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Sample records for pichia stipitis cbs

  1. Detoxification of corn stover prehydrolyzate by trialkylamine extraction to improve the ethanol production with Pichia stipitis CBS 5776.

    PubMed

    Zhu, Junjun; Yong, Qiang; Xu, Yong; Yu, Shiyuan

    2011-01-01

    In order to realize the separated ethanol fermentation of glucose and xylose, prehydrolysis of corn stover with sulfuric acid at moderate temperature was applied, while inhibitors were produced inevitably. A complex extraction was adopted to detoxify the prehydrolyzate before fermentation to ethanol with Pichia stipitis CBS 5776. The best proportion of mixed extractant was 30% trialkylamine-50% n-octanol -20% kerosene. Detoxification results indicated that 73.3% of acetic acid, 45.7% of 5-hydroxymethylfurfural and 100% of furfural could be removed. Compared with the undetoxified prehydrolyzate, the fermentability of the detoxified prehydrolyzate was significantly improved. After 48 h fermentation of the detoxified prehydrolyzate containing 7.80 g/l of glucose and 52.8 g/l of xylose, the sugar utilization ratio was 93.2%; the ethanol concentration reached its peak value of 21.8 g/l, which was corresponding to 82.3% of the theoretical value.

  2. 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.

  3. Cause analysis of the effects of acid-catalyzed steam-exploded corn stover prehydrolyzate on ethanol fermentation by Pichia stipitis CBS 5776.

    PubMed

    Zhu, Junjun; Yang, Jinlong; Zhu, Yuanyuan; Zhang, Lingling; Yong, Qiang; Xu, Yong; Li, Xin; Yu, Shiyuan

    2014-11-01

    The prehydrolyzate obtained from acid-catalyzed steam-exploded corn stover (ASC) mainly contains xylose and a number of inhibitory compounds that inhibit ethanol fermentation by Pichia stipitis. In this study, the effects of the ASC prehydrolyzate, specifically those of the carbohydrate-degradation products, lignin-degradation products (which were extracted from ASC prehydrolyzate using ethyl acetate), and six major phenolic compounds (added to pure-sugar media individually or in combination), on ethanol fermentation were investigated. Results indicate that the effects of the carbohydrate-degradation products were negligible (10 h delayed) compared with those of pure-sugar fermentation, whereas the effects of the lignin-degradation products were significant (52 h delayed). Meanwhile, the inhibitory effects of the major phenolic compounds were not caused by certain types of inhibitors, but were due to the synergistic effects of various inhibitors.

  4. 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...

  5. 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...

  6. Evaluation of Engineered Pichia stipitis Strains for Ethanol Production

    USDA-ARS?s Scientific Manuscript database

    We evaluated the fermentation capabilities of five strains of Pichia stipitis that had been engineered for xylose fermentation to ethanol by USDA, ARS, National Center for Agricultural Utilization Research. The strains tested were P. stipitis WT-1-11, WT-1-2, 14-2-6, 22-1-1, and 22-1-12. Strains w...

  7. 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...

  8. Production of ethanol from corn stover hemicellulose hydrolyzate using Pichia stipitis.

    PubMed

    Agbogbo, Frank K; Wenger, Kevin S

    2007-11-01

    Hemicellulose liquid hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol using Pichia stipitis CBS 6054. The fermentation rate increased with aeration but the pH also increased due to consumption of acetic acid by Pichia stipitis. Hemicellulose hydrolyzate containing 34 g/L xylose, 8 g/L glucose, 8 g/L Acetic acid, 0.73 g/L furfural, and 1 g/L hydroxymethyl furfural was fermented to 15 g/L ethanol in 72 h. The yield in all the hemicellulose hydrolyzates was 0.37-0.44 g ethanol/g (glucose + xylose). Nondetoxified hemicellulose hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol with high yields, and this has the potential to improve the economics of the biomass to ethanol process.

  9. Genetic transformation of xylose-fermenting yeast Pichia stipitis

    SciTech Connect

    Ho, N.W.Y.; Petros, D.; Deng, X.X.

    1991-12-31

    A plasmid-mediated transformation system has been developed for the xylose-fermenting yeast Pichia stipitis. We found that plasmid vectors containing the Saccharomyces cerevisiae 2 p replicon and the kanamycin resistance gene (KmR) could be introduced into the Pichia cells and maintained as extrachromosomal elements. Pichia transformants containing such vectors will be resistant to the antibiotic geneticin that can be inactivated by the protein product of KmR. Plasmids identical to those used for transformation can be recovered from the Pichia transformants. Protocols for transformation of P. stipitis by the CaCl{sub 2}-polyethylene glycol-protoplast process or by direct electroporation of intact Pichia cells have both been developed.

  10. Effect of oxygenation on xylose fermentation by Pichia stipitis

    SciTech Connect

    Skoog, K.; Hahn-Haegerdal, B. )

    1990-11-01

    The effect of oxygen limitation on xylose fermentation by Pichia stipitis (CBS 6054) was investigated in continuous culture. The maximum specific ethanol productivity (0.20 g of ethanol g dry weight{sup {minus}1} h{sup {minus}1}) and ethanol yield (0.48 g/g) was reached at an oxygen transfer rate below 1 mmol/liter per h. In the studied range of oxygenation, the xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) activities were constant as well as the ratio between the NADPH and NADH activities of xylose reductase. No xylitol production was found. The pyruvate decarboxylase (EC 4.1.1.1) activity increased and the malate dehydrogenase (EC 1.1.1.37) activity decreased with decreasing oxygenation. With decreasing oxygenation, the intracellular intermediary metabolites sedoheptulose 7-phosphate, glucose 6-phosphate, fructose 1,6-diphosphate, and malate accumulated slightly while pyruvate decreased. The ratio of the xylose uptake rate under aerobic conditions, in contrast to that under anaerobic assay conditions, increased with increasing oxygenation in the culture. The results are discussed in relation to the energy level in the cell, the redox balance, and the mitochondrial function.

  11. 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...

  12. 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....

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

    SciTech Connect

    Toivola, A.; Yarrow, D.; van den Bosch, E.; van Dijken, J.P.; Scheffers, W.A.

    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 good fermenter of both xylose and cellobiose under the test conditions used.

  14. The switch from xylose to glucose stalled by repression of xylose-utilizing enzymes during exposure of Scheffersomyces (Pichia) stipitis to high ethanol concentrations

    USDA-ARS?s Scientific Manuscript database

    During the fermentation of lignocellulosic hydrolyzates to ethanol by Scheffersomyces (Pichia) stipitis NRRL Y-7124 (CBS 5773), the switch from glucose to xylose uptake results in a diauxic lag unless process strategies to prevent this are applied. When cells were grown on glucose, the length of th...

  15. Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and elimination of diauxic lag with xylose-grown populations

    USDA-ARS?s Scientific Manuscript database

    During the fermentation of lignocellulosic hydrolyzates to ethanol by native pentose-fermenting yeasts such as Scheffersomyces (Pichia) stipitis NRRL Y-7124 (CBS 5773) and Pachysolen tannophilus NRRL Y-2460, the switch from glucose to xylose uptake results in a diauxic lag unless process strategies ...

  16. Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminate diauxic lag

    USDA-ARS?s Scientific Manuscript database

    During the fermentation of lignocellulosic hydrolyzates to ethanol by native pentose-fermenting yeasts such as Scheffersomyces (Pichia) stipitis NRRL Y-7124 (CBS 5773) and Pachysolen tannophilus NRRL Y-2460, the switch from glucose to xylose uptake results in a diauxic lag unless process strategies ...

  17. 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...

  18. 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...

  19. 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...

  20. The Effect of Initial Cell Concentration on Xylose Fermentation by Pichia stipitis

    NASA Astrophysics Data System (ADS)

    Agbogbo, Frank K.; Coward-Kelly, Guillermo; Torry-Smith, Mads; Wenger, Kevin; Jeffries, Thomas W.

    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 produced when the initial cell concentrations were high, cell density had no effect on the final ethanol yield. A two-parameter mathematical model was used to predict the cell population dynamics at the different initial cell concentrations. The model parameters, a and b correlate with the initial cell concentrations used with an R 2 of 0.99.

  1. 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...

  2. Culture Nutrition and Physiology Impact the Inhibitor Tolerance of the Yeast Pichia stipitis NRRL Y-7124

    USDA-ARS?s Scientific Manuscript database

    Robust microorganisms are needed to consume both hexose and pentose sugars and to withstand, survive, and function in the presence of stress factors common to fermentations of lignocellulose hydrolysates, including the inhibitors furfural, 5-hydroxymethylfurfural (HMF), and ethanol. Pichia stipitis...

  3. Molecular cloning of XYL3 (D-xylulokinase) from Pichia stipitis and characterization of its physiological function.

    PubMed

    Jin, Yong-Su; Jones, Sharon; Shi, Nian-Qing; Jeffries, Thomas W

    2002-03-01

    XYL3, which encodes a D-xylulokinase (EC 2.7.1.17), was isolated from Pichia stipitis CBS 6054 genomic DNA by using primers designed against conserved motifs. Disruption of XYL3 eliminated D-xylulokinase activity, but D-ribulokinase activity was still present. Southern analysis of P. stipitis genomic DNA with XYL3 as a probe confirmed the disruption and did not reveal additional related genes. Disruption of XYL3 stopped ethanol production from xylose, but the resulting mutant still assimilated xylose slowly and formed xylitol and arabinitol. These results indicate that XYL3 is critical for ethanol production from xylose but that P. stipitis has another pathway for xylose assimilation. Expression of XYL3 using its P. stipitis promoter increased Saccharomyces cerevisiae D-xylulose consumption threefold and enabled the transformants to produce ethanol from a mixture of xylose and xylulose, whereas the parental strain only accumulated xylitol. In vitro, D-xylulokinase activity in recombinant S. cerevisiae was sixfold higher with a multicopy than with a single-copy XYL3 plasmid, but ethanol production decreased with increased copy number. These results confirmed the function of XYL3 in S. cerevisiae.

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

    SciTech Connect

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

    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 of co-localized genes with related functions,and evidence of how P. stipitis manages to achieve redox balance whilegrowing on xylose under microaerobic conditions.

  5. Pichia stipitis Genes for Alcohol Dehydrogenase with Fermentative and Respiratory Functions

    PubMed Central

    Cho, Jae-yong; Jeffries, Thomas W.

    1998-01-01

    Two genes coding for isozymes of alcohol dehydrogenase (ADH); designated PsADH1 and PsADH2, have been identified and isolated from Pichia stipitis CBS 6054 genomic DNA by Southern hybridization to Saccharomyces cerevisiae ADH genes, and their physiological roles have been characterized through disruption. The amino acid sequences of the PsADH1 and PsADH2 isozymes are 80.5% identical to one another and are 71.9 and 74.7% identical to the S. cerevisiae ADH1 protein. They also show a high level identity with the group I ADH proteins from Kluyveromyces lactis. The PsADH isozymes are presumably localized in the cytoplasm, as they do not possess the amino-terminal extension of mitochondrion-targeted ADHs. Gene disruption studies suggest that PsADH1 plays a major role in xylose fermentation because PsADH1 disruption results in a lower growth rate and profoundly greater accumulation of xylitol. Disruption of PsADH2 does not significantly affect ethanol production or aerobic growth on ethanol as long as PsADH1 is present. The PsADH1 and PsADH2 isozymes appear to be equivalent in the ability to convert ethanol to acetaldehyde, and either is sufficient to allow cell growth on ethanol. However, disruption of both genes blocks growth on ethanol. P. stipitis strains disrupted in either PsADH1 or PsADH2 still accumulate ethanol, although in different amounts, when grown on xylose under oxygen-limited conditions. The PsADH double disruptant, which is unable to grow on ethanol, still produces ethanol from xylose at about 13% of the rate seen in the parental strain. Thus, deletion of both PsADH1 and PsADH2 blocks ethanol respiration but not production, implying a separate path for fermentation. PMID:9546172

  6. Diminished respirative growth and enhanced assimilative sugar uptake result in higher specific fermentation rates by the mutant Pichia stipitis FPL-061

    SciTech Connect

    Sreenath, H.K.; Jeffries, T.W. |

    1997-12-31

    A mutant strain of Pichia stipitis, FPL-061, was obtained by selecting for growth on L-xylose in the presence of respiratory inhibitors. The specific fermentation rate of FPL-061, was higher than that of the parent, Pichia stipitis CBS 6054, because of its lower cell yield and growth rate and higher specific substrate uptake rate. With a mixture of glucose and xylose, the mutant strain FPL-061 produced 29.4 g ethanol/L with a yield of 0.42 g ethanol/g sugar consumed. By comparison, CBS 6054 produced 25.7 g ethanol/L with a yield of 0.35 g/g. The fermentation was most efficient at an aeration rate of 9.2 mmoles O{sub 2} L{sup -1} h{sup -1}. At high aeration rates (22 mmoles O{sub 2} L{sup -1} h{sup -1}), the mutant cell yield was less than that of the parent. At low aeration rates, (1.1 to 2.5 O{sub 2} L{sup -1} h{sup -1}), cell yields were similar, the ethanol formation rates were low, and xylitol accumulation was observed in both the strains. Both strains respired the ethanol once sugar was exhausted. We infer from the results that the mutant, P. stipitis FPL-061, diverts a larger fraction of its metabolic energy from cell growth into ethanol production. 21 refs., 5 figs.

  7. 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...

  8. 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...

  9. 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...

  10. Enhancing ethanol production from cellulosic sugars using Scheffersomyces (Pichia) stipitis.

    PubMed

    Okonkwo, C C; Azam, M M; Ezeji, T C; Qureshi, N

    2016-07-01

    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. Use of xylose alone resulted in the production of 20.68 ± 0.44 g L(-1) ethanol with a productivity of 0.17 ± 0.00 g L(-1) h(-1), while xylose plus 3 g L(-1) CaCO3 resulted in the production of 24.68 ± 0.75 g L(-1) ethanol with a productivity of 0.21 ± 0.01 g L(-1) h(-1). Use of xylose plus glucose in combination with 3 g L(-1) CaCO3 resulted in the production of 47.37 ± 0.55 g L(-1) ethanol (aerobic culture), thus resulting in an ethanol productivity of 0.39 ± 0.00 g L(-1) h(-1). These values are 229 % of that achieved in xylose medium. Supplementation of xylose and glucose medium with 0.40 g L(-1) CaCl2 resulted in the production of 44.84 ± 0.28 g L(-1) ethanol with a productivity of 0.37 ± 0.02 g L(-1) h(-1). Use of glucose plus 3 g L(-1) CaCO3 resulted in the production of 57.39 ± 1.41 g L(-1) ethanol under micro-aerophilic conditions. These results indicate that supplementation of cellulosic sugars in the fermentation medium with CaCO3 and CaCl2 would improve economics of ethanol production from agricultural residues.

  11. An improved process of ethanol production from hemicellulose: bioconversion of undetoxified hemicellulosic hydrolyzate from steam-exploded corn stover with a domesticated Pichia stipitis.

    PubMed

    Yong, Qiang; Li, Xin; Yuan, Yun; Lai, Chenhuan; Zhang, Nannan; Chu, Qiulu; Xu, Yong; Yu, Shiyuan

    2012-08-01

    Bioconversion of undetoxified hemicellulosic hydrolyzate from steam-exploded corn stover was investigated with a domesticated Pichia stipitis CBS 5776. The countercurrent washing was applied to recover sugars from the steam-exploded corn stover, which could enrich sugars in washing liquor and give an efficient saving of water. Acid concentration, reaction temperature, and time were optimized for the acid post-hydrolysis of oligosaccharides in steam-exploded prehydrolyzate by a central composite design and response surface methodology. The domestication of P. stipitis to the hydrolyzate resulted in improving sugar consumption and ethanol yield by gradually increasing the ratio of hydrolyzate in the medium. Recycling utilization of the domesticated yeast demonstrated that the yeast kept a stable ability of fermenting both hexose and pentose in the undetoxified hydrolyzate. The sugar consumption and ethanol yield were over 90 and 80 %, respectively.

  12. Alcohol fermentation of enzymatic hydrolysate of exploded rice straw by Pichia stipitis.

    PubMed

    Moniruzzaman, M

    1995-11-01

    The xylose in an enzymatic hydrolysate of steam-exploded rice straw was not consumed by Pichia stipitis until the glucose was almost exhausted. A diauxic lag of 2 to 3 h in both cell growth and ethanol production occurred as metabolism switched from glucose to xylose utilization. Ethanol production was maximal [6 g ethano/l from 15 g reducing sugars/l (78% theoretical yield)] at an aeration rate of 0.2 vol/vol. min.

  13. Cellulosic ethanol production using the naturally occurring xylose-fermenting yeast, Pichia stipitis.

    PubMed

    Agbogbo, Frank K; Coward-Kelly, Guillermo

    2008-09-01

    Rising crude oil prices and environmental concerns have renewed interest in renewable energy. Cellulosic ethanol promises to deliver a renewable fuel from non-food feedstocks. One technical challenge producing cellulosic ethanol economically is a robust organism to utilize the different sugars present in cellulosic biomass. Unlike starch where glucose is the only sugar present, cellulosic biomass has other sugars such as xylose and arabinose, usually called C5 sugars. This review examines the most promising naturally occurring C5 fermenting organism, Pichia stipitis. In this work, the properties that make P. stipitis unique from other organisms, its physiology and fermentation results on lignocellulosic substrates have been reviewed. P. stipitis can produce 41 g ethanol/l with a potential to cleanup some of the most concentrated toxins. These results coupled with the less stringent nutritional requirements, great resistance to contamination and its thick cell walls makes P. stipitis a viable organism for scale-up. However, P. stipitis has a slower sugar consumption rate compared to Saccharomyces cerevisiae and requires microaerophilic condition for ethanol production. Finally, future studies to enhance fermentation capabilities of this yeast have been discussed.

