is the most prevalent agent in such infections, Aspergillus is at the second order. Primary cutaneous aspergillosis is most common in immunocompromised patients but can rarely be seen in healthy hosts as well. We report a case of posttraumatic primary cutaneous aspergillosis and Candida guilliermondii coinfection in a 70-years-old healthy man. The patient had an ulcerous lesion which developed in the site of a trauma on the middle finger of the right hand. Histopathological examination of the biopsy specimens revealed septate hyphae with dichotomous branching small circular blastospores. The cultures of the biopsy specimen yielded yellow-green colored, granular mold colonies and creamy white yeast colonies. Microscopic examination of the lactophenol cotton blue stained mold colonies indicated long conidiophores with vesicles surrounded by uniseriate phialides, compatible with Aspergillus flavus. Yeast colonies were identified as Candida guilliermondii by ID32C (BioMerieux, France) and by their microscopical morphology detected in corn meal-Tween 80 agar incubated at 25°C for 72 hours. The patient was treated properly with surgical debridement and itraconazole therapy. Since the immune system is compressed as a consequence of aging, cutaneous opportunistic fungal infections should be considered in the differential diagnosis of posttraumatic necrotic ulcers and black eschar in aged patients.

21 CFR 173.160 - Candida guilliermondii.

Code of Federal Regulations, 2010 CFR

2010-04-01

... guilliermondii may be safely used as the organism for fermentation production of citric acid in accordance with... guilliermondii and its concomitant metabolites produced during the fermentation process. (b)(1) The nonpathogenic... pure culture in the fermentation process for the production of citric acid using an acceptable aqueous...

Microbiological Characteristics of Wild Yeast Strain Pichia anomala Y197-13 for Brewing Makgeolli

PubMed Central

Kim, Hye Ryun; Kim, Jae-Ho; Bai, Dong-Hoon

2013-01-01

Makgeolli is a traditional cloudy-white Korean rice wine with an alcohol content of 6~7%. The present study investigated the morphological characteristics, carbon-utilizing ability, fatty acid composition, alcohol resistance, glucose tolerance, and flocculence of Saccharomyces cerevisiae Y98-5 and Pichia anomala Y197-13, non-S. cerevisiae isolated from Nuruk, which is used in brewing Makgeolli. Similar morphological characteristics were observed for both isolated wild yeast strains; and the carbon source assimilation of Y197-13 differed from that of other P. anomala strains. Strain Y197-13 was negative for D-trehalose, mannitol, arbutin, I-erythritol, and succinic acid. The major cellular fatty acids of strain Y197-13 included C18:2n6c (33.94%), C18:1n9c (26.97%) and C16:0 (20.57%). Strain Y197-13 was Crabtree-negative, with 60% cell viability at 12% (v/v) ethanol. The flocculation level of strain Y197-13 was 8.38%, resulting in its classification as a non-flocculent yeast. PMID:24198668

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.

Intracellular trehalose and sorbitol synergistically promoting cell viability of a biocontrol yeast, Pichia anomala, for aflatoxin reduction.

PubMed

Hua, Sui Sheng T; Hernlem, Bradley J; Yokoyama, Wallace; Sarreal, Siov Bouy L

2015-05-01

Pichia anomala (Wickerhamomyces anomalus) WRL-076 was discovered by a visual screening bioassay for its antagonism against Aspergillus flavus. The yeast was shown to significantly inhibit aflatoxin production and the growth of A. flavus. P. anomala is a potential biocontrol agent for reduction of aflatoxin in the food chain. Maintaining the viability of biocontrol agents in formulated products is a great challenge for commercial applications. Four media, NYG, NYGS, NYGT and NYGST are described which support good growth of yeast cells and were tested as storage formulations. Post growth supplement of 5 % trehalose to NYGST resulted in 83 % viable yeast cells after 12 months in cold storage. Intracellular sorbitol and trehalose concentrations were determined by HPLC analysis at the beginning of the storage and at the end of 12 month. Correlation of cell viability to both trehalose and sorbitol suggested a synergistic effect. Bonferroni (Dunn) t Test, Tukey's Studentized Range (HSD) Test and Duncan's Multiple Range Test, all showed that yeast cell viability in samples with both intracellular trehalose and sorbitol were significantly higher than those with either or none, at a 95 % confidence level. DiBAC4(5) and CFDA-AM were used as the membrane integrity fluorescent stains to create a two-color vital staining scheme with red and green fluorescence, respectively. Yeast cells stored in formulations NYG and NYGS with no detectable trehalose, displayed mostly red fluorescence. Yeast cells in NYGST+5T showed mostly green fluorescence.

Normal yeast flora of the upper digestive tract of some wild columbids

USGS Publications Warehouse

Kocan, R.M.; Hasenclever, H.F.

1972-01-01

Seven species of pigeons and doves were cultured for yeasts in the upper digestive tract. The following list gives the isolation rate for each columbid species and the yeasts cultured from them: feral pigeon Columba Livia (Gmelin) 95% -Candida albicans (Robin) Berkhout, C. tropicalis (Castellani) Berkhout, C. krusei (Cast.) Berkhout, C. guilliermondii (Cast.) Langeron et Guerra, Torulopsis glabrata (Anderson) Lodder et De Vries, Saccharomyces telluris Van der Walt, and Geotrichum sp.; white-crowned pigeon (C. leucocephala Linnaeus) 56% -- S. telluris; mourning dove (Zenaidura rnacroura Linnaeus) 24% -- C. albicans, C. tropicalis, C. guilliermondii, and Geotrichurn sp.; passerine ground dove (Collumbigallina passerina Linnaeus) 20% -- C. parapsilosis (Ashford) Langeron et Talice, Kloeckera apiculata (Reess Emend. Klocker) Janke; zenaida dove (Zenaida aurita Temminck) 16% -- C. albicans, C. guilliermondii, and T. glabrata; one moustasche dove (Geotrygon mystacea Gosse) -- C. guillierrnondii; ringed turtle dove (Streptopelia rizoria Linnaeus) 14% -- C. albicans and Geotrichurn sp. No signs of disease could be seen in the 139 birds that were examined, and it was concluded that these yeasts comprise a part of the columbid's normal microbial flora.

Biodegradation and detoxification of aliphatic and aromatic hydrocarbons by new yeast strains.

PubMed

Hashem, Mohamed; Alamri, Saad A; Al-Zomyh, Sharefah S A A; Alrumman, Sulaiman A

2018-04-30

Seeking new efficient hydrocarbon-degrading yeast stains was the main goal of this study. Because microorganisms are greatly affected by the environmental factors, the biodegradation potentiality of the microorganisms varies from climatic area to another. This induces research to develop and optimize the endemic organisms in bioremediation technology. In this study, 67 yeast strains were tested for their growth potentiality on both aliphatic and aromatic hydrocarbons. The most efficient six strains were identified using sequence analysis of the variable D1/D2 domain of the large subunit 26S ribosomal DNA. The identity of these strains was confirmed as Yamadazyma mexicana KKUY-0160, Rhodotorula taiwanensis KKUY-0162, Pichia kluyveri KKUY-0163, Rhodotorula ingeniosa KKUY-0170, Candida pseudointermedia KKUY-0192 and Meyerozyma guilliermondii KKUY-0214. These species are approved for their ability to degrade both aliphatic and aromatic hydrocarbons for the first time in this study. Although, all of them were able to utilize and grow on both hydrocarbons, Rhodotorula taiwanensis KKUY-0162 emerged as the best degrader of octane, and Rhodotorula ingeniosa KKUY-170 was the best degrader of pyrene. GC-MS analysis approved the presence of many chemical compounds that could be transitional or secondary metabolites during the utilization of the hydrocarbons. Our results recommend the application of these yeast species on large scale to approve their efficiency in bioremediation of oil-contamination of the environment. Using these yeasts, either individually or in consortia, could offer a practical solution for aquatic or soil contamination with the crude oil and its derivatives in situ. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast Pichia pastoris

PubMed Central

Hwang, Jung-Shan; Yamada, Kazunori; Honda, Ayae; Nakade, Kohji; Ishihama, Akira

2000-01-01

Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris. Simultaneous expression of all three P proteins in the yeast P. pastoris was achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P. pastoris cell lysate, we partially purified a 3P complex by Ni2+-agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris. PMID:10756019

SEM study of the effects of bacteria and yeasts on wood decay by brown and white-rot fungi. [Enterobacter, Cryptococcus Pichia, and Saccharomyces

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

Blanchette, R.A.; Shaw, C.G.; Cohen, A.L.

The scanning electron microscope was used to 1) examine the associations among microorganisms during wood decay and 2) observe the effect of these organisms on degradation of cell wall components. Bacteria (Enterobacter) and yeasts (Cryptococcus Pichia, and Saccharomyces) were found to have a mutualistic association with a white-rot fungus during decay of coniferous wood. Coriolus (Polyporus versicolar) degraded cell wall components in a typical ''erosion trough'' manner (i.e., by lysing zones around fungal hyphae). Bacteria and yeasts were seen only in these lysed zones. Typical gross decay patterns caused by the white-rot fungus were unaltered by bacteria and yeasts. Themore » SEM study suggests that the decay process is enhanced when these organisms are associated. In contrast, the same bacteria and yeasts were inhibitory when combined with a brown-rot fungus.« less

Diversity and killer activity of yeasts in Malaysian fermented food samples.

PubMed

Lim, S L; Tay, S T

2011-08-01

The biodiversity and the killer activity of yeasts isolated from various types of fermented food in Malaysia were investigated in this study. Of 252 yeasts isolated from 48 fermented food samples in this study, 19 yeast species were identified based on sequence analysis of the ITS1-5.8S-ITS2 partial fragments of the yeasts. A total of 29 (11.5%) of the yeast isolates demonstrated killer activity to at least one Candida species tested in this study; including 22 isolates of Trichosporon asahii, 4 isolates of Pichia anomala, and one isolate each of Pichia norvegensis, Pichia fermentans and Issatchenkia orientalis, respectively. The presence of killer yeasts reflects antagonism that occurs during microbial interaction in the fermented food, whereby certain yeasts produce killer toxins and possibly other toxic substances in competition for limited nutrients and space. The anti-Candida activity demonstrated by killer yeasts in this study should be further explored for development of alternative therapy against candidiasis.

Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on L-arabinose and D-xylose.

PubMed

Knoshaug, Eric P; Vidgren, Virve; Magalhães, Frederico; Jarvis, Eric E; Franden, Mary Ann; Zhang, Min; Singh, Arjun

2015-10-01

Genes encoding L-arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on L-arabinose was dependent on a functioning L-arabinose transporter, or by screening a differential display library, respectively. These transporters also transport D-xylose and were designated KmAXT1 (arabinose-xylose transporter) and PgAXT1, respectively. Transport assays using L-arabinose showed that KmAxt1p has K(m) 263 mM and V(max) 57 nM/mg/min, and PgAxt1p has K(m) 0.13 mM and V(max) 18 nM/mg/min. Glucose, galactose and xylose significantly inhibit L-arabinose transport by both transporters. Transport assays using D-xylose showed that KmAxt1p has K(m) 27 mM and V(max) 3.8 nM/mg/min, and PgAxt1p has K(m) 65 mM and V(max) 8.7 nM/mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed K(m) 371 mM and V(max) 341 nM/mg/min for L-arabinose, and K(m) 25 mM and V(max) 76 nM/mg/min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both L-arabinose and D-xylose, one scenario for the complete usage of biomass-derived pentose sugars would require only the low-affinity, high-throughput transporter Gal2p and one additional high-affinity general pentose transporter, rather than dedicated D-xylose or L-arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering. Copyright © 2015 John Wiley & Sons, Ltd.

Wild Grape-Associated Yeasts as Promising Biocontrol Agents against Vitis vinifera Fungal Pathogens.

PubMed

Cordero-Bueso, Gustavo; Mangieri, Nicola; Maghradze, David; Foschino, Roberto; Valdetara, Federica; Cantoral, Jesús M; Vigentini, Ileana

2017-01-01

The increasing level of hazardous residues in the environment and food chains has led the European Union to restrict the use of chemical fungicides. Thus, exploiting new natural antagonistic microorganisms against fungal diseases could serve the agricultural production to reduce pre- and post-harvest losses, to boost safer practices for workers and to protect the consumers' health. The main aim of this work was to evaluate the antagonistic potential of epiphytic yeasts against Botrytis cinerea, Aspergillus carbonarius , and Penicillium expansum pathogen species. In particular, yeast isolation was carried out from grape berries of Vitis vinifera ssp sylvestris populations, of the Eurasian area, and V. vinifera ssp vinifera cultivars from three different farming systems (organic, biodynamic, and conventional). Strains able to inhibit or slow the growth of pathogens were selected by in vitro and in vivo experiments. The most effective antagonist yeast strains were subsequently assayed for their capability to colonize the grape berries. Finally, possible modes of action, such as nutrients and space competition, iron depletion, cell wall degrading enzymes, diffusible and volatile antimicrobial compounds, and biofilm formation, were investigated as well. Two hundred and thirty-one yeast strains belonging to 26 different species were isolated; 20 of them, ascribed to eight species, showed antagonistic action against all molds. Yeasts isolated from V. vinifera ssp sylvestris were more effective (up to 50%) against B. cinerea rather than those isolated from V. vinifera ssp vinifera. Six strains, all isolated from wild vines, belonging to four species ( Meyerozyma guilliermondii, Hanseniaspora uvarum, Hanseniaspora clermontiae , and Pichia kluyveri ) revealed one or more phenotypical characteristics associated to the analyzed modes of antagonistic action.

Wild Grape-Associated Yeasts as Promising Biocontrol Agents against Vitis vinifera Fungal Pathogens

PubMed Central

Cordero-Bueso, Gustavo; Mangieri, Nicola; Maghradze, David; Foschino, Roberto; Valdetara, Federica; Cantoral, Jesús M.; Vigentini, Ileana

2017-01-01

The increasing level of hazardous residues in the environment and food chains has led the European Union to restrict the use of chemical fungicides. Thus, exploiting new natural antagonistic microorganisms against fungal diseases could serve the agricultural production to reduce pre- and post-harvest losses, to boost safer practices for workers and to protect the consumers' health. The main aim of this work was to evaluate the antagonistic potential of epiphytic yeasts against Botrytis cinerea, Aspergillus carbonarius, and Penicillium expansum pathogen species. In particular, yeast isolation was carried out from grape berries of Vitis vinifera ssp sylvestris populations, of the Eurasian area, and V. vinifera ssp vinifera cultivars from three different farming systems (organic, biodynamic, and conventional). Strains able to inhibit or slow the growth of pathogens were selected by in vitro and in vivo experiments. The most effective antagonist yeast strains were subsequently assayed for their capability to colonize the grape berries. Finally, possible modes of action, such as nutrients and space competition, iron depletion, cell wall degrading enzymes, diffusible and volatile antimicrobial compounds, and biofilm formation, were investigated as well. Two hundred and thirty-one yeast strains belonging to 26 different species were isolated; 20 of them, ascribed to eight species, showed antagonistic action against all molds. Yeasts isolated from V. vinifera ssp sylvestris were more effective (up to 50%) against B. cinerea rather than those isolated from V. vinifera ssp vinifera. Six strains, all isolated from wild vines, belonging to four species (Meyerozyma guilliermondii, Hanseniaspora uvarum, Hanseniaspora clermontiae, and Pichia kluyveri) revealed one or more phenotypical characteristics associated to the analyzed modes of antagonistic action. PMID:29163377

Evaluating Potential Risks of Food Allergy and Toxicity of Soy Leghemoglobin Expressed in Pichia pastoris

PubMed Central

Jin, Yuan; He, Xiaoyun; Andoh‐Kumi, Kwame; Fraser, Rachel Z.; Lu, Mei

2017-01-01

Scope The Soybean (Glycine max) leghemoglobin c2 (LegHb) gene was introduced into Pichia pastoris yeast for sustainable production of a heme‐carrying protein, for organoleptic use in plant‐based meat. The potential allergenicity and toxicity of LegHb and 17 Pichia host‐proteins each representing ≥1% of total protein in production batches are evaluated by literature review, bioinformatics sequence comparisons to known allergens or toxins, and in vitro pepsin digestion. Methods and results Literature searches found no evidence of allergenicity or toxicity for these proteins. There are no significant sequence matches of LegHb to known allergens or toxins. Eleven Pichia proteins have modest identity matches to minor environmental allergens and 13 Pichia proteins have significant matches to proteins from toxic sources. Yet the matched allergens and toxins have similar matches to proteins from the commonly consumed yeast Saccharomyces cerevisiae, without evidence of food allergy or toxicity. The demonstrated history of safe use indicates additional tests for allergenicity and toxicity are not needed. The LegHb and Pichia sp. proteins were rapidly digested by pepsin at pH 2. Conclusion These results demonstrate that foods containing recombinant soy LegHb produced in Pichia sp. are unlikely to present an unacceptable risk of allergenicity or toxicity to consumers. PMID:28921896

Expression and purification of soluble porcine CTLA-4 in yeast Pichia pastoris

PubMed Central

Peraino, Jaclyn; Zhang, Huiping; Hermanrud, Christina E.; Li, Guoying; Sachs, David H.; Huang, Christene A.; Wang, Zhirui

2012-01-01

Co-stimulation blockade can be used to modulate the immune response for induction of organ transplantation tolerance, treatment of autoimmune disease as well as cancer treatment. Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4), also known as CD152, is an important co-stimulatory molecule which serves as a negative regulator for T cell proliferation and differentiation. CTLA-4/CD28-CD80/CD86 pathway is a critical co-stimulatory pathway for adaptive immune response. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for CD80 and CD86. MGH MHC-defined miniature swine provide a unique large animal model useful for preclinical studies of transplantation tolerance and immune regulation. In this study, we have expressed the codon-optimized soluble porcine CTLA-4 in the yeast Pichia pastoris system. The secreted porcine CTLA-4 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50HQ. Glycosylation analysis using PNGase F demonstrated the N-linked glycosylation on Pichia pastoris expressed soluble porcine CTLA-4. To improve the expression level and facilitate the downstream purification we mutated the two potential N-linked glycosylation sites with non-polarized alanines by site-directed mutagenesis. Removal of the two N-glycosylation sites significantly improved the production level from ~2 mg/L to ~8 mg/L. Biotinylated glycosylated and non-N-glycosylated soluble porcine CTLA-4 both bind to a porcine CD80-expressing B-cell lymphoma cell line (KD = 13 nM) and competitively inhibit the binding of an anti-CD80 monoclonal antibody. The availability of soluble porcine CTLA-4, especially the non-N-glycosylated CTLA-4, will provide a very valuable tool for assessing co-stimulatory blockade treatment for translational studies in the clinically relevant porcine model. PMID:22326797

Transcriptional Regulation of Aerobic Metabolism in Pichia pastoris Fermentation

PubMed Central

Zhang, Biao; Li, Baizhi; Chen, Dai; Zong, Jie; Sun, Fei; Qu, Huixin; Liang, Chongyang

2016-01-01

In this study, we investigated the classical fermentation process in Pichia pastoris based on transcriptomics. We utilized methanol in pichia yeast cell as the focus of our study, based on two key steps: limiting carbon source replacement (from glycerol to methonal) and fermentative production of exogenous proteins. In the former, the core differential genes in co-expression net point to initiation of aerobic metabolism and generation of peroxisome. The transmission electron microscope (TEM) results showed that yeast gradually adapted methanol induction to increased cell volume, and decreased density, via large number of peroxisomes. In the fermentative production of exogenous proteins, the Gene Ontology (GO) mapping results show that PAS_chr2-1_0582 played a vital role in regulating aerobic metabolic drift. In order to confirm the above results, we disrupted PAS_chr2-1_0582 by homologous recombination. Alcohol consumption was equivalent to one fifth of the normal control, and fewer peroxisomes were observed in Δ0582 strain following methanol induction. In this study we determined the important core genes and GO terms regulating aerobic metabolic drift in Pichia, as well as developing new perspectives for the continued development within this field. PMID:27537181

Pichia stipitis genomics, transcriptomics, and gene clusters

Treesearch

Thomas W. Jeffries; Jennifer R. Headman Van Vleet

2009-01-01

Genome sequencing and subsequent global gene expression studies have advanced our understanding of the lignocellulose-fermenting yeast Pichia stipitis. These studies have provided an insight into its central carbon metabolism, and analysis of its genome has revealed numerous functional gene clusters and tandem repeats. Specialized physiological traits are often the...

From grape berries to wine: population dynamics of cultivable yeasts associated to "Nero di Troia" autochthonous grape cultivar.

PubMed

Garofalo, Carmela; Tristezza, Mariana; Grieco, Francesco; Spano, Giuseppe; Capozzi, Vittorio

2016-04-01

The aim of this work was to study the biodiversity of yeasts isolated from the autochthonous grape variety called "Uva di Troia", monitoring the natural diversity from the grape berries to wine during a vintage. Grapes were collected in vineyards from two different geographical areas and spontaneous alcoholic fermentations (AFs) were performed. Different restriction profiles of ITS-5.8S rDNA region, corresponding to Saccharomyces cerevisiae, Issatchenkia orientalis, Metschnikowia pulcherrima, Hanseniaspora uvarum, Candida zemplinina, Issatchenkia terricola, Kluyveromyces thermotolerans, Torulaspora delbrueckii, Metschnikowia chrysoperlae, Pichia fermentans, Hanseniaspora opuntiae and Hanseniaspora guilliermondii, were observed. The yeast occurrences varied significantly from both grape berries and grape juices, depending on the sampling location. Furthermore, samples collected at the end of AF revealed the great predominance of Saccharomyces cerevisiae, with a high intraspecific biodiversity. This is the first report on the population dynamics of 'cultivable' microbiota diversity of "Uva di Troia" cultivar from the grape to the corresponding wine ("Nero di Troia"), and more general for Southern Italian oenological productions, allowing us to provide the basis for an improved management of wine yeasts (with both non-Saccharomyces and Saccharomyces) for the production of typical wines with desired unique traits. A certain geographical-dependent variability has been reported, suggesting the need of local based formulation for autochthonous starter cultures, especially in the proportion of the different species/strains in the design of mixed microbial preparations.

Storage lipids of yeasts: a survey of nonpolar lipid metabolism in Saccharomyces cerevisiae, Pichia pastoris, and Yarrowia lipolytica.

PubMed

Koch, Barbara; Schmidt, Claudia; Daum, Günther

2014-09-01

Biosynthesis and storage of nonpolar lipids, such as triacylglycerols (TG) and steryl esters (SE), have gained much interest during the last decades because defects in these processes are related to severe human diseases. The baker's yeast Saccharomyces cerevisiae has become a valuable tool to study eukaryotic lipid metabolism because this single-cell microorganism harbors many enzymes and pathways with counterparts in mammalian cells. In this article, we will review aspects of TG and SE metabolism and turnover in the yeast that have been known for a long time and combine them with new perceptions of nonpolar lipid research. We will provide a detailed insight into the mechanisms of nonpolar lipid synthesis, storage, mobilization, and degradation in the yeast S. cerevisiae. The central role of lipid droplets (LD) in these processes will be addressed with emphasis on the prevailing view that this compartment is more than only a depot for TG and SE. Dynamic and interactive aspects of LD with other organelles will be discussed. Results obtained with S. cerevisiae will be complemented by recent investigations of nonpolar lipid research with Yarrowia lipolytica and Pichia pastoris. Altogether, this review article provides a comprehensive view of nonpolar lipid research in yeast. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Evaluating Potential Risks of Food Allergy and Toxicity of Soy Leghemoglobin Expressed in Pichia pastoris.

PubMed

Jin, Yuan; He, Xiaoyun; Andoh-Kumi, Kwame; Fraser, Rachel Z; Lu, Mei; Goodman, Richard E

2018-01-01

The Soybean (Glycine max) leghemoglobin c2 (LegHb) gene was introduced into Pichia pastoris yeast for sustainable production of a heme-carrying protein, for organoleptic use in plant-based meat. The potential allergenicity and toxicity of LegHb and 17 Pichia host-proteins each representing ≥1% of total protein in production batches are evaluated by literature review, bioinformatics sequence comparisons to known allergens or toxins, and in vitro pepsin digestion. Literature searches found no evidence of allergenicity or toxicity for these proteins. There are no significant sequence matches of LegHb to known allergens or toxins. Eleven Pichia proteins have modest identity matches to minor environmental allergens and 13 Pichia proteins have significant matches to proteins from toxic sources. Yet the matched allergens and toxins have similar matches to proteins from the commonly consumed yeast Saccharomyces cerevisiae, without evidence of food allergy or toxicity. The demonstrated history of safe use indicates additional tests for allergenicity and toxicity are not needed. The LegHb and Pichia sp. proteins were rapidly digested by pepsin at pH 2. These results demonstrate that foods containing recombinant soy LegHb produced in Pichia sp. are unlikely to present an unacceptable risk of allergenicity or toxicity to consumers. © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purification and characterization of a novel extracellular inulinase from a new yeast species Candida kutaonensis sp. nov. KRF1(T).

PubMed

Yuan, Bo; Hu, Nan; Sun, Juan; Wang, Shi-An; Li, Fu-Li

2012-12-01

A novel extracellular exoinulinase was purified and characterized from a new yeast strain KRF1(T), and the gene encoding the enzyme was successfully cloned. The enzyme was stable at low pH between 3.0 and 6.5. The K (m) and V (max) values of the purified enzyme for inulin were 2.3 mg/mL and 4.8 mg/min, respectively. The optimum temperature of the inulinase was 50 °C, and the enzyme remained 78 % of activity at 60 °C for 2 h. The inulinase showed an amino acid sequence identity of 58 % to its closest homolog in Meyerozyma (Pichia) guilliermondii. In the secondary structure, the domain G (VMEVH) of the enzyme contained three unique residues (V, M, and H). Compared with previously reported inulinases, the enzyme from strain KRF1(T) displayed strong acid resistance, notable thermostability, and high affinity for the substrate of inulin. Based on sequence analysis of the 26S rDNA D1/D2 domain and phenotypic characterization, the yeast strain KRF1(T) was found to represent a novel anamorphic, ascomycetous yeast species. A complete description of the species is given and the name Candida kutaonensis sp. nov (type strain = KRF1(T) = AS 2.4027(T) = CBS 11388(T)) is proposed.

Expression and characterization of human CB1 cannabinoid receptor in methylotrophic yeast Pichia pastoris.

PubMed

Kim, Tae-Kang; Zhang, Rundong; Feng, Wenke; Cai, Jian; Pierce, William; Song, Zhao-Hui

2005-03-01

For the purpose of purification and structural characterization, the CB1 cannabinoid receptors are expressed in methylotrophic yeast Pichia pastoris. The expression plasmid was constructed in which the CB1 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase I gene. To facilitate easy detection and purification, a FLAG tag was introduced at the N-terminal, a c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB1. In membrane preparations of CB1 gene transformed yeast cells, Western blot analysis detected the expression of CB1 proteins. Radioligand binding assays demonstrated that the tagged CB1 receptors expressed in P. pastoris have a pharmacological profile similar to that of the untagged CB1 receptors expressed in mammalian systems. Furthermore, the tagged CB1 receptors were purified by anti-FLAG M2 affinity chromatography and the identity of the purified CB1 receptor proteins was confirmed by Western blot analysis. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions of purified CB1 preparations detected 17 peptide fragments derived from the CB1, thus further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope tagged, functional CB1 cannabinoid receptors can be expressed in P. pastoris for purification and mass spectrometry characterization.

Performance evaluation of Pichia kluyveri, Kluyveromyces marxianus and Saccharomyces cerevisiae in industrial tequila fermentation.

PubMed

Amaya-Delgado, L; Herrera-López, E J; Arrizon, Javier; Arellano-Plaza, M; Gschaedler, A

2013-05-01

Traditionally, industrial tequila production has used spontaneous fermentation or Saccharomyces cerevisiae yeast strains. Despite the potential of non-Saccharomyces strains for alcoholic fermentation, few studies have been performed at industrial level with these yeasts. Therefore, in this work, Agave tequilana juice was fermented at an industrial level using two non-Saccharomyces yeasts (Pichia kluyveri and Kluyveromyces marxianus) with fermentation efficiency higher than 85 %. Pichia kluyveri (GRO3) was more efficient for alcohol and ethyl lactate production than S. cerevisiae (AR5), while Kluyveromyces marxianus (GRO6) produced more isobutanol and ethyl-acetate than S. cerevisiae (AR5). The level of volatile compounds at the end of fermentation was compared with the tequila standard regulation. All volatile compounds were within the allowed range except for methanol, which was higher for S. cerevisiae (AR5) and K. marxianus (GRO6). The variations in methanol may have been caused by the Agave tequilana used for the tests, since this compound is not synthesized by these yeasts.

Expression of the Laccase Gene from a White Rot Fungus in Pichia pastoris Can Enhance the Resistance of This Yeast to H2O2-Mediated Oxidative Stress by Stimulating the Glutathione-Based Antioxidative System

PubMed Central

Fan, Fangfang; Zhuo, Rui; Ma, Fuying; Gong, Yangmin; Wan, Xia; Jiang, Mulan

2012-01-01

Laccase is a copper-containing polyphenol oxidase that has great potential in industrial and biotechnological applications. Previous research has suggested that fungal laccase may be involved in the defense against oxidative stress, but there is little direct evidence supporting this hypothesis, and the mechanism by which laccase protects cells from oxidative stress also remains unclear. Here, we report that the expression of the laccase gene from white rot fungus in Pichia pastoris can significantly enhance the resistance of yeast to H2O2-mediated oxidative stress. The expression of laccase in yeast was found to confer a strong ability to scavenge intracellular H2O2 and to protect cells from lipid oxidative damage. The mechanism by which laccase gene expression increases resistance to oxidative stress was then investigated further. We found that laccase gene expression in Pichia pastoris could increase the level of glutathione-based antioxidative activity, including the intracellular glutathione levels and the enzymatic activity of glutathione peroxidase, glutathione reductase, and γ-glutamylcysteine synthetase. The transcription of the laccase gene in Pichia pastoris was found to be enhanced by the oxidative stress caused by exogenous H2O2. The stimulation of laccase gene expression in response to exogenous H2O2 stress further contributed to the transcriptional induction of the genes involved in the glutathione-dependent antioxidative system, including PpYAP1, PpGPX1, PpPMP20, PpGLR1, and PpGSH1. Taken together, these results suggest that the expression of the laccase gene in Pichia pastoris can enhance the resistance of yeast to H2O2-mediated oxidative stress by stimulating the glutathione-based antioxidative system to protect the cell from oxidative damage. PMID:22706050

Lindnera (Pichia) fabianii blood infection after mesenteric ischemia.

PubMed

Gabriel, Frederic; Noel, Thierry; Accoceberry, Isabelle

2012-04-01

Lindnera (Pichia) fabianii (teleomorph of Candida fabianii) is a yeast species rarely involved in human infections. This report describes the first known human case of a Lindnera fabianii blood infection after mesenteric ischemia. The 53-year-old patient was hospitalized in the intensive care unit after a suicide attempt and was suffering from a mesenteric ischemia and acute renal failure. Lindnera fabianii was recovered from an oropharyngeal swab, then isolated from stool and urine samples before the diagnosis of the blood infection. Caspofungin intravenous treatment was associated with a successful outcome. Final unequivocal identification of the strain was done by sequencing the internal transcribed spacer (ITS) region, and regions of 18S rDNA gene and of the translation elongation factor-1α gene. Until our work, the genomic databases did not contain the complete ITS region of L. fabianii as a single nucleotide sequence (encompassing ITS1, the 5.8S rDNA and ITS2), and misidentification with other yeast species, e.g., Lindnera (Pichia) mississippiensis, could have occurred. Our work demonstrates that the usual DNA barcoding method based on sequencing of the ITS region may fail to provide the correct identification of some taxa, and that partial sequencing of the EF1α gene may be much more effective for the accurate delineation and molecular identification of new emerging opportunistic yeast pathogens.

Candida guilliermondii and Other Species of Candida Misidentified as Candida famata: Assessment by Vitek 2, DNA Sequencing Analysis, and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry in Two Global Antifungal Surveillance Programs

PubMed Central

Woosley, Leah N.; Diekema, Daniel J.; Jones, Ronald N.; Pfaller, Michael A.

2013-01-01

Candida famata (teleomorph Debaryomyces hansenii) has been described as a medically relevant yeast, and this species has been included in many commercial identification systems that are currently used in clinical laboratories. Among 53 strains collected during the SENTRY and ARTEMIS surveillance programs and previously identified as C. famata (includes all submitted strains with this identification) by a variety of commercial methods (Vitek, MicroScan, API, and AuxaColor), DNA sequencing methods demonstrated that 19 strains were C. guilliermondii, 14 were C. parapsilosis, 5 were C. lusitaniae, 4 were C. albicans, and 3 were C. tropicalis, and five isolates belonged to other Candida species (two C. fermentati and one each C. intermedia, C. pelliculosa, and Pichia fabianni). Additionally, three misidentified C. famata strains were correctly identified as Kodomaea ohmeri, Debaryomyces nepalensis, and Debaryomyces fabryi using intergenic transcribed spacer (ITS) and/or intergenic spacer (IGS) sequencing. The Vitek 2 system identified three isolates with high confidence to be C. famata and another 15 with low confidence between C. famata and C. guilliermondii or C. parapsilosis, displaying only 56.6% agreement with DNA sequencing results. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) results displayed 81.1% agreement with DNA sequencing. One strain each of C. metapsilosis, C. fermentati, and C. intermedia demonstrated a low score for identification (<2.0) in the MALDI Biotyper. K. ohmeri, D. nepalensis, and D. fabryi identified by DNA sequencing in this study were not in the current database for the MALDI Biotyper. These results suggest that the occurrence of C. famata in fungal infections is much lower than previously appreciated and that commercial systems do not produce accurate identifications except for the newly introduced MALDI-TOF instruments. PMID:23100350

21 CFR 173.160 - Candida guilliermondii.

Code of Federal Regulations, 2014 CFR

2014-04-01

...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and...: (a) The food additive is the enzyme system of the viable organism Candida guilliermondii and its...

Low-pH production of D-lactic acid using newly isolated acid tolerant yeast Pichia kudriavzevii NG7.

PubMed

Park, Hyun Joo; Bae, Jung-Hoon; Ko, Hyeok-Jin; Lee, Sun-Hee; Sung, Bong Hyun; Han, Jong-In; Sohn, Jung-Hoon

2018-06-13

Lactic acid is a platform chemical for the sustainable production of various materials. To develop a robust yeast platform for low-pH production of D-lactic acid, an acid-tolerant yeast strain was isolated from grape skins and named Pichia kudriavzevii NG7 by ribosomal RNA sequencing. This strain was able to grow at pH 2.0 and 50°C. For the commercial application of P. kudriavzevii NG7 as a lactic acid producer, the ethanol fermentation pathway was redirected to lactic acid by replacing pyruvate decarboxylase 1 gene (PDC1) with D-lactate dehydrogenase gene (D-LDH) derived from Lactobacillus plantarum. To enhance lactic acid tolerance, this engineered strain was adapted to high lactic acid concentrations, and a new transcriptional regulator, PAR1, responsible for acid tolerance, was identified by whole-genome resequencing. The final engineered strain produced 135 g/L and 154 g/L of D-lactic acid with productivity over 3.66 g/L/h at pH 3.6 and 4.16 g/L/h at pH 4.7, respectively. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

21 CFR 173.160 - Candida guilliermondii.

Code of Federal Regulations, 2013 CFR

2013-04-01

... CONSUMPTION Enzyme Preparations and Microorganisms § 173.160 Candida guilliermondii. The food additive Candida... the following conditions: (a) The food additive is the enzyme system of the viable organism Candida...

21 CFR 173.160 - Candida guilliermondii.

Code of Federal Regulations, 2012 CFR

2012-04-01

... CONSUMPTION Enzyme Preparations and Microorganisms § 173.160 Candida guilliermondii. The food additive Candida... the following conditions: (a) The food additive is the enzyme system of the viable organism Candida...

21 CFR 173.160 - Candida guilliermondii.

Code of Federal Regulations, 2011 CFR

2011-04-01

... CONSUMPTION Enzyme Preparations and Microorganisms § 173.160 Candida guilliermondii. The food additive Candida... the following conditions: (a) The food additive is the enzyme system of the viable organism Candida...

Engineering of Methane Metabolism in Pichia Pastoris Through Methane Monooxygenase Expression

NASA Technical Reports Server (NTRS)

Fleury, Samantha T.; Neff, Lily S.; Galazka, Jonathan M.

2017-01-01

Exploration of the solar system is constrained by the cost of moving mass off Earth. Producing materials in situ will reduce the mass that must be delivered from earth. CO2 is abundant on Mars and manned spacecraft. On the ISS, NASA reacts excess CO2 with H2 to generate CH4 and H2O using the Sabatier System. The resulting water is recovered into the ISS, but the methane is vented to space. Thus, there is a capability need for systems that convert methane into valuable materials. Methanotrophic bacteria consume methane but these are poor synthetic biology platforms. Thus, there is a knowledge gap in utilizing methane in a robust and flexible synthetic biology platform. The yeast Pichia pastoris is a refined microbial factory that is used widely by industry because it efficiently secretes products. Pichia could produce a variety of useful products in space. Pichia does not consume methane but robustly consumes methanol, which is one enzymatic step removed from methane. Our goal is to engineer Pichia to consume methane thereby creating a powerful methane-consuming microbial factory.

Taxonomic structure of the yeasts and lactic acid bacteria microbiota of pineapple (Ananas comosus L. Merr.) and use of autochthonous starters for minimally processing.

PubMed

Di Cagno, Raffaella; Cardinali, Gainluigi; Minervini, Giovanna; Antonielli, Livio; Rizzello, Carlo Giuseppe; Ricciuti, Patrizia; Gobbetti, Marco

2010-05-01

Pichia guilliermondii was the only identified yeast in pineapple fruits. Lactobacillus plantarum and Lactobacillus rossiae were the main identified species of lactic acid bacteria. Typing of lactic acid bacteria differentiated isolates depending on the layers. L. plantarum 1OR12 and L. rossiae 2MR10 were selected within the lactic acid bacteria isolates based on the kinetics of growth and acidification. Five technological options, including minimal processing, were considered for pineapple: heating at 72 degrees C for 15 s (HP); spontaneous fermentation without (FP) or followed by heating (FHP), and fermentation by selected autochthonous L. plantarum 1OR12 and L. rossiae 2MR10 without (SP) or preceded by heating (HSP). After 30 days of storage at 4 degrees C, HSP and SP had a number of lactic acid bacteria 1000 to 1,000,000 times higher than the other processed pineapples. The number of yeasts was the lowest in HSP and SP. The Community Level Catabolic Profiles of processed pineapples indirectly confirmed the capacity of autochthonous starters to dominate during fermentation. HSP and SP also showed the highest antioxidant activity and firmness, the better preservation of the natural colours and were preferred for odour and overall acceptability. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Evaluation of chitosans and Pichia guillermondii as growth inhibitors of Penicillium digitatum.

PubMed

Pacheco, Neith; Larralde-Corona, C Patricia; Sepulveda, Jose; Trombotto, Stéphan; Domard, Alain; Shirai, Keiko

2008-07-01

Chitosans were obtained by room-temperature-homogeneous-deacetylation (RTHD) and freeze-pump-out-thaw-heterogeneous-deacetylation (FPT) from chitins purified from fermentations. Commercial chitosan was deacetylated by three-FPT-cycles. Chitosans and Pichia guillermondii were evaluated on the growth of Penicillium digitatum. Medium molecular weight (M(W)) chitosans displayed higher inhibitory activity against the yeast than low M(W) biopolymers. Chitosans with low degree of acetylation (DA) were inhibitory for yeast and mould. Therefore, a low M(W) and high DA chitosan was selected for use against moulds combined with yeasts. Biopolymer and yeasts presented an additive effect, since chitosans were effective to delay spore germination, whereas yeast decreased apical fungal growth.

Production and Its Anti-hyperglycemic Effects of γ-Aminobutyric Acid from the Wild Yeast Strain Pichia silvicola UL6-1 and Sporobolomyces carnicolor 402-JB-1.

PubMed

Han, Sang-Min; Lee, Jong-Soo

2017-09-01

This study was done to produce γ-aminobutyric acid (GABA) from wild yeast as well as investigate its anti-hyperglycemic effects. Among ten GABA-producing yeast strains, Pichia silvicola UL6-1 and Sporobolomyces carnicolor 402-JB-1 produced high GABA concentration of 134.4 µg/mL and 179.2 µg/mL, respectively. P. silvicola UL6-1 showed a maximum GABA yield of 136.5 µg/mL and 200.8 µg/mL from S. carnicolor 402-JB-1 when they were cultured for 30 hr at 30℃ in yeast extract-peptone-dextrose medium. The cell-free extract from P. silvicola UL6-1 and S. carnicolor 402-JB-1 showed very high anti-hyperglycemic α-glucosidase inhibitory activity of 72.3% and 69.9%, respectively. Additionally, their cell-free extract-containing GABA showed the anti-hyperglycemic effect in streptozotocin-induced diabetic Sprague-Dawley rats.

Characterization of newly isolated oleaginous yeasts - Cryptococcus podzolicus, Trichosporon porosum and Pichia segobiensis

PubMed Central

2014-01-01

The yeast strains Cryptococcus podzolicus, Trichosporon porosum and Pichia segobiensis were isolated from soil samples and identified as oleaginous yeast strains beneficial for the establishment of microbial production processes for sustainable lipid production suitable for several industrial applications. When cultured in bioreactors with glucose as the sole carbon source C. podzolicus yielded 31.8% lipid per dry biomass at 20°C, while T. porosum yielded 34.1% at 25°C and P. segobiensis 24.6% at 25°C. These amounts correspond to lipid concentrations of 17.97 g/L, 17.02 g/L and 12.7 g/L and volumetric productivities of 0.09 g/Lh, 0.1 g/Lh and 0.07 g/Lh, respectively. During the culture of C. podzolicus 30 g/l gluconic acid was detected as by-product in the culture broth and 12 g/L gluconic acid in T. porosum culture. The production of gluconic acid was eliminated for both strains when glucose was substituted by xylose as the carbon source. Using xylose lipid yields were 11.1 g/L and 13.9 g/L, corresponding to 26.8% and 33.4% lipid per dry biomass and a volumetric productivity of 0.07 g/Lh and 0.09 g/Lh, for C. podzolicus and T. porosum respectively. The fatty acid profile analysis showed that oleic acid was the main component (39.6 to 59.4%) in all three strains and could be applicable for biodiesel production. Palmitic acid (18.4 to 21.1%) and linolenic acid (7.5 to 18.7%) are valuable for cosmetic applications. P. segobiensis had a considerable amount of palmitoleic acid (16% content) and may be suitable for medical applications. PMID:24949259

Alcoholic Fermentation of d-Xylose by Yeasts

PubMed Central

Toivola, Ansa; Yarrow, David; van den Bosch, Eduard; van Dijken, Johannes P.; Scheffers, W. Alexander

1984-01-01

Type strains of 200 species of yeasts able to ferment glucose and grow on xylose were screened for fermentation of d-xylose. In most of the strains tested, ethanol production was negligible. Nineteen were found to produce between 0.1 and 1.0 g of ethanol per liter. Strains of the following species produce more than 1 g of ethanol per liter in the fermentation test with 2% xylose: Brettanomyces naardenensis, Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis. Subsequent screening of these yeasts for their capacity to ferment d-cellobiose revealed that only Candida tenuis CBS 4435 was a good fermenter of both xylose and cellobiose under the test conditions used. PMID:16346558

Production and Its Anti-hyperglycemic Effects of γ-Aminobutyric Acid from the Wild Yeast Strain Pichia silvicola UL6-1 and Sporobolomyces carnicolor 402-JB-1

PubMed Central

Han, Sang-Min

2017-01-01

This study was done to produce γ-aminobutyric acid (GABA) from wild yeast as well as investigate its anti-hyperglycemic effects. Among ten GABA-producing yeast strains, Pichia silvicola UL6-1 and Sporobolomyces carnicolor 402-JB-1 produced high GABA concentration of 134.4 µg/mL and 179.2 µg/mL, respectively. P. silvicola UL6-1 showed a maximum GABA yield of 136.5 µg/mL and 200.8 µg/mL from S. carnicolor 402-JB-1 when they were cultured for 30 hr at 30℃ in yeast extract-peptone-dextrose medium. The cell-free extract from P. silvicola UL6-1 and S. carnicolor 402-JB-1 showed very high anti-hyperglycemic α-glucosidase inhibitory activity of 72.3% and 69.9%, respectively. Additionally, their cell-free extract-containing GABA showed the anti-hyperglycemic effect in streptozotocin-induced diabetic Sprague-Dawley rats. PMID:29138625

Characteristics and Applicability of Phytase of the Yeast Pichia anomala in Synthesizing Haloperoxidase.

PubMed

Joshi, Swati; Satyanarayana, Tulasi

2015-07-01

The phytase of the yeast Pichia anomala is a histidine acid phosphatase based on signature sequences and catalytic amino acids identified by site-directed mutagenesis. Among modulators, N-bromosuccinimide and butanedione inhibit phytase, while Ca(2+) and Ni(2+) stimulate slightly. Vanadate exhibits competitive inhibition of phytase, making it bifunctional to act as haloperoxidase. Molecular docking supports vanadate to share its binding site with phytate. The T 1/2, activation energy (E a ), temperature quotient (Q 10), activation energy of thermal inactivation (Ed), and enthalpy (ΔH d (0) ) of the enzyme are 4.0 min (80 °C), 27.72 kJ mol(-1), 2.1, 410.62 kJ mol(-1), and ∼407.8 kJ mol(-1) (65-80 °C), respectively. The free energy of the process (ΔG d (o) ) increases from 49.56 to 71.58 kJ mol(-1) with rise in temperature, while entropy of inactivation (ΔS d (0) ) remains constant at ∼1.36 kJ mol(-1) K(-1). The supplementation of whole wheat dough with rPPHY resulted in 72.5 % reduction in phytic acid content of bread. These characteristics confirm that the phytase has adequate thermostability for its applicability as a food and feed additive.

PMKT2, a new killer toxin from Pichia membranifaciens, and its promising biotechnological properties for control of the spoilage yeast Brettanomyces bruxellensis.

PubMed

Santos, A; San Mauro, M; Bravo, E; Marquina, D

2009-02-01

Pichia membranifaciens CYC 1086 secretes a killer toxin (PMKT2) that is inhibitory to a variety of spoilage yeasts and fungi of agronomical interest. The killer toxin in the culture supernatant was concentrated by ultrafiltration and purified to homogeneity by two successive steps, including native electrophoresis and HPLC gel filtration. Biochemical characterization of the toxin showed it to be a protein with an apparent molecular mass of 30 kDa and an isoelectric point of 3.7. At pH 4.5, optimal killer activity was observed at temperatures up to 20 degrees C. Above approximately this pH, activity decreased sharply and was barely noticeable at pH 6. The toxin concentrations present in the supernatant during optimal production conditions exerted a fungicidal effect on a variety of fungal and yeast strains. The results obtained suggest that PMKT2 has different physico-chemical properties from PMKT as well as different potential uses in the biocontrol of spoilage yeasts. PMKT2 was able to inhibit Brettanomyces bruxellensis while Saccharomyces cerevisiae was fully resistant, indicating that PMKT2 could be used in wine fermentations to avoid the development of the spoilage yeast without deleterious effects on the fermentative strain. In small-scale fermentations, PMKT2, as well as P. membranifaciens CYC 1086, was able to inhibit B. bruxellensis, verifying the biocontrol activity of PMKT2 in simulated winemaking conditions.

Microbiota during fermentation of chum salmon (Oncorhynchus keta) sauce mash inoculated with halotolerant microbial starters: analyses using the plate count method and PCR-denaturing gradient gel electrophoresis (DGGE).

PubMed

Yoshikawa, Shuji; Yasokawa, Daisuke; Nagashima, Koji; Yamazaki, Koji; Kurihara, Hideyuki; Ohta, Tomoki; Kawai, Yuji

2010-06-01

Nine different combinations of mugi koji (barley steamed and molded with Aspergillus oryzae) and halotolerant microorganisms (HTMs), Zygosaccharomyces rouxii, Candida versatilis, and Tetragenococcus halophilus, were inoculated into chum salmon sauce mash under a non-aseptic condition used in industrial fish sauce production and fermented at 35 +/- 2.5 degrees C for 84 days to elucidate the microbial dynamics (i.e., microbial count and microbiota) during fermentation. The viable count of halotolerant yeast (HTY) in fermented chum salmon sauce (FCSS) mash showed various time courses dependent on the combination of the starter microorganisms. Halotolerant lactic acid bacteria (HTL) were detected morphologically and physiologically only from FCSS mash inoculated with T. halophilus alone or with T. halophilus and C. versatilis during the first 28 days of fermentation. Only four fungal species, Z. rouxii, C. versatilis, Pichia guilliermondii, and A. oryzae, were detected throughout the fermentation by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). In FCSS mash, dominant HTMs, especially eumycetes, were nonexistent. However, under the non-aseptic conditions, undesirable wild yeast such as P. guilliermondii grew fortuitously. Therefore, HTY inoculation into FCSS mash at the beginning of fermentation is effective in preventing the growth of wild yeast and the resultant unfavorable flavor. 2009 Elsevier Ltd. All rights reserved.

[The regulation of peroxisomal matrix enzymes (alcohol oxidase and catalase) formation by the product of the gene Mth1 in methylotrophic yeast Pichia methanolica].

PubMed

Leonovich, O A; Kurales, Iu A; Dutova, T A; Isakova, E P; Deriabina, Iu I; Rabinovich, Ia M

2009-01-01

Two independent mutant strains of methylotrophic yeast Pichia methanolica (mth1 arg1 and mth2 arg4) from the initial line 616 (ade1 ade5) were investigated. The mutant strains possessed defects in genes MTH1 and MTH2 which resulted in the inability to assimilate methanol as a sole carbon source and the increased activity of alcohol oxidase (AO). The function of the AUG2 gene encoding one of the subunits of AO and CTA1, a probable homolog of peroxisomal catalase of Saccharomyces cereviseae, was investigated by analyses of the molecular forms of isoenzymes. It was shown that optimal conditions for the expression of the AUG2 gene on a medium supplemented with 3% of methanol leads to an increasing synthesis of peroxisomal catalase. The mutant mth1 possessed a dominant formation of AO isoform with electrophoretic mobility which is typical for isogenic form 9, the product of the AUG2 gene, and a decreased level of peroxisomal catalase. The restoration of growth of four spontaneous revertants of the mutant mth1 (Rmth1) on the methanol containing medium was accompanied by an increase in activity of AO isogenic form 9 and peroxisomal catalase. The obtained results confirmed the functional continuity of the structural gene AUG2 in mutant mth1. The correlation of activity of peroxisomal catalase and AO isogenic form 1 in different conditions evidenced the existence of common regulatory elements for genes AUG2 and CTA1 in methilotrophic yeast Pichia methanolica.

Cloning, production, and functional expression of the bacteriocin enterocin A, produced by Enterococcus faecium T136, by the yeasts Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Arxula adeninivorans.

PubMed

Borrero, Juan; Kunze, Gotthard; Jiménez, Juan J; Böer, Erik; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

2012-08-01

The bacteriocin enterocin A (EntA) produced by Enterococcus faecium T136 has been successfully cloned and produced by the yeasts Pichia pastoris X-33EA, Kluyveromyces lactis GG799EA, Hansenula polymorpha KL8-1EA, and Arxula adeninivorans G1212EA. Moreover, P. pastoris X-33EA and K. lactis GG799EA produced EntA in larger amounts and with higher antimicrobial and specific antimicrobial activities than the EntA produced by E. faecium T136.

A Novel Production Method for High-Fructose Glucose Syrup from Sucrose-Containing Biomass by a Newly Isolated Strain of Osmotolerant Meyerozyma guilliermondii.

PubMed

Khattab, Sadat Mohammad Rezq; Kodaki, Tsutomu

2016-04-28

One osmotolerant strain from among 44 yeast isolates was selected based on its growth abilities in media containing high concentrations of sucrose. This selected strain, named SKENNY, was identified as Meyerozyma guilliermondii by sequencing the internal transcribed spacer regions and partial D1/D2 large-subunit domains of the 26S ribosomal RNA. SK-ENNY was utilized to produce high-fructose glucose syrup (HFGS) from sucrose-containing biomass. Conversion rates to HFGS from 310-610 g/l of pure sucrose and from 75-310 g/l of sugar beet molasses were 73.5-94.1% and 76.2-91.1%, respectively. In the syrups produced, fructose yields were 89.4-100% and 96.5-100% and glucose yields were 57.6-82.5% and 55.3-79.5% of the theoretical values for pure sucrose and molasses sugars, respectively. This is the first report of employing M. guilliermondii for production of HFGS from sucrose-containing biomass.

Nutrient limitation leads to penetrative growth into agar and affects aroma formation in Pichia fabianii, P. kudriavzevii and Saccharomyces cerevisiae.

PubMed

van Rijswijck, Irma M H; Dijksterhuis, Jan; Wolkers-Rooijackers, Judith C M; Abee, Tjakko; Smid, Eddy J

2015-01-01

Among fermentative yeast species, Saccharomyces cerevisiae is most frequently used as a model organism, although other yeast species may have special features that make them interesting candidates to apply in food-fermentation processes. In this study, we used three yeast species isolated from fermented masau (Ziziphus mauritiana) fruit, S. cerevisiae 131, Pichia fabianii 65 and Pichia kudriavzevii 129, and determined the impact of nitrogen and/or glucose limitation on surface growth mode and the production of volatile organic compounds (VOCs). All three species displayed significant changes in growth mode in all nutrient-limited conditions, signified by the formation of metafilaments or pseudohyphae. The timing of the transition was found to be species-specific. Transition in growth mode is suggested to be linked to the production of certain fusel alcohols, such as phenylethyl alcohol, which serve as quorum-sensing molecules. Interestingly, we did not observe concomitant increased production of phenylethyl alcohol and filamentous growth. Notably, a broader range of esters was found only for the Pichia spp. grown on nitrogen-limited agar for 21 days compared to nutrient-rich agar, and when grown on glucose- and glucose- plus nitrogen-limited agar. Our data suggest that for the Pichia spp., the formation of esters may play an important role in the switch in growth mode upon nitrogen limitation. Further biological or ecological implications of ester formation are discussed. Copyright © 2014 John Wiley & Sons, Ltd.

Differential gene expression during the pathogenic interaction between Pichia fermentans and peach fruit

USDA-ARS?s Scientific Manuscript database

A biofilm-forming strain of Pichia fermentans was found to be a very strong antagonist against brown rot and grey mold in artificially wounded apple fruit when co-inoculated with either Monilinia fructicola or Botrytis cinerea, respectively. The same strain of yeast; however, was an aggressive path...

Assessment of the Factors Contributing to the Growth or Spoilage of Meyerozyma guilliermondii in Organic Yogurt: Comparison of Methods for Strain Differentiation

PubMed Central

Wrent, Petra; Rivas, Eva-María; Gil de Prado, Elena; Peinado, José M.; de Silóniz, María-Isabel

2015-01-01

In this work we analyze the spoiling potential of Meyerozyma guilliermondii in yogurt. The analysis was based on contaminated samples sent to us by an industrial laboratory over two years. All the plain and fruit yogurt packages were heavily contaminated by yeasts, but only the last ones, containing fermentable sugars besides lactose, were spoiled by gas swelling. These strains were unable to grow and ferment lactose (as the type strain); they did grow on lactate plus galactose, fermented glucose and sucrose, and galactose (weakly), but did not compete with lactic acid bacteria for lactose. This enables them to grow in any yogurt, although only those with added jam were spoiled due to the fermentation of the fruit sugars. Fermentation, but not growth, was strongly inhibited at 8 °C. In consequence, in plain yogurt as well as in any yogurt maintained at low temperature, yeast contamination would not be detected by the consumer. The risk could be enhanced because the species has been proposed for biological control of fungal infections in organic agriculture. The combination of the IGS PCR-RFLP (amplification of the intergenic spacer region of rDNA followed by restriction fragment length polymorphism analysis) method and mitochondrial DNA-RFLP makes a good tool to trace and control the contamination by M. guilliermondii. PMID:27682098

A case of Candida guilliermondii abortion in an Arab mare

PubMed Central

Stefanetti, Valentina; Marenzoni, Maria Luisa; Lepri, Elvio; Coletti, Mauro; Casagrande Proietti, Patrizia; Agnetti, Francesco; Crotti, Silvia; Pitzurra, Lucia; Del Sero, Andrea; Passamonti, Fabrizio

2014-01-01

Ascending infections of equine uterus frequently result in placentitis and abortions; most of these infections are bacterial and are less commonly due to fungi. This report describes an abortion case in an Arab mare due to Candida guilliermondii that was diagnosed via cytological, histological, cultural and biomolecular assays. The histological lesions found were severe necrotizing placentitis associated with fetal pneumonia. To our knowledge this is the first case of C. guilliermondii abortion reported in equine species. PMID:24707460

Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review

PubMed Central

Cos, Oriol; Ramón, Ramón; Montesinos, José Luis; Valero, Francisco

2006-01-01

The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for heterologous protein production. The use of different phenotypes under PAOX promoter, other alternative promoters, culture medium, and operational strategies with the objective to maximize either yield or productivity of the heterologous protein, but also to obtain a repetitive product batch to batch to get a robust process for the final industrial application have been reported. Medium composition, kinetics growth, fermentation operational strategies from fed-batch to continuous cultures using different phenotypes with the most common PAOX promoter and other novel promoters (GAP, FLD, ICL), the use of mixed substrates, on-line monitoring of the key fermentation parameters (methanol) and control algorithms applied to the bioprocess are reviewed and discussed in detail. PMID:16600031

[Comparison of methods for the identification of the most common yeasts in the clinical microbiology laboratory].

PubMed

Guelfand, L; Grisolía, P; Bozzano, C; Kaufman, S

2003-01-01

We evaluated different methods for the routine identification of medically important yeasts. A total of 150 clinical isolates: 25 C. albicans, 25 C. tropicalis, 25 C. glabrata, 25 C. parapsilosis, 8 C. guilliermondii, 11 C. krusei and 31 Cryptococcus neoformans were tested by Yeast Biochemical Card bioMerieux Vitek (YBC), CHROMagar Candida (CHR). The addition of yeast morphology in Corn Meal agar-Tween 80 (AM) to YBC and CHR was also evaluated. The reference methods used were: API 20C, germ tube formation, AM, Christensen urea and Birdseed agar. YBC identified 135 yeasts with an overall accuracy of 90%. Sensitivity (S) and specificity (E) were: 92-98% for C. albicans and C. tropicalis; 84-99% for C. papapsilosis; 100-99% for C. glabrata; 91-100% for C. krusei; 63-98% for C. guilliermondii and 90-99% for Cryptococcus neoformans, respectively. CHR identified correctly 100% for C. albicans, 92% for C. tropicalis and 91% for C. krusei. Both methods combined with AM provided 100% S and E. We found that YBC system was appropriate for identification of yeasts isolated from human sources. CHR was effective and easy to use for identification of C. albicans, C. tropicalis and C. krusei. The routine use of AM with both methods is recommended.

Regulation of intracellular formaldehyde toxicity during methanol metabolism of the methylotrophic yeast Pichia methanolica.

PubMed

Wakayama, Keishi; Yamaguchi, Sakiko; Takeuchi, Akihito; Mizumura, Tasuku; Ozawa, Shotaro; Tomizuka, Noboru; Hayakawa, Takashi; Nakagawa, Tomoyuki

2016-11-01

In this study we found that the methylotrophic yeast Pichia methanolica showed impaired growth on high methanol medium (>5%, or 1.56 M, methanol). In contrast, P. methanolica grew well on glucose medium containing 5% methanol, but the growth defects reappeared on glucose medium supplemented with 5 mM formaldehyde. During methanol growth of P. methanolica, formaldehyde accumulated in the medium up to 0.3 mM before it was consumed rapidly based on cell growth. These findings indicate that the growth defect of P. methanolica on high methanol media is not caused directly by methanol toxicity, but rather by formaldehyde, which is a key toxic intermediate of methanol metabolism. Moreover, during methanol growth of P. methanolica, expression of enzymes in the methanol-oxidation pathway were induced before the alcohol oxidase isozymes Mod1p and Mod2p, and Mod1p expression was induced before Mod2p. These results suggest that to avoid excess accumulation of formaldehyde-the toxic intermediate of methanol metabolism-P. methanolica grown on methanol strictly regulates the order in which methanol-metabolizing enzymes are expressed. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

“In vitro” antifungal activity of ozonized sunflower oil on yeasts from onychomycosis

PubMed Central

Guerrer, L.V.; Cunha, K. C.; Nogueira, M. C. L.; Cardoso, C. C.; Soares, M. M. C. N.; Almeida, M. T. G.

2012-01-01

The “in vitro” antifungal activity of ozonized sunflower oil (Bioperoxoil®) was tested on 101 samples of yeasts originating from onychomycosis using the disk diffusion method. The oil was efficacious against several clinical fungal strains: Candida parapsilosis, Candida albicans, Trichosporon asahii, Candida tropicalis and Candida guilliermondii. PMID:24031958

Candida ficus sp. nov., a novel yeast species from the gut of Apriona germari larvae.

PubMed

Hui, Feng-Li; Niu, Qiu-Hong; Ke, Tao; Liu, Zheng

2012-11-01

A novel yeast species is described based on three strains from the gut of wood-boring larvae collected in a tree trunk of Ficus carica cultivated in parks near Nanyang, central China. Phylogenetic analysis based on sequences of the D1/D2 domains of the large subunit rRNA gene showed that these strains occurred in a separate clade that was genetically distinct from all known ascomycetous yeasts. In terms of pairwise sequence divergence, the novel strains differed by 15.3% divergence from the type strain of Pichia terricola, and by 15.8% divergence from the type strains of Pichia exigua and Candida rugopelliculosa in the D1/D2 domains. All three are ascomycetous yeasts in the Pichia clade. Unlike P. terricola, P. exigua and C. rugopelliculosa, the novel isolates did not ferment glucose. The name Candida ficus sp. nov. is proposed to accommodate these highly divergent organisms, with STN-8(T) (=CICC 1980(T)=CBS 12638(T)) as the type strain.