  14. Cell density-correlated induction of pyruvate decarboxylase under aerobic conditions in the yeast Pichia stipitis.

    PubMed

    Mergler, M; Klinner, U

    2001-01-01

    During the aerobic batch cultivation of P. stipitis CBS 5776 with glucose, pyruvate decarboxylase was activated in a cell number-correlated manner. Activation started when a cell number between 7 x 10(7) and x 10(8) cells ml(-1) was reached and the enzyme activity increased during further cultivation. This induction might have been triggered either by an unknown quorum sensing system or by a shortage of cytoplasmic acetyl-CoA.

  15. Isolation of xylose reductase gene of Pichia stipitis and its expression in Saccharomyces cerevisiae

    SciTech Connect

    Takuma, Shinya; Nakashima, Noriyuki; Tantirungkij, Manee

    1991-12-31

    A NADPH/NADH-dependent xylose reductase gene was isolated from the xylose-assimilating yeast, Pichia stipitis. DNA sequence analysis showed that the gene consists of 951 bp. The gene introduced in Saccharomyces cerevisiae was transcribed to mRNA, and a considerable amount of enzyme activity was observed constitutively, whereas transcription and translation in P steps were inducible. S. cerevisiae carrying the xylose reductase gene could not, however, grow on xylose medium, and could not produce ethanol from xylose. Since xylose uptake and accumulation of xylitol by S. cerevisiae were observed, the conversion of xylitol to xylulose seemed to be limited.

  16. Characterization of the Genetic System of the Xylose-Fermenting Yeast Pichia stipitis

    PubMed

    Melake; Passoth; Klinner

    1996-10-01

    High mutant frequencies indicated that the wild-type strains of Pichia stipitis are haploid. Sporulation ability of these clones pointed to a homothallic life cycle. Mating was induced by cultivation under nutritionally poor conditions on malt extract medium. Conjugation was followed immediately by sporulation. However, hybrids could be rescued by transferring the nascent zygotes to complete medium before meiosis had started. Under rich nutritional conditions, hybrids were mitotically stable and did not sporulate. The segregation pattern of auxotrophic markers of diploid zygotes indicated regular meiosis, although asci contained preferentially spore dyads.

  17. Aerobic induction of respiro-fermentative growth by decreasing oxygen tensions in the respiratory yeast Pichia stipitis.

    PubMed

    Klinner, U; Fluthgraf, S; Freese, S; Passoth, V

    2005-04-01

    The fermentative and respiratory metabolism of Pichia stipitis wild-type strain CBS 5774 and the derived auxotrophic transformation recipient PJH53 trp5-10 his3-1 were examined in differentially oxygenated glucose cultures in the hermetically sealed Sensomat system. There was a good agreement of the kinetics of gas metabolism, growth, ethanol formation and glucose utilisation, proving the suitability of the Sensomat system for rapid and inexpensive investigation of strains and mutants for their respiratory and fermentative metabolism. Our study revealed respiro-fermentative growth by the wild-type strain, although the cultures were not oxygen-limited. The induction of respiro-fermentative behaviour was obviously due to the decrease in oxygen tension but not falling below a threshold of oxygen tension. The responses differed depending on the velocity of the decrease in oxygen tension. At high oxygenation (slow decrease in oxygen tension), ethanol production was induced but glucose uptake was not influenced. At low oxygenation, glucose uptake and ethanol formation increased during the first hours of cultivation. The transformation recipient PJH53 most probably carries a mutation that influences the response to a slow decrease in oxygen tension, since almost no ethanol formation was found under these conditions.

  18. Metabolic-flux profiling of the yeasts Saccharomyces cerevisiae and Pichia stipitis.

    PubMed

    Fiaux, Jocelyne; Cakar, Z Petek; Sonderegger, Marco; Wüthrich, Kurt; Szyperski, Thomas; Sauer, Uwe

    2003-02-01

    The so far largely uncharacterized central carbon metabolism of the yeast Pichia stipitis was explored in batch and glucose-limited chemostat cultures using metabolic-flux ratio analysis by nuclear magnetic resonance. The concomitantly characterized network of active metabolic pathways was compared to those identified in Saccharomyces cerevisiae, which led to the following conclusions. (i) There is a remarkably low use of the non-oxidative pentose phosphate (PP) pathway for glucose catabolism in S. cerevisiae when compared to P. stipitis batch cultures. (ii) Metabolism of P. stipitis batch cultures is fully respirative, which contrasts with the predominantly respiro-fermentative metabolic state of S. cerevisiae. (iii) Glucose catabolism in chemostat cultures of both yeasts is primarily oxidative. (iv) In both yeasts there is significant in vivo malic enzyme activity during growth on glucose. (v) The amino acid biosynthesis pathways are identical in both yeasts. The present investigation thus demonstrates the power of metabolic-flux ratio analysis for comparative profiling of central carbon metabolism in lower eukaryotes. Although not used for glucose catabolism in batch culture, we demonstrate that the PP pathway in S. cerevisiae has a generally high catabolic capacity by overexpressing the Escherichia coli transhydrogenase UdhA in phosphoglucose isomerase-deficient S. cerevisiae.

  19. Two novel gene expression systems based on the yeasts Schwanniomyces occidentalis and Pichia stipitis.

    PubMed

    Piontek, M; Hagedorn, J; Hollenberg, C P; Gellissen, G; Strasser, A W

    1998-09-01

    Two non-Saccharomyces yeasts have been developed as hosts for heterologous gene expression. The celD gene from Clostridium thermocellum, encoding a heat-stable cellulase, served as the test sequence. The first system is based on the amylolytic species Schwanniomyces occidentalis, the second on the xylolytic species Pichia stipitis. The systems comprise auxotrophic host strains (trp5 in the case of S. occidentalis; trp5-10, his3 in the case of P. stipitis) and suitable transformation vectors. Vector components consist of an S. occidentalis-derived autonomously replicating sequence (SwARS) and the Saccharomyces cerevisiae-derived TRP5 sequence for plasmid propagation and selection in the yeast hosts, an ori and an ampicillin-resistance sequence for propagation and selection in a bacterial host. A range of vectors has been engineered employing different promoter elements for heterologous gene expression control in both species. Homologous elements derived from highly expressed genes of the respective hosts appeared to be of superior quality: in the case of S. occidentalis that of the GAM1 gene, in the case of P. stipitis that of the XYL1 gene. Further elements tested are the S. cerevisiae-derived ADH1 and PDC1 promoter sequences.

  20. Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars

    USDA-ARS?s Scientific Manuscript database

    Yeast strains for anaerobic conversion of lignocellulosic sugars to ethanol were produced from Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 using UV-C mutagenesis. Random UV-C mutagenesis potentially produces large numbers of mutations broadly and uniformly over the whole genome to genera...

  1. High solid loading hydrolyzate-tolerant strains of Scheffersomyces (Pichia) stipitis exhibiting reduced diauxic lag and higher ethanol productivity

    USDA-ARS?s Scientific Manuscript database

    During the fermentation of lignocellulosic hydrolyzates to ethanol by native pentose-fermenting yeasts such as Scheffersomyces (Pichia) stipitis NRRL Y-7124, the switch from glucose to xylose uptake results in a diauxic lag unless process strategies to prevent this are applied. Further investigation...

  2. Fermentation kinetics for xylitol production by a Pichia stipitis D-xylulokinase mutant previously grown in spent sulfite liquor

    Treesearch

    Rita C.L.B. Rodrigues; Chenfeng Lu; Bernice Liu; Thomas W. Jeffries

    2008-01-01

    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 (xyl3[delta]) to convert these sugars into useful products. FPL-YS30 produces a...

  3. Continuous fermentation of D-xylose by immobilized Pichia stipitis

    SciTech Connect

    Nunez, M.J.; Dominguez, C.H.; Sanroman, A.; Lema, J.M.

    1991-12-31

    The main purpose of this work was to compare the performance of two different kinds of reactors (CSTR and CPFR) in order to enhance the ethanol productivity in the fermentation of D-xylose by Pichia steps immobilized in {kappa}-carrageenan. Immobilization was carried out in a 4% aqueous suspension of {kappa}-carrageenan, which was mixed with the inoculum. The bioparticles were treated with Al(NO{sub 3}){sub 3} as hardening agent. The fermenters operated during a long period of time (about 30 d). Best results were obtained in the packed-bed reactor (CPFR), which allowed operation at high final ethanol concentrations, this fact having been explained because of the observed strong product inhibition. The overall productivity reached values higher than 3.8 g/(L{circ}h). This supposed an interesting improvement with relation to the productivities found in the literature, which as an average did not exceed 1 g/(L{circ}h). However, the specific productivities of yeast in the continuous stirred tank reactor (CSTR) were always greater because the bioparticles were kept in close contact with the broth, whereas in the CPFR, there were at least two problems: (a) the possibility that the produced gas could prevent the intimate contact between the substrate and the particles and (b) the possible existence of preferential paths.

  4. Peculiarities of the regulation of fermentation and respiration in the crabtree-negative, xylose-fermenting yeast Pichia stipitis.

    PubMed

    Passoth, V; Zimmermann, M; Klinner, U

    1996-01-01

    The respiration of Pichia stipitis was not repressed by either high concentrations of fermentable sugars or oxygen limitation. Fermentation was not induced by high sugar concentrations, but was inactivated by aerobic conditions. The activity of pyruvate dehydrogenase was constitutive. In contrast, pyruvate decarboxylase, alcohol dehydrogenase, and aldehyde dehydrogenase were induced by a reduction in the oxygen tension. It was demonstrated that in P. stipitis, the pyruvate decarboxylase is not induced by a signal from glycolysis. Contrary to Saccharomyces cerevisiae, the pyruvate decarboxylase was not inhibited by phosphate.

  5. Bioconversion of novel substrate Saccharum spontaneum, a weedy material, into ethanol by Pichia stipitis NCIM3498.

    PubMed

    Chandel, Anuj K; Singh, Om V; Rao, L Venkateswar; Chandrasekhar, G; Narasu, M Lakshmi

    2011-01-01

    Rising fuel prices and environmental issues have paved the way for the exploration of cellulosic ethanol. However, challenges involving substrate hydrolysis and cost-effectiveness still limit the efficient bioconversion and utilization of cellulosic ethanol. We aimed to evaluate a cheaper and abundantly available wild sugarcane variety, Saccharum spontaneum, as the raw substrate for bioconversion of ethanol by Pichia stipitis NCIM3498. Three different strategies for substrate hydrolysis using acid (dilute sulfuric acid) and alkali (dilute sodium hydroxide) and aqueous ammonia (AA) treatment followed by enzymatic hydrolysis were studied. A maximum of 631.5±3.25 mg/g sugars with 89.38% hydrolytic efficiency (HE) could be achieved after enzymatic hydrolysis of AA-pretreated S. spontaneum. All the substrate hydrolysates were evaluated for ethanol conversion in batches by P. stipitis. The microbial fermentation of released sugars into ethanol showed (g/g) 0.36±0.011, 0.384±0.022, 0.391±0.02, and 0.40±0.01 yield from detoxified acid hydrolysate and acid-, NaOH- and AA-pretreated substrate S. spontaneum enzymatic hydrolysates, respectively.

  6. Effects of pretreatment methods for hazelnut shell hydrolysate fermentation with Pichia Stipitis to ethanol.

    PubMed

    Arslan, Yeşim; Eken-Saraçoğlu, Nurdan

    2010-11-01

    In this study, we investigated the use of hazelnut shell as a renewable and low cost lignocellulosic material for bioethanol production for the first time. High lignin content of hazelnut shell is an important obstacle for such a biotransformation. Biomass hydrolysis with acids yields reducing sugar with several inhibitors which limit the fermentability of sugars. The various conditioning methods for biomass and hydrolysate were performed to overcome the toxicity and their effects on the subsequent fermentation of hazelnut shell hydrolysate by Pichia stipitis were evaluated with shaking flasks experiments. Hazelnut shells hydrolysis with 0.7M H(2)SO(4) yielded 49 gl(-1) total reducing sugars and fermentation inhibitors in untreated hydrolysate. First, it was shown that several hydrolysate detoxification methods were solely inefficient in achieving cell growth and ethanol production in the fermentation of hazelnut shell hydrolysates derived from non-delignified biomass. Next, different pretreatments of hazelnut shells were considered for delignification and employed before hydrolysis in conjunction with hydrolysate detoxification to improve alcohol fermentation. Among six delignification methods, the most effective pretreatment regarding to ethanol concentration includes the treatment of shells with 3% (w/v) NaOH at room temperature, which was integrated with sequential hydrolysate detoxification by overliming and then treatment with charcoal twice at 60 degrees C. This treatment brought about a total reduction of 97% in furans and 88.4% in phenolics. Almost all trialed treatments caused significant sugar loss. Under the best assayed conditions, ethanol concentration of 16.79gl(-1) was reached from a hazelnut shell hyrolysate containing initial 50g total reducing sugar l(-1) after partial synthetic xylose supplementation. This value is equal to 91.25% of ethanol concentration that was obtained from synthetic d-xylose under same conditions. The present study

  7. Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminate diauxic lag.

    PubMed

    Slininger, Patricia J; Thompson, Stephanie R; Weber, Scott; Liu, Z Lewis; Moon, Jaewoong

    2011-08-01

    During the fermentation of lignocellulosic hydrolyzates to ethanol by native pentose-fermenting yeasts such as Scheffersomyces (Pichia) stipitis NRRL Y-7124 (CBS 5773) and Pachysolen tannophilus NRRL Y-2460, the switch from glucose to xylose uptake results in a diauxic lag unless process strategies to prevent this are applied. When yeast were grown on glucose and resuspended in mixed sugars, the length of this lag was observed to be a function of the glucose concentration consumed (and consequently, the ethanol concentration accumulated) prior to the switch from glucose to xylose fermentation. At glucose concentrations of 95 g/L, the switch to xylose utilization was severely stalled such that efficient xylose fermentation could not occur. Further investigation focused on the impact of ethanol on cellular xylose transport and the induction and maintenance of xylose reductase and xylitol dehydrogenase activities when large cell populations of S. stipitis NRRL Y-7124 were pre-grown on glucose or xylose and then presented mixtures of glucose and xylose for fermentation. Ethanol concentrations around 50 g/L fully repressed enzyme induction although xylose transport into the cells was observed to be occurring. Increasing degrees of repression were documented between 15 and 45 g/L ethanol. Repitched cell populations grown on xylose resulted in faster fermentation rates, particularly on xylose but also on glucose, and eliminated diauxic lag and stalling during mixed sugar conversion by P. tannophilus or S. stipitis, despite ethanol accumulations in the 60 or 70 g/L range, respectively. The process strategy of priming cells on xylose was key to the successful utilization of high mixed sugar concentrations because specific enzymes for xylose utilization could be induced before ethanol concentration accumulated to an inhibitory level. Copyright © 2011 Wiley Periodicals, Inc.

  8. Altering coenzyme specificity of Pichia stipitis xylose reductase by the semi-rational approach CASTing

    PubMed Central

    Liang, Ling; Zhang, Jingqing; Lin, Zhanglin

    2007-01-01

    Background The NAD(P)H-dependent Pichia stipitis xylose reductase (PsXR) is one of the key enzymes for xylose fermentation, and has been cloned into the commonly used ethanol-producing yeast Saccharomyces cerevisiae. In order to eliminate the redox imbalance resulting from the preference of this enzyme toward NADPH, efforts have been made to alter the coenzyme specificity of PsXR by site-directed mutagenesis, with limited success. Given the industrial importance of PsXR, it is of interest to investigate further ways to create mutants of PsXR that prefers NADH rather than NADPH, by the alternative directed evolution approach. Results Based on a homology model of PsXR, six residues were predicted to interact with the adenine ribose of NAD(P)H in PsXR and altered using a semi-rational mutagenesis approach (CASTing). Three rounds of saturation mutagenesis were carried to randomize these residues, and a microplate-based assay was applied in the screening. A best mutant 2-2C12, which carried four mutations K270S, N272P, S271G and R276F, was obtained. The mutant showed a preference toward NADH over NADPH by a factor of about 13-fold, or an improvement of about 42-fold, as measured by the ratio of the specificity constant kcat/Kmcoenzyme. Compared with the wild-type, the kcatNADH for the best mutant was only slightly lower, while the kcatNADPH decreased by a factor of about 10. Furthermore, the specific activity of 2-2C12 in the presence of NADH was 20.6 U·mg-1, which is highest among PsXR mutants reported. Conclusion A seemingly simplistic and yet very effective mutagenesis approach, CASTing, was applied successfully to alter the NAD(P)H preference for Pichia stipitis xylose reductase, an important enzyme for xylose-fermenting yeast. The observed change in the NAD(P)H preference for this enzyme seems to have resulted from the altered active site that is more unfavorable for NADPH than NADH in terms of both Km and kcat. There are potentials for application of our PsXR in

  9. Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeast Pichia stipitis.