Characterization and Reclassification of Yeasts Used for Biological Control of Postharvest Diseases of Fruits and Vegetables

PubMed Central

McLaughlin, R. J.; Wilson, C. L.; Chalutz, E.; Kurtzman, C. P.; Fett, W. F.; Osman, S. F.

1990-01-01

In previous studies workers have shown that three yeast strains (strains US-7, 82, and 101) have biological control activity against various postharvest fungal pathogens of fruits and vegetables, including Penicillium rots of apples and citrus and Botrytis rot of apples. In these reports the researchers have described these strains as Debaryomyces hansenii (anamorph, Candida famata) or Candida sp. strains. In this study we performed additional physiological, DNA reassociation, and mannan characterization tests that clearly established a new taxonomic classification for these strains, Candida guilliermondii. We also propose amendment of the physiological test profile in the taxonomic description of C. guilliermondii. PMID:16348361

Regulation of aflatoxin biosynthesis and branched-chain amino acids metabolism in Aspergillus flavus by 2-phenylethanol reveal biocontrol mechanism of Pichia anomala

USDA-ARS?s Scientific Manuscript database

Pichia anomala WRL-076 is a biocontrol yeast which has been shown to inhibit growth and aflatoxin production of A. flavus. Using the SPME-GC/MS analysis we identified that the volatile, 2-phenylethanol (2-PE) produced by this yeast and demonstrated that the compound inhibited aflatoxin production. W...

The environmental and intrinsic yeast diversity of Cuban cocoa bean heap fermentations.

PubMed

Fernández Maura, Yurelkys; Balzarini, Tom; Clapé Borges, Pablo; Evrard, Pierre; De Vuyst, Luc; Daniel, H-M

2016-09-16

The environmental yeast diversity of spontaneous cocoa bean fermentations in east Cuba was investigated. Seven fermentations, 25 equipment- and handling-related samples, and 115 environmental samples, such as flowers, leaf and cocoa pod surfaces, as well as drosophilid insects, were analysed. The basic fermentation parameters temperature and pH were recorded during five fermentations for at least six days. A total of 435 yeast isolates were identified by a combination of PCR-fingerprinting of genomic DNA with the M13 primer and sequence analysis of DNA from representative isolates, using the internal transcribed spacer region, the D1/D2 region of the large subunit rRNA gene, and an actin gene-encoding fragment, as required. Among 65 yeast species detected, Pichia manshurica and Hanseniaspora opuntiae were the most frequently isolated species, obtained from five and four fermentations, followed in frequency by Pichia kudriavzevii from two fermentations. Saccharomyces cerevisiae was isolated only occasionally. Cocoa fermentation yeast species were also present on processing equipment. The repeated isolation of a preliminarily as Yamadazyma sp. classified species, a group of strains similar to Saccharomycopsis crataegensis from fermentations and equipment, and the isolation of fifteen other potentially novel yeast species in low numbers provides material for further studies. Environmental samples showed higher yeast diversity compared to the fermentations, included the most frequent fermentation species, whereas the most frequently isolated environmental species were Candida carpophila, Candida conglobata, and Candida quercitrusa. Potential selective advantages of the most frequently isolated species were only partly explained by the physiological traits tested. For instance, tolerance to higher ethanol concentrations was more frequent in strains of Pichia spp. and S. cerevisiae compared to Hanseniaspora spp.; the ability to also assimilate ethanol might have

Identification of yeasts isolated from raffia wine (Raphia hookeri) produced in Côte d'Ivoire and genotyping of Saccharomyces cerevisiae strains by PCR inter-delta.

PubMed

Tra Bi, Charles Y; N'guessan, Florent K; Kouakou, Clémentine A; Jacques, Noemie; Casaregola, Serge; Djè, Marcellin K

2016-08-01

Raffia wine is a traditional alcoholic beverage produced in several African countries where it plays a significant role in traditional customs and population diet. Alcoholic fermentation of this beverage is ensured by a complex natural yeast flora which plays a decisive role in the quality of the final product. This present study aims to evaluate the distribution and the diversity of the yeast strains isolated in raffia wine from four sampling areas (Abengourou, Alépé, Grand-Lahou and Adzopé) in Côte d'Ivoire. Based on the D1/D2 domain of the LSU rDNA sequence analysis, nine species belonging to six genera were distinguished. With a percentage of 69.5 % out of 171 yeast isolates, Saccharomyces cerevisiae was the predominant species in the raffia wine, followed by Kodamaea ohmeri (20.4 %). The other species isolated were Candida haemulonii (4.1 %), Candida phangngensis (1.8 %), Pichia kudriavzevii (1.2 %), Hanseniaspora jakobsenii (1.2 %), Candida silvae (0.6 %), Hanseniaspora guilliermondii (0.6 %) and Meyerozyma caribbica (0.6 %). The molecular characterization of S. cerevisiae isolates at the strain level using the PCR-interdelta method revealed the presence of 21 profiles (named I to XXI) within 115 isolates. Only four profiles (I, III, V and XI) were shared by the four areas under study. Phenotypic characterization of K. ohmeri strains showed two subgroups for sugar fermentation and no diversity for the nitrogen compound assimilations and the growth at different temperatures.

Expression and purification of soluble murine CD40L monomers and polymers in yeast Pichia pastoris

PubMed Central

Hermanrud, Christina E.; Lucas, Carrie L.; Sykes, Megan; Huang, Christene A.; Wang, Zhirui

2010-01-01

The anti-murine CD40L monoclonal antibody MR1 has been widely used in immunology research to block the CD40-CD40L interaction for induction of transplantation tolerance and to abrogate autoimmune diseases. The availability of recombinant CD40L with high binding capacity for MR1 would provide a valuable immunological research tool. In this study, we constructed the single chain murine soluble CD40L monomer, dimer, trimer and successfully expressed them in yeast Pichia pastoris under the control of the alcohol oxidase promoter. The secreted single chain murine soluble CD40L monomers, dimers, and trimers were initially enriched through histidine tag capture by Ni-Sepharose 6 fast flow resin and further purified on a cation exchange resin. Purity reached more than 95% for the monomer and dimer forms and more than 90% for the trimer. Protein yield following purification was 16 mg/L for the monomer and dimer, and 8 mg/L for the trimer. ELISA analysis demonstrated that the CD40L dimers and trimers correctly folded in conformations exposing the MR1 antigenic determinant. PMID:21074618

Management of Candida guilliermondii joint infection in a dog.

PubMed

Bufalari, Antonello; Maggio, Chiara; Moretti, Giulia; Crovace, Alberto; Stefanetti, Valentina; Straubinger, Reinhard Konrad; Passamonti, Fabrizio

2016-07-08

Candida spp. are dimorphic fungi in the family Cryptococcaceae. Infections with Candida spp. are usually rare conditions in dogs, but immunocompromised patients have a higher risk for developing invasive candidal infections. A 5-year-old male Boxer, positive to Leishmania infantum, was referred to the Veterinary Teaching Hospital of the Department of Veterinary Medicine, University of Perugia, Italy for examination of a non-weight bearing left hind limb lameness of a duration of at least 3 months. During this period, treatment involved systemic anti-inflammatory medications and intra-articular corticosteroid administration. On presentation, clinical examination and radiographic findings were suggestive of cranial cruciate ligament deficiency. To support this diagnosis a stifle arthroscopy was performed: it confirmed a partial rupture of cranial cruciate ligament. Samples culture of synovial fluid and membrane was routinely collected as well, and revealed Candida guilliermondii joint infection. Treatment for the C. guilliermondii joint infection involved systemic anti-fungal therapy, joint lavage and intra-articular administration of antifungal drugs. Lameness improved markedly during this treatment, but lameness did not resolve completely, probably due to cranial cruciate ligament deficiency. Tibial tuberosity advancement (TTA) was chosen in order to treat stifle instability and was performed 4 weeks following cessation of treatment of the C. guilliermondii joint infection. Six month after TTA the dog showed a completely recovery with no lameness. To the authors' knowledge, this is the first case of Candida spp. joint infection reported in dogs. The cause of the progression of the joint C. guilliermondii infection remains unclear but it may be associated with leishmaniasis or intra-articular corticosteroid injections. Treatment with systemic and intra-articular anti-fungal therapies was successful. In the evaluation of hind limb lameness in a chronically

Heterologous expression of bovine lactoferricin in Pichia methanolica.

PubMed

Wang, Haikuan; Zhao, Xinhuai; Lu, Fuping

2007-06-01

According to the bias of codon utilization of Pichia methanolica, a fragment encoding bovine lactoferricin has been cloned and expressed in the P. methanolica under the control of the alcohol oxidase promoter, which was followed by the Saccharomyces cerevisiae alpha-factor signal peptide. The alpha-factor signal peptide efficiently directed the secretion of bovine lactoferricin from the recombinant yeast cell. The recombinant bovine lactoferricin appears to be successfully expressed, as it displays antibacterial activity (antibacterial assay). Moreover, the identity of the recombinant product was estimated by Tricine-SDS-PAGE.

Use of the VITEK 2 system to identify and test the antifungal susceptibility of clinically relevant yeast species

PubMed Central

Melhem, MSC; Bertoletti, A; Lucca, HRL; Silva, RBO; Meneghin, FA; Szeszs, MW

2013-01-01

Eleven quality control isolates (Candida albicans ATCC 64548, C. tropicalis ATCC 200956, C. glabrata ATCC 90030, C. lusitaniae ATCC 200951, C. parapsilosis ATCC 22019, C. krusei ATCC 6258, C. dubliniensis ATCC 6330, Saccharomyces cerevisiae ATCC 9763, Cryptococcus neoformans ATCC 90012, C. gattii FIOCRUZ-CPF 60, and Trichosporon mucoides ATCC 204094) and 32 bloodstream isolates, including C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, C. pelliculosa (Pichia anomala), C. haemulonii, C. lusitaniae, and C. kefyr were identified at the species level by the VITEK 2 system. A set of clinical isolates (32 total) were used as challenge strains to evaluate the ability of the VITEK 2 system to determine the antifungal susceptibility of yeasts compared with the CLSI and EUCAST BMD reference standards. The VITEK 2 system correctly identified 100% of the challenge strains. The identification of yeast species and the evaluation of their susceptibility profiles were performed in an automated manner by the VITEK 2 system after approximately 15 h of growth for most species of Candida. The VITEK 2 system ensures that each test is performed in a standardized manner and provides quantitative MIC results that are reproducible and accurate when compared with the BMD reference methods. This system was able to determine the MICs of amphotericin B, flucytosine, voriconazole, and fluconazole in 15 h or less for the most common clinically relevant Candida species. In addition, the VITEK 2 system could reliably identify resistance to flucytosine, voriconazole, and fluconazole and exhibits excellent quantitative and qualitative agreement with the CLSI or EUCAST broth microdilution reference methods. PMID:24688520

Experimental Systems to Study Yeast Pexophagy.

PubMed

Yamashita, Shun-Ichi; Oku, Masahide; Sakai, Yasuyoshi; Fujiki, Yukio

2017-01-01

Peroxisome abundance is tightly regulated according to the physiological contexts, through regulations of both proliferation and degradation of the organelles. Here, we describe detailed methods to analyze processes for autophagic degradation of peroxisomes, termed pexophagy, in yeast organisms. The assay systems include a method for biochemical detection of pexophagy completion, and one for microscopic visualization of specialized membrane structures acting in pexophagy. As a model yeast organism utilized in studies of pexophagy, the methylotrophic yeast Komagataella phaffii (Pichia pastoris) is referred to in this chapter and related information on the studies with baker's yeast (Saccharomyces cerevisiae) is also included. The described techniques facilitate elucidation of molecular machineries for pexophagy and understanding of peroxisome-selective autophagic pathways.

Identification of yeast and bacteria involved in the mezcal fermentation of Agave salmiana.

PubMed

Escalante-Minakata, P; Blaschek, H P; Barba de la Rosa, A P; Santos, L; De León-Rodríguez, A

2008-06-01

To identify the yeast and bacteria present in the mezcal fermentation from Agave salmiana. The restriction and sequence analysis of the amplified region, between 18S and 28S rDNA and 16S rDNA genes, were used for the identification of yeast and bacteria, respectively. Eleven different micro-organisms were identified in the mezcal fermentation. Three of them were the following yeast: Clavispora lusitaniae, Pichia fermentans and Kluyveromyces marxianus. The bacteria found were Zymomonas mobilis subsp. mobilis and Zymomonas mobilis subsp. pomaceae, Weissella cibaria, Weissella paramesenteroides, Lactobacillus pontis, Lactobacillus kefiri, Lactobacillus plantarum and Lactobacillus farraginis. The phylogenetic analysis of 16S rDNA and ITS sequences showed that microbial diversity present in mezcal is dominated by bacteria, mainly lactic acid bacteria species and Zymomonas mobilis. Pichia fermentans and K. marxianus could be micro-organisms with high potential for the production of some volatile compounds in mezcal. We identified the community of bacteria and yeast present in mezcal fermentation from Agave salmiana.

Individual and interaction effects of vanillin and syringaldehyde on the xylitol formation by Candida guilliermondii.

PubMed

Cortez, Daniela Vieira; Roberto, Inês Conceição

2010-03-01

The effect of lignin degradation products liberated during chemical hydrolysis of lignocellulosic materials on xylose-to-xylitol bioconversion by Candida guilliermondii FTI 20037 was studied. Two aromatic aldehydes (vanillin and syringaldehyde) were selected as model compounds. A two-level factorial design was employed to evaluate the effects of pH (5.5-7.0), cell concentration (1.0-3.0 g l(-1)), vanillin concentration (0-2.0 g l(-1)) and syringaldehyde concentration (0-2.0 g l(-1)) on this bioprocess. The results showed that in the presence of vanillin or syringaldehyde (up to 2.0 g l(-1)) the cell growth was inhibited to different degrees with a complete inhibition of the yeast growth when the mixture of both (at 2.0 g l(-1) each) was added to the fermentation medium. The xylitol yield was not significantly influenced by vanillin, but was strongly reduced by syringaldehyde, which showed a more pronounced inhibitor effect at pH 7.0. The yeast was also able to convert vanillin and syringaldehyde to the corresponding aromatic acids or alcohols and their formation was dependent of the experimental conditions employed. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Probiotic potentials of yeasts isolated from some cereal-based Nigerian traditional fermented food products.

PubMed

Ogunremi, O R; Sanni, A I; Agrawal, R

2015-09-01

To determine the starter culture and multifunctional potentials of yeast strains from some cereal-based Nigerian traditional fermented food products. Yeast isolates were screened for enzyme production and identified by sequencing the D1/D2 region of 26S rDNA. Pichia kluyveri LKC17, Issatchenkia orientalis OSL11, Pichia kudriavzevii OG32, Pichia kudriavzevii ROM11 and Candida tropicalis BOM21 exhibited the highest protease, lipase and phytase activity. They were selected and further evaluated for gastrointestinal survival and adherence ability. Although strain-specific, they retained viability at 37°C and showed survival at pH 2·0., I. orientalis OSL11 showed the highest survival at 2% bile salts concentration and P. kudriavzevii ROM11 showed the least survival. The yeast strains showed strong autoaggregation ability (81·24-91·85%) and hydrophobicity to n-hexadecane (33·61-42·30%). The highest co-aggregation ability was detected for P. kudriavzevii OG32 and Escherichia coli (71·57%). All the yeast strains removed cholesterol in the range of 49·03-74·05% over 48 h and scavenged for free radicals in methanol reaction system. In this study, we isolated new yeast strains with multifunctional potentials that can be used as functional starter cultures to produce cereal-based probiotic products. The development of probiotic yeast strains as starter culture to improve the quality attributes and confer functional value on cereal-based traditional fermented foods is beneficial. © 2015 The Society for Applied Microbiology.

Pigeons and their droppings as reservoirs of Candida and other zoonotic yeasts.

PubMed

Rosario Medina, Inmaculada; Román Fuentes, Lorena; Batista Arteaga, Miguel; Real Valcárcel, Fernando; Acosta Arbelo, Félix; Padilla Del Castillo, Daniel; Déniz Suárez, Soraya; Ferrer Quintana, Otilia; Vega Gutiérrez, Belinda; Silva Sergent, Freddy; Acosta-Hernández, Begoña

The importance of pigeons as reservoirs and carriers of Cryptococcus neoformans and other species of this genus is well-known; however, less is known about their role as reservoirs and carriers of other yeasts that impact public health. The present study was performed on Gran Canaria Island to define yeasts other than Cryptococcus spp. that have been reported to impact public health and which could be carried by pigeons. Samples were obtained from 83 pigeon lofts (Columba livia); moreover, 331 crop samples, 331 cloacal samples and 174 dropping samples were collected. In addition, 17 dropping samples were taken from a total of 17 public squares. Samples were inoculated on Sabouraud dextrose agar with chloramphenicol. Different yeast species, i.e. Candida guilliermondii (24.36%), Candida kefyr (1.21%), Saccharomyces cerevisiae (2.43%), and Trichosporon asahii (1.21%) were isolated for the first time from the cloaca. The most frequently isolated yeast from the crop, cloaca and dropping samples from lofts was C. guilliermondii (30.46%, 24.36% and 49.37%, respectively). In addition, for the first time, C. kefyr (3.65%), Candida pelliculosa (2.43%), Candida rugosa (1.21%), T. asahii (3.65%), Trichosporon mucoides (3.65%) and Prototheca wickerhamii (1.21%) were obtained from crop samples; Candida pelliculosa (1.20%), T. asahii (9.63%) and T. mucoides (7.22%) were isolated from dropping samples in the lofts. Candida albicans was the most frequently isolated yeast in dropping samples collected in public squares. It can be assumed that pigeons and their droppings act as carriers and reservoirs of Candida spp. and other zoonotic yeasts. Copyright © 2017 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.

Development of cereal-based functional food using cereal-mix substrate fermented with probiotic strain - Pichia kudriavzevii OG32.

PubMed

Ogunremi, Omotade R; Agrawal, Renu; Sanni, Abiodun I

2015-11-01

Probiotic strains contribute to the functionality of foods during fermentation. In this present work, cereal-mix was fermented with probiotic Pichia kudriavzevii OG32. Selected fermentation parameters and functional properties of the product were determined. The growth of Pichia kudriavzevii OG32 was supported by the cereal-mix containing 1% salt and 0.2% red chili powder to counts of between 7.46 and 8.22 Log10 cfu/mL within 24 h. Pichia kudriavzevii OG32 increased the viscosity of cereal-mix with the highest inoculum size (1.84x105cfu/ml) giving the highest viscosity of 1793.6 mPa.S. An inoculum size of 1.98 × 10(4) cfu/mL gave the most acceptable product based on the sensory evaluation by the panelist. Forty volatile compounds were identified in the fermented product, while acids (32.21%) and esters (32.37%) accounted for the largest proportions. The cereal-based fermented product scavenged DPPH from 200 μmol/L methanolic solution by 55.71%. Probiotic yeast improved the sensory and some functional properties of cereal-based substrate during fermentation. This is one of the first reports on the volatile composition of cereal-based functional food produced with probiotic yeast.

Evaluation of a kinetic model for computer simulation of growth and fermentation by Scheffersomyces (Pichia) stipitis fed D-xylose

USDA-ARS?s Scientific Manuscript database

Scheffersomyces (formly Pichia) stipitis is a potential biocatalyst for converting lignocelluloses to ethanol because the yeast natively ferments xylose. An unstructured kinetic model based upon a system of linear differential equations has been formulated that describes growth and ethanol productio...

Seasonal variation of the upper digestive tract yeast flora of feral pigeons

USGS Publications Warehouse

Kocan, R.M.; Hasenclever, H.F.

1974-01-01

Feral pigeons were sampled over a 16-month period to determine whether their normal yeast flora varied according to season. Candida albicans and Saccharomyces telluris occurred during the entire sampling period, with C. albicans reaching its highest levels between August and January and S. telluris peaking from March through May. Candida krusei was present for 10 months but exhibited no predictable variation in density. Candida tropicalis, C. guilliermondii and Geotrichum were isolated on several occasions while C. lusitaniae, C. pseudotropicalis and Torulopsis glabrata were each isolated once. The high levels of infection and frequency of occurrence of some yeast species make the feral pigeon highly suspect as a carrier and disseminator of potentially pathogenic yeast.

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

Candida dajiaensis sp. nov., Candida yuanshanicus sp. nov., Candida jianshihensis sp. nov., and Candida sanyiensis sp. nov., four anamorphic, ascomycetous yeast species isolated from soil in Taiwan.

PubMed

Liu, Chun-Hao; Young, Shuh-Sen; Chang, Tsung-Chain; Lee, Ching-Fu

2008-08-01

Nine anamorphic, ascomycetous yeast strains belonging to the Pichia anomala clade were recovered from forest soil in 2006 in Taiwan. The nine yeast strains represent four novel yeast species based on the sequences of their D1/D2 domain of the large subunit (LSU) rRNA gene and their physiological characteristics. The scientific names of Candida dajiaensis sp. nov., Candida yuanshanicus sp. nov., Candida jianshihensis sp. nov., and Candida sanyiensis sp. nov. are proposed for these novel yeast species. The type strains are C. dajiaensis SM11S03(T) (=CBS 10590(T)=BCRC 23099(T)), C. yuanshanicus SY3S02(T) (=CBS 10589(T)=BCRC 23100(T)), C. jianshihensis SM8S04(T) (=CBS 10591(T)=BCRC 23096(T)), and C. sanyiensis SA1S06(T) (=CBS 10592(T)=BCRC 23094(T)). Sequence analysis of the D1/D2 of the LSU rRNA gene revealed that the three species, C. dajiaensis, C. yuanshanicus and Pichia onychis, shared a separate branch in the phylogenetic tree, C. jianshihensis is phylogenetically related to Candida ulmi and Pichia alni, and the phylogenetically closest relative of C. sanyiensis is Pichia populi.

Purification and characterization of an alpha-amylase of Pichia burtonii isolated from the traditional starter "murcha" in Nepal.

PubMed

Takeuchi, Akiko; Shimizu-Ibuka, Akiko; Nishiyama, Yoshitaka; Mura, Kiyoshi; Okada, Sanae; Tokue, Chiyoko; Arai, Soichi

2006-12-01

Among more than 20 yeast strains isolated from the traditional starter "murcha" in Nepal, we characterized a yeast that might be involved in saccharification. This strain, identified as Pichia burtonii, produced an extracellular amylolytic enzyme when cultured in the presence of starch in the medium. Since no amylase secreted by P. burtonii has yet been reported, we purified the enzyme and determined its N-terminal amino acid sequence. Together with the results of a hydrolyzing activity assay toward various substrates, it was found to be an alpha-amylase. The purified enzyme, named Pichia burtonii alpha-amylase (PBA), was a glycoprotein with an apparent molecular mass of 51 kDa. Enzyme activity was optimal at pH 5.0 at 40 degrees C. The enzyme retained 80% of its original activity after incubation under the optimal pH condition at 50 degrees C for 30 min. The activity was inhibited by metal ions such as Cd(2+), Cu(2+), Hg(2+), Al(3+), and Zn(2+).

Biological Control of Botrytis cinerea: Interactions with Native Vineyard Yeasts from Washington State.

PubMed

Wang, Xuefei; Glawe, Dean A; Kramer, Elizabeth; Weller, David; Okubara, Patricia A

2018-06-01

Native yeasts are of increasing interest to researchers, grape growers, and vintners because of their potential for biocontrol activity and their contributions to the aroma, flavor, and mouthfeel qualities of wines. To assess biocontrol activity, we tested 11 yeasts from Washington vineyards, representing isolates of Candida saitoana, Curvibasidium pallidicorallinum, Metschnikowia chrysoperlae, M. pulcherrima, Meyerozyma guilliermondii, Saccharomyces cerevisiae, and Wickerhamomyces anomalus, for ability to colonize Thompson Seedless grape berries, inhibit the growth of Botrytis cinerea in vitro, and suppress disease symptoms on isolated berries. The yeast-like fungus Aureobasidium pullulans was also included based on its known biocontrol activity against B. cinerea in studies on apple and grape. All yeast strains multiplied rapidly in grape berries and reached densities of over log 6 cells per wound as early as 2 days after inoculation with 200 cells. One of the Botrytis isolates used in this study was much less virulent than the others and was provisionally identified as B. prunorum based on multilocus sequence analysis. Suppression of the growth of B. cinerea isolates 111bb, 207a, 207cb, and 407cb occurred on berries treated with A. pullulans P01A006, Metschnikowia chrysoperlae P34A004 and P40A002, M. pulcherrima P01A016 and P01C004, Meyerozyma guilliermondii P34D003, and S. cerevisiae HNN11516. Inhibition of Botrytis isolates by the yeast strains was more common on berries than in vitro, suggesting the possibility that niche competition was a more likely biocontrol mechanism than antibiosis in planta. Metabolic profiling of yeast strains and B. cinerea isolates using Biolog YT plates revealed seven distinct metabolic groups. Furthermore, the yeast strains showed partial to complete tolerance to the commonly used fungicides fluopyram, triflumizole, metrafenone, pyraclostrobin, and boscalid. Implications of these findings for field deployment of native Washington

The Biology of Pichia membranifaciens Killer Toxins

PubMed Central

Belda, Ignacio; Ruiz, Javier; Alonso, Alejandro; Marquina, Domingo; Santos, Antonio

2017-01-01

The killer phenomenon is defined as the ability of some yeast to secrete toxins that are lethal to other sensitive yeasts and filamentous fungi. Since the discovery of strains of Saccharomyces cerevisiae capable of secreting killer toxins, much information has been gained regarding killer toxins and this fact has substantially contributed knowledge on fundamental aspects of cell biology and yeast genetics. The killer phenomenon has been studied in Pichia membranifaciens for several years, during which two toxins have been described. PMKT and PMKT2 are proteins of low molecular mass that bind to primary receptors located in the cell wall structure of sensitive yeast cells, linear (1→6)-β-d-glucans and mannoproteins for PMKT and PMKT2, respectively. Cwp2p also acts as a secondary receptor for PMKT. Killing of sensitive cells by PMKT is characterized by ionic movements across plasma membrane and an acidification of the intracellular pH triggering an activation of the High Osmolarity Glycerol (HOG) pathway. On the contrary, our investigations showed a mechanism of killing in which cells are arrested at an early S-phase by high concentrations of PMKT2. However, we concluded that induced mortality at low PMKT2 doses and also PMKT is indeed of an apoptotic nature. Killer yeasts and their toxins have found potential applications in several fields: in food and beverage production, as biocontrol agents, in yeast bio-typing, and as novel antimycotic agents. Accordingly, several applications have been found for P. membranifaciens killer toxins, ranging from pre- and post-harvest biocontrol of plant pathogens to applications during wine fermentation and ageing (inhibition of Botrytis cinerea, Brettanomyces bruxellensis, etc.). PMID:28333108

Application of Response Surface Methodology for Optimization of Extracellular Glucoamylase Production by Candida guilliermondii.

PubMed

Mohamed, Lagzouli; Kettani, Youssfi El; Ali, Aitounejjar; Mohamed, Elyachioui; Mohamed, Jadal

2017-01-01

Glucoamylase is among the most important enzymes in biotechnology. The present study aims to determine better conditions for growth and glucoamylase production by Candida guilliermondii and to reduce the overall cost of the medium using Box-Behnken design with one central point and response surface methodology. Box-Behnken factorial design based on three levels was carried out to obtain optimal medium combination of five independent variables such as initial pH, soluble starch, CH4N2O, yeast extract and MgSO4. Forty one randomized mediums were incubated in flask on a rotary shaker at 105 rpm for 72 h at 30°C. The production of biomass was found to be pH and starch dependent, maximum production when the starch concentration was 8 g L-1 and the initial pH was 6, while maximum glucoamylase production was found at 6.5 of initial pH, 4 g L-1 yeast extract and 6 g L-1 starch, whereas yeast extract and urea were highly significant, but interacted negatively. Box-Behnken factorial design used for the analysis of treatment combinations gave a second-order polynomial regression model with R2 = 0.976 for Biomass and R2 = 0.981 for glucoamylase. The final biomass and glucoamylase activity obtained was very close to the calculated parameters according to the p-values (p<0.001), the predicted optimal parameters were confirmed and provides a basis for further studies in baking additives and in the valuation of starch waste products.

Yeast microbiota of natural cavities of manatees (Trichechus inunguis and Trichechus manatus) in Brazil and its relevance for animal health and management in captivity.

PubMed

Sidrim, José Júlio Costa; Carvalho, Vitor Luz; Castelo-Branco, Débora de Souza Collares Maia; Brilhante, Raimunda Sâmia Nogueira; Bandeira, Tereza de Jesus Pinheiro Gomes; Cordeiro, Rossana de Aguiar; Guedes, Gláucia Morgana de Melo; Barbosa, Giovanna Riello; Lazzarini, Stella Maris; Oliveira, Daniella Carvalho Ribeiro; de Meirelles, Ana Carolina Oliveira; Attademo, Fernanda Löffler Niemeyer; Freire, Augusto Carlos da Bôaviagem; Moreira, José Luciano Bezerra; Monteiro, André Jalles; Rocha, Marcos Fábio Gadelha

2015-10-01

The aim of this study was to characterize the yeast microbiota of natural cavities of manatees kept in captivity in Brazil. Sterile swabs from the oral cavity, nostrils, genital opening, and rectum of 50 Trichechus inunguis and 26 Trichechus manatus were collected. The samples were plated on Sabouraud agar with chloramphenicol and incubated at 25 °C for 5 days. The yeasts isolated were phenotypically identified by biochemical and micromorphological tests. Overall, 141 strains were isolated, of which 112 were from T. inunguis (Candida albicans, Candida parapsilosis sensu stricto, Candida orthopsilosis, Candida metapsilosis, Candida guilliermondii, Candida pelliculosa, Candida tropicalis, Candida glabrata, Candida famata, Candida krusei, Candida norvegensis, Candida ciferri, Trichosporon sp., Rhodotorula sp., Cryptococcus laurentii) and 29 were from T. manatus (C. albicans, C. tropicalis, C. famata, C. guilliermondii, C. krusei, Rhodotorula sp., Rhodotorula mucilaginosa, Rhodotorula minuta, Trichosporon sp.). This was the first systematic study to investigate the importance of yeasts as components of the microbiota of sirenians, demonstrating the presence of potentially pathogenic species, which highlights the importance of maintaining adequate artificial conditions for the health of captive manatees.

The impact of yeast starter cultures on the microbial communities and volatile compounds in cocoa fermentation and the resulting sensory attributes of chocolate.

PubMed

Batista, Nádia Nara; Ramos, Cíntia Lacerda; Dias, Disney Ribeiro; Pinheiro, Ana Carla Marques; Schwan, Rosane Freitas

2016-02-01

Theobroma cacao seeds are the main raw material for chocolate production. During their fermentation, a succession of microorganisms are responsible for the physicochemical changes occurring in the pulp and inside the beans. The aim of this study was to investigate the effects of yeast inoculation (Saccharomyces cerevisiae UFLA CA11, Pichia kluivery CCMA0237, and Hanseniaspora uvarum CCMA0236) on the profile of the volatile compounds and microbial communities in cocoa fermentation. The resulting chocolate was also evaluated by temporal dominance of sensations (TDS) analyses. The dominant microorganisms during spontaneous fermentation were S. cerevisiae, H. uvarum, H. guilliermondii, Lactobacillus fermentum, Pediococcus sp., and Acetobacter pasteurianus. Similarly, S. cerevisiae, P. kluyveri, Candida sp., Pediococcus sp., and A. pasteurianus were the predominant microorganisms assessed by Denaturing Gradient Gel Electrophoresis (DGGE) in inoculated fermentation. Sixty-seven volatile compounds were detected and quantified by gas chromatography/mass spectrometry (GC/MS) at the end of fermentation and chocolates. The main group of volatile compound found after the inoculated and spontaneous fermentations was esters (41 and 39 %, respectively). In the chocolates, the main group was acids (73 and 44 % from the inoculated and spontaneous fermentations, respectively). The TDS analyses showed a dominance of bitter and cocoa attributes in both chocolates. However, in the inoculated chocolate, lingering fruity notes were more intense, while the chocolate produced by spontaneous fermentation was more astringent. Thus, the inoculation of yeast influenced the microbial profile, which likely affected the volatile compounds that affect sensory characteristics, resulting in chocolate with dominant bitter, cocoa, and fruity attributes.

Hansenula polymorpha (Pichia angusta): Biology and Applications

NASA Astrophysics Data System (ADS)

Kunze, Gotthard; Kang, Hyun Ah; Gellissen, Gerd

Hansenula polymorpha (Pichia angusta) belongs to a limited number of methylotrophic yeast species. It is able to assimilate nitrate and can grow on a range of carbon sources. Furthermore, H. polymorpha is a thermo-tolerant microorganism with some strains growing at temperatures up to 50° C and more. These unusual characteristics render H. polymorpha attractive as a model organism to study the development and functions of peroxisomes and the biochemistry of nitrate assimilation. H. polymorpha provides an established platform for heterologous gene expression and is distinguished by an impressive track record as producer of recom-binant proteins that include commercially available pharmaceuticals like hepatitis B vaccine, insulin and the IFN α-2a

Detection and identification of wild yeasts in Champús, a fermented Colombian maize beverage.

PubMed

Osorio-Cadavid, Esteban; Chaves-López, Clemencia; Tofalo, Rosanna; Paparella, Antonello; Suzzi, Giovanna

2008-09-01

The aim of this study was to identify and characterise the predominant yeasts in Champús, a traditional Colombian cereal-based beverage with a low alcoholic content. Samples of Champús from 20 production sites in the Cauca Valley region were analysed. A total of 235 yeast isolates were identified by conventional microbiological analyses and by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of ITS1-5.8S rDNA-ITS2. The dominant species were: Saccharomyces cerevisiae, Issatchenkia orientalis, Pichia fermentans, Pichia kluyveri var. kluyveri, Zygosaccharomyces fermentati, Torulospora delbruekii, Galactomyces geotrichum and Hanseniaspora spp. Model Champús systems were inoculated with single strains of some isolated sporogenus species and the aromatic profiles were analysed by SPME. Analysis of data showed that Champús strains produced high amounts of esters. The aromatic compounds produced by Saccharomyces and non-Saccharomyces yeasts from Champús can exert a relevant influence on the sensory characteristics of the fermented beverage. The Champús strains could thus represent an important source for new yeast biotypes with potential industrial applications.

Tolerance of pentose utilising yeast to hydrogen peroxide-induced oxidative stress.

PubMed

Spencer, Jennifer; Phister, Trevor G; Smart, Katherine A; Greetham, Darren

2014-03-17

Bioethanol fermentations follow traditional beverage fermentations where the yeast is exposed to adverse conditions such as oxidative stress. Lignocellulosic bioethanol fermentations involve the conversion of pentose and hexose sugars into ethanol. Environmental stress conditions such as osmotic stress and ethanol stress may affect the fermentation performance; however, oxidative stress as a consequence of metabolic output can also occur. However, the effect of oxidative stress on yeast with pentose utilising capabilities has yet to be investigated. Assaying for the effect of hydrogen peroxide-induced oxidative stress on Candida, Pichia and Scheffersomyces spp. has demonstrated that these yeast tolerate hydrogen peroxide-induced oxidative stress in a manner consistent with that demonstrated by Saccharomyces cerevisiae. Pichia guillermondii appears to be more tolerant to hydrogen peroxide-induced oxidative stress when compared to Candida shehatae, Candida succiphila or Scheffersomyces stipitis. Sensitivity to hydrogen peroxide-induced oxidative stress increased in the presence of minimal media; however, addition of amino acids and nucleobases was observed to increase tolerance. In particular adenine increased tolerance and methionine reduced tolerance to hydrogen peroxide-induced oxidative stress.

Tolerance of pentose utilising yeast to hydrogen peroxide-induced oxidative stress

PubMed Central

2014-01-01

Background Bioethanol fermentations follow traditional beverage fermentations where the yeast is exposed to adverse conditions such as oxidative stress. Lignocellulosic bioethanol fermentations involve the conversion of pentose and hexose sugars into ethanol. Environmental stress conditions such as osmotic stress and ethanol stress may affect the fermentation performance; however, oxidative stress as a consequence of metabolic output can also occur. However, the effect of oxidative stress on yeast with pentose utilising capabilities has yet to be investigated. Results Assaying for the effect of hydrogen peroxide-induced oxidative stress on Candida, Pichia and Scheffersomyces spp. has demonstrated that these yeast tolerate hydrogen peroxide-induced oxidative stress in a manner consistent with that demonstrated by Saccharomyces cerevisiae. Pichia guillermondii appears to be more tolerant to hydrogen peroxide-induced oxidative stress when compared to Candida shehatae, Candida succiphila or Scheffersomyces stipitis. Conclusions Sensitivity to hydrogen peroxide-induced oxidative stress increased in the presence of minimal media; however, addition of amino acids and nucleobases was observed to increase tolerance. In particular adenine increased tolerance and methionine reduced tolerance to hydrogen peroxide-induced oxidative stress. PMID:24636079

Characterisation of the gene cluster for L-rhamnose catabolism in the yeast Scheffersomyces (Pichia) stipitis

Treesearch

Outi M. Koivistoinen; Mikko Arvas; Jennifer R. Headman; Martina Andberg; Merja Penttilä; Thomas W. Jeffries; Peter Richard

2012-01-01

In Scheffersomyces (Pichia) stipitis and related fungal species the genes for L-rhamnose catabolism RHA1, LRA2, LRA3 and LRA4 but not LADH are clustered. We find that located next to the cluster is a transcription...

Taming wild yeast: potential of conventional and nonconventional yeasts in industrial fermentations.

PubMed

Steensels, Jan; Verstrepen, Kevin J

2014-01-01

Yeasts are the main driving force behind several industrial food fermentation processes, including the production of beer, wine, sake, bread, and chocolate. Historically, these processes developed from uncontrolled, spontaneous fermentation reactions that rely on a complex mixture of microbes present in the environment. Because such spontaneous processes are generally inconsistent and inefficient and often lead to the formation of off-flavors, most of today's industrial production utilizes defined starter cultures, often consisting of a specific domesticated strain of Saccharomyces cerevisiae, S. bayanus, or S. pastorianus. Although this practice greatly improved process consistency, efficiency, and overall quality, it also limited the sensorial complexity of the end product. In this review, we discuss how Saccharomyces yeasts were domesticated to become the main workhorse of food fermentations, and we investigate the potential and selection of nonconventional yeasts that are often found in spontaneous fermentations, such as Brettanomyces, Hanseniaspora, and Pichia spp.

Engineering of biotin-prototrophy in Pichia pastoris for robust production processes.

PubMed

Gasser, Brigitte; Dragosits, Martin; Mattanovich, Diethard

2010-11-01

Biotin plays an essential role as cofactor for biotin-dependent carboxylases involved in essential metabolic pathways. The cultivation of Pichia pastoris, a methylotrophic yeast that is successfully used as host for the production of recombinant proteins, requires addition of high dosage of biotin. As biotin is the only non-salt media component used during P. pastoris fermentation (apart from the carbon source), nonconformities during protein production processes are usually attributed to poor quality of the added biotin. In order to avoid dismissed production runs due to biotin quality issues, we engineered the biotin-requiring yeast P. pastoris to become a biotin-prototrophic yeast. Integration of four genes involved in the biotin biosynthesis from brewing yeast into the P. pastoris genome rendered P. pastoris biotin-prototrophic. The engineered strain has successfully been used as production host for both intracellular and secreted heterologous proteins in fed-batch processes, employing mineral media without vitamins. Another field of application for these truly prototrophic hosts is the production of biochemicals and small metabolites, where defined mineral media leads to easier purification procedures. Copyright © 2010 Elsevier Inc. All rights reserved.

Improved cell disruption of Pichia pastoris utilizing aminopropyl magnesium phyllosilicate (AMP) clay.

PubMed

Kim, Sun-Il; Wu, Yuanzheng; Kim, Ka-Lyun; Kim, Geun-Joong; Shin, Hyun-Jae

2013-06-01

An efficient method for Pichia cell disruption that employs an aminopropyl magnesium phyllosilicate (AMP) clay-assisted glass beads mill is presented. AMP clay is functionalized nanocomposite resembling the talc parent structure Si8Mg6O20(OH)4 that has been proven to permeate the bacterial membrane and cause cell lysis. The recombinant capsid protein of cowpea chlorotic mottle virus (CCMV) expressed in Pichia pastoris GS115 was used as demonstration system for their ability of self-assembly into icosahedral virus-like particles (VLPs). The total protein concentration reached 4.24 mg/ml after 4 min treatment by glass beads mill combined with 0.2 % AMP clay, which was 11.2 % higher compared to glass beads mill only and the time was half shortened. The stability of purified CCMV VLPs illustrated AMP clay had no influence on virus assembly process. Considering the tiny amount added and simple approach of AMP clay, it could be a reliable method for yeast cell disruption.

The Fermentative and Aromatic Ability of Kloeckera and Hanseniaspora Yeasts

NASA Astrophysics Data System (ADS)

Díaz-Montaño, Dulce M.; de Jesús Ramírez Córdova, J.

Spontaneous alcoholic fermentation from grape, agave and others musts into an alcoholic beverage is usually characterized by the presence of several non-Saccharomyces yeasts. These genera yeasts are dominant in the early stages of the alcoholic fermentation. However the genera Hanseniaspora and Kloeckera may survive at a significant level during fermentation and can influence the chemical composition of the beverage. Several strains belonging to the species Kloeckera api-culata and Hanseniaspora guilliermondii have been extensively studied in relation to the formation of some metabolic compounds affecting the bouquet of the final product. Indeed some apiculate yeast showed positive oenological properties and their use in the alcoholic fermentations has been suggested to enhance the aroma and flavor profiles. The non- Saccharomyces yeasts have the capability to produce and secrete enzymes in the medium, such as β -glucosidases, which release monoterpenes derived from their glycosylated form. These compounds contribute to the higher fruit-like characteristic of final product. This chapter reviews metabolic activity of Kloeckera and Hanseniaspora yeasts in several aspects: fermentative capability, aromatic compounds production and transformation of aromatic precursor present in the must, also covers the molecular methods for identifying of the yeast

A strain of Meyerozyma guilliermondii isolated from sugarcane juice is able to grow and ferment pentoses in synthetic and bagasse hydrolysate media.

PubMed

Martini, Cristina; Tauk-Tornisielo, Sâmia Maria; Codato, Carolina Brito; Bastos, Reinaldo Gaspar; Ceccato-Antonini, Sandra Regina

2016-05-01

The search for new microbial strains that are able to withstand inhibitors released from hemicellulosic hydrolysis and are also still able to convert sugars in ethanol/xylitol is highly desirable. A yeast strain isolated from sugarcane juice and identified as Meyerozyma guilliermondii was evaluated for the ability to grow and ferment pentoses in synthetic media and in sugarcane bagasse hydrolysate. The yeast grew in xylose, arabinose and glucose at the same rate at an initial medium pH of 5.5. At pH 4.5, the yeast grew more slowly in arabinose. There was no sugar exhaustion within 60 h. At higher xylose concentrations with a higher initial cell concentration, sugar was exhausted within 96 h at pH 4.5. An increase of 350 % in biomass was obtained in detoxified hydrolysates, whereas supplementation with 3 g/L yeast extract increased biomass production by approximately 40 %. Ethanol and xylitol were produced more significantly in supplemented hydrolysates regardless of detoxification. Xylose consumption was enhanced in supplemented hydrolysates and arabinose was consumed only when xylose and glucose were no longer available. Supplementation had a greater impact on ethanol yield and productivity than detoxification; however, the product yields obtained in the present study are still much lower when compared to other yeast species in bagasse hydrolysate. By the other hand, the fermentation of both xylose and arabinose and capability of withstanding inhibitors are important characteristics of the strain assayed.

The primary and subunit structure of a novel type killer toxin produced by a halotolerant yeast, Pichia farinosa.

PubMed

Suzuki, C; Nikkuni, S

1994-01-28

A halotolerant yeast, Pichia farinosa KK1 strain, produces a unique killer toxin termed SMK toxin (salt-mediated killer toxin) which shows its maximum killer activity in the presence of 2 M NaCl. The toxin consists of two distinct subunits, alpha and beta, which are tightly linked without a disulfide bond under acidic conditions, even in the presence of 6 M urea. Under neutral conditions, however, the alpha subunit precipitates, resulting in the dissociation of the subunits and the loss of killer activity. The nucleotide sequence of the SMK1 gene predicts a 222 amino acid preprotoxin with a typical signal sequence, the hydrophobic alpha, an interstitial gamma polypeptide with a putative glycosylation site, and the hydrophilic beta. Amino acid sequence analyses of peptide fragments including the carboxyl-terminal peptides fragments including the carboxyl-terminal peptides from each subunit suggest that the alpha and beta subunits consist of amino acid residues 19-81 and 146-222 of the preprotoxin, respectively, and the molecular weight of the mature alpha beta dimer is 14,214. The KEX2-like endopeptidase and KEX1-like carboxypeptidase may be involved in the stepwise processing of the SMK preprotoxin. The maturation process and the functions of the SMK toxin are compared with the K1 toxin of Saccharomyces cerevisiae.

Use of yeast (Pichia kudriavzevii) as a novel feed additive to ameliorate the effects of aflatoxin B1 on broiler chicken performance.

PubMed

Magnoli, A P; Rodriguez, M C; González Pereyra, M L; Poloni, V L; Peralta, M F; Nilson, A J; Miazzo, R D; Bagnis, G; Chiacchiera, S M; Cavaglieri, L R

2017-11-01

The aim of this study was to evaluate the efficacy of autochthonous Pichia kudriavzevii as a novel bioadsorbent for aflatoxin B 1 (AFB 1 ). The selection of this yeast was based on the AFB 1 adsorption capacity previously demonstrated in vitro (Magnoli et al. 2016). One-day-old Cobb broilers (n = 160) were randomly assigned to four dietary treatments (T1: basal diet (B); T2: B + 0.1% yeast; T3: B + AFB 1 , 100 μg/kg; T4: B + 0.1% yeast + AFB 1 , 100 μg/kg). Performance parameters (average daily weight gain body, average daily consumption, feed conversion ratio, carcass weight, and dead weight), biochemical parameters (albumin, globulin, and albumin/globulin), liver pathological changes, and AFB 1 residual levels in the liver and excreta were evaluated. Significant differences (P < 0.05) in performance parameters were observed among treatments and controls: T3 group showed the lowest average daily body weight gain value while in T4 group, the value of this parameter increased significantly (P < 0.05). T3 and T4 groups showed the lowest and highest values for average daily feed consumption, respectively. The feed conversion ratio (FC) showed no significant differences among treatments. T3 group showed the lowest dead weight and carcass weight compared with T1 group. The biochemical parameters showed no significant differences among treatments. T3 group showed macroscopic and microscopic liver changes compared to the control. Aflatoxin B 1 levels (μg/g) were detected in broiler livers and showed significant differences among treatments (P < 0.05). In conclusion, native P. kudriavzevii incorporation (0.1%) in broiler diets containing AFB 1 was shown to be effective in ameliorating the adverse effects of AFB 1 on production.

Identification of the major yeasts isolated from high moisture corn and corn silages in the United States using genetic and biochemical methods.

PubMed

Santos, M C; Golt, C; Joerger, R D; Mechor, G D; Mourão, Gerson B; Kung, L

2017-02-01

The objective of this study was to identify species of yeasts in samples of high moisture corn (HMC) and corn silage (CS) collected from farms throughout the United States. Samples were plated and colonies were isolated for identification using DNA analysis. Randomly selected colonies were also identified by fatty acid methyl esters (FAME) and by physiological substrate profiling (ID 32C). For CS, Candida ethanolica, Saccharomyces bulderi, Pichia anomala, Kazachstania unispora, and Saccharomyces cerevisiae were the predominant yeasts. Pichia anomala, Issatchenkia orientalis, S. cerevisiae, and Pichia fermentans were the prevalent species in HMC. The 3 identification methods were in agreement at the species level for 16.6% of the isolates and showed no agreement for 25.7%. Agreement in species identification between ID 32C and DNA analysis, FAME and ID 32C, and FAME and DNA analysis was 41.1, 14.4, and 2.2%, respectively. Pichia anomala and I. orientalis were able to grow on lactic acid, whereas S. cerevisiae metabolized sugars (galactose, sucrose, and glucose) but failed to use lactic acid. The yeast diversity in CS and HMC varied due to type of feed and location. Differences in species assignments were seen among methods, but identification using substrate profiling generally corresponded with that based on DNA analysis. These findings provide information about the species that may be expected in silages, and this knowledge may lead to interventions that control unwanted yeasts. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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

In situ microscopy for online monitoring of cell concentration in Pichia pastoris cultivations.

PubMed

Marquard, D; Enders, A; Roth, G; Rinas, U; Scheper, T; Lindner, P

2016-09-20

In situ Microscopy (ISM) is an optical non-invasive technique to monitor cells in bioprocesses in real-time. Pichia pastoris is one of the most promising protein expression systems. This yeast combines fast growth on simple media and important eukaryotic features such as glycosylation. In this work, the ISM technology was applied to Pichia pastoris cultivations for online monitoring of the cell concentration during cultivation. Different ISM settings were tested. The acquired images were analyzed with two image processing algorithms. In seven cultivations the cell concentration was monitored by the applied algorithms and offline samples were taken to determine optical density (OD) and dry cell mass (DCM). Cell concentrations up to 74g/L dry cell mass could be analyzed via the ISM. Depending on the algorithm and the ISM settings, an accuracy between 0.3 % and 12 % was achieved. The overall results show that for a robust measurement a combination of the two described algorithms is required. Copyright © 2016 Elsevier B.V. All rights reserved.

Biosorption of nickel by yeasts in an osmotically unsuitable environment.

PubMed

Breierová, Emilia; Certík, Milan; Kovárová, Annamaria; Gregor, Tomas

2008-01-01

The tolerance, sorption of nickel(II) ions, and changes in the production and composition of exopolymers of eight yeast strains grown under nickel presence with/without NaCl were studied. Strains of Pichia anomala and Candida maltosa known as the most resistant yeasts against nickel tolerated up to 3 mM Ni2+. NaCl addition decreased both the resistance of the yeast strains toward nickel ions and the sorption of metal ions into cells. All yeasts absorbed nickel predominantly into exopolymers (glycoproteins) and on the surface of cells. However, while the amount of polysaccharide moieties of exoglycoproteins of most of the resistant yeasts was induced by stress conditions, the ratio polysaccharide/protein in the exopolymers remained unchanged in the sensitive species Cystofilobasidium. The exopolymer composition might play a key role in yeast adaptation to stress conditions caused by heavy metal ions.

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.

Use of the Yeast Pichia pastoris as an Expression Host for Secretion of Enterocin L50, a Leaderless Two-Peptide (L50A and L50B) Bacteriocin from Enterococcus faecium L50▿

PubMed Central

Basanta, Antonio; Gómez-Sala, Beatriz; Sánchez, Jorge; Diep, Dzung B.; Herranz, Carmen; Hernández, Pablo E.; Cintas, Luis M.

2010-01-01

In this work, we report the expression and secretion of the leaderless two-peptide (EntL50A and EntL50B) bacteriocin enterocin L50 from Enterococcus faecium L50 by the methylotrophic yeast Pichia pastoris X-33. The bacteriocin structural genes entL50A and entL50B were fused to the Saccharomyces cerevisiae gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s) and cloned, separately and together (entL50AB), into the P. pastoris expression and secretion vector pPICZαA, which contains the methanol-inducible alcohol oxidase promoter (PAOX1) to express the fusion genes. After transfer into the yeast, the recombinant plasmids were integrated into the genome, resulting in three bacteriocinogenic yeast strains able to produce and secrete the individual bacteriocin peptides EntL50A and EntL50B separately and together. The secretion was efficiently directed by MFα1s through the Sec system, and the precursor peptides were found to be correctly processed to form mature and active bacteriocin peptides. The present work describes for the first time the heterologous expression and secretion of a two-peptide non-pediocin-like bacteriocin by a yeast. PMID:20348300

Towards systems metabolic engineering in Pichia pastoris.

PubMed

Schwarzhans, Jan-Philipp; Luttermann, Tobias; Geier, Martina; Kalinowski, Jörn; Friehs, Karl

2017-11-01

The methylotrophic yeast Pichia pastoris is firmly established as a host for the production of recombinant proteins, frequently outperforming other heterologous hosts. Already, a sizeable amount of systems biology knowledge has been acquired for this non-conventional yeast. By applying various omics-technologies, productivity features have been thoroughly analyzed and optimized via genetic engineering. However, challenging clonal variability, limited vector repertoire and insufficient genome annotation have hampered further developments. Yet, in the last few years a reinvigorated effort to establish P. pastoris as a host for both protein and metabolite production is visible. A variety of compounds from terpenoids to polyketides have been synthesized, often exceeding the productivity of other microbial systems. The clonal variability was systematically investigated and strategies formulated to circumvent untargeted events, thereby streamlining the screening procedure. Promoters with novel regulatory properties were discovered or engineered from existing ones. The genetic tractability was increased via the transfer of popular manipulation and assembly techniques, as well as the creation of new ones. A second generation of sequencing projects culminated in the creation of the second best functionally annotated yeast genome. In combination with landmark physiological insights and increased output of omics-data, a good basis for the creation of refined genome-scale metabolic models was created. The first application of model-based metabolic engineering in P. pastoris showcased the potential of this approach. Recent efforts to establish yeast peroxisomes for compartmentalized metabolite synthesis appear to fit ideally with the well-studied high capacity peroxisomal machinery of P. pastoris. Here, these recent developments are collected and reviewed with the aim of supporting the establishment of systems metabolic engineering in P. pastoris. Copyright © 2017. Published

Bioprospection of cold-adapted yeasts with biotechnological potential from Antarctica.

PubMed

Martorell, María Martha; Ruberto, Lucas Adolfo Mauro; Fernández, Pablo Marcelo; Castellanos de Figueroa, Lucía Inés; Mac Cormack, Walter Patricio

2017-06-01

The aim of this study was to investigate the ability to produce extracellular hydrolytic enzymes at low temperature of yeasts isolated from 25 de Mayo island, Antarctica, and to identify those exhibiting one or more of the evaluated enzymatic activities. A total of 105 yeast isolates were obtained from different samples and 66 were identified. They belonged to 12 basidiomycetous and four ascomycetous genera. Most of the isolates were ascribed to the genera Cryptococcus, Mrakia, Cystobasidium, Rhodotorula, Gueomyces, Phenoliferia, Leucosporidium, and Pichia. Results from enzymes production at low temperatures revealed that the Antarctic environment contains metabolically diverse cultivable yeasts, which represent potential tools for biotechnological applications. While most the isolates proved to produce 2-4 of the investigated exoenzymes, two of them evidenced the six evaluated enzymatic activities: Pichia caribbica and Guehomyces pullulans, which were characterized as psycrotolerant and psycrophilic, respectively. In addition, P. caribbica could assimilate several n-alkanes and diesel fuel. The enzyme production profile and hydrocarbons assimilation capacity, combined with its high level of biomass production and the extended exponential growth phase make P. caribbica a promising tool for cold environments biotechnological purposes in the field of cold-enzymes production and oil spills bioremediation as well. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Investigation on culturable microflora in Tibetan kefir grains from different areas of China.

PubMed

Gao, Jie; Gu, Fengying; Abdella, Nesredin H; Ruan, Hui; He, Guoqing

2012-08-01

Four samples of Tibetan kefir grains (TK-ZJUJ 01-04) from Tibet and surrounding areas were investigated via phenotypic and genotypic methods to compare and analyze the diversity of culturable microflora among different origins. As a result, 4 genera of microorganisms from TK-ZJUJ01: Bacillus subtilis (2.9 × 10(7) cfu/mL), Lactococcus lactis (8.2 × 10(7) cfu/mL), Kluyveromyces marxianus (3.0 × 10(6) cfu/mL), Saccharomyces cerevisiae (9.0 × 10(6) cfu/mL); 4 genera from TK-ZJUJ02: Lactobacillus kefiri (1.0 × 10(8) cfu/mL), Pichia kudriavzevii (5.0 × 10(6) cfu/mL), K. marxianus (1.9 × 10(7) cfu/mL), Kazachstania unispora (6.2 × 10(7) cfu/mL); 6 genera from TK-ZJUJ03: Leuconostoc lactis (4.6 × 10(7) cfu/mL), L. lactis (3.0 × 10(7) cfu/mL), Lactobacillus plantarum (3.0 × 10(7) cfu/mL), K. unispora (3.0 × 10(6) cfu/mL), K. marxianus (2.0 × 10(6) cfu/mL), (1.7 × 10(7) cfu/mL); and 4 genera from TK-ZJUJ04: L. plantarum (1.8 × 10(7) cfu/mL), Acetobacter fabarum (5.0 × 10(6) cfu/mL), K. unispora (6.2 × 10(7) cfu/mL), Pichia guilliermondii (6.2 × 10(7) cfu/mL) were identified. Yeasts like P. kudriavzevii and P. guilliermondii isolated in this study were the first time reported in Tibetan kefir grains. For TK-ZJUJ 01-03, lactic acid bacteria were the major microorganisms, which accounted for more than 50% of all the microbial population, while for TK-ZJUJ04, the largest microbial group was yeasts which accounted for more than 50%. In a word, study of diversity and composition of microflora provided us theoretical foundation for further investigation and application of Tibetan kefir grains. This is the basic research in order to develop and industrialize a new kind of yogurt starter which is naturally formed microbiota with both lactic acid bacteria and yeasts in it. © 2012 Institute of Food Technologists®

Effect of GAPDH-derived antimicrobial peptides on sensitive yeasts cells: membrane permeability, intracellular pH and H+-influx/-efflux rates.

PubMed

Branco, Patrícia; Albergaria, Helena; Arneborg, Nils; Prista, Catarina

2018-05-01

Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which induce the death of several non-Saccharomyces yeasts. Previously, we demonstrated that the naturally secreted GAPDH-derived AMPs (i.e. saccharomycin) caused a loss of culturability and decreased the intracellular pH (pHi) of Hanseniaspora guilliermondii cells. In this study, we show that chemically synthesised analogues of saccharomycin also induce a pHi drop and loss of culturability in H. guilliermondii, although to a lesser extent than saccharomycin. To assess the underlying causes of the pHi drop, we evaluated the membrane permeability to H+ cations of H. guilliermondii cells, after being exposed to saccharomycin or its synthetic analogues. Results showed that the H+-efflux decreased by 75.6% and the H+-influx increased by 66.5% in cells exposed to saccharomycin at pH 3.5. Since H+-efflux via H+-ATPase is energy dependent, reduced glucose consumption would decrease ATP production and consequently H+-ATPase activity. However, glucose uptake rates were not affected, suggesting that the AMPs rather than affecting glucose transporters may affect directly the plasma membrane H+-ATPase or increase ATP leakage due to cell membrane disturbance. Thus, our study revealed that both saccharomycin and its synthetic analogues induced cell death of H. guilliermondii by increasing the proton influx and inhibiting the proton efflux.

21 CFR 573.750 - Pichia pastoris dried yeast.

Code of Federal Regulations, 2010 CFR

2010-04-01

... the following percent-by-weight specifications: (1) Crude protein, not less than 60 percent. (2) Crude fat, not less than 2 percent. (3) Crude fiber, not more than 2 percent. (4) Ash, not more than 13... pastoris dried yeast may be used in feed formulations of broiler chickens as a source of protein not to...

21 CFR 573.750 - Pichia pastoris dried yeast.

Code of Federal Regulations, 2011 CFR

2011-04-01

... the following percent-by-weight specifications: (1) Crude protein, not less than 60 percent. (2) Crude fat, not less than 2 percent. (3) Crude fiber, not more than 2 percent. (4) Ash, not more than 13... pastoris dried yeast may be used in feed formulations of broiler chickens as a source of protein not to...

21 CFR 573.750 - Pichia pastoris dried yeast.

Code of Federal Regulations, 2012 CFR

2012-04-01

... the following percent-by-weight specifications: (1) Crude protein, not less than 60 percent. (2) Crude fat, not less than 2 percent. (3) Crude fiber, not more than 2 percent. (4) Ash, not more than 13... pastoris dried yeast may be used in feed formulations of broiler chickens as a source of protein not to...

21 CFR 573.750 - Pichia pastoris dried yeast.

Code of Federal Regulations, 2014 CFR

2014-04-01

... the following percent-by-weight specifications: (1) Crude protein, not less than 60 percent. (2) Crude fat, not less than 2 percent. (3) Crude fiber, not more than 2 percent. (4) Ash, not more than 13... pastoris dried yeast may be used in feed formulations of broiler chickens as a source of protein not to...

21 CFR 573.750 - Pichia pastoris dried yeast.

Code of Federal Regulations, 2013 CFR

2013-04-01

... the following percent-by-weight specifications: (1) Crude protein, not less than 60 percent. (2) Crude fat, not less than 2 percent. (3) Crude fiber, not more than 2 percent. (4) Ash, not more than 13... pastoris dried yeast may be used in feed formulations of broiler chickens as a source of protein not to...

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'.

Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris.

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 methylotrophic yeast used as an efficient expression system for heterologous protein production. In order to evaluate the effects of temperature (10 and 30 °C) and methanol (1 and 3% (v/v)) on genetically-modified Pichia pastoris, different biomarkers were evaluated: Heat stress (HSF-1 and Hsp70), oxidative stress (OGG1 and TBARS) and antioxidant (GLR). 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. Ours results show that at 3% methanol -30 °C there is an increase of mitochondrial OGG1 (mtOGG1), Glutathione Reductase (GLR) and TBARS. In addition, there was a cytosolic expression of HSF-1 and HSP-70, which indicates a deprotection against nucleolar fragmentation (apoptosis). On the other hand, at 3% methanol -10 °C and 1% and at methanol -10 °C conditions there was nuclear expression of OGG1, lower levels of TBARS and lower expression of GLR, cytosolic expression of HSF-1 and nuclear expression HSP-70. In conclusion, our results suggest that 3% methanol-30 °C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris.

Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris

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 methylotrophic yeast used as an efficient expression system for heterologous protein production. In order to evaluate the effects of temperature (10 and 30 °C) and methanol (1 and 3% (v/v)) on genetically-modified Pichia pastoris, different biomarkers were evaluated: Heat stress (HSF-1 and Hsp70), oxidative stress (OGG1 and TBARS) and antioxidant (GLR). 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. Ours results show that at 3% methanol −30 °C there is an increase of mitochondrial OGG1 (mtOGG1), Glutathione Reductase (GLR) and TBARS. In addition, there was a cytosolic expression of HSF-1 and HSP-70, which indicates a deprotection against nucleolar fragmentation (apoptosis). On the other hand, at 3% methanol −10 °C and 1% and at methanol −10 °C conditions there was nuclear expression of OGG1, lower levels of TBARS and lower expression of GLR, cytosolic expression of HSF-1 and nuclear expression HSP-70. In conclusion, our results suggest that 3% methanol-30 °C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris. PMID:25242930

Kinetic behavior of Candida guilliermondii yeast during xylitol production from Brewer's spent grain hemicellulosic hydrolysate.

PubMed

Mussatto, Solange I; Dragone, Giuliano; Roberto, Inês C

2005-01-01

Brewer's spent grain, the main byproduct of breweries, was hydrolyzed with dilute sulfuric acid to produce a hemicellulosic hydrolysate (containing xylose as the main sugar). The obtained hydrolysate was used as cultivation medium by Candidaguilliermondii yeast in the raw form (containing 20 g/L xylose) and after concentration (85 g/L xylose), and the kinetic behavior of the yeast during xylitol production was evaluated in both media. Assays in semisynthetic media were also performed to compare the yeast performance in media without toxic compounds. According to the results, the kinetic behavior of the yeast cultivated in raw hydrolysate was as effective as in semisynthetic medium containing 20 g/L xylose. However, in concentrated hydrolysate medium, the xylitol production efficiency was 30.6% and 42.6% lower than in raw hydrolysate and semisynthetic medium containing 85 g/L xylose, respectively. In other words, the xylose-to-xylitol bioconversion from hydrolysate medium was strongly affected when the initial xylose concentration was increased; however, similar behavior did not occur from semisynthetic media. The lowest efficiency of xylitol production from concentrated hydrolysate can be attributed to the high concentration of toxic compounds present in this medium, resulting from the hydrolysate concentration process.

An endogenous factor enhances ferulic acid decarboxylation catalyzed by phenolic acid decarboxylase from Candida guilliermondii

PubMed Central

2012-01-01

The gene for a eukaryotic phenolic acid decarboxylase of Candida guilliermondii was cloned, sequenced, and expressed in Escherichia coli for the first time. The structural gene contained an open reading frame of 504 bp, corresponding to 168 amino acids with a calculated molecular mass of 19,828 Da. The deduced amino sequence exhibited low similarity to those of functional phenolic acid decarboxylases previously reported from bacteria with 25-39% identity and to those of PAD1 and FDC1 proteins from Saccharomyces cerevisiae with less than 14% identity. The C. guilliermondii phenolic acid decarboxylase converted the main substrates ferulic acid and p-coumaric acid to the respective corresponding products. Surprisingly, the ultrafiltrate (Mr 10,000-cut-off) of the cell-free extract of C. guilliermondii remarkably activated the ferulic acid decarboxylation by the purified enzyme, whereas it was almost without effect on the p-coumaric acid decarboxylation. Gel-filtration chromatography of the ultrafiltrate suggested that an endogenous amino thiol-like compound with a molecular weight greater than Mr 1,400 was responsible for the activation. PMID:22217315

Identification of yeast strains isolated from marcha in Sikkim, a microbial starter for amylolytic fermentation.

PubMed

Tsuyoshi, Naoko; Fudou, Ryosuke; Yamanaka, Shigeru; Kozaki, Michio; Tamang, Namrata; Thapa, Saroj; Tamang, Jyoti P

2005-03-15

Marcha or murcha is a traditional amylolytic starter used to produce sweet-sour alcoholic drinks, commonly called jaanr in the Himalayan regions of India, Nepal, Bhutan, and Tibet (China). The aim of this study was to examine the microflora of marcha collected from Sikkim in India, focusing on yeast flora and their roles. Twenty yeast strains were isolated from six samples of marcha and identified by genetic and phenotypic methods. They were first classified into four groups (Group I, II, III, and IV) based on physiological features using an API test. Phylogenetic, morphological, and physiological characterization identified the isolates as Saccharomyces bayanus (Group I); Candida glabrata (Group II); Pichia anomala (Group III); and Saccharomycopsis fibuligera, Saccharomycopsis capsularis, and Pichia burtonii (Group IV). Among them, the Group I, II, and III strains produced ethanol. The isolates of Group IV had high amylolytic activity. Because all marcha samples tested contained both starch degraders and ethanol producers, it was hypothesized that all four groups of yeast (Group I, II, III, and IV) contribute to starch-based alcohol fermentation.

Secretion metabolites of probiotic yeast, Pichia kudriavzevii AS-12, induces apoptosis pathways in human colorectal cancer cell lines.

PubMed

Saber, Amir; Alipour, Beitollah; Faghfoori, Zeinab; Mousavi Jam, Ali; Yari Khosroushahi, Ahmad

2017-05-01

There is a common agreement on the important role of the gastrointestinal microbiota in the etiology of cancer. Benign probiotic yeast strains are able to ameliorate intestinal microbiota and regulate the host metabolism, physiology, and immune system through anti-inflammatory, antiproliferative, and anticancer effects. We hypothesized that Pichia kudriavzevii AS-12 secretion metabolites possess anticancer activity on human colorectal cancer cells (HT-29, Caco-2) via inhibiting growth and inducing apoptosis. This study aimed to assess the anticancer effect of P. kudriavzevii AS-12 secretion metabolites and the underlying mechanisms. The cytotoxicity evaluations were performed via 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay; 4',6-diamidino-2-phenylindole staining; and FACS-flow cytometry tests. Also, the effects of P. kudriavzevii AS-12 secretion metabolites on the expression level of 6 important genes (BAD, Bcl-2, Caspase-3, Caspase-8, Caspase-9 and Fas-R) involved in the extrinsic and intrinsic apoptosis pathways were studied by real-time polymerase chain reaction method. P. kudriavzevii AS-12 secretion metabolites showed significant (P < .0001) cytotoxic effects on HT-29 cells (57.5%) and Caco-2 (32.5%) compared to KDR/293 normal cells (25%). Moreover, the cytotoxic effects of examined yeast supernatant on HT-29 cells were comparable with 5-fluorouracil, as a positive control (57.5% versus 62.2% respectively). Flow cytometric results showed that the induction of apoptosis is the main mechanism of the anticancer effects. Also, according to the reverse transcriptase polymerase chain reaction results, the expression level of proapoptotic genes (BAD, Caspase-3, Caspase-8, Caspase-9, and Fas-R) in treated HT-29 and Caco-2 cells was higher than untreated and normal cells, whereas the antiapoptotic gene (Bcl-2) was downregulated. P. kudriavzevii AS-12 secretion metabolites exert its anticancer effects by inhibiting cell proliferation and inducing

Persistence of Two Non-Saccharomyces Yeasts (Hanseniaspora and Starmerella) in the Cellar

PubMed Central

Grangeteau, Cédric; Gerhards, Daniel; von Wallbrunn, Christian; Alexandre, Hervé; Rousseaux, Sandrine; Guilloux-Benatier, Michèle

2016-01-01

Different genera and/or species of yeasts present on grape berries, in musts and wines are widely described. Nevertheless, the community of non-Saccharomyces yeasts present in the cellar is still given little attention. Thus it is not known if the cellar is a real ecological niche for these yeasts or if it is merely a transient habitat for populations brought in by grape berries during the winemaking period. This study focused on three species of non-Saccharomyces yeasts commonly encountered during vinification: Starmerella bacillaris (synonymy with Candida zemplinina), Hanseniaspora guilliermondii and Hanseniaspora uvarum. More than 1200 isolates were identified at the strain level by FT-IR spectroscopy (207 different FTIR strain pattern). Only a small proportion of non-Saccharomyces yeasts present in musts came directly from grape berries for the three species studied. Some strains were found in the must in two consecutive years and some of them were also found in the cellar environment before the arrival of the harvest of second vintage. This study demonstrates for the first time the persistence of non-Saccharomyces yeast strains from year to year in the cellar. Sulfur dioxide can affect yeast populations in the must and therefore their persistence in the cellar environment. PMID:27014199

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

Valorization of kitchen biowaste for ethanol production via simultaneous saccharification and fermentation using co-cultures of the yeasts Saccharomyces cerevisiae and Pichia stipitis.

PubMed

Ntaikou, Ioanna; Menis, Nikolaos; Alexandropoulou, Maria; Antonopoulou, Georgia; Lyberatos, Gerasimos

2018-04-30

The biotransformation of the pre-dried and shredded organic fraction of kitchen waste to ethanol was investigated, via co-cultures of the yeasts Saccharomyces cerevisiae and Pichia stipitis (Scheffersomyces stipitis). Preliminary experiments with synthetic media were performed, in order to investigate the effect of different operational parameters on the ethanol production efficiency of the co-culture. The control of the pH and the supplementation with organic nitrogen were shown to be key factors for the optimization of the process. Subsequently, the ethanol production efficiency from the waste was assessed via simultaneous saccharification and fermentation experiments. Different loadings of cellulolytic enzymes and mixtures of cellulolytic with amylolytic enzymatic blends were tested in order to enhance the substrate conversion efficiency. It was further shown that for solids loading up to 40% waste on dry mass basis, corresponding to 170 g.L -1 initial concentration of carbohydrates, no substrate inhibition occurred, and ethanol concentration up to 45 g.L -1 was achieved. Copyright © 2018 Elsevier Ltd. All rights reserved.

Pichia anomala DBVPG 3003 Secretes a Ubiquitin-Like Protein That Has Antimicrobial Activity▿

PubMed Central

De Ingeniis, Jessica; Raffaelli, Nadia; Ciani, Maurizio; Mannazzu, Ilaria

2009-01-01

The yeast strain Pichia anomala DBVPG 3003 secretes a killer toxin (Pikt) that has antifungal activity against Brettanomyces/Dekkera sp. yeasts. Pikt interacts with β-1,6-glucan, consistent with binding to the cell wall of sensitive targets. In contrast to that of toxin K1, secreted by Saccharomyces cerevisiae, Pikt killer activity is not mediated by an increase in membrane permeability. Purification of the toxin yielded a homogeneous protein of about 8 kDa, which showed a marked similarity to ubiquitin in terms of molecular mass and N-terminal sequences. Pikt is also specifically recognized by anti-bovine ubiquitin antibodies and, similar to ubiquitin-like peptides, is not absorbed by DEAE-cellulose. However, Pikt differs from ubiquitin in its sensitivity to proteolytic enzymes. Therefore, Pikt appears to be a novel ubiquitin-like peptide that has killer activity. PMID:19114528

South Brazilian wines: culturable yeasts associated to bottled wines produced in Rio Grande do Sul and Santa Catarina.

PubMed

Ramírez-Castrillón, Mauricio; Mendes, Sandra Denise Camargo; Valente, Patricia

2017-04-01

A comprehensive understanding of the presence and role of yeasts in bottled wines helps to know and control the organoleptic quality of the final product. The South Region of Brazil is an important wine producer, and the state of "Rio Grande do Sul" (RS) accounts for 90% of Brazilian wines. The state of "Santa Catarina" (SC) started the production in 1975, and is currently the fifth Brazilian producer. As there is little information about yeasts present in Brazilian wines, our main objective was to assess the composition of culturable yeasts associated to bottled wines produced in RS and SC, South of Brazil. We sampled 20 RS and 29 SC bottled wines produced between 2003 and 2011, and we isolated culturable yeasts in non-selective agar plates. We identified all isolates by sequencing of the D1/D2 domain of LSU rDNA or ITS1-5.8 S-ITS2 region, and comparison with type strain sequences deposited in GenBank database. Six yeast species were shared in the final product in both regions. We obtained two spoilage yeast profiles: RS with Zygosaccharomyces bailii and Pichia membranifaciens (Dekkera bruxellensis was found only in specific table wines); and SC with Dekkera bruxellensis and Pichia manshurica. Knowledge concerning the different spoilage profiles is important for winemaking practices in both regions.

Evaluation of a kinetic model for computer simulation of growth and fermentation by Scheffersomyces (Pichia) stipitis fed D-xylose.

PubMed

Slininger, P J; Dien, B S; Lomont, J M; Bothast, R J; Ladisch, M R; Okos, M R

2014-08-01

Scheffersomyces (formerly Pichia) stipitis is a potential biocatalyst for converting lignocelluloses to ethanol because the yeast natively ferments xylose. An unstructured kinetic model based upon a system of linear differential equations has been formulated that describes growth and ethanol production as functions of ethanol, oxygen, and xylose concentrations for both growth and fermentation stages. The model was validated for various growth conditions including batch, cell recycle, batch with in situ ethanol removal and fed-batch. The model provides a summary of basic physiological yeast properties and is an important tool for simulating and optimizing various culture conditions and evaluating various bioreactor designs for ethanol production. © 2014 Wiley Periodicals, Inc.

Expression and characterization of the antimicrobial peptide ABP-dHC-cecropin A in the methylotrophic yeast Pichia pastoris.

PubMed

Sang, Ming; Wei, Hui; Zhang, Jiaxin; Wei, Zhiheng; Wu, Xiaolong; Chen, Yan; Zhuge, Qiang

2017-12-01

ABP-dHC-cecropin A is a linear cationic peptide that exhibits antimicrobial properties. To explore a new approach for expression of ABP-dHC-cecropin A using the methylotrophic yeast Pichia pastoris, we cloned the ABP-dHC-cecropin A gene into the vector pPICZαA. The SacI-linearized plasmid pPICZαA-ABP-dHC-cecropin A was then transformed into P. pastoris GS115 by electroporation. Expression was induced after a 96-h incubation with 0.5% methanol at 20 °C in a culture supplied with 2% casamino acids to avoid proteolysis. Under these conditions, approximately 48 mg of ABP-dHC-cecropin A was secreted into 1L (4 × 250-mL)of medium. Recombinant ABP-dHC-cecropin A was purified using size-exclusion chromatography, and 21 mg of pure active ABP-dHC-cecropin A was obtained from 1L (4 × 250-mL)of culture. Electrophoresis on 4-20% gradient gels indicated that recombinant ABP-dHC-cecropin A was secreted as a protein approximately 4 kDa in size. Recombinant ABP-dHC-cecropin A was successfully expressed, as the product displayed antibacterial and antifungal activities (based on an antibacterial assay, scanning electron microscopy, and antifungal assay) indistinguishable from those of the synthesized protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Production, characteristics and applications of the cell-bound phytase of Pichia anomala.

PubMed

Vohra, Ashima; Kaur, Parvinder; Satyanarayana, T

2011-01-01

Among several yeasts isolated from dried flowers of Woodfordia fruticosa, Pichia anomala produced a high titre of cell-bound phytase. The optimization of fermentation variables led to formulation of media and selection of cultural variables that supported enhanced phytase production. The enzyme productivity was very high in fed batch fermentation in air-lift fermentor as compared to that in stirred tank fermentor. Amelioration in the cell-bound phytase activity was observed when yeast cells were permeabilized with Triton-X-100. The enzyme is thermostable and acid stable with broad substrate specificity, the characteristics that are desirable for enzymes to be used in the animal feed industry. The phytase-encoding gene was cloned and sequenced. The 3D structure of the enzyme was proposed by comparative modeling using phytase of Debaryomyces occidentalis (50% sequence identity) as template. When broiler chicks, and fresh water and marine fishes were fed with the feed supplemented with yeast biomass containing phytase, improvement in growth and phosphorus retention, and decrease in the excretion of phosphorus in the faeces were recorded. The cell-bound phytase of P. anomala could effectively dephytinize wheat flour and soymilk.

Identification of uncommon oral yeasts from cancer patients by MALDI-TOF mass spectrometry.

PubMed

Aslani, Narges; Janbabaei, Ghasem; Abastabar, Mahdi; Meis, Jacques F; Babaeian, Mahasti; Khodavaisy, Sadegh; Boekhout, Teun; Badali, Hamid

2018-01-08

Opportunistic infections due to Candida species occur frequently in cancer patients because of their inherent immunosuppression. The aim of the present study was to investigate the epidemiology of yeast species from the oral cavity of patients during treatment for oncological and haematological malignancies. MALDI-TOF was performed to identify yeasts isolated from the oral cavity of 350 cancer patients. Moreover, antifungal susceptibility testing was performed in according to CLSI guidelines (M27-A3). Among 162 yeasts and yeast-like fungi isolated from the oral cavity of cancer patients, Candida albicans was the most common species (50.6%), followed by Candida glabrata (24.7%), Pichia kudriavzevii (Candida krusei (9.9%)), Candida tropicalis (4.3%), Candida dubliniensis (3.7%), Kluyveromyces marxianus (Candida kefyr (3.7%)) and Candida parapsilosis (1%). In addition, uncommon yeast species i.e., Saprochaete capitata, Saccharomyces cerevisiae, Clavispora lusitaniae (C. lusitaniae) and Pichia kluyveri (C. eremophila) were recovered from oral lesions. Oral colonization by C. albicans, non-albicans Candida species and uncommon yeasts were as follow; 55%, 44% and 1%, whereas oral infection due to C. albicans was 33.3%, non-albicans Candida species 60.6%, and uncommon yeasts 6.1%. Poor oral hygiene and xerostomia were identified as independent risk factors associated with oral yeast colonization. The overall resistance to fluconazole was 11.7% (19/162). Low MIC values were observed for anidulafungin for all Candida and uncommon yeast species. This current study provides insight into the prevalence and susceptibility profiles of Candida species, including emerging Candida species and uncommon yeasts, isolated from the oral cavity of Iranian cancer patients. The incidence of oral candidiasis was higher amongst patients with hematological malignancies. The majority of oral infections were caused by non-albicans Candida species which were often more resistant to anti

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.

Expression of enzymes for the usage in food and feed industry with Pichia pastoris.

PubMed

Spohner, Sebastian C; Müller, Hagen; Quitmann, Hendrich; Czermak, Peter

2015-05-20

The methylotrophic yeast Pichia pastoris is an established protein expression host for the production of industrial enzymes. This yeast can be grown to very high cell densities and produces high titers of recombinant protein, which can be expressed intercellularly or be secreted to the fermentation medium. P. pastoris offers some advantages over other established expression systems especially in protein maturation. In food and feed production many enzymatically catalyzed processes are reported and the demand for new enzymes grows continuously. For instance the unique catalytic properties of enzymes are used to improve resource efficiency, maintain quality, functionalize food, and to prevent off-flavors. This review aims to provide an overview on recent developments in heterologous production of enzymes with P. pastoris and their application within the food sector. Copyright © 2015 Elsevier B.V. All rights reserved.

Recent advances of molecular toolbox construction expand Pichia pastoris in synthetic biology applications.

PubMed

Kang, Zhen; Huang, Hao; Zhang, Yunfeng; Du, Guocheng; Chen, Jian

2017-01-01

Pichia pastoris: (reclassified as Komagataella phaffii), a methylotrophic yeast strain has been widely used for heterologous protein production because of its unique advantages, such as readily achievable high-density fermentation, tractable genetic modifications and typical eukaryotic post-translational modifications. More recently, P. pastoris as a metabolic pathway engineering platform has also gained much attention. In this mini-review, we addressed recent advances of molecular toolboxes, including synthetic promoters, signal peptides, and genome engineering tools that established for P. pastoris. Furthermore, the applications of P. pastoris towards synthetic biology were also discussed and prospected especially in the context of genome-scale metabolic pathway analysis.

Occurrence of mycotoxins and yeasts and moulds identification in corn silages in tropical climate.

PubMed

Carvalho, B F; Ávila, C L S; Krempser, P M; Batista, L R; Pereira, M N; Schwan, R F

2016-05-01

This study was aimed to identify yeasts and moulds as well as to detect mycotoxin in corn silages in southern Minas Gerais, Brazil. Corn silages from 36 farms were sampled to analyse dry matter, crude protein, ether extract, ash, neutral detergent fibre, nonfibre carbohydrates and mycotoxins contents, yeasts and moulds population, pH and temperature values. The mycotoxins found in high frequency were aflatoxin in 77·7% of analysed samples, ochratoxin (33·3%) and zearalenone (22·2%). There was no significant correlation between the mycotoxin concentration and the presence of moulds. The pH was negatively correlated with ochratoxin concentration. Aspergillus fumigatus was identified in all silages that presented growth of moulds. Ten different yeast species were identified using the culture-dependent method: Candida diversa, Candida ethanolica, Candida rugosa, Issatchenkia orientalis, Kluyveromyces marxianus, Pichia manshurica, Pichia membranifaciens, Saccharomyces cerevisiae, Trichosporon asahii and Trichosporon japonicum. Another six different yeast species were identified using the culture-independent method. A high mycotoxin contamination rate (91·6% of the analysed silages) was observed. The results indicated that conventional culturing and PCR-DGGE should be combined to optimally describe the microbiota associated with corn silage. This study provides information about the corn silage fermentation dynamics and our findings are relevant to optimization of this silage fermentation. © 2016 The Society for Applied Microbiology.

Inoculation of starter cultures in a semi-dry coffee (Coffea arabica) fermentation process.

PubMed

Evangelista, Suzana Reis; Miguel, Maria Gabriela da Cruz Pedrozo; Cordeiro, Cecília de Souza; Silva, Cristina Ferreira; Pinheiro, Ana Carla Marques; Schwan, Rosane Freitas

2014-12-01

The aim of this study was to evaluate the use of yeasts as starter cultures in coffee semi-dry processing. Arabica coffee was inoculated with one of the following starter cultures: Saccharomyces cerevisiae UFLA YCN727, S. cerevisiae UFLA YCN724, Candida parapsilosis UFLA YCN448 and Pichia guilliermondii UFLA YCN731. The control was not inoculated with a starter culture. Denaturing gradient gel electrophoresis (DGGE) was used to assess the microbial population, and organic acids and volatile compounds were quantified by HPLC and HS-SPME/GC, respectively. Sensory analyses were evaluated using the Temporal Dominance of Sensations (TDS). DGGE analysis showed that the inoculated yeasts were present throughout the fermentation. Other yeast species were also detected, including Debaryomyces hansenii, Cystofilobasidium ferigula and Trichosporon cavernicola. The bacterial population was diverse and was composed of the following genera: Weissella, Leuconostoc, Gluconobacter, Pseudomonas, Pantoea, Erwinia and Klebsiella. Butyric and propionic acids, were not detected in any treatment A total of 47 different volatiles compounds have been identified. The coffee inoculated with yeast had a caramel flavor that was not detected in the control, as assessed by TDS. The use of starter cultures during coffee fermentation is an interesting alternative for obtaining a beverage quality with distinctive flavor. Copyright © 2014 Elsevier Ltd. All rights reserved.

Expression and Purification of Rat Glucose Transporter 1 in Pichia pastoris.

PubMed

Venskutonytė, Raminta; Elbing, Karin; Lindkvist-Petersson, Karin

2018-01-01

Large amounts of pure and homogenous protein are a prerequisite for several biochemical and biophysical analyses, and in particular if aiming at resolving the three-dimensional protein structure. Here we describe the production of the rat glucose transporter 1 (GLUT1), a membrane protein facilitating the transport of glucose in cells. The protein is recombinantly expressed in the yeast Pichia pastoris. It is easily maintained and large-scale protein production in shaker flasks, as commonly performed in academic research laboratories, results in relatively high yields of membrane protein. The purification protocol describes all steps needed to obtain a pure and homogenous GLUT1 protein solution, including cell growth, membrane isolation, and chromatographic purification methods.

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.

Cloning of the Pichia anomala SEC61 gene and its expression in a Saccharomyces cerevisiae sec61 mutant.

PubMed

Ruíz, Teresa; De la Rosa, José M; Domínguez, Angel; Rodríguez, Luis

2003-05-01

In several organisms, including Saccharomyces cerevisiae and other yeast species, the product encoded by the SEC61 gene is considered to be the core element of the translocation apparatus within the endoplasmic reticulum membrane through which translocation of secretory and membrane proteins occurs. In this study, we have cloned and characterized the homolog of the SEC61 gene from the yeast Pichia anomala. The cloned gene includes an ORF, interrupted after the first ten nucleotides by an intron of 131 bp, encoding a 479-amino acid putative polypeptide exhibiting homology to the products encoded by different eukaryotic SEC61 genes, particularly to those from other yeast species. We show that the P. anomala SEC61 gene is correctly processed (intron splicing) when expressed in S. cerevisiae and that it is able to complement the thermosensitive phenotype associated with a mutation in the S. cerevisiae SEC61 gene.

Analysis of the yeast short-term Crabtree effect and its origin

PubMed Central

Hagman, Arne; Säll, Torbjörn; Piškur, Jure

2014-01-01

The short-term Crabtree effect is defined as the immediate occurrence of aerobic alcoholic fermentation in response to provision of a pulse of excess sugar to sugar-limited yeast cultures. Here we have characterized ten yeast species with a clearly defined phylogenetic relationship. Yeast species were cultivated under glucose-limited conditions, and we studied their general carbon metabolism in response to a glucose pulse. We generated an extensive collection of data on glucose and oxygen consumption, and ethanol and carbon dioxide generation. We conclude that the Pichia,Debaryomyces,Eremothecium and Kluyveromyces marxianus yeasts do not exhibit any significant ethanol formation, while Kluyveromyces lactis behaves as an intermediate yeast, and Lachancea,Torulaspora,Vanderwaltozyma and Saccharomyces yeasts exhibit rapid ethanol accumulation. Based on the present data and our previous data relating to the presence of the long-term Crabtree effect in over 40 yeast species, we speculate that the origin of the short-term effect may coincide with the origin of the long-term Crabtree effect in the Saccharomycetales lineage, occurring ∼ 150 million years ago. PMID:25161062

The secretory pathway: exploring yeast diversity.

PubMed

Delic, Marizela; Valli, Minoska; Graf, Alexandra B; Pfeffer, Martin; Mattanovich, Diethard; Gasser, Brigitte

2013-11-01

Protein secretion is an essential process for living organisms. In eukaryotes, this encompasses numerous steps mediated by several hundred cellular proteins. The core functions of translocation through the endoplasmic reticulum membrane, primary glycosylation, folding and quality control, and vesicle-mediated secretion are similar from yeasts to higher eukaryotes. However, recent research has revealed significant functional differences between yeasts and mammalian cells, and even among diverse yeast species. This review provides a current overview of the canonical protein secretion pathway in the model yeast Saccharomyces cerevisiae, highlighting differences to mammalian cells as well as currently unresolved questions, and provides a genomic comparison of the S. cerevisiae pathway to seven other yeast species where secretion has been investigated due to their attraction as protein production platforms, or for their relevance as pathogens. The analysis of Candida albicans, Candida glabrata, Kluyveromyces lactis, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, and Schizosaccharomyces pombe reveals that many - but not all - secretion steps are more redundant in S. cerevisiae due to duplicated genes, while some processes are even absent in this model yeast. Recent research obviates that even where homologous genes are present, small differences in protein sequence and/or differences in the regulation of gene expression may lead to quite different protein secretion phenotypes. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Carbohydrate and energy-yielding metabolism in non-conventional yeasts.

PubMed

Flores, C L; Rodríguez, C; Petit, T; Gancedo, C

2000-10-01

Sugars are excellent carbon sources for all yeasts. Since a vast amount of information is available on the components of the pathways of sugar utilization in Saccharomyces cerevisiae it has been tacitly assumed that other yeasts use sugars in the same way. However, although the pathways of sugar utilization follow the same theme in all yeasts, important biochemical and genetic variations on it exist. Basically, in most non-conventional yeasts, in contrast to S. cerevisiae, respiration in the presence of oxygen is prominent for the use of sugars. This review provides comparative information on the different steps of the fundamental pathways of sugar utilization in non-conventional yeasts: glycolysis, fermentation, tricarboxylic acid cycle, pentose phosphate pathway and respiration. We consider also gluconeogenesis and, briefly, catabolite repression. We have centered our attention in the genera Kluyveromyces, Candida, Pichia, Yarrowia and Schizosaccharomyces, although occasional reference to other genera is made. The review shows that basic knowledge is missing on many components of these pathways and also that studies on regulation of critical steps are scarce. Information on these points would be important to generate genetically engineered yeast strains for certain industrial uses.

Metabolism of the Fusarium mycotoxins zearalenone and deoxynivalenol by yeast strains of technological relevance.

PubMed

Böswald, C; Engelhardt, G; Vogel, H; Wallnöfer, P R

1995-01-01

The Fusarium mycotoxin zearalenone (ZEA), added at a level of 2 micrograms/ml, was reduced stereoselectively by cultures of Candida tropicalis, Torulaspora delbrückii, Zygosaccharomyces rouxii, and 7 Saccharomyces strains to both alpha- and beta-zearalenol. In contrast, only alpha-zearalenol was produced from ZEA by Pichia fermentans and several yeast strains of the genera Candida, Hansenula, Brettanomyces, Schizosaccharomyces, and Saccharomycopsis. No glucose conjugates of ZEA (zearalenone-4-beta-D-glucopyranoside) were detected. The trichothecene mycotoxin deoxynivalenol (DON) was not metabolized by any of the yeast strains that were used for analysis.

Methanol Expression Regulator 1 (Mxr1p) Is Essential for the Utilization of Amino Acids as the Sole Source of Carbon by the Methylotrophic Yeast, Pichia pastoris.

PubMed

Sahu, Umakant; Rangarajan, Pundi N

2016-09-23

Unlike Saccharomyces cerevisiae, the methylotrophic yeast Pichia pastoris can assimilate amino acids as the sole source of carbon and nitrogen. It can grow in media containing yeast extract and peptone (YP), yeast nitrogen base (YNB) + glutamate (YNB + Glu), or YNB + aspartate (YNB + Asp). Methanol expression regulator 1 (Mxr1p), a zinc finger transcription factor, is essential for growth in these media. Mxr1p regulates the expression of several genes involved in the utilization of amino acids as the sole source of carbon and nitrogen. These include the following: (i) GDH2 encoding NAD-dependent glutamate dehydrogenase; (ii) AAT1 and AAT2 encoding mitochondrial and cytosolic aspartate aminotransferases, respectively; (iii) MDH1 and MDH2 encoding mitochondrial and cytosolic malate dehydrogenases, respectively; and (iv) GLN1 encoding glutamine synthetase. Synthesis of all these enzymes is regulated by Mxr1p at the level of transcription except GDH2, whose synthesis is regulated at the level of translation. Mxr1p activates the transcription of AAT1, AAT2, and GLN1 in cells cultured in YP as well as in YNB + Glu media, whereas transcription of MDH1 and MDH2 is activated in cells cultured in YNB + Glu but not in YP. A truncated Mxr1p composed of 400 N-terminal amino acids activates transcription of target genes in cells cultured in YP but not in YNB + Glu. Mxr1p binds to Mxr1p response elements present in the promoters of AAT2, MDH2, and GLN1 We conclude that Mxr1p is essential for utilization of amino acids as the sole source of carbon and nitrogen, and it is a global regulator of multiple metabolic pathways in P. pastoris. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Genomic sequence of the xylose fermenting, insect-inhabitingyeast, Pichia stipitis

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

Jeffries, Thomas W.; Grigoriev, Igor; Grimwood, Jane

2007-06-25

Xylose is a major constituent of angiosperm lignocellulose,so its fermentation is important for bioconversion to fuels andchemicals. Pichia stipitis is the best-studied native xylose fermentingyeast. Genes from P. stipitis have been used to engineer xylosemetabolism in Saccharomycescerevisiae, and the regulation of the P.stipitis genome offers insights into the mechanisms of xylose metabolismin yeasts. We have sequenced, assembled and finished the genome ofP.stipitis. As such, it is one of only a handful of completely finishedeukaryotic organisms undergoing analysis and manual curation. Thesequence has revealed aspects of genome organization, numerous genes forbiocoversion, preliminary insights into regulation of central metabolicpathways, numerous examples ofmore » co-localized genes with related functions,and evidence of how P. stipitis manages to achieve redox balance whilegrowing on xylose under microaerobic conditions.« less

Expression of CB2 cannabinoid receptor in Pichia pastoris.

PubMed

Feng, Wenke; Cai, Jian; Pierce, William M; Song, Zhao-Hui

2002-12-01

To facilitate purification and structural characterization, the CB2 cannabinoid receptor is expressed in methylotrophic yeast Pichia pastoris. The expression plasmids were constructed in which the CB2 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase 1 gene. A c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB2 to permit easy detection and purification. In membrane preparations of CB2 gene transformed yeast cells, Western blot analysis detected the expression of CB2 proteins. Radioligand binding assays demonstrated that the CB2 receptors expressed in P. pastoris have a pharmacological profile similar to that of the receptors expressed in mammalian systems. Furthermore, the epitope-tagged receptor was purified by metal chelating chromatography and the purified CB2 preparations were subjected to digestion by trypsin. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions detected 14 peptide fragments derived from the CB2 receptor. ESI mass spectrometry was used to sequence one of these peptide fragments, thus, further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope-tagged, functional CB2 cannabinoid receptor can be expressed in P. pastoris for purification.

[Effect of phenolic ketones on ethanol fermentation and cellular lipid composition of Pichia stipitis].

PubMed

Yang, Jinlong; Cheng, Yichao; Zhu, Yuanyuan; Zhu, Junjun; Chen, Tingting; Xu, Yong; Yong, Qiang; Yu, Shiyuan

2016-02-01

Lignin degradation products are toxic to microorganisms, which is one of the bottlenecks for fuel ethanol production. We studied the effects of phenolic ketones (4-hydroxyacetophenone, 4-hydroxy-3-methoxy-acetophenone and 4-hydroxy-3,5-dimethoxy-acetophenone) derived from lignin degradation on ethanol fermentation of xylose and cellular lipid composition of Pichia stipitis NLP31. Ethanol and the cellular fatty acid of yeast were analyzed by high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). Results indicate that phenolic ketones negatively affected ethanol fermentation of yeast and the lower molecular weight phenolic ketone compound was more toxic. When the concentration of 4-hydroxyacetophenone was 1.5 g/L, at fermentation of 24 h, the xylose utilization ratio, ethanol yield and ethanol concentration decreased by 42.47%, 5.30% and 9.76 g/L, respectively, compared to the control. When phenolic ketones were in the medium, the ratio of unsaturated fatty acids to saturated fatty acids (UFA/SFA) of yeast cells was improved. When 1.5 g/L of three aforementioned phenolic ketones was added to the fermentation medium, the UFA/SFA ratio of yeast cells increased to 3.03, 3.06 and 3.61, respectively, compared to 2.58 of the control, which increased cell membrane fluidity and instability. Therefore, phenolic ketones can reduce the yeast growth, increase the UFA/SFA ratio of yeast and lower ethanol productivity. Effectively reduce or remove the content of lignin degradation products is the key to improve lignocellulose biorefinery.

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.

[Yeast diversity in Bulnesia retama and Larrea divaricata canopies and associated soils].

PubMed

Toro, M E; Oro, N P; Vega, A D; Maturano, Y P; Nally, M C; Fernandez, E; Pucheta, E; Vázquez, F

2005-01-01

Bush like vegetation dominates arid environments, and there is nutrients accumulation under shrub canopies and relatively unfertile soils between vegetal patches areas. Plants are one of the most common habitats for yeasts. There are many reports about yeasts inhabiting different plant components. Nevertheless, there are no reports about yeasts associated with Zygophyllaceae, an important shrub family of the Argentinean Province of Monte. The objective of this work was to analyzed yeast biodiversity of Bulnesia retama and Larrea divaricata canopies and associated soils, at Medanos Grandes of Caucete, San Juan, Argentina. Eighty seven (87) isolated yeasts were identified. From B. retama canopy and associated soil was observed a larger taxonomical diversity respect to L. divaricata. Nine (9) and ten (10) species were isolated from canopy and associated soil of B. retama, respectively. From L. divaricata canopy were 4 species and 3 species from its associated soil isolated. Identified genera were: Candida, Debaryomyces, Dekkera, Saccharomyces, Torulaspora, Sporidiobolus and Pichia. Fourteen (14) species were found at all microenvironments.

Identification of differentially expressed genes associated with changes in the morphology of Pichia fermentans on apple and peach fruit.

PubMed

Fiori, Stefano; Scherm, Barbara; Liu, Jia; Farrell, Robert; Mannazzu, Ilaria; Budroni, Marilena; Maserti, Bianca E; Wisniewski, Michael E; Migheli, Quirico

2012-11-01

Pichia fermentans (strain DISAABA 726) is an effective biocontrol agent against Monilinia fructicola and Botrytis cinerea when inoculated in artificially wounded apple fruit but is an aggressive pathogen when inoculated on wounded peach fruit, causing severe fruit decay. Pichia fermentans grows as budding yeast on apple tissue and exhibits pseudohyphal growth on peach tissue, suggesting that dimorphism may be associated with pathogenicity. Two complementary suppressive subtractive hybridization (SSH) strategies, that is, rapid subtraction hybridization (RaSH) and PCR-based subtraction, were performed to identify genes differentially expressed by P. fermentans after 24-h growth on apple vs. peach fruit. Gene products that were more highly expressed on peach than on apple tissue, or vice versa, were sequenced and compared with available yeast genome sequence databases. Several of the genes more highly expressed, when P. fermentans was grown on peach, were related to stress response, glycolysis, amino acid metabolism, and alcoholic fermentation but surprisingly not to cell wall degrading enzymes such as pectinases or cellulases. The dual activity of P. fermentans as both a biocontrol agent and a pathogen emphasizes the need for a thorough risk analysis of potential antagonists to avoid unpredictable results that could negatively impact the safe use of postharvest biocontrol strategies. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Nutrient Effects on Biocontrol of Penicillium roqueforti by Pichia anomala J121 during Airtight Storage of Wheat

PubMed Central

Druvefors, Ulrika Ädel; Passoth, Volkmar; Schnürer, Johan

2005-01-01

The biocontrol yeast Pichia anomala inhibits the growth of a variety of mold species. We examined the mechanism underlying the inhibition of the grain spoilage mold Penicillium roqueforti by the biocontrol yeast P. anomala J121 during airtight storage. The biocontrol effect in a model grain silo with moist wheat (water activity of 0.96) was enhanced when complex medium, maltose, or glucose was added. Supplementation with additional nitrogen or vitamin sources did not affect the biocontrol activity of the yeast. The addition of complex medium or glucose did not significantly influence the yeast cell numbers in the silos, whether in the presence or absence of P. roqueforti. Mold growth was not influenced by the addition of nutrients, if cultivated without yeast. The products of glucose metabolism, mainly ethanol and ethyl acetate, increased after glucose addition to P. anomala-inoculated treatments. Our results suggest that neither competition for nutrients nor production of a glucose-repressible cell wall lytic enzyme is the main mode of action of biocontrol by P. anomala in this grain system. Instead, the mold-inhibiting effect probably is due to the antifungal action of metabolites, most likely a combination of ethyl acetate and ethanol, derived from glycolysis. The discovery that sugar amendments enhance the biocontrol effect of P. anomala suggests novel ways of formulating biocontrol yeasts. PMID:15812013

Feasibility of brewing makgeolli using Pichia anomala Y197-13, a non-Saccharomyces cerevisiae.

PubMed

Kim, Hye Ryun; Kim, Jae-Ho; Bai, Dong-Hoon; Ahn, ByungHak

2012-12-01

Makgeolli is a traditional rice wine favored by the general public in Korea. This study investigated the fermentation and sensory characteristics of using wild yeast strains for brewing makgeolli. A non-Saccharomyces cerevisiae strain was isolated from nuruk and termed Y197-13. It showed 98% similarity to Pichia anomala and had an optimal growth temperature of 25 degrees C. Makgeolli was manufactured using koji, jinju nuruk, and improved nuruk as fermentation agents. Y197-13 makgeolli brewed with koji had alcohol and solids contents of 11.1% and 13.9%, respectively. Sweet sensory characteristics were attributed to residual sugars in makgeolli with 6% alcohol. The makgeolli had a fresh sour taste and carbonated taste. Volatile component analysis showed the isoamyl alcohol, phenylethyl alcohol, isoamyl acetate, and fatty acid, including ethyl oleate and ethyl linoleate, relative peak area was higher in Y197-13 makgeolli than in makgeolli with Saccharomyces cerevisiae. These results suggest the wild yeast, Y197-13, as a candidate for brewing makgeolli.

Technical note: use of internal transcribed spacer for ruminal yeast identification in dairy cows.

PubMed

Vargas-Bello-Pérez, E; Cancino-Padilla, N; Romero, J

2016-12-01

Molecular techniques are important tools for microbiological studies in different habitats, and the internal transcribed spacer (ITS) has been proved to be useful for analyzing fungal diversity. The aim of this study was to use the ITS region to generate ruminal yeast profile and to identify ruminal yeast. DNA from ruminal digesta was extracted to amplify the ribosomal ITS region. The profile from the PCR products was visualized and the excised bands from the profile were identified as the genera Millerozyma, Pichia, Rhizomucor and Hyphopichia. Overall, the ITS resulted to be a simple, fast and sensitive approach that allowed profiling and identification of ruminal yeast that have not been previously described (Millerozyma and Hyphopichia) in the rumen microbial community.

Extraction of genomic DNA from yeasts for PCR-based applications.

PubMed

Lõoke, Marko; Kristjuhan, Kersti; Kristjuhan, Arnold

2011-05-01

We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)-SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 10(7) cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤ 3500 bp.

Xylose reductase and xylitol dehydrogenase activities of Candida guilliermondii as a function of different treatments of sugarcane bagasse hemicellulosic hydrolysate employing experimental design.

PubMed

Alves, Lourdes A; Vitolo, Michele; Felipe, Maria das Graças A; de Almeida e Silva, João Batista

2002-01-01

The sugarcane bagasse hydrolysate, which is rich in xylose, can be used as culture medium for Candida guilliermondii in xylitol production. However, the hydrolysate obtained from bagasse by acid hydrolysis at 120 degrees C for 20 min has by-products (acetic acid and furfural, among others), which are toxic to the yeast over certain concentrations. So, the hydrolysate must be pretreated before using in fermentation. The pretreatment variables considered were: adsorption time (15,37.5, and 60 min), type of acid used (H2So4 and H3Po4), hydrolysate concentration (original, twofold, and fourfold concentrated), and active charcoal (0.5, 1.75 and 3.0%). The suitability of the pretreatment was followed by measuring the xylose reductase (XR) and xylitol dehydrogenase (XD) activity of yeast grown in each treated hydrolysate. The response surface methodology (2(4) full factorial design with a centered face) indicated that the hydrolysate might be concentrated fourfold and the pH adjusted to 7.0 with CaO, followed by reduction to 5.5 with H3PO4. After that it was treated with active charcoal (3.0%) by 60 min. This pretreated hydrolysate attained the high XR/XD ratio of 4.5.

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris.

PubMed

Vogl, Thomas; Gebbie, Leigh; Palfreyman, Robin W; Speight, Robert

2018-03-15

Pichia pastoris (syn. Komagataella phaffii ) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to Saccharomyces cerevisiae , where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in P. pastoris and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of >700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in P. pastoris Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (<10%) harboring ectopically integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs). IMPORTANCE Yeasts are commonly used as biotechnological production hosts for proteins and

Native Killer Yeasts as Biocontrol Agents of Postharvest Fungal Diseases in Lemons.

PubMed

Perez, María Florencia; Contreras, Luciana; Garnica, Nydia Mercedes; Fernández-Zenoff, María Verónica; Farías, María Eugenia; Sepulveda, Milena; Ramallo, Jacqueline; Dib, Julián Rafael

2016-01-01

Economic losses caused by postharvest diseases represent one of the main problems of the citrus industry worldwide. The major diseases affecting citrus are the "green mold" and "blue mold", caused by Penicillium digitatum and P. italicum, respectively. To control them, synthetic fungicides are the most commonly used method. However, often the emergence of resistant strains occurs and their use is becoming more restricted because of toxic effects and environmental pollution they generate, combined with trade barriers to international markets. The aim of this work was to isolate indigenous killer yeasts with antagonistic activity against fungal postharvest diseases in lemons, and to determine their control efficiency in in vitro and in vivo assays. Among 437 yeast isolates, 8.5% show to have a killer phenotype. According to molecular identification, based on the 26S rDNA D1/D2 domain sequences analysis, strains were identified belonging to the genera Saccharomyces, Wickerhamomyces, Kazachstania, Pichia, Candida and Clavispora. Killers were challenged with pathogenic molds and strains that caused the maximum in vitro inhibition of P. digitatum were selected for in vivo assays. Two strains of Pichia and one strain of Wickerhamomyces depicted a significant protection (p <0.05) from decay by P. digitatum in assays using wounded lemons. Thus, the native killer yeasts studied in this work showed to be an effective alternative for the biocontrol of postharvest fungal infections of lemons and could be promising agents for the development of commercial products for the biological control industry.

Characterization of culturable yeast species associating with whole crop corn and total mixed ration silage

PubMed Central

Wang, Huili; Hao, Wei; Ning, Tingting; Zheng, Mingli; Xu, Chuncheng

2018-01-01

Objective This study investigated the association of yeast species with improved aerobic stability of total mixed ration (TMR) silages with prolonged ensiling, and clarified the characteristics of yeast species and their role during aerobic deterioration. Methods Whole crop corn (WCC) silages and TMR silages formulated with WCC were ensiled for 7, 14, 28, and 56 d and used for an aerobic stability test. Predominant yeast species were isolated from different periods and identified by sequencing analyses of the 26S rRNA gene D1/D2 domain. Characteristics (assimilation and tolerance) of the yeast species and their role during aerobic deterioration were investigated. Results In addition to species of Candida glabrata and Pichia kudriavzevii (P. kudriavzevii) previously isolated in WCC and TMR, Pichia manshurica (P. manshurica), Candida ethanolica (C. ethanolica), and Zygosaccharomyces bailii (Z. bailii) isolated at great frequency during deterioration, were capable of assimilating lactic or acetic acid and tolerant to acetic acid and might function more in deteriorating TMR silages at early fermentation (7 d and 14 d). With ensiling prolonged to 28 d, silages became more (p<0.01) stable when exposed to air, coinciding with the inhibition of yeast to below the detection limit. Species of P. manshurica that were predominant in deteriorating WCC silages were not detectable in TMR silages. In addition, the predominant yeast species of Z. bailii in deteriorating TMR silages at later fermentation (28 d and 56 d) were not observed in both WCC and WCC silages. Conclusion The inhibition of yeasts, particularly P. kudriavzevii, probably account for the improved aerobic stability of TMR silages at later fermentation. Fewer species seemed to be involved in aerobic deterioration of silages at later fermentation and Z. bailii was most likely to initiate the aerobic deterioration of TMR silages at later fermentation. The use of WCC in TMR might not influence the predominant yeast

Characterization of culturable yeast species associating with whole crop corn and total mixed ration silage.

PubMed

Wang, Huili; Hao, Wei; Ning, Tingting; Zheng, Mingli; Xu, Chuncheng

2018-02-01

This study investigated the association of yeast species with improved aerobic stability of total mixed ration (TMR) silages with prolonged ensiling, and clarified the characteristics of yeast species and their role during aerobic deterioration. Whole crop corn (WCC) silages and TMR silages formulated with WCC were ensiled for 7, 14, 28, and 56 d and used for an aerobic stability test. Predominant yeast species were isolated from different periods and identified by sequencing analyses of the 26S rRNA gene D1/D2 domain. Characteristics (assimilation and tolerance) of the yeast species and their role during aerobic deterioration were investigated. In addition to species of Candida glabrata and Pichia kudriavzevii ( P. kudriavzevii ) previously isolated in WCC and TMR, Pichia manshurica ( P. manshurica ), Candida ethanolica ( C. ethanolica ), and Zygosaccharomyces bailii ( Z. bailii ) isolated at great frequency during deterioration, were capable of assimilating lactic or acetic acid and tolerant to acetic acid and might function more in deteriorating TMR silages at early fermentation (7 d and 14 d). With ensiling prolonged to 28 d, silages became more (p<0.01) stable when exposed to air, coinciding with the inhibition of yeast to below the detection limit. Species of P. manshurica that were predominant in deteriorating WCC silages were not detectable in TMR silages. In addition, the predominant yeast species of Z. bailii in deteriorating TMR silages at later fermentation (28 d and 56 d) were not observed in both WCC and WCC silages. The inhibition of yeasts, particularly P. kudriavzevii , probably account for the improved aerobic stability of TMR silages at later fermentation. Fewer species seemed to be involved in aerobic deterioration of silages at later fermentation and Z. bailii was most likely to initiate the aerobic deterioration of TMR silages at later fermentation. The use of WCC in TMR might not influence the predominant yeast species during aerobic

Genomic and probiotic characterization of SJP-SNU strain of Pichia kudriavzevii.

PubMed

Hong, Seung-Min; Kwon, Hyuk-Joon; Park, Se-Joon; Seong, Won-Jin; Kim, Ilhwan; Kim, Jae-Hong

2018-05-17

The yeast strain SJP-SNU was investigated as a probiotic and was characterized with respect to growth temperature, bile salt resistance, hydrogen sulfide reducing activity, intestinal survival ability and chicken embryo pathogenicity. In addition, we determined the complete genomic and mitochondrial sequences of SJP-SNU and conducted comparative genomics analyses. SJP-SNU grew rapidly at 37 °C and formed colonies on MacConkey agar containing bile salt. SJP-SNU reduced hydrogen sulfide produced by Salmonella serotype Enteritidis and, after being fed to 4-week-old chickens, could be isolated from cecal feces. SJP-SNU did not cause mortality in 10-day-old chicken embryos. From 13 initial contigs, 11 were finally assembled and represented 10 chromosomal sequences and 1 mitochondrial DNA sequence. Comparative genomic analyses revealed that SJP-SNU was a strain of Pichia kudriavzevii. Although SJP-SNU possesses pathogenicity-related genes, they showed very low amino acid sequence identities to those of Candida albicans. Furthermore, SJP-SNU possessed useful genes, such as phytases and cellulase. Thus, SJP-SNU is a useful yeast possessing the basic traits of a probiotic, and further studies to demonstrate its efficacy as a probiotic in the future may be warranted.

Distribution of dimorphic yeast species in commercial extra virgin olive oil.

PubMed

Zullo, B A; Cioccia, G; Ciafardini, G

2010-12-01

Recent microbiological research has demonstrated the presence of a rich microflora mainly composed of yeasts in the suspended fraction of freshly produced olive oil. Some of the yeasts are considered useful as they improve the organoleptic characteristics of the oil during preservation, whereas others are considered harmful as they can damage the quality of the oil through the hydrolysis of the triglycerides. However, some dimorphic species can also be found among the unwanted yeasts present in the oil, considered to be opportunistic pathogens to man as they have often been isolated from immunocompromised hospital patients. Present research demonstrates the presence of dimorphic yeast forms in 26% of the commercial extra virgin olive oil originating from different geographical areas, where the dimorphic yeasts are represented by 3-99.5% of the total yeasts. The classified isolates belonged to the opportunistic pathogen species Candida parapsilosis and Candida guilliermondii, while among the dimorphic yeasts considered not pathogenic to man, the Candida diddensiae species was highlighted for the first time in olive oil. The majority of the studied yeast strains resulted lipase positive, and can consequently negatively influence the oil quality through the hydrolysis of the triglycerides. Furthermore, all the strains showed a high level of affinity with some organic solvents and a differing production of biofilm in "vitro" corresponded to a greater or lesser hydrophobia of their cells. Laboratory trials indicated that the dimorphic yeasts studied are sensitive towards some components of the oil among which oleic acid, linoleic acid and triolein, whereas a less inhibiting effect was observed with tricaprilin or when the total polyphenols extracted from the oil were used. The observations carried out on a scanning electron microscope (SEM), demonstrated the production of long un-branched pseudohyphae in all the tested dimorphic yeasts when cultivated on nutrient

Identification of yeasts during alcoholic fermentation of tchapalo, a traditional sorghum beer from Côte d'Ivoire.

PubMed

N'guessan, Kouadio Florent; Brou, Kouakou; Jacques, Noémie; Casaregola, Serge; Dje, Koffi Marcellin

2011-05-01

This study investigated the diversity and dynamics of yeasts involved in alcoholic fermentation of a traditional sorghum beer from Côte d'Ivoire, tchapalo. A total of 240 yeast strains were isolated from fermenting sorghum wort inoculated with dry yeast from two geographic regions of Côte d'Ivoire (Abidjan and Bondoukou). Initial molecular identification to the species level was carried out using RFLP of PCR-amplified internal transcribed spacers of rDNA (ITS1-5.8S-ITS2). Ten different profiles were obtained from the restriction of PCR products with the three endonucleases HaeIII, CfoI and HinfI. Sequence analysis of the D1/D2 domain of the 26S rDNA and the ACT1 gene allowed us to assign these groups to six different species: Saccharomyces cerevisiae-like, Candida tropicalis, Pichia kudriavzevii, Pichia kluyveri, Kodamaea ohmeri and Meyerozyma caribbica. The most frequent species associated with tchapalo fermentation was S. cerevisiae-like (87.36%), followed by C. tropicalis (5.45%) and M. caribbica (2.71%). S. cerevisiae-like strains were diploid heterozygotes and exhibited three to four nucleotides divergence from the type strain in the D1/D2 domain and several indels in the more discriminant sequence of the intron of the ACT1 gene. During the process, the yeast species isolated and their frequencies varied according to the geographic origin of the dry yeast. The occurrence of some species was sporadic and only two non-Saccharomyces species were found in the final product.

Yeast Biomass Production in Brewery's Spent Grains Hemicellulosic Hydrolyzate

NASA Astrophysics Data System (ADS)

Duarte, Luís C.; Carvalheiro, Florbela; Lopes, Sónia; Neves, Ines; Gírio, Francisco M.

Yeast single-cell protein and yeast extract, in particular, are two products which have many feed, food, pharmaceutical, and biotechnological applications. However, many of these applications are limited by their market price. Specifically, the yeast extract requirements for culture media are one of the major technical hurdles to be overcome for the development of low-cost fermentation routes for several top value chemicals in a biorefinery framework. A potential biotechnical solution is the production of yeast biomass from the hemicellulosic fraction stream. The growth of three pentose-assimilating yeast cell factories, Debaryomyces hansenii, Kluyveromyces marxianus, and Pichia stipitis was compared using non-detoxified brewery's spent grains hemicellulosic hydrolyzate supplemented with mineral nutrients. The yeasts exhibited different specific growth rates, biomass productivities, and yields being D. hansenii as the yeast species that presented the best performance, assimilating all sugars and noteworthy consuming most of the hydrolyzate inhibitors. Under optimized conditions, D. hansenii displayed a maximum specific growth rate, biomass yield, and productivity of 0.34 h-1, 0.61 g g-1, and 0.56 g 1-1 h-1, respectively. The nutritional profile of D. hansenii was thoroughly evaluated, and it compares favorably to others reported in literature. It contains considerable amounts of some essential amino acids and a high ratio of unsaturated over saturated fatty acids.

Rare opportunistic (non-Candida, non-Cryptococcus) Yeast Bloodstream Infections in Patients with Cancer

PubMed Central

Chitasombat, Maria N.; Kofteridis, Diamantis P.; Jiang, Ying; Tarrand, Jeffrey; Lewis, Russell E.; Kontoyiannis, Dimitrios P.

2013-01-01

Background Rare opportunistic (non-Candida, non-Cryptococcus) yeast bloodstream infections (ROYBSIs) are rare, even in cancer patients. Methods We retrospectively reviewed all episodes of ROYBSIs occurring from 1998 to 2010 in our cancer center. Results Of 2984 blood cultures positive for Candida and non-Candida yeasts, 94 (3.1%) were positive for non-Candida yeasts, representing 41 ROYBSIs (incidence, 2.1 cases/100,000 patient-days). Catheter-associated fungemia occurred in 21 (51%) patients. Breakthrough ROYBSIs occurred in 20 (49%) patients. The yeast species distribution was Rhodotorula in 21 (51%) patients, Trichosporon in 8 (20%) patients, Saccharomyces cerevisiae in 8 (20%) patients, Geotrichum in 2 (5%) patients, Pichia anomala, and Malassezia furfur in 1 patient each. All tested Trichosporon, Geotrichum, and Pichia isolates were azole-susceptible, whereas the Rhodotorula isolates were mostly azole-resistant. We noted echinocandin nonsusceptibility (minimal inhibitory concentration ≥ 2 mg/L) in all but the S. cerevisiae isolates. Most of the isolates (28/33 [85%]) were susceptible to amphotericin B. The mortality rate in all patients at 30 days after ROYBSIs diagnosis was 34%. Multivariate survival analysis revealed increased risk of death in patients with S. cerevisiae infections (hazard ratio, 3.7), Geotrichum infections (hazard ratio, 111.3), or disseminated infections (hazard ratio, 33.4) and reduced risk in patients who had catheter removal (hazard ratio, 0.1). Conclusions ROYBSIs are uncommon in patients with cancer, and catheters are common sources of them. Half of the ROYBSIs occurred as breakthrough infections, and in vitro species-specific resistance to echinocandins and azoles was common. Disseminated infections resulted in the high mortality rate. PMID:22101079

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.

Yeast diversity of sourdoughs and associated metabolic properties and functionalities.

PubMed

De Vuyst, Luc; Harth, Henning; Van Kerrebroeck, Simon; Leroy, Frédéric

2016-12-19

Together with acidifying lactic acid bacteria, yeasts play a key role in the production process of sourdough, where they are either naturally present or added as a starter culture. Worldwide, a diversity of yeast species is encountered, with Saccharomyces cerevisiae, Candida humilis, Kazachstania exigua, Pichia kudriavzevii, Wickerhamomyces anomalus, and Torulaspora delbrueckii among the most common ones. Sourdough-adapted yeasts are able to withstand the stress conditions encountered during their growth, including nutrient starvation as well as the effects of acidic, oxidative, thermal, and osmotic stresses. From a technological point of view, their metabolism primarily contributes to the leavening and flavour of sourdough products. Besides ethanol and carbon dioxide, yeasts can produce metabolites that specifically affect flavour, such as organic acids, diacetyl, higher alcohols from branched-chain amino acids, and esters derived thereof. Additionally, several yeast strains possess functional properties that can potentially lead to nutritional and safety advantages. These properties encompass the production of vitamins, an improvement of the bioavailability of phenolic compounds, the dephosphorylation of phytic acid, the presence of probiotic potential, and the inhibition of fungi and their mycotoxin production. Strains of diverse species are new candidate functional starter cultures, offering opportunities beyond the conventional use of baker's yeast. Copyright © 2016 Elsevier B.V. All rights reserved.

Cloning of gene-encoded stem bromelain on system coming from Pichia pastoris as therapeutic protein candidate

NASA Astrophysics Data System (ADS)

Yusuf, Y.; Hidayati, W.

2018-01-01

The process of identifying bacterial recombination using PCR, and restriction, and then sequencing process was done after identifying the bacteria. This research aimed to get a yeast cell of Pichia pastoris which has an encoder gene of stem bromelain enzyme. The production of recombinant stem bromelain enzymes using yeast cells of P. pastoris can produce pure bromelain rod enzymes and have the same conformation with the enzyme’s conformation in pineapple plants. This recombinant stem bromelain enzyme can be used as a therapeutic protein in inflammatory, cancer and degenerative diseases. This study was an early stage of a step series to obtain bromelain rod protein derived from pineapple made with genetic engineering techniques. This research was started by isolating the RNA of pineapple stem which was continued with constructing cDNA using reserve transcriptase-PCR technique (RT-PCR), doing the amplification of bromelain enzyme encoder gene with PCR technique using a specific premiere couple which was designed. The process was continued by cloning into bacterium cells of Escherichia coli. A vector which brought the encoder gene of stem bromelain enzyme was inserted into the yeast cell of P. pastoris and was continued by identifying the yeast cell of P. pastoris which brought the encoder gene of stem bromelain enzyme. The research has not found enzyme gene of stem bromelain in yeast cell of P. pastoris yet. The next step is repeating the process by buying new reagent; RNase inhibitor, and buying liquid nitrogen.

Citrobacter amalonaticus Phytase on the Cell Surface of Pichia pastoris Exhibits High pH Stability as a Promising Potential Feed Supplement

PubMed Central

Li, Cheng; Lin, Ying; Huang, Yuanyuan; Liu, Xiaoxiao; Liang, Shuli

2014-01-01

Phytase expressed and anchored on the cell surface of Pichia pastoris avoids the expensive and time-consuming steps of protein purification and separation. Furthermore, yeast cells with anchored phytase can be used as a whole-cell biocatalyst. In this study, the phytase gene of Citrobacter amalonaticus was fused with the Pichia pastoris glycosylphosphatidylinositol (GPI)-anchored glycoprotein homologue GCW61. Phytase exposed on the cell surface exhibits a high activity of 6413.5 U/g, with an optimal temperature of 60°C. In contrast to secreted phytase, which has an optimal pH of 5.0, phytase presented on the cell surface is characterized by an optimal pH of 3.0. Moreover, our data demonstrate that phytase anchored on the cell surface exhibits higher pH stability than its secreted counterpart. Interestingly, our in vitro digestion experiments demonstrate that phytase attached to the cell surface is a more efficient enzyme than secreted phytase. PMID:25490768

Citrobacter amalonaticus phytase on the cell surface of Pichia pastoris exhibits high pH stability as a promising potential feed supplement.