    PubMed

    Maassen, Nicole; Freese, Stefan; Schruff, Barbara; Passoth, Volkmar; Klinner, Ulrich

    2008-08-01

    Pichia stipitis integrates linear homologous DNA fragments mainly ectopically. High rates of randomly occurring integration allow tagging mutagenesis with high efficiency using simply PCR amplificates of suitable selection markers from the P. stipitis genome. Linearization of an autonomously replicating vector caused a distinct increase of the transformation efficiency compared with the circular molecule. Cotransformation of a restriction endonuclease further enhanced the transformation efficiency. This effect was also observed with integrative vector DNA. In most cases vector integration in chromosomal targets did not depend on microhomologies, indicating that restriction-enzyme-mediated integration (REMI) does not play an essential role in P. stipitis. Small deletions were observed at the ends of the integrated vectors and in the target sites. Disruption of the PsKU80 gene increased the frequency of homologous integration considerably but resulted in a remarkable decrease of the transformation efficiency. These results suggest that in P. stipitis the nonhomologous end joining (NHEJ) pathway obviously predominates the homologous recombination pathway of double-strand break repair.

  10. Ethanol production from acid hydrolysates based on the construction and demolition wood waste using Pichia stipitis.

    PubMed

    Cho, Dae Haeng; Shin, Soo-Jeong; Bae, Yangwon; Park, Chulhwan; Kim, Yong Hwan

    2011-03-01

    The feasibility of ethanol production from the construction and demolition (C&D) wood waste acid hydrolysates was investigated. The chemical compositions of the classified C&D wood waste were analyzed. Concentrated sulfuric acid hydrolysis was used to obtain the saccharide hydrolysates and the inhibitors in the hydrolysates were also analyzed. The C&D wood waste composed of lumber, plywood, particleboard, and medium density fiberboard (MDF) had polysaccharide (cellulose, xylan, and glucomannan) fractions of 60.7-67.9%. The sugar composition (glucose, xylose, and mannose) of the C&D wood wastes varied according to the type of wood. The additives used in the wood processing did not appear to be released into the saccharide solution under acid hydrolysis. Although some fermentation inhibitors were detected in the hydrolysates, they did not affect the ethanol production by Pichia stipitis. The hexose sugar-based ethanol yield and ethanol yield efficiency were 0.42-0.46 g ethanol/g substrate and 84.7-90.7%, respectively. Therefore, the C&D wood wastes dumped in landfill sites could be used as a raw material feedstock for the production of bioethanol.

  11. Effect of inhibitors formed during wheat straw pretreatment on ethanol fermentation by Pichia stipitis.

    PubMed

    Bellido, Carolina; Bolado, Silvia; Coca, Mónica; Lucas, Susana; González-Benito, Gerardo; García-Cubero, María Teresa

    2011-12-01

    The inhibitory effect of the main inhibitors (acetic acid, furfural and 5-hydroxymethylfurfural) formed during steam explosion of wheat straw was studied through ethanol fermentations of model substrates and hydrolysates from wheat straw by Pichia stipitis. Experimental results showed that an increase in acetic acid concentration led to a reduction in ethanol productivity and complete inhibition was observed at 3.5 g/L. Furfural produced a delay on sugar consumption rates with increasing concentration and HMF did not exert a significant effect. Fermentations of the whole slurry from steam exploded wheat straw were completely inhibited by a synergistic effect due to the presence of 1.5 g/L acetic acid, 0.15 g/L furfural and 0.05 g/L HMF together with solid fraction. When using only the solid fraction from steam explosion, hydrolysates presented 0.5 g/L of acetic acid, whose fermentations have submitted promising results, providing an ethanol yield of 0.45 g ethanol/g sugars and the final ethanol concentration reached was 12.2 g/L (10.9 g ethanol/100 g DM).

  12. Induction of Xylose Reductase and Xylitol Dehydrogenase Activities in Pachysolen tannophilus and Pichia stipitis on Mixed Sugars

    PubMed Central

    Bicho, Paul A.; Runnals, P. Lynn; Cunningham, J. Douglas; Lee, Hung

    1988-01-01

    The induction of xylose reductase and xylitol dehydrogenase activities on mixed sugars was investigated in the yeasts Pachysolen tannophilus and Pichia stipitis. Enzyme activities induced on d-xylose served as the controls. In both yeasts, d-glucose, d-mannose, and 2-deoxyglucose inhibited enzyme induction by d-xylose to various degrees. Cellobiose, l-arabinose, and d-galactose were not inhibitory. In liquid batch culture, P. tannophilus utilized d-glucose and d-mannose rapidly and preferentially over d-xylose, while d-galactose consumption was poor and lagged behind that of the pentose sugar. In P. stipitis, all three hexoses were used preferentially over d-xylose. The results showed that the repressibility of xylose reductase and xylitol dehydrogenase may limit the potential of yeast fermentation of pentose sugars in hydrolysates of lignocellulosic substrates. PMID:16347538

  13. [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.

  14. Molecular cloning of alcohol dehydrogenase genes of the yeast Pichia stipitis and identification of the fermentative ADH.

    PubMed

    Passoth, V; Schäfer, B; Liebel, B; Weierstall, T; Klinner, U

    1998-10-01

    Two Pichia stipitis ADH genes (PsADH1 and PsADH2) were isolated by complementation of a Saccharomyces cerevisiae Adh(-)-mutant. The genes enabled the transformants to grow in the presence of antimycin A on glucose, to use ethanol as sole carbon source and made them sensitive to allylalcohol. The sequences of the genes showed similarities of 70-77% to sequences of ADH genes of Candida albicans, Kluyveromyces lactis, K. marxianus, and S. cerevisiae and about 60% homology to those of Schizosaccharomyces pombe and Aspergillus flavus. Southern hybridization experiments suggested that P. stipitis has only these two ADH genes. Both genes are located on the largest chromosome of P. stipitis. PsADH2 encodes for the ADH activity that is responsible for ethanol formation at oxygen limitation. The gene is regulated at the transcriptional level. Moreover, also in cells grown on ethanol, only PsADH2 transcript was found. PsADH1 transcript was detected under aerobic conditions on fermentable carbon sources.

  15. The electrophoretic banding pattern of the chromosomes of Pichia stipitis and Candida shehatae.

    PubMed

    Passoth, V; Hansen, M; Klinner, U; Emeis, C C

    1992-11-01

    The electrophoretic karyotype of fu1ur strains of P. stipitis and five strains of C. shehatae were compared by means of OFAGE and TAFE techniques. Although the number of chromosomal bands was six in all cases except one, P. stipitis revealed a clearly distinct pattern in comparison to C. shehatae. Both yeasts showed remarkable chromosome length polymorphism.

  16. Alcoholic fermentation of Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis in the presence of inhibitory compounds and seawater.

    PubMed

    Gonçalves, Fabiano Avelino; dos Santos, Everaldo Silvino; de Macedo, Gorete Ribeiro

    2015-06-01

    Production of cellulosic ethanol and holocellulosic ethanol from vegetable or microbial biomass starts with a hydrolysate containing compounds which may produce negative effects in the enzymatic hydrolysis and fermentation stages due to the need of pretreatment of the materials. In this way, the simultaneous presence of hydroxymethylfurfural (HMF), furfural, acetic acid, levulinic acid, and formic acid in different concentrations was tested in the fermentation using Saccharomyces cerevisiae, Pichia stipitis, and Zymomonas mobilis. The substitution of freshwater by seawater in the culture medium was also analyzed. Thus, inhibitory effects were stronger in the fermentation using P. stipitis, followed by Z. mobilis and S. cerevisiae. Formic acid and acetic acid presented more significant effects among the inhibitory compounds, followed by HMF, furfural and levulinic acid. Fermentation performed in culture medium with seawater showed promising results, especially in the ethanol yield using S. cerevisiae (0.50 g ethanol/g glucose) and Z. mobilis (0.49 g ethanol/g glucose). Whereas the production of cellulosic ethanol and holocellulosic ethanol are in early stages of development on an industrial scale, and that the availability and use of freshwater may cause socio-environmental problems for expansion of ethanol production, the use of seawater appears as an alternative to mitigate this problem.

  17. Evaluation of the tolerance of acetic acid and 2-furaldehyde on the growth of Pichia stipitis and its respiratory deficient.

    PubMed

    Ortiz-Muñiz, B; Rasgado-Mellado, J; Solis-Pacheco, J; Nolasco-Hipólito, C; Domínguez-González, J M; Aguilar-Uscanga, M G

    2014-10-01

    The use of lignocellulosic residues for ethanol production is limited by toxic compounds in fermenting yeasts present in diluted acid hydrolysates like acetic acid and 2-furaldehyde. The respiratory deficient phenotype gives the cell the ability to resist several toxic compounds. So the aim of this work was to evaluate the tolerance to toxic compounds present in lignocellulosic hydrolysates like acetic acid and 2-furaldehyde in Pichia stipitis and its respiratory deficient strains. The respiratory deficient phenotype was induced by exposure to chemical agents such as acriflavine, acrylamide and rhodamine; 23 strains were obtained. The selection criterion was based on increasing specific ethanol yield (g ethanol g(-1) biomass) with acetic acid and furaldehyde tolerance. The screening showed that P. stipitis NRRL Y-7124 ACL 2-1RD (lacking cytochrome c), obtained using acrylamide, presented the highest specific ethanol production rate (1.82 g g(-1 )h(-1)). Meanwhile, the ACF8-3RD strain showed the highest acetic acid tolerance (7.80 g L(-1)) and the RHO2-3RD strain was able to tolerate up to 1.5 g L(-1) 2-furaldehyde with a growth and ethanol production inhibition of 23 and 22 %, respectively. The use of respiratory deficient yeast phenotype is a strategy for ethanol production improvement in a medium with toxic compounds such as hydrolysed sugarcane bagasse amongst others.

  18. Particle properties of sugar maple hemicellulose hydrolysate and its influence on growth and metabolic behavior of Pichia stipitis.

    PubMed

    Sun, Zhijie; Shupe, Alan; Liu, Tingjun; Hu, Ruofei; Amidon, Thomas E; Liu, Shijie

    2011-01-01

    In this study the influence of the insoluble solids in nano-filtrated sugar maple hemicellulosic hydrolysate on the metabolic behavior of Pichia stiptis was investigated. The particle properties of hemicellulosic hydrolysate were analyzed. Phosphoric acid and ammonium (PA) were applied to remove the particles. The metabolic behavior and growth property of P. stipitis in particle--removed hydrolysate was measured. Results demonstrated that the average particle size and zeta potential of the untreated hydrolysate were 2266.9±78.2 nm and -6.09±0.49 mV. Xylose consumption and ethanol production rate were significantly decreased when particle content is greater than 1.63 g/L. Because the majority of particles (34 g/L) were removed from hydrolysates by phosphoric acid and ammonium treatment, the fermentability of the hydrolysate was significantly improved. These results indicated particles play an important role in hydrolysate inhibition effect.

  19. Reversal of coenzyme specificity and improvement of catalytic efficiency of Pichia stipitis xylose reductase by rational site-directed mutagenesis.

    PubMed

    Zeng, Qi-Kai; Du, Hong-Li; Wang, Jing-Fang; Wei, Dong-Qing; Wang, Xiao-Ning; Li, Yi-Xue; Lin, Ying

    2009-07-01

    A major problem when xylose is used for ethanol production is the intercellular redox imbalance arising from different coenzyme specificities of xylose reductase (XR) and xylitol dehydrogenase. The residue Lys21 in XR from Pichia stipitis was subjected to site-directed mutagenesis to alter its coenzyme specificity. The N272D mutant exhibited improved catalytic efficiency when NADH was the coenzyme. Both K21A and K21A/N272D preferred NADH to NADPH, their catalytic efficiencies for NADPH were almost zero. The catalytic efficiency of K21A/N272D for NADH was almost 9-fold and 2-fold that of K21A and the wild-type enzyme, respectively. Complete reversal of coenzyme specificity toward NADH and improved catalytic efficiency were achieved.

  20. Bioconversion of lignocellulosic fraction of water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate to ethanol by Pichia stipitis.

    PubMed

    Kumar, Ashish; Singh, L K; Ghosh, Sanjoy

    2009-07-01

    Fermentation of acid hydrolysate of water-hyacinth (Eichhornia crassipes), a free floating aquatic plant has been investigated for ethanol production. The dilute acid treatment has been applied to utilize the maximum hemicellulosic content of the water-hyacinth. The goal of this work was to investigate, both experimentally and theoretically using mathematical tools, a fermentative system utilizing water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate as a substrate for ethanol production using Pichia stipitis. It was found that 72.83% of xylose was converted to ethanol with a yield of 0.425 g(p)/g(s) and productivity of 0.176 g(p)/L/h. An appropriate mathematical model was developed to explain theoretically the bioconversion of this hemicellulose acid hydrolysate to ethanol and the model was tested statistically to check the validity of the model.

  1. 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.

  2. Evaluation of fermentation kinetics of acid-treated corn cob hydrolysate for xylose fermentation in the presence of acetic acid by Pichia stipitis.

    PubMed

    Kashid, Mohan; Ghosalkar, Anand

    2017-08-01

    The efficient utilization of lignocellulosic biomass for ethanol production depends on the fermentability of the biomass hydrolysate obtained after pretreatment. In this work we evaluated the kinetics of ethanol production from xylose using Pichia stipitis in acid-treated corn cob hydrolysate. Acetic acid is one of the main inhibitors in corn cob hydrolysate that negatively impacts kinetics of xylose fermentation by P. stipitis. Unstructured kinetic model has been formulated that describes cell mass growth and ethanol production as a function of xylose, oxygen, ethanol, and acetic acid concentration. Kinetic parameters were estimated under different operating conditions affecting xylose fermentation. This is the first report on kinetics of xylose fermentation by P. stipitis which includes inhibition of acetic acid on growth and product formation. In the presence of acetic acid in the hydrolysate, the model accurately predicted reduction in maximum specific growth rate (from 0.23 to 0.15 h(-1)) and increase in ethanol yield per unit biomass (from 3 to 6.2 gg(-1)), which was also observed during experimental trials. Presence of acetic acid in the fermentation led to significant reduction in the cell growth rate, reduction in xylose consumption and ethanol production rate. The developed model accurately described physiological state of P. stipitis during corn cob hydrolysate fermentation. Proposed model can be used to predict the influence of xylose, ethanol, oxygen, and acetic acid concentration on cell growth and ethanol productivity in industrial fermentation.

  3. Enzymatic hydrolysis of sodium dodecyl sulphate (SDS)-pretreated newspaper for cellulosic ethanol production by Saccharomyces cerevisiae and Pichia stipitis.

    PubMed

    Xin, Fengxue; Geng, Anli; Chen, Ming Li; Gum, Ming Jun Marcus

    2010-10-01

    Fermentation of enzymatic hydrolysate of waste newspaper was investigated for cellulosic ethanol production in this study. Various nonionic and ionic surfactants were applied for waste newspaper pretreatment to increase the enzymatic digestibility. The surfactant-pretreated newspaper was enzymatically digested in 0.05 M sodium citrate buffer (pH 4.8) with varying solid content, filter paper unit loading (FPU/g newspaper), and ratio of filter paper unit/beta-glucosidase unit (FPU/CBU). Newspaper pretreated with the anionic surfactant sodium dodecyl sulphate (SDS) demonstrated the highest sugar yield. The addition of Tween-80 in the enzymatic hydrolysis process enhanced the enzymatic digestibility of newspaper pretreated with all of the surfactants. Enzymatic hydrolysis of SDS-pretreated newspaper with 15% solid content, 15 FPU/g newspaper, and FPU/CBU of 1:4 resulted in a newspaper hydrolysate conditioning 29.07 g/L glucose and 4.08 g/L xylose after 72 h of incubation at 50 degrees C. The fermentation of the enzymatic hydrolysate with Saccharomyces cerevisiae, Pichia stipitis, and their co-culture produced 14.29, 13.45, and 14.03 g/L of ethanol, respectively. Their corresponding ethanol yields were 0.43, 0.41, and 0.42 g/g.

  4. Bioethanol fermentation of concentrated rice straw hydrolysate using co-culture of Saccharomyces cerevisiae and Pichia stipitis.

    PubMed

    Yadav, K Srilekha; Naseeruddin, Shaik; Prashanthi, G Sai; Sateesh, Lanka; Rao, L Venkateswar

    2011-06-01

    Rice straw is one of the abundant lignocellulosic feed stocks in the world and has been selected for producing ethanol at an economically feasible manner. It contains a mixture of sugars (hexoses and pentoses). Biphasic acid hydrolysis was carried out with sulphuric acid using rice straw. After acid hydrolysis, the sugars, furans and phenolics were estimated. The initial concentration of sugar was found to be 16.8 g L(-1). However to increase the ethanol yield, the initial sugar concentration of the hydrolysate was concentrated to 31 g L(-1) by vacuum distillation. The concentration of sugars, phenols and furans was checked and later detoxified by over liming to use for ethanol fermentation. Ethanol concentration was found to be 12 g L(-1), with a yield, volumetric ethanol productivity and fermentation efficiency of 0.33 g L(-1)h(-1), 0.4 g g(-1) and 95%, respectively by co-culture of OVB 11 (Saccharomyces cerevisiae) and Pichia stipitis NCIM 3498.

  5. Nitrogen source and mineral optimization enhance D: -xylose conversion to ethanol by the yeast Pichia stipitis NRRL Y-7124.