PubMed

Li, Cheng; Lin, Ying; Huang, Yuanyuan; Liu, Xiaoxiao; Liang, Shuli

2014-01-01

Phytase expressed and anchored on the cell surface of Pichia pastoris avoids the expensive and time-consuming steps of protein purification and separation. Furthermore, yeast cells with anchored phytase can be used as a whole-cell biocatalyst. In this study, the phytase gene of Citrobacter amalonaticus was fused with the Pichia pastoris glycosylphosphatidylinositol (GPI)-anchored glycoprotein homologue GCW61. Phytase exposed on the cell surface exhibits a high activity of 6413.5 U/g, with an optimal temperature of 60°C. In contrast to secreted phytase, which has an optimal pH of 5.0, phytase presented on the cell surface is characterized by an optimal pH of 3.0. Moreover, our data demonstrate that phytase anchored on the cell surface exhibits higher pH stability than its secreted counterpart. Interestingly, our in vitro digestion experiments demonstrate that phytase attached to the cell surface is a more efficient enzyme than secreted phytase.

[Studies on antigencity of human immunodeficiency virus type 1 (HIV-1) external glycoprotein as well as its expression in Pichia pastoris].

PubMed

Zhao, Li-Hui; Yu, Xiang-Hui; Jiang, Chun-Lai; Wu, Yong-Ge; Shen, Jia-Cong; Kong, Wei

2007-05-01

Based on the computer simulation, we analyzed hydrophobicity, potential epitope of recombined subtypes HIV-1 Env protein (851 amino acids) from Guangxi in China. Compared with conservative peptides of other subtypes in env protein, three sequences (469-511aa, 538-674aa, 700-734aa) were selected to recombine into a chimeric gene that codes three conservative epitope peptides with stronger antigencity, and was constructed in the yeast expression plasmid pPICZB. Chimeric proteins were expressed in Pichia pastoris under the induction of methanol, and were analyzed by SDS-PAGE and Westernblot. The results showed that fusion proteins of three-segment antigen were expressed in Pichia pastoris and that specific protein band at the site of 40kD was target protein, which is interacted with HIV-1 serum. The target proteins were purified by metal Ni-sepharose 4B, and were demonstrated to possess good antigenic specificity from the data of ELISA. This chimeric antigen may be used as research and developed into HIV diagnostic reagents.

Native Killer Yeasts as Biocontrol Agents of Postharvest Fungal Diseases in Lemons

PubMed Central

Garnica, Nydia Mercedes; Fernández-Zenoff, María Verónica; Farías, María Eugenia; Sepulveda, Milena; Ramallo, Jacqueline; Dib, Julián Rafael

2016-01-01

Economic losses caused by postharvest diseases represent one of the main problems of the citrus industry worldwide. The major diseases affecting citrus are the "green mold" and "blue mold", caused by Penicillium digitatum and P. italicum, respectively. To control them, synthetic fungicides are the most commonly used method. However, often the emergence of resistant strains occurs and their use is becoming more restricted because of toxic effects and environmental pollution they generate, combined with trade barriers to international markets. The aim of this work was to isolate indigenous killer yeasts with antagonistic activity against fungal postharvest diseases in lemons, and to determine their control efficiency in in vitro and in vivo assays. Among 437 yeast isolates, 8.5% show to have a killer phenotype. According to molecular identification, based on the 26S rDNA D1/D2 domain sequences analysis, strains were identified belonging to the genera Saccharomyces, Wickerhamomyces, Kazachstania, Pichia, Candida and Clavispora. Killers were challenged with pathogenic molds and strains that caused the maximum in vitro inhibition of P. digitatum were selected for in vivo assays. Two strains of Pichia and one strain of Wickerhamomyces depicted a significant protection (p <0.05) from decay by P. digitatum in assays using wounded lemons. Thus, the native killer yeasts studied in this work showed to be an effective alternative for the biocontrol of postharvest fungal infections of lemons and could be promising agents for the development of commercial products for the biological control industry. PMID:27792761

Interactions between yeasts and bacteria in the smear surface-ripened cheeses.

PubMed

Corsetti, A; Rossi, J; Gobbetti, M

2001-09-19

In the initial phase of ripening, the microflora of bacterial smear surface-ripened cheeses such as Limburger, Taleggio, Brick, Münster and Saint-Paulin and that of surface mould-ripened cheeses such as Camembert and Brie may be similar, but at the end of the ripening, bacteria such as Brevibacterium spp., Arthrobacter spp., Micrococcus spp., Corynebacterium spp. and moulds such as Penicillium camemberti are, respectively, the dominant microorganisms. Yeasts such as Candida spp., Cryptococcus spp., Debaryomyces spp., Geotrichum candidum, Pichia spp., Rhodotorula spp., Saccharomyces spp. and Yarrowia lipolytica are often and variably isolated from the smear surface-ripened cheeses. Although not dominant within the microorganisms of the smear surface-ripened cheeses, yeasts establish significant interactions with moulds and especially bacteria, including surface bacteria and lactic acid bacteria. Some aspects of the interactions between yeasts and bacteria in such type of cheeses are considered in this paper.

[Onychomycosis by yeast not common in diabetics of a health center].

PubMed

Imbert, J L; G Gomez, J V; Escudero, R B; Blasco, J L

2016-10-01

Mexican diabetic population frequently presents mycosis under foot hyperkeratosis; however, in another type of onychomycosis as the ones that is assumed Candida albicans is the causal agent, it is unknown the frequency, the prevalence and if another Candida species or other yeasts are found. Evaluate the frequency of yeasts causing onychomycosis in diabetic patients looked after in public institutions of health of the State of Hidalgo, Mexico, and its association with clinical epidemiological variables. An observational, descriptive and transversal study was made on 261 patients, from which one nail sample of each one was obtained, used to isolate and identify dermatophytes and yeasts; the results were statistically correlated with 24 epidemiological parameters. The clinical study was done through interrogation and by medical exploration in order to evaluate Tinea pedis and onychomycosis. Onychomycosis were caused by Candida guilliermondii, Candida parapsilosis, Candida glabrata, Candida krusei, Candida spp., Kodamaea ohmeri, Prototheca wickerhamii and unidentified yeasts. The prevalence for general onychomycosis, by dermatophytes, mixed onychomycosis and by yeasts were: 24.1, 19.5, 2.3 and 14.6%, respectively. Patients with significant probability to be diagnosed as having onychomycosis by yeasts are those wearing open shoes (2.59%); technicians and professionals (10.49%) and alcohol drinkers (3.72%). The fact that Candida albicans is not present in this study as causal agent of onychomycosis, and emerging and non-common yeasts were indeed isolated, creates new challenges. It is remarked the clinical criterion that when onychomycosis is suspected in diabetics, the diagnosis for culturing dermatophytes and yeasts should be included. Copyright © 2015 Sociedad Española de Médicos de Atención Primaria (SEMERGEN). Publicado por Elsevier España, S.L.U. All rights reserved.

Genome and metabolic engineering in non-conventional yeasts: Current advances and applications.

PubMed

Löbs, Ann-Kathrin; Schwartz, Cory; Wheeldon, Ian

2017-09-01

Microbial production of chemicals and proteins from biomass-derived and waste sugar streams is a rapidly growing area of research and development. While the model yeast Saccharomyces cerevisia e is an excellent host for the conversion of glucose to ethanol, production of other chemicals from alternative substrates often requires extensive strain engineering. To avoid complex and intensive engineering of S. cerevisiae, other yeasts are often selected as hosts for bioprocessing based on their natural capacity to produce a desired product: for example, the efficient production and secretion of proteins, lipids, and primary metabolites that have value as commodity chemicals. Even when using yeasts with beneficial native phenotypes, metabolic engineering to increase yield, titer, and production rate is essential. The non-conventional yeasts Kluyveromyces lactis, K. marxianus, Scheffersomyces stipitis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris have been developed as eukaryotic hosts because of their desirable phenotypes, including thermotolerance, assimilation of diverse carbon sources, and high protein secretion. However, advanced metabolic engineering in these yeasts has been limited. This review outlines the challenges of using non-conventional yeasts for strain and pathway engineering, and discusses the developed solutions to these problems and the resulting applications in industrial biotechnology.

Stereoselective reduction of aromatic ketones by a new ketoreductase from Pichia glucozyma.

PubMed

Contente, Martina Letizia; Serra, Immacolata; Brambilla, Marta; Eberini, Ivano; Gianazza, Elisabetta; De Vitis, Valerio; Molinari, Francesco; Zambelli, Paolo; Romano, Diego

2016-01-01

A new NADPH-dependent benzil reductase (KRED1-Pglu) was identified from the genome of the non-conventional yeast Pichia glucozyma CBS 5766 and overexpressed in E. coli. The new protein was characterised and reaction parameters were optimised for the enantioselective reduction of benzil to (S)-benzoin. A thorough study of the substrate range of KRED1-Pglu was conducted; in contrast to most other known ketoreductases, KRED1-Pglu prefers space-demanding substrates, which are often converted with high stereoselectivity. A molecular modelling study was carried out for understanding the structural determinants involved in the stereorecognition experimentally observed and unpredictable on the basis of steric properties of the substrates. As a result, a new useful catalyst was identified, enabling the enantioselective preparation of different aromatic alcohols and hydroxyketones.

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.

Yeast Diversity and Persistence in Botrytis-Affected Wine Fermentations

PubMed Central

Mills, David A.; Johannsen, Eric A.; Cocolin, Luca

2002-01-01

Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected (“botrytized”) wine fermentations carried out at high (∼30°C) and ambient (∼20°C) temperatures. Fermentations at both temperatures possessed similar populations of Saccharomyces, Hanseniaspora, Pichia, Metschnikowia, Kluyveromyces, and Candida species. However, higher populations of non-Saccharomyces yeasts persisted in ambient-temperature fermentations, with Candida and, to a lesser extent, Kluyveromyces species remaining long after the fermentation was dominated by Saccharomyces. In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data. The direct molecular methods also revealed a Hanseniaspora osmophila population not identified in the plating analysis. rRNA analysis also indicated a large population (>106 cells per ml) of a nonculturable Candida strain in the high-temperature fermentation. Monoculture analysis of the Candida isolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations of Candida. Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations. PMID:12324335

Industrially Important Carbohydrate Degrading Enzymes from Yeasts: Pectinases, Chitinases, and β-1,3-Glucanases

NASA Astrophysics Data System (ADS)

Gummadi, Sathyanarayana N.; Kumar, D. Sunil; Dash, Swati S.; Sahu, Santosh Kumar

Polysaccharide degrading enzymes are hydrolytic enzymes, which have a lot of industrial potential and also play a crucial role in carbon recycling. Pectinases, chitinases and glucanases are the three major polysaccharide degrading enzymes found abundantly in nature and these enzymes are mainly produced by fungal strains. Production of these enzymes by yeasts is advantageous over fungi, because the former are easily amenable to genetic manipulations and time required for growth and production is less than that of the latter. Several yeasts belonging to Saccharomyces, Pichia, Rhodotorula and Cryptococcus produce extracellular pectinases, glucanases and chitinases. This chapter emphasizes on the biological significance of these enzymes, their production and their industrial applications.

MALDI-TOF MS as a tool to identify foodborne yeasts and yeast-like fungi.

PubMed

Quintilla, Raquel; Kolecka, Anna; Casaregola, Serge; Daniel, Heide M; Houbraken, Jos; Kostrzewa, Markus; Boekhout, Teun; Groenewald, Marizeth

2018-02-02

Since food spoilage by yeasts causes high economic losses, fast and accurate identifications of yeasts associated with food and food-related products are important for the food industry. In this study the efficiency of the matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify food related yeasts was evaluated. A CBS in-house MALDI-TOF MS database was created and later challenged with a blinded test set of 146 yeast strains obtained from food and food related products. Ninety eight percent of the strains were correctly identified with log score values>1.7. One strain, Mrakia frigida, gained a correct identification with a score value<1.7. Two strains could not be identified at first as they represented a mix of two different species. These mixes were Rhodotorula babjevae with Meyerozyma caribbica and Clavispora lusitaniae with Debaryomyces hansenii. After separation, all four species could be correctly identified with scores>1.7. Ambiguous identifications were observed due to two incorrect reference mass spectra's found in the commercial database BDAL v.4.0, namely Candida sake DSM 70763 which was re-identified as Candida oleophila, and Candida inconspicua DSM 70631 which was re-identified as Pichia membranifaciens. MALDI-TOF MS can distinguish between most of the species, but for some species complexes, such as the Kazachstania telluris and Mrakia frigida complexes, MALDI-TOF MS showed limited resolution and identification of sibling species was sometimes problematic. Despite this, we showed that the MALDI-TOF MS is applicable for routine identification and validation of foodborne yeasts, but a further update of the commercial reference databases is needed. Copyright © 2017 Elsevier B.V. All rights reserved.

Exogenous calcium improves viability of biocontrol yeasts under heat stress by reducing ROS accumulation and oxidative damage of cellular protein.

PubMed

An, Bang; Li, Boqiang; Qin, Guozheng; Tian, Shiping

2012-08-01

In this article, we investigated the effect of exogenous calcium on improving viability of Debaryomyces hansenii and Pichia membranaefaciens under heat stress, and evaluated the role of calcium in reducing oxidant damage of proteins in the yeast cells. The results indicated that high concentration of exogenous calcium in culture medium was beneficial for enhancing the tolerance of the biocontrol yeasts to heat stress. The possible mechanism of calcium improving the viability of yeasts was attributed to enhancement of antioxidant enzyme activities, decrease in ROS accumulation and reduction of oxidative damage of intracellular protein in yeast cells under heat stress. D. hansenii is more sensitive to calcium as compared to P. membranaefaciens. Our results suggest that application of exogenous calcium combined with biocontrol yeasts is a practical approach for the control of postharvest disease in fruit.

Yeast: An Overlooked Component of Bactrocera tryoni (Diptera: Tephritidae) Larval Gut Microbiota.

PubMed

Deutscher, Ania T; Reynolds, Olivia L; Chapman, Toni A

2017-02-01

Yeasts, often in hydrolyzed form, are key ingredients in the larval and adult diets of tephritid fruit fly colonies. However, very little is known about the presence or role of yeasts in the diets of tephritid fruit flies in nature. Previous studies have identified bacteria but not detected yeasts in the gut of Queensland fruit fly, Bactrocera tryoni (Froggatt), one of Australia's most economically damaging insect pests of horticultural crops and of significant biosecurity concern domestically and internationally. Here we demonstrate that cultivable yeasts are commonly found in the gut of B. tryoni larvae from fruit hosts. Analysis of the ITS1, 5.8S rRNA gene, and ITS2 sequences of randomly selected isolates identified yeasts and yeast-like fungi of the genera Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Pichia, and Starmerella. The prevalence of these yeasts in fruits suggests that larvae consume the yeasts as part of their diet. This work highlights that yeasts should be considered in future tephritid larval gut microbiota studies. Understanding tephritid-microbial symbiont interactions will lead to improvements in artificial diets and the quality of mass-reared tephritids for the sterile insect technique. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The Genetics of Non-conventional Wine Yeasts: Current Knowledge and Future Challenges

PubMed Central

Masneuf-Pomarede, Isabelle; Bely, Marina; Marullo, Philippe; Albertin, Warren

2016-01-01

Saccharomyces cerevisiae is by far the most widely used yeast in oenology. However, during the last decade, several other yeasts species has been purposed for winemaking as they could positively impact wine quality. Some of these non-conventional yeasts (Torulaspora delbrueckii, Metschnikowia pulcherrima, Pichia kluyveri, Lachancea thermotolerans, etc.) are now proposed as starters culture for winemakers in mixed fermentation with S. cerevisiae, and several others are the subject of various studies (Hanseniaspora uvarum, Starmerella bacillaris, etc.). Along with their biotechnological use, the knowledge of these non-conventional yeasts greatly increased these last 10 years. The aim of this review is to describe the last updates and the current state-of-art of the genetics of non-conventional yeasts (including S. uvarum, T. delbrueckii, S. bacillaris, etc.). We describe how genomics and genetics tools provide new data into the population structure and biodiversity of non-conventional yeasts in winemaking environments. Future challenges will lie on the development of selection programs and/or genetic improvement of these non-conventional species. We discuss how genetics, genomics and the advances in next-generation sequencing will help the wine industry to develop the biotechnological use of non-conventional yeasts to improve the quality and differentiation of wines. PMID:26793188

The Genetics of Non-conventional Wine Yeasts: Current Knowledge and Future Challenges.

PubMed

Masneuf-Pomarede, Isabelle; Bely, Marina; Marullo, Philippe; Albertin, Warren

2015-01-01

Saccharomyces cerevisiae is by far the most widely used yeast in oenology. However, during the last decade, several other yeasts species has been purposed for winemaking as they could positively impact wine quality. Some of these non-conventional yeasts (Torulaspora delbrueckii, Metschnikowia pulcherrima, Pichia kluyveri, Lachancea thermotolerans, etc.) are now proposed as starters culture for winemakers in mixed fermentation with S. cerevisiae, and several others are the subject of various studies (Hanseniaspora uvarum, Starmerella bacillaris, etc.). Along with their biotechnological use, the knowledge of these non-conventional yeasts greatly increased these last 10 years. The aim of this review is to describe the last updates and the current state-of-art of the genetics of non-conventional yeasts (including S. uvarum, T. delbrueckii, S. bacillaris, etc.). We describe how genomics and genetics tools provide new data into the population structure and biodiversity of non-conventional yeasts in winemaking environments. Future challenges will lie on the development of selection programs and/or genetic improvement of these non-conventional species. We discuss how genetics, genomics and the advances in next-generation sequencing will help the wine industry to develop the biotechnological use of non-conventional yeasts to improve the quality and differentiation of wines.

Evaluation of sorghum straw hemicellulosic hydrolysate for biotechnological production of xylitol by Candida guilliermondii

PubMed Central

Sene, L.; Arruda, P.V.; Oliveira, S.M.M.; Felipe, M.G.A.

2011-01-01

A preliminary study on xylitol production by Candida guilliermondii in sorghum straw hemicellulosic hydrolysate was performed. Hydrolysate had high xylose content and inhibitors concentrations did not exceed the commonly found values in other hemicellulosic hydrolysates. The highest xylitol yield (0.44 g/g) and productivity (0.19 g/Lh) were verified after 72 hours. PMID:24031733

Expression and characterization of camel chymosin in Pichia pastoris.

PubMed

Wang, Nan; Wang, Kevin Yueju; Li, GangQiang; Guo, WenFang; Liu, DeHu

2015-07-01

Chymosin efficiently coagulates milk and so is widely used in commercial cheese production. Traditional chymosin production requires the slaughter of a large numbers of unweaned calves. In the present study, a full-length camel prochymosin gene was synthesized and cloned into the pPIC9K vector, which was then inserted into the yeast strain, Pichia pastoris GS115. Expression of the chymosin gene in yeast was under the control of an AOX1 inducible promoter. The yeast system produced approximately 37mg/L of recombinant enzyme under lab conditions. SDS-PAGE of the raw supernatant revealed two molecular bands, which were approximately 42kDa and 45kDa in size. The 45kDa band disappeared after treatment of the supernatant with N-glycosidase F (PNGase F), indicating that the recombinant protein was partially glycosylated. When subjected to a low pH, recombinant prochymosin was converted into mature and active chymosin. The active chymosin was capable of specifically hydrolyzing κ-casein. A pH of 5.04, and temperature range of 45-50°C, was optimum for milk clotting activity. Maximum milk clotting activity was detected with the inclusion of 20-40mM CaCl2. The recombinant enzyme was highly active and stable over a wide pH range (from 2.5 to 6.5) at 20°C for 8h. Thermostability of the recombinant enzyme was also analyzed. Pilot-scale production (300mg/L) was attained using a 5L fermenter. We demonstrated that expression of the camel chymosin gene in P. pastoris could represent an excellent system for producing active camel chymosin for potential use in the commercial production of cheese. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparison of laccase production levels in Pichia pastoris and Cryptococcus sp. S-2.

PubMed

Nishibori, Nahoko; Masaki, Kazuo; Tsuchioka, Hiroaki; Fujii, Tsutomu; Iefuji, Haruyuki

2013-04-01

The heterologous expression of the laccase gene from Trametes versicolor and Gaeumannomyces graminis was evaluated in the yeasts Pichia pastoris and Cryptococcus sp. S-2. The expression levels of both laccase genes in Cryptococcus sp. S-2 were considerably higher than those in P. pastoris. The codon usage of Cryptococcus sp. S-2 as well as the GC content were similar to those of T. versicolor and G. graminis. These results suggest that using a host with a similar codon usage for the expressed gene may improve protein expression. The use of Cryptococcus sp. S-2 as a host may be advantageous for the heterologous expression of genes with high GC content. Moreover, this yeast provides the same advantages as P. pastoris for the production of recombinant proteins, such as growth on minimal medium, capacity for high-density growth during fermentation, and capability for post-translational modifications. Therefore, we propose that Cryptococcus sp. S-2 be used as an expression host to improve enzyme production levels when other hosts have not yielded good results. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Different effects of sodium chloride preincubation on cadmium tolerance of Pichia kudriavzevii and Saccharomyces cerevisiae.

PubMed

Ma, Ning; Li, Chunsheng; Dong, Xiaoyan; Wang, Dongfeng; Xu, Ying

2015-08-01

Application of growing microorganisms for cadmium removal is restricted by high cadmium toxicity. The effects of sodium chloride (NaCl) preincubation on the cadmium tolerance and removal ability of Pichia kudriavzevii and Saccharomyces cerevisiae were investigated in this study. NaCl preincubation improved the biomass of P. kudriavzevii under cadmium stress, while no obvious effect was observed in S. cerevisiae. The improved activities of peroxidase (POD) and catalase (CAT) after NaCl preincubation might be an important reason for the decrease of the reactive oxygen species (ROS) accumulation, cell death, and oxidative damage of proteins and lipids induced by cadmium, contributing to the improvement of the yeast growth. The cadmium bioaccumulation capacity of P. kudriavzevii decreased significantly after NaCl preincubation, which played an important role in mitigating the cadmium toxicity to the yeast. The cadmium removal rate of P. kudriavzevii was obviously higher than S. cerevisiae and was significantly enhanced after NaCl preincubation. The results suggested that NaCl preincubation improved the cadmium tolerance and removal ability of P. kudriavzevii. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthetic biology and molecular genetics in non-conventional yeasts: Current tools and future advances.

PubMed

Wagner, James M; Alper, Hal S

2016-04-01

Coupling the tools of synthetic biology with traditional molecular genetic techniques can enable the rapid prototyping and optimization of yeast strains. While the era of yeast synthetic biology began in the well-characterized model organism Saccharomyces cerevisiae, it is swiftly expanding to include non-conventional yeast production systems such as Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica. These yeasts already have roles in the manufacture of vaccines, therapeutic proteins, food additives, and biorenewable chemicals, but recent synthetic biology advances have the potential to greatly expand and diversify their impact on biotechnology. In this review, we summarize the development of synthetic biological tools (including promoters and terminators) and enabling molecular genetics approaches that have been applied in these four promising alternative biomanufacturing platforms. An emphasis is placed on synthetic parts and genome editing tools. Finally, we discuss examples of synthetic tools developed in other organisms that can be adapted or optimized for these hosts in the near future. Copyright © 2015 Elsevier Inc. All rights reserved.

Production, characterization and gene cloning of the extracellular enzymes from the marine-derived yeasts and their potential applications.

PubMed

Chi, Zhenming; Chi, Zhe; Zhang, Tong; Liu, Guanglei; Li, Jing; Wang, Xianghong

2009-01-01

In this review article, the extracellular enzymes production, their properties and cloning of the genes encoding the enzymes from marine yeasts are overviewed. Several yeast strains which could produce different kinds of extracellular enzymes were selected from the culture collection of marine yeasts available in this laboratory. The strains selected belong to different genera such as Yarrowia, Aureobasidium, Pichia, Metschnikowia and Cryptococcus. The extracellular enzymes include cellulase, alkaline protease, aspartic protease, amylase, inulinase, lipase and phytase, as well as killer toxin. The conditions and media for the enzyme production by the marine yeasts have been optimized and the enzymes have been purified and characterized. Some genes encoding the extracellular enzymes from the marine yeast strains have been cloned, sequenced and expressed. It was found that some properties of the enzymes from the marine yeasts are unique compared to those of the homologous enzymes from terrestrial yeasts and the genes encoding the enzymes in marine yeasts are different from those in terrestrial yeasts. Therefore, it is of very importance to further study the enzymes and their genes from the marine yeasts. This is the first review on the extracellular enzymes and their genes from the marine yeasts.

Effect of zinc formulations, sodium chloride, and hydroxytyrosol on the growth/no-growth boundaries of table olive related yeasts.

PubMed

Romero-Gil, V; Rejano-Zapata, L; Garrido-Fernández, A; Arroyo-López, F N

2016-08-01

This study uses a mathematical approach to assessing the inhibitory effect of Zn(2)(+)(0-10 mM, obtained from ZnCl2 and ZnSO4) in presence of NaCl (0-8%) and hydroxytyrosol (0-2588 mg/L), on a yeast cocktail formed by species Pichia galeiformis, Pichia kudriavzevii, Pichia manshurica and Candida thaimueangensis obtained from spoilt green olive packages. The logistic/probabilistic models were built in laboratory medium using a total of 1980 responses (1188 for NaCl and 792 for hydroxytyrosol). ZnCl2 showed significantly higher inhibitory effect than ZnSO4 in the presence of both NaCl (p < 0.033) and hydroxytyrosol (p < 0.009). NaCl did not interfere the effect of Zn(2)(+)while hydroxytyrosol, at high levels, had a slight antagonistic effect. According to models, Zn(2)(+)inhibits (p = 0.01) the yeast cocktail in the range 4.5-5.0 mM for ZnCl2, or 8.5-9.5 mM for ZnSO4. Therefore, this work confirms the fungicidal activity of zinc compounds (mainly ZnCl2) in synthetic medium, and also shows that the loss of zinc effectiveness in real green Spanish-style olive packaging is not due to the presence of NaCl or hydroxytyrosol, two of the most abundant chemical compounds in the product. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ascorbic acid enhances oxidative stress tolerance and biological control efficacy of Pichia caribbica against postharvest blue mold decay of apples.

PubMed

Li, Chaolan; Zhang, Hongyin; Yang, Qiya; Komla, Mahunu Gustav; Zhang, Xiaoyun; Zhu, Shuyun

2014-07-30

The effect of ascorbic acid (VC) on improving oxidative stress tolerance of Pichia caribbica and biocontrol efficacy against blue mold caused by Penicillium expansum on apples was investigated. P. caribbica showed susceptibility to the oxidative stress in vitro test, and 250 μg/mL VC treatment improved its oxidative stress tolerance. The higher viability exhibited by VC-treated yeast was associated with a lower intracellular ROS level. The activities of antioxidant enzymes of P. caribbica were improved by VC treatment, including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX). Additionally, VC-treated yeast exhibited greater biocontrol activity against P. expansum and faster growth when stored at 25 and 4 °C, respectively, compared to the performance of the non-VC-treated yeast. In response to the VC treatment under oxidative stress, several differentially expressed proteins were identified in P. caribbica, and most of the poteins were confirmed to be related to basic metabolism. Therefore, the application of ascorbic acid is a useful approach to improve oxidative stress tolerance of P. caribbica and its biocontrol efficacy on apples.

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.

Yeast Derived LysA2 Can Control Bacterial Contamination in Ethanol Fermentation.

PubMed

Kim, Jun-Seob; Daum, M Angela; Jin, Yong-Su; Miller, Michael J

2018-05-24

Contamination of fuel-ethanol fermentations continues to be a significant problem for the corn and sugarcane-based ethanol industries. In particular, members of the Lactobacillaceae family are the primary bacteria of concern. Currently, antibiotics and acid washing are two major means of controlling contaminants. However, antibiotic use could lead to increased antibiotic resistance, and the acid wash step stresses the fermenting yeast and has limited effectiveness. Bacteriophage endolysins such as LysA2 are lytic enzymes with the potential to contribute as antimicrobials to the fuel ethanol industries. Our goal was to evaluate the potential of yeast-derived LysA2 as a means of controlling Lactobacillaceae contamination. LysA2 intracellularly produced by Pichia pastoris showed activity comparable to Escherichia coli produced LysA2. Lactic Acid Bacteria (LAB) with the A4α peptidoglycan chemotype (L-Lys-D-Asp crosslinkage) were the most sensitive to LysA2, though a few from that chemotype were insensitive. Pichia -expressed LysA2, both secreted and intracellularly produced, successfully improved ethanol productivity and yields in glucose (YPD60) and sucrose-based (sugarcane juice) ethanol fermentations in the presence of a LysA2 susceptible LAB contaminant. LysA2 secreting Sacharomyces cerevisiae did not notably improve production in sugarcane juice, but it did control bacterial contamination during fermentation in YPD60. Secretion of LysA2 by the fermenting yeast, or adding it in purified form, are promising alternative tools to control LAB contamination during ethanol fermentation. Endolysins with much broader lytic spectrums than LysA2 could supplement or replace the currently used antibiotics or the acidic wash.

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.

Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation.

PubMed

Crafack, Michael; Mikkelsen, Morten B; Saerens, Sofie; Knudsen, Morten; Blennow, Andreas; Lowor, Samuel; Takrama, Jemmy; Swiegers, Jan H; Petersen, Gert B; Heimdal, Hanne; Nielsen, Dennis S

2013-10-01

The potential impact of aromatic and pectinolytic yeasts on cocoa flavour was investigated using two defined mixed starter cultures encompassing strains of Pichia kluyveri and Kluyveromyces marxianus for inoculating cocoa beans in small scale tray fermentations. Samples for microbial and metabolite analysis were collected at 12-24 hour intervals during 120 h of fermentation. Yeast isolates were grouped by (GTG)5-based rep-PCR fingerprinting and identified by sequencing of the D1/D2 region of the 26S rRNA gene and the actin gene. Pulsed Field Gel Electrophoresis (PFGE) was conducted on isolates belonging to the species P. kluyveri and K. marxianus to verify strain level identity with the inoculated strains. Furthermore, Denaturing Gradient Gel Electrophoresis (DGGE) was performed to follow yeast and bacterial dynamics over time including the presence of the bacterial inoculum consisting of Lactobacillus fermentum and Acetobacter pasteurianus. Yeast cell counts peaked after 12 h of fermentation with the predominant species being identified as Hanseniaspora opuntiae and Hanseniaspora thailandica. P. kluyveri and K. marxianus were found to compose 9.3% and 13.5% of the yeast population, respectively, after 12 h of fermentation whilst PFGE showed that ~88% of all P. kluyveri isolates and 100% of all K. marxianus isolates were identical to the inoculated strains. Despite never being the dominant yeast species at any stage of fermentation, the un-conched chocolates produced from the two inoculated fermentations were judged by sensory analysis to differ in flavour profile compared to the spontaneously fermented control. This could indicate that yeasts have a greater impact on the sensory qualities of cocoa than previously assumed. © 2013.

Association between Grape Yeast Communities and the Vineyard Ecosystems

PubMed Central

Drumonde-Neves, João; Lima, Teresa; Schuller, Dorit; Pais, Célia

2017-01-01

The grape yeast biota from several wine-producing areas, with distinct soil types and grapevine training systems, was assessed on five islands of Azores Archipelago, and differences in yeast communities composition associated with the geographic origin of the grapes were explored. Fifty-seven grape samples belonging to the Vitis vinifera grapevine cultivars Verdelho dos Açores (Verdelho), Arinto da Terceira (Arinto) and Terrantez do Pico (Terrantez) were collected in two consecutive years and 40 spontaneous fermentations were achieved. A total of 1710 yeast isolates were obtained from freshly crushed grapes and 1200 from final stage of fermentations. Twenty-eight species were identified, Hanseniaspura uvarum, Pichia terricola and Metschnikowia pulcherrima being the three most representative species isolated. Candida carpophila was encountered for the first time as an inhabitant of grape or wine-associated environments. In both sampling years, a higher proportion of H. uvarum in fresh grapes from Verdelho cultivar was observed, in comparison with Arinto cultivar. Qualitatively significant differences were found among yeast communities from several locations on five islands of the Archipelago, particularly in locations with distinctive agro-ecological compositions. Our results are in agreement with the statement that grape-associated microbial biogeography is non-randomly associated with interactions of climate, soil, cultivar, and vine training systems in vineyard ecosystems. Our observations strongly support a possible linkage between grape yeast and wine typicality, reinforcing the statement that different viticultural terroirs harbor distinctive yeast biota, in particular in vineyards with very distinctive environmental conditions. PMID:28085916

Inoculation of Pichia kudriavzevii RB1 degrades the organic acids present in raw compost material and accelerates composting.

PubMed

Nakasaki, Kiyohiko; Araya, Shogo; Mimoto, Hiroshi

2013-09-01

In this study, the yeast strain Pichia kudriavzevii RB1 was used as an inoculum to accelerate organic matter degradation of rabbit food with added organic acids, which was used as a model food waste for composting. The RB1 strain rapidly degraded the organic acids present in the raw compost material, leading to an increase in pH beyond the neutral level, within 2 days. Both mesophilic and thermophilic bacteria proliferated faster in the compost with RB1 inoculation than in that without inoculation. Although the yeast died with the increase in compost temperature, it affected the early stages of composting prior to the thermophilic stage and accelerated the composting process by 2 days by eliminating the initial lag phase seen in the growth of other microorganisms. Moreover, populations of Bacillus thermoamylovorans, Bacillus foraminis, and Bacillus coagulans became dominant during the thermophilic stages of both composting with and without RB1 inoculation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Characterization of Human Bone Alkaline Phosphatase in Pichia Pastoris

NASA Technical Reports Server (NTRS)

Malone, Christine C.; Ciszak, Eva; Karr, Laurel J.

1999-01-01

A soluble form of human bone alkaline phosphatase has been expressed in a recombinant strain of the methylotrophic yeast Pichia pastoris. We constructed a plasmid containing cDNA encoding for human bone alkaline phosphatase, with the hydrophobic carboxyl terminal portion deleted. Alkaline phosphatase was secreted into the medium to a level of 32mg/L when cultured in shake flasks, and enzyme activity was 12U/mg, as measured by a spectrophotometric assay. By conversion to a fermentation system, a yield of 880mg/L has been achieved with an enzyme activity of 968U/mg. By gel electrophoresis analysis, it appears that greater than 50% of the total protein in the fermentation media is alkaline phosphatase. Although purification procedures are not yet completely optimized, they are expected to include filtration, ion exchange and affinity chromatography. Our presentation will focus on the purification and crystallization results up to the time of the conference. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

Effect of Pyruvate Decarboxylase Knockout on Product Distribution Using Pichia pastoris (Komagataella phaffii) Engineered for Lactic Acid Production.

PubMed

Melo, Nadiele T M; Mulder, Kelly C L; Nicola, André Moraes; Carvalho, Lucas S; Menino, Gisele S; Mulinari, Eduardo; Parachin, Nádia S

2018-02-16

Lactic acid is the monomer unit of the bioplastic poly-lactic acid (PLA). One candidate organism for lactic acid production is Pichia pastoris , a yeast widely used for heterologous protein production. Nevertheless, this yeast has a poor fermentative capability that can be modulated by controlling oxygen levels. In a previous study, lactate dehydrogenase (LDH) activity was introduced into P. pastoris, enabling this yeast to produce lactic acid. The present study aimed to increase the flow of pyruvate towards the production of lactic acid in P. pastoris . To this end, a strain designated GLp was constructed by inserting the bovine lactic acid dehydrogenase gene (LDHb) concomitantly with the interruption of the gene encoding pyruvate decarboxylase (PDC). Aerobic fermentation, followed by micro-aerophilic culture two-phase fermentations, showed that the GLp strain achieved a lactic acid yield of 0.65 g/g. The distribution of fermentation products demonstrated that the acetate titer was reduced by 20% in the GLp strain with a concomitant increase in arabitol production: arabitol increased from 0.025 g/g to 0.174 g/g when compared to the GS115 strain. Taken together, the results show a significant potential for P. pastoris in producing lactic acid. Moreover, for the first time, physiological data regarding co-product formation have indicated the redox balance limitations of this yeast.

Peroxisomal Targeting, Import, and Assembly of Alcohol Oxidase in Pichia pastoris

PubMed Central

Waterham, Hans R.; Russell, Kimberly A.; de Vries, Yne; Cregg, James M.

1997-01-01

Alcohol oxidase (AOX), the first enzyme in the yeast methanol utilization pathway is a homooctameric peroxisomal matrix protein. In peroxisome biogenesis-defective (pex) mutants of the yeast Pichia pastoris, AOX fails to assemble into active octamers and instead forms inactive cytoplasmic aggregates. The apparent inability of AOX to assemble in the cytoplasm contrasts with other peroxisomal proteins that are able to oligomerize before import. To further investigate the import of AOX, we first identified its peroxisomal targeting signal (PTS). We found that sequences essential for targeting AOX are primarily located within the four COOH-terminal amino acids of the protein leucine-alanine-arginine-phenylalanine COOH (LARF). To examine whether AOX can oligomerize before import, we coexpressed AOX without its PTS along with wild-type AOX and determined whether the mutant AOX could be coimported into peroxisomes. To identify the mutant form of AOX, the COOH-terminal LARF sequence of the protein was replaced with a hemagglutinin epitope tag (AOX–HA). Coexpression of AOX–HA with wild-type AOX (AOX-WT) did not result in an increase in the proportion of AOX–HA present in octameric active AOX, suggesting that newly synthesized AOX–HA cannot oligomerize with AOX-WT in the cytoplasm. Thus, AOX cannot initiate oligomerization in the cytoplasm, but must first be targeted to the organelle before assembly begins. PMID:9396748

Comparative Lipidomic Profiling of S. cerevisiae and Four Other Hemiascomycetous Yeasts

PubMed Central

Hein, Eva-Maria; Hayen, Heiko

2012-01-01

Glycerophospholipids (GP) are the building blocks of cellular membranes and play essential roles in cell compartmentation, membrane fluidity or apoptosis. In addition, GPs are sources for multifunctional second messengers. Whereas the genome and proteome of the most intensively studied eukaryotic model organism, the baker’s yeast (Saccharomyces cerevisiae), are well characterized, the analysis of its lipid composition is still at the beginning. Moreover, different yeast species can be distinguished on the DNA, RNA and protein level, but it is currently unknown if they can also be differentiated by determination of their GP pattern. Therefore, the GP compositions of five different yeast strains, grown under identical environmental conditions, were elucidated using high performance liquid chromatography coupled to negative electrospray ionization-hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry in single and multistage mode. Using this approach, relative quantification of more than 100 molecular species belonging to nine GP classes was achieved. The comparative lipidomic profiling of Saccharomyces cerevisiae, Saccharomyces bayanus, Kluyveromyces thermotolerans, Pichia angusta, and Yarrowia lipolytica revealed characteristic GP profiles for each strain. However, genetically related yeast strains show similarities in their GP compositions, e.g., Saccharomyces cerevisiae and Saccharomyces bayanus. PMID:24957378

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.

Predominant yeasts in Chinese traditional sourdough and their influence on aroma formation in Chinese steamed bread.

PubMed

Liu, Tongjie; Li, Yang; Sadiq, Faizan A; Yang, Huanyi; Gu, Jingsi; Yuan, Lei; Lee, Yuan Kun; He, Guoqing

2018-03-01

A total of 105 yeast isolates was obtained from 15 sourdough samples collected from different regions in China and subjected to random amplified polymorphic DNA (RAPD) analysis. Six species were identified including Pichia membranifaciens, which has not previously been reported in Chinese sourdoughs. Different species of yeast were used in single-culture fermentation to make Chinese steamed bread (CSB). The volatiles of the CSB were captured by solid-phase microextraction method, separated and identified by gas chromatography-mass spectrometry. In total, 41 volatile compounds were found in all the steamed breads. All CSBs showed a similar volatile profile; however, significant differences in the quantity of some volatile compounds were seen among the CSB fermented by different yeast species. A partial least squares discriminant analysis showed that the CSBs could be separated by their characteristic volatile profiles. The study suggested that the aromatic properties of CSB are determined by the yeast used. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural characterization of the α-mating factor prepro-peptide for secretion of recombinant proteins in Pichia pastoris.

PubMed

Chahal, Sabreen; Wei, Peter; Moua, Pachai; Park, Sung Pil James; Kwon, Janet; Patel, Arth; Vu, Anthony T; Catolico, Jason A; Tsai, Yu Fang Tina; Shaheen, Nadia; Chu, Tiffany T; Tam, Vivian; Khan, Zill-E-Huma; Joo, Hyun Henry; Xue, Liang; Lin-Cereghino, Joan; Tsai, Jerry W; Lin-Cereghino, Geoff P

2017-01-20

The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation, the ability to provide complex posttranslational modifications and the capacity for efficient protein secretion. The most successful and commonly used secretion signal leader in Pichia pastoris has been the alpha mating factor (MATα) prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently, leading to strategies to enhance secretion efficiency by modifying the secretion signal leader. Based on a Jpred secondary structure prediction and knob-socket modeling of tertiary structure, numerous deletions and duplications of the MATα prepro leader were engineered to evaluate the correlation between predicted secondary structure and the secretion level of the reporters horseradish peroxidase (HRP) and Candida antarctica lipase B. In addition, circular dichroism analyses were completed for the wild type and several mutant pro-peptides to evaluate actual differences in secondary structure. The results lead to a new model of MATα pro-peptide signal leader, which suggests that the N and C-termini of MATα pro-peptide need to be presented in a specific orientation for proper interaction with the cellular secretion machinery and for efficient protein secretion. Copyright © 2016 Elsevier B.V. All rights reserved.

Ethanol production from sugarcane bagasse hydrolysate using Pichia stipitis.

PubMed

Canilha, Larissa; Carvalho, Walter; Felipe, Maria das Graças de Almeida; Silva, João Batista de Almeida e; Giulietti, Marco

2010-05-01

The objective of this study was to evaluate the ethanol production from the sugars contained in the sugarcane bagasse hemicellulosic hydrolysate with the yeast Pichia stipitis DSM 3651. The fermentations were carried out in 250-mL Erlenmeyers with 100 mL of medium incubated at 200 rpm and 30 degrees C for 120 h. The medium was composed by raw (non-detoxified) hydrolysate or by hydrolysates detoxified by pH alteration followed by active charcoal adsorption or by adsorption into ion-exchange resins, all of them supplemented with yeast extract (3 g/L), malt extract (3 g/L), and peptone (5 g/L). The initial concentration of cells was 3 g/L. According to the results, the detoxification procedures removed inhibitory compounds from the hemicellulosic hydrolysate and, thus, improved the bioconversion of the sugars into ethanol. The fermentation using the non-detoxified hydrolysate led to 4.9 g/L ethanol in 120 h, with a yield of 0.20 g/g and a productivity of 0.04 g L(-1) h(-1). The detoxification by pH alteration and active charcoal adsorption led to 6.1 g/L ethanol in 48 h, with a yield of 0.30 g/g and a productivity of 0.13 g L(-1) h(-1). The detoxification by adsorption into ion-exchange resins, in turn, provided 7.5 g/L ethanol in 48 h, with a yield of 0.30 g/g and a productivity of 0.16 g L(-1) h(-1).

Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris

PubMed Central

Bazan, Silvia Boschi; de Alencar Muniz Chaves, Agtha; Aires, Karina Araújo; Cianciarullo, Aurora Marques; Garcea, Robert L.; Ho, Paulo Lee

2013-01-01

Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin–sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines. PMID:19756360

Bacaba beverage produced by Umutina Brazilian Amerindians: Microbiological and chemical characterization

PubMed Central

Puerari, Cláudia; Magalhães-Guedes, Karina Teixeira; Schwan, Rosane Freitas

2015-01-01

Bacaba chicha is a beverage prepared by the indigenous Umutina people from the bacaba fruit (Oenocarpus bacaba), a purple berry that is rich in fat and carbohydrates, as well as a source of phenolic compounds. In this study, samples of bacaba chicha beverage were collected, and the microbial community was assessed using culture-dependent and -independent techniques. The nutritional composition and metabolite profiles were analyzed, and species belonging to lactic acid bacteria (LAB) and yeasts were detected. The LAB group detected by culture-dependent analysis included Enterococcus hormaechei and Leuconostoc lactis. Polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) detected additional Propionibacterium avidum, Acetobacter spp., and uncultured bacteria. Pichia caribbica and Pichia guilliermondii were detected in a culture-dependent method, and Pichia caribbica was confirmed by PCR-DGGE analysis. The pH value of the beverage was 6.2. The nutritional composition was as follows: 16.47 ± 0.73 g 100 mL-1 dry matter, 2.2 ± 0.0 g 100 mL-1 fat, 3.36 ± 0.44 g 100 mL-1 protein, and 10.87 ± 0.26 g 100 mL-1 carbohydrate. The metabolites detected were 2.69 g L-1 succinic acid, 0.9 g L-1 acetic acid, 0.49 g L-1 citric acid, 0.52 g L-1 ethanol, and 0.4 g L-1 glycerol. This is the first study to identify microbial diversity in bacaba chicha spontaneous fermentation. This study is also the starting step in the immaterial record of this Brazilian indigenous beverage prepared from bacaba fruit. PMID:26691483

Bacaba beverage produced by Umutina Brazilian Amerindians: Microbiological and chemical characterization.

PubMed

Puerari, Cláudia; Magalhães-Guedes, Karina Teixeira; Schwan, Rosane Freitas

2015-01-01

Bacaba chicha is a beverage prepared by the indigenous Umutina people from the bacaba fruit (Oenocarpus bacaba), a purple berry that is rich in fat and carbohydrates, as well as a source of phenolic compounds. In this study, samples of bacaba chicha beverage were collected, and the microbial community was assessed using culture-dependent and -independent techniques. The nutritional composition and metabolite profiles were analyzed, and species belonging to lactic acid bacteria (LAB) and yeasts were detected. The LAB group detected by culture-dependent analysis included Enterococcus hormaechei and Leuconostoc lactis. Polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) detected additional Propionibacterium avidum, Acetobacter spp., and uncultured bacteria. Pichia caribbica and Pichia guilliermondii were detected in a culture-dependent method, and Pichia caribbica was confirmed by PCR-DGGE analysis. The pH value of the beverage was 6.2. The nutritional composition was as follows: 16.47 ± 0.73 g 100 mL-1 dry matter, 2.2 ± 0.0 g 100 mL-1 fat, 3.36 ± 0.44 g 100 mL-1 protein, and 10.87 ± 0.26 g 100 mL-1 carbohydrate. The metabolites detected were 2.69 g L-1 succinic acid, 0.9 g L-1 acetic acid, 0.49 g L-1 citric acid, 0.52 g L-1 ethanol, and 0.4 g L-1 glycerol. This is the first study to identify microbial diversity in bacaba chicha spontaneous fermentation. This study is also the starting step in the immaterial record of this Brazilian indigenous beverage prepared from bacaba fruit.

Characterization and Dynamic Behavior of Wild Yeast during Spontaneous Wine Fermentation in Steel Tanks and Amphorae

PubMed Central

Díaz, Cecilia; Molina, Ana María; Nähring, Jörg; Fischer, Rainer

2013-01-01

We studied the dynamic behavior of wild yeasts during spontaneous wine fermentation at a winery in the Valais region of Switzerland. Wild yeasts in the winery environment were characterized using a PCR-RFLP method. Up to 11 different yeast species were isolated from the vineyard air, whereas only seven were recovered from the grapes surface. We initially investigated a cultureindependent method in pilot-scale steel fermentation tanks and found a greater diversity of yeasts in the musts from two red grape varieties compared to three white grape varieties. We found that the yeasts Metschnikowia pulcherrima, Rhodotorula mucilaginosa, Pichia kluyveri, P. membranifaciens and Saccharomyces cerevisiae remained active at the end of the fermentation. We also studied the dynamic behavior of yeasts in Qvevris for the first time using a novel, highlysensitive quantitative real-time PCR method. We found that non-Saccharomyces yeasts were present during the entire fermentation process, with R. mucilaginosa and P. anomala the most prominent species. We studied the relationship between the predominance of different species and the output of the fermentation process. We identified so-called spoilage yeasts in all the fermentations, but high levels of acetic acid accumulated only in those fermentations with an extended lag phase. PMID:23738327

Species Distribution and Susceptibility to Azoles of Vaginal Yeasts Isolated Prostitutes

PubMed Central

Gross, Norma T.; Arias, M. L.; Moraga, M.; Baddasarow, Y.; Jarstrand, C.

2007-01-01

Objective. We investigated the use of miconazole among female prostitutes in Costa Rica as well as the distribution of vaginal yeasts and the susceptibility pattern to azoles of strains obtained from this population. Our intention was to relate a frequent use of miconazole to occurrence of vaginal yeasts resistant to azoles. Methods. Vaginal samples were taken from 277 patients that have previously used azoles. Vaginal swabs were obtained for direct microscopy and culture. Yeast isolates were identified by germ tube test and assimilation pattern. Susceptibility testing was determined using a tablet diffusion method. Results. The number of clinical Candida isolates (one from each patient) was 57 (20.6%). C. albicans was the predominant species (70%), followed by C. parapsilosis (12%), C. tropicalis (5.3%), C. glabrata and C. famata (3.5% each), C. krusei, C. inconspicua and C. guilliermondii (1.7% each). The majority of vaginal Candida isolates were susceptible to ketoconazole (91%), fluconazole (96.5%), and itraconazole (98%). A lower susceptibility of some isolates to miconazole (63%) was observed as compared to the other azoles tested. Moreover, the strains, nonsusceptible to miconazole, were more often obtained from patients that have used this antifungal at least four times within the last year before taking the samples as compared to those with three or less treatments (P<.01). Conclusion. An indiscriminate use of miconazole, such as that observed among female prostitutes in Costa Rica, results in a reduced susceptibility of vaginal yeasts to miconazole but not to other azoles. PMID:18273407

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

Yeast community associated with the solid state fermentation of traditional Chinese Maotai-flavor liquor.

PubMed

Wu, Qun; Chen, Liangqiang; Xu, Yan

2013-09-02

Yeasts are the most important group of microorganisms contributing to liquor quality in the solid-state fermentation process of Chinese Maotai-flavor liquor. There occurred a complex yeast community structure during this process, including stages of Daqu (the starter) making, stacking fermentation on the ground and liquor fermentation in the pits. In the Daqu making stage, few yeast strains accumulated. However, the stacking fermentation stage accumulated nine yeast species with different physio-biochemical characteristics. But only four species kept dominant until liquor fermentation, which were Zygosaccharomyces bailii, Saccharomyces cerevisiae, Pichia membranifaciens, and Schizosaccharomyces pombe, implying their important functions in liquor making. The four species tended to inhabit in different locations of the stack and pits during stacking and liquor fermentation, due to the condition heterogeneity of the solid-state fermentation, including the different fermentation temperature profiles and oxygen density in different locations. Moreover, yeast population was much larger in the upper layer than that in the middle and bottom layers in liquor fermentation, which was in accordance with the profile of reducing sugar consumption and ethanol production. This was a systematical investigation of yeast community structure dynamics in the Maotai-flavor liquor fermentation process. It would be of help to understand the fermentative mechanism in solid-state fermentation for Maotai-flavor liquor. © 2013.

Return to Glacier Bay

USGS Publications Warehouse

Bodkin, James L.

2003-01-01

Seven species of pigeons and doves were cultured for yeasts in the upper digestive tract. The following list gives the isolation rate for each columbid species and the yeasts cultured from them: feral pigeon Columba Livia (Gmelin) 95% -Candida albicans (Robin) Berkhout, C. tropicalis (Castellani) Berkhout, C. krusei (Cast.) Berkhout, C. guilliermondii (Cast.) Langeron et Guerra, Torulopsis glabrata (Anderson) Lodder et De Vries, Saccharomyces telluris Van der Walt, and Geotrichum sp.; white-crowned pigeon (C. leucocephala Linnaeus) 56% -- S. telluris; mourning dove (Zenaidura rnacroura Linnaeus) 24% -- C. albicans, C. tropicalis, C. guilliermondii, and Geotrichurn sp.; passerine ground dove (Collumbigallina passerina Linnaeus) 20% -- C. parapsilosis (Ashford) Langeron et Talice, Kloeckera apiculata (Reess Emend. Klocker) Janke; zenaida dove (Zenaida aurita Temminck) 16% -- C. albicans, C. guilliermondii, and T. glabrata; one moustasche dove (Geotrygon mystacea Gosse) -- C. guillierrnondii; ringed turtle dove (Streptopelia rizoria Linnaeus) 14% -- C. albicans and Geotrichurn sp. No signs of disease could be seen in the 139 birds that were examined, and it was concluded that these yeasts comprise a part of the columbid's normal microbial flora.

Chemical signaling and insect attraction is a conserved trait in yeasts.

PubMed

Becher, Paul G; Hagman, Arne; Verschut, Vasiliki; Chakraborty, Amrita; Rozpędowska, Elżbieta; Lebreton, Sébastien; Bengtsson, Marie; Flick, Gerhard; Witzgall, Peter; Piškur, Jure

2018-03-01

Yeast volatiles attract insects, which apparently is of mutual benefit, for both yeasts and insects. However, it is unknown whether biosynthesis of metabolites that attract insects is a basic and general trait, or if it is specific for yeasts that live in close association with insects. Our goal was to study chemical insect attractants produced by yeasts that span more than 250 million years of evolutionary history and vastly differ in their metabolism and lifestyle. We bioassayed attraction of the vinegar fly Drosophila melanogaster to odors of phylogenetically and ecologically distinct yeasts grown under controlled conditions. Baker's yeast Saccharomyces cerevisiae , the insect-associated species Candida californica , Pichia kluyveri and Metschnikowia andauensis , wine yeast Dekkera bruxellensis , milk yeast Kluyveromyces lactis , the vertebrate pathogens Candida albicans and Candida glabrata , and oleophilic Yarrowia lipolytica were screened for fly attraction in a wind tunnel. Yeast headspace was chemically analyzed, and co-occurrence of insect attractants in yeasts and flowering plants was investigated through a database search. In yeasts with known genomes, we investigated the occurrence of genes involved in the synthesis of key aroma compounds. Flies were attracted to all nine yeasts studied. The behavioral response to baker's yeast was independent of its growth stage. In addition to Drosophila , we tested the basal hexapod Folsomia candida (Collembola) in a Y-tube assay to the most ancient yeast, Y. lipolytica, which proved that early yeast signals also function on clades older than neopteran insects. Behavioral and chemical data and a search for selected genes of volatile metabolites underline that biosynthesis of chemical signals is found throughout the yeast clade and has been conserved during the evolution of yeast lifestyles. Literature and database reviews corroborate that yeast signals mediate mutualistic interactions between insects and yeasts

Bioflavoring by non-conventional yeasts in sequential beer fermentations.

PubMed

Holt, Sylvester; Mukherjee, Vaskar; Lievens, Bart; Verstrepen, Kevin J; Thevelein, Johan M

2018-06-01

Non-conventional yeast species have great capacity for producing diverse flavor profiles in production of alcoholic beverages, but their potential for beer brewing, in particular in consecutive fermentations with Saccharomyces cerevisiae, has only poorly been explored. We have screened 17 non-conventional yeast species for production of an appealing profile of flavor esters and phenolics in the first phase of alcoholic fermentation, followed by inoculation with S. cerevisiae to complete the fermentation. For measurement of phenolic compounds and their precursors we developed an improved and highly sensitive methodology. The results show that non-conventional yeast species possess promising potential for enhancement of desirable flavors in beer production. Notable examples are increasing isoamyl acetate (fruity, banana flavor) by application of P. kluyverii, augmenting ethyl phenolic compounds (spicy notes) with Brettanomyces species and enhancing 4-vinyl guaiacol (clove-like aroma) with T. delbrueckii. All Pichia strains also produced high levels of ethyl acetate (solvent-like flavor). This might be selectively counteracted by selection of an appropriate S. cerevisiae strain for the second fermentation phase, which lowers total ester profile. Hence, optimization of the process conditions and/or proper strain selection in sequentially inoculated fermentations are required to unlock the full potential for aroma improvement by the non-conventional yeast species. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Kinetic modelling and optimisation of antimicrobial compound production by Candida pyralidae KU736785 for control of Candida guilliermondii.

PubMed

Mewa-Ngongang, Maxwell; du Plessis, Heinrich W; Hutchinson, Ucrecia F; Mekuto, Lukhanyo; Ntwampe, Seteno Ko

2017-06-01

Biological antimicrobial compounds from yeast can be used to address the critical need for safer preservatives in food, fruit and beverages. The inhibition of Candida guilliermondii, a common fermented beverage spoilage organism, was achieved using antimicrobial compounds produced by Candida pyralidae KU736785. The antimicrobial production system was modelled and optimised using response surface methodology, with 22.5 ℃ and pH of 5.0 being the optimum conditions. A new concept for quantifying spoilage organism inhibition was developed. The inhibition activity of the antimicrobial compounds was observed to be at a maximum after 17-23 h of fermentation, with C. pyralidae concentration being between 0.40 and 1.25 × 10 9 CFU ml -1 , while its maximum specific growth rate was 0.31-0.54 h -1 . The maximum inhibitory activity was between 0.19 and 1.08 l contaminated solidified media per millilitre of antimicrobial compound used. Furthermore, the antimicrobial compound formation rate was 0.037-0.086 l VZI ml -1 ACU h -1 , respectively. The response surface methodology analysis showed that the model developed sufficiently described the antimicrobial compound formation rate 1.08 l VZI ml -1 ACU, as 1.17 l VZI ml -1 ACU, predicted under the optimum production conditions.

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.

Safety and regulation of yeasts used for biocontrol or biopreservation in the food or feed chain.

PubMed

Sundh, Ingvar; Melin, Petter

2011-01-01

Yeasts have been important components of spontaneous fermentations in food and beverage processing for millennia. More recently, the potential of utilising antagonistic yeasts, e.g. Pichia anomala and Candida spp., for post-harvest biological control of spoilage fungi during storage of plant-derived produce ('biopreservation') has been clearly demonstrated. Although some yeast species are among the safest microorganisms known, several have been reported in opportunistic infections in humans, including P. anomala and bakers' yeast, Saccharomyces cerevisiae. More research is needed about the dominant pathogenicity and virulence factors in opportunistic yeasts, and whether increased utilisation of biopreservative yeasts in general could contribute to an increased prevalence of yeast infections. The regulatory situation for yeasts used in post-harvest biocontrol is complex and the few products that have reached the market are mainly registered as biological pesticides. The qualified presumption of safety (QPS) approach to safety assessments of microorganisms intentionally added to food or feed, recently launched by the European Food Safety Authority, can lead to more efficient evaluations of new products containing microbial species with a sufficient body of knowledge or long-term experience on their safety. P. anomala is one of several yeast species that have been given QPS status, although the status is restricted to use of this yeast for enzyme and metabolite production purposes. With regard to authorisation of new biopreservative yeasts, we recommend that the possibility to regulate microorganisms for food biopreservation as food additives be considered.

Genetic improvement of native xylose-fermenting yeasts for ethanol production.

PubMed

Harner, Nicole K; Wen, Xin; Bajwa, Paramjit K; Austin, Glen D; Ho, Chi-Yip; Habash, Marc B; Trevors, Jack T; Lee, Hung

2015-01-01

Lignocellulosic substrates are the largest source of fermentable sugars for bioconversion to fuel ethanol and other valuable compounds. To improve the economics of biomass conversion, it is essential that all sugars in potential hydrolysates be converted efficiently into the desired product(s). While hexoses are fermented into ethanol and some high-value chemicals, the bioconversion of pentoses in hydrolysates remains inefficient. This remains one of the key challenges in lignocellulosic biomass conversion. Native pentose-fermenting yeasts can ferment both glucose and xylose in lignocellulosic biomass to ethanol. However, they perform poorly in the presence of hydrolysate inhibitors, exhibit low ethanol tolerance and glucose repression, and ferment pentoses less efficiently than the main hexoses glucose and mannose. This paper reviews classical and molecular strain improvement strategies applied to native pentose-fermenting yeasts for improved ethanol production from xylose and lignocellulosic substrates. We focus on Pachysolen tannophilus, Scheffersomyces (Candida) shehatae, Scheffersomyces (Pichia) stipitis, and Spathaspora passalidarum which are good ethanol producers among the native xylose-fermenting yeasts. Strains obtained thus far are not robust enough for efficient ethanol production from lignocellulosic hydrolysates and can benefit from further improvements.

Decreased Killing Activity of Micafungin Against Candida guilliermondii, Candida lusitaniae, and Candida kefyr in the Presence of Human Serum.

PubMed

Saleh, Qasem; Kovács, Renátó; Kardos, Gábor; Gesztelyi, Rudolf; Kardos, Tamás; Bozó, Aliz; Majoros, László

2017-09-01

Currently, echinocandins are first-line drugs for treatment of invasive candidiasis. However, data on how serum influences killing activity of echinocandins against uncommon Candida species are limited. Therefore, the killing activity of micafungin in RPMI-1640 and in 50% serum was compared against Candida guilliermondii, Candida lusitaniae, and Candida kefyr. Minimum inhibitory concentration (MIC) ranges in RPMI-1640 were 0.5-1, 0.12-0.25, and 0.06-0.12 mg/L, respectively. In 50% serum, MICs increased 32- to 256-fold. In RPMI-1640 ≥ 0.25, ≥4, and 32 mg/L micafungin was fungicidal against all four C. kefyr (≤4.04 hours), two of three C. lusitaniae (≤16.10 hours), and two of three C. guilliermondii (≤12.30 hours), respectively. In 50% serum, all three species grew at ≤4 mg/L. Micafungin at 16-32 mg/L was fungicidal against all C. kefyr isolates (≤3.03 hours) and at 32 mg/L was fungistatic against one of three C. lusitaniae isolates. Two C. lusitaniae isolates and all three C. guilliermondii grew at all tested concentrations. Adding human serum to susceptibility test media drew attention to loss of fungicidal or fungistatic activity of micafungin in the presence of serum proteins, which is not predicted by MICs in case of C. kefyr and C. lusitaniae in RPMI-1640. Our results strongly suggest that micafungin and probably other echinocandins should be used with caution against rare Candida species.