    PubMed

    Slininger, Patricia J; Dien, Bruce S; Gorsich, Steven W; Liu, Zonglin L

    2006-10-01

    Nutrition-based strategies to optimize xylose to ethanol conversion by Pichia stipitis were identified in growing and stationary-phase cultures provided with a defined medium varied in nitrogen, vitamin, purine/pyrimidine, and mineral content via full or partial factorial designs. It is surprising to note that stationary-phase cultures were unable to ferment xylose (or glucose) to ethanol without the addition of a nitrogen source, such as amino acids. Ethanol accumulation increased with arginine, alanine, aspartic acid, glutamic acid, glycine, histidine, leucine, and tyrosine, but declined with isoleucine. Ethanol production from 150 g/l xylose was maximized (61+/-9 g/l) by providing C:N in the vicinity of approximately 57-126:1 and optimizing the combination of urea and amino acids to supply 40-80 % nitrogen from urea and 60-20 % from amino acids (casamino acids supplemented with tryptophan and cysteine). When either urea or amino acids were used as sole nitrogen source, ethanol accumulation dropped to 11 or 24 g/l, respectively, from the maximum of 46 g/l for the optimal nitrogen combination. The interaction of minerals with amino acids and/or urea was key to optimizing ethanol production by cells in both growing and stationary-phase cultures. In nongrowing cultures supplied with nitrogen as amino acids, ethanol concentration increased from 24 to 54 g/l with the addition of an optimized mineral supplement of Fe, Mn, Mg, Ca, Zn, and others.

  6. Fermentation of Acid-pretreated Corn Stover to Ethanol Without Detoxification Using Pichia stipitis

    NASA Astrophysics Data System (ADS)

    Agbogbo, Frank K.; Haagensen, Frank D.; Milam, David; Wenger, Kevin S.

    In this work, the effect of adaptation on P. stipitis fermentation using acidpretreated corn stover hydrolyzates without detoxification was examined. Two different types of adaptation were employed, liquid hydrolyzate and solid state agar adaptation. Fermentation of 12.5% total solids undetoxified acid-pretreated corn stover was performed in shake flasks at different rotation speeds. At low rotation speed (100 rpm), both liquid hydrolyzate and solid agar adaptation highly improved the sugar consumption rate as well as ethanol production rate compared to the wild-type strains. The fermentation rate was higher for solid agar-adapted strains compared to liquid hydrolyzate-adapted strains. At a higher rotation speed (150 rpm), there was a faster sugar consumption and ethanol production for both the liquid-adapted and the wild-type strains. However, improvements in the fermentation rate between the liquid-adapted and wild strains were less pronounced at the high rotation speed.

  7. 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)

    SciTech Connect

    Dominguez, 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{sub 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{sub L}a{approx_equal} 16 h{sup {minus}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{sub L}a = 15.8 h{sup {minus}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.

  8. Reconstruction and visualization of carbohydrate, N-glycosylation pathways in Pichia pastoris CBS7435 using computational and system biology approaches.

    PubMed

    Srivastava, Akriti; Somvanshi, Pallavi; Mishra, Bhartendu Nath

    2013-06-01

    Pichia pastoris is an efficient expression system for production of recombinant proteins. To understand its physiology for building novel applications it is important to understand and reconstruct its metabolic network. The metabolic reconstruction approach connects genotype with phenotype. Here, we have attempted to reconstruct carbohydrate metabolism pathways responsible for high biomass density and N-glycosylation pathways involved in the post translational modification of proteins of P. pastoris CBS7435. Both these metabolic pathways play a crucial role in heterologous protein production. We report novel, missing and unannotated enzymes involved in the target metabolic pathways. A strong possibility of cellulose and xylose metabolic processes in P. pastoris CBS7435 suggests its use in the area of biofuels. The reconstructed metabolic networks can be used for increased yields and improved product quality, for designing appropriate growth medium, for production of recombinant therapeutics and for making biofuels.

  9. Analysis of the hypoxia-induced ADH2 promoter of the respiratory yeast Pichia stipitis reveals a new mechanism for sensing of oxygen limitation in yeast.

    PubMed

    Passoth, Volkmar; Cohn, Marita; Schäfer, Bernd; Hahn-Hägerdal, Bärbel; Klinner, Ulrich

    2003-01-15

    We introduced a reporter gene system into Pichia stipitis using the gene for the artificial green fluorescent protein (GFP), variant yEGFP. This system was used to analyse hypoxia-dependent PsADH2 regulation. Reporter gene activity was only found under oxygen limitation on a fermentable carbon source. The promoter was not induced by oxygen limitation in the Crabtree-positive yeast Saccharomyces cerevisiae. Promoter deletions revealed that a region of 15 bp contained the essential site for hypoxic induction. This motif was different from the known hypoxia response elements of S. cerevisiae but showed some similarity to the mammalian HIF-1 binding site. Electrophoretic mobility shift assays demonstrated specific protein binding to this region under oxygen limitation. Similar to the S. cerevisiae heme sensor system, the promoter was induced by Co(2+). Cyanide was not able to mimic the effect of oxygen limitation. The activation mechanism of PsADH2 also, in this respect, has similarities to the mammalian HIF-1 system, which is inducible by Co(2+) but not by cyanide. Thus, the very first promoter analysis in P. stipitis revealed a hitherto unknown mechanism of oxygen sensing in yeast.

  10. Biovalorization potential of peels of Ananas cosmosus (L.) Merr. for ethanol production by Pichia stipitis NCIM 3498 & Pachysolen tannophilus MTCC 1077.

    PubMed

    Bhatia, Latika; Johri, Sonia

    2015-12-01

    Bioethanol, is a potential alternate source of energy, renewable and safe. Ethanol production from value added food and feedstock has also not shown growth as estimated. Of late, the second generation processes of production of ethanol, such as from lignocellulosic biomass out of agricultural/domestic waste has been gaining considerable momentum. Here, we explored a new approach for optimizing the conditions of physiochemical pretreatment as well as fermentation process using peels of Ananas cosmosus as substrate and immobilized yeast Pachysolen tannophilus MTCC.1077 and Pichia stipitis NCIM 3498. We have also studied the influence of process variables such as incubation temperature, inoculum concentration and different nutrients on ethanol production. Pulverized peels of A. cosmosus recorded 25 ± 0.31% cellulose, 28 ± 0.18% hemicellulose and 8 ± 0.07% of lignin on dry solid (DS) basis. Peels of A. cosmosus delignified with 1% H2SO4 yielded 18.89% glucose, 38.81% xylose and 29.31% fructose under thermochemical pretreatment using autoclave (121 degrees C, 20 min.), with a hydrolytic efficiency of 75.52 ± 0.45%. FTIR spectroscopy results not only indicated the penetration of H2SO4 in the amorphous region of the biomass and degradation of hemicelluloses but also showed the structural differences before and after pretreatment. The enzymes required for hydrolysis were prepared from culture supernatants of Trichoderma reesei NCIM 1052 using wheat bran as carbon source under submerged fermentation conditions on rotatory shaker incubator (at 28 degrees C for 10 days). Enzyme activity (U/ml) of crude cellulase produced by T. reesei NCIM 1052 was 311.1 μmole/ml/min. Delignified A. cosmosus peel yielded 51.71 ± 0.44 g/l glucose when enzymatically hydrolysed by crude cellulase at the substrate enzyme ratio of 1:5. Simultaneous saccharification and fermentation (SSF) of peels of A. cosmosus by crude cellulase and separately entrapped Pichia stipitis NCIM 3498 (now known

  11. Two-step process using immobilized Saccharomyces cerevisiae and Pichia stipitis for ethanol production from Ulva pertusa Kjellman hydrolysate.

    PubMed

    Lee, Sang-Eun; Kim, Yi-Ok; Choi, Woo Yong; Kang, Do-Hyung; Lee, Hyeon-Yong; Jung, Kyung-Hwan

    2013-10-28

    We established a two-step production process using immobilized S. cerevisiae and P. stipitis yeast to produce ethanol from seaweed (U. pertusa Kjellman) hydrolysate. The process was designed to completely consume both glucose and xylose. In particular, the yeasts were immobilized using DEAE-corncob and DEAE-cotton, respectively. The first step of the process included a continuous column reactor using immobilized S. cerevisiae, and the second step included a repeated-batch reactor using immobilized P. stipitis. It was verified that the glucose and xylose in 20 L of medium containing the U. pertusa Kjellman hydrolysate was converted completely to about 5.0 g/l ethanol through the two-step process, in which the overall ethanol yield from total reducing sugar was 0.37 and the volumetric ethanol productivity was 0.126 g/ l/h. The volumetric ethanol productivity of the two-step process was about 2.7 times greater than that when P. stipitis was used alone for ethanol production from U. pertusa Kjellman hydrolysate. In addition, the overall ethanol yield from glucose and xylose was superior to that when P. stipitis was used alone for ethanol production. This two-step process will not only contribute to the development of an integrated process for ethanol production from glucose and xylose-containing biomass hydrolysates, but could also be used as an alternative method for ethanol production.

  12. Cloning novel sugar transporters from Scheffersomyces (Pichia) stipitis allowing D-xylose fermentation by recombinant Saccharomyces cerevisiae.

    PubMed

    de Sales, Belisa B; Scheid, Bruna; Gonçalves, Davi L; Knychala, Marilia M; Matsushika, Akinori; Bon, Elba P S; Stambuk, Boris U

    2015-10-01

    Since uptake of xylose limits its fermentation, we aimed to identify novel sugar transporters from Scheffersomyces stipitis that allow xylose uptake and fermentation by engineered Saccharomyces cerevisiae. An hxt-null S. cerevisiae strain, lacking the major hexose transporters (hxt1Δ-hxt7Δ and gal2Δ) but having high xylose reductase, xylitol dehydrogenase and xylulokinase activities, was transformed with a genomic DNA library from S. stipitis. Four plasmids allowing growth on xylose contained three genes encoding sugar transporters: the previously characterized XUT1 permease, and two new genes (HXT2.6 and QUP2) not previously identified as xylose transporters. High cell density fermentations with the recombinant strains showed that the XUT1 gene allowed ethanol production from xylose or xylose plus glucose as carbon sources, while the HXT2.6 permease produced both ethanol and xylitol, and the strain expressing the QUP2 gene produced mainly xylitol during xylose consumption. Cloning novel sugar transporters not previously identified in the S. stipitis genome using an hxt-null S. cerevisiae strain with a high xylose-utilizing pathway provides novel promising target genes for improved lignocellulosic ethanol production by yeasts.

  13. Insights from modeling the 3D structure of NAD(P)H-dependent D-xylose reductase of Pichia stipitis and its binding interactions with NAD and NADP.

    PubMed

    Wang, Jing-Fang; Wei, Dong-Qing; Lin, Ying; Wang, Yong-Hua; Du, Hong-Li; Li, Yi-Xve; Chou, Kuo-Chen

    2007-07-27

    NAD(P)H-dependent d-xylose reductase is a homodimeric oxidoreductase that belongs to the aldo-keto reductase superfamily. The enzyme has the special function to catalyze the first step in the assimilation of xylose into yeast metabolic pathways. Performing this function via reducing the open chain xylose to xylitol, the xylose reductase of Pichia stipitis is one of the most important enzymes that can be used to construct recombinant Saccharomyces cerevisiae strain for utilizing xylose and producing alcohol. To investigate into the interaction mechanism of the enzyme with its ligand NAD and NADP, the 3D structure was developed for the NAD(P)H-dependent d-xylose reductase from P. stipitis. With the 3D structure, the molecular docking operations were conducted to find the most stable bindings of the enzyme with NAD and NADP, respectively. Based on these results, the binding pockets of the enzyme for NAD and NADP have been explicitly defined. It has been found that the residues in forming the binding pockets for both NAD and NADP are almost the same and mainly hydrophilic. These findings may be used to guide mutagenesis studies, providing useful clues to modify the enzyme to improve the utilization of xylose for producing alcohol. Also, because human aldose reductases have the function to reduce the open chain form of glucose to sorbitol, a process physiologically significant for diabetic patients at the time that their blood glucose levels are elevated, the information gained through this study may also stimulate the development of new strategies for therapeutic treatment of diabetes.

  14. Bioconversion of Saccharum spontaneum (wild sugarcane) hemicellulosic hydrolysate into ethanol by mono and co-cultures of Pichia stipitis NCIM3498 and thermotolerant Saccharomyces cerevisiae-VS₃.

    PubMed

    Chandel, Anuj K; Singh, Om V; Narasu, M Lakshmi; Rao, L Venkateswar

    2011-10-01

    The lignocellulosic biomass is a low-cost renewable resource for eco-benign liquid fuel 'ethanol'. To resolve the hydrolysis of mixed sugars in lignocellulosic substrate Saccharum spontaneum, the microbial co-cultures of Pichia stipitis NCIM 3498 and thermotolerant Saccharomyces cerevisiae-VS(3) were analyzed for efficient bioconversion of mixed sugars into ethanol. Among the hydrolysis conditions, the acid hydrolysis released maximum sugars along with furans, phenolics and acetic acid. The acidic hydrolysate was detoxified and fermented by monocultures of P. stipitis NCIM3498 (P.S.) and thermotolerant S. cerevisiae VS(3) (S.C.), and co-culture of P.S. (7.5 mL) and S.C. (2.5 mL). Before the fermentation of hemicellulose acid hydrolysate, both the monocultures (P.S. and S.C.), and varying ratios of P.S. and S.C. microorganisms in co-cultures #1, #2 and #3 were grown on simulated synthetic medium. The ethanol yield from monocultures of P.S. (0.44 ± 0.021 g/g), S.C. (0.22 ± 0.01 g/g) and co-culture #3 (0.49 ± 0.02 g/g) revealed unique characteristics of each mono and co-culture technology. The fermentation of hemicellulose acid hydrolysate with monocultures of P.S., S.C. and co-culture #3 produced 12.08 ± 0.72 g/L, 1.40 ± 0.07 g/L, and 15.0 ± -0.92 g/L ethanol, respectively.

  15. Evaluation of ethanol production from corncob using Scheffersomyces (Pichia) stipitis CBS 6054 by volumetric scale-up.

    PubMed

    Lee, Jae-Won; Zhu, J Y; Scordia, Danilo; Jeffries, Thomas W

    2011-10-01

    In scale-up, the potential of ethanol production by dilute sulfuric acid pretreatment using corncob was investigated. Pretreatments were performed at 170 °C with various acid concentrations ranging from 0% to 1.656% based on oven dry weight. Following pretreatment, pretreated biomass yield ranged from 59% to 67%. More than 90% of xylan was removed at 0.828% of sulfuric acid. At same pretreatment condition, the highest glucose yield obtained from pretreated biomass by enzymatic hydrolysis was about 76%, based on a glucan content of 37/100 g. In hydrolysate obtained by pretreatment, glucose concentration was low, while xylose concentration was significantly increased above 0.368% of sulfuric acid. At 1.656% of sulfuric acid, xylose and glucose concentration was highest. In subsequent, fermentation with hydrolysate, maximal ethanol yield was attained after 24 h with 0.368% of sulfuric acid. The fermentation efficiency of hydrolysate obtained by enzymatic hydrolysis reached a maximum of 75% at an acid charge of 0.368%.

  16. Evaluation of ethanol production from corncob using Scheffersomyces (Pichia) stipitis CBS 6054 by volumetric scale-up

    Treesearch

    Jae-Won Lee; J.Y. Zhu; Danilo Scordia; Thomas W. Jeffries

    2011-01-01

    In scale-up, the potential of ethanol production by dilute sulfuric acid pretreatment using corncob was investigated. Pretreatments were performed at 170 °C with various acid concentrations ranging from 0% to 1.656% based on oven dry weight. Following pretreatment, pretreated biomass yield ranged from 59% to 67%. More than 90% of xylan was removed at 0.828% of sulfuric...

  17. Separate hydrolysis and fermentation (SHF) of Prosopis juliflora, a woody substrate, for the production of cellulosic ethanol by Saccharomyces cerevisiae and Pichia stipitis-NCIM 3498.

    PubMed

    Gupta, Rishi; Sharma, Krishna Kant; Kuhad, Ramesh Chander

    2009-02-01

    Prosopis juliflora (Mesquite) is a raw material for long-term sustainable production of cellulosics ethanol. In this study, we used acid pretreatment, delignification and enzymatic hydrolysis to evaluate the pretreatment to produce more sugar, to be fermented to ethanol. Dilute H(2)SO(4) (3.0%,v/v) treatment resulted in hydrolysis of hemicelluloses from lignocellulosic complex to pentose sugars along with other byproducts such as furfural, hydroxymethyl furfural (HMF), phenolics and acetic acid. The acid pretreated substrate was delignified to the extent of 93.2% by the combined action of sodium sulphite (5.0%,w/v) and sodium chlorite (3.0%,w/v). The remaining cellulosic residue was enzymatically hydrolyzed in 0.05 M citrate phosphate buffer (pH 5.0) using 3.0 U of filter paper cellulase (FPase) and 9.0 U of beta-glucosidase per mL of citrate phosphate buffer. The maximum enzymatic saccharification of cellulosic material (82.8%) was achieved after 28 h incubation at 50 degrees C. The fermentation of both acid and enzymatic hydrolysates, containing 18.24 g/L and 37.47 g/L sugars, with Pichia stipitis and Saccharomyces cerevisiae produced 7.13 g/L and 18.52 g/L of ethanol with corresponding yield of 0.39 g/g and 0.49 g/g, respectively.

  18. Curation of the genome annotation of Pichia pastoris (Komagataella phaffii) CBS7435 from gene level to protein function.

    PubMed

    Valli, Minoska; Tatto, Nadine E; Peymann, Armin; Gruber, Clemens; Landes, Nils; Ekker, Heinz; Thallinger, Gerhard G; Mattanovich, Diethard; Gasser, Brigitte; Graf, Alexandra B

    2016-09-01

    As manually curated and non-automated BLAST analysis of the published Pichia pastoris genome sequences revealed many differences between the gene annotations of the strains GS115 and CBS7435, RNA-Seq analysis, supported by proteomics, was performed to improve the genome annotation. Detailed analysis of sequence alignment and protein domain predictions were made to extend the functional genome annotation to all P. pastoris sequences. This allowed the identification of 492 new ORFs, 4916 hypothetical UTRs and the correction of 341 incorrect ORF predictions, which were mainly due to the presence of upstream ATG or erroneous intron predictions. Moreover, 175 previously erroneously annotated ORFs need to be removed from the annotation. In total, we have annotated 5325 ORFs. Regarding the functionality of those genes, we improved all gene and protein descriptions. Thereby, the percentage of ORFs with functional annotation was increased from 48% to 73%. Furthermore, we defined functional groups, covering 25 biological cellular processes of interest, by grouping all genes that are part of the defined process. All data are presented in the newly launched genome browser and database available at www.pichiagenome.org In summary, we present a wide spectrum of curation of the P. pastoris genome annotation from gene level to protein function.

  19. Bioethanol production from ball milled bagasse using an on-site produced fungal enzyme cocktail and xylose-fermenting Pichia stipitis.

    PubMed

    Buaban, Benchaporn; Inoue, Hiroyuki; Yano, Shinichi; Tanapongpipat, Sutipa; Ruanglek, Vasimon; Champreda, Verawat; Pichyangkura, Rath; Rengpipat, Sirirat; Eurwilaichitr, Lily

    2010-07-01

    Sugarcane bagasse is one of the most promising agricultural by-products for conversion to biofuels. Here, ethanol fermentation from bagasse has been achieved using an integrated process combining mechanical pretreatment by ball milling, with enzymatic hydrolysis and fermentation. Ball milling for 2 h was sufficient for nearly complete cellulose structural transformation to an accessible amorphous form. The pretreated cellulosic residues were hydrolyzed by a crude enzyme preparation from Penicillium chrysogenum BCC4504 containing cellulase activity combined with Aspergillus flavus BCC7179 preparation containing complementary beta-glucosidase activity. Saccharification yields of 84.0% and 70.4% for glucose and xylose, respectively, were obtained after hydrolysis at 45 degrees C, pH 5 for 72 h, which were slightly higher than those obtained with a commercial enzyme mixture containing Acremonium cellulase and Optimash BG. A high conversion yield of undetoxified pretreated bagasse (5%, w/v) hydrolysate to ethanol was attained by separate hydrolysis and fermentation processes using Pichia stipitis BCC15191, at pH 5.5, 30 degrees C for 24 h resulting in an ethanol concentration of 8.4 g/l, corresponding to a conversion yield of 0.29 g ethanol/g available fermentable sugars. Comparable ethanol conversion efficiency was obtained by a simultaneous saccharification and fermentation process which led to production of 8.0 g/l ethanol after 72 h fermentation under the same conditions. This study thus demonstrated the potential use of a simple integrated process with minimal environmental impact with the use of promising alternative on-site enzymes and yeast for the production of ethanol from this potent lignocellulosic biomass.

  20. Effect of ozonolysis parameters on the inhibitory compound generation and on the production of ethanol by Pichia stipitis and acetone-butanol-ethanol by Clostridium from ozonated and water washed sugarcane bagasse.

    PubMed

    Travaini, Rodolfo; Barrado, Enrique; Bolado-Rodríguez, Silvia

    2016-10-01

    Sugarcane bagasse (SCB) was ozone pretreated and detoxified by water washing, applying a L9(3)(4) orthogonal array (OA) design of experiments to study the effect of pretreatment parameters (moisture content, ozone concentration, ozone/oxygen flow and particle size) on the generation of inhibitory compounds and on the composition of hydrolysates of ozonated-washed samples. Ozone concentration resulted the highest influence process parameter on delignification and sugar release after washing; while, for inhibitory compound formation, moisture content also had an important role. Ozone expended in pretreatment related directly with sugar release and inhibitory compound formation. Washing detoxification was effective, providing non-inhibitory hydrolysates. Maximum glucose and xylose release yields obtained were 84% and 67%, respectively, for ozonated-washed SCB. Sugar concentration resulted in the decisive factor for biofuels yields. Ethanol production achieved an 88% yield by Pichia stipitis, whereas Clostridium acetobutylicum produced 0.072gBUTANOL/gSUGAR and 0.188gABE/gSUGAR, and, Clostridium beijerinckii 0.165gBUTANOL/gSUGAR and 0.257gABE/gSUGAR.

  1. 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...

  2. A thermotolerant and cold-active mannan endo-1,4-β-mannosidase from Aspergillus niger CBS 513.88: Constitutive overexpression and high-density fermentation in Pichia pastoris.

    PubMed

    Zhao, Wei; Zheng, Jia; Zhou, Hong-bo

    2011-08-01

    The mannan endo-1,4-β-mannosidase gene man26A from Aspergillus niger CBS 513.88 was optimized according to the codon usage bias in Pichia pastoris and synthesized by splicing overlap extension PCR. It was successfully expressed in P. pastoris using constitutive expression vector pGAPzαA. The recombinant endo-beta-1,4-mannanase could work in an extremely board temperature range and over 30% relative activity were retained in the temperature range of 5-60°C. The optimal pH value and temperature for activity were 5.0 and 45°C, respectively. It was highly thermotolerant with a half-life time of 15min at 90°C. A novel fed-batch strategy was developed successfully for high cell-density fermentation and mannanase activity reached 5069U/mL after cultivation for 56h in 50L fermenter. The broad working temperature range, high thermotolerance and efficient expression made this enzyme possible to be applied in food, animal feed and the production of biofuels.

  3. Cell-recycle batch process of Scheffersomyces stipitis and Saccharomyces cerevisiae co-culture for second generation bioethanol production.

    PubMed

    Ashoor, Selim; Comitini, Francesca; Ciani, Maurizio

    2015-11-01

    To achieve an optimized co-culture ratio of Scheffersomyces stipitis and Saccharomyces cerevisiae for the production of second generation bioethanol under a cell-recycle batch process. Three Sacc. cerevisiae strains were evaluated in co-culture with Sch. stipitis CBS 5773 at different ratios using synthetic medium containing glucose and xylose. Bioreactor trials indicated that the optimal condition for ethanol production using Sacc. cerevisiae EC1118 and Sch. stipitis co-culture was 1 % of O2 concentration. To increase ethanol production with Sacc. cerevisiae/Sch. stipitis co-culture a cell-recycle batch process was evaluated. Using this process, the maximum ethanol production (9.73 g l(-1)) and ethanol yield (0.42 g g(-1)) were achieved exhibiting a tenfold increase in ethanol productivity in comparison with batch process (2.1 g l(-1) h(-1)). In these conditions a stabilization of the cells ratio Sacc. cerevisiae/Sch. stipitis (1:5) at steady state condition was obtained. Batch cells recycling fermentation is an effective process to use Sch. stipitis/Sacc. cerevisiae co-culture for second generation ethanol production.

  4. Reconstruction and analysis of a genome-scale metabolic model for Scheffersomyces stipitis

    PubMed Central

    2012-01-01

    Background Fermentation of xylose, the major component in hemicellulose, is essential for economic conversion of lignocellulosic biomass to fuels and chemicals. The yeast Scheffersomyces stipitis (formerly known as Pichia stipitis) has the highest known native capacity for xylose fermentation and possesses several genes for lignocellulose bioconversion in its genome. Understanding the metabolism of this yeast at a global scale, by reconstructing the genome scale metabolic model, is essential for manipulating its metabolic capabilities and for successful transfer of its capabilities to other industrial microbes. Results We present a genome-scale metabolic model for Scheffersomyces stipitis, a native xylose utilizing yeast. The model was reconstructed based on genome sequence annotation, detailed experimental investigation and known yeast physiology. Macromolecular composition of Scheffersomyces stipitis biomass was estimated experimentally and its ability to grow on different carbon, nitrogen, sulphur and phosphorus sources was determined by phenotype microarrays. The compartmentalized model, developed based on an iterative procedure, accounted for 814 genes, 1371 reactions, and 971 metabolites. In silico computed growth rates were compared with high-throughput phenotyping data and the model could predict the qualitative outcomes in 74% of substrates investigated. Model simulations were used to identify the biosynthetic requirements for anaerobic growth of Scheffersomyces stipitis on glucose and the results were validated with published literature. The bottlenecks in Scheffersomyces stipitis metabolic network for xylose uptake and nucleotide cofactor recycling were identified by in silico flux variability analysis. The scope of the model in enhancing the mechanistic understanding of microbial metabolism is demonstrated by identifying a mechanism for mitochondrial respiration and oxidative phosphorylation. Conclusion The genome-scale metabolic model developed for

  5. GRE2 from Scheffersomyces stipitis as an aldehyde reductase contributes tolerance to aldehyde inhibitors derived from lignocellulosic biomass.

    PubMed

    Wang, Xu; Ma, Menggen; Liu, Z Lewis; Xiang, Quanju; Li, Xi; Liu, Na; Zhang, Xiaoping

    2016-08-01

    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 reduction enzymes involved in this reaction remain largely unknown. In this study, we reported that an uncharacterized open reading frame PICST_72153 (putative GRE2) from S. stipitis was highly induced in response to furfural and HMF stresses. Overexpression of this gene in Saccharomyces cerevisiae improved yeast tolerance to furfural and HMF. GRE2 was identified as an aldehyde reductase which can reduce furfural to FM with either NADH or NADPH as the co-factor and reduce HMF to FDM with NADPH as the co-factor. This enzyme can also reduce multiple aldehydes to their corresponding alcohols. Amino acid sequence analysis indicated that it is a member of the subclass "intermediate" of the short-chain dehydrogenase/reductase (SDR) superfamily. Although GRE2 from S. stipitis is similar to GRE2 from S. cerevisiae in a three-dimensional structure, some differences were predicted. GRE2 from S. stipitis forms loops at D133-E137 and T143-N145 locations with two α-helices at E154-K157 and E252-A254 locations, different GRE2 from S. cerevisiae with an α-helix at D133-E137 and a β-sheet at T143-N145 locations, and two loops at E154-K157 and E252-A254 locations. This research provided guidelines for the study of other SDR enzymes from S. stipitis and other yeasts on tolerant mechanisms to aldehyde inhibitors derived from lignocellulosic biomass.

  6. C- and N-catabolic utilization of tricarboxylic acid cycle-related amino acids by Scheffersomyces stipitis and other yeasts.

    PubMed

    Freese, Stefan; Vogts, Tanja; Speer, Falk; Schäfer, Bernd; Passoth, Volkmar; Klinner, Ulrich

    2011-05-01

    Scheffersomyces stipitis and the closely related yeast Candida shehatae assimilated the L-amino acids glutamate, aspartate and proline as both carbon and nitrogen sole sources. We also found this rarely investigated ability in ascomycetous species such as Candida glabrata, C. reukaufii, C. utilis, Debaryomyces hansenii, Kluyveromyces lactis, K. marxianus, Candida albicans, L. elongisporus, Meyerozyma guilliermondii, C. maltosa, Pichia capsulata and Yarrowia lipolytica and in basidiomycetous species such as Rhodotorula rubra and Trichosporon beigelii. Glutamate was a very efficient carbon source for Sc. stipitis, which enabled a high biomass yield/mole, although the growth rate was lower when compared to growth on glucose medium. The cells secreted waste ammonium during growth on glutamate alone. In Sc. stipitis cultures grown in glucose medium containing glutamate as the nitrogen source the biomass yield was maximal, and ethanol concentration and specific ethanol formation rate were significantly higher than in glucose medium containing ammonium as the nitrogen source. Mainly C-assimilation of glutamate but also N-assimilation in glucose-containing medium correlated with enhanced activity of the NAD-dependent glutamate dehydrogenase 2 (GDH2). A Δgdh2 disruptant was unable to utilize glutamate as either a carbon or a nitrogen source; moreover, this disruptant was also unable to utilize aspartate as a carbon source. The mutation was complemented by retransformation of the GDH2 ORF into the Δgdh2 strain. The results show that Gdh2p plays a dual role in Sc. stipitis as both C- and N-catabolic enzyme, which indicates its role as an interface between the carbon and nitrogen metabolism of this yeast.

  7. Pichia guilliermondii

    NASA Astrophysics Data System (ADS)

    Sibirny, Andriy A.; Boretsky, Yuriy R.

    Pichia guilliermondii (asporogenous strains of this species are designated as Candida guilliermondii ) is the model organism of a group so named “ flavinogenic yeasts ” capable of riboflavin oversynthesis during starvation for iron. Besides, some strains of this species efficiently convert xylose to xylitol, an anti-caries sweetener. However, there are also pathogenic C. guilliermondii strains. This species has been used for studying enzymology of riboflavin synthesis due to overproduction of participating enzymes and intermediates under iron-limiting conditions as well as for identification of genes of negative and positive action involved in such a regulation. Besides, P. guilliermondii was used for identification and studying the properties of the systems for active transport of riboflavin in the cell (riboflavin permease) and out of the cell (riboflavin “ excretase ” ). The genetic line of P. guilliermondii with high fertility has been selected and the methods of classic genetics (hybridization and analysis of meiotic segregation) have been developed. More recently, tools for molecular genetic studies of P. guilliermondii have been developed which include collection of host strains, vectors with recessive and dominant markers, several transformation protocols including that for gene knock out. Recently, the genome of this yeast species was sequenced and become publicly available ( http://www.broad.mit.edu )

  8. Efficient Production of l-Lactic Acid from Xylose by Pichia stipitis▿

    PubMed Central

    Ilmén, Marja; Koivuranta, Kari; Ruohonen, Laura; Suominen, Pirkko; Penttilä, Merja

    2007-01-01

    Microbial conversion of renewable raw materials to useful products is an important objective in industrial biotechnology. Pichia stipitis, a yeast that naturally ferments xylose, was genetically engineered for l-(+)-lactate production. We constructed a P. stipitis strain that expressed the l-lactate dehydrogenase (LDH) from Lactobacillus helveticus under the control of the P. stipitis fermentative ADH1 promoter. Xylose, glucose, or a mixture of the two sugars was used as the carbon source for lactate production. The constructed P. stipitis strain produced a higher level of lactate and a higher yield on xylose than on glucose. Lactate accumulated as the main product in xylose-containing medium, with 58 g/liter lactate produced from 100 g/liter xylose. Relatively efficient lactate production also occurred on glucose medium, with 41 g/liter lactate produced from 94 g/liter glucose. In the presence of both sugars, xylose and glucose were consumed simultaneously and converted predominantly to lactate. Lactate was produced at the expense of ethanol, whose production decreased to ∼15 to 30% of the wild-type level on xylose-containing medium and to 70 to 80% of the wild-type level on glucose-containing medium. Thus, LDH competed efficiently with the ethanol pathway for pyruvate, even though the pathway from pyruvate to ethanol was intact. Our results show, for the first time, that lactate production from xylose by a yeast species is feasible and efficient. This is encouraging for further development of yeast-based bioprocesses to produce lactate from lignocellulosic raw material. PMID:17071782

  9. Prediction of CBS tidal evolution

    NASA Astrophysics Data System (ADS)

    Dryomova, G. N.

    The time series of basic processes, accompanying the tidal evolution of star components of Close Binary Systems (CBS) are predicted in the framework of evolutionary stellar models by Claret (2004). The series includes the apsidal motion period, timescale of synchronization of axial rotation of a star with the orbital revolution, the orbit circularization timescale, and the age. Data from the catalogues by Svechnikov & Perevozkina (1999) and by Torres, Andersen, Gimenez (2010) are used for testing the sensitivity of the numerical prediction algorithm.

  10. Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid- and base-pretreated biomass hydrolyzate at high solids loading.

    PubMed

    Slininger, Patricia J; Shea-Andersh, Maureen A; Thompson, Stephanie R; Dien, Bruce S; Kurtzman, Cletus P; Balan, Venkatesh; da Costa Sousa, Leonardo; Uppugundla, Nirmal; Dale, Bruce E; Cotta, Michael A

    2015-01-01

    Lignocellulosic biomass is an abundant, renewable feedstock useful for the production of fuel-grade ethanol via the processing steps of pretreatment, enzyme hydrolysis, and microbial fermentation. Traditional industrial yeasts do not ferment xylose and are not able to grow, survive, or ferment in concentrated hydrolyzates that contain enough sugar to support economical ethanol recovery since they are laden with toxic byproducts generated during pretreatment. Repetitive culturing in two types of concentrated hydrolyzates was applied along with ethanol-challenged xylose-fed continuous culture to force targeted evolution of the native pentose fermenting yeast Scheffersomyces (Pichia) stipitis strain NRRL Y-7124 maintained in the ARS Culture Collection, Peoria, IL. Isolates collected from various enriched populations were screened and ranked based on relative xylose uptake rate and ethanol yield. Ranking on hydrolyzates with and without nutritional supplementation was used to identify those isolates with best performance across diverse conditions. Robust S. stipitis strains adapted to perform very well in enzyme hydrolyzates of high solids loading ammonia fiber expansion-pretreated corn stover (18% weight per volume solids) and dilute sulfuric acid-pretreated switchgrass (20% w/v solids) were obtained. Improved features include reduced initial lag phase preceding growth, significantly enhanced fermentation rates, improved ethanol tolerance and yield, reduced diauxic lag during glucose-xylose transition, and ability to accumulate >40 g/L ethanol in <167 h when fermenting hydrolyzate at low initial cell density of 0.5 absorbance units and pH 5 to 6.