Isolation of Yeasts from Guajillo Pepper (Capsicum annuum L.) Fermentation and Study of Some Probiotic Characteristics.

PubMed

Lara-Hidalgo, C E; Dorantes-Álvarez, L; Hernández-Sánchez, H; Santoyo-Tepole, F; Martínez-Torres, A; Villa-Tanaca, L; Hernández-Rodríguez, C

2018-04-25

Three yeast strains were isolated from the spontaneous fermentation of guajillo pepper: Hanseniaspora opuntiae, Pichia kudriavzevii, and Wickerhamomyces anomalus, which were identified by amplification of the ITS/5.8S ribosomal DNA. Some probiotic characteristics of these strains were evaluated and compared with one commercial probiotic yeast (Saccharomyces boulardii). The survival percentage of all the yeasts was similar to that of the commercial product. They showed different hydrophobicity characteristics with hydrocarbons, autoaggregation > 90%, and characteristics of co-aggregation with pathogenic microorganisms. The adhesion capacity to mucin of the three yeast samples was similar to the reference yeast. The antioxidant activity of the yeasts varied between 155 and 178 μM Trolox equivalents. All exhibited cholesterol reduction capacity, and W. anomalus was able to decrease up to 83% of cholesterol after 48 h of incubation. The 7.5-fold concentrated H. opuntiae supernatant had antimicrobial activity against Salmonella enterica ser. Typhimurium ATCC 14028 and Candida albicans ENCBDM2; tests suggest this activity against S. Typhimurium is due to a proteinaceous metabolite with a weight between 10 and 30 kDa. Among the yeasts, P. kudriavzevii exhibited the highest protective effect on the viability of Lactobacillus casei Shirota in gastric and intestinal conditions. These results suggest that yeasts isolated from guajillo pepper may have a probiotic potential.

Constitutive expression of the active fragment of human vasostatin Vs30 in Pichia pastoris SMD1168H.

PubMed

Calderon-Salais, Sergio; Velazquez-Bernardino, Prisiliana; Balderas-Hernandez, Victor E; Barba de la Rosa, Ana P; De Leon-Rodriguez, Antonio

2018-04-01

Vasostatin 30 (Vs30) is an active fragment derived from the N-terminal region (135-164 aa) of human calreticulin and has the ability to inhibit angiogenesis. In this work, the expression of Vs30 was performed using a protease-deficient strain of the methylotrophic yeast Pichia pastoris. The vs30 gene was optimized for P. pastoris preferential codon usage and inserted into constitutive expression vector pGAPZαA. In addition, a plasmid with four copies of the expression cassette was obtained and transformed into P. pastoris. The flask fermentation conditions were: culture volume of 25 mL in 250 mL baffled flasks at 28 °C, pH 6 and harvest time of 48 h. Up to 21.07 mg/L Vs30 were attained and purified by ultrafiltration with a 30-kDa cut-off membrane and the recovery was 49.7%. Bioactivity of Vs30 was confirmed by the inhibition of cell proliferation, as well as the inhibition of the capillary-like structures formation of EA.hy926 cells in vitro. This work constitutes the first report on the expression of Vs30 in Pichia pastoris using a constitutive promoter and multi-copy approach such as strategies to improve the recombinant Vs30 expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Ethanol production from xylose with the yeast Pichia stipitis and simultaneous product recovery by gas stripping using a gas-lift loop fermentor with attached side-arm (GLSA).

PubMed

Domínguez, J M; Cao, N; Gong, C S; Tsao, G T

2000-02-05

The bioconversion of xylose into ethanol with the yeast Pichia stipitis CBS 5773 is inhibited when 20 g/L of ethanol are present in the fermentation broth. In order to avoid this limitation, the fermentation was carried out with simultaneous recovery of product by CO(2) stripping. The fermentation was also improved by attaching a side-arm to the main body of a classical gas-lift loop fermentor. This side-arm increases the liquid circulation, mass transfer, and gas distribution, reducing the amount of oxygen in the inlet gas necessary to perform the fermentation of xylose under microaerobic conditions (K(L)a approximately 16 h(-1)). The continuous stripping of ethanol from the fermentation broth in this new bioreactor system allowed the consumption of higher xylose concentrations than using Erlenmeyer shaker flasks, improved significantly the process productivity and provided a clean ethanol solution by using an ice-cooled condenser system. Finally, a fed-batch fermentation was carried out with a K(L)a = 15.8 h(-1). Starting with 248.2 g of xylose, 237.6 g of xylose was consumed to produce 88.1 g of ethanol which represents 72.6% of the theoretical yield (47.2 g/L of ethanol was recovered in the condenser, while 9.6 g/L remained in the fermentation broth). Copyright 2000 John Wiley & Sons, Inc.

Biodegradable Bioadherent Microcapsules for Orally Administered Sustained Release Vaccines. Phase 1.

DTIC Science & Technology

1995-10-23

based on homology with the yeast alcohol oxidase (AOX) or histidine (his 4) loci and also upon the site of linearization of the plasmid since DNA ends...and Quantitation of Recombinant Fasciola Protein from the Pichia Yeast Expression System ...... 9 B. Production of Microcarriers...Recombinant Fasciola Protein from the Pichia Yeast Expression System ..... 15 B. Production of Microcarriers

Expression of lignocellulolytic enzymes in Pichia pastoris

PubMed Central

2012-01-01

Background Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes. Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages. Results In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes we could develop strains capable of secreting gram quantities of enzyme per liter in fed-batch cultivations. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. Conclusion In our experiments we could clearly show the importance of gene optimization and strain characterization for successfully improving secretion levels. We also present a basic guideline how to correctly interpret the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris. PMID:22583625

Cloning and sequence analysis of the LEU2 homologue gene from Pichia anomala.

PubMed

De la Rosa, J M; Pérez, J A; Gutiérrez, F; González, J M; Ruiz, T; Rodríguez, L

2001-11-01

The Pichia anomala LEU2 gene (PaLEU2) was isolated by complementation of a leu2 Saccharomyces cerevisiae mutant. The cloned gene also allowed growth of a Escherichia coli leuB mutant in leucine-lacking medium, indicating that it encodes a product able to complement the beta-isopropylmalate dehydrogenase deficiency of the mutants. The sequenced DNA fragment contains a complete ORF of 1092 bp, and the deduced polypeptide shares significant homologies with the products of the LEU2 genes from S. cerevisiae (84% identity) and other yeast species. A sequence resembling the GC-rich palindrome motif identified in the 5' region of S. cerevisiae LEU2 gene as the binding site for the transcription activating factor encoded by the LEU3 gene was found at the promoter region. In addition, upstream of the PaLEU2 the 3'-terminal half of a gene of the same orientation, encoding a homologue of the S. cerevisiae NFS1/SPL1 gene that encodes a mitochondrial cysteine desulphurase involved in both tRNA processing and mitochondrial metabolism, was found. The genomic organization of the PaNFS1-PaLEU2 gene pair is similar to that found in several other yeast species, including S. cerevisiae and Candida albicans, except that in some of them the LEU2 gene appears in the reverse orientation. Copyright 2001 John Wiley & Sons, Ltd.

Potential benefits of the application of yeast starters in table olive processing.

PubMed

Arroyo-López, Francisco N; Romero-Gil, Verónica; Bautista-Gallego, Joaquín; Rodríguez-Gómez, Francisco; Jiménez-Díaz, Rufino; García-García, Pedro; Querol, Amparo; Garrido-Fernández, Antonio

2012-01-01

Yeasts play an important role in the food and beverage industry, especially in products such as bread, wine, and beer, among many others. However, their use as a starter in table olive processing has not yet been studied in detail. The candidate yeast strains should be able to dominate fermentation, together with lactic acid bacteria, but should also provide a number of beneficial advantages. Technologically, yeasts should resist low pH and high salt concentrations, produce desirable aromas, improve lactic acid bacteria growth, and inhibit spoilage microorganisms. Nowadays, they are being considered as probiotic agents because many species are able to resist the passage through the gastrointestinal tract and show favorable effects on the host. In this way, yeasts may improve the health of consumers by means of the degradation of non-assimilated compounds (such as phytate complexes), a decrease in cholesterol levels, the production of vitamins and antioxidants, the inhibition of pathogens, an adhesion to intestinal cell line Caco-2, and the maintenance of epithelial barrier integrity. Many yeast species, usually found in table olive processing (Wickerhamomyces anomalus, Saccharomyces cerevisiae, Pichia membranifaciens, and Kluyveromyces lactis, among others), have exhibited some of these properties. Thus, the selection of the most appropriate strains to be used as starters in this fermented vegetable, alone or in combination with lactic acid bacteria, is a promising research line to develop in the near future.

Potential benefits of the application of yeast starters in table olive processing

PubMed Central

Arroyo-López, Francisco N.; Romero-Gil, Verónica; Bautista-Gallego, Joaquín; Rodríguez-Gómez, Francisco; Jiménez-Díaz, Rufino; García-García, Pedro; Querol, Amparo; Garrido-Fernández, Antonio

2012-01-01

Yeasts play an important role in the food and beverage industry, especially in products such as bread, wine, and beer, among many others. However, their use as a starter in table olive processing has not yet been studied in detail. The candidate yeast strains should be able to dominate fermentation, together with lactic acid bacteria, but should also provide a number of beneficial advantages. Technologically, yeasts should resist low pH and high salt concentrations, produce desirable aromas, improve lactic acid bacteria growth, and inhibit spoilage microorganisms. Nowadays, they are being considered as probiotic agents because many species are able to resist the passage through the gastrointestinal tract and show favorable effects on the host. In this way, yeasts may improve the health of consumers by means of the degradation of non-assimilated compounds (such as phytate complexes), a decrease in cholesterol levels, the production of vitamins and antioxidants, the inhibition of pathogens, an adhesion to intestinal cell line Caco-2, and the maintenance of epithelial barrier integrity. Many yeast species, usually found in table olive processing (Wickerhamomyces anomalus, Saccharomyces cerevisiae, Pichia membranifaciens, and Kluyveromyces lactis, among others), have exhibited some of these properties. Thus, the selection of the most appropriate strains to be used as starters in this fermented vegetable, alone or in combination with lactic acid bacteria, is a promising research line to develop in the near future.

The genome of wine yeast Dekkera bruxellensis provides a tool to explore its food-related properties

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

Piskur, Jure; Ling, Zhihao; Marcet-Houben, Marina

2012-03-14

The yeast Dekkera/Brettanomyces bruxellensis can cause enormous economic losses in wine industry due to production of phenolic off-flavor compounds. D. bruxellensis is a distant relative of baker's yeast Saccharomyces cerevisiae. Nevertheless, these two yeasts are often found in the same habitats and share several food-related traits, such as production of high ethanol levels and ability to grow without oxygen. In some food products, like lambic beer, D. bruxellensis can importantly contribute to flavor development. We determined the 13.4 Mb genome sequence of the D. bruxellensis strain Y879 (CBS2499) and deduced the genetic background of several ?food-relevant? properties and evolutionary historymore » of this yeast. Surprisingly, we find that this yeast is phylogenetically distant to other food-related yeasts and most related to Pichia (Komagataella) pastoris, which is an aerobic poor ethanol producer. We further show that the D. bruxellensis genome does not contain an excess of lineage specific duplicated genes nor a horizontally transferred URA1 gene, two crucial events that promoted the evolution of the food relevant traits in the S. cerevisiae lineage. However, D. bruxellensis has several independently duplicated ADH and ADH-like genes, which are likely responsible for metabolism of alcohols, including ethanol, and also a range of aromatic compounds.« less

Changes in volatile profile of soybean residue (okara) upon solid-state fermentation by yeasts.

PubMed

Vong, Weng Chan; Liu, Shao-Quan

2017-01-01

Soybean residue (okara), a by-product of soymilk, is produced in large volumes by the soy food industry and is often discarded due to its undesirable flavour. As it contains a considerable amount of protein and fats, biotransformation of okara to improve its flavour presents an opportunity for alternative utilisation. This paper evaluated 10 yeasts in the solid-state fermentation of okara based on their volatile profiles as analysed with HS-SPME GC-MS/FID. Four 'dairy yeasts' (Geotrichum candidum, Yarrowia lipolytica, Debaryomyces hansenii and Kluyveromyces lactis) and six 'wine yeasts' (Saccharomyces cerevisiae, Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia kluyveri, Torulaspora delbrueckii, and Williopsis saturnus) were studied. The main off-odourants in okara, hexanal and trans-2-hexenal, significantly decreased after fermentation due to their bioconversion into methyl ketones and/or esters. The okara fermented by dairy yeasts contained greater proportions of methyl ketones, while that by wine yeasts contained more ethyl and acetyl esters. Notably, the okara fermented by W. saturnus contained 13 esters and the total GC-FID peak area of esters was about 380 times that in fresh okara, leading to a perceptible fruity note. Okara can be exploited as an inexpensive substrate for bioflavour extraction and/or a more pleasant food ingredient via yeast fermentation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Denaturing gradient gel electrophoresis profiles of bacteria from the saliva of twenty four different individuals form clusters that showed no relationship to the yeasts present.

PubMed

M Weerasekera, Manjula; H Sissons, Chris; Wong, Lisa; A Anderson, Sally; R Holmes, Ann; D Cannon, Richard

2017-10-01

The aim was to investigate the relationship between groups of bacteria identified by cluster analysis of the DGGE fingerprints and the amounts and diversity of yeast present. Bacterial and yeast populations in saliva samples from 24 adults were analysed using denaturing gradient gel electrophoresis (DGGE) of the bacteria present and by yeast culture. Eubacterial DGGE banding patterns showed considerable variation between individuals. Seventy one different amplicon bands were detected, the band number per saliva sample ranged from 21 to 39 (mean±SD=29.3±4.9). Cluster and principal component analysis of the bacterial DGGE patterns yielded three major clusters containing 20 of the samples. Seventeen of the 24 (71%) saliva samples were yeast positive with concentrations up to 10 3 cfu/mL. Candida albicans was the predominant species in saliva samples although six other yeast species, including Candida dubliniensis, Candida tropicalis, Candida krusei, Candida guilliermondii, Candida rugosa and Saccharomyces cerevisiae, were identified. The presence, concentration, and species of yeast in samples showed no clear relationship to the bacterial clusters. Despite indications of in vitro bacteria-yeast interactions, there was a lack of association between the presence, identity and diversity of yeasts and the bacterial DGGE fingerprint clusters in saliva. This suggests significant ecological individual-specificity of these associations in highly complex in vivo oral biofilm systems under normal oral conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Bio-prospecting of distillery yeasts as bio-control and bio-remediation agents.

PubMed

Ubeda, Juan F; Maldonado, María; Briones, Ana I; Francisco, J Fernández; González, Francisco J

2014-05-01

This work constitutes a preliminary study in which the capacity of non-Saccharomyces yeasts isolated from ancient distilleries as bio-control agents against moulds and in the treatment of waste waters contaminated by heavy metals-i.e. bio-remediation-is shown. In the first control assays, antagonist effect between non-Saccharomyces yeasts, their extracts and supernatants against some moulds, analysing the plausible (not exhaustive) involved factors were qualitatively verified. In addition, two enzymatic degrading properties of cell wall plant polymers, quitinolitic and pectinolitic, were screened. Finally, their use as agents of bio-remediation of three heavy metals (cadmium, chromium and lead) was analysed semi-quantitatively. The results showed that all isolates belonging to Pichia species effectively inhibited all moulds assayed. Moreover, P. kudriavzevii is a good candidate for both bio-control and bio-remediation because it inhibited moulds and accumulated the major proportion of the three tested metals.

Occurrence of yeasts in psittacines droppings from captive birds in Italy.

PubMed

Mancianti, Francesca; Nardoni, Simona; Ceccherelli, Renato

2002-01-01

Three-hundred twenty five droppings from parrots raised in the premises of 4 breeders and in several private households were cultured for yeasts. One-hundred sixty droppings (49.2%) resulted positive. From these specimens 212 isolates belonging to 27 different species were obtained. Mainly Candida species such as C. albicans, C. catenulata, C. curvata, C. famata, C. glabrata, C. guilliermondi, C. holmii, C. intermedia, C. krusei, C. lambica, C. lusitaniae, C. membranaefaciens, C. parapsilosis, C. pelliculosa, C. sake and C. valida were isolated. Debarvomyces marama, D. polymorphus, Geotrichum sp., Pichia etchelsii, P. ohmeri, Rhodotorula glutinis, R. rubra, Rhodotorula sp., Saccharomyces cerevisiae, S. kluyiveri and Zygosaccharomyces sp. were also obtained. Dark colonies on Staib medium were never observed. The psittacine birds apparently serve as carriers for several Candida species or their perfect states and to a lesser extent for other opportunistic yeasts such as Rhodotorula, Trichosporon and Saccharomyces spp., which are considered part of the transient microbiota of the gastrointestinal tract. The most striking finding was the absence of Cryptococcus spp. among the isolates. The present survey confirms the role of pet birds in carrying potential zoonotic yeasts.

Pichia pastoris Exhibits High Viability and a Low Maintenance Energy Requirement at Near-Zero Specific Growth Rates

PubMed Central

Rebnegger, Corinna; Vos, Tim; Graf, Alexandra B.; Valli, Minoska; Pronk, Jack T.

2016-01-01

ABSTRACT The yeast Pichia pastoris is a widely used host for recombinant protein production. Understanding its physiology at extremely low growth rates is a first step in the direction of decoupling product formation from cellular growth and therefore of biotechnological relevance. Retentostat cultivation is an excellent tool for studying microbes at extremely low specific growth rates but has so far not been implemented for P. pastoris. Retentostat feeding regimes were based on the maintenance energy requirement (mS) and maximum biomass yield on glucose (YX/Smax) estimated from steady-state glucose-limited chemostat cultures. Aerobic retentostat cultivation enabled reproducible, smooth transitions from a specific growth rate (μ) of 0.025 h−1 to near-zero specific growth rates (μ < 0.001 h−1). At these near-zero specific growth rates, viability remained at least 97%. The value of mS at near-zero growth rates was 3.1 ± 0.1 mg glucose per g biomass and h, which was 3-fold lower than the mS estimated from faster-growing chemostat cultures. This difference indicated that P. pastoris reduces its maintenance energy requirement at extremely low μ, a phenomenon not previously observed in eukaryotes. Intracellular levels of glycogen and trehalose increased, while μ progressively declined during retentostat cultivation. Transcriptional reprogramming toward zero growth included the upregulation of many transcription factors as well as stress-related genes and the downregulation of cell cycle genes. This study underlines the relevance of comparative analysis of maintenance energy metabolism, which has an important impact on large-scale industrial processes. IMPORTANCE The yeast Pichia pastoris naturally lives on trees and can utilize different carbon sources, among them glucose, glycerol, and methanol. In biotechnology, it is widely used for the production of recombinant proteins. For both the understanding of life in its natural habitat and optimized production

Ethanol production from eucalyptus wood hemicellulose hydrolysate by Pichia stipitis.

PubMed

Ferrari, M D; Neirotti, E; Albornoz, C; Saucedo, E

1992-10-05

Ethanol production was evaluated from eucalyptus wood hemicellulose acid hydrolysate using Pichia stipitis NRRL Y-7124. An initial lag phase characterized by flocculation and viability loss of the yeast inoculated was observed. Subsequently, cell regrowth occurred with sequential consumption of sugars and production of ethanol. Polyol formation was detected. Acetic acid present in the hydrolysate was an important inhibitor of the fermentation, reducing the rate and the yield. Its toxic effect was due essentially to its undissociated form. The fermentation was more effective at an oxygen transfer rate between 1.2 and 2.4 mmol/L h and an initial pH of 6.5. The hydrolysate used in the experiences had the following composition (expressed in grams per liter): xylose 30, arabinose 2.8, glucose 1.5, galactose 3.7, mannose 1.0, cellobiose 0.5, acetic acid 10, glucuronic acid 1.5, and galacturonic acid 1.0. The best values obtained were maximum ethanol concentration 12.6 g/L, fermentation time 75 h, fermentable sugar consumption 99% ethanol yield 0.35 g/g sugars consumed, and volumetric ethanol productivity 4 g/L day. ( (c) 1992 John Wiley & Sons, Inc.

Evolution of microbiological and chemical parameters during red wine making with extended post-fermentation maceration.

PubMed

Francesca, Nicola; Romano, Raffaele; Sannino, Ciro; Le Grottaglie, Laura; Settanni, Luca; Moschetti, Giancarlo

2014-02-03

The aim of the present work was to investigate the microbiological, chemical, and sensory characteristics of red wine subjected to post-fermentation maceration that was extended to 90 days. For this purpose, the 'Aglianico di Taurasi' grape was used as a case study. The total yeast concentration increased until day 40 of maceration and decreased thereafter, whereas the concentration of lactic acid bacteria slightly increased. Dekkera/Brettanomyces spp. and acetic acid bacteria were not detected. The yeast community was composed of Saccharomyces cerevisiae, Zygosaccharomyces bisporus, Metschnikowia pulcherrima, Hanseniaspora guilliermondii, Hanseniaspora uvarum, Pichia guilliermondii, Aureobasidium pullulans and Debaryomyces carsonii. Nine S. cerevisiae strains were detected at high levels at different times of maceration. The results of all the conventional chemical analyses of the wines were in agreement with the regulations of commercial production and, interestingly, the changes in terms of concentration demonstrated the presence of yeast and LAB populations that were not only alive but also in a metabolically active state until day 90 of maceration. The alcohol and glycerol contents slightly increased until day 90. The concentrations of malic acid decreased, whereas those of lactic acid slightly increased throughout the maceration process. Furthermore, different durations of maceration resulted in significant differences in the total polyphenol content, which was higher at 40-50 days. The main phenolic compounds were benzoic and cinnamic acids and catechins. Interestingly, the highest ratio between (+)-catechin and (-)-epicatechin was found on day 40. In addition, the highest antioxidant activity was observed between days 40 and 50. The concentration of volatile organic compounds, which were mainly represented by alcohols, increased until the end of the maceration process. Sensory analysis revealed that samples that were subjected to maceration for a long

Yeast identification in floral nectar of Mimulus aurantiacus (Invited)

NASA Astrophysics Data System (ADS)

Kyauk, C.; Belisle, M.; Fukami, T.

2009-12-01

Nectar is such a sugar-rich resource that serves as a natural habitat in which microbes thrive. As a result, yeasts arrive to nectar on the bodies of pollinators such as hummingbirds and bees. Yeasts use the sugar in nectar for their own needs when introduced. This research focuses on the identification of different types of yeast that are found in the nectar of Mimulus aurantiacus (commonly known as sticky monkey-flower). Unopened Mimulus aurantiacus flower buds were tagged at Jasper Ridge and bagged three days later. Floral nectar was then extracted and plated on potato dextrose agar. Colonies on the plates were isolated and DNA was extracted from each sample using QIAGEN DNeasy Plant Mini Kit. The DNA was amplified through PCR and ran through gel electrophoresis. The PCR product was used to clone the nectar samples into an E.coli vector. Finally, a phylogenetic tree was created by BLAST searching sequences in GenBank using the Internal Transcribed Space (ITS) locus. It was found that 18 of the 50 identified species were Candida magnifica, 14 was Candida rancensis, 6 were Crytococcus albidus and there were 3 or less of the following: Starmella bombicola, Candida floricola, Aureobasidium pullulans, Pichia kluyvera, Metschnikowa cibodaserisis, Rhodotorua colostri, and Malassezia globosa. The low diversity of the yeast could have been due to several factors: time of collection, demographics of Jasper Ridge, low variety of pollinators, and sugar concentration of the nectar. The results of this study serve as a necessary first step for a recently started research project on ecological interactions between plants, pollinators, and nectar-living yeast. More generally, this research studies the use of the nectar-living yeast community as a natural microcosm for addressing basic questions about the role of dispersal and competitive and facilitative interactions in ecological succession.

Immobilization of Pichia pastoris cells containing alcohol oxidase activity

PubMed Central

Maleknia, S; Ahmadi, H; Norouzian, D

2011-01-01

Background and Objectives The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. Materials and Methods Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran (CSMPI). Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al. Results At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide (CTAB) and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD 420. There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. Conclusion Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media. PMID:22530090

Production of arabitol by yeasts: current status and future prospects.

PubMed

Kordowska-Wiater, M

2015-08-01

Arabitol belongs to the pentitol family and is used in the food industry as a sweetener and in the production of human therapeutics as an anticariogenic agent and an adipose tissue reducer. It can also be utilized as a substrate for chemical products such as arabinoic and xylonic acids, propylene, ethylene glycol, xylitol and others. It is included on the list of 12 building block C3-C6 compounds, designated for further biotechnological research. This polyol can be produced by yeasts in the processes of bioconversion or biotransformation of waste materials from agriculture, the forest industry (l-arabinose, glucose) and the biodiesel industry (glycerol). The present review discusses research on native yeasts from the genera Candida, Pichia, Debaryomyces and Zygosaccharomyces as well as genetically modified strains of Saccharomyces cerevisiae which are able to utilize biomass hydrolysates to effectively produce L- or D-arabitol. The metabolic pathways of these yeasts leading from sugars and glycerol to arabitol are presented. Although the number of reports concerning microbial production of arabitol is rather limited, the research on this topic has been growing for the last several years, with researchers looking for new micro-organisms, substrates and technologies. © 2015 The Society for Applied Microbiology.

Production of Delta(1)-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris.

PubMed

Taura, Futoshi; Dono, Emi; Sirikantaramas, Supaart; Yoshimura, Kohji; Shoyama, Yukihiro; Morimoto, Satoshi

2007-09-28

Delta(1)-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of Delta(1)-tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted approximately 1.32nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a approximately 98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure-function studies because it displayed much more activity than the previously reported native enzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.

High efficient expression of a functional humanized single-chain variable fragment (scFv) antibody against CD22 in Pichia pastoris.

PubMed

Zarei, Najmeh; Vaziri, Behrouz; Shokrgozar, Mohammad Ali; Mahdian, Reza; Fazel, Ramin; Khalaj, Vahid

2014-12-01

Single-chain variable fragments (scFvs) have recently emerged as attractive candidates in targeted immunotherapy of various malignancies. The anti-CD22 scFv is able to target CD22, on B cell surface and is being considered as a promising molecule in targeted immunotherapy of B cell malignancies. The recombinant anti-CD22 scFv has been successfully expressed in Escherichia coli; however, the insufficient production yield has been a major bottleneck for its therapeutic application. The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the production of a wide variety of recombinant proteins such as antibody fragments. In this study, we used the Pichia expression system to express a humanized scFv antibody against CD22. The full-length humanized scFv gene was codon optimized, cloned into the pPICZαA and expressed in GS115 strain. The maximum production level of the scFv (25 mg/L) were achieved at methanol concentration, 1 %; pH 6.0; inoculum density, OD600 = 3 and the induction time of 72 h. The correlation between scFv gene dosage and expression level was also investigated by real-time PCR, and the results confirmed the presence of such correlation up to five gene copies. Immunofluorescence and flow cytometry studies and Biacore analysis demonstrated binding to CD22 on the surface of human lymphoid cell line Raji and recombinant soluble CD22, respectively. Taken together, the presented data suggest that the Pichia pastoris can be considered as an efficient host for the large-scale production of anti-CD22 scFv as a promising carrier for targeted drug delivery in treatment of CD22(+) B cell malignancies.

Differential secretion pathways of proteins fused to the Escherichia coli maltose binding protein (MBP) in Pichia pastoris.

PubMed

Moua, Pachai S; Gonzalez, Alfonso; Oshiro, Kristin T; Tam, Vivian; Li, Zhiguo Harry; Chang, Jennifer; Leung, Wilson; Yon, Amy; Thor, Der; Venkatram, Sri; Franz, Andreas H; Risser, Douglas D; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

2016-08-01

The Escherichia coli maltose binding protein (MBP) is an N-terminal fusion partner that was shown to enhance the secretion of some heterologous proteins from the yeast Pichia pastoris, a popular host for recombinant protein expression. The amount of increase in secretion was dependent on the identity of the cargo protein, and the fusions were proteolyzed prior to secretion, limiting its use as a purification tag. In order to overcome these obstacles, we used the MBP as C-terminal partner for several cargo peptides. While the Cargo-MBP proteins were no longer proteolyzed in between these two moieties when the MBP was in this relative position, the secretion efficiency of several fusions was lower than when MBP was located at the opposite end of the cargo protein (MBP-Cargo). Furthermore, fluorescence analysis suggested that the MBP-EGFP and EGFP-MBP proteins followed different routes within the cell. The effect of several Pichia pastoris beta-galactosidase supersecretion (bgs) strains, mutants showing enhanced secretion of select reporters, was also investigated on both MBP-EGFP and EGFP-MBP. While the secretion efficiency, proteolysis and localization of the MBP-EGFP was influenced by the modified function of Bgs13, EGFP-MBP behavior was not affected in the bgs strain. Taken together, these results indicate that the location of the MBP in a fusion affects the pathway and trans-acting factors regulating secretion in P. pastoris. Copyright © 2016 Elsevier Inc. All rights reserved.

The diversity and antifungal susceptibility of the yeasts isolated from coconut water and reconstituted fruit juices in Brazil.

PubMed

Maciel, Natália O P; Piló, Fernanda B; Freitas, Larissa F D; Gomes, Fátima C O; Johann, Susana; Nardi, Regina M D; Lachance, Marc-André; Rosa, Carlos A

2013-01-01

The aims of this study were to characterise the yeasts present in the reconstituted fruit juices and coconut water extracted with "coconut machines", both collected from commercial outlets in a Brazilian city, and to investigate the antifungal resistance of isolates from these beverages that were able to grow at 37°C. The yeast population counts in the coconut water samples ranged from 1.7 to >6.5logcfu/ml, and in the reconstituted fruit juices, the counts ranged from 1.5 to >5.5logcfu/ml. Aureobasidium pullulans, Candida boidinii, Candidaintermedia, Candidaoleophila, Candidaparapsilosis, Candidasantamariae, Candidatropicalis, Clavispora lusitaniae, Kloeckera apis, Lachancea fermentati, Pichia fermentans and Rhodotorula mucilaginosa were the most frequent species isolated from these beverages. At least 18 yeast species isolated from these beverages have been reported as opportunistic pathogens. Eight yeast isolates were resistant to fluconazole, seven were resistant to itraconazole, and 26 to amphotericin B. Some yeast species were resistant to more than one of the antifungal drugs tested. Two isolates of C. tropicalis from the reconstituted fruit juices exhibited resistance to all three drugs. The presence of yeast strains that are resistant to commonly used antifungal drugs suggests a potential risk, at least to immunocompromised individuals who consume these beverages. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification of yeasts and evaluation of their distribution in Taiwanese Kefir and Viili starters.

PubMed

Wang, S Y; Chen, H C; Liu, J R; Lin, Y C; Chen, M J

2008-10-01

The objective of the present study was to investigate yeast communities in kefir grains and viili starters in Taiwan through conventional microbiological cultivation and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The DNA sequencing was used as a validity technique to ensure that all isolates within each group belonged to just one species, and to confirm the identified results of PCR-DGGE. Results indicated that a combination of conventional microbiological cultivation with PCR-DGGE and sequencing could successfully identify 4 yeast species from both types of cultures in Taiwan. Kluyveromyces marxianus, Saccharomyces turicensis, and Pichia fermentans were found in Taiwanese kefir grains with a distribution of 76, 22, and 2%, respectively, whereas Klu. marxianus, Saccharomyces unisporus and P. fermentans were identified in viili starters corresponding to 58, 11, and 31% of the total cell counts, respectively. Furthermore, the culture-independent method was applied to identify the yeast species using DGGE. Only 2 yeast species, Klu. marxianus and S. turicensis, were found in kefir grains and 2, Klu. marxianus and P. fermentans, in viili starters. These results suggest that in samples containing multiple species, PCR-DGGE may fail to detect some species. Sequences of yeast isolates reported in this study have been deposited in the GenBank database under accession nos. DQ139802, AF398485, DQ377652, and AY007920.

Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.

PubMed

Baghban, Roghayyeh; Gargari, Seyed Latif Mousavi; Rajabibazl, Masoumeh; Nazarian, Shahram; Bakherad, Hamid

2016-01-01

Botulinum neurotoxins (BoNTs) result in severe and often fatal disease, botulism. Common remedial measures such as equine antitoxin and human botulism immunoglobulin in turn are problematic and time-consuming. Therefore, diagnosis and therapy of BoNTs are vital. The variable domain of heavy-chain antibodies (VHH) has unique features, such as the ability to identify and bind specifically to target epitopes and ease of production in bacteria and yeast. The Pichia pastoris is suitable for expression of recombinant antibody fragments. Disulfide bond formation and correct folds of protein with a high yield are some of the advantages of this eukaryotic host. In this study, we have expressed and purified the camelid VHH against BoNT/E in P. pastoris. The final yield of P. pastoris-expressed antibody was estimated to be 16 mg/l, which is higher than that expressed by Escherichia coli. The nanobody expressed in P. pastoris neutralized 4LD50 of the BoNT/E upon i.p. injection in 25% of mice. The nanobody expressed in E. coli extended the mice's survival to 1.5-fold compared to the control. This experiment indicated that the quality of expressed protein in the yeast is superior to that of the bacterial expression. Favorable protein folding by P. pastoris seems to play a role in its better toxin-binding property. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

The genome of wine yeast Dekkera bruxellensis provides a tool to explore its food-related properties.

PubMed

Piškur, Jure; Ling, Zhihao; Marcet-Houben, Marina; Ishchuk, Olena P; Aerts, Andrea; LaButti, Kurt; Copeland, Alex; Lindquist, Erika; Barry, Kerrie; Compagno, Concetta; Bisson, Linda; Grigoriev, Igor V; Gabaldón, Toni; Phister, Trevor

2012-07-02

The yeast Dekkera/Brettanomyces bruxellensis can cause enormous economic losses in wine industry due to production of phenolic off-flavor compounds. D. bruxellensis is a distant relative of baker's yeast Saccharomyces cerevisiae. Nevertheless, these two yeasts are often found in the same habitats and share several food-related traits, such as production of high ethanol levels and ability to grow without oxygen. In some food products, like lambic beer, D. bruxellensis can importantly contribute to flavor development. We determined the 13.4 Mb genome sequence of the D. bruxellensis strain Y879 (CBS2499) and deduced the genetic background of several "food-relevant" properties and evolutionary history of this yeast. Surprisingly, we find that this yeast is phylogenetically distant to other food-related yeasts and most related to Pichia (Komagataella) pastoris, which is an aerobic poor ethanol producer. We further show that the D. bruxellensis genome does not contain an excess of lineage specific duplicated genes nor a horizontally transferred URA1 gene, two crucial events that promoted the evolution of the food relevant traits in the S. cerevisiae lineage. However, D. bruxellensis has several independently duplicated ADH and ADH-like genes, which are likely responsible for metabolism of alcohols, including ethanol, and also a range of aromatic compounds. Copyright © 2012 Elsevier B.V. All rights reserved.

Microbial terroir and food innovation: The case of yeast biodiversity in wine.

PubMed

Capozzi, Vittorio; Garofalo, Carmela; Chiriatti, Maria Assunta; Grieco, Francesco; Spano, Giuseppe

2015-12-01

Saccharomyces and non-Saccharomyces represents a heterogeneous class in the grape/must/wine environments including several yeast genera (e.g., Saccharomyces, Hanseniaspora, Pichia, Candida, Metschnikowia, Kluyveromyces, Zygosaccharomyces, Torulaspora, Dekkera and Schizosaccharomyces) and species. Since, each species may differently contribute to the improvement/depreciation of wine qualities, it appears clear the reason why species belong to non-Saccharomyces are also considered a biotechnological resource in wine fermentation. Here, we briefly review the oenological significance of this specific part of microbiota associated with grapes/musts/wine. Moreover, the diversity of cultivable non-Saccharomyces genera and their contribute to typical wines fermentations will be discussed. Copyright © 2015 Elsevier GmbH. All rights reserved.

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

Monitoring of Yeast Communities and Volatile Flavor Changes During Traditional Korean Soy Sauce Fermentation.

PubMed

Song, Young-Ran; Jeong, Do-Youn; Baik, Sang-Ho

2015-09-01

Flavor development in soy sauce is significantly related to the diversity of yeast species. Due to its unique fermentation with meju, the process of making Korean soy sauce gives rise to a specific yeast community and, therefore, flavor profile; however, no detailed analysis of the identifying these structure has been performed. Changes in yeast community structure during Korean soy sauce fermentation were examined using both culture-dependent and culture-independent methods with simultaneous analysis of the changes in volatile compounds by GC-MS analysis. During fermentation, Candida, Pichia, and Rhodotorula sp. were the dominant species, whereas Debaryomyces, Torulaspora, and Zygosaccharomyces sp. were detected only at the early stage. In addition, Cryptococcus, Microbotryum, Tetrapisispora, and Wickerhamomyces were detected as minor strains. Among the 62 compounds identified in this study, alcohols, ketones, and pyrazines were present as the major groups during the initial stages, whereas the abundance of acids with aldehydes increased as the fermentation progressed. Finally, the impacts of 10 different yeast strains found to participate in fermentation on the formation of volatile compounds were evaluated under soy-based conditions. It was revealed that specific species produced different profiles of volatile compounds, some of which were significant flavor contributors, especially volatile alcohols, aldehydes, esters, and ketones. © 2015 Institute of Food Technologists®

Non-Saccharomyces Yeasts Nitrogen Source Preferences: Impact on Sequential Fermentation and Wine Volatile Compounds Profile

PubMed Central

Gobert, Antoine; Tourdot-Maréchal, Raphaëlle; Morge, Christophe; Sparrow, Céline; Liu, Youzhong; Quintanilla-Casas, Beatriz; Vichi, Stefania; Alexandre, Hervé

2017-01-01

Nitrogen sources in the must are important for yeast metabolism, growth, and performance, and wine volatile compounds profile. Yeast assimilable nitrogen (YAN) deficiencies in grape must are one of the main causes of stuck and sluggish fermentation. The nitrogen requirement of Saccharomyces cerevisiae metabolism has been described in detail. However, the YAN preferences of non-Saccharomyces yeasts remain unknown despite their increasingly widespread use in winemaking. Furthermore, the impact of nitrogen consumption by non-Saccharomyces yeasts on YAN availability, alcoholic performance and volatile compounds production by S. cerevisiae in sequential fermentation has been little studied. With a view to improving the use of non-Saccharomyces yeasts in winemaking, we studied the use of amino acids and ammonium by three strains of non-Saccharomyces yeasts (Starmerella bacillaris, Metschnikowia pulcherrima, and Pichia membranifaciens) in grape juice. We first determined which nitrogen sources were preferentially used by these yeasts in pure cultures at 28 and 20°C (because few data are available). We then carried out sequential fermentations at 20°C with S. cerevisiae, to assess the impact of the non-Saccharomyces yeasts on the availability of assimilable nitrogen for S. cerevisiae. Finally, 22 volatile compounds were quantified in sequential fermentation and their levels compared with those in pure cultures of S. cerevisiae. We report here, for the first time, that non-Saccharomyces yeasts have specific amino-acid consumption profiles. Histidine, methionine, threonine, and tyrosine were not consumed by S. bacillaris, aspartic acid was assimilated very slowly by M. pulcherrima, and glutamine was not assimilated by P. membranifaciens. By contrast, cysteine appeared to be a preferred nitrogen source for all non-Saccharomyces yeasts. In sequential fermentation, these specific profiles of amino-acid consumption by non-Saccharomyces yeasts may account for some of the

Non-Saccharomyces Yeasts Nitrogen Source Preferences: Impact on Sequential Fermentation and Wine Volatile Compounds Profile.

PubMed

Gobert, Antoine; Tourdot-Maréchal, Raphaëlle; Morge, Christophe; Sparrow, Céline; Liu, Youzhong; Quintanilla-Casas, Beatriz; Vichi, Stefania; Alexandre, Hervé

2017-01-01

Nitrogen sources in the must are important for yeast metabolism, growth, and performance, and wine volatile compounds profile. Yeast assimilable nitrogen (YAN) deficiencies in grape must are one of the main causes of stuck and sluggish fermentation. The nitrogen requirement of Saccharomyces cerevisiae metabolism has been described in detail. However, the YAN preferences of non- Saccharomyces yeasts remain unknown despite their increasingly widespread use in winemaking. Furthermore, the impact of nitrogen consumption by non- Saccharomyces yeasts on YAN availability, alcoholic performance and volatile compounds production by S. cerevisiae in sequential fermentation has been little studied. With a view to improving the use of non- Saccharomyces yeasts in winemaking, we studied the use of amino acids and ammonium by three strains of non- Saccharomyces yeasts ( Starmerella bacillaris, Metschnikowia pulcherrima , and Pichia membranifaciens ) in grape juice. We first determined which nitrogen sources were preferentially used by these yeasts in pure cultures at 28 and 20°C (because few data are available). We then carried out sequential fermentations at 20°C with S. cerevisiae , to assess the impact of the non- Saccharomyces yeasts on the availability of assimilable nitrogen for S. cerevisiae . Finally, 22 volatile compounds were quantified in sequential fermentation and their levels compared with those in pure cultures of S. cerevisiae . We report here, for the first time, that non- Saccharomyces yeasts have specific amino-acid consumption profiles. Histidine, methionine, threonine, and tyrosine were not consumed by S. bacillaris , aspartic acid was assimilated very slowly by M. pulcherrima , and glutamine was not assimilated by P. membranifaciens . By contrast, cysteine appeared to be a preferred nitrogen source for all non- Saccharomyces yeasts. In sequential fermentation, these specific profiles of amino-acid consumption by non- Saccharomyces yeasts may account for

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

Orally Administered Bioadherent Sustained Release Microencapsulated Vaccines

DTIC Science & Technology

1996-09-01

ml flask culture (MMY media) was inoculated with a recombinant Pichia yeast clone containing the vpB gene integrated at the alcohol oxidase (AOX...measured. 6 DAMD17-95-C-5099 2. Materials and Methods 2.1. Materials vpB is expressed in Pichia yeast and isolated and purified as described below. SEA...locus in the Pichia genome. The culture was grown at 30"C for approximately 48 hours until an optical density ( OD 650) of 3.0 was reached. The culture

Temperature control strategy to enhance the activity of yeast inoculated into compost raw material for accelerated composting.

PubMed

Nakasaki, Kiyohiko; Hirai, Hidehira

2017-07-01

The effects of inoculating the mesophilic yeast Pichia kudriavzevii RB1, which is able to degrade organic acids, on organic matter degradation in composting were elucidated. When model food waste with high carbohydrate content (C/N=22.3) was used, fluctuation in the inoculated yeast cell density was observed, as well as fluctuation in the composting temperature until day 5 when the temperature rose to 60°C, which is lethal for the yeast. After the decrease in yeast, acetic acid accumulated to levels as high as 20mg/g-ds in the composting material and vigorous organic matter degradation was inhibited. However, by maintaining the temperature at 40°C for 2days during the heating phase in the early stage of composting, both the organic acids originally contained in the raw material and acetic acid produced during the heating phase were degraded by the yeast. The concentration of acetic acid was kept at a relatively low level (10.1mg/g-ds at the highest), thereby promoting the degradation of organic matter by other microorganisms and accelerating the composting process. These results indicate that temperature control enhances the effects of microbial inoculation into composts. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of MALDI-TOF mass spectrometry for differentiation of Pichia kluyveri strains isolated from traditional fermentation processes.

PubMed

De la Torre González, Francisco Javier; Gutiérrez Avendaño, Daniel Oswaldo; Gschaedler Mathis, Anne Christine; Kirchmayr, Manuel Reinhart

2018-06-06

Non- Saccharomyces yeasts are widespread microorganisms and some time ago were considered contaminants in the beverage industry. However, nowadays they have gained importance for their ability to produce aromatic compounds, which in alcoholic beverages improves aromatic complexity and therefore the overall quality. Thus, identification and differentiation of the species involved in fermentation processes is vital and can be classified in traditional methods and techniques based on molecular biology. Traditional methods, however, can be expensive, laborious and/or unable to accurately discriminate on strain level. In the present study, a total of 19 strains of Pichia kluyveri isolated from mezcal, tejuino and cacao fermentations were analyzed with rep-PCR fingerprinting and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The comparative analysis between MS spectra and rep-PCR patterns obtained from these strains showed a high similarity between both methods. However, minimal differences between the obtained rep-PCR and MALDI-TOF MS clusters could be observed. The data shown suggests that MALDI-TOF MS is a promising alternative technique for rapid, reliable and cost-effective differentiation of natives yeast strains isolated from different traditional fermented foods and beverages. This article is protected by copyright. All rights reserved.

The Effect of α-Mating Factor Secretion Signal Mutations on Recombinant Protein Expression in Pichia pastoris

PubMed Central

Lin-Cereghino, Geoff P.; Stark, Carolyn M.; Kim, Daniel; Chang, Jennifer; Shaheen, Nadia; Poerwanto, Hansel; Agari, Kimiko; Moua, Pachai; Low, Lauren K.; Tran, Namphuong; Huang, Amy D.; Nattestad, Maria; Oshiro, Kristin T.; Chang, John William; Chavan, Archana; Tsai, Jerry W.; Lin-Cereghino, Joan

2013-01-01

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins are initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future. PMID:23454485

Wickerhamomyces xylosica sp. nov. and Candida phayaonensis sp. nov., two xylose-assimilating yeast species from soil.

PubMed

Limtong, Savitree; Nitiyon, Sukanya; Kaewwichian, Rungluk; Jindamorakot, Sasitorn; Am-In, Somjit; Yongmanitchai, Wichien

2012-11-01

Two strains (NT29(T) and NT31(T)) of xylose-assimilating yeasts were obtained from soils collected in northern Thailand. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and sequence analysis of the D1/D2 domain of the large subunit rRNA gene and the internal transcribed spacer region, the two strains were found to represent two novel ascomycete yeast species. Strain NT29(T) was assigned to the genus Candida belonging to the Pichia clade as a representative of Candida phayaonensis sp. nov.; the type strain is NT29(T) (=BCC 47634(T)=NBRC 108868(T)=CBS 12319(T)). Strain NT31(T) represented a novel Wickerhamomyces species, which was named Wickerhamomyces xylosica sp. nov.; the type strain is NT31(T) (=BCC 47635(T)=NBRC 108869(T)=CBS 12320(T)).

Yeast community in traditional Portuguese Serpa cheese by culture-dependent and -independent DNA approaches.

PubMed

Gonçalves Dos Santos, Maria Teresa P; Benito, María José; Córdoba, María de Guía; Alvarenga, Nuno; Ruiz-Moyano Seco de Herrera, Santiago

2017-12-04

This study investigated the yeast community present in the traditional Portuguese cheese, Serpa, by culture-dependent and -independent methods. Sixteen batches of Serpa cheeses from various regional industries registered with the Protected Designation of Origin (PDO) versus non-PDO registered, during spring and winter, were used. Irrespective of the producer, the yeast counts were around 5log CFU/g in winter and, overall, were lower in spring. The yeast species identified at the end of ripening (30days), using PCR-RFLP analysis and sequencing of the 26S rRNA, mainly corresponded to Debaryomyces hansenii and Kluyveromyces marxianus, with Candida spp. and Pichia spp. present to a lesser extent. The culture-independent results, obtained using high-throughput sequencing analysis, confirmed the prevalence of Debaryomyces spp. and Kluyveromyces spp. but, also, that Galactomyces spp. was relevant for three of the five producers, which indicates its importance during the early stages of the cheese ripening process, considering it was not found among the dominant viable yeast species. In addition, differences between the identified yeast isolated from cheeses obtained from PDO and non-PDO registered industries, showed that the lack of regulation of the cheese-making practice, may unfavourably influence the final yeast microbiota. The new knowledge provided by this study of the yeast diversity in Serpa cheese, could be used to modify the cheese ripening conditions, to favour desirable yeast species. Additionally, the prevalent yeast isolates identified, Debaryomyces hansenii and Kluyveromyces spp., may have an important role during cheese ripening and in the final sensorial characteristics. Thus, the study of their technological and functional properties could be relevant, in the development of an autochthonous starter culture, to ensure final quality and safety of the cheese. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Screening and characterizing of xylanolytic and xylose-fermenting yeasts isolated from the wood-feeding termite, Reticulitermes chinensis

PubMed Central

Xie, Rongrong; Zhou, Feng; Huang, Miao

2017-01-01

The effective fermentation of xylose remains an intractable challenge in bioethanol industry. The relevant xylanase enzyme is also in a high demand from industry for several biotechnological applications that inevitably in recent times led to many efforts for screening some novel microorganisms for better xylanase production and fermentation performance. Recently, it seems that wood-feeding termites can truly be considered as highly efficient natural bioreactors. The highly specialized gut systems of such insects are not yet fully realized, particularly, in xylose fermentation and xylanase production to advance industrial bioethanol technology as well as industrial applications of xylanases. A total of 92 strains from 18 yeast species were successfully isolated and identified from the gut of wood-feeding termite, Reticulitermes chinensis. Of these yeasts and strains, seven were identified for new species: Candida gotoi, Candida pseudorhagii, Hamamotoa lignophila, Meyerozyma guilliermondii, Sugiyamaella sp.1, Sugiyamaella sp. 2, and Sugiyamaella sp.3. Based on the phylogenetic and phenotypic characterization, the type strain of C. pseudorhagii sp. nov., which was originally designated strain SSA-1542T, was the most frequently occurred yeast from termite gut samples, showed the highly xylanolytic activity as well as D-xylose fermentation. The highest xylanase activity was recorded as 1.73 and 0.98 U/mL with xylan or D-xylose substrate, respectively, from SSA-1542T. Among xylanase-producing yeasts, four novel species were identified as D-xylose-fermenting yeasts, where the yeast, C. pseudorhagii SSA-1542T, showed the highest ethanol yield (0.31 g/g), ethanol productivity (0.31 g/L·h), and its fermentation efficiency (60.7%) in 48 h. Clearly, the symbiotic yeasts isolated from termite guts have demonstrated a competitive capability to produce xylanase and ferment xylose, suggesting that the wood-feeding termite gut is a promising reservoir for novel xylanases

Isolation, identification and growth determination of lactic acid-utilizing yeasts from the ruminal fluid of dairy cattle.

PubMed

Sirisan, V; Pattarajinda, V; Vichitphan, K; Leesing, R

2013-08-01

Ruminal organic acid production, especially lactic acid, can be modified by feeding cattle highly concentrated diets, which have been shown to adversely affect dairy cattle health. Therefore, the use of lactic acid-utilizing organisms is considered to be a potential method for controlling lactic acid levels. This study was conducted to isolate and identify lactic acid-utilizing yeasts from the ruminal fluid of dairy cattle and to determine the specific growth rate and generation time when using lactic acid as a carbon source instead of glucose. Seventeen yeast isolates were examined in this study. Yeasts isolated from dairy cattle that were fed a high cassava pulp diet (HCP) had higher specific growth rates and shorter generation times than yeasts isolated from dairy cattle that were fed a high-concentrate diet (HC) and a mixed diet (M). The three most effective yeasts in terms of specific growth rate and generation time were Pichia kudriavzevii, Candida rugosa and Kodamaea ohmeri, with 99, 100 and 99% nucleotide identities, respectively. These three isolates could be used as potential probiotics in dairy cattle diets. This study demonstrates that yeasts isolated from the ruminal fluid of dairy cattle can utilize lactic acid as a carbon and energy source for growth. The isolated yeasts can be used as probiotic supplements for dairy cattle that are fed highly concentrated diets to reduce ruminal lactic acid production. © 2013 The Society for Applied Microbiology.

Assessment of the yeast species composition of cocoa bean fermentations in different cocoa-producing regions using denaturing gradient gel electrophoresis.

PubMed

Papalexandratou, Zoi; De Vuyst, Luc

2011-11-01

The yeast species composition of 12 cocoa bean fermentations carried out in Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-independently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments, obtained through polymerase chain reaction with universal eukaryotic primers, was carried out with two different commercial apparatus (the DCode and CBS systems). In general, this molecular method allowed a rapid monitoring of the yeast species prevailing during fermentation. Under similar and optimal denaturing gradient gel electrophoresis conditions, the CBS system allowed a better separated band pattern than the DCode system and an unambiguous detection of the prevailing species present in the fermentation samples. The most frequent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates a restricted yeast species composition of the cocoa bean fermentation process. Exceptionally, the Ivorian cocoa bean box fermentation samples showed a wider yeast species composition, with Hyphopichia burtonii and Meyerozyma caribbica among the main representatives. Yeasts were not detected in the samples when the temperature inside the fermenting cocoa pulp-bean mass reached values higher than 45 °C or under early acetic acid production conditions. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Lactic acid bacteria and yeasts associated with gowé production from sorghum in Bénin.

PubMed

Vieira-Dalodé, G; Jespersen, L; Hounhouigan, J; Moller, P L; Nago, C M; Jakobsen, M

2007-08-01

To identify the dominant micro-organisms involved in the production of gowé, a fermented beverage, and to select the most appropriate species for starter culture development. Samples of sorghum gowé produced twice at three different production sites were taken at different fermentation times. DNA amplification by internal transcribed spacer-polymerase chain reaction of 288 lactic acid bacteria (LAB) isolates and 16S rRNA gene sequencing of selected strains revealed that the dominant LAB responsible for gowé fermentation were Lactobacillus fermentum, Weissella confusa, Lactobacillus mucosae, Pediococcus acidilactici, Pediococcus pentosaceus and Weissella kimchii. DNA from 200 strains of yeasts was amplified and the D1/D2 domain of the 26S rRNA gene was sequenced for selected isolates, revealing that the yeasts species were Kluyveromyces marxianus, Pichia anomala, Candida krusei and Candida tropicalis. Gowé processing is characterized by a mixed fermentation dominated by Lact. fermentum, W. confusa and Ped. acidilactici for the LAB and by K. marxianus, P. anomala and C. krusei for the yeasts. The diversity of the LAB and yeasts identified offers new opportunities for technology upgrading and products development in gowé production. The identified species can be used as possible starter for a controlled fermentation of gowé.

(1-->6)-beta-D-glucan as cell wall receptor for Pichia membranifaciens killer toxin.

PubMed

Santos, A; Marquina, D; Leal, J A; Peinado, J M

2000-05-01

The killer toxin from Pichia membranifaciens CYC 1106, a yeast isolated from fermenting olive brines, binds primarily to the (1-->6)-beta-D-glucan of the cell wall of a sensitive yeast (Candida boidinii IGC 3430). The (1-->6)-beta-D-glucan was purified from cell walls of C. boidinii by alkali and hot-acetic acid extraction, a procedure which solubilizes glucans. The major fraction of receptor activity remained with the alkali-insoluble (1-->6)-beta- and (1-->3)-beta-D-glucans. The chemical (gas-liquid chromatography) and structural (periodate oxidation, infrared spectroscopy, and (1)H nuclear magnetic resonance) analyses of the fractions obtained showed that (1-->6)-beta-D-glucan was a receptor. Adsorption of most of the killer toxin to the (1-->6)-beta-D-glucan was complete within 2 min. Killer toxin adsorption to the linear (1-->6)-beta-D-glucan, pustulan, and a glucan from Penicillium allahabadense was observed. Other polysaccharides with different linkages failed to bind the killer toxin. The specificity of the killer toxin for its primary receptor provides an effective means to purify the killer toxin, which may have industrial applications for fermentations in which salt is present as an adjunct, such as olive brines. This toxin shows its maximum killer activity in the presence of NaCl. This report is the first to identify the (1-->6)-beta-D-glucan as a receptor for this novel toxin.

Toward systems metabolic engineering of Aspergillus and Pichia species for the production of chemicals and biofuels.

PubMed

Caspeta, Luis; Nielsen, Jens

2013-05-01

Recently genome sequence data have become available for Aspergillus and Pichia species of industrial interest. This has stimulated the use of systems biology approaches for large-scale analysis of the molecular and metabolic responses of Aspergillus and Pichia under defined conditions, which has resulted in much new biological information. Case-specific contextualization of this information has been performed using comparative and functional genomic tools. Genomics data are also the basis for constructing genome-scale metabolic models, and these models have helped in the contextualization of knowledge on the fundamental biology of Aspergillus and Pichia species. Furthermore, with the availability of these models, the engineering of Aspergillus and Pichia is moving from traditional approaches, such as random mutagenesis, to a systems metabolic engineering approach. Here we review the recent trends in systems biology of Aspergillus and Pichia species, highlighting the relevance of these developments for systems metabolic engineering of these organisms for the production of hydrolytic enzymes, biofuels and chemicals from biomass. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An efficient screen for peroxisome-deficient mutants of Pichia pastoris.

PubMed Central

Liu, H; Tan, X; Veenhuis, M; McCollum, D; Cregg, J M

1992-01-01

We describe a rapid and efficient screen for peroxisome-deficient (per) mutants in the yeast Pichia pastoris. The screen relies on the unusual ability of P. pastoris to grow on two carbon sources, methanol and oleic acid, both of which absolutely require peroxisomes to be metabolized. A collection of 280 methanol utilization-defective (Mut-) P. pastoris mutants was isolated, organized into 46 complementation groups, and tested for those that were also oleate-utilization defective (Out-) but still capable of growth on ethanol and glucose. Mutants in 10 groups met this phenotypic description, and 8 of these were observed by electron microscopy to be peroxisome deficient (Per-). In each per mutant, Mut-, Out-, and Per- phenotypes were tightly linked and therefore were most likely due to a mutation at a single locus. Subcellular fractionation experiments indicated that the peroxisomal marker enzyme catalase was mislocalized to the cytosol in both methanol- and oleate-induced cultures of the mutants. In contrast, alcohol oxidase, a peroxisomal methanol utilization pathway enzyme, was virtually absent from per mutant cells. The relative ease of per mutant isolation in P. pastoris, in conjunction with well-developed procedures for its molecular and genetic manipulation, makes this organism an attractive system for studies on peroxisome biogenesis. Images PMID:1629154

Detergent Screening and Purification of the Human Liver ABC Transporters BSEP (ABCB11) and MDR3 (ABCB4) Expressed in the Yeast Pichia pastoris

PubMed Central

Stindt, Jan; Smits, Sander H. J.; Schmitt, Lutz

2013-01-01

The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters. PMID:23593265

Display of adenoregulin with a novel Pichia pastoris cell surface display system.

PubMed

Ren, Ren; Jiang, Zhengbing; Liu, Meiyun; Tao, Xinyi; Ma, Yushu; Wei, Dongzhi

2007-02-01

Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo1p with its own secretion signal sequence or the alpha-factor secretion signal sequence, a polyhistidine (6xHis) tag for detection, an enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR.

Yeast communities associated with artisanal mezcal fermentations from Agave salmiana.

PubMed

Verdugo Valdez, A; Segura Garcia, L; Kirchmayr, M; Ramírez Rodríguez, P; González Esquinca, A; Coria, R; Gschaedler Mathis, A

2011-11-01

The aims of this work were to characterize the fermentation process of mezcal from San Luis Potosi, México and identify the yeasts present in the fermentation using molecular culture-dependent methods (RFLP of the 5.8S-ITS and sequencing of the D1/D2 domain) and also by using a culture-independent method (DGGE). The alcoholic fermentations of two separate musts obtained from Agave salmiana were analyzed. Sugar, ethanol and major volatile compounds concentrations were higher in the first fermentation, which shows the importance of having a quality standard for raw materials, particularly in the concentration of fructans, in order to produce fermented Agave salmiana must with similar characteristics. One hundred ninety-two (192) different yeast colonies were identified, from those present on WL agar plates, by RFLP analysis of the ITS1-5.8S- ITS2 from the rRNA gene, with restriction endonucleases, HhaI, HaeIII and HinfI. The identified yeasts were: Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia kluyveri, Zygosaccharomyces bailii, Clavispora lusitaniae, Torulaspora delbrueckii, Candida ethanolica and Saccharomyces exiguus. These identifications were confirmed by sequencing the D1-D2 region of the 26S rRNA gene. With the PCR-DGGE method, bands corresponding to S. cerevisiae, K. marxianus and T. delbrueckii were clearly detected, confirming the results obtained with classic techniques.

Ethyl Carbamate Formation Regulated by Lactic Acid Bacteria and Nonconventional Yeasts in Solid-State Fermentation of Chinese Moutai-Flavor Liquor.

PubMed

Du, Hai; Song, Zhewei; Xu, Yan

2018-01-10

This study aimed to identify specific microorganisms related to the formation of precursors of EC (ethyl carbamate) in the solid-state fermentation of Chinese Moutai-flavor liquor. The EC content was significantly correlated with the urea content during the fermentation process (R 2 = 0.772, P < 0.01). Differences in urea production and degradation were found at both species and functional gene levels by metatranscriptomic sequencing and culture-dependent analysis. Lactobacillus spp. could competitively degrade arginine through the arginine deiminase pathway with yeasts, and most Lactobacillus species were capable of degrading urea. Some dominant nonconventional yeasts, such as Pichia, Schizosaccharomyces, and Zygosaccharomyces species, were shown to produce low amounts of urea relative to Saccharomyces cerevisiae. Moreover, unusual urea degradation pathways (urea carboxylase, allophanate hydrolase, and ATP-independent urease) were identified. Our results indicate that EC precursor levels in the solid-state fermentation can be controlled using lactic acid bacteria and nonconventional yeasts.

Combination of Pichia membranifaciens and ammonium molybdate for controlling blue mould caused by Penicillium expansum in peach fruit.