  11. Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid- and base-pretreated biomass hydrolyzate at high solids loading

    DOE PAGES

    Slininger, Patricia J.; Shea-Andersh, Maureen A.; Thompson, Stephanie R.; ...

    2015-04-09

    Lignocellulosic biomass is an abundant, renewable feedstock useful for the production of fuel-grade ethanol via the processing steps of pretreatment, enzyme hydrolysis, and microbial fermentation. Traditional industrial yeasts do not ferment xylose and are not able to grow, survive, or ferment in concentrated hydrolyzates that contain enough sugar to support economical ethanol recovery since they are laden with toxic byproducts generated during pretreatment. Repetitive culturing in two types of concentrated hydrolyzates was applied along with ethanol challenged xylose-fed continuous culture to force targeted evolution of the native pentose fermenting yeast Scheffersomyces (Pichia) stipitis strain NRRL Y-7124 maintained in the ARSmore » Culture Collection, Peoria, IL. Isolates collected from various enriched populations were screened and ranked based on relative xylose uptake rate and ethanol yield. Ranking on hydrolyzates with and without nutritional supplementation was used to identify those isolates with best performance across diverse conditions. Robust S. stipitis strains adapted to perform very well in enzyme hydrolyzates of high solids loading ammonia fiber expansion-pretreated corn stover (18% weight per volume solids) and dilute sulfuric acid-pretreated switchgrass (20% w/v solids) were obtained. Improved features include reduced initial lag phase preceding growth, significantly enhanced fermentation rates, improved ethanol tolerance and yield, reduced diauxic lag during glucose-xylose transition, and ability to accumulate >40 g/L ethanol in <167 h when fermenting hydrolyzate at low initial cell density of 0.5 absorbance units and pH 5 to 6.« less

  12. Draft Genome Sequence of Rubrivivax gelatinosus CBS

    SciTech Connect

    Hu, P. S.; Lang, J.; Wawrousek, K.; Yu, J. P.; Maness, P. C.; Chen, J.

    2012-06-01

    Rubrivivax gelatinosus CBS, a purple nonsulfur photosynthetic bacterium, can grow photosynthetically using CO and N{sub 2} as the sole carbon and nitrogen nutrients, respectively. R. gelatinosus CBS is of particular interest due to its ability to metabolize CO and yield H{sub 2}. We present the 5-Mb draft genome sequence of R. gelatinosus CBS with the goal of providing genetic insight into the metabolic properties of this bacterium.

  13. 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

  14. Pichia hawaiiensis sp. nov., occurring in decaying bark of Charpentiera trees in the Hawaiian archipelago.

    PubMed

    Phaff, H J; Starmer, W T; Kurtzman, C P

    2000-07-01

    A description is given for Pichia hawaiiensis sp. nov., a nitrate-utilizing member of the genus Pichia E. C. Hansen emend. Kurtzman. Seven strains of the new species were isolated during the years 1972, 1973 and 1978 from rotting bark of the Hawaiian tree genera Charpentiera, Pisonia and Cheirodendron. P. hawaiiensis is heterothallic but appears to occur in nature mainly in the diploid state. Asci are deliquescent and produce up to four hat-shaped spores per ascus. Phylogenetic analysis of the 600 nucleotide D1/D2 domain of the 26S rDNA showed that P. hawaiiensis is most closely related to Pichia populi and Williopsis californica (syn. Hansenula californica). The type strain of P. hawaiiensis, isolated on the island of Hawaii from the rotting bark of Charpentiera sp. containing insect larvae, is strain UCD-FST 72-181T (= ATCC MYA-137T = CBS 8760T = NRRL Y-27270T).

  15. Ogataea chonburiensis sp. nov. and Ogataea nakhonphanomensis sp. nov., thermotolerant, methylotrophic yeast species isolated in Thailand, and transfer of Pichia siamensis and Pichia thermomethanolica to the genus Ogataea.

    PubMed

    Limtong, Savitree; Srisuk, Nantana; Yongmanitchai, Wichien; Yurimoto, Hiroya; Nakase, Takashi

    2008-01-01

    Two thermotolerant, methylotrophic yeast strains, PT44(T) and S051(T), were respectively isolated from a tree exudate and soil collected in Thailand. They were categorized as thermotolerant strains on the basis of their good growth below 20 degrees C and up to a relatively high temperature (37 degrees C). The major characteristics of the two strains that place them in the genus Ogataea are the formation of four helmet- or hat-shaped ascospores in a deliquescent ascus that may be produced parthenogenetically or by conjugation between a cell and its bud or between independent cells; multilateral budding; assimilation of nitrate; the presence of ubiquinone Q7; negative for Diazonium blue B colour and urease reactions; and the absence of arthroconidia and ballistoconidia. Analysis of the D1/D2 domains of the large-subunit rDNA sequence revealed that strain PT44(T) was differentiated from the strain S051(T) by 25 nucleotide substitutions and 1 gap in 554 nt, which was sufficient to justify the description of two separate species. The closest recognized species in terms of pairwise sequences similarity to PT44(T) was Pichia (Ogataea) dorogensis, with 13 nucleotide substitutions and 1 gap in 554 nt. Strain S051(T) was closest to Pichia thermomethanolica, with 7 nucleotide substitutions in 566 nt. Phenotypic characteristics of strains PT44(T) and S051(T) allowed them to be differentiated from each other and from the closest related species. On the basis of the above finding, the two strains represent two novel species of the genus Ogataea, for which the names Ogataea chonburiensis sp. nov. (type strain PT44(T) =BCC 21227(T) =NBRC 101965(T) =CBS 10363(T)) and Ogataea nakhonphanomensis sp. nov. (type strain S051(T) =BCC 21228(T) =NBRC 101966(T) =CBS 10362(T)) are proposed. We also propose the transfer of two thermotolerant methylotrophic members of the genus Pichia described previously to the genus Ogataea: Pichia siamensis is renamed Ogataea siamensis (Limtong, Srisuk

  16. Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid- and base-pretreated biomass hydrolyzate at high solids loading

    SciTech Connect

    Slininger, Patricia J.; Shea-Andersh, Maureen A.; Thompson, Stephanie R.; Dien, Bruce S.; Kurtzman, Cletus P.; Balan, Venkatesh; da Costa Sousa, Leonardo; Uppugundla, Nirmal; Dale, Bruce E; Cotta, Michael A

    2015-04-09

    Lignocellulosic biomass is an abundant, renewable feedstock useful for the production of fuel-grade ethanol via the processing steps of pretreatment, enzyme hydrolysis, and microbial fermentation. Traditional industrial yeasts do not ferment xylose and are not able to grow, survive, or ferment in concentrated hydrolyzates that contain enough sugar to support economical ethanol recovery since they are laden with toxic byproducts generated during pretreatment. Repetitive culturing in two types of concentrated hydrolyzates was applied along with ethanol challenged xylose-fed continuous culture to force targeted evolution of the native pentose fermenting yeast Scheffersomyces (Pichia) stipitis strain NRRL Y-7124 maintained in the ARS Culture Collection, Peoria, IL. Isolates collected from various enriched populations were screened and ranked based on relative xylose uptake rate and ethanol yield. Ranking on hydrolyzates with and without nutritional supplementation was used to identify those isolates with best performance across diverse conditions. Robust S. stipitis strains adapted to perform very well in enzyme hydrolyzates of high solids loading ammonia fiber expansion-pretreated corn stover (18% weight per volume solids) and dilute sulfuric acid-pretreated switchgrass (20% w/v solids) were obtained. Improved features include reduced initial lag phase preceding growth, significantly enhanced fermentation rates, improved ethanol tolerance and yield, reduced diauxic lag during glucose-xylose transition, and ability to accumulate >40 g/L ethanol in <167 h when fermenting hydrolyzate at low initial cell density of 0.5 absorbance units and pH 5 to 6.

  17. Pichia garciniae sp. nov., isolated from a rotten mangosteen fruit (Garcinia mangostana L., Clusiaceae).

    PubMed

    Bhadra, Bhaskar; Begum, Zareena; Shivaji, Sisinthy

    2008-11-01

    Ascogenous yeasts were isolated from a decaying mangosteen fruit (Garcinia mangostana L., Clusiaceae). Based on colony morphology and RAPD analysis, the strains were grouped into four groups (G-I, G-II, G-III and G-IV). Phenotypic properties and analysis of the D1/D2 domain sequence of the 26S rRNA gene identified representative members of groups G-II, G-III and G-IV as Pichia manshurica (=Pichia galeiformis), Pichia fermentans and Pichia anomala, respectively. Representatives of group G-I, YS110(T) and YS111, showed similar phenotypic traits, 99.9 % similarity in the sequence of the D1/D2 domain of the 26S rRNA gene and ITS1-5.8S rRNA gene-ITS2 sequence and 92 % DNA-DNA relatedness. Hence, YS110(T) and YS111 are regarded as members of the same species. Based on D1/D2 domain and ITS region sequences, the nearest phylogenetic neighbours of YS110(T) and YS111 were identified as Pichia membranifaciens and P. manshurica. However, YS110(T) differs from these two nearest phylogenetic neighbours by >3 % in the D1/D2 domain sequence and by >18 % with respect to the ITS region sequence. In addition, YS110(T) and YS111 differ from P. membranifaciens NRRL Y-2026(T) and P. manshurica NRRL Y-27978(T) with respect to a number of phenotypic traits. The strains show 52-57 % DNA-DNA relatedness with P. membranifaciens NRRL Y-2026(T) and P. manshurica NRRL Y-27978(T). Strains YS110(T) and YS111 are proposed as two strains of a novel species, for which the name Pichia garciniae sp. nov. is proposed. The type strain is YS110(T) (=NRRL Y-48422(T) =CBS 10758(T)).

  18. Simultaneous saccharification and ethanol fermentation of oxalic acid pretreated corncob assessed with response surface methodology

    Treesearch

    Jae-Won Lee; Rita C.L.B. Rodrigues; Thomas W. Jeffries

    2009-01-01

    Response surface methodology was used to evaluate optimal time, temperature and oxalic acid concentration for simultaneous saccharification and fermentation (SSF) of corncob particles by Pichia stipitis CBS 6054. Fifteen different conditions for pretreatment were examined in a 23 full factorial design with six axial points. Temperatures ranged from 132 to 180º...

  19. Pichia dushanensis sp. nov. and Hyphopichia paragotoi sp. nov., two sexual yeast species associated with insects and rotten wood.

    PubMed

    Ren, Yong-Cheng; Liu, Si-Tong; Li, Ying; Hui, Feng-Li

    2015-09-01

    Seven yeast strains were isolated from the gut of insect larvae and decayed wood, which were collected from three localities near Nanyang, Henan Province, China. These strains were identified as two novel species through comparison of sequences in the D1/D2 domains of the large subunit (LSU) rRNA gene and other taxonomic characteristics. Pichia dushanensis sp. nov. was closely related to species in the Pichia clade and produced one to four spheroid ascospores in a deliquescent ascus. The D1/D2 sequence of P. dushanensis sp. nov. differed from its closest relative, Issatchenkia (Pichia) sp. NRRL Y-12824, by 3.6% sequence divergence (16 substitutions and 4 gaps). The species also differed from its four closest known species, Candida rugopelliculosa, Pichia occidentalis, Pichia exigua and Candida phayaonensis, by 4.1-4.4% sequence divergence (22-24 substitutions and 0-2 gaps) in the D1/D2 sequences. Hyphopichia paragotoi sp. nov. belonged to the Hyphopichia clade, and its nearest phylogenetic neighbours were Candida gotoi, Candida pseudorhagii, Candida rhagii and Hyphopichia heimii with 3.2-4.2% sequence divergence (16-21 substitutions and 1 gap) in the D1/D2 sequences. In comparison with previously established species, H. paragotoi sp. nov. formed one hat-shaped ascospore in a persistent ascus. The type strain of P. dushanensis sp. nov. is NYNU 14658(T) ( = CICC 33049(T) = CBS 13912(T)), and the type strain of H. paragotoi sp. nov. is NYNU 14666(T) ( = CICC 33048(T) = CBS 13913(T)).

  20. Pichia thermomethanolica sp. nov., a novel thermotolerant, methylotrophic yeast isolated in Thailand.

    PubMed

    Limtong, Savitree; Srisuk, Nantana; Yongmanitchai, Wichien; Yurimoto, Hiroya; Nakase, Takashi; Kato, Nobuo

    2005-09-01

    Three strains (N002, N069 and PT31(T)) of a novel thermotolerant methylotrophic yeast species belonging to the genus Pichia were isolated from soil collected in Thailand by three consecutive enrichments in methanol broth at room temperature. They were categorized as thermotolerant strains on the basis of their good growth below 20 degrees C and up to a high temperature (37 degrees C). The major characteristics of the three strains included the following and placed them in the genus Pichia: the formation of four helmet-/hat-shaped ascospores in a deliquescent ascus that might be unconjugated or produced by conjugation between a cell and its bud or between independent cells; multilateral budding; the presence of ubiquinone Q-7; negative for Diazonium blue B colour and urease reactions; and the absence of arthrospores and ballistospores. The three strains differed by one to three nucleotide substitutions in the sequences of the D1/D2 domain of the large-subunit rDNA sequence. Phylogenetic analysis revealed that their closest species was Pichia dorogensis, but with 11-13 nucleotide substitutions in 554 nt. The phenotypic characteristics of the three strains were the same. The strains could be distinguished from P. dorogensis by a number of phenotypic characteristics. On the basis of the above findings, these three strains were assigned to a single novel species of Pichia, for which the name Pichia thermomethanolica sp. nov. is proposed. The type strain is PT31(T) (=BCC 16875(T)=JCM 12984(T)=CBS 10098(T)).

  1. A new source of resistance to 2-furaldehyde from Scheffersomyces (Pichia) stipitis for sustainable lignocellulose-to-biofuel conversion

    USDA-ARS?s Scientific Manuscript database

    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 ...

  2. CBS domains: structure, function, and pathology in human proteins.

    PubMed

    Ignoul, Sofie; Eggermont, Jan

    2005-12-01

    The cystathionine-beta-synthase (CBS) domain is an evolutionarily conserved protein domain that is present in the proteome of archaebacteria, prokaryotes, and eukaryotes. CBS domains usually come in tandem repeats and are found in cytosolic and membrane proteins performing different functions (metabolic enzymes, kinases, and channels). Crystallographic studies of bacterial CBS domains have shown that two CBS domains form an intramolecular dimeric structure (CBS pair). Several human hereditary diseases (homocystinuria, retinitis pigmentosa, hypertrophic cardiomyopathy, myotonia congenital, etc.) can be caused by mutations in CBS domains of, respectively, cystathionine-beta-synthase, inosine 5'-monophosphate dehydrogenase, AMP kinase, and chloride channels. Despite their clinical relevance, it remains to be established what the precise function of CBS domains is and how they affect the structural and/or functional properties of an enzyme, kinase, or channel. Depending on the protein in which they occur, CBS domains have been proposed to affect multimerization and sorting of proteins, channel gating, and ligand binding. However, recent experiments revealing that CBS domains can bind adenosine-containing ligands such ATP, AMP, or S-adenosylmethionine have led to the hypothesis that CBS domains function as sensors of intracellular metabolites.

  3. Pichia bruneiensis sp. nov., a biofilm-producing dimorphic yeast species isolated from flowers in Borneo.

    PubMed

    Sipiczki, Matthias

    2012-12-01

    Taxonomic analysis of five yeast strains isolated from Hibiscus flowers in Brunei (Borneo) is described. The strains represent a dimorphic, biofilm-producing, anamorphic budding yeast species for which the name Pichia bruneiensis is proposed. P. bruneiensis alternates between yeast and pseudohyphal modes of growth. The pseudohyphae form biofilms on the surface of liquid media and penetrate into solid substrates. The sequences of the D1/D2 domains of the large subunit rRNA genes, the internal transcribed spacer (ITS) regions and the 18S rRNA genes were identical in the five strains and indicated a close phylogenetic relationship with teleomorph species of the genus Pichia. In a phylogenetic analysis of these sequences, the closest relative of the new species was Pichia fermentans (6% nucleotide substitutions and indels in the D1/D2 domain). The type strain is 11-485(T) and has been deposited in the Centralbureau voor Schimmelcultures (Utrecht, the Netherlands) as CBS 12611(T), the National Collection of Agricultural and Industrial Micro-organisms (Budapest, Hungary) as NCAIM Y.02019(T) and the Culture Collection of Yeasts (Bratislava, Slovakia) as CCY 29-189-1(T). Mycobank no. MB800537.

  4. Evaluation of UV-C mutagenized Scheffersomyces stipitis strains for ethanol production

    USDA-ARS?s Scientific Manuscript database

    We evaluated fermentation capabilities of five strains of Scheffersomyces stipitis (WT-2-1, WT-1-11, 14-2-6, 22-1-1, and 22-1-12) that had been produced by UV-C mutagenesis and selection for improved xylose fermentation to ethanol using an integrated automated robotic work cell. They were incubated ...