PubMed

Cao, Shifeng; Yuan, Yongjun; Hu, Zhichao; Zheng, Yonghua

2010-07-15

The potential enhancement of Pichia membranifaciens by ammonium molybdate (NH(4)Mo) to control blue mould caused by Penicillium expansum on peach fruit was investigated. Combining P. membranifaciens at 1x10(8) cell/ml with 1 mM NH(4)Mo provided a more effective control of blue mould rot than applying the yeast or NH(4)Mo alone. Addition of 1 mM NH(4)Mo significantly increased the growth of P. membranifaciens in peach wounds, but did not affect the population in nutrient yeast dextrose broth medium. The in vitro experiment showed that the combined treatment inhibited spore germination and germ tube elongation of P. expansum in comparison with the treatment of P. membranifaciens or NH(4)Mo alone. Moreover, P. membranifaciens, NH(4)Mo, and the combination of them did not impair the quality parameters including fruit firmness and content of total soluble solids, titratable acidity and vitamin C of peach fruit after 6 days of storage at 20 degrees C. These results suggested that the use of NH(4)Mo is a useful approach to improve the efficacy of P. membranifaciens for postharvest disease control in peach fruit. 2010 Published by Elsevier B.V. All rights reserved.

Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

PubMed Central

Zhang, Li; Liang, Shuli; Zhou, Xinying; Jin, Zi; Jiang, Fengchun; Han, Shuangyan; Zheng, Suiping

2013-01-01

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface. PMID:23835174

Identification of a novel interspecific hybrid yeast from a metagenomic spontaneously inoculated beer sample using Hi-C.

PubMed

Smukowski Heil, Caiti; Burton, Joshua N; Liachko, Ivan; Friedrich, Anne; Hanson, Noah A; Morris, Cody L; Schacherer, Joseph; Shendure, Jay; Thomas, James H; Dunham, Maitreya J

2018-01-01

Interspecific hybridization is a common mechanism enabling genetic diversification and adaptation; however, the detection of hybrid species has been quite difficult. The identification of microbial hybrids is made even more complicated, as most environmental microbes are resistant to culturing and must be studied in their native mixed communities. We have previously adapted the chromosome conformation capture method Hi-C to the assembly of genomes from mixed populations. Here, we show the method's application in assembling genomes directly from an uncultured, mixed population from a spontaneously inoculated beer sample. Our assembly method has enabled us to de-convolute four bacterial and four yeast genomes from this sample, including a putative yeast hybrid. Downstream isolation and analysis of this hybrid confirmed its genome to consist of Pichia membranifaciens and that of another related, but undescribed, yeast. Our work shows that Hi-C-based metagenomic methods can overcome the limitation of traditional sequencing methods in studying complex mixtures of genomes. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Efficacy and putative mode of action of native and commercial antagonistic yeasts against postharvest pathogens of pear.

PubMed

Lutz, M Cecilia; Lopes, Christian A; Rodriguez, M Eugenia; Sosa, M Cristina; Sangorrín, Marcela P

2013-06-17

Putative mechanisms of action associated with the biocontrol capacity of four yeast strains (Cryptoccocus albidus NPCC 1248, Pichia membranifaciens NPCC 1250, Cryptoccocus victoriae NPCC 1263 and NPCC 1259) against Penicillium expansum and Botrytis cinerea were studied by means of in vitro and in situ assays. C. albidus(YP), a commercial yeast was also evaluated for comparative purposes. The yeast strains exhibited a variety of different mechanisms including: wound colonization, germination inhibition, biofilm formation, secretion of killer toxins, competition for nutrient and secretion of hydrolytic enzymes (protease, chitinase and glucanase). The relationship between strains (and their associated antagonist mechanisms) and in situ antagonist activity was also evaluated. Results indicate that mechanisms such as production of hydrolytic enzymes, the ability for colonization of wounds, production of killer toxin and inhibition of germination are the most important for biocontrol activity. Our study indicate that multiple modes of action may explain why P. membranifaciens NPCC 1250 and C. victoriae NPCC 1263 provided excellent control of postharvest pears disease. Copyright © 2013 Elsevier B.V. All rights reserved.

Constitutive expression of human pancreatic lipase-related protein 1 in Pichia pastoris.

PubMed

Aloulou, Ahmed; Grandval, Philippe; De Caro, Josiane; De Caro, Alain; Carrière, Frédéric

2006-06-01

High-level constitutive expression of the human pancreatic lipase-related protein 1 (HPLRP1) was achieved using the methylotrophic yeast Pichia pastoris. The HPLRP1 cDNA, including its original leader sequence, was subcloned into the pGAPZB vector and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. A major protein with a molecular mass of 50 kDa was found to be secreted into the culture medium and was identified using anti-HPLRP1 polyclonal antibodies as HPLRP1 recombinant protein. The level of expression reached 100-120 mg of HPLRP1 per liter of culture medium after 40 h, as attested by specific and quantitative enzyme-linked immunosorbent assay. A single cation-exchange chromatography sufficed to obtain a highly purified recombinant HPLRP1 after direct batch adsorption onto S-Sepharose of the HPLRP1 present in the culture medium, at pH 5.5. N-terminal sequencing and mass spectrometry analysis were carried out to monitor the production of the mature protein and to confirm that its signal peptide was properly processed.

A differential medium for the enumeration of the spoilage yeast Zygosaccharomyces bailii in wine.

PubMed

Schuller, D; Côrte-Real, M; Leão, C

2000-11-01

A collection of yeasts, isolated mostly from spoiled wines, was used in order to develop a differential medium for Zygosaccharomyces bailii. The 118 selected strains of 21 species differed in their origin and resistance to preservatives and belonged to the genera Pichia, Torulaspora, Dekkera, Debaryomyces, Saccharomycodes, Issatchenkia, Kluyveromyces, Kloeckera, Lodderomyces, Schizosaccharomyces, Rhodotorula, Saccharomyces, and Zygosaccharomyces. The design of the culture medium was based on the different ability of the various yeast species to grow in a mineral medium with glucose and formic acid (mixed-substrate medium) as the only carbon and energy sources and supplemented with an acid-base indicator. By manipulating the concentration of the acid and the sugar it was possible to select conditions where only Z. bailii strains gave rise to alkalinization, associated with a color change of the medium (positive response). The final composition of the mixed medium was adjusted as a compromise between the percentage of recovery and selectivity for Z. bailii. This was accomplished by the use of pure or mixed cultures of the yeast strains and applying the membrane filtration methodology. The microbiological analysis of two samples of contaminated Vinho Verde showed that the new medium can be considered as a differential medium to distinguish Z. bailii from other contaminating yeasts, having potential application in the microbiological control of wines and probably other beverages and foods.

Isolation and Identification of the Indigenous Yeast Population during Spontaneous Fermentation of Isabella (Vitis labrusca L.) Grape Must.

PubMed

Raymond Eder, María L; Reynoso, Cristina; Lauret, Santiago C; Rosa, Alberto L

2017-01-01

Grape must harbors a complex community of yeast species responsible for spontaneous alcoholic fermentation. Although there are detailed studies on the microbiota of Vitis vinifera L. grapes, less is known about the diversity and behavior of yeast communities present on fermenting grape must from other species of Vitis . In this work, we used a culture-dependent method to study the identity and dynamics of the indigenous yeast population present during the spontaneous fermentation of Isabella ( Vitis labrusca L.) grape must. Alcoholic fermentation was conducted using standard enological practices, and the associated non- Saccharomyces and S. cerevisiae yeast community was analyzed using selective growth media and 5.8-ITS DNA sequencing. Candida californica, Candida hellenica, Starmerella bacillaris (synonym Candida zemplinina ), Hanseniaspora uvarum , and Hanseniaspora vineae were the main non- Saccharomyces species identified on Isabella fermenting must. Issatchenkia hanoiensis , a yeast species rarely found on Vitis vinifera L. grapes, was also recognized on Isabella grape must. Candida azymoides, Candida californica and Pichia cecembensis , identified in this work on Isabella fermenting must, have not previously been found on Vitis vinifera L. grape must. Interestingly, C. azymoides, I. hanoiensis and P. cecembensis have recently been isolated from the surface of Vitis labrusca L. grapes from vineyards in the Azores archipelago, suggesting that specific Vitis -yeast species associations are formed independently of geographic origin. We suggest that C. azymoides, C. californica , and P. cecembensis are yeast species preferentially associated with Vitis labrusca L. grapes. Specific biological interactions between grapevines and yeast species may underlie the assembly of differential Vitis -microbial communities.

Isolation and Identification of the Indigenous Yeast Population during Spontaneous Fermentation of Isabella (Vitis labrusca L.) Grape Must

PubMed Central

Raymond Eder, María L.; Reynoso, Cristina; Lauret, Santiago C.; Rosa, Alberto L.

2017-01-01

Grape must harbors a complex community of yeast species responsible for spontaneous alcoholic fermentation. Although there are detailed studies on the microbiota of Vitis vinifera L. grapes, less is known about the diversity and behavior of yeast communities present on fermenting grape must from other species of Vitis. In this work, we used a culture-dependent method to study the identity and dynamics of the indigenous yeast population present during the spontaneous fermentation of Isabella (Vitis labrusca L.) grape must. Alcoholic fermentation was conducted using standard enological practices, and the associated non-Saccharomyces and S. cerevisiae yeast community was analyzed using selective growth media and 5.8-ITS DNA sequencing. Candida californica, Candida hellenica, Starmerella bacillaris (synonym Candida zemplinina), Hanseniaspora uvarum, and Hanseniaspora vineae were the main non-Saccharomyces species identified on Isabella fermenting must. Issatchenkia hanoiensis, a yeast species rarely found on Vitis vinifera L. grapes, was also recognized on Isabella grape must. Candida azymoides, Candida californica and Pichia cecembensis, identified in this work on Isabella fermenting must, have not previously been found on Vitis vinifera L. grape must. Interestingly, C. azymoides, I. hanoiensis and P. cecembensis have recently been isolated from the surface of Vitis labrusca L. grapes from vineyards in the Azores archipelago, suggesting that specific Vitis-yeast species associations are formed independently of geographic origin. We suggest that C. azymoides, C. californica, and P. cecembensis are yeast species preferentially associated with Vitis labrusca L. grapes. Specific biological interactions between grapevines and yeast species may underlie the assembly of differential Vitis-microbial communities. PMID:28424672

Yeast species associated with orange juice: evaluation of different identification methods.

PubMed

Arias, Covadonga R; Burns, Jacqueline K; Friedrich, Lorrie M; Goodrich, Renee M; Parish, Mickey E

2002-04-01

Five different methods were used to identify yeast isolates from a variety of citrus juice sources. A total of 99 strains, including reference strains, were identified using a partial sequence of the 26S rRNA gene, restriction pattern analysis of the internal transcribed spacer region (5.8S-ITS), classical methodology, the RapID Yeast Plus system, and API 20C AUX. Twenty-three different species were identified representing 11 different genera. Distribution of the species was considerably different depending on the type of sample. Fourteen different species were identified from pasteurized single-strength orange juice that had been contaminated after pasteurization (PSOJ), while only six species were isolated from fresh-squeezed, unpasteurized orange juice (FSOJ). Among PSOJ isolates, Candida intermedia and Candida parapsilosis were the predominant species. Hanseniaspora occidentalis and Hanseniaspora uvarum represented up to 73% of total FSOJ isolates. Partial sequence of the 26S rRNA gene yielded the best results in terms of correct identification, followed by classical techniques and 5.8S-ITS analysis. The commercial identification kits RapID Yeast Plus system and API 20C AUX were able to correctly identify only 35 and 13% of the isolates, respectively. Six new 5.8S-ITS profiles were described, corresponding to Clavispora lusitaniae, Geotrichum citri-aurantii, H. occidentalis, H. vineae, Pichia fermentans, and Saccharomycopsis crataegensis. With the addition of these new profiles to the existing database, the use of 5.8S-ITS sequence became the best tool for rapid and accurate identification of yeast isolates from orange juice.

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

Quantification and characterization of microbial biofilm community attached on the surface of fermentation vessels used in green table olive processing.

PubMed

Grounta, Athena; Doulgeraki, Agapi I; Panagou, Efstathios Z

2015-06-16

The aim of the present study was the quantification of biofilm formed on the surface of plastic vessels used in Spanish-style green olive fermentation and the characterization of the biofilm community by means of molecular fingerprinting. Fermentation vessels previously used in green olive processing were subjected to sampling at three different locations, two on the side and one on the bottom of the vessel. Prior to sampling, two cleaning treatments were applied to the containers, including (a) washing with hot tap water (60 °C) and household detergent (treatment A) and (b) washing with hot tap water, household detergent and bleach (treatment B). Population (expressed as log CFU/cm(2)) of total viable counts (TVC), lactic acid bacteria (LAB) and yeasts were enumerated by standard plating. Bulk cells (whole colonies) from agar plates were isolated for further characterization by PCR-DGGE. Results showed that regardless of the cleaning treatment no significant differences were observed between the different sampling locations in the vessel. The initial microbial population before cleaning ranged between 3.0-4.5 log CFU/cm(2) for LAB and 4.0-4.6 log CFU/cm(2) for yeasts. Cleaning treatments exhibited the highest effect on LAB that were recovered at 1.5 log CFU/cm(2) after treatment A and 0.2 log CFU/cm(2) after treatment B, whereas yeasts were recovered at approximately 1.9 log CFU/cm(2) even after treatment B. High diversity of yeasts was observed between the different treatments and sampling spots. The most abundant species recovered belonged to Candida genus, while Wickerhamomyces anomalus, Debaryomyces hansenii and Pichia guilliermondii were frequently detected. Among LAB, Lactobacillus pentosus was the most abundant species present on the abiotic surface of the vessels. Copyright © 2015 Elsevier B.V. All rights reserved.

Applications of recombinant Pichia pastoris in the healthcare industry.

PubMed

Weinacker, Daniel; Rabert, Claudia; Zepeda, Andrea B; Figueroa, Carolina A; Pessoa, Adalberto; Farías, Jorge G

2013-12-01

Since the 1970s, the establishment and development of the biotech industry has improved exponentially, allowing the commercial production of biopharmaceutical proteins. Nowadays, new recombinant protein production is considered a multibillion-dollar market, in which about 25% of commercial pharmaceuticals are biopharmaceuticals. But to achieve a competitive production process is not an easy task. Any production process has to be highly productive, efficient and economic. Despite that the perfect host is still not discovered, several research groups have chosen Pichia pastoris as expression system for the production of their protein because of its many features. The attempt of this review is to embrace several research lines that have adopted Pichia pastoris as their expression system to produce a protein on an industrial scale in the health care industry.

Applications of recombinant Pichia pastoris in the healthcare industry

PubMed Central

Weinacker, Daniel; Rabert, Claudia; Zepeda, Andrea B.; Figueroa, Carolina A.; Pessoa, Adalberto; Farías, Jorge G.

2013-01-01

Since the 1970s, the establishment and development of the biotech industry has improved exponentially, allowing the commercial production of biopharmaceutical proteins. Nowadays, new recombinant protein production is considered a multibillion-dollar market, in which about 25% of commercial pharmaceuticals are biopharmaceuticals. But to achieve a competitive production process is not an easy task. Any production process has to be highly productive, efficient and economic. Despite that the perfect host is still not discovered, several research groups have chosen Pichia pastoris as expression system for the production of their protein because of its many features. The attempt of this review is to embrace several research lines that have adopted Pichia pastoris as their expression system to produce a protein on an industrial scale in the health care industry. PMID:24688491

Analysis of the 5′ untranslated region (5′UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris

PubMed Central

Staley, Chris A.; Huang, Amy; Nattestad, Maria; Oshiro, Kristin T.; Ray, Laura E.; Mulye, Tejas; Li, Zhiguo Harry; Le, Thu; Stephens, Justin J.; Gomez, Seth R.; Moy, Allison D.; Nguyen, Jackson C.; Franz, Andreas H.; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P.

2012-01-01

Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express over one thousand heterologous proteins valued for industrial, pharmaceutical and basic research purposes. In most cases, the 5′ untranslated region (UTR) of the alcohol oxidase 1 (AOX1) gene is fused to the coding sequence of the recombinant gene for protein expression in this yeast. Because the effect of the AOX1 5′UTR on protein expression is not known, site-directed mutagenesis was performed in order to decrease or increase the length of this region. Both of these types of changes were shown to affect translational efficiency, not transcript stability. While increasing the length of the 5′UTR clearly decreased expression of a β-galactosidase reporter in a proportional manner, a deletion analysis demonstrated that the AOX1 5′UTR contains a complex mixture of both positive and negative cis-acting elements, suggesting that the construction of a synthetic 5′UTR optimized for a higher level of expression may be challenging. PMID:22285974

Cloning and expression of Pectobacterium carotovorum endo-polygalacturonase gene in Pichia pastoris for production of oligogalacturonates

USDA-ARS?s Scientific Manuscript database

A bacterial endo-polygalacturonase (endo-PGase) gene from the plant pathogen Pectobacterium carotovorum was cloned into pGAPZaA vector and constitutively expressed in Pichia pastoris. The recombinant endo-PGase secreted by the Pichia clone showed a 1.7 fold increase when the culture medium included ...

Cloning and Expression of Yak Active Chymosin in Pichia pastoris.

PubMed

Luo, Fan; Jiang, Wei Hua; Yang, Yuan Xiao; Li, Jiang; Jiang, Ming Feng

2016-09-01

Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZαA, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production.

Identification and characterisation of xylanolytic yeasts isolated from decaying wood and sugarcane bagasse in Brazil.

PubMed

Lara, Carla A; Santos, Renata O; Cadete, Raquel M; Ferreira, Carla; Marques, Susana; Gírio, Francisco; Oliveira, Evelyn S; Rosa, Carlos A; Fonseca, César

2014-06-01

In this study, yeasts associated with lignocellulosic materials in Brazil, including decaying wood and sugarcane bagasse, were isolated, and their ability to produce xylanolytic enzymes was investigated. A total of 358 yeast isolates were obtained, with 198 strains isolated from decaying wood and 160 strains isolated from decaying sugarcane bagasse samples. Seventy-five isolates possessed xylanase activity in solid medium and were identified as belonging to nine species: Candida intermedia, C. tropicalis, Meyerozyma guilliermondii, Scheffersomyces shehatae, Sugiyamaella smithiae, Cryptococcus diffluens, Cr. heveanensis, Cr. laurentii and Trichosporon mycotoxinivorans. Twenty-one isolates were further screened for total xylanase activity in liquid medium with xylan, and five xylanolytic yeasts were selected for further characterization, which included quantitative analysis of growth in xylan and xylose and xylanase and β-D-xylosidase activities. The yeasts showing the highest growth rate and cell density in xylan, Cr. laurentii UFMG-HB-48, Su. smithiae UFMG-HM-80.1 and Sc. shehatae UFMG-HM-9.1a, were, simultaneously, those exhibiting higher xylanase activity. Xylan induced the highest level of (extracellular) xylanase activity in Cr. laurentii UFMG-HB-48 and the highest level of (intracellular, extracellular and membrane-associated) β-D-xylosidase activity in Su. smithiae UFMG-HM-80.1. Also, significant β-D-xylosidase levels were detected in xylan-induced cultures of Cr. laurentii UFMG-HB-48 and Sc. shehatae UFMG-HM-9.1a, mainly in extracellular and intracellular spaces, respectively. Under xylose induction, Cr. laurentii UFMG-HB-48 showed the highest intracellular β-D-xylosidase activity among all the yeast tested. C. tropicalis UFMG-HB 93a showed its higher (intracellular) β-D-xylosidase activity under xylose induction and higher at 30 °C than at 50 °C. This study revealed different xylanolytic abilities and strategies in yeasts to metabolise xylan and

Biotechnological advances towards an enhanced peroxidase production in Pichia pastoris.

PubMed

Krainer, Florian W; Gerstmann, Michaela A; Darnhofer, Barbara; Birner-Gruenberger, Ruth; Glieder, Anton

2016-09-10

Horseradish peroxidase (HRP) is a high-demand enzyme for applications in diagnostics, bioremediation, biocatalysis and medicine. Current HRP preparations are isolated from horseradish roots as mixtures of biochemically diverse isoenzymes. Thus, there is a strong need for a recombinant production process enabling a steady supply with enzyme preparations of consistent high quality. However, most current recombinant production systems are limited at titers in the low mg/L range. In this study, we used the well-known yeast Pichia pastoris as host for recombinant HRP production. To enhance recombinant enzyme titers we systematically evaluated engineering approaches on the secretion process, coproduction of helper proteins, and compared expression from the strong methanol-inducible PAOX1 promoter, the strong constitutive PGAP promoter, and a novel bidirectional promoter PHTX1. Ultimately, coproduction of HRP and active Hac1 under PHTX1 control yielded a recombinant HRP titer of 132mg/L after 56h of cultivation in a methanol-independent and easy-to-do bioreactor cultivation process. With regard to the many versatile applications for HRP, the establishment of a microbial host system suitable for efficient recombinant HRP production was highly overdue. The novel HRP production platform in P. pastoris presented in this study sets a new benchmark for this medically relevant enzyme. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Electrochemical studies of a truncated laccase produced in Pichia pastoris

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

Gelo-Pujic, M.; Kim, H.H.; Butlin, N.G.

1999-12-01

The cDNA that encodes an isoform is laccase from Trametes versicolor (LCCI), as well as a truncated version (LCCIa), was subcloned and expressed by using the yeast Pichia pastoris as the heterologous host. The amino acid sequence of LCCIa is identical to that of LCCI except that the final 11 amino acids at the C terminus of LCCI are replaced with a single cysteine residue. This modification was introduced for the purpose of improving the kinetics of electron transfer between an electrode and the copper-containing active site of laccase. The two laccases (LCCI and LCCIa) are compared in terms ofmore » their relative activity with two substrates that have different redox potentials. Results from electrochemical studies on solutions containing LCCI and LCCIa indicate that the redox potential of the active site of LCCIa is shifted to more negative values (411 mV versus normal hydrogen electrode voltage) than that found in other fungal laccases. In addition, replacing the 11 codons at the C terminus of the laccase gene with a single cysteine codon influences the rate of heterogeneous electron transfer between and electrode and the copper-containing active site. These results demonstrate for the first time that the rate of electron transfer between an oxidoreductase and an electrode can be enhanced by changes to the primary structure of a protein via site-directed mutagenesis.« less

High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity

PubMed Central

Iglesias-Figueroa, Blanca; Valdiviezo-Godina, Norberto; Siqueiros-Cendón, Tania; Sinagawa-García, Sugey; Arévalo-Gallegos, Sigifredo; Rascón-Cruz, Quintín

2016-01-01

In this study, bovine lactoferrin (bLf), an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf) in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin) demonstrated antibacterial activity against Escherichia coli (E. coli) BL21DE3, Staphylococcus aureus (S. aureus) FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa) ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly. PMID:27294912

Comparative behaviour of yeast strains for ethanolic fermentation of culled apple juice.

PubMed

Modi, D R; Garg, S K; Johri, B N

1998-07-01

The culled apple juice contained (% w/v): nitrogen, 0.036; total sugars, 11.6 and was of pH 3.9. Saccharomyces cerevisiae NCIM 3284, Pichia kluyeri and Candida krusei produced more ethanol from culled apple juice at its optimum initial pH 4.5, whereas S. cerevisiae NCIM 3316 did so at pH 5.0. An increase in sugar concentration of apple juice from natural 11.6% to 20% exhibited enhanced ethanol production and improved fermentation efficiency of both the S. cerevisiae strains, whereas P. kluyveri and C. krusei produced high ethanol at 11.6% and 16.0% sugar levels, respectively. Urea was stimulatory for ethanol production as well as fermentation efficiency of the yeast strains under study.

Chromosome VIII disomy influences the nonsense suppression efficiency and transition metal tolerance of the yeast Saccharomyces cerevisiae.

PubMed

Zadorsky, S P; Sopova, Y V; Andreichuk, D Y; Startsev, V A; Medvedeva, V P; Inge-Vechtomov, S G

2015-06-01

The SUP35 gene of the yeast Saccharomyces cerevisiae encodes the translation termination factor eRF3. Mutations in this gene lead to the suppression of nonsense mutations and a number of other pleiotropic phenotypes, one of which is impaired chromosome segregation during cell division. Similar effects result from replacing the S. cerevisiae SUP35 gene with its orthologues. A number of genetic and epigenetic changes that occur in the sup35 background result in partial compensation for this suppressor effect. In this study we showed that in S. cerevisiae strains in which the SUP35 orthologue from the yeast Pichia methanolica replaces the S. cerevisiae SUP35 gene, chromosome VIII disomy results in decreased efficiency of nonsense suppression. This antisuppressor effect is not associated with decreased stop codon read-through. We identified SBP1, a gene that localizes to chromosome VIII, as a dosage-dependent antisuppressor that strongly contributes to the overall antisuppressor effect of chromosome VIII disomy. Disomy of chromosome VIII also leads to a change in the yeast strains' tolerance of a number of transition metal salts. Copyright © 2015 John Wiley & Sons, Ltd.

Expression of recombinant myostatin propeptide pPIC9K-Msp plasmid in Pichia pastoris.

PubMed

Du, W; Xia, J; Zhang, Y; Liu, M J; Li, H B; Yan, X M; Zhang, J S; Li, N; Zhou, Z Y; Xie, W Z

2015-12-28

Myostatin propeptide can inhibit the biological activity of myostatin protein and promote muscle growth. To express myostatin propeptide in vitro with a higher biological activity, we performed codon optimization on the sheep myostatin propeptide gene sequence, and mutated aspartic acid-76 to alanine based on the codon usage bias of Pichia pastoris and the enhanced biological activity of myostatin propeptide mutant. Modified myostatin propeptide gene was cloned into the pPIC9K plasmid to form the recombinant plasmid pPIC9K-Msp. Recombinant plasmid pPIC9K-Msp was transformed into Pichia pastoris GS115 by electrotransformation. Transformed cells were screened, and methanol was used to induce expression. SDS-PAGE and western blotting were used to verify the successful expression of myostatin propeptide with biological activity in Pichia pastoris, providing the basis for characterization of this protein.

Phenotypic landscape of non-conventional yeast species for different stress tolerance traits desirable in bioethanol fermentation.

PubMed

Mukherjee, Vaskar; Radecka, Dorota; Aerts, Guido; Verstrepen, Kevin J; Lievens, Bart; Thevelein, Johan M

2017-01-01

potential of non- Saccharomyces species to emerge either as alternative host species or as a source of valuable genetic information for construction of more robust industrial S. serevisiae bioethanol production yeasts. Striking examples include yeast species like Pichia kudriavzevii and Wickerhamomyces anomalus that show very high tolerance to diverse stress factors. This large-scale phenotypic analysis has yielded a detailed database useful as a resource for future studies to understand and benefit from the molecular mechanisms underlying the extreme phenotypes of non-conventional yeast species.

Expression, purification, and antibacterial activity of bovine lactoferrampin-lactoferricin in Pichia pastoris.

PubMed

Tang, Xiang-Shan; Tang, Zhi-Ru; Wang, Sheng-Ping; Feng, Ze-Meng; Zhou, Dong; Li, Tie-Jun; Yin, Yu-Long

2012-02-01

Bovine lactoferrampin (LFA) and bovine lactoferricin (LFC) are two antimicrobial peptides located in the N(1) domain of bovine lactoferrin. The bactericidal activity of the fused peptide LFA-LFC is stronger than that of either LFA or LFC. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current study, the expression, purification, and antibacterial activity of LFA-LFC using the Pichia pastoris expression system are reported. The linearized expression vector pPICZaA-LFA-LFC was transformed into P. pastoris KM71 by electroporation, and positive colonies harboring the target genes were screened out and used for fermentation. The recombinant LFA-LFC peptide was purified via two-step column chromatography and identified by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate that P. pastoris is a suitable system for secreting LFA-LFC. The fermentation supernate and the purified LFA-LFC show high antimicrobial activities. The current study is the first to report on the expression and purification of LFA-LFC in P. pastoris and may have potential practical applications in microbial peptide production.

Cloning and Expression of Yak Active Chymosin in Pichia pastoris

PubMed Central

Luo, Fan; Jiang, Wei Hua; Yang, Yuan Xiao; Li, Jiang; Jiang, Ming Feng

2016-01-01

Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZαA, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production. PMID:27004812

High-level expression and characterization of the Bacillus subtilis subsp. subtilis str. BSP1 YwaD aminopeptidase in Pichia pastoris.

PubMed

Tang, Wei; Li, Zhezhe; Li, Chunhua; Yu, Xianhong; Wang, Fei; Wan, Xin; Wang, Yaping; Ma, Lixin

2016-06-01

Aminopeptidases are widely used for creating protein hydrolysates and peptide sequencing. The ywaD gene from a new Bacillus isolate, named Bacillus subtilis subsp. subtilis str. BSP1, was cloned into the yeast expression vector pHBM905A and expressed and secreted by Pichia pastoris strain GS115. The deduced amino acid sequence of the aminopeptidase encoded by the ywaD gene shared up to 98% identity with aminopeptidases from B. subtilis strains 168 and zj016. The yield (3.81 g/l) and specific activity (788 U/mg) of recombinant YwaD in high-density fermentation were extremely high. And 829.83 mg of the purified enzyme (4089.72 U/mg) were harvested. YwaD was glycosylated, and its activity decreased after deglycosylation, which was similar to that of the aminopeptidase from B. subtilis strain zj016. YwaD was most active toward l-arginine-4-nitroanilide. Moreover, it exhibited high resistance to carbamide, which was not true for aminopeptidases from B. subtilis strains 168 and zj016, which could simplify the purification of YwaD. Moreover, the expression and parts of characterization of the aminopeptidase from B. subtilis strain 168 in Pichia pastoris were added as supplementary material. The sequence and other characteristics of YwaD were compared with those of aminopeptidases from B. subtilis strains 168 and zj016, and they will provide a solid foundation for further research on the influence of amino acid mutations on the function of aminopeptidases. Copyright © 2016 Elsevier Inc. All rights reserved.

Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using the Pichia pastoris PGK1 Promoter.

PubMed

Mariz, F C; Coimbra, E C; Jesus, A L S; Nascimento, L M; Torres, F A G; Freitas, A C

2015-01-01

The human papillomavirus (HPV) L1 major capsid protein, which forms the basis of the currently available vaccines against cervical cancer, self-assembles into virus-like particles (VLPs) when expressed heterologously. We report the development of a biotechnology platform for HPV16 L1 protein expression based on the constitutive PGK1 promoter (PPGK1) from the methylotrophic yeast Pichia pastoris. The L1 gene was cloned under regulation of PPGK1 into pPGKΔ3 expression vector to achieve intracellular expression. In parallel, secretion of the L1 protein was obtained through the use of an alternative vector called pPGKΔ3α, in which a codon optimized α-factor signal sequence was inserted. We devised a work-flow based on the detection of the L1 protein by dot blot, colony blot, and western blot to classify the positive clones. Finally, intracellular HPV VLPs assembly was demonstrated for the first time in yeast cells. This study opens up perspectives for the establishment of an innovative platform for the production of HPV VLPs or other viral antigens for vaccination purposes, based on constitutive expression in P. pastoris.

The strange case of a biofilm-forming strain of Pichia fermentans, which controls Monilinia brown rot on apple but is pathogenic on peach fruit.

PubMed

Giobbe, Sara; Marceddu, Salvatore; Scherm, Barbara; Zara, Giacomo; Mazzarello, Vittorio L; Budroni, Marilena; Migheli, Quirico

2007-12-01

A biofilm-forming strain of Pichia fermentans proved to be most effective in controlling brown rot on apple fruit when coinoculated into artificial wounds with a phytopathogenic isolate of Monilinia fructicola. Culture filtrates and autoclaved cells had no significant influence on the disease. When sprayed onto the apple fruit surface, this yeast formed a thin biofilm but failed to colonize the underlying tissues. When inoculated into wounds artificially inflicted to peach fruit or when sprayed onto the surface of peach fruit, the same strain showed an unexpected pathogenic behaviour, causing rapid decay of fruit tissues even in the absence of M. fructicola. Both optical and scanning electron microscopy were used to evaluate the pattern of fruit tissue colonization by P. fermentans. While on apple surface and within the apple wound the antagonist retained its yeast-like shape, colonization of peach fruit tissue was always characterized by a transition from budding growth to pseudohyphal growth. These results suggest that pseudohyphal growth plays a major role in governing the potential pathogenicity of P. fermentans, further emphasizing the importance of a thorough risk assessment for the safe use of any novel biocontrol agent.

Isolation and Characterization of the Diatom Phaeodactylum Δ5-Elongase Gene for Transgenic LC-PUFA Production in Pichia pastoris

PubMed Central

Jiang, Mulan; Guo, Bing; Wan, Xia; Gong, Yangmin; Zhang, Yinbo; Hu, Chuanjiong

2014-01-01

The diatom Phaeodactylum tricornutum can accumulate eicosapentaenoic acid (EPA) up to 30% of the total fatty acids. This species has been targeted for isolating gene encoding desaturases and elongases for long-chain polyunsaturated fatty acid (LC-PUFA) metabolic engineering. Here we first report the cloning and characterization of Δ5-elongase gene in P. tricornutum. A full-length cDNA sequence, designated PhtELO5, was shown to contain a 1110 bp open reading frame encoding a 369 amino acid polypeptide. The putative protein contains seven transmembrane regions and two elongase characteristic motifs of FLHXYHH and MYSYY, the latter being typical for microalgal Δ5-elongases. Phylogenetic analysis indicated that PhtELO5 belongs to the ELO5 group, tightly clustered with the counterpart of Thalassiosira pseudonana. Heterologous expression of PhtELO5 in Pichia pastoris confirmed that it encodes a specific Δ5-elongase capable of elongating arachidonic acid and eicosapentaenoic acid. Co-expression of PhtELO5 and IsFAD4 (a ∆4-desaturase from Isochrysis sphaerica) demonstrated that the high-efficiency biosynthetic pathway of docosahexaenoic acid was assembled in the transgenic yeast. Substrate competition revealed that PhtELO5 exhibited higher activity towards n-3 PUFA than n-6 PUFA. It is hypothesized that Phaeodactylum ELO5 may preferentially participate in biosynthesis of transgenic LC-PUFA via a n-3 pathway in the yeast host. PMID:24608969

In Situ Analysis of Metabolic Characteristics Reveals the Key Yeast in the Spontaneous and Solid-State Fermentation Process of Chinese Light-Style Liquor

PubMed Central

Kong, Yu; Wu, Qun; Zhang, Yan

2014-01-01

The in situ metabolic characteristics of the yeasts involved in spontaneous fermentation process of Chinese light-style liquor are poorly understood. The covariation between metabolic profiles and yeast communities in Chinese light-style liquor was modeled using the partial least square (PLS) regression method. The diversity of yeast species was evaluated by sequence analysis of the 26S ribosomal DNA (rDNA) D1/D2 domains of cultivable yeasts, and the volatile compounds in fermented grains were analyzed by gas chromatography (GC)-mass spectrometry (MS). Eight yeast species and 58 volatile compounds were identified, respectively. The modulation of 16 of these volatile compounds was associated with variations in the yeast population (goodness of prediction [Q2] > 20%). The results showed that Pichia anomala was responsible for the characteristic aroma of Chinese liquor, through the regulation of several important volatile compounds, such as ethyl lactate, octanoic acid, and ethyl tetradecanoate. Correspondingly, almost all of the compounds associated with P. anomala were detected in a pure culture of this yeast. In contrast to the PLS regression results, however, ethyl lactate and ethyl isobutyrate were not detected in the same pure culture, which indicated that some metabolites could be generated by P. anomala only when it existed in a community with other yeast species. Furthermore, different yeast communities provided different volatile patterns in the fermented grains, which resulted in distinct flavor profiles in the resulting liquors. This study could help identify the key yeast species involved in spontaneous fermentation and provide a deeper understanding of the role of individual yeast species in the community. PMID:24727269

Chitosan inactivates spoilage yeasts but enhances survival of Escherichia coli O157:H7 in apple juice.

PubMed

Kiskó, G; Sharp, R; Roller, S

2005-01-01

To develop new measures for controlling both spoilage and pathogenic micro-organisms in unpasteurized apple juice using chitosan. Micro-organisms were isolated and identified from apple juice treated or untreated with chitosan using enrichment, selective media, microscopy, substrate assimilation patterns and ribosomal DNA profiling. Chitosan (0.05-0.1%) delayed spoilage by yeasts at 25 degrees C for up to 12 days but the effect was species specific: Kloeckera apiculata and Metschnikowia pulcherrima were inactivated but Saccharomyces cerevisiae and Pichia spp. multiplied slowly. In challenge experiments at 25 degrees C, total yeast counts were 3-5 log CFU ml(-1) lower in chitosan-treated juices than in the controls for 4 days but the survival of Escherichia coli O157:H7 was extended from 1 to 2 days; at 4 degrees C, chitosan reduced the yeast counts by 2-3 log CFU ml(-1) for up to 10 days but survival of the pathogen was prolonged from 3 to 5 days. The survival of Salmonella enterica serovar Typhimurium was unaffected by chitosan at either temperature. The addition of chitosan to apple juice delayed spoilage by yeasts but enhanced the survival of E. coli O157:H7. The results suggest that the use of chitosan in the treatment of fruit juices may potentially lead to an increased risk of food poisoning from E. coli O157:H7.

Influence of the farming system and vine variety on yeast communities associated with grape berries.

PubMed

Cordero-Bueso, Gustavo; Arroyo, Teresa; Serrano, Ana; Tello, Javier; Aporta, Irene; Vélez, María Dolores; Valero, Eva

2011-01-31

Wine production in most countries is based on the use of commercial strains leading to the colonisation of the wineries by these strains with the consequent reduction of autochthonous biodiversity. This implies that wine styles could therefore become standardised. The vineyard could be an important source of native yeasts of oenological interest. For this reason the objective of this study was to compare two agronomic conditions with the aim of preserving yeast biodiversity in the vineyard. A three year sampling plan was designed to evaluate the influence of different agronomic parameters on the biodiversity of fermentative grape yeasts. Thus two vineyards, one organic and one conventional, with three different grape varieties (Shiraz, Grenache and Barbera) were chosen. In total, 27 samples were collected from both vineyards. Of these, 1080 colonies were isolated and a total of 9 species were identified. The strains identified as Saccharomyces cerevisiae were genotyped by microsatellite analysis obtaining nine different electrophoretic patterns. Classical ecology indexes were used to obtain the richness (S), the biodiversity (H') and the dominance (D) of the species studied. The results indicated a clear influence on grape associated yeast diversity of the phytosanitary treatment used in the vineyard. This is the first time that classical ecology indexes have been used to study the ecology of the spontaneous fermentation of grape musts and the species Candida sorbosa and Pichia toletana have been described in vineyards of the Madrid winegrowing region. Copyright © 2010 Elsevier B.V. All rights reserved.

Electrochemical Studies of a Truncated Laccase Produced in Pichia pastoris

PubMed Central

Gelo-Pujic, Mirjana; Kim, Hyug-Han; Butlin, Nathan G.; Palmore, G. Tayhas R.

1999-01-01

The cDNA that encodes an isoform of laccase from Trametes versicolor (LCCI), as well as a truncated version (LCCIa), was subcloned and expressed by using the yeast Pichia pastoris as the heterologous host. The amino acid sequence of LCCIa is identical to that of LCCI except that the final 11 amino acids at the C terminus of LCCI are replaced with a single cysteine residue. This modification was introduced for the purpose of improving the kinetics of electron transfer between an electrode and the copper-containing active site of laccase. The two laccases (LCCI and LCCIa) are compared in terms of their relative activity with two substrates that have different redox potentials. Results from electrochemical studies on solutions containing LCCI and LCCIa indicate that the redox potential of the active site of LCCIa is shifted to more negative values (411 mV versus normal hydrogen electrode voltage) than that found in other fungal laccases. In addition, replacing the 11 codons at the C terminus of the laccase gene with a single cysteine codon (i.e., LCCI→LCCIa) influences the rate of heterogeneous electron transfer between an electrode and the copper-containing active site (khet for LCCIa = 1.3 × 10−4 cm s−1). These results demonstrate for the first time that the rate of electron transfer between an oxidoreductase and an electrode can be enhanced by changes to the primary structure of a protein via site-directed mutagenesis. PMID:10584012

A functional Kv1.2-hERG chimaeric channel expressed in Pichia pastoris

PubMed Central

Dhillon, Mandeep S.; Cockcroft, Christopher J.; Munsey, Tim; Smith, Kathrine J.; Powell, Andrew J.; Carter, Paul; Wrighton, David C.; Rong, Hong-lin; Yusaf, Shahnaz P.; Sivaprasadarao, Asipu

2014-01-01

Members of the six-transmembrane segment family of ion channels share a common structural design. However, there are sequence differences between the members that confer distinct biophysical properties on individual channels. Currently, we do not have 3D structures for all members of the family to help explain the molecular basis for the differences in their biophysical properties and pharmacology. This is due to low-level expression of many members in native or heterologous systems. One exception is rat Kv1.2 which has been overexpressed in Pichia pastoris and crystallised. Here, we tested chimaeras of rat Kv1.2 with the hERG channel for function in Xenopus oocytes and for overexpression in Pichia. Chimaera containing the S1–S6 transmembrane region of HERG showed functional and pharmacological properties similar to hERG and could be overexpressed and purified from Pichia. Our results demonstrate that rat Kv1.2 could serve as a surrogate to express difficult-to-overexpress members of the six-transmembrane segment channel family. PMID:24569544

Fermentation Kinetics for Xylitol Production by a Pichia stipitis d-Xylulokinase Mutant Previously Grown in Spent Sulfite Liquor

NASA Astrophysics Data System (ADS)

Rodrigues, Rita C. L. B.; Lu, Chenfeng; Lin, Bernice; Jeffries, Thomas W.

Spent sulfite pulping liquor (SSL) contains lignin, which is present as lignosulfonate, and hemicelluloses that are present as hydrolyzed carbohydrates. To reduce the biological oxygen demand of SSL associated with dissolved sugars, we studied the capacity of Pichia stipitis FPL-YS30 (xyl3Δ) to convert these sugars into useful products. FPL-YS30 produces a negligible amount of ethanol while converting xylose into xylitol. This work describes the xylose fermentation kinetics of yeast strain P.stipitis FPL-YS30. Yeast was grown in rich medium supplemented with different carbon sources: glucose, xylose, or ammonia-base SSL. The SSL and glucose-acclimatized cells showed similar maximum specific growth rates (0.146 h-1). The highest xylose consumption at the beginning of the fermentation process occurred using cells precultivated in xylose, which showed relatively high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49). However, the maximum specific rates of xylose consumption (0.19 gxylose/gcel h) and xylitol production (0.059 gxylitol/gcel h) were obtained with cells acclimatized in glucose, in which the ratio between xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) was kept at higher level (0.82). In this case, xylitol production (31.6 g/l) was 19 and 8% higher than in SSL and xylose-acclimatized cells, respectively. Maximum glycerol (6.26 g/l) and arabitol (0.206 g/l) production were obtained using SSL and xylose-acclimatized cells, respectively. The medium composition used for the yeast precultivation directly reflected their xylose fermentation performance. The SSL could be used as a carbon source for cell production. However, the inoculum condition to obtain a high cell concentration in SSL needs to be optimized.

Constitutive expression of human gastric lipase in Pichia pastoris and site-directed mutagenesis of key lid-stabilizing residues.

PubMed

Sams, Laura; Amara, Sawsan; Chakroun, Almahdi; Coudre, Sébastien; Paume, Julie; Giallo, Jacqueline; Carrière, Frédéric

2017-10-01

The cDNA encoding human gastric lipase (HGL) was integrated into the genome of Pichia pastoris using the pGAPZα A transfer vector. The HGL signal peptide was replaced by the yeast α-factor to achieve an efficient secretion. Active rHGL was produced by the transformed yeast but its levels and stability were dependent on the pH. The highest activity was obtained upon buffering the culture medium at pH5, a condition that allowed preserving enzyme activity over time. A large fraction (72±2%) of secreted rHGL remained however bound to the yeast cells, and was released by washing the cell pellet with an acid glycine-HCl buffer (pH2.2). This procedure allowed establishing a first step of purification that was completed by size exclusion chromatography. N-terminal sequencing and MALDI-ToF mass spectrometry revealed that rHGL was produced in its mature form, with a global mass of 50,837±32Da corresponding to a N-glycosylated form of HGL polypeptide (43,193Da). rHGL activity was characterized as a function of pH, various substrates and in the presence of bile salts and pepsin, and was found similar to native HGL, except for slight changes in pH optima. We then studied by site-directed mutagenesis the role of three key residues (K4, E225, R229) involved in salt bridges stabilizing the lid domain that controls the access to the active site and is part of the interfacial recognition site. Their substitution has an impact on the pH-dependent activity of rHGL and its relative activities on medium and long chain triglycerides. Copyright © 2017 Elsevier B.V. All rights reserved.

Influence of Pichia pastoris cellular material on polymerase chain reaction performance as a synthetic biology standard for genome monitoring.

PubMed

Templar, Alexander; Woodhouse, Stefan; Keshavarz-Moore, Eli; Nesbeth, Darren N

2016-08-01

Advances in synthetic genomics are now well underway in yeasts due to the low cost of synthetic DNA. These new capabilities also bring greater need for quantitating the presence, loss and rearrangement of loci within synthetic yeast genomes. Methods for achieving this will ideally; i) be robust to industrial settings, ii) adhere to a global standard and iii) be sufficiently rapid to enable at-line monitoring during cell growth. The methylotrophic yeast Pichia pastoris (P. pastoris) is increasingly used for industrial production of biotherapeutic proteins so we sought to answer the following questions for this particular yeast species. Is time-consuming DNA purification necessary to obtain accurate end-point polymerase chain reaction (e-pPCR) and quantitative PCR (qPCR) data? Can the novel linear regression of efficiency qPCR method (LRE qPCR), which has properties desirable in a synthetic biology standard, match the accuracy of conventional qPCR? Does cell cultivation scale influence PCR performance? To answer these questions we performed e-pPCR and qPCR in the presence and absence of cellular material disrupted by a mild 30s sonication procedure. The e-pPCR limit of detection (LOD) for a genomic target locus was 50pg (4.91×10(3) copies) of purified genomic DNA (gDNA) but the presence of cellular material reduced this sensitivity sixfold to 300pg gDNA (2.95×10(4) copies). LRE qPCR matched the accuracy of a conventional standard curve qPCR method. The presence of material from bioreactor cultivation of up to OD600=80 did not significantly compromise the accuracy of LRE qPCR. We conclude that a simple and rapid cell disruption step is sufficient to render P. pastoris samples of up to OD600=80 amenable to analysis using LRE qPCR which we propose as a synthetic biology standard. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Characterization of an ethanol-tolerant 1,4-β-xylosidase produced by Pichia membranifaciens.

PubMed

Romero, A M; Mateo, J J; Maicas, S

2012-11-01

The purification and biochemical properties of the 1,4-β-xylosidase of an oenological yeast were investigated. An ethanol-tolerant 1,4-β-xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G-100. The relative molecular mass of the enzyme was determined to be 50kDa by SDS-PAGE. The activity of 1,4-β-xylosidase was optimum at pH 6·0 and at 35°C. The activity had a Km of 0·48±0·06mmol l(-1) and a Vmax of 7·4±0·1μmol min(-1)mg(-1) protein for p-nitrophenyl-β-d-xylopyranoside. The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. This study may be useful for assessing the ability of the 1,4-β-xylosidase from P. membranifaciens to be used in the bioethanol production process. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

Outlining a future for non-Saccharomyces yeasts: selection of putative spoilage wine strains to be used in association with Saccharomyces cerevisiae for grape juice fermentation.

PubMed

Domizio, Paola; Romani, Cristina; Lencioni, Livio; Comitini, Francesca; Gobbi, Mirko; Mannazzu, Ilaria; Ciani, Maurizio

2011-06-30

The use of non-Saccharomyces yeasts that are generally considered as spoilage yeasts, in association with Saccharomyces cerevisiae for grape must fermentation was here evaluated. Analysis of the main oenological characteristics of pure cultures of 55 yeasts belonging to the genera Hanseniaspora, Pichia, Saccharomycodes and Zygosaccharomyces revealed wide biodiversity within each genus. Moreover, many of these non-Saccharomyces strains had interesting oenological properties in terms of fermentation purity, and ethanol and secondary metabolite production. The use of four non-Saccharomyces yeasts (one per genus) in mixed cultures with a commercial S. cerevisiae strain at different S. cerevisiae/non-Saccharomyces inoculum ratios was investigated. This revealed that most of the compounds normally produced at high concentrations by pure cultures of non-Saccharomyces, and which are considered detrimental to wine quality, do not reach threshold taste levels in these mixed fermentations. On the other hand, the analytical profiles of the wines produced by these mixed cultures indicated that depending on the yeast species and the S. cerevisiae/non-Saccharomyces inoculum ratio, these non-Saccharomyces yeasts can be used to increase production of polysaccharides and to modulate the final concentrations of acetic acid and volatile compounds, such as ethyl acetate, phenyl-ethyl acetate, 2-phenyl ethanol, and 2-methyl 1-butanol. Copyright © 2011 Elsevier B.V. All rights reserved.

Expression of endoglucanases in Pichia pastoris under control of the GAP promoter

PubMed Central

2014-01-01

Background Plant-derived biomass is a potential alternative to fossil feedstocks for a greener economy. Enzymatic saccharification of biomass has been studied extensively and endoglucanases have been found to be a prerequisite for quick initial liquefaction of biomass under industrial conditions. Pichia pastoris, widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. The recently marketed PichiaPink™ expression system allows for rapid clone selection, and employs the methanol inducible AOX1 promoter to ensure high protein expression levels. However, methanol is toxic and poses a fire hazard, issues which become more significant at an industrial scale. It is possible to eliminate these risks and still maintain high productivity by switching to the constitutive GAP promoter. Results In the present study, a plasmid carrying the constitutive GAP promoter was created for PichiaPink™. We then studied expression of two endoglucanases, AfCel12A from Aspergillus fumigatus and TaCel5A from Thermoascus aurantiacus, regulated by either the AOX1 promoter or the GAP promoter. Initial experiments in tubes and small bioreactors showed that the levels of AfCel12A obtained with the constitutive promoter were similar or higher, compared to the AOX1 promoter, whereas the levels of TaCel5A were somewhat lower. After optimization of cultivation conditions using a 15-l bioreactor, the recombinant P. pastoris strains utilizing the GAP promoter produced ca. 3–5 g/l of total secreted protein, with CMCase activity equivalent to 1200 nkat/ml AfCel12A and 170 nkat/ml TaCel5A. Conclusions We present a strategy for constitutive recombinant protein expression in the novel PichiaPink™ system. Both AfCel12A and TaCel5A were successfully expressed constitutively in P. pastoris under the GAP promoter. Reasonable protein levels were reached after optimizing cultivation conditions. PMID:24742273

Expression of endoglucanases in Pichia pastoris under control of the GAP promoter.

PubMed

Várnai, Anikó; Tang, Campbell; Bengtsson, Oskar; Atterton, Andrew; Mathiesen, Geir; Eijsink, Vincent G H

2014-04-18

Plant-derived biomass is a potential alternative to fossil feedstocks for a greener economy. Enzymatic saccharification of biomass has been studied extensively and endoglucanases have been found to be a prerequisite for quick initial liquefaction of biomass under industrial conditions. Pichia pastoris, widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. The recently marketed PichiaPink™ expression system allows for rapid clone selection, and employs the methanol inducible AOX1 promoter to ensure high protein expression levels. However, methanol is toxic and poses a fire hazard, issues which become more significant at an industrial scale. It is possible to eliminate these risks and still maintain high productivity by switching to the constitutive GAP promoter. In the present study, a plasmid carrying the constitutive GAP promoter was created for PichiaPink™. We then studied expression of two endoglucanases, AfCel12A from Aspergillus fumigatus and TaCel5A from Thermoascus aurantiacus, regulated by either the AOX1 promoter or the GAP promoter. Initial experiments in tubes and small bioreactors showed that the levels of AfCel12A obtained with the constitutive promoter were similar or higher, compared to the AOX1 promoter, whereas the levels of TaCel5A were somewhat lower. After optimization of cultivation conditions using a 15-l bioreactor, the recombinant P. pastoris strains utilizing the GAP promoter produced ca. 3-5 g/l of total secreted protein, with CMCase activity equivalent to 1200 nkat/ml AfCel12A and 170 nkat/ml TaCel5A. We present a strategy for constitutive recombinant protein expression in the novel PichiaPink™ system. Both AfCel12A and TaCel5A were successfully expressed constitutively in P. pastoris under the GAP promoter. Reasonable protein levels were reached after optimizing cultivation conditions.

Choosing Between Yeast and Bacterial Expression Systems: Yield Dependent

NASA Technical Reports Server (NTRS)

Miller, Rebecca S.; Malone, Christine C.; Moore, Blake P.; Burk, Melissa; Crawford, Lisa; Karr, Laurel J.; Curreri, Peter A. (Technical Monitor)

2002-01-01

Green fluorescent protein (GFP) is a naturally occurring fluorescent protein isolated from the jellyfish Aequorea victoria. The intrinsic fluorescence of the protein is due to a chromophore located in the center of the molecule. Its usefulness has been established as a marker for gene expression and localization of gene products. GFP has recently been utilized as a model protein for crystallization studies at NASA/MSFC, both in earth-based and in microgravity experiments. Because large quantities of purified protein were needed, the cDNA of GFP was cloned into the Pichia pastoris pPICZ(alpha) C strain, with very little protein secreted into the media. Microscopic analysis prior to harvest showed gigantic green fluorescent yeast, but upon harvesting most protein was degraded. Trial fermentations of GFP cloned into pPICZ A for intracellular expression provided unsatisfactory yield. GFP cloned into E, coli was overexpressed at greater than 150 mg/liter, with purification yields at greater than 100mg/liter.

Dynamic study of yeast species and Saccharomyces cerevisiae strains during the spontaneous fermentations of Muscat blanc in Jingyang, China.

PubMed

Wang, Chunxiao; Liu, Yanlin

2013-04-01

The evolution of yeast species and Saccharomyces cerevisiae genotypes during spontaneous fermentations of Muscat blanc planted in 1957 in Jingyang region of China was followed in this study. Using a combination of colony morphology on Wallerstein Nutrient (WLN) medium, sequence analysis of the 26S rDNA D1/D2 domain and 5.8S-ITS-RFLP analysis, a total of 686 isolates were identified at the species level. The six species identified were S. cerevisiae, Hanseniaspora uvarum, Hanseniaspora opuntiae, Issatchenkia terricola, Pichia kudriavzevii (Issatchenkia orientalis) and Trichosporon coremiiforme. This is the first report of T. coremiiforme as an inhabitant of grape must. Three new colony morphologies on WLN medium and one new 5.8S-ITS-RFLP profile are described. Species of non-Saccharomyces, predominantly H. opuntiae, were found in early stages of fermentation. Subsequently, S. cerevisiae prevailed followed by large numbers of P. kudriavzevii that dominated at the end of fermentations. Six native genotypes of S. cerevisiae were determined by interdelta sequence analysis. Genotypes III and IV were predominant. As a first step in exploring untapped yeast resources of the region, this study is important for monitoring the yeast ecology in native fermentations and screening indigenous yeasts that will produce wines with regional characteristics. Copyright © 2012 Elsevier Ltd. All rights reserved.

Characterization of Osmotolerant Yeasts and Yeast-Like Molds from Apple Orchards and Apple Juice Processing Plants in China and Investigation of Their Spoilage Potential.

PubMed

Wang, Huxuan; Hu, Zhongqiu; Long, Fangyu; Niu, Chen; Yuan, Yahong; Yue, Tianli

2015-08-01

Yeasts and yeast-like fungal isolates were recovered from apple orchards and apple juice processing plants located in the Shaanxi province of China. The strains were evaluated for osmotolerance by growing them in 50% (w/v) glucose. Of the strains tested, 66 were positive for osmotolerance and were subsequently identified by 26S or 5.8S-ITS ribosomal RNA (rRNA) gene sequencing. Physiological tests and RAPD-PCR analysis were performed to reveal the polymorphism of isolates belonging to the same species. Further, the spoilage potential of the 66 isolates was determining by evaluating their growth in 50% to 70% (w/v) glucose and measuring gas generation in 50% (w/v) glucose. Thirteen osmotolerant isolates representing 9 species were obtained from 10 apple orchards and 53 target isolates representing 19 species were recovered from 2 apple juice processing plants. In total, members of 14 genera and 23 species of osmotolerant isolates including yeast-like molds were recovered from all sources. The commonly recovered osmotolerant isolates belonged to Kluyveromyces marxianus, Hanseniaspora uvarum, Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Candida tropicalis, and Pichia kudriavzevii. The polymorphism of isolates belonging to the same species was limited to 1 to 3 biotypes. The majority of species were capable of growing within a range of glucose concentration, similar to sugar concentrations found in apple juice products with a lag phase from 96 to 192 h. Overall, Z. rouxii was particularly the most tolerant to high glucose concentration with the shortest lag phase of 48 h in 70% (w/v) glucose and the fastest gas generation rate in 50% (w/v) glucose. © 2015 Institute of Food Technologists®

Identification and characterization of yeasts causing chalk mould defects on par-baked bread.

PubMed

Deschuyffeleer, N; Audenaert, K; Samapundo, S; Ameye, S; Eeckhout, M; Devlieghere, F

2011-08-01

Pichia anomala, Hyphopichia burtonii and Saccharomycopsis fibuligera are spoilage yeasts causing chalk mould defects on par-baked breads packaged under modified atmosphere. The first objective of this study was to identify yeasts isolated from spoiled par-baked breads by means of a RAPD protocol and to determine the dominant spoilers amongst identified strains. The second objective was to determine the effects of water activity (a(w)) and pH value on the growth rates and lag phase durations of P. anomala, H. burtonii and S. fibuligera. 95% of the yeasts tested were identified as P. anomala and 5% as S. fibuligera, H. burtonii was not detected. In order to investigate the effect of a(w) and pH the growth of the three yeasts was tested within an a(w) range of 0.88-0.98 and a pH range of 2.8-8.0. P. anomala was able to grow from pH 2.8 to 8 without a clear optimum. S. fibuligera and H. burtonii showed a pH optimum for growth of 5. The optimum water activity for growth was different for the three strains and varied between 0.96 and 0.98. These growth data were further used to develop secondary models that describe the relationship between a(w) and the radial or colony growth rate (g, mm/d) or the lag phase duration (λ, d). The identification of the spoilage organisms and a good understanding of the effects of a(w) and pH on the growth behavior is essential for future development of adequate conservation strategies against chalk mould defects. Copyright © 2011 Elsevier Ltd. All rights reserved.

The effect of lactic acid bacteria on cocoa bean fermentation.

PubMed

Ho, Van Thi Thuy; Zhao, Jian; Fleet, Graham

2015-07-16

Cocoa beans (Theobroma cacao L.) are the raw material for chocolate production. Fermentation of cocoa pulp by microorganisms is crucial for developing chocolate flavor precursors. Yeasts conduct an alcoholic fermentation within the bean pulp that is essential for the production of good quality beans, giving typical chocolate characters. However, the roles of bacteria such as lactic acid bacteria and acetic acid bacteria in contributing to the quality of cocoa bean and chocolate are not fully understood. Using controlled laboratory fermentations, this study investigated the contribution of lactic acid bacteria to cocoa bean fermentation. Cocoa beans were fermented under conditions where the growth of lactic acid bacteria was restricted by the use of nisin and lysozyme. The resultant microbial ecology, chemistry and chocolate quality of beans from these fermentations were compared with those of indigenous (control) fermentations. The yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii, Kluyveromyces marxianus and Saccharomyces cerevisiae, the lactic acid bacteria Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus fermentum and the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateurii were the major species found in control fermentations. In fermentations with the presence of nisin and lysozyme, the same species of yeasts and acetic acid bacteria grew but the growth of lactic acid bacteria was prevented or restricted. These beans underwent characteristic alcoholic fermentation where the utilization of sugars and the production of ethanol, organic acids and volatile compounds in the bean pulp and nibs were similar for beans fermented in the presence of lactic acid bacteria. Lactic acid was produced during both fermentations but more so when lactic acid bacteria grew. Beans fermented in the presence or absence of lactic acid bacteria were fully fermented, had similar shell weights and gave acceptable chocolates with no differences

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.

Postharvest application of a novel chitinase cloned from Metschnikowia fructicola and overexpressed in Pichia pastoris to control brown rot of peaches.

PubMed

Banani, Houda; Spadaro, Davide; Zhang, Dianpeng; Matic, Slavica; Garibaldi, Angelo; Gullino, Maria Lodovica

2015-04-16

Metschnikowia fructicola strain AP47 is a yeast antagonist against postharvest pathogens of fruits. The yeast was able to produce chitinase enzymes in the presence of pathogen cell wall. A novel chitinase gene MfChi (GenBank accession number HQ113461) was amplified from the genomic DNA of Metschnikowia fructicola AP47. Sequence analysis showed lack of introns, an open reading frame (ORF) of 1098 bp encoding a 365 amino acid protein with a calculated molecular weight of 40.9 kDa and a predicted pI of 5.27. MfChi was highly induced in Metschnikowia fructicola after interaction with Monilinia fructicola cell wall, suggesting a primary role of MfChi chitinase in the antagonistic activity of the yeast. The MfChi gene overexpressed in the heterologous expression system of Pichia pastoris KM71 and the recombinant chitinase showed high endochitinase activity towards 4-Nitrophenyl β-d-N,N',N″-triacetylchitotriose substrate. The antifungal activity of the recombinant chitinase was investigated against Monilinia fructicola and Monilinia laxa in vitro and on peaches. The chitinase significantly controlled the spore germination and the germ tube length of the tested pathogens in PDB medium and the mycelium diameter in PDA. The enzyme, when applied on peaches cv. Redhaven, successfully reduced brown rot severity. This work shows that the chitinase MfChi could be developed as a postharvest treatment with antimicrobial activity for fruit undergoing a short shelf life, and confirms that P. pastoris KM71 is a suitable microorganism for cost-effective large-scale production of recombinant chitinases. Copyright © 2015 Elsevier B.V. All rights reserved.

Enhancing ethanol production from cellulosic sugars using Scheffersomyces (Pichia) stipitis

USDA-ARS?s Scientific Manuscript database

Studies were performed on the effect of CaCO3 and CaCl2 supplementation to fermentation medium for ethanol production from xylose, glucose, or their mixtures using Scheffersomyces (Pichia) stipitis. Both of these chemicals were found to improve maximum ethanol concentration and ethanol productivity....

Pichia pastoris: a recombinant microfactory for antibodies and human membrane proteins.

PubMed

Gonçalves, A M; Pedro, A Q; Maia, C; Sousa, F; Queiroz, J A; Passarinha, L A

2013-05-01

During the last few decades, it has become evident that the compatibility of the yeast biochemical environment with the ability to process and translate the RNA transcript, along with its capacity to modify a translated protein, are relevant requirements for selecting this host cell for protein expression in several pharmaceutical and clinical applications. In particular, Pichia pastoris is used as an industrial host for recombinant protein and metabolite production, showing a powerful capacity to meet required biomolecular target production levels in high-throughput assays for functional genomics and drug screening. In addition, there is a great advantage to using P. pastoris for protein secretion, even at high molecular weights, since the recovery and purification steps are simplified owing to relatively low levels of endogenous proteins in the extracellular medium. Clearly, no single microexpression system can provide all of the desired properties for human protein production. Moreover, chemical and physical bioprocess parameters, including culture medium formulation, temperature, pH, agitation, aeration rates, induction, and feeding strategies, can highly influence product yield and quality. In order to benefit from the currently available wide range of biosynthesis strategies using P. pastoris, this mini review focuses on the developments and technological fermentation achievements, providing both a comparative and an overall integration analysis. The main aim is to highlight the relevance and versatility of the P. pastoris biosystem to the design of more cost-effective microfactories to meet the increasing demands for recombinant membrane proteins and clinical antibodies for several therapeutic applications.

Improved bioethanol production using fusants of Saccharomyces cerevisiae and xylose-fermenting yeasts.

PubMed

Kumari, Rajni; Pramanik, K

2012-06-01

The present research deals with the development of a hybrid yeast strain with the aim of converting pentose and hexose sugar components of lignocellulosic substrate to bioethanol by fermentation. Different fusant strains were obtained by fusing protoplasts of Saccharomyces cerevisiae and xylose-fermenting yeasts such as Pachysolen tannophilus, Candida shehatae and Pichia stipitis. The fusants were sorted by fluorescent-activated cell sorter and further confirmed by molecular characterization. The fusants were evaluated by fermentation of glucose-xylose mixture and the highest ethanol producing fusant was used for further study to ferment hydrolysates produced by acid pretreatment and enzymatic hydrolysis of cotton gin waste. Among the various fusant and parental strains used under present study, RPR39 was found to be stable and most efficient strain giving maximum ethanol concentration (76.8 ± 0.31 g L(-1)), ethanol productivity (1.06 g L(-1) h(-1)) and ethanol yield (0.458 g g(-1)) by fermentation of glucose-xylose mixture under test conditions. The fusant has also shown encouraging result in fermenting hydrolysates of cotton gin waste with ethanol concentration of 7.08 ± 0.142 g L(-1), ethanol yield of 0.44 g g(-1), productivity of 0.45 g L(-1) h(-1) and biomass yield of 0.40 g g(-1).

Description of Komagataella mondaviorum sp. nov., a new sibling species of Komagataella (Pichia) pastoris.

PubMed

Naumov, Gennadi I; Naumova, Elena S; Boundy-Mills, Kyria L

2018-07-01

Five methylotrophic strains (UCDFST 71-1024 T , UCDFST 54-11.16, UCDFST 54-11.141, UCDFST 68-967.1 and UCDFST 74-1030) from the Phaff Yeast Culture Collection (University of California Davis, USA) that were originally designated as Pichia pastoris were found to represent a novel Komagataella species. Strains of Komagataella mondaviorum sp. nov. UCDFST 71-1024 T (type strain) = CBS 15017, UCDFST 54-11.16, UCDFST 54-11.141, UCDFST 68-967.1, and UCDFST 74-1030 were isolated in USA, respectively, from cottonwood tree Populus deltoides in 1971 (Davis, CA), slime flux of Quercus sp. in 1954 (CA), exudate of black oak Q. kelloggii in 1954 (Central Sierra Nevada. CA), dry frass from Salix sp. in 1968 (Soleduck Road, Olympic National Park, WA) and from flux of hackberry tree Celtis sp. in 1974 (CA). The new species was differentiated from Komagataella kurtzmanii, Komagataella pastoris, Komagataella phaffii, Komagataella populi, Komagataella pseudopastoris and Komagataella ulmi by divergence in gene sequences for D1/D2 LSU rRNA, ITS1-5.8S-ITS2, RNA polymerase subunit I and translation elongation factor-1α. Komagataella mondaviorum sp. nov. is registered in MycoBank under MB 821789.

Effect of Furfural, Vanillin and Syringaldehyde on Candida guilliermondii Growth and Xylitol Biosynthesis

NASA Astrophysics Data System (ADS)

Kelly, Christine; Jones, Opal; Barnhart, Christopher; Lajoie, Curtis

Xylitol is a five-carbon sugar alcohol with established commercial use as an alternative sweetener and can be produced from hemicellulose hydrolysate. However, there are difficulties with microbiological growth and xylitol biosynthesis on hydrolysate because of the inhibitors formed from hydrolysis of hemicellulose. This research focused on the effect of furfural, vanillin, and syringaldehyde on growth of Candida guilliermondii and xylitol accumulation from xylose in a semi-synthetic medium in microwell plate and bioreactor cultivations. All three compounds reduced specific growth rate, increased lag time, and reduced xylitol production rate. In general, increasing concentration of inhibitor increased the severity of inhibition, except in the case of 0.5 g vanillin per liter, which resulted in a faster late batch phase growth rate and increased biomass yield. At concentrations of 1 g/1 or higher, furfural was the least inhibitory to growth, followed by syringaldehyde. Vanillin most severely reduced specific growth rate. All three inhibitors reduced xylitol production rate approximately to the same degree.

Effect of furfural, vanillin and syringaldehyde on Candida guilliermondii growth and xylitol biosynthesis.

PubMed

Kelly, Christine; Jones, Opal; Barnhart, Christopher; Lajoie, Curtis

2008-03-01

Xylitol is a five-carbon sugar alcohol with established commercial use as an alternative sweetener and can be produced from hemicellulose hydrolysate. However, there are difficulties with microbiological growth and xylitol biosynthesis on hydrolysate because of the inhibitors formed from hydrolysis of hemicellulose. This research focused on the effect of furfural, vanillin, and syringaldehyde on growth of Candida guilliermondii and xylitol accumulation from xylose in a semi-synthetic medium in microwell plate and bioreactor cultivations. All three compounds reduced specific growth rate, increased lag time, and reduced xylitol production rate. In general, increasing concentration of inhibitor increased the severity of inhibition, except in the case of 0.5 g vanillin per liter, which resulted in a faster late batch phase growth rate and increased biomass yield. At concentrations of 1 g/l or higher, furfural was the least inhibitory to growth, followed by syringaldehyde. Vanillin most severely reduced specific growth rate. All three inhibitors reduced xylitol production rate approximately to the same degree.

Reduction of saliva-promoted adhesion of Streptococcus mutans MT8148 and dental biofilm development by tragacanth gum and yeast-derived phosphomannan.

PubMed

Shimotoyodome, A; Kobayashi, H; Nakamura, J; Tokimitsu, I; Hase, T; Inoue, T; Matsukubo, T; Takaesu, Y

2006-01-01

The aim of this study was to investigate materials which reduce saliva-promoted adhesion of Streptococcus mutans onto enamel surfaces, and their potential in preventing dental biofilm development. The effects of hydroxyapatite (HA) surface pretreatment with hydrophilic polysaccharides on saliva-promoted S. mutans adhesion in vitro and de novo dental biofilm deposition in vivo were examined. Saliva-promoted adhesion of S. mutans MT8148 was significantly reduced by pretreatment of the HA surface with tragacanth gum (TG) and yeast-derived phosphoglycans. Extracellular phosphomannan (PM) from Pichia capsulata NRRL Y-1842 and TG reduced biofilm development on lower incisors in plaque-susceptible rats when administered via drinking water at concentrations of 0.5% and 0.01%, respectively. The inhibitory effect of TG on de novo dental biofilm formation was also demonstrated when administered via mouthwash in humans. It is concluded that TG and yeast-derived PM have the potential for use as anti-adherent agents and are effective in reducing de novo dental biofilm formation.

Genetic engineering of Pichia stipitis for fermentation of xylose

Treesearch

Thomas W. Jeffries; N. Q. Shi; J. Y. Cho; P. Lu; K. Dahn; J. Hendrick; H. K. Sreenath

1998-01-01

A useful genetic system has been developed for the transformation of Pichia stipitis. This includes two selectable markers (URA3 and LEU2), integrating and autonomous replication vectors, a pop-out cassette that enables multiple targeted disruptions, and a genomic X-library for rapid cloning. Using this system we have cloned two genes for alcohol dehydrogenase (PsADH1...

The Role of ABC Proteins in Drug-Resistant Breast Cancer Cells

DTIC Science & Technology

2007-04-01

and a biotin acceptor domain) under control of the alcohol oxidase promoter (Figure 2). Upon methanol induction, the yeast expressed high levels of...as native cDNA. Therefore, we backtranslated the protein into a nucleotide sequence codon-optimized for expression in Pichia pastoris yeast. Yeast

Optimized extraction by cetyl trimethyl ammonium bromide reversed micelles of xylose reductase and xylitol dehydrogenase from Candida guilliermondii homogenate.

PubMed

Cortez, Ely Vieira; Pessoa, Adalberto; das Graças de Almeida Felipe, Maria; Roberto, Inês Conceição; Vitolo, Michele

2004-07-25

The intracellular enzymes xylose reductase (XR, EC 1.1.1.21) and xylitol dehydrogenase (XD, EC 1.1.1.9) from Candida guilliermondii, grown in sugar cane bagasse hydrolysate, were separated by reversed micelles of cetyl trimethyl ammonium bromide (CTAB) cationic surfactant. An experimental design was employed to optimize the extraction conditions of both enzymes. Under these conditions (temperature = 5 degree C, hexanol: isooctane proportion = 5% (v/v), 22 %, surfactant concentration = 0.15M, pH = 7.0 and electrical conductivity = 14 mScm(-1)) recovery values of about 100 and 80% were achieved for the enzymes XR and XD, respectively. The purity of XR and XD increased 5.6- and 1.8-fold, respectively. The extraction process caused some structural modifications in the enzymes molecules, as evidenced by the alteration of K(M) values determined before and after extraction, either in regard to the substrate (up 35% for XR and down 48% for XD) or cofactor (down 29% for XR and up 11% for XD). However, the average variation of V(max) values for both enzymes was not higher than 7%, indicating that the modified affinity of enzymes for their respective substrates and cofactors, as consequence of structural modifications suffered by them during the extraction, are compensated in some extension. This study demonstrated that liquid-liquid extraction by CTAB reversed micelles is an efficient process to separate the enzymes XR and XD present in the cell extract, and simultaneously increase the enzymatic activity and the purity of both enzymes produced by C. guilliermondii.

Indigenous Georgian Wine-Associated Yeasts and Grape Cultivars to Edit the Wine Quality in a Precision Oenology Perspective.

PubMed

Vigentini, Ileana; Maghradze, David; Petrozziello, Maurizio; Bonello, Federica; Mezzapelle, Vito; Valdetara, Federica; Failla, Osvaldo; Foschino, Roberto

2016-01-01

In Georgia, one of the most ancient vine-growing environment, the homemade production of wine is still very popular in every rural family and spontaneous fermentation of must, without addition of chemical preservatives, is the norm. The present work investigated the yeast biodiversity in five Georgian areas (Guria, Imereti, Kakheti, Kartli, Ratcha-Lechkhumi) sampling grapes and wines from 22 different native cultivars, in 26 vineyards and 19 family cellars. One hundred and eighty-two isolates were ascribed to 15 different species by PCR-ITS and RFLP, and partial sequencing of D1/D2 domain 26S rDNA gene. Metschnikowia pulcherrima (F' = 0.56, I' = 0.32), Hanseniaspora guilliermondii (F' = 0.49, I' = 0.27), and Cryptococcus flavescens (F' = 0.31, I' = 0.11) were the dominant yeasts found on grapes, whereas Saccharomyces cerevisiae showed the highest prevalence into wine samples. Seventy four isolates with fermentative potential were screened for oenological traits such as ethanol production, resistance to SO2, and acetic acid, glycerol and H2S production. Three yeast strains (Kluyveromyces marxianus UMY207, S. cerevisiae UMY255, Torulaspora delbrueckii UMY196) were selected and separately inoculated in vinifications experiments at a Georgian cellar. Musts were prepared from healthy grapes of local varieties, Goruli Mtsvane (white berry cultivar) and Saperavi (black berry cultivar). Physical (°Brix) and microbial analyses (plate counts) were performed to monitor the fermentative process. The isolation of indigenous S. cerevisiae yeasts beyond the inoculated strains indicated that a co-presence occurred during the vinification tests. Results from quantitative GC-FID analysis of volatile compounds revealed that the highest amount of fermentation flavors, such as 4-ethoxy-4-oxobutanoic acid (monoethyl succinate), 2-methylpropan-1-ol, ethyl 2-hydroxypropanoate, and 2-phenylethanol, were significantly more produced in fermentation conducted in Saperavi variety inoculated

Unravelling the contribution of lactic acid bacteria and acetic acid bacteria to cocoa fermentation using inoculated organisms.

PubMed

Ho, Van Thi Thuy; Fleet, Graham H; Zhao, Jian

2018-08-20

Cocoa beans (Theobroma cacao L.) are the raw material for chocolate production. Fermentation of the bean pulp by microorganisms is essential for developing the precursors of chocolate flavour. Currently, the cocoa fermentation is still conducted by an uncontrolled traditional process via a consortium of indigenous species of yeasts, lactic acid bacteria and acetic acid bacteria. Although the essential contribution of yeasts to the production of good quality beans and, typical chocolate character is generally agreed, the roles of lactic acid bacteria and acetic acid bacteria are less certain. The objective of this study was to investigate the contribution of LAB and AAB in cocoa bean fermentation by conducting small scale laboratory fermentations under aseptic conditions, inoculated with different groups of microorganisms previously isolated from spontaneous cocoa fermentations. The inoculation protocols were: (1) four yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii, Kluyveromyces marxianus and Saccharomyces cerevisiae; (2) four yeasts plus the lactic acid bacteria Lactobacillus plantarum and Lactobacillus fermentum; (3) four yeasts plus the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateuri and (4) four yeasts plus two lactic acid bacteria and two acetic acid bacteria. Only the inoculated species were detected in the microbiota of their respective fermentations. Beans from the inoculated fermentations showed no significant differences in colour, shell weights and concentrations of residual sugars, alcohols and esters (p>0.05), but they were slightly different in contents of lactic acid and acetic acid (p<0.05). All beans were fully brown and free of mould. Residual sugar levels were less than 2.6 mg/g while the shell contents and ethanol were in the range of 11-13.4% and 4.8-7 mg/g, respectively. Beans fermented in the presence of LAB contained higher levels of lactic acid (0.6-1.2 mg/g) whereas higher concentrations of acetic acid

The Zinc Finger Proteins Mxr1p and Repressor of Phosphoenolpyruvate Carboxykinase (ROP) Have the Same DNA Binding Specificity but Regulate Methanol Metabolism Antagonistically in Pichia pastoris*

PubMed Central

Kumar, Nallani Vijay; Rangarajan, Pundi N.

2012-01-01

The methanol-inducible alcohol oxidase I (AOXI) promoter of the methylotrophic yeast, Pichia pastoris, is used widely for the production of recombinant proteins. AOXI transcription is regulated by the zinc finger protein Mxr1p (methanol expression regulator 1). ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) is a methanol- and biotin starvation-inducible zinc finger protein that acts as a negative regulator of PEPCK in P. pastoris cultured in biotin-deficient, glucose-ammonium medium. The function of ROP during methanol metabolism is not known. In this study, we demonstrate that ROP represses methanol-inducible expression of AOXI when P. pastoris is cultured in a nutrient-rich medium containing yeast extract, peptone, and methanol (YPM). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion whereas overexpression of ROP results in repression of AOXI and growth retardation of P. pastoris cultured in YPM medium. Surprisingly, deletion or overexpression of ROP has no effect on AOXI gene expression and growth of P. pastoris cultured in a minimal medium containing yeast nitrogen base and methanol (YNBM). Subcellular localization studies indicate that ROP translocates from cytosol to nucleus of cells cultured in YPM but not YNBM. In vitro DNA binding studies indicate that AOXI promoter sequences containing 5′ CYCCNY 3′ motifs serve as binding sites for Mxr1p as well as ROP. Thus, Mxr1p and ROP exhibit the same DNA binding specificity but regulate methanol metabolism antagonistically in P. pastoris. This is the first report on the identification of a transcriptional repressor of methanol metabolism in any yeast species. PMID:22888024

Structural and functional characterization of recombinant napin-like protein of Momordica charantia expressed in methylotrophic yeast Pichia pastoris.

PubMed

Yadav, Shailesh Kumar R; Sahu, Tejram; Dixit, Aparna

2016-08-01

Napin and napin-like proteins belong to the 2S albumin seed storage family of proteins and have been shown to display a variety of biological activities. However, due to a high degree of polymorphism, purification of a single napin or napin-like protein exhibiting biological activity is extremely difficult. In the present study, we have produced the napin-like protein of Momordica charantia using the methylotrophic Pichia pastoris expression system. The recombinant napin-like protein (rMcnapin) secreted in the extracellular culture supernatant was enriched by ammonium sulfate precipitation, and purified using size exclusion chromatography at a yield of ∼290 mg/L of culture. Secondary structure analysis of the purified rMcnapin revealed it to be predominantly α-helical with minimal β strand content. CD spectroscopic and fluorescence spectroscopic analyses revealed the rMcnapin to be stable at a wide range of temperatures and pH. The rMcnapin exhibited antifungal activity against Trichoderma viride with an IC50 of ∼3.7 μg/ml and trypsin inhibitor activity with an IC50 of 4.2 μM. Thus, large amounts of homogenous preparations of the biologically active rMcnapin could be obtained at shake flask level, which is otherwise difficult from its natural source.

Glycerophosphate as a phosphorus source in a defined medium for Pichia pastoris fermentation.

PubMed

Zhang, Wenhui; Sinha, Jayanta; Meagher, Michael M

2006-08-01

Pichia pastoris has emerged as a commercially important yeast for the production of a vast majority of recombinant therapeutic proteins and vaccines. The organism can be grown to very high cell densities using a defined basal salts media (BSM). However, BSM contains bi-cation or tri-cation phosphate, which precipitates out of the medium at pH above 5.5, although the optimal fermentation pH of most recombinant protein fermentation varies between 5.5 and 7.0. In this article, the application of glycerophosphates was investigated as a substitute phosphate source in an effort to eliminate precipitation. The solubility of BSM containing sodium or potassium glycerophosphates was examined before and after autoclaving at various pHs. Sodium glycerophosphate was found stable at autoclave temperature but formed complexes with coexisting magnesium and calcium ions that were insoluble above pH 7.0. Medium where sodium glycerophosphate was autoclaved separately and then added to the growth medium did not produce any precipitate up to pH 10.5. The performance of P. pastoris fermentations expressing alpha-galactosidase and ovine interferon-tau using a glycerolphosphate-based medium was found to be comparable to a conventional BSM. The results from this work demonstrate that sodium glycerophosphate can be assimilated by the P. pastoris strains and can be employed as a reliable phosphorus source for both cell growth and recombinant protein production.

Novel endophytic yeast Rhodotorula mucilaginosa strain PTD3 I: production of xylitol and ethanol.

PubMed

Bura, Renata; Vajzovic, Azra; Doty, Sharon L

2012-07-01

An endophytic yeast, Rhodotorula mucilaginosa strain PTD3, that was isolated from stems of hybrid poplar was found to be capable of production of xylitol from xylose, of ethanol from glucose, galactose, and mannose, and of arabitol from arabinose. The utilization of 30 g/L of each of the five sugars during fermentation by PTD3 was studied in liquid batch cultures. Glucose-acclimated PTD3 produced enhanced yields of xylitol (67% of theoretical yield) from xylose and of ethanol (84, 86, and 94% of theoretical yield, respectively) from glucose, galactose, and mannose. Additionally, this yeast was capable of metabolizing high concentrations of mixed sugars (150 g/L), with high yields of xylitol (61% of theoretical yield) and ethanol (83% of theoretical yield). A 1:1 glucose:xylose ratio with 30 g/L of each during double sugar fermentation did not affect PTD3's ability to produce high yields of xylitol (65% of theoretical yield) and ethanol (92% of theoretical yield). Surprisingly, the highest yields of xylitol (76% of theoretical yield) and ethanol (100% of theoretical yield) were observed during fermentation of sugars present in the lignocellulosic hydrolysate obtained after steam pretreatment of a mixture of hybrid poplar and Douglas fir. PTD3 demonstrated an exceptional ability to ferment the hydrolysate, overcome hexose repression of xylose utilization with a short lag period of 10 h, and tolerate sugar degradation products. In direct comparison, PTD3 had higher xylitol yields from the mixed sugar hydrolysate compared with the widely studied and used xylitol producer Candida guilliermondii.

Competition assays and physiological experiments of soil and phyllosphere yeasts identify Candida subhashii as a novel antagonist of filamentous fungi.

PubMed

Hilber-Bodmer, Maja; Schmid, Michael; Ahrens, Christian H; Freimoser, Florian M

2017-01-05

While recent advances in next generation sequencing technologies have enabled researchers to readily identify countless microbial species in soil, rhizosphere, and phyllosphere microbiomes, the biological functions of the majority of these species are unknown. Functional studies are therefore urgently needed in order to characterize the plethora of microorganisms that are being identified and to point out species that may be used for biotechnology or plant protection. Here, we used a dual culture assay and growth analyses to characterise yeasts (40 different isolates) and their antagonistic effect on 16 filamentous fungi; comprising plant pathogens, antagonists, and saprophytes. Overall, this competition screen of 640 pairwise combinations revealed a broad range of outcomes, ranging from small stimulatory effects of some yeasts up to a growth inhibition of more than 80% by individual species. On average, yeasts isolated from soil suppressed filamentous fungi more strongly than phyllosphere yeasts and the antagonistic activity was a species-/isolate-specific property and not dependent on the filamentous fungus a yeast was interacting with. The isolates with the strongest antagonistic activity were Metschnikowia pulcherrima, Hanseniaspora sp., Cyberlindnera sargentensis, Aureobasidium pullulans, Candida subhashii, and Pichia kluyveri. Among these, the soil yeasts (C. sargentensis, A. pullulans, C. subhashii) assimilated and/or oxidized more di-, tri- and tetrasaccharides and organic acids than yeasts from the phyllosphere. Only the two yeasts C. subhashii and M. pulcherrima were able to grow with N-acetyl-glucosamine as carbon source. The competition assays and physiological experiments described here identified known antagonists that have been implicated in the biological control of plant pathogenic fungi in the past, but also little characterised species such as C. subhashii. Overall, soil yeasts were more antagonistic and metabolically versatile than yeasts from

Surface display and bioactivity of Bombyx mori acetylcholinesterase on Pichia pastoris

USDA-ARS?s Scientific Manuscript database

To construct the Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE), the gene for the anchor protein (AGa1) was obtained from Saccharomyces cerevisiae and was fused with the modified Bombyx mori acetylcholinesterase gene (bmace) and transformed int...

Improved fermentation performance to produce bioethanol from Gelidium amansii using Pichia stipitis adapted to galactose.

PubMed

Sukwong, Pailin; Ra, Chae Hun; Sunwoo, In Yung; Tantratian, Sumate; Jeong, Gwi-Taek; Kim, Sung-Koo

2018-03-23

This study employed a statistical method to obtain optimal hyper thermal acid hydrolysis conditions using Gelidium amansii (red seaweed) as a source of biomass. The optimal hyper thermal acid hydrolysis using G. amansii as biomass was determined as 12% (w/v) slurry content, 358.3 mM H 2 SO 4 , and temperature of 142.6 °C for 11 min. After hyper thermal acid hydrolysis, enzymatic saccharification was carried out. The total monosaccharide concentration was 45.1 g/L, 72.2% of the theoretical value of the total fermentable monosaccharides of 62.4 g/L based on 120 g dry weight/L in the G. amansii slurry. To increase ethanol production, 3.8 g/L 5-hydroxymethylfurfural (HMF) in the hydrolysate was removed by treatment with 3.5% (w/v) activated carbon for 2 min and fermented with Pichia stipitis adapted to high galactose concentrations via separate hydrolysis and fermentation. With complete HMF removal and the use of P. stipitis adapted to high galactose concentrations, 22 g/L ethanol was produced (yield 0.50). Fermentation with total HMF removal and yeast adapted to high galactose concentrations increased the fermentation performance and decreased the fermentation time from 96 to 36 h compared to traditional fermentation.

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.

Production of in vivo biotinylated scFv specific to almond (Prunus dulcis) proteins by recombinant Pichia pastoris.

PubMed

de la Cruz, Silvia; Alcocer, Marcos; Madrid, Raquel; García, Aina; Martín, Rosario; González, Isabel; García, Teresa

2016-06-10

The methylotropic yeast Pichia pastoris has demonstrated its suitability for large-scale production of recombinant proteins. As an eukaryotic organism P. pastoris presents a series of advantages at expression and processing of heterologous proteins when compared with Escherichia coli. In this work, P. pastoris has been used to express a scFv from a human synthetic library previously shown to bind almond proteins. In order to facilitate purification and post processing manipulations, the scFv was engineered with a C-terminal tag and biotinylated in vivo. After purification, biotinylated scFv were bound to avidin conjugated with HRP producing a multimeric scFv. The multimeric scFv showed to maintain their ability to recognize almond protein when assayed in ELISA, reaching a LOD of 470mgkg(-1). This study describes an easy method to produce large quantities of in vivo biotinylated scFv in P. pastoris. By substituting the enzyme or fluorochromes linked to avidin, it will be possible to generate a diverse number of multimeric scFv as probes to suit different analytical platforms in the detection of almond in food products. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Xylitol production by a Pichia stipitis D-xylulokinase mutant

Treesearch

Yong-Su Jin; Jose Cruz; Thomas W. Jeffries

2005-01-01

Xylitol production by Pichia stipitis FPL-YS30, a xyl3-Ä1 mutant that metabolizes xylose using an alternative metabolic pathway, was investigated under aerobic and oxygen-limited culture conditions. Under both culture conditions, FPL-YS30 (xyl3-Ä1) produced a negligible amount of ethanol and converted xylose mainly into xylitol with comparable yields (0.30 and 0.27 g...

A model for the effect of pH on the growth of chalk yeasts.

PubMed

Dantigny, Philippe; Burgain, Anaïs; Deniel, Franck; Bensoussan, Maurice

2014-09-01

Hyphopichia burtonii, Pichia anomala, and Saccharomycopsis fibuligera were isolated from spoiled packaged sliced bread. These chalk yeasts were characterized by a wide range of pH for which growth was almost optimum. Thus, the curve growth vs pH exhibited plateau and sharp profiles close to the minimum and the maximum pH. This study described a chalk yeast model (CYM) for the effect of pH derived from a new germination model for fungi (Dantigny, P., Nanguy, S., P.-M., Judet-Correia, D., and Bensoussan, M. 2011, International Journal of Food Microbiology, 146, 176-181). The CYM is asymmetric, versatile, based on parameters with biological significance, and compatible with the gamma concept. The CYM was compared to the cardinal pH model (CPM) which is widely used to describe the effect of pH on microbial growth. The CYM exhibited RMSE values two fold less than those obtained with the CPM for H. burtonii, and S. fibuligera for which plateaus were clearly observed. For P. anomala, the plateau was less obvious, but the RMSE value obtained with the CYM was similar to that found with the CPM. The CYM could extend its use to represent the effect of pH on mold growth. Copyright © 2014 Elsevier B.V. All rights reserved.

Secretion expression of human neutrophil peptide 1 (HNP1) in Pichia pastoris and its functional analysis against antibiotic-resistant Helicobacter pylori.

PubMed

Zhang, Xiaolin; Jiang, Anmin; Qi, Banghua; Yu, Hao; Xiong, Youyi; Zhou, Guoliang; Qin, Meisong; Dou, Jinfeng; Wang, Jianfei

2018-06-01

Human neutrophil peptide 1 (HNP1) is a small (3.44 kDa) cationic peptide that is a distinct member of the defensin family. HNP1 plays a crucial role in controlling bacterial infections, particularly by antibiotic-resistant bacteria, through membrane perforation patterns. The structural characteristics of HNP1's three intramolecular disulfide bridges cause difficulty in its synthesis via chemical methods. In this study, bioactive recombinant HNP1 was produced using the Pichia pastoris (P. Pichia) expression system. HNP1 was fused with the polyhedrin of Bombyx mori and enhanced green fluorescent protein (EGFP) to prevent HNP1 toxicity in yeast host cells under direct expression. An enterokinase protease cleavage site (amino acid sequence DDDDK) was designed upstream of the HNP1 peptide to obtain the antibacterial peptide HNP1 with native structure after it was cleaved by the enterokinase. The fusion HNP1 protein (FHNP1) was successfully expressed and had a molecular mass of approximately 62.6 kDa, as determined using SDS-PAGE and Western blot. Then, the recovered FHNP1 was digested and purified; Tricine-SDS-PAGE results showed that HNP1 was successfully released from FHNP1. Functional analysis of induction against antibiotic-resistant Helicobacter pylori (H. pylori) showed that it was challenging for HNP1 to acquire resistance to the antibiotic-resistant H. pylori. Moreover, in vitro studies showed that HNP1 exerted a strong effect against antibiotic-resistant H. pylori activity. Furthermore, the animal model of H. pylori infection established in vivo showed that HNP1 significantly reduced the colonization of antibiotic-resistant H. pylori in the stomach. Our study indicated that this could be a new potential avenue for large-scale production of HNP1 for therapeutic application against the antibiotic-resistant H. pylori infection in humans.

Saccharomycotina and Taphrinomycotina: the yeasts and yeast-like fungi of the Ascomycota

USDA-ARS?s Scientific Manuscript database

The phylum Ascomycota has been resolved into three major phylogenetic lineages: the subphyla Saccharomycotina (e.g., Saccharomyces, Pichia, Candida), Taphrinomycotina (e.g., Protomyces, Taphrina, Pneumocystis), and the Pezizomycotina (e.g., Aspergillus, Neurospora, Peziza). We discuss the ecology, ...

Integrating metabolic modeling and population heterogeneity analysis into optimizing recombinant protein production by Komagataella (Pichia) pastoris.

PubMed

Theron, Chrispian W; Berrios, Julio; Delvigne, Frank; Fickers, Patrick

2018-01-01

The methylotrophic yeast Komagataella (Pichia) pastoris has become one of the most utilized cell factories for the production of recombinant proteins over the last three decades. This success story is linked to its specific physiological traits, i.e., the ability to grow at high cell density in inexpensive culture medium and to secrete proteins at high yield. Exploiting methanol metabolism is at the core of most P. pastoris-based processes but comes with its own challenges. Co-feeding cultures with glycerol/sorbitol and methanol is a promising approach, which can benefit from improved understanding and prediction of metabolic response. The development of profitable processes relies on the construction and selection of efficient producing strains from less efficient ones but also depends on the ability to master the bioreactor process itself. More specifically, how a bioreactor processes could be monitored and controlled to obtain high yield of production. In this review, new perspectives are detailed regarding a multi-faceted approach to recombinant protein production processes by P. pastoris; including gaining improved understanding of the metabolic pathways involved, accounting for variations in transcriptional and translational efficiency at the single cell level and efficient monitoring and control of methanol levels at the bioreactor level.

A new source of resistance to 2-furaldehyde from Scheffersomyces (Pichia) stipitis for sustainable lignocellulose-to-biofuel conversion.

PubMed

Wang, Xu; Lewis Liu, Z; Zhang, Xiaoping; Ma, Menggen

2017-06-01

Aldehyde inhibitory compounds derived from lignocellulosic biomass pretreatment have been identified as a major class of toxic chemicals that interfere with microbial growth and subsequent fermentation for advanced biofuel production. Development of robust next-generation biocatalyst is a key for a low-cost biofuel production industry. Scheffersomyces (Pichia) stipitis is a naturally occurring C-5 sugar utilization yeast; however, little is known about the genetic background underlying its potential tolerance to biomass conversion inhibitors. We investigated and identified five uncharacterized putative aryl-alcohol dehydrogenase genes (SsAADs) from this yeast as a new source of resistance against biomass fermentation inhibitor 2-furaldehyde (furfural) by gene expression, gene cloning, and direct enzyme assay analysis using partially purified proteins. All five proteins from S. stipitis showed furfural reduction using cofactor NADH. An optimum active temperature was observed at 40 °C for SsAad1p; 30 °C for SsAad3p, SsAad4p, and SsAad5p; and 20 °C for SsAad2p. SsAad2p, SsAad3p, and SsAad4p showed tolerance to a wide range of pH from 4.5 to 8, but SsAad1p and SsAad5p were sensitive to pH changes beyond 7. Genes SsAAD2, SsAAD3, and SsAAD4 displayed significantly enhanced higher levels of expression in response to the challenge of furfural. Their encoding proteins also showed higher levels of specific activity toward furfural and were suggested as core functional enzymes contributing aldehyde resistance in S. stipitis.

Pineapple (Ananas comosus L. Merr.) wine production in Angola: Characterisation of volatile aroma compounds and yeast native flora.

PubMed

Dellacassa, Eduardo; Trenchs, Oriol; Fariña, Laura; Debernardis, Florencia; Perez, Gabriel; Boido, Eduardo; Carrau, Francisco

2017-01-16

A pineapple vinification process was conducted through inoculated and spontaneous fermentation to develop a process suitable for a quality beverage during two successive vintages in Huambo, Angola. Wines obtained with the conventional Saccharomyces cerevisiae strain, were analysed by gas chromatography, and a total of 61 volatile constituents were detected in the volatile fraction and 18 as glycosidically bound aroma compounds. Concentration levels of carbonyl and sulphur compounds were in agreement with the limited information reported about pineapple fruits of other regions. We report, for the first time in pineapple wines, the presence of significant concentrations of lactones, ketones, terpenes, norisoprenoids and a variety of volatile phenols. Eight native yeast strains were isolated from spontaneous batches. Further single-strain fermentations allowed us to characterise their suitability for commercial fermentation. Three native strains (Hanseniaspora opuntiae, H. uvarum and Meyerozyma guilliermondii) were selected with sensory potential to ferment pineapple fruits with increased flavour diversity. Results obtained here contribute to a better understanding of quality fermentation alternatives of this tropical fruit in subtropical regions. Copyright © 2016 Elsevier B.V. All rights reserved.

Ethanol production from the seaweed Gelidium amansii, using specific sugar acclimated yeasts.

PubMed

Cho, Hyeyoung; Ra, Chae-Hun; Kim, Sung-Koo

2014-02-28

For the production of ethanol from seaweed as the source material, thermal acid hydrolysis and enzymatic saccharification were carried out for monosugars production of 25.5 g/l galactose and 7.6 g/l glucose using Gelidium amansii. The fermentation was performed with Pichia stipitis KCTC 7228 or Saccharomyces cerevisiae KCCM 1129. When wild P. stipitis and S. cerevisiae were used, the ethanol productions of 11.2 g/l and 6.9 g/l were produced, respectively. The ethanol productions of 16.6 g/l and 14.6 g/l were produced using P. stipitis and S. cerevisiae acclimated to high concentration of galactose, respectively. The yields of ethanol fermentation increased to 0.5 and 0.44 from 0.34 and 0.21 using acclimated P. stipitis and S. cerevisiae, respectively. Therefore, acclimation of yeasts to a specific sugar such as galactose reduced the glucose-induced repression on the transport of galactose.

Pichia pastoris secretes recombinant proteins less efficiently than Chinese hamster ovary cells but allows higher space-time yields for less complex proteins

PubMed Central

Maccani, Andreas; Landes, Nils; Stadlmayr, Gerhard; Maresch, Daniel; Leitner, Christian; Maurer, Michael; Gasser, Brigitte; Ernst, Wolfgang; Kunert, Renate; Mattanovich, Diethard

2014-01-01

Chinese hamster ovary (CHO) cells are currently the workhorse of the biopharmaceutical industry. However, yeasts such as Pichia pastoris are about to enter this field. To compare their capability for recombinant protein secretion, P. pastoris strains and CHO cell lines producing human serum albumin (HSA) and the 3D6 single chain Fv-Fc anti-HIV-1 antibody (3D6scFv-Fc) were cultivated in comparable fed batch processes. In P. pastoris, the mean biomass-specific secretion rate (qp) was 40-fold lower for 3D6scFv-Fc compared to HSA. On the contrary, qp was similar for both proteins in CHO cells. When comparing both organisms, the mean qp of the CHO cell lines was 1011-fold higher for 3D6scFv-Fc and 26-fold higher for HSA. Due to the low qp of the 3D6scFv-Fc producing strain, the space-time yield (STY) was 9.6-fold lower for P. pastoris. In contrast, the STY of the HSA producer was 9.2-fold higher compared to CHO cells because of the shorter process time and higher biomass density. The results indicate that the protein secretion machinery of P. pastoris is much less efficient and the secretion rate strongly depends on the complexity of the recombinant protein. However, process efficiency of the yeast system allows higher STYs for less complex proteins. PMID:24390926

Yeast population dynamics reveal a potential 'collaboration' between Metschnikowia pulcherrima and Saccharomyces uvarum for the production of reduced alcohol wines during Shiraz fermentation.

PubMed

Contreras, A; Curtin, C; Varela, C

2015-02-01

The wine sector is actively seeking strategies and technologies that facilitate the production of wines with lower alcohol content. One of the simplest approaches to achieve this aim would be the use of wine yeast strains which are less efficient at transforming grape sugars into ethanol; however, commercial wine yeasts have very similar ethanol yields. We recently demonstrated that Metschnikowia pulcherrima AWRI1149 was able to produce wine with reduced alcohol concentration when used in sequential inoculation with a wine strain of Saccharomyces cerevisiae. Here, different inoculation regimes were explored to study the effect of yeast population dynamics and potential yeast interactions on the metabolism of M. pulcherrima AWRI1149 during fermentation of non-sterile Shiraz must. Of all inoculation regimes tested, only ferments inoculated with M. pulcherrima AWRI1149 showed reduced ethanol concentration. Population dynamics revealed the presence of several indigenous yeast species and one of these, Saccharomyces uvarum (AWRI 2846), was able to produce wine with reduced ethanol concentration in sterile conditions. Both strains however, were inhibited when a combination of three non-Saccharomyces strains, Hanseniaspora uvarum AWRI863, Pichia kluyveri AWRI1896 and Torulaspora delbrueckii AWRI2845 were inoculated into must, indicating that the microbial community composition might impact on the growth of M. pulcherrima AWRI1149 and S. uvarum AWRI 2846. Our results indicate that mixed cultures of M. pulcherrima AWRI1149 and S. uvarum AWRI2846 enable an additional reduction of wine ethanol concentration compared to the same must fermented with either strain alone. This work thus provides a foundation to develop inoculation regimes for the successful application of non-cerevisiae yeast to the production of wines with reduced alcohol.

Cloning of novel cellulases from cellulolytic fungi: heterologous expression of a family 5 glycoside hydrolase from Trametes versicolor in Pichia pastoris.

PubMed

Salinas, Alejandro; Vega, Marcela; Lienqueo, María Elena; Garcia, Alejandro; Carmona, Rene; Salazar, Oriana

2011-12-10

Total cDNA isolated from cellulolytic fungi cultured in cellulose was examined for the presence of sequences encoding for endoglucanases. Novel sequences encoding for glycoside hydrolases (GHs) were identified in Fusarium oxysporum, Ganoderma applanatum and Trametes versicolor. The cDNA encoding for partial sequences of GH family 61 cellulases from F. oxysporum and G. applanatum shares 58 and 68% identity with endoglucanases from Glomerella graminicola and Laccaria bicolor, respectively. A new GH family 5 endoglucanase from T. versicolor was also identified. The cDNA encoding for the mature protein was completely sequenced. This enzyme shares 96% identity with Trametes hirsuta endoglucanase and 22% with Trichoderma reesei endoglucanase II (EGII). The enzyme, named TvEG, has N-terminal family 1 carbohydrate binding module (CBM1). The full length cDNA was cloned into the pPICZαB vector and expressed as an active, extracellular enzyme in the methylotrophic yeast Pichia pastoris. Preliminary studies suggest that T. versicolor could be useful for lignocellulose degradation. Copyright © 2011 Elsevier Inc. All rights reserved.

The response to unfolded protein is involved in osmotolerance of Pichia pastoris

PubMed Central

2010-01-01

Background The effect of osmolarity on cellular physiology has been subject of investigation in many different species. High osmolarity is of importance for biotechnological production processes, where high cell densities and product titers are aspired. Several studies indicated that increased osmolarity of the growth medium can have a beneficial effect on recombinant protein production in different host organisms. Thus, the effect of osmolarity on the cellular physiology of Pichia pastoris, a prominent host for recombinant protein production, was studied in carbon limited chemostat cultures at different osmolarities. Transcriptome and proteome analyses were applied to assess differences upon growth at different osmolarities in both, a wild type strain and an antibody fragment expressing strain. While our main intention was to analyze the effect of different osmolarities on P. pastoris in general, this was complemented by studying it in context with recombinant protein production. Results In contrast to the model yeast Saccharomyces cerevisiae, the main osmolyte in P. pastoris was arabitol rather than glycerol, demonstrating differences in osmotic stress response as well as energy metabolism. 2D Fluorescence Difference Gel electrophoresis and microarray analysis were applied and demonstrated that processes such as protein folding, ribosome biogenesis and cell wall organization were affected by increased osmolarity. These data indicated that upon increased osmolarity less adaptations on both the transcript and protein level occurred in a P. pastoris strain, secreting the Fab fragment, compared with the wild type strain. No transcriptional activation of the high osmolarity glycerol (HOG) pathway was observed at steady state conditions. Furthermore, no change of the specific productivity of recombinant Fab was observed at increased osmolarity. Conclusion These data point out that the physiological response to increased osmolarity is different to S. cerevisiae

Yeast Hog1 proteins are sequestered in stress granules during high-temperature stress.

PubMed

Shiraishi, Kosuke; Hioki, Takahiro; Habata, Akari; Yurimoto, Hiroya; Sakai, Yasuyoshi

2018-01-09

The yeast high-osmolarity glycerol (HOG) pathway plays a central role in stress responses. It is activated by various stresses, including hyperosmotic stress, oxidative stress, high-temperature stress and exposure to arsenite. Hog1, the crucial MAP kinase of the pathway, localizes to the nucleus in response to high osmotic concentrations, i.e. high osmolarity; but, otherwise, little is known about its intracellular dynamics and regulation. By using the methylotrophic yeast Candida boidinii , we found that CbHog1-Venus formed intracellular dot structures after high-temperature stress in a reversible manner. Microscopic observation revealed that CbHog1-mCherry colocalized with CbPab1-Venus, a marker protein of stress granules. Hog1 homologs in Pichia pastoris and Schizosaccharomyces pombe also exhibited similar dot formation under high-temperature stress, whereas Saccharomyces cerevisiae Hog1 (ScHog1)-GFP did not. Analysis of CbHog1-Venus in C. boidinii revealed that a β-sheet structure in the N-terminal region was necessary and sufficient for its localization to stress granules. Physiological studies revealed that sequestration of activated Hog1 proteins in stress granules was responsible for downregulation of Hog1 activity under high-temperature stress.This article has an associated First Person interview with the first author of the paper. © 2018. Published by The Company of Biologists Ltd.

Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris

PubMed Central

Capone, Simona; Ćorajević, Lejla; Bonifert, Günther; Murth, Patrick; Maresch, Daniel; Altmann, Friedrich; Herwig, Christoph; Spadiut, Oliver

2015-01-01

Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1) was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins. PMID:26404235

The Expression and Characterization of Functionally Active Soluble CD83 by Pichia pastoris Using High-Density Fermentation

PubMed Central

Song, Xiaoping; Zhong, Yongjun; Wang, Chenguang; Jia, Hao; Wu, Lidan; Wang, Dong; Fang, Fang; Ma, Jiajia; Kang, Wenyao; Sun, Jie; Tian, Zhigang; Xiao, Weihua

2014-01-01

CD83 is a highly glycosylated type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD83 is upregulated during dendritic cell (DC) maturation, which is critical for the initiation of adaptive immune responses. The soluble isoform of CD83 (sCD83) is encoded by alternative splicing from full-length CD83 mRNA and inhibits DC maturation, which suggests that sCD83 acts as a potential immune suppressor. In this study, we developed a sound strategy to express functional sCD83 from Pichia pastoris in extremely high-density fermentation. Purified sCD83 was expressed as a monomer at a yield of more than 200 mg/L and contained N-linked glycosylation sites that were characterized by PNGase F digestion. In vitro tests indicated that recombinant sCD83 bound to its putative counterpart on monocytes and specifically blocked the binding of anti-CD83 antibodies to cell surface CD83 on DCs. Moreover, sCD83 from yeast significantly suppressed ConA-stimulated PBMC proliferation. Therefore, sCD83 that was expressed from the P. pastoris was functionally active and may be used for in vivo and in vitro studies as well as future clinical applications. PMID:24586642

Free fatty acids reduce metabolic stress and favor a stable production of heterologous proteins in Pichia pastoris.

PubMed

Zepeda, Andrea B; Figueroa, Carolina A; Pessoa, Adalberto; Farías, Jorge G

2018-04-12

The growth of yeasts in culture media can be affected by many factors. For example, methanol can be metabolized by other pathways to produce ethanol, which acts as an inhibitor of the heterologous protein production pathway; oxygen concentration can generate aerobic or anaerobic environments and affects the fermentation rate; and temperature affects the central carbon metabolism and stress response protein folding. The main goal of this study was determine the implication of free fatty acids on the production of heterologous proteins in different culture conditions in cultures of Pichia pastoris. We evaluated cell viability using propidium iodide by flow cytometry and thiobarbituric acid reactive substances to measure cell membrane damage. The results indicate that the use of low temperatures and low methanol concentrations favors the decrease in lipid peroxidation in the transition phase from glycerol to methanol. In addition, a temperature of 14°C+1%M provided the most stable viability. By contrast, the temperature of 18°C+1.5%M favored the production of a higher antibody fragment concentration. In summary, these results demonstrate that the decrease in lipid peroxidation is related to an increased production of free fatty acids. Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

[Optimization on the production of analgesic peptide from Buthus martensii Karsch in Pichia pastoris].

PubMed

Yang, Jin-ling; He, Hui-xia; Zhu, Hui-xin; Cheng, Ke-di; Zhu, Ping

2009-01-01

The technology of liquid fermentation for producing the recombinant analgesic peptide BmK AngM1 from Buthus martensii Karsch in Pichia pastoris was studied by single-factor and orthogonal test. The results showed that the optimal culture conditions were as follows: 1.2% methanol, 0.6% casamino acids, initial pH 6.0, and three times of basal inoculation volume. Under the above culture conditions, the expression level of recombinant BmK AngM1 in Pichia pastoris was above 500 mg x L(-1), which was more than three times of the control. The study has laid a foundation for the large-scale preparation of BmK AngM1 to meet the needs of theoretical research of BmK AngM1 and development of new medicines.

Bioprocess for efficient production of recombinant Pichia anomala phytase and its applicability in dephytinizing chick feed and whole wheat flat Indian breads.

PubMed

Joshi, Swati; Satyanarayana, T

2015-10-01

The phytase of the yeast Pichia anomala (PPHY) is a suitable biocatalyst as a food and feed additive because of its adequate thermostability, acid stability, protease insensitivity and broad substrate spectrum. The cell-bound nature and low phytase titres are the main bottlenecks for its utility in food and feed industries. In this investigation, we have overcome the problems by constitutive secretory expression of PPHY under glyceraldehyde phosphate dehydrogenase (GAP) promoter. A ~44-fold increase in rPPHY titre has been achieved after optimization of cultural variables by one-variable-at-a-time approach and two factorial statistical design. The use of GAP promoter makes the cultivation of the recombinant P. pastoris straight forward and eliminates the requirement of methanol for induction and hazards associated with its storage. Among metal-phytate complexes, Ca(2+) phytate is hydrolyzed more efficiently by rPPHY than Co(2+), Mn(2+), Mg(2+), Fe(3+) and Zn(2+) phytates. The enzyme is effective in dephytinizing whole wheat unleavened flat Indian breads (naan and tandoori) and different broiler feeds, thus mitigating anti-nutritional effects of phytates.

Species Differences in Human and Rodent PEPT2-Mediated Transport of Glycylsarcosine and Cefadroxil in Pichia Pastoris Transformants

PubMed Central

Song, Feifeng; Hu, Yongjun; Jiang, Huidi

2017-01-01

The proton-coupled oligopeptide transporter PEPT2 (SLC15A2) plays an important role in the disposition of di/tripeptides and peptide-like drugs in kidney and brain. However, unlike PEPT1 (SLC15A1), there is little information about species differences in the transport of PEPT2-mediated substrates. The purpose of this study was to determine whether PEPT2 exhibited a species-dependent uptake of glycylsarcosine (GlySar) and cefadroxil using yeast Pichia pastoris cells expressing cDNA from human, mouse, and rat. In such a system, the functional activity of PEPT2 was evaluated with [3H]GlySar as a function of time, pH, substrate concentration, and specificity, and with [3H]cefadroxil as a function of concentration. We observed that the uptake of GlySar was pH-dependent with an optimal uptake at pH 6.5 for all three species. Moreover, GlySar showed saturable uptake kinetics, with Km values in human (150.6 µM) > mouse (42.8 µM) ≈ rat (36.0 µM). The PEPT2-mediated uptake of GlySar in yeast transformants was specific, being inhibited by di/tripeptides and peptide-like drugs, but not by amino acids and nonsubstrate compounds. Cefadroxil also showed a saturable uptake profile in all three species, with Km values in human (150.8 μM) > mouse (15.6 μM) ≈ rat (11.9 μM). These findings demonstrated that the PEPT2-mediated uptake of GlySar and cefadroxil was specific, species dependent, and saturable. Furthermore, based on the Km values, mice appeared similar to rats but both were less than optimal as animal models in evaluating the renal reabsorption and pharmacokinetics of peptides and peptide-like drugs in humans. PMID:27836942

Priceomyuces M. Suzuki & Kurtzman (2010)

USDA-ARS?s Scientific Manuscript database

This chapter describes the ascomycete yeast genus Priceomyces and is to be published in "The Yeasts, A Taxonomic Study, 5th edition." The genus Priceomyces has five described species that were earlier assigned to the genus Pichia, but gene sequence analysis showed that the species, now reclassified...

The combined rapid detection and species-level identification of yeasts in simulated blood culture using a colorimetric sensor array.

PubMed

Shrestha, Nabin K; Lim, Sung H; Wilson, Deborah A; SalasVargas, Ana Victoria; Churi, Yair S; Rhodes, Paul A; Mazzone, Peter J; Procop, Gary W

2017-01-01

A colorimetric sensor array (CSA) has been demonstrated to rapidly detect and identify bacteria growing in blood cultures by obtaining a species-specific "fingerprint" of the volatile organic compounds (VOCs) produced during growth. This capability has been demonstrated in prokaryotes, but has not been reported for eukaryotic cells growing in culture. The purpose of this study was to explore if a disposable CSA could differentially identify 7 species of pathogenic yeasts growing in blood culture. Culture trials of whole blood inoculated with a panel of clinically important pathogenic yeasts at four different microorganism loads were performed. Cultures were done in both standard BacT/Alert and CSA-embedded bottles, after adding 10 mL of spiked blood to each bottle. Color changes in the CSA were captured as images by an optical scanner at defined time intervals. The captured images were analyzed to identify the yeast species. Time to detection by the CSA was compared to that in the BacT/Alert system. One hundred sixty-two yeast culture trials were performed, including strains of several species of Candida (Ca. albicans, Ca. glabrata, Ca. parapsilosis, and Ca. tropicalis), Clavispora (synonym Candida) lusitaniae, Pichia kudriavzevii (synonym Candida krusei) and Cryptococcus neoformans, at loads of 8.2 × 105, 8.3 × 103, 8.5 × 101, and 1.7 CFU/mL. In addition, 8 negative trials (no yeast) were conducted. All negative trials were correctly identified as negative, and all positive trials were detected. Colorimetric responses were species-specific and did not vary by inoculum load over the 500000-fold range of loads tested, allowing for accurate species-level identification. The mean sensitivity for species-level identification by CSA was 74% at detection, and increased with time, reaching almost 95% at 4 hours after detection. At an inoculum load of 1.7 CFU/mL, mean time to detection with the CSA was 6.8 hours (17%) less than with the BacT/Alert platform. The CSA

Development of a high-throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design.

PubMed

Bláha, Benjamin A F; Morris, Stephen A; Ogonah, Olotu W; Maucourant, Sophie; Crescente, Vincenzo; Rosenberg, William; Mukhopadhyay, Tarit K

2018-01-01

The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high-throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high-throughput disruption methods exist. The development of an automated, miniaturized, high-throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high-pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA-based methods to mimic large-scale homogenization processes. These results demonstrate that AFA-mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:130-140, 2018. © 2017 American Institute of Chemical Engineers.

Molecular cloning and characterization of an alpha-amylase from Pichia burtonii 15-1.

PubMed

Kato, Saemi; Shimizu-Ibuka, Akiko; Mura, Kiyoshi; Takeuchi, Akiko; Tokue, Chiyoko; Arai, Soichi

2007-12-01

An alpha-amylase secreted by Pichia burtonii 15-1 isolated from a traditional starter murcha of Nepal, named Pichia burtonii alpha-amylase (PBA), was studied. The gene was cloned and its nucleotide sequence was determined. PBA was deduced to consist of 494 amino acid residues. It shared certain degrees of amino acid sequence identity with other homologous proteins: 60% with Schwanniomyces occidentalis alpha-amylase, 58% with Saccharomycopsis sp. alpha-amylase, and 47% with Taka-amylase A from Aspergillus oryzae. A three-dimensional structural model of PBA generated using the known three-dimensional structure of Taka-amylase A as a template suggested high structural similarity between them. Kinetic analysis revealed that the K(m) values of PBA were lower than those of Taka-amylase A for the oligosaccharides. Although the k(cat) values of PBA were lower than those of Taka-amylase A for the oligosaccharide substrates, the k(cat)/K(m) values of PBA were higher.

[Evaluation of common commercial systems for the identification of yeast isolates in microbiology laboratories: a multicenter study].

PubMed

Karabıçak, Nilgün; Uludağ Altun, Hatice; Karatuna, Onur; Hazırolan, Gülşen; Aksu, Neriman; Adiloğlu, Ali; Akyar, Işın

2015-04-01

: 3/104) by both of the systems. For rarely detected yeast isolates, misidentification or unidentification were most frequently observed in species of C.pelliculosa (Vitek: 3/11; API: 6/11) and C.dubliniensis (API and Vitek: 2/5) isolates. Candida guilliermondii (API: 2/5) isolates had lower rate of identification with API compared to other species. Blastoschizomyces capitatus and Saccharomyces cerevisiae isolates could not be identified by both of the systems. As a result, the accurate diagnosis of Vitek and API systems were similar in terms of consistency (86.3%). Two systems performed well in correct identification of common clinical yeast species (at least 95%), while the identification of rare species was more challenging indicating that they require further morphological and physiological testing. The addition of morphological identification to commercial systems will be useful for accurate diagnosis and treatment of mixed infections.

Stoichiometric network constraints on xylose metabolism by recombinant Saccharomyces cerevisiae

Treesearch

Yong-Su Jin; Thomas W. Jeffries

2004-01-01

Metabolic pathway engineering is constrained by the thermodynamic and stoichiometric feasibility of enzymatic activities of introduced genes. Engineering of xylose metabolism in Saccharomyces cerevisiae has focused on introducing genes for the initial xylose assimilation steps from Pichia stipitis, a xylose-fermenting yeast, into S. cerevisiae, a yeast raditionally...

Multicenter Study of Epidemiological Cutoff Values and Detection of Resistance in Candida spp. to Anidulafungin, Caspofungin, and Micafungin Using the Sensititre YeastOne Colorimetric Method

PubMed Central

Alvarez-Fernandez, M.; Cantón, E.; Carver, P. L.; Chen, S. C.-A.; Eschenauer, G.; Getsinger, D. L.; Gonzalez, G. M.; Grancini, A.; Hanson, K. E.; Kidd, S. E.; Klinker, K.; Kubin, C. J.; Kus, J. V.; Lockhart, S. R.; Meletiadis, J.; Morris, A. J.; Pelaez, T.; Rodriguez-Iglesias, M.; Sánchez-Reus, F.; Shoham, S.; Wengenack, N. L.; Borrell Solé, N.; Echeverria, J.; Esperalba, J.; Gómez-G. de la Pedrosa, E.; García García, I.; Linares, M. J.; Marco, F.; Merino, P.; Pemán, J.; Pérez del Molino, L.; Roselló Mayans, E.; Rubio Calvo, C.; Ruiz Pérez de Pipaon, M.; Yagüe, G.; Garcia-Effron, G.; Perlin, D. S.; Sanguinetti, M.; Shields, R.; Turnidge, J.

2015-01-01

Neither breakpoints (BPs) nor epidemiological cutoff values (ECVs) have been established for Candida spp. with anidulafungin, caspofungin, and micafungin when using the Sensititre YeastOne (SYO) broth dilution colorimetric method. In addition, reference caspofungin MICs have so far proven to be unreliable. Candida species wild-type (WT) MIC distributions (for microorganisms in a species/drug combination with no detectable phenotypic resistance) were established for 6,007 Candida albicans, 186 C. dubliniensis, 3,188 C. glabrata complex, 119 C. guilliermondii, 493 C. krusei, 205 C. lusitaniae, 3,136 C. parapsilosis complex, and 1,016 C. tropicalis isolates. SYO MIC data gathered from 38 laboratories in Australia, Canada, Europe, Mexico, New Zealand, South Africa, and the United States were pooled to statistically define SYO ECVs. ECVs for anidulafungin, caspofungin, and micafungin encompassing ≥97.5% of the statistically modeled population were, respectively, 0.12, 0.25, and 0.06 μg/ml for C. albicans, 0.12, 0.25, and 0.03 μg/ml for C. glabrata complex, 4, 2, and 4 μg/ml for C. parapsilosis complex, 0.5, 0.25, and 0.06 μg/ml for C. tropicalis, 0.25, 1, and 0.25 μg/ml for C. krusei, 0.25, 1, and 0.12 μg/ml for C. lusitaniae, 4, 2, and 2 μg/ml for C. guilliermondii, and 0.25, 0.25, and 0.12 μg/ml for C. dubliniensis. Species-specific SYO ECVs for anidulafungin, caspofungin, and micafungin correctly classified 72 (88.9%), 74 (91.4%), 76 (93.8%), respectively, of 81 Candida isolates with identified fks mutations. SYO ECVs may aid in detecting non-WT isolates with reduced susceptibility to anidulafungin, micafungin, and especially caspofungin, since testing the susceptibilities of Candida spp. to caspofungin by reference methodologies is not recommended. PMID:26282428

2-deoxyglucose as a selective agent for derepressed mutants of Pichia stipitis

Treesearch

Hassan K. Sreenath; Thomas W. Jeffries

1998-01-01

The glucose analog 2-deoxyglucose (2-DOG) has been used to obtain mutants derepressed for pentose metabolism. Some researchers have used 2-DOG alone whereas others have used it in the presence of a glucoserepressible carbon source. We examined both methods and screened mutant strains for improved use of xylose in the presence of glucose. Pichia stipitis mutants...