  5. Characterization of a S-adenosyl-l-methionine (SAM)-accumulating strain of Scheffersomyces stipitis.

    PubMed

    Križanović, Stela; Butorac, Ana; Mrvčić, Jasna; Krpan, Maja; Cindrić, Mario; Bačun-Družina, Višnja; Stanzer, Damir

    2015-06-01

    S-adenosyl-l-methionine (SAM) is an important molecule in the cellular metabolism of mammals. In this study, we examined several of the physiological characteristics of a SAM-accumulating strain of the yeast Scheffersomyces stipitis (M12), including SAM production, ergosterol content, and ethanol tolerance. S. stipitis M12 accumulated up to 52.48 mg SAM/g dry cell weight. Proteome analyses showed that the disruption of C-24 methylation in ergosterol biosynthesis, a step mediated by C-24 sterol methyltransferase (Erg6p), results in greater SAM accumulation by S. stipitis M12 compared to the wild-type strain. A comparative proteome-wide analysis identified 25 proteins that were differentially expressed by S. stipitis M12. These proteins are involved in ribosome biogenesis, translation, the stress response, ubiquitin-dependent catabolic processes, the cell cycle, ethanol tolerance, posttranslational modification, peroxisomal membrane stability, epigenetic regulation, the actin cytoskeleton and cell morphology, iron and copper homeostasis, cell signaling, and energy metabolism. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

  6. Scheffersomyces stipitis: a comparative systems biology study with the Crabtree positive yeast Saccharomyces cerevisiae

    PubMed Central

    2012-01-01

    Background Scheffersomyces stipitis is a Crabtree negative yeast, commonly known for its capacity to ferment pentose sugars. Differently from Crabtree positive yeasts such as Saccharomyces cerevisiae, the onset of fermentation in S. stipitis is not dependent on the sugar concentration, but is regulated by a decrease in oxygen levels. Even though S. stipitis has been extensively studied due to its potential application in pentoses fermentation, a limited amount of information is available about its metabolism during aerobic growth on glucose. Here, we provide a systems biology based comparison between the two yeasts, uncovering the metabolism of S. stipitis during aerobic growth on glucose under batch and chemostat cultivations. Results Starting from the analysis of physiological data, we confirmed through 13C-based flux analysis the fully respiratory metabolism of S. stipitis when growing both under glucose limited or glucose excess conditions. The patterns observed showed similarity to the fully respiratory metabolism observed for S. cerevisiae under chemostat cultivations however, intracellular metabolome analysis uncovered the presence of several differences in metabolite patterns. To describe gene expression levels under the two conditions, we performed RNA sequencing and the results were used to quantify transcript abundances of genes from the central carbon metabolism and compared with those obtained with S. cerevisiae. Interestingly, genes involved in central pathways showed different patterns of expression, suggesting different regulatory networks between the two yeasts. Efforts were focused on identifying shared and unique families of transcription factors between the two yeasts through in silico transcription factors analysis, suggesting a different regulation of glycolytic and glucoenogenic pathways. Conclusions The work presented addresses the impact of high-throughput methods in describing and comparing the physiology of Crabtree positive and Crabtree

  7. Scheffersomyces stipitis: a comparative systems biology study with the Crabtree positive yeast Saccharomyces cerevisiae.

    PubMed

    Papini, Marta; Nookaew, Intawat; Uhlén, Mathias; Nielsen, Jens

    2012-10-09

    Scheffersomyces stipitis is a Crabtree negative yeast, commonly known for its capacity to ferment pentose sugars. Differently from Crabtree positive yeasts such as Saccharomyces cerevisiae, the onset of fermentation in S. stipitis is not dependent on the sugar concentration, but is regulated by a decrease in oxygen levels. Even though S. stipitis has been extensively studied due to its potential application in pentoses fermentation, a limited amount of information is available about its metabolism during aerobic growth on glucose. Here, we provide a systems biology based comparison between the two yeasts, uncovering the metabolism of S. stipitis during aerobic growth on glucose under batch and chemostat cultivations. Starting from the analysis of physiological data, we confirmed through 13C-based flux analysis the fully respiratory metabolism of S. stipitis when growing both under glucose limited or glucose excess conditions. The patterns observed showed similarity to the fully respiratory metabolism observed for S. cerevisiae under chemostat cultivations however, intracellular metabolome analysis uncovered the presence of several differences in metabolite patterns. To describe gene expression levels under the two conditions, we performed RNA sequencing and the results were used to quantify transcript abundances of genes from the central carbon metabolism and compared with those obtained with S. cerevisiae. Interestingly, genes involved in central pathways showed different patterns of expression, suggesting different regulatory networks between the two yeasts. Efforts were focused on identifying shared and unique families of transcription factors between the two yeasts through in silico transcription factors analysis, suggesting a different regulation of glycolytic and glucoenogenic pathways. The work presented addresses the impact of high-throughput methods in describing and comparing the physiology of Crabtree positive and Crabtree negative yeasts. Based on

  8. Asteroseismology of the DBV star CBS 114

    NASA Astrophysics Data System (ADS)

    Chen, Yan-Hui

    2016-08-01

    Asteroseismology is a unique and powerful tool to investigate the internal structure of stars. CBS 114 is the sixth known pulsating DBV star. It was observed by Handler, Metcalfe, & Wood at the South African Astronomical Observatory over three weeks in 2001. Then, it was observed by Metcalfe et al. for seven nights (2004 Feb. 19-25) on the 1.8 m telescope at the Bohyunsan Optical Astronomy Observatory and seven nights (2004 Feb. 21-27) on the 2.1 m telescope at the McDonald Observatory. Totally two triplets, four doublets, and five singlets were identified. The frequency splitting values are very different, from 5.2 μHz to 11.9 μHz, which may reflect differential rotations. We evolve grids of white dwarf models by MESA. Cores, added with He/C envelopes, of those white dwarf models are inserted into WDEC to evolve grids of DBV star models. With those DBV star models, we calculate eigenperiods. Those calculated periods are used to fit observed periods. A best-fitting model is selected. The parameters are T eff = 25000 K, M * = 0.740 M ⊙ and log(M He/M *) = —4.5. With the relatively large stellar mass, the effective temperature is close to the previous spectroscopic result. In addition, kinetic energy distributions are calculated for the best-fitting model. We find that the observed modes with large frequency splitting values are fitted by the calculated modes with a large amount of kinetic energy distributed in the C/O core. After preliminary analysis, we suggest that the C/O core may rotate at least two times faster than the helium layer for CBS 114.

  9. The Child Behavior Scale (CBS) Revisited: A Longitudinal Evaluation of CBS Subscales with Children, Preadolescents, and Adolescents

    ERIC Educational Resources Information Center

    Ladd, Gary W.; Herald-Brown, Sarah L.; Andrews, Rebecca K.

    2009-01-01

    The Child Behavior Scale (CBS) is a teacher-report instrument that was developed over a decade ago as an alternative to more costly methods for assessing children's behavior and peer relations in school contexts. This investigation was undertaken to clarify how well the CBS achieves its aims with a broader age spectrum of youth (6- to…

  10. Occurrence and diversity of Pichia spp. in marine environments

    NASA Astrophysics Data System (ADS)

    Li, Jing; Chi, Zhenming; Wang, Xianghong; Wang, Lin; Sheng, Jun; Gong, Fang

    2008-08-01

    A total of 328 yeast strains from seawater, sediments, mud of salterns, the guts of marine fish and marine algae were obtained. The results of routine identification and molecular methods show that five yeast strains obtained in this study belonged to Pichia spp., including Pichia guilliermondii 1uv-small, Pichia ohmeri YF04d, Pichia fermentans YF12b, Pichia burtonii YF11A and Pichia anomala YF07b. Further studies revealed that Pichia anomala YF07b could produce killer toxin against pathogenic yeasts in crabs while Pichia guilliermondii 1uv-small could produce high activity of extracellular inulinase. It is advisable to test if Pichia ohmeri YF04d obtained in this study is related to central-venous-catheter-associated infection.

  11. RNA-Seq of the xylose-fermenting yeast Scheffersomyces stipitis cultivated in glucose or xylose.

    PubMed

    Yuan, Tiezheng; Ren, Yan; Meng, Kun; Feng, Yun; Yang, Peilong; Wang, Shaojing; Shi, Pengjun; Wang, Lei; Xie, Daoxin; Yao, Bin

    2011-12-01

    Xylose is the second most abundant lignocellulosic component besides glucose, but it cannot be fermented by the widely used ethanol-producing yeast Saccharomyces cerevisiae. The yeast Scheffersomyces stipitis, however, is well known for its high native capacity to ferment xylose. Here, we applied next-generation sequencing technology for RNA (RNA-Seq) to generate two high-resolution transcriptional maps of the S. stipitis genome when this yeast was grown using glucose or xylose as the sole carbon source. RNA-Seq revealed that 5,176 of 5,816 annotated open reading frames had a uniform transcription and that 214 open reading frames were differentially transcribed. Differential expression analysis showed that, compared with other biological processes, carbohydrate metabolism and oxidation-reduction reactions were highly enhanced in yeast grown on xylose. Measurement of metabolic indicators of fermentation showed that, in yeast grown on xylose, the concentrations of cysteine and ornithine were twofold higher and the concentrations of unsaturated fatty acids were also increased. Analysis of metabolic profiles coincided with analysis of certain differentially expressed genes involved in metabolisms of amino acid and fatty acid. In addition, we predicted protein-protein interactions of S. stipitis through integration of gene orthology and gene expression. Further analysis of metabolic and protein-protein interactions networks through integration of transcriptional and metabolic profiles predicted correlations of genes involved in glycolysis, the tricarboxylic acid cycle, gluconeogenesis, sugar uptake, amino acid metabolism, and fatty acid β-oxidation. Our study reveals potential target genes for xylose fermentation improvement and provides insights into the mechanisms underlying xylose fermentation in S. stipitis.

  12. Evaluation of CBS 600 carburized steel as a gear material

    NASA Technical Reports Server (NTRS)

    Townsend, D. P.; Parker, R. J.; Zaretsky, E. V.

    1979-01-01

    Gear endurance tests were conducted with one lot of consumable-electrode vacuum-melted (CVM) AISI 9310 gears and one lot of air-melt CBS 600 gears. The gears were 8 pitch with a pitch diameter of 8.89 centimeters (3.5 in.). Bench-type rolling-element fatigue tests were also conducted with one lot of CVM AISI 9310, three lots of CVM CBS 600, and one of air-melt CBS 600 material. The rolling-element bars were 0.952 centimeter (0.375 in.) in diameter. The CBS 600 material exhibited pitting fatigue lives in both rolling-element specimens and gears at least equivalent to that of CVM AISI 9310. Tooth fracture failure occurred with the CBS 600 gears after overrunning a fatigue spall, but it did not occur with the CVM AISI 9310 gears. Tooth fracture in the CBS 600 was attributed to excessive carbon content in the case, excessive case depth, and a higher than normal core hardness.

  13. Lack of global epigenetic methylation defects in CBS deficient mice.

    PubMed

    Lee, Hyung-Ok; Wang, Liqun; Kuo, Yin-Ming; Gupta, Sapna; Slifker, Michael J; Li, Yue-Sheng; Andrews, Andrew J; Kruger, Warren D

    2017-01-01

    Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of metabolism in which patients have extremely elevated plasma total homocysteine and have clinical manifestations in the vascular, visual, skeletal, and nervous systems. Homocysteine is an intermediary metabolite produced from the hydrolysis of S-adenosylhomocysteine (SAH), which is a by-product of methylation reactions involving the methyl-donor S-adenosylmethionine (SAM). Here, we have measured SAM, SAH, DNA and histone methylation status in an inducible mouse model of CBS deficiency to test the hypothesis that homocysteine-related phenotypes are caused by inhibition of methylation due to elevated SAH and reduced SAM/SAH ratio. We found that mice lacking CBS have elevated cellular SAH and reduced SAM/SAH ratios in both liver and kidney, but this was not associated with alterations in the level of 5-methylcytosine or various histone modifications. Using methylated DNA immunoprecipitation in combination with microarray, we found that of the 241 most differentially methylated promoter probes, 89 % were actually hypermethylated in CBS deficient mice. In addition, we did not find that changes in DNA methylation correlated well with changes in RNA expression in the livers of induced and uninduced CBS mice. Our data indicates that reduction in the SAM/SAH ratio, due to loss of CBS activity, does not result in overall hypomethylation of either DNA or histones.

  14. Bioethanol production from mixed sugars by Scheffersomyces stipitis free and immobilized cells, and co-cultures with Saccharomyces cerevisiae.

    PubMed

    De Bari, Isabella; De Canio, Paola; Cuna, Daniela; Liuzzi, Federico; Capece, Angela; Romano, Patrizia

    2013-09-25

    Bioethanol can be produced from several biomasses including lignocellulosic materials. Besides 6-carbon sugars that represent the prevalent carbohydrates, some of these feedstocks contain significant amounts of 5-carbon sugars. One common limit of the major part of the xylose-fermenting yeasts is the diauxic shift between the uptake of glucose and xylose during the fermentation of mixed syrups. Thus, optimized fermentation strategies are required. In this paper the ability of Scheffersomyces stipitis strain NRRLY-11544 to ferment mixed syrups with a total sugar concentration in the range 40-80 g/L was investigated by using mono cultures, co-cultures with Saccharomyces cerevisiae strain Bakers Yeast Type II and single cultures immobilized in silica-hydrogel films. The experimental design for the fermentations with immobilized cells included the process analysis in function of two parameters: the fraction of the gel in the broth and the concentration of the cells loaded in the gel. Furthermore, for each total sugars level, the fermentative course of S. stipitis was analyzed at several glucose-to xylose ratios. The results indicated that the use of S. stipitis and S. cerevisiae in free co-cultures ensured faster processes than single cultures of S. stipitis either free or immobilized. However, the rapid production of ethanol by S. cerevisiae inhibited S. stipitis and caused a stuck of the process. Immobilization of S. stipitis in silica-hydrogel increased the relative consumption rate of xylose-to-glucose by 2-6 times depending on the composition of the fermentation medium. Furthermore the films performances appeared stable over three weeks of continuous operations. However, on the whole, the final process yields obtained with the immobilized cells were not meaningfully different from that of the free cells. This was probably due to concurrent fermentations operated by the cells released in the broth. Optimization of the carrier characteristics could improve the

  15. 21 CFR 573.750 - Pichia pastoris dried yeast.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.750 Pichia pastoris dried yeast. (a) Identity. The food additive Pichia... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Pichia pastoris dried yeast. 573.750 Section 573...

  16. 21 CFR 573.750 - Pichia pastoris dried yeast.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.750 Pichia pastoris dried yeast. (a) Identity. The food additive Pichia... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Pichia pastoris dried yeast. 573.750 Section 573...

  17. 21 CFR 573.750 - Pichia pastoris dried yeast.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.750 Pichia pastoris dried yeast. (a) Identity. The food additive Pichia... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Pichia pastoris dried yeast. 573.750 Section 573...

  18. Organization of specific genomic regions of Zygosaccharomyces rouxii and Pichia sorbitophila: comparison with Saccharomyces cerevisiae.

    PubMed

    Sychrova, H; Braun, V; Potier, S; Souciet, J L

    2000-11-01

    The genomes of Zygosaccharomyces rouxii and Pichia sorbitophila were partially explored. The genome of Z. rouxii CBS 732 consists of seven chromosomes with an approximate size of 1.0-2.75 Mb, 12.8 Mb in total. Five of the chromosomes were labelled with specific probes. Three Z. rouxii genomic DNA fragments were sequenced; all 10 ORFs found were without introns and they have homologues in S. cerevisiae. Gene order comparison revealed that the organization is partially conserved in both species. The genome of P. sorbitophila CBS 7064 consists of seven chromosomes with an approximate size of 1.0-2.9 Mb, 13.9 Mb in total. Three of the chromosomes were labelled with specific probes. The sequencing of a 5.2 kb genomic DNA fragment revealed three ORFs, but no conservation of their organization was found, although all of them have their respective homologues in S. cerevisiae. According to our results, the presence of two overlapping ORFs in S. cerevisiae (YJL107c-YJL108c) could be interpreted as the result of a frameshift mutation.

  19. Comparative Xylose Metabolism among the Ascomycetes C. albicans, S. stipitis and S. cerevisiae

    PubMed Central

    Lépine, Guylaine; Askew, Chris; Raymond, Martine; Whiteway, Malcolm; Wu, Cunle

    2013-01-01

    The ascomycetes Candida albicans, Saccharomyces cerevisiae and Scheffersomyces stipitis metabolize the pentose sugar xylose very differently. S. cerevisiae fails to grow on xylose, while C. albicans can grow, and S. stipitis can both grow and ferment xylose to ethanol. However, all three species contain highly similar genes that encode potential xylose reductases and xylitol dehydrogenases required to convert xylose to xylulose, and xylulose supports the growth of all three fungi. We have created C. albicans strains deleted for the xylose reductase gene GRE3, the xylitol dehydrogenase gene XYL2, as well as the gre3 xyl2 double mutant. As expected, all the mutant strains cannot grow on xylose, while the single gre3 mutant can grow on xylitol. The gre3 and xyl2 mutants are efficiently complemented by the XYL1 and XYL2 from S. stipitis. Intriguingly, the S. cerevisiae GRE3 gene can complement the Cagre3 mutant, while the ScSOR1 gene can complement the Caxyl2 mutant, showing that S. cerevisiae contains the enzymatic capacity for converting xylose to xylulose. In addition, the gre3 xyl2 double mutant of C. albicans is effectively rescued by the xylose isomerase (XI) gene of either Piromyces or Orpinomyces, suggesting that the XI provides an alternative to the missing oxido-reductase functions in the mutant required for the xylose-xylulose conversion. Overall this work suggests that C. albicans strains engineered to lack essential steps for xylose metabolism can provide a platform for the analysis of xylose metabolism enzymes from a variety of species, and confirms that S. cerevisiae has the genetic potential to convert xylose to xylulose, although non-engineered strains cannot proliferate on xylose as the sole carbon source. PMID:24236198

  20. [Expression of Vitreoscilla hemoglobin improves recombinant lipase production in Pichia pastoris].