A novel method to prepare L-Arabinose from xylose mother liquor by yeast-mediated biopurification

PubMed Central

2011-01-01

Background L-arabinose is an important intermediate for anti-virus drug synthesis and has also been used in food additives for diets-controlling in recent years. Commercial production of L-arabinose is a complex progress consisting of acid hydrolysis of gum arabic, followed by multiple procedures of purification, thus making high production cost. Therefore, there is a biotechnological and commercial interest in the development of new cost-effective and high-performance methods for obtaining high purity grade L-arabinose. Results An alternative, economical method for purifying L-arabinose from xylose mother liquor was developed in this study. After screening 306 yeast strains, a strain of Pichia anomala Y161 was selected as it could effectively metabolize other sugars but not L-arabinose. Fermentation in a medium containing xylose mother liquor permitted enrichment of L-arabinose by a significant depletion of other sugars. Biochemical analysis of this yeast strain confirmed that its poor capacity for utilizing L-arabinose was due to low activities of the enzymes required for the metabolism of this sugar. Response surface methodology was employed for optimization the fermentation conditions in shake flask cultures. The optimum conditions were: 75 h fermentation time, at 32.5°C, in a medium containing 21% (v/v) xylose mother liquor. Under these conditions, the highest purity of L-arabinose reached was 86.1% of total sugar, facilitating recovery of white crystalline L-arabinose from the fermentation medium by simple methods. Conclusion Yeast-mediated biopurification provides a dynamic method to prepare high purity of L-arabinose from the feedstock solution xylose mother liqour, with cost-effective and high-performance properties. PMID:21649890

Indigenous Georgian Wine-Associated Yeasts and Grape Cultivars to Edit the Wine Quality in a Precision Oenology Perspective

PubMed Central

Vigentini, Ileana; Maghradze, David; Petrozziello, Maurizio; Bonello, Federica; Mezzapelle, Vito; Valdetara, Federica; Failla, Osvaldo; Foschino, Roberto

2016-01-01

In Georgia, one of the most ancient vine-growing environment, the homemade production of wine is still very popular in every rural family and spontaneous fermentation of must, without addition of chemical preservatives, is the norm. The present work investigated the yeast biodiversity in five Georgian areas (Guria, Imereti, Kakheti, Kartli, Ratcha-Lechkhumi) sampling grapes and wines from 22 different native cultivars, in 26 vineyards and 19 family cellars. One hundred and eighty-two isolates were ascribed to 15 different species by PCR-ITS and RFLP, and partial sequencing of D1/D2 domain 26S rDNA gene. Metschnikowia pulcherrima (F’ = 0.56, I’ = 0.32), Hanseniaspora guilliermondii (F’ = 0.49, I’ = 0.27), and Cryptococcus flavescens (F’ = 0.31, I’ = 0.11) were the dominant yeasts found on grapes, whereas Saccharomyces cerevisiae showed the highest prevalence into wine samples. Seventy four isolates with fermentative potential were screened for oenological traits such as ethanol production, resistance to SO2, and acetic acid, glycerol and H2S production. Three yeast strains (Kluyveromyces marxianus UMY207, S. cerevisiae UMY255, Torulaspora delbrueckii UMY196) were selected and separately inoculated in vinifications experiments at a Georgian cellar. Musts were prepared from healthy grapes of local varieties, Goruli Mtsvane (white berry cultivar) and Saperavi (black berry cultivar). Physical (°Brix) and microbial analyses (plate counts) were performed to monitor the fermentative process. The isolation of indigenous S. cerevisiae yeasts beyond the inoculated strains indicated that a co-presence occurred during the vinification tests. Results from quantitative GC-FID analysis of volatile compounds revealed that the highest amount of fermentation flavors, such as 4-ethoxy-4-oxobutanoic acid (monoethyl succinate), 2-methylpropan-1-ol, ethyl 2-hydroxypropanoate, and 2-phenylethanol, were significantly more produced in fermentation conducted in Saperavi variety

Development of Synthetic Spider Silk Fibers for High Performance Applications

DTIC Science & Technology

2013-08-08

complete with N- and C-termini. • Transformed all protein variants into a proprietary yeast strain and screened for expression. While all encoded...mammals1- 6,10-12. Among the most successfully expressing organisms has been the methylotropic yeast Pichia pastoris. Yeast are an attractive...modifications, recombinant proteins can be secreted into their culture media, and they are well adapted to high density fermentation . In addition, P

High-level extracellular production and characterization of Candida antarctica lipase B in Pichia pastoris.

PubMed

Eom, Gyeong Tae; Lee, Seung Hwan; Song, Bong Keun; Chung, Keun-Wo; Kim, Young-Wun; Song, Jae Kwang

2013-08-01

The gene encoding lipase B from Candida antarctica (CalB) was expressed in Pichia pastoris after it was synthesized by the recursive PCR and cloned into the Pichia expression plasmid, pPICZαA. The CalB was successfully secreted in the recombinant P. pastoris strain X-33 with an apparent molecular weight of 34 kDa. For 140 h flask culture, the dry cell weight and the extracellular lipase activity reached at 5.4 g/l and 57.9 U/l toward p-nitrophenyl palmitate, respectively. When we performed the fed-batch fermentation using a methanol feeding strategy for 110 h, the dry cell weight and the extracellular lipase activity were increased to 135.7 g/l and 11,900 U/l; the CalB protein concentration was 1.18 g/l of culture supernatant. The characteristics of CalB recovered from the P. pastoris culture were compared with the commercial form of CalB produced in Aspergillus oryzae. The kinetic constants and specific activity, the effects of activity and stability on temperature and pH, the glycosylation extent, the degree of immobilization on macroporous resin and the yield of esterification reaction between oleic acid and n-butanol were almost identical to each other. Therefore, we successfully proved that the Pichia-based expression system for CalB in this study was industrially promising compared with one of the most efficient production systems. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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.

Biotransformation of β‐hydroxypyruvate and glycolaldehyde to l‐erythrulose by Pichia pastoris strain GS115 overexpressing native transketolase

PubMed Central

Wei, Yu‐Chia; Braun‐Galleani, Stephanie; Henríquez, Maria José; Bandara, Sahan

2017-01-01

Transketolase is a proven biocatalytic tool for asymmetric carbon‐carbon bond formation, both as a purified enzyme and within bacterial whole‐cell biocatalysts. The performance of Pichia pastoris as a host for transketolase whole‐cell biocatalysis was investigated using a transketolase‐overexpressing strain to catalyze formation of l‐erythrulose from β‐hydroxypyruvic acid and glycolaldehyde substrates. Pichia pastoris transketolase coding sequence from the locus PAS_chr1‐4_0150 was subcloned downstream of the methanol‐inducible AOX1 promoter in a plasmid for transformation of strain GS115, generating strain TK150. Whole and disrupted TK150 cells from shake flasks achieved 62% and 65% conversion, respectively, under optimal pH and methanol induction conditions. In a 300 μL reaction, TK150 samples from a 1L fed‐batch fermentation achieved a maximum l‐erythrulose space time yield (STY) of 46.58 g L−1 h−1, specific activity of 155 U gCDW−1, product yield on substrate (Yp/s) of 0.52 mol mol−1 and product yield on catalyst (Yp/x) of 2.23g gCDW−1. We have successfully exploited the rapid growth and high biomass characteristics of Pichia pastoris in whole cell biocatalysis. At high cell density, the engineered TK150 Pichia pastoris strain tolerated high concentrations of substrate and product to achieve high STY of the chiral sugar l‐erythrulose. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:99–106, 2018 PMID:29086489

Use of synthetic genes for cloning, production and functional expression of the bacteriocins enterocin A and bacteriocin E 50-52 by Pichia pastoris and Kluyveromyces lactis.

PubMed

Jiménez, Juan J; Borrero, Juan; Gútiez, Loreto; Arbulu, Sara; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

2014-06-01

The use of synthetic genes may constitute a successful approach for the heterologous production and functional expression of bacterial antimicrobial peptides (bacteriocins) by recombinant yeasts. In this work, synthetic genes with adapted codon usage designed from the mature amino acid sequence of the bacteriocin enterocin A (EntA), produced by Enterococcus faecium T136, and the mature bacteriocin E 50-52 (BacE50-52), produced by E. faecium NRRL B-32746, were synthesized. The synthetic entA and bacE50-52 were cloned into the protein expression vectors pPICZαA and pKLAC2 for transformation of derived vectors into Pichia pastoris X-33 and Kluyveromyces lactis GG799, respectively. The recombinant vectors were linearized and transformed into competent cells selecting for P. pastoris X-33EAS (entA), P. pastoris X-33BE50-52S (bacE50-52), K. lactis GG799EAS (entA), and K. lactis GG799BE50-52S (bacE50-52). P. pastoris X-33EAS and K. lactis GG799EAS, but not P. pastoris X-33BE50-52S and K. lactis GG799BE50-52S, showed antimicrobial activity in their supernatants. However, purification of the supernatants of the producer yeasts permitted recovery of the bacteriocins EntA and BacE50-52. Both purified bacteriocins were active against Gram-positive bacteria such as Listeria monocytogenes but not against Gram-negative bacteria, including Campylobacter jejuni.

Biosynthesis of silver and zinc oxide nanoparticles using Pichia fermentans JA2 and their antimicrobial property

NASA Astrophysics Data System (ADS)

Chauhan, Ritika; Reddy, Arpita; Abraham, Jayanthi

2015-01-01

The development of eco-friendly alternative to chemical synthesis of metal nanoparticles is of great challenge among researchers. The present study aimed to investigate the biological synthesis, characterization, antimicrobial study and synergistic effect of silver and zinc oxide nanoparticles against clinical pathogens using Pichia fermentans JA2. The extracellular biosynthesis of silver and zinc oxide nanoparticles was investigated using Pichia fermentans JA2 isolated from spoiled fruit pulp bought in Vellore local market. The crystalline and stable metallic nanoparticles were characterized evolving several analytical techniques including UV-visible spectrophotometer, X-ray diffraction pattern analysis and FE-scanning electron microscope with EDX-analysis. The biosynthesized metallic nanoparticles were tested for their antimicrobial property against medically important Gram positive, Gram negative and fungal pathogenic microorganisms. Furthermore, the biosynthesized nanoparticles were also evaluated for their increased antimicrobial activities with various commercially available antibiotics against clinical pathogens. The biosynthesized silver nanoparticles inhibited most of the Gram negative clinical pathogens, whereas zinc oxide nanoparticles were able to inhibit only Pseudomonas aeruginosa. The combined effect of standard antibiotic disc and biosynthesized metallic nanoparticles enhanced the inhibitory effect against clinical pathogens. The biological synthesis of silver and zinc oxide nanoparticles is a novel and cost-effective approach over harmful chemical synthesis techniques. The metallic nanoparticles synthesized using Pichia fermentans JA2 possess potent inhibitory effect that offers valuable contribution to pharmaceutical associations.

Dietary supplementation with bovine lactoferrampin-lactoferricin produced by Pichia pastoris fed-batch fermentation affects intestinal microflora in weaned piglets.

PubMed

Tang, Xiang-Shan; Shao, Hua; Li, Tie-Jun; Tang, Zhi-Ru; Huang, Rui-Ling; Wang, Sheng-Ping; Kong, Xiang-Feng; Wu, Xin; Yin, Yu-Long

2012-10-01

This work is aimed at investigating the effects of recombinant bovine lactoferrampin-lactoferricin (LFA-LFC) instead of chlortetracycline on intestinal microflora in weaned piglets. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current study, recombinant LFA-LFC was produced via fed-batch fermentation in recombinant strain Pichia pastoris (KM71) XS10. Uniform design U6(6(4)) was used to optimize the fermentation conditions. The target peptide purified via cation-exchange and size-exclusion chromatography was added into the dietary of weaned piglets. After 21 days, the Lactobacilli, Bifidobacteria, and Enterobacteria in the chyme of the gut were quantified using real-time polymerase chain reaction. The results showed that approximately 82 mg of LFA-LFC was secreted into 1 L of medium under optimized conditions. Moreover, purified peptide showed strong antimicrobial activities against all the tested microorganisms. Compared with the control group, the LFA-LFC group increased the amount of Lactobacilli and Bifidobacteria (P<0.05) in the chyme of the stomach, duodenum, jejunum, ileum, colon, and caecum. These results show that dietary supplementation with LFA-LFC can affect intestinal microflora in weaned piglets.

Characterization of deamidation at Asn138 in L-chain of recombinant humanized Fab expressed from Pichia pastoris.

PubMed

Ohkuri, Takatoshi; Murase, Eri; Sun, Shu-Lan; Sugitani, Jun; Ueda, Tadashi

2013-10-01

A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ∼30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.

Oral Mycobiome Analysis of HIV-Infected Patients: Identification of Pichia as an Antagonist of Opportunistic Fungi

PubMed Central

Mukherjee, Pranab K.; Chandra, Jyotsna; Retuerto, Mauricio; Sikaroodi, Masoumeh; Brown, Robert E.; Jurevic, Richard; Salata, Robert A.; Lederman, Michael M.; Gillevet, Patrick M.; Ghannoum, Mahmoud A.

2014-01-01

Oral microbiota contribute to health and disease, and their disruption may influence the course of oral diseases. Here, we used pyrosequencing to characterize the oral bacteriome and mycobiome of 12 HIV-infected patients and matched 12 uninfected controls. The number of bacterial and fungal genera in individuals ranged between 8–14 and 1–9, among uninfected and HIV-infected participants, respectively. The core oral bacteriome (COB) comprised 14 genera, of which 13 were common between the two groups. In contrast, the core oral mycobiome (COM) differed between HIV-infected and uninfected individuals, with Candida being the predominant fungus in both groups. Among Candida species, C. albicans was the most common (58% in uninfected and 83% in HIV-infected participants). Furthermore, 15 and 12 bacteria-fungi pairs were correlated significantly within uninfected and HIV-infected groups, respectively. Increase in Candida colonization was associated with a concomitant decrease in the abundance of Pichia, suggesting antagonism. We found that Pichia spent medium (PSM) inhibited growth of Candida, Aspergillus and Fusarium. Moreover, Pichia cells and PSM inhibited Candida biofilms (P = .002 and .02, respectively, compared to untreated controls). The mechanism by which Pichia inhibited Candida involved nutrient limitation, and modulation of growth and virulence factors. Finally, in an experimental murine model of oral candidiasis, we demonstrated that mice treated with PSM exhibited significantly lower infection score (P = .011) and fungal burden (P = .04) compared to untreated mice. Moreover, tongues of PSM-treated mice had few hyphae and intact epithelium, while vehicle- and nystatin-treated mice exhibited extensive fungal invasion of tissue with epithelial disruption. These results showed that PSM was efficacious against oral candidiasis in vitro and in vivo. The inhibitory activity of PSM was associated with secretory protein/s. Our findings provide the

Isolation, Identification and Characterization of Yeasts from Fermented Goat Milk of the Yaghnob Valley in Tajikistan

PubMed Central

Qvirist, Linnea A.; De Filippo, Carlotta; Strati, Francesco; Stefanini, Irene; Sordo, Maddalena; Andlid, Thomas; Felis, Giovanna E.; Mattarelli, Paola; Cavalieri, Duccio

2016-01-01

The geographically isolated region of the Yaghnob Valley, Tajikistan, has allowed its inhabitants to maintain a unique culture and lifestyle. Their fermented goat milk constitutes one of the staple foods for the Yaghnob population, and is produced by backslopping, i.e., using the previous fermentation batch to inoculate the new one. This study addresses the yeast composition of the fermented milk, assessing genotypic, and phenotypic properties. The 52 isolates included in this study revealed small species diversity, belonging to Kluyveromyces marxianus, Pichia fermentans, Saccharomyces cerevisiae, and one Kazachstania unispora. The K. marxianus strains showed two different genotypes, one of which never described previously. The two genetically different groups also differed significantly in several phenotypic characteristics, such as tolerance toward high temperatures, low pH, and presence of acid. Microsatellite analysis of the S. cerevisiae strains from this study, compared to 350 previously described strains, attributed the Yaghnobi S. cerevisiae to two different ancestry origins, both distinct from the wine and beer strains, and similar to strains isolated from human and insects feces, suggesting a peculiar origin of these strains, and the existence of a gut reservoir for S. cerevisiae. Our work constitutes a foundation for strain selection for future applications as starter cultures in food fermentations. This work is the first ever on yeast diversity from fermented milk of the previously unexplored area of the Yaghnob Valley. PMID:27857705

In Vitro Activities of Terbinafine against Cutaneous Isolates of Candida albicans and Other Pathogenic Yeasts

PubMed Central

Ryder, Neil S.; Wagner, Sonja; Leitner, Ingrid

1998-01-01

Terbinafine is active in vitro against a wide range of pathogenic fungi, including dermatophytes, molds, dimorphic fungi, and some yeasts, but earlier studies indicated that the drug had little activity against Candida albicans. In contrast, clinical studies have shown topical and oral terbinafine to be active in cutaneous candidiasis and Candida nail infections. In order to define the anti-Candida activity of terbinafine, we tested the drug against 350 fresh clinical isolates and additional strains by using a broth dilution assay standardized according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) M27-A assay. Terbinafine was found to have an MIC of 1 μg/ml for reference C. albicans strains. For 259 clinical isolates, the MIC at which 50% of the isolates are inhibited (MIC50) of terbinafine was 1 μg/ml (fluconazole, 0.5 μg/ml), and the MIC90 was 4 μg/ml (fluconazole, 1 μg/ml). Terbinafine was highly active against Candida parapsilosis (MIC90, 0.125 μg/ml) and showed potentially interesting activity against isolates of Candida dubliniensis, Candida guilliermondii, Candida humicola, and Candida lusitaniae. It was not active against the Candida glabrata, Candida krusei, and Candida tropicalis isolates in this assay. Cryptococcus laurentii and Cryptococcus neoformans were highly susceptible to terbinafine, with MICs of 0.06 to 0.25 μg/ml. The NCCLS macrodilution assay provides reproducible in vitro data for terbinafine against Candida and other yeasts. The MICs for C. albicans and C. parapsilosis are compatible with the known clinical efficacy of terbinafine in cutaneous infections, while the clinical relevance of its activities against the other species has yet to be determined. PMID:9593126

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

Validation of a predictive model for the growth of chalk yeasts on bread.

PubMed

Burgain, Anaïs; Bensoussan, Maurice; Dantigny, Philippe

2015-07-02

The present study focused on the effects of temperature, T, and water activity, aw, on the growth of Hyphopichia burtonii, Pichia anomala, and Saccharomycopsis fibuligera on Sabouraud Agar Medium. Cardinal values were estimated by means of cardinal models with inflection. All the yeasts were xerophilic, and they exhibited growth at 0.85 aw. The combined effects of T, aw, and pH on the growth of these species were described by the gamma-concept and validated on bread in the range of 15-25 °C, 0.91-0.97 aw, and pH 4.6-6.8. The optimum growth rates on bread were 2.88, 0.259, and 1.06 mm/day for H. burtonii, P. anomala, and S. fibuligera, respectively. The optimal growth rate of S. fibuligera on bread was about 2 fold that obtained on Sabouraud. Due to reproduction by budding, P. anomala exhibited low growth on Sabouraud and bread. However, this species is of major concern in the baker's industry because of the production of ethyl acetate in bread. Copyright © 2015 Elsevier B.V. All rights reserved.

The effect of initial cell concentration on xylose fermentation by Pichia stipitis

Treesearch

Frank K. Agbogbo; Guillermo Coward-Kelly; Mads Torry-Smith; Kevin Wenger; Thomas W. Jeffries

2007-01-01

Xylose was fermented using Pichia stipitis CBS 6054 at different initial cell concentrations. A high initial cell concentration increased the rate of xylose utilization, ethanol formation, and the ethanol yield. The highest ethanol concentration of 41.0 g/L and a yield of 0.38 g/g was obtained using an initial cell concentration of 6.5 g/L. Even though more xylitol was...

Xylitol production from DEO hydrolysate of corn stover by Pichia stipitis YS-30

Treesearch

Rita C.L.B. Rodrigues; William R. Kenealy; Thomas W. Jeffries

2011-01-01

Corn stover that had been treated with vapor-phase diethyl oxalate released a mixture of mono-and oligosaccharides consisting mainly of xylose and glucose. Following overliming and neutralization, a D-xylulokinase mutant of Pichia stipitis, FPL-YS30 (xyl3 -Ä1), converted the stover hydrolysate into xylitol. This research examined the effects of phosphoric or gluconic...

Molecular characterization of the Hansenula polymorpha FLD1 gene encoding formaldehyde dehydrogenase.

Treesearch

Richard J. Baerends; Grietje J. Sulter; Thomas W. Jeffries; James M. Cregg; Marten Veenhuis

2002-01-01

Glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required for the catabolism of methanol as a carbon source and certain primary amines, such as methylamine as nitrogen sources in methylotrophic yeasts. Here we describe the molecular characterization of the FLD1 gene from the yeast Hansenula polymorpha. Unlike the recently described Pichia pastoris...

Cytosine DNA Methylation Is Found in Drosophila melanogaster but Absent in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Other Yeast Species

PubMed Central

2014-01-01

The methylation of cytosine to 5-methylcytosine (5-meC) is an important epigenetic DNA modification in many bacteria, plants, and mammals, but its relevance for important model organisms, including Caenorhabditis elegans and Drosophila melanogaster, is still equivocal. By reporting the presence of 5-meC in a broad variety of wild, laboratory, and industrial yeasts, a recent study also challenged the dogma about the absence of DNA methylation in yeast species. We would like to bring to attention that the protocol used for gas chromatography/mass spectrometry involved hydrolysis of the DNA preparations. As this process separates cytosine and 5-meC from the sugar phosphate backbone, this method is unable to distinguish DNA- from RNA-derived 5-meC. We employed an alternative LC–MS/MS protocol where by targeting 5-methyldeoxycytidine moieties after enzymatic digestion, only 5-meC specifically derived from DNA is quantified. This technique unambiguously identified cytosine DNA methylation in Arabidopsis thaliana (14.0% of cytosines methylated), Mus musculus (7.6%), and Escherichia coli (2.3%). Despite achieving a detection limit at 250 attomoles (corresponding to <0.00002 methylated cytosines per nonmethylated cytosine), we could not confirm any cytosine DNA methylation in laboratory and industrial strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Saccharomyces boulardii, Saccharomyces paradoxus, or Pichia pastoris. The protocol however unequivocally confirmed DNA methylation in adult Drosophila melanogaster at a value (0.034%) that is up to 2 orders of magnitude below the detection limit of bisulphite sequencing. Thus, 5-meC is a rare DNA modification in drosophila but absent in yeast. PMID:24640988

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.

Altered Fermentation Performances, Growth, and Metabolic Footprints Reveal Competition for Nutrients between Yeast Species Inoculated in Synthetic Grape Juice-Like Medium

PubMed Central

Rollero, Stephanie; Bloem, Audrey; Ortiz-Julien, Anne; Camarasa, Carole; Divol, Benoit

2018-01-01

The sequential inoculation of non-Saccharomyces yeasts and Saccharomyces cerevisiae in grape juice is becoming an increasingly popular practice to diversify wine styles and/or to obtain more complex wines with a peculiar microbial footprint. One of the main interactions is competition for nutrients, especially nitrogen sources, that directly impacts not only fermentation performance but also the production of aroma compounds. In order to better understand the interactions taking place between non-Saccharomyces yeasts and S. cerevisiae during alcoholic fermentation, sequential inoculations of three yeast species (Pichia burtonii, Kluyveromyces marxianus, Zygoascus meyerae) with S. cerevisiae were performed individually in a synthetic medium. Different species-dependent interactions were evidenced. Indeed, the three sequential inoculations resulted in three different behaviors in terms of growth. P. burtonii and Z. meyerae declined after the inoculation of S. cerevisiae which promptly outcompeted the other two species. However, while the presence of P. burtonii did not impact the fermentation kinetics of S. cerevisiae, that of Z. meyerae rendered the overall kinetics very slow and with no clear exponential phase. K. marxianus and S. cerevisiae both declined and became undetectable before fermentation completion. The results also demonstrated that yeasts differed in their preference for nitrogen sources. Unlike Z. meyerae and P. burtonii, K. marxianus appeared to be a competitor for S. cerevisiae (as evidenced by the uptake of ammonium and amino acids), thereby explaining the resulting stuck fermentation. Nevertheless, the results suggested that competition for other nutrients (probably vitamins) occurred during the sequential inoculation of Z. meyerae with S. cerevisiae. The metabolic footprint of the non-Saccharomyces yeasts determined after 48 h of fermentation remained until the end of fermentation and combined with that of S. cerevisiae. For instance

Altered Fermentation Performances, Growth, and Metabolic Footprints Reveal Competition for Nutrients between Yeast Species Inoculated in Synthetic Grape Juice-Like Medium.

PubMed

Rollero, Stephanie; Bloem, Audrey; Ortiz-Julien, Anne; Camarasa, Carole; Divol, Benoit

2018-01-01

The sequential inoculation of non- Saccharomyces yeasts and Saccharomyces cerevisiae in grape juice is becoming an increasingly popular practice to diversify wine styles and/or to obtain more complex wines with a peculiar microbial footprint. One of the main interactions is competition for nutrients, especially nitrogen sources, that directly impacts not only fermentation performance but also the production of aroma compounds. In order to better understand the interactions taking place between non-Saccharomyces yeasts and S. cerevisiae during alcoholic fermentation, sequential inoculations of three yeast species ( Pichia burtonii, Kluyveromyces marxianus, Zygoascus meyerae ) with S. cerevisiae were performed individually in a synthetic medium. Different species-dependent interactions were evidenced. Indeed, the three sequential inoculations resulted in three different behaviors in terms of growth. P. burtonii and Z. meyerae declined after the inoculation of S. cerevisiae which promptly outcompeted the other two species. However, while the presence of P. burtonii did not impact the fermentation kinetics of S. cerevisiae , that of Z. meyerae rendered the overall kinetics very slow and with no clear exponential phase. K. marxianus and S. cerevisiae both declined and became undetectable before fermentation completion. The results also demonstrated that yeasts differed in their preference for nitrogen sources. Unlike Z. meyerae and P. burtonii, K. marxianus appeared to be a competitor for S. cerevisiae (as evidenced by the uptake of ammonium and amino acids), thereby explaining the resulting stuck fermentation. Nevertheless, the results suggested that competition for other nutrients (probably vitamins) occurred during the sequential inoculation of Z. meyerae with S. cerevisiae . The metabolic footprint of the non- Saccharomyces yeasts determined after 48 h of fermentation remained until the end of fermentation and combined with that of S. cerevisiae . For instance

FT-IR spectroscopy: A powerful tool for studying the inter- and intraspecific biodiversity of cultivable non-Saccharomyces yeasts isolated from grape must.

PubMed

Grangeteau, Cédric; Gerhards, Daniel; Terrat, Sebastien; Dequiedt, Samuel; Alexandre, Hervé; Guilloux-Benatier, Michèle; von Wallbrunn, Christian; Rousseaux, Sandrine

2016-02-01

The efficiency of the FT-IR technique for studying the inter- and intra biodiversity of cultivable non-Saccharomyces yeasts (NS) present in different must samples was examined. In first, the capacity of the technique FT-IR to study the global diversity of a given sample was compared to the pyrosequencing method, used as a reference technique. Seven different genera (Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Issatchenkia, Metschnikowia and Pichia) were identified by FT-IR and also by pyrosequencing. Thirty-eight other genera were identified by pyrosequencing, but together they represented less than 6% of the average total population of 6 musts. Among the species identified, some of them present organoleptic potentials in winemaking, particularly Starmerella bacillaris (synonym Candidazemplinina). So in a second time, we evaluated the capacity of the FT-IR technique to discriminate the isolates of this species because few techniques were able to study intraspecific NS yeast biodiversity. The results obtained were validated by using a classic method as ITS sequencing. Biodiversity at strain level was high: 19 different strains were identified from 58 isolates. So, FT-IR spectroscopy seems to be an accurate and reliable method for identifying major genera present in the musts. The two biggest advantages of the FT-IR are the capacity to characterize intraspecific biodiversity of non-Saccharomyces yeasts and the possibility to discriminate a lot of strains. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of Metabolic Pathways Expressed by Pichia anomala Kh6 in the Presence of the Pathogen Botrytis cinerea on Apple: New Possible Targets for Biocontrol Improvement

PubMed Central

Kwasiborski, Anthony; Bajji, Mohammed; Renaut, Jenny; Delaplace, Pierre; Jijakli, M. Haissam

2014-01-01

Yeast Pichia anomala strain Kh6 Kurtzman (Saccharomycetales: Endomycetaceae) exhibits biological control properties that provide an alternative to the chemical fungicides currently used by fruit or vegetable producers against main post-harvest pathogens, such as Botrytis cinerea (Helotiales: Sclerotiniaceae). Using an in situ model that takes into account interactions between organisms and a proteomic approach, we aimed to identify P. anomala metabolic pathways influenced by the presence of B. cinerea. A total of 105 and 60 P. anomala proteins were differentially represented in the exponential and stationary growth phases, respectively. In the exponential phase and in the presence of B. cinerea, the pentose phosphate pathway seems to be enhanced and would provide P. anomala with the needed nucleic acids and energy for the wound colonisation. In the stationary phase, P. anomala would use alcoholic fermentation both in the absence and presence of the pathogen. These results would suggest that the competitive colonisation of apple wounds could be implicated in the mode of action of P. anomala against B. cinerea. PMID:24614090

Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

PubMed Central

2011-01-01

Background Various protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies. Results The ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells. Conclusion Compared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all

The combined rapid detection and species-level identification of yeasts in simulated blood culture using a colorimetric sensor array

PubMed Central

Lim, Sung H.; Wilson, Deborah A.; SalasVargas, Ana Victoria; Churi, Yair S.; Rhodes, Paul A.; Mazzone, Peter J.; Procop, Gary W.

2017-01-01

Background A colorimetric sensor array (CSA) has been demonstrated to rapidly detect and identify bacteria growing in blood cultures by obtaining a species-specific “fingerprint” of the volatile organic compounds (VOCs) produced during growth. This capability has been demonstrated in prokaryotes, but has not been reported for eukaryotic cells growing in culture. The purpose of this study was to explore if a disposable CSA could differentially identify 7 species of pathogenic yeasts growing in blood culture. Methods Culture trials of whole blood inoculated with a panel of clinically important pathogenic yeasts at four different microorganism loads were performed. Cultures were done in both standard BacT/Alert and CSA-embedded bottles, after adding 10 mL of spiked blood to each bottle. Color changes in the CSA were captured as images by an optical scanner at defined time intervals. The captured images were analyzed to identify the yeast species. Time to detection by the CSA was compared to that in the BacT/Alert system. Results One hundred sixty-two yeast culture trials were performed, including strains of several species of Candida (Ca. albicans, Ca. glabrata, Ca. parapsilosis, and Ca. tropicalis), Clavispora (synonym Candida) lusitaniae, Pichia kudriavzevii (synonym Candida krusei) and Cryptococcus neoformans, at loads of 8.2 × 105, 8.3 × 103, 8.5 × 101, and 1.7 CFU/mL. In addition, 8 negative trials (no yeast) were conducted. All negative trials were correctly identified as negative, and all positive trials were detected. Colorimetric responses were species-specific and did not vary by inoculum load over the 500000-fold range of loads tested, allowing for accurate species-level identification. The mean sensitivity for species-level identification by CSA was 74% at detection, and increased with time, reaching almost 95% at 4 hours after detection. At an inoculum load of 1.7 CFU/mL, mean time to detection with the CSA was 6.8 hours (17%) less than with the

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.

Effect of trehalose on the biocontrol efficacy of Pichia caribbica against post-harvest grey mould and blue mould decay of apples.

PubMed

Zhao, Lina; Zhang, Hongyin; Lin, Hetong; Zhang, Xiaoyun; Ren, Xiaofeng

2013-08-01

The influence of adding trehalose to the culture medium on the efficacy of Pichia caribbica (JSU-1) in controlling post-harvest grey mould and blue mould decay of apples and the possible mechanisms involved were investigated. The antagonistic activity of P. caribbica harvested from nutrient yeast dextrose broth (NYDB) amended with trehalose at 5 mg mL(-1) to post-harvest grey mould and blue mould decay was improved greatly compared with that without trehalose. The intracellular trehalose content of P. caribbica harvested from NYDB amended with trehalose at 5 mg mL(-1) was higher than that of P. caribbica harvested from NYDB. The population of P. caribbica could rapidly colonise and grow in apple wounds. Moreover, the application of P. caribbica cultivated in the culture medium of the trehalose supplement (5 mg mL(-1) ) induced higher polyphenoloxidase (PPO) and peroxidase (POD) activity of apples compared with that cultivated by P. caribbica in the NYDB. SEM results suggested that P. caribbica harvested from NYDB amended with trehalose at 5 mg mL(-1) showed stronger inhibition efficacy to Botrytis cinerea than P. caribbica harvested from NYDB. The antagonistic activity of P. caribbica can be enhanced by adding trehalose to the medium. © 2012 Society of Chemical Industry.

Sh ble and Cre adapted for functional genomics and metabolic engineering of Pichia stipitis

Treesearch

Jose M. Laplaza; Beatriz Rivas Torres; Yong-Su Jin; Thomas W. Jeffries

2006-01-01

Pichia stipitis is widely studied for its capacity to ferment d-xylose to ethanol. Strain improvement has been facilitated by recent completion of the P. stipitis genome. P. stipitis uses CUG to code for serine rather than leucine, as is the case for the universal genetic code thereby limiting the availability of heterologous drug resistance markers for transformation...

Efficient chemical and enzymatic saccharification of the lignocellulosic residue from Agave tequilana bagasse to produce ethanol by Pichia caribbica.

PubMed

Saucedo-Luna, Jaime; Castro-Montoya, Agustin Jaime; Martinez-Pacheco, Mauro Manuel; Sosa-Aguirre, Carlos Ruben; Campos-Garcia, Jesus

2011-06-01

Bagasse of Agave tequilana (BAT) is the residual lignocellulosic waste that remains from tequila production. In this study we characterized the chemical composition of BAT, which was further saccharified and fermented to produce ethanol. BAT was constituted by cellulose (42%), hemicellulose (20%), lignin (15%), and other (23%). Saccharification of BAT was carried out at 147 °C with 2% sulfuric acid for 15 min, yielding 25.8 g/l of fermentable sugars, corresponding to 36.1% of saccharificable material (cellulose and hemicellulose contents, w/w). The remaining lignocellulosic material was further hydrolyzed by commercial enzymes, ~8.2% of BAT load was incubated for 72 h at 40 °C rendering 41 g/l of fermentable sugars corresponding to 73.6% of the saccharificable material (w/w). Mathematic surface response analysis of the acid and enzymatic BAT hydrolysis was used for process optimization. The results showed a satisfactory correlation (R (2) = 0.90) between the obtained and predicted responses. The native yeast Pichia caribbica UM-5 was used to ferment sugar liquors from both acid and enzymatic hydrolysis to ethanol yielding 50 and 87%, respectively. The final optimized process generated 8.99 g ethanol/50 g of BAT, corresponding to an overall 56.75% of theoretical ethanol (w/w). Thus, BAT may be employed as a lignocellulosic raw material for bioethanol production and can contribute to BAT residue elimination from environment.

Optimization of a protective medium for freeze-dried Pichia membranifaciens and application of this biocontrol agent on citrus fruit.

PubMed

Niu, X; Deng, L; Zhou, Y; Wang, W; Yao, S; Zeng, K

2016-07-01

To optimize a protective medium for freeze-dried Pichia membranifaciens and to evaluate biocontrol efficacies of agents against blue and green mould and anthracnose in citrus fruit. Based on the screening assays of saccharides and antioxidants, response surface methodology was used to optimize sucrose, sodium glutamate and skim milk to improve viability of freeze-dried Pi. membranifaciens. Biocontrol assays were conducted between fresh and freeze-dried Pi. membranifaciens against Penicillium italicum, Penicillium digitatum and Colletotrichum gloeosporioides in citrus fruit. Solving the regression equation indicated that the optimal protective medium was 6·06% (w/v) sucrose combined with 3·40% (w/v) sodium glutamate and 5·43% (w/v) skim milk. Pi. membranifaciens freeze-dried in the optimal protective medium showed 76·80% viability, and retained biocontrol efficacy against Pe. italicum, Pe. digitatum and Co. gloeosporioides in citrus fruit. The optimal protective medium showed more effective protective properties than each of the three protectants used alone. The viability of freeze-dried Pi. membranifaciens finally reached 76·80%. Meanwhile, the biocontrol efficacies showed no significant difference between fresh and freeze-dried yeast against Pe. italicum, Pe. digitatum and Co. gloeosporioides in citrus fruit. The results showed the potential value of Pi. membranifaciens CICC 32259 for commercialization. © 2016 The Society for Applied Microbiology.

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.

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 ...

The requirement of sterol glucoside for pexophagy in yeast is dependent on the species and nature of peroxisome inducers.

PubMed

Nazarko, Taras Y; Polupanov, Andriy S; Manjithaya, Ravi R; Subramani, Suresh; Sibirny, Andriy A

2007-01-01

Sterol glucosyltransferase, Ugt51/Atg26, is essential for both micropexophagy and macropexophagy of methanol-induced peroxisomes in Pichia pastoris. However, the role of this protein in pexophagy in other yeast remained unclear. We show that oleate- and amine-induced peroxisomes in Yarrowia lipolytica are degraded by Atg26-independent macropexophagy. Surprisingly, Atg26 was also not essential for macropexophagy of oleate- and amine-induced peroxisomes in P. pastoris, suggesting that the function of sterol glucoside (SG) in pexophagy is both species and peroxisome inducer specific. However, the rates of degradation of oleate- and amine-induced peroxisomes in P. pastoris were reduced in the absence of SG, indicating that P. pastoris specifically uses sterol conversion by Atg26 to enhance selective degradation of peroxisomes. However, methanol-induced peroxisomes apparently have lost the redundant ability to be degraded without SG. We also show that the P. pastoris Vac8 armadillo repeat protein is not essential for macropexophagy of methanol-, oleate-, or amine-induced peroxisomes, which makes PpVac8 the first known protein required for the micropexophagy, but not for the macropexophagy, machinery. The uniqueness of Atg26 and Vac8 functions under different pexophagy conditions demonstrates that not only pexophagy inducers, such as glucose or ethanol, but also the inducers of peroxisomes, such as methanol, oleate, or primary amines, determine the requirements for subsequent pexophagy in yeast.

[Pichia pastoris as an expression system for recombinant protein production].

PubMed

Ciarkowska, Anna; Jakubowska, Anna

2013-01-01

Pichia pastoris has become increasingly popular as a host for recombinant protein production in recent years. P. pastoris is more cost effective and allows achieving higher expression levels than insect and mammalian cells. It also offers some significant advantages over E. coli expression systems, such as avoiding problems with proper protein folding. Also, P. pastoris as an eukaryotic organism can carry out posttranslational modifications of produced proteins. Additionally, P. pastoris can produce high levels of recombinant proteins in extracellular medium which simplifies protein purification. Having many advantages over other expression systems makes P. pastoris an organism of choice for industrial protein production.

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.

Bioconversion of giant reed (Arundo donax L.) hemicellulose hydrolysate to ethanol by Scheffersomyces stipitis CBS6054

Treesearch

Danilo Scordia; Salvatore L. Cosentino; Jae-Won Lee; Thomas W. Jeffries

2012-01-01

The objective of this study was to evaluate the production of ethanol by Scheffersomyces (Pichia) stipitis CBS6054, a native xylose fermenting yeast, from sugars contained in the giant reed (Arundo donax L.) hemicellulosic hydrolysate.

Expression of recombinant Arabian camel lactoferricin-related peptide in Pichia pastoris and its antimicrobial identification.

PubMed

Chahardooli, Mahmood; Niazi, Ali; Aram, Farzaneh; Sohrabi, Seyyed Mohsen

2016-01-30

Lactoferricin (LFcin) is a strong cationic peptide released from the N-terminus of lactoferrin by gastric pepsin digestion. LFcin has some important properties, including high antimicrobial activity. To date, lactoferricins have been isolated and characterised from various animal species, but not from camel. The aim of this study was to characterise and express recombinant camel lactoferricin (LFcinC) in Pichia pastoris and investigate its antimicrobial activity. After methanol induction, LFcinC was expressed and secreted into a culture broth medium and the results determined by concentrated supernatant culture medium showed high antimicrobial activity against the following microorganisms: Escherichia coli PTCC 1330 (ATCC 8739), Staphylococcus aureus PTCC 1112 (ATCC 6538), Pseudomonas aeruginosa PTCC 1074 (ATCC 9027), Bacillus subtilis PTCC 1023 (ATCC 6633), and Candida albicans PTCC 5027 (ATCC 10231). Thermal stability was clarified with antibacterial activity against Escherichia coli PTCC 1330 (ATCC 8739). Results confirmed that camel lactoferricin had suitable antimicrobial activity and its production by Pichia pastoris can be used for recombinant production. © 2015 Society of Chemical Industry.

Expression of plectasin in Pichia pastoris and its characterization as a new antimicrobial peptide against Staphyloccocus and Streptococcus.

PubMed

Zhang, Jun; Yang, Yalin; Teng, Da; Tian, Zigang; Wang, Shaoran; Wang, Jianhua

2011-08-01

Recombinant plectasin, the first fungus defensin, was expressed in Pichia pastoris and purified, and its physical, chemical and antimicrobial characteristics were studied. Following a 120 h induction of recombinant yeast, the amount of total secreted protein reached 748.63 μg/ml. The percentage of recombinant plectasin was estimated to be 71.79% of the total protein. After purification with a Sephadex G-25 column and RP-HPLC, the identity of plectasin was verified by MALDI-TOF MS. Plectasin exhibited strong antimicrobial activity against the Gram-positive bacteria Staphyloccocusaureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Streptococcus suis. At a concentration of 2560 μg/ml, this peptide showed approximately equal activity against S. aureus, S. epidermidis, S. suis, and S. pneumoniae, when compared to 320 μg/ml vancomycin, 640 μg/ml penicillin, 320 μg/ml vancomycin and 160 μg/ml vancomycin, respectively. In addition, plectasin showed anti-S. aureus activity over a wide pH range of 2.0 and 10.0, a high thermal stability at 100 °C for 1h and remarkable resistance to papain and pepsin. The expression and characterization of recombinant plectasin in P. pastoris has potential to treat Streptococcus and Staphyloccocus infections when most traditional antibiotics show no effect on them. Our results indicate that plectasin can be produced in large quantities, and that it has pharmaceutical importance for the prevention and clinical treatment of Staphyloccocus and Streptococcus infections. Copyright © 2011 Elsevier Inc. All rights reserved.

A new type of gene-disruption cassette with a rescue gene for Pichia pastoris.

PubMed

Shibui, Tatsuro; Hara, Hiroyoshi

2017-09-01

Pichia pastoris has been used for the production of many recombinant proteins, and many useful mutant strains have been created. However, the efficiency of mutant isolation by gene-targeting is usually low and the procedure is difficult for those inexperienced in yeast genetics. In order to overcome these issues, we developed a new gene-disruption system with a rescue gene using an inducible Cre/mutant-loxP system. With only short homology regions, the gene-disruption cassette of the system replaces its target-gene locus containing a mutation with a compensatory rescue gene. As the cassette contains the AOX1 promoter-driven Cre gene, when targeted strains are grown on media containing methanol, the DNA fragment, i.e., the marker, rescue and Cre genes, between the mutant-loxP sequences in the cassette is excised, leaving only the remaining mutant-loxP sequence in the genome, and consequently a target gene-disrupted mutant can be isolated. The system was initially validated on ADE2 gene disruption, where the disruption can easily be detected by color-change of the colonies. Then, the system was applied for knocking-out URA3 and OCH1 genes, reported to be difficult to accomplish by conventional gene-targeting methods. All three gene-disruption cassettes with their rescue genes replaced their target genes, and the Cre/mutant-loxP system worked well to successfully isolate their knock-out mutants. This study identified a new gene-disruption system that could be used to effectively and strategically knock out genes of interest, especially whose deletion is detrimental to growth, without using special strains, e.g., deficient in nonhomologous end-joining, in P. pastoris. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1201-1208, 2017. © 2017 American Institute of Chemical Engineers.

Overall Key Performance Indicator to Optimizing Operation of High-Pressure Homogenizers for a Reliable Quantification of Intracellular Components in Pichia pastoris.

PubMed

Garcia-Ortega, Xavier; Reyes, Cecilia; Montesinos, José Luis; Valero, Francisco

2015-01-01

The most commonly used cell disruption procedures may present lack of reproducibility, which introduces significant errors in the quantification of intracellular components. In this work, an approach consisting in the definition of an overall key performance indicator (KPI) was implemented for a lab scale high-pressure homogenizer (HPH) in order to determine the disruption settings that allow the reliable quantification of a wide sort of intracellular components. This innovative KPI was based on the combination of three independent reporting indicators: decrease of absorbance, release of total protein, and release of alkaline phosphatase activity. The yeast Pichia pastoris growing on methanol was selected as model microorganism due to it presents an important widening of the cell wall needing more severe methods and operating conditions than Escherichia coli and Saccharomyces cerevisiae. From the outcome of the reporting indicators, the cell disruption efficiency achieved using HPH was about fourfold higher than other lab standard cell disruption methodologies, such bead milling cell permeabilization. This approach was also applied to a pilot plant scale HPH validating the methodology in a scale-up of the disruption process. This innovative non-complex approach developed to evaluate the efficacy of a disruption procedure or equipment can be easily applied to optimize the most common disruption processes, in order to reach not only reliable quantification but also recovery of intracellular components from cell factories of interest.

Overall Key Performance Indicator to Optimizing Operation of High-Pressure Homogenizers for a Reliable Quantification of Intracellular Components in Pichia pastoris

PubMed Central

Garcia-Ortega, Xavier; Reyes, Cecilia; Montesinos, José Luis; Valero, Francisco

2015-01-01

The most commonly used cell disruption procedures may present lack of reproducibility, which introduces significant errors in the quantification of intracellular components. In this work, an approach consisting in the definition of an overall key performance indicator (KPI) was implemented for a lab scale high-pressure homogenizer (HPH) in order to determine the disruption settings that allow the reliable quantification of a wide sort of intracellular components. This innovative KPI was based on the combination of three independent reporting indicators: decrease of absorbance, release of total protein, and release of alkaline phosphatase activity. The yeast Pichia pastoris growing on methanol was selected as model microorganism due to it presents an important widening of the cell wall needing more severe methods and operating conditions than Escherichia coli and Saccharomyces cerevisiae. From the outcome of the reporting indicators, the cell disruption efficiency achieved using HPH was about fourfold higher than other lab standard cell disruption methodologies, such bead milling cell permeabilization. This approach was also applied to a pilot plant scale HPH validating the methodology in a scale-up of the disruption process. This innovative non-complex approach developed to evaluate the efficacy of a disruption procedure or equipment can be easily applied to optimize the most common disruption processes, in order to reach not only reliable quantification but also recovery of intracellular components from cell factories of interest. PMID:26284241

Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars.

PubMed

Gimeno-Pérez, María; Linde, Dolores; Fernández-Arrojo, Lucía; Plou, Francisco J; Fernández-Lobato, María

2015-04-01

The β-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous is the largest microbial enzyme producing neo-fructooligosaccharides (neo-FOS) known to date. It mainly synthesizes neokestose and neonystose, oligosaccharides with potentially improved prebiotic properties. The Xd-INV gene comprises an open reading frame of 1995 bp, which encodes a 665-amino acid protein. Initial N-terminal sequencing of Xd-INV pointed to a majority extracellular protein of 595 amino acids lacking the first 70 residues (potential signal peptide). Functionality of the last 1785 bp of Xd-INV gene was previously proved in Saccharomyces cerevisiae but only weak β-fructofuranosidase activity was quantified. In this study, different strategies to improve this enzyme level in a heterologous system have been used. Curiously, best results were obtained by increasing the protein N-terminus sequence in 39 amino acids, protein of 634 residues. The higher β-fructofuranosidase activity detected in this study, about 15 U/mL, was obtained using Pichia pastoris and represents an improvement of about 1500 times the level previously obtained in a heterologous organism and doubles the best level of activity obtained by the natural producer. Heterologously expressed protein was purified and characterized biochemically and kinetically. Except by its glycosylation degree (10 % lower) and thermal stability (4-5 °C lower in the 60-85 °C range), the properties of the heterologous enzyme, including ability to produce neo-FOS, remained unchanged. Interestingly, besides the neo-FOS referred before blastose was also detected (8-22 g/L) in the reaction mixtures, making Xd-INV the first yeast enzyme producing this non-conventional disaccharide reported to date.

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.

Enzymatic Synthesis of Structured Lipids using a Novel Cold-Active Lipase from Pichia lynferdii NRRL Y-7723

USDA-ARS?s Scientific Manuscript database

Structured lipids (SL) were synthesized by the acidolysis of borage oil with caprylic acid using lipases. Six commercial lipases from different sources and a novel lipase from Pichia lynferdii NRRL Y-7723 were screened for their acidolysis activities and Lipozyme RM IM and NRRL Y-7723 lipase were s...

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

Yeasts from Scarlet ibises (Eudocimus ruber): A focus on monitoring the antifungal susceptibility of Candida famata and closely related species.

PubMed

Brilhante, Raimunda Sâmia Nogueira; Silva, Aline Lobão da; Monteiro, Frederico Ozanan Barros; Guedes, Glaucia Morgana de Melo; Sales, Jamille Alencar; Oliveira, Jonathas Sales de; Maia Junior, José Erisvaldo; Miranda, Stefânia Araújo; Sidrim, José Júlio Costa; Alencar, Lucas Pereira de; Castelo-Branco, Débora Souza Collares Maia; Cordeiro, Rossana de Aguiar; Pereira Neto, Waldemiro de Aquino; Rocha, Marcos Fábio Gadelha

2017-10-01

This study aimed to identify yeasts from the gastrointestinal tract of scarlet ibises (Eudocimus ruber) and from plant material collected from the environment where they live. Then, the isolates phenotypically identified as Candida famata were submitted to molecular identification of their closely related species and evaluated for their antifungal susceptibility and possible resistance mechanisms to antifungal drugs. Cloacal swabs from 20 scarlet ibises kept in captivity at Mangal das Garças Park (Brazil), pooled stool samples (n = 20) and samples of trunks and hollow of trees (n = 20) obtained from their enclosures were collected. The samples were seeded on Sabouraud agar supplemented with chloramphenicol. The 48 recovered isolates were phenotypically identified as 15 Candida famata, 13 Candida catenulata, 2 Candida intermedia, 1 Candida lusitaniae, 2 Candida guilliermondii, 1 Candida kefyr, 1 Candida amapae, 1 Candida krusei, 8 Trichosporon spp., and 4 Rhodotorula spp. The C. famata isolates were further identified as 3 C. famata, 8 Debaryomyces nepalensis, and 4 C. palmioleophila. All C. famata and C. palmioleophila were susceptible to caspofungin and itraconazole, while one D. nepalensis was resistant to fluconazole and voriconazole. This same isolate and another D. nepalensis had lower amphotericin B susceptibility. The azole resistant strain had an increased efflux of rhodamine 6G and an alteration in the membrane sterol content, demonstrating multifactorial resistance mechanism. Finally, this research shows that scarlet ibises and their environment harbor C. famata and closely related species, including antifungal resistant isolates, emphasizing the need of monitoring the antifungal susceptibility of these yeast species. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

GRE2 from Scheffersomyces stipitis as an aldehyde reductase contributes tolerance to aldehyde inhibitors derived from lignocellulosic biomass

USDA-ARS?s Scientific Manuscript database

Scheffersomyces (Pichia) stipitis is one of the most promising yeasts for industrial bioethanol production from lignocellulosic biomass. S. stipitis is able to in situ detoxify aldehyde inhibitors [such as furfural and 5-hydroxymethylfurfural (HMF)] to less toxic corresponding alcohols. However, the...

Overproduction, purification and characterization of human interferon alpha2a-human serum albumin fusion protein produced in methilotropic yeast Pichia pastoris

NASA Astrophysics Data System (ADS)

Ningrum, R. A.; Santoso, A.; Herawati, N.

2017-05-01

Human interferon alpha2a (hIFNα2a) is a therapeutic protein that used in cancer and hepatitis B/C therapy. The main problem of using hIFNα-2a is its short elimination half life due to its low molecular weight. Development of higher molecular weight protein by albumin fusion technology is a rational strategy to solve the problem. In our previous research we constructed an open reading frame (ORF) encoding hIFNα2a-human serum albumin (HSA) fusion protein that expressed in Pichia pastoris (P. pastoris) protease deficient strain SMD1168. This research was performed to overproduce, purify and characterize the fusion protein. To overproduce the protein, cultivation was performed in buffered complex medium containing glyserol (BMGY) for 24 h and protein overproduction was applied in buffered complex medium containing methanol (BMMY) for 48 hours at 30°C. The fusion protein was purified by blue sepharose affinity chromatography. Molecular weight characterization by SDS PAGE corresponds with its theoretical size, 85 kDa. Western blot analysis demonstrated that the fusion protein was recognized by anti hIFNα2 and anti HSA monoclonal antibody as well. Amino acid sequence of the fusion protein was determined by LC MS/MS2 mass spectrometry with trypsin as proteolitic enzyme. There were three fragments that identified as hIFNα2a and seven fragments that identified as HSA. Total identified amino acids were 150 residues with 20% coverage from total residues. To conclude, hIFNα2a-HSA fusion protein was overproduced, purified and characterized. Characterization based on molecular weight, antibody recognition and amino acid sequence confirmed that the fusion protein has correct identity as theoretically thought.

Combination of antagonistic yeasts with two food additives for control of brown rot caused by Monilinia fructicola on sweet cherry fruit.

PubMed

Qin, G Z; Tian, S P; Xu, Y; Chan, Z L; Li, B Q

2006-03-01

To evaluate beneficial effect of two food additives, ammonium molybdate (NH4-Mo) and sodium bicarbonate (NaBi), on antagonistic yeasts for control of brown rot caused by Monilinia fructicola in sweet cherry fruit under various storage conditions. The mechanisms of action by which food additives enhance the efficacy of antagonistic yeasts were also evaluated. Biocontrol activity of Pichia membranefaciens and Cryptococcus laurentii against brown rot in sweet cherry fruit was improved by addition of 5 mmol l(-1) NH4-Mo or 2% NaBi when stored in air at 20 and 0 degrees C, and in controlled atmosphere (CA) storage with 10% O2 + 10% CO2 at 0 degrees C. Population dynamics of P. membranefaciens in the wounds of fruit were inhibited by NH4-Mo at 20 degrees C after 1 day of incubation and growth of C. laurentii was inhibited by NH4-Mo at 0 degrees C in CA storage after 60 days. In contrast, NaBi did not significantly influence growth of the two yeasts in fruit wounds under various storage conditions except that the growth of P. membranefaciens was stimulated after storage for 45 days at 0 degrees C in CA storage. When used alone, the two additives showed effective control of brown rot in sweet cherry fruit and the efficacy was closely correlated with the concentrations used. The result of in vitro indicated that growth of M. fructicola was significantly inhibited by NH4-Mo and NaBi. Application of additives improved biocontrol of brown rot on sweet cherry fruit under various storage conditions. It is postulated that the enhancement of disease control is directly because of the inhibitory effects of additives on pathogen growth, and indirectly because of the relatively little influence of additives on the growth of antagonistic yeasts. The results obtained in this study suggest that an integration of NH4-Mo or NaBi with biocontrol agents has great potential in commercial management of postharvest diseases of fruit.

Constitutive expression, purification and characterisation of pectin methylesterase from Aspergillus niger in Pichia pastoris for potential application in the fruit juice industry.

PubMed

Jiang, Xiuping; Chen, Peng; Yin, Maolu; Yang, Qing

2013-01-01

Pectin methylesterase (PME) catalyses the hydrolysis of the methyl ester of pectin, yielding free carboxyl groups and methanol. PME is widely used in the food, cosmetic and pharmaceutical industries. PME from Aspergillus niger was constitutively expressed to a high level in the yeast Pichia pastoris. The recombinant PME was purified by a combination of ammonium sulfate fractionation and ion exchange chromatography, giving an overall yield of 28.0%. It appeared as a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis, with a molecular mass of about 45 kDa. Optimal activity of the enzyme occurred at a temperature of 50 °C and a pH of 4.7. The K(m), V(max) and k(cat) values of the enzyme with respect to pectin were 8.6 mmol L⁻¹ [Formula: See Text], 1.376 mmol min⁻¹ mg⁻¹ and 8.26 × 10² s⁻¹ respectively. Cations such as K⁺, Mg²⁺, Ni²⁺, Mn²⁺ and Co²⁺ slightly inhibited its activity, whereas Na⁺ had no effect. PME from A. niger was constitutively expressed to a high level in P. pastoris without methanol induction. The recombinant PME was purified and characterised and shown to be a good candidate for potential application in the fruit juice industry. Copyright © 2012 Society of Chemical Industry.

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.

rRNA fragmentation induced by a yeast killer toxin.

PubMed

Kast, Alene; Klassen, Roland; Meinhardt, Friedhelm

2014-02-01

Virus like dsDNA elements (VLE) in yeast were previously shown to encode the killer toxins PaT and zymocin, which target distinct tRNA species via specific anticodon nuclease (ACNase) activities. Here, we characterize a third member of the VLE-encoded toxins, PiT from Pichia inositovora, and identify PiOrf4 as the cytotoxic subunit by conditional expression in Saccharomyces cerevisiae. In contrast to the tRNA targeting toxins, however, neither a change of the wobble uridine modification status by introduction of elp3 or trm9 mutations nor tRNA overexpression rescued from PiOrf4 toxicity. Consistent with a distinct RNA target, expression of PiOrf4 causes specific fragmentation of the 25S and 18S rRNA. A stable cleavage product comprising the first ∼ 130 nucleotides of the 18S rRNA was purified and characterized by linker ligation and subsequent reverse transcription; 3'-termini were mapped to nucleotide 131 and 132 of the 18S rRNA sequence, a region showing some similarity to the anticodon loop of tRNA(Glu)(UUC), the zymocin target. PiOrf4 residues Glu9 and His214, corresponding to catalytic sites Glu9 and His209 in the ACNase subunit of zymocin are essential for in vivo toxicity and rRNA fragmentation, raising the possibility of functionally conserved RNase modules in both proteins. © 2013 John Wiley & Sons Ltd.

A chloride tolerant laccase from the plant pathogen ascomycete Botrytis aclada expressed at high levels in Pichia pastoris.

PubMed

Kittl, Roman; Mueangtoom, Kitti; Gonaus, Christoph; Khazaneh, Shima Tahvilda; Sygmund, Christoph; Haltrich, Dietmar; Ludwig, Roland

2012-01-20

Fungal laccases from basidiomycetous fungi are thoroughly investigated in respect of catalytic mechanism and industrial applications, but the number of reported and well characterized ascomycetous laccases is much smaller although they exhibit interesting catalytic properties. We report on a highly chloride tolerant laccase produced by the plant pathogen ascomycete Botrytis aclada, which was recombinantly expressed in Pichia pastoris with an extremely high yield and purified to homogeneity. In a fed-batch fermentation, 495 mg L(-1) of laccase was measured in the medium, which is the highest concentration obtained for a laccase by a yeast expression system. The recombinant B. aclada laccase has a typical molecular mass of 61,565 Da for the amino acid chain. The pI is approximately 2.4, a very low value for a laccase. Glycosyl residues attached to the recombinant protein make up for approximately 27% of the total protein mass. B. aclada laccase exhibits very low K(M) values and high substrate turnover numbers for phenolic and non-phenolic substrates at acidic and near neutral pH. The enzyme's stability increases in the presence of chloride ions and, even more important, its substrate turnover is only weakly inhibited by chloride ions (I(50)=1.4M), which is in sharp contrast to most other described laccases. This high chloride tolerance is mandatory for some applications such as implantable biofuel cells and laccase catalyzed reactions, which suffer from the presence of chloride ions. The high expression yield permits fast and easy production for further basic and applied research. Copyright © 2011 Elsevier B.V. All rights reserved.

Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris.

PubMed

Yang, Xiaoxue; Cong, Hua; Song, Jinzhu; Zhang, Junzheng

2013-11-01

Trichoderma asperellum parasitizes a large variety of phytopathogenic fungi. The mycoparasitic activity of T. asperellum depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall and proteases which are a group of enzymes capable of degrading proteins from host. In this study, a full-length cDNA clone of aspartic protease gene, TaAsp, from T. asperellum was obtained and sequenced. The 1,185 bp long cDNA sequence was predicted to encode a 395 amino acid polypeptide with molecular mass of 42.3 kDa. The cDNA of TaAsp was inserted into the pPIC9K vector and transformed into yeast Pichia pastoris GS115 for heterologous expression. A clearly visible band with molecular mass about 42 kDa in the SDS-PAGE gel indicated that the transformant harboring the gene TaAsp had been successfully translated in P. pastoris and produced a recombinant protein. Enzyme characterization test showed that the optimum fermentation time for P. pastoris GS115 transformant was 72 h. Enzyme activity of the recombinant aspartic proteinase remained relatively stable at 25-60 °C and pH 3.0-9.0, which indicated its good prospect of application in biocontrol. The optimal pH value and temperature of the enzyme activity were pH 4.0 and 40 °C, and under this condition, with casein as the substrate, the recombinant protease activity was 18.5 U mL(-1). In order to evaluate antagonistic activity of the recombinant protease against pathogenic fungi, five pathogenic fungi, Fusarium oxysporum, Alternaria alternata, Cytospora chrysosperma, Sclerotinia sclerotiorum and Rhizoctonia solani, were applied to the test of in vitro inhibition of their mycelial growth by culture supernatant of P. pastoris GS115 transformant.

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

Enhancement of heterogeneous alkaline xylanase production in Pichia pastoris GS115

NASA Astrophysics Data System (ADS)

Zheng, Wei

2017-08-01

A series of strategies were applied to improve expression level of the recombinant alkaline xylanase from Bacillus pumilus G1-3 in Pichia pastoris GS115. Codon optimization of xylanase gene xynG1-3 from B. pumilus G1-3 were carried out for its heterogeneous expression in P. pastoris. The activity of xylanase encoded by optimized gene (xynG1-3-opt) was up to 33641 U/mL, which was 37% higher than that by wild-type (xynG1-3) gene. The results will greatly contribute to increasing the production of recombinant proteins in P. pastoris and improving the industrial production of the alkaline xylanase.

Secretory overexpression and isotopic labeling of the chimeric relaxin family peptide R3/I5 in Pichia pastoris.

PubMed

Guo, Yu-Qi; Wu, Qing-Ping; Shao, Xiao-Xia; Shen, Ting; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

2015-06-01

Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficient approach for preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression in Pichia pastoris and in vitro enzymatic maturation. A designed single-chain R3/I5 precursor containing the B-chain of human relaxin-3 and the A-chain of human INSL5 was overexpressed in PichiaPink strain 1 by high-density fermentation in a two-liter fermenter, and approximately 200 mg of purified precursor was obtained from one liter of the fermentation supernatant. We also developed an economical approach for preparation of the uniformly (15)N-labeled R3/I5 precursor by culturing in shaking flasks, and approximately 15 mg of purified (15)N-labeled precursor was obtained from one liter of the culture supernatant. After purification by cation ion-exchange chromatography and reverse-phase high performance liquid chromatography, the R3/I5 precursor was converted to the mature two-chain form by sequential treatment with endoproteinase Lys-C and carboxypeptidase B. The mature R3/I5 peptide had an α-helix-dominated conformation and retained full receptor-binding and receptor activation activities. Thus, Pichia overexpression was an efficient approach for sample preparation and isotopic labeling of the chimeric R3/I5 peptide. This approach could also be extended to the preparation of other relaxin family peptides in future studies.

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.