    PubMed

    Wang, Xiaofeng; Sun, Yongchuan; Shen, Xuguang; Ke, Feng; Xu, Li; Liu, Yun; Yan, Yunjun

    2011-12-01

    Yarrowia lipolytica lipase Lip2 (YlLip2) is an important industrial enzyme with many potential applications. To alleviate the dissolved oxygen (DO) limitation and improve YlLip2 production during high-cell density fermentation, the YlLip2 gene lip2 and Vitreoscilla hemoglobin (VHb) gene vgb were co-expressed in Pichiapastoris under the control of AOX1 and PsADH2 promoter, respectively. The PsADH2 promoter from Pichia stipitis could be activated under oxygen limitation. The SDS-PAGE and CO-difference spectrum analysis indicated that VHb and YlLip2 had successfully co-expressed in recombinant strains. Compared with the control cells (VHb-, GS115/9Klip2), the expression levels of YlLip2 in VHb-expressing cells (VHb+, GS115/9Klip2-pZPVT) under oxygen limitation were improved 25% in shake-flask culture and 83% in a 10 L fermentor. Moreover, the VHb+ cells displayed higher biomass than VHb- cells at lower DO levels in a 10 L fermentor. In this study, we also achieved a VHb-expressing clone harboring multicopy lip2 gene (GS115/9Klip2-pZPVTlip2 49#), which showed the maximum lipolytic activity of 33 900 U/mL in a 10 L fermentor under lower DO conditions. Therefore, it can be seen that expression of VHb with PsADH2 promoter in P. pastoris combined with increasing copies of lip2 gene is an effective strategy to improve YlLip2 production.

  1. Impact of pseudo-continuous fermentation on the ethanol tolerance of Scheffersomyces stipitis.

    PubMed

    Liang, Meng; Kim, Min Hea; He, Qinghua Peter; Wang, Jin

    2013-09-01

    In this work we conducted the pseudo-continuous fermentation, i.e., continuous fermentation with cell retention, using Scheffersomyces stipitis, and studied its effect on ethanol tolerance of the strain. During the fermentation experiments, S. stipitis was adapted to a mild concentration of ethanol (20-26 g/L) for two weeks. Two substrates (glucose and xylose) were used in different fermentation experiments. After fermentation, various experiments were performed to evaluate the ethanol tolerance of adapted cells and unadapted cells. Compared to the unadapted cells, the viability of adapted cells increased by 8 folds with glucose as the carbon source and 6 folds with xylose as the carbon source following exposure to 60 g/L ethanol for 2 h. Improved ethanol tolerance of the adapted cells was also revealed in the effects of ethanol on plasma membrane permeability, extracellular alkalization and acidification. The mathematical modeling of cell leakage, extracellular alkalization and acidification revealed that cells cultured on glucose show better ethanol tolerance than cells cultured on xylose but the differences become smaller for adapted cells. The results show that pseudo-continuous fermentation can effectively improve cell's ethanol tolerance due to the environmental pressure during the fermentation process. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. FUS and TDP43 genetic variability in FTD and CBS.

    PubMed

    Huey, Edward D; Ferrari, Raffaele; Moreno, Jorge H; Jensen, Christopher; Morris, Christopher M; Potocnik, Felix; Kalaria, Rajesh N; Tierney, Michael; Wassermann, Eric M; Hardy, John; Grafman, Jordan; Momeni, Parastoo

    2012-05-01

    This study aimed to evaluate genetic variability in the FUS and TDP-43 genes, known to be mainly associated with amyotrophic lateral sclerosis (ALS), in patients with the diagnoses of frontotemporal lobar degeneration (FTLD) and corticobasal syndrome (CBS). We screened the DNA of 228 patients for all the exons and flanking introns of FUS and TDP-43 genes. We identified 2 novel heterozygous missense mutations in FUS: P106L (g.22508384C>T) in a patient with behavioral variant frontotemporal dementia (bvFTD) and Q179H in several members of a family with behavioral variant FTD. We also identified the N267S mutation in TDP-43 in a CBS patient, previously only reported in 1 ALS family and 1 FTD patient. Additionally, we identified 2 previously reported heterozygous insertion and deletion mutations in Exon 5 of FUS; Gly174-Gly175 del GG (g. 4180-4185 delGAGGTG) in an FTD patient and Gly175-Gly176 ins GG (g. 4185-4186 insGAGGTG) in a patient with diagnosis of CBS. Not least, we have found a series of variants in FUS also in neurologically normal controls. In summary, we report that genetic variability in FUS and TDP-43 encompasses a wide range of phenotypes (including ALS, FTD, and CBS) and that there is substantial genetic variability in FUS gene in neurologically normal controls.

  3. User's manual for CBS3DS, version 1.0

    NASA Technical Reports Server (NTRS)

    Reddy, C. J.; Deshpande, M. D.

    1995-01-01

    CBS3DS is a computer code written in FORTRAN 77 to compute the backscattering radar cross section of cavity backed apertures in infinite ground plane and slots in thick infinite ground plane. CBS3DS implements the hybrid Finite Element Method (FEM) and Method of Moments (MoM) techniques. This code uses the tetrahedral elements, with vector edge basis functions for FEM in the volume of the cavity/slot and the triangular elements with the basis functions for MoM at the apertures. By virtue of FEM, this code can handle any arbitrarily shaped three-dimensional cavities filled with inhomogeneous lossy materials; due to MoM, the apertures can be of any arbitrary shape. The User's Manual is written to make the user acquainted with the operation of the code. The user is assumed to be familiar with the FORTRAN 77 language and the operating environment of the computer the code is intended to run.

  4. User's manual for CBS3DS, version 1.0

    NASA Astrophysics Data System (ADS)

    Reddy, C. J.; Deshpande, M. D.

    1995-10-01

    CBS3DS is a computer code written in FORTRAN 77 to compute the backscattering radar cross section of cavity backed apertures in infinite ground plane and slots in thick infinite ground plane. CBS3DS implements the hybrid Finite Element Method (FEM) and Method of Moments (MoM) techniques. This code uses the tetrahedral elements, with vector edge basis functions for FEM in the volume of the cavity/slot and the triangular elements with the basis functions for MoM at the apertures. By virtue of FEM, this code can handle any arbitrarily shaped three-dimensional cavities filled with inhomogeneous lossy materials; due to MoM, the apertures can be of any arbitrary shape. The User's Manual is written to make the user acquainted with the operation of the code. The user is assumed to be familiar with the FORTRAN 77 language and the operating environment of the computer the code is intended to run.

  5. 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

  6. An efficient extrapolation to the (T)/CBS limit

    NASA Astrophysics Data System (ADS)

    Ranasinghe, Duminda S.; Barnes, Ericka C.

    2014-05-01

    We extrapolate to the perturbative triples (T)/complete basis set (CBS) limit using double ζ basis sets without polarization functions (Wesleyan-1-Triples-2ζ or "Wes1T-2Z") and triple ζ basis sets with a single level of polarization functions (Wesleyan-1-Triples-3ζ or "Wes1T-3Z"). These basis sets were optimized for 102 species representing the first two rows of the Periodic Table. The species include the entire set of neutral atoms, positive and negative atomic ions, as well as several homonuclear diatomic molecules, hydrides, rare gas dimers, polar molecules, such as oxides and fluorides, and a few transition states. The extrapolated Wes1T-(2,3)Z triples energies agree with (T)/CBS benchmarks to within ±0.65 mEh, while the rms deviations of comparable model chemistries W1, CBS-APNO, and CBS-QB3 for the same test set are ±0.23 mEh, ±2.37 mEh, and ±5.80 mEh, respectively. The Wes1T-(2,3)Z triples calculation time for the largest hydrocarbon in the G2/97 test set, C6H5Me+, is reduced by a factor of 25 when compared to W1. The cost-effectiveness of the Wes1T-(2,3)Z extrapolation validates the usefulness of the Wes1T-2Z and Wes1T-3Z basis sets which are now available for a more efficient extrapolation of the (T) component of any composite model chemistry.

  7. An efficient extrapolation to the (T)/CBS limit

    SciTech Connect

    Ranasinghe, Duminda S.; Barnes, Ericka C.

    2014-05-14

    We extrapolate to the perturbative triples (T)/complete basis set (CBS) limit using double ζ basis sets without polarization functions (Wesleyan-1-Triples-2ζ or “Wes1T-2Z”) and triple ζ basis sets with a single level of polarization functions (Wesleyan-1-Triples-3ζ or “Wes1T-3Z”). These basis sets were optimized for 102 species representing the first two rows of the Periodic Table. The species include the entire set of neutral atoms, positive and negative atomic ions, as well as several homonuclear diatomic molecules, hydrides, rare gas dimers, polar molecules, such as oxides and fluorides, and a few transition states. The extrapolated Wes1T-(2,3)Z triples energies agree with (T)/CBS benchmarks to within ±0.65 mE{sub h}, while the rms deviations of comparable model chemistries W1, CBS-APNO, and CBS-QB3 for the same test set are ±0.23 mE{sub h}, ±2.37 mE{sub h}, and ±5.80 mE{sub h}, respectively. The Wes1T-(2,3)Z triples calculation time for the largest hydrocarbon in the G2/97 test set, C{sub 6}H{sub 5}Me{sup +}, is reduced by a factor of 25 when compared to W1. The cost-effectiveness of the Wes1T-(2,3)Z extrapolation validates the usefulness of the Wes1T-2Z and Wes1T-3Z basis sets which are now available for a more efficient extrapolation of the (T) component of any composite model chemistry.

  8. Impact of culture nutrition on tolerance of furan inhibitors and the conversion of high xylose concentrations to ethanol by Pichia stipitis NRRL Y-7124

    USDA-ARS?s Scientific Manuscript database

    Efficient fermentation processes to produce ethanol from both the hexose and pentose sugars available in low-cost lignocellulosic biomass are sought to support the expansion of the biofuels industry. Such an expansion is expected to strengthen our nation by lessening dependence on foreign sources o...

  9. Description of Komagataella phaffii sp. nov. and the transfer of Pichia pseudopastoris to the methylotrophic yeast genus Komagataella.

    PubMed

    Kurtzman, Cletus P

    2005-03-01

    The new methanol-assimilating yeast species Komagataella phaffii Kurtzman sp. nov. (type strain NRRL Y-7556(T)=CBS 2612(T)) is described. Of the four known strains of this species, two were isolated from black oak trees in California, USA, one from an Emory oak in Arizona, USA, and one from an unidentified source in Mexico. The species forms hat-shaped ascospores in deliquescent asci and appears to be homothallic. Analysis of nucleotide sequences from domains D1/D2 of large-subunit (26S) rDNA separates the new species from Komagataella pastoris, the type species of the genus, and from Pichia pseudopastoris, which is here renamed Komagataella pseudopastoris (Dlauchy, Tornai-Lehoczki, Fulop & Peter) Kurtzman comb. nov. (type strain NRRL Y-27603(T)=CBS 9187(T)=NCAIM Y 01541(T)). On the basis of D1/D2 26S rDNA sequence analysis, the three species now assigned to the genus Komagataella represent a clade that is phylogenetically isolated from other ascomycetous yeast genera.

  10. Biotransformation of 5-hydroxymethylfurfural (HMF) by Scheffersomyces stipitis during ethanol fermentation of hydrolysate of the seaweed Gelidium amansii.

    PubMed

    Ra, Chae Hun; Jeong, Gwi-Taek; Shin, Myung Kyo; Kim, Sung-Koo

    2013-07-01

    The seaweed, Gelidium amansii, was fermented to produce bioethanol. Optimal pretreatment condition was determined as 94 mM H2SO4 and 10% (w/v) seaweed slurry at 121°C for 60 min. The mono sugars of 43.5 g/L with 57.4% of conversion from total carbohydrate of 75.8 g/L with G. amansii slurry 100g dcw/L were obtained by thermal acid hydrolysis pretreatment and enzymatic saccharification. G. amansii hydrolysate was used as the substrate for ethanol production by separate hydrolysis and fermentation (SHF). The ethanol concentration of 20.5 g/L was produced by Scheffersomyces stipitis KCTC 7228. The effect of HMF on ethanol production by S. stipitis KCTC 7228 was evaluated and 5-hydroxymethylfurfural (HMF) was converted to 2,5-bis-hydroxymethylfuran. The accumulated 2,5-bis-hydroxymethylfuran in the medium did not affect galactose and glucose uptakes and ethanol production. Biotransformation of HMF to less inhibitory compounds by S. stipitis KCTC 7228 could enhance overall fermentation yields of seaweed hydrolysates to ethanol.

  11. Characterization of the Collagen-Binding S-Layer Protein CbsA of Lactobacillus crispatus

    PubMed Central

    Sillanpää, Jouko; Martínez, Beatriz; Antikainen, Jenni; Toba, Takahiro; Kalkkinen, Nisse; Tankka, Sanna; Lounatmaa, Kari; Keränen, Jaakko; Höök, Magnus; Westerlund-Wikström, Benita; Pouwels, Peter H.; Korhonen, Timo K.

    2000-01-01

    The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus. PMID:11053389

  12. Ethanol production improvement driven by genome-scale metabolic modeling and sensitivity analysis in Scheffersomyces stipitis

    PubMed Central

    2017-01-01

    The yeast Scheffersomyces stipitis naturally produces ethanol from xylose, however reaching high ethanol yields is strongly dependent on aeration conditions. It has been reported that changes in the availability of NAD(H/+) cofactors can improve fermentation in some microorganisms. In this work genome-scale metabolic modeling and phenotypic phase plane analysis were used to characterize metabolic response on a range of uptake rates. Sensitivity analysis was used to assess the effect of ARC on ethanol production indicating that modifying ARC by inhibiting the respiratory chain ethanol production can be improved. It was shown experimentally in batch culture using Rotenone as an inhibitor of the mitochondrial NADH dehydrogenase complex I (CINADH), increasing ethanol yield by 18%. Furthermore, trajectories for uptakes rates, specific productivity and specific growth rate were determined by modeling the batch culture, to calculate ARC associated to the addition of CINADH inhibitor. Results showed that the increment in ethanol production via respiratory inhibition is due to excess in ARC, which generates an increase in ethanol production. Thus ethanol production improvement could be predicted by a change in ARC. PMID:28658270

  13. Ethanol production improvement driven by genome-scale metabolic modeling and sensitivity analysis in Scheffersomyces stipitis.

    PubMed

    Acevedo, Alejandro; Conejeros, Raúl; Aroca, Germán

    2017-01-01

    The yeast Scheffersomyces stipitis naturally produces ethanol from xylose, however reaching high ethanol yields is strongly dependent on aeration conditions. It has been reported that changes in the availability of NAD(H/+) cofactors can improve fermentation in some microorganisms. In this work genome-scale metabolic modeling and phenotypic phase plane analysis were used to characterize metabolic response on a range of uptake rates. Sensitivity analysis was used to assess the effect of ARC on ethanol production indicating that modifying ARC by inhibiting the respiratory chain ethanol production can be improved. It was shown experimentally in batch culture using Rotenone as an inhibitor of the mitochondrial NADH dehydrogenase complex I (CINADH), increasing ethanol yield by 18%. Furthermore, trajectories for uptakes rates, specific productivity and specific growth rate were determined by modeling the batch culture, to calculate ARC associated to the addition of CINADH inhibitor. Results showed that the increment in ethanol production via respiratory inhibition is due to excess in ARC, which generates an increase in ethanol production. Thus ethanol production improvement could be predicted by a change in ARC.

  14. Alcohol dehydrogenases from Scheffersomyces stipitis involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion.

    PubMed

    Ma, Menggen; Wang, Xu; Zhang, Xiaoping; Zhao, Xianxian

    2013-09-01

    Aldehyde inhibitors such as furfural and 5-hydroxymethylfurfural (HMF) are generated from biomass pretreatment. Scheffersomyces stipitis is able to reduce furfural and HMF to less toxic furanmethanol and furan-2,5-dimethanol; however, the enzymes involved in the reductive reaction still remain unknown. In this study, transcription responses of two known and five putative alcohol dehydrogenase genes from S. stipitis were analyzed under furfural and HMF stress conditions. All the seven alcohol dehydrogenase genes were also cloned and overexpressed for their activity analyses. Our results indicate that transcriptions of SsADH4 and SsADH6 were highly induced under furfural and HMF stress conditions, and the proteins encoded by them exhibited NADH- and/or NADPH-dependent activities for furfural and HMF reduction, respectively. For furfural reduction, NADH-dependent activity was also observed in SsAdh1p and NAD(P)H-dependent activities were also observed in SsAdh5p and SsAdh7p. For HMF reduction, NADPH-dependent activities were also observed in SsAdh5p and SsAdh7p. SsAdh4p displayed the highest NADPH-dependent specific activity and catalytic efficiency for reduction of both furfural and HMF among the seven alcohol dehydrogenases. Enzyme activities of all SsADH proteins were more stable under acidic condition. For most SsADH proteins, the optimum temperature for enzyme activities was 30 °C and more than 50 % enzyme activities remained at 60 °C. Reduction activities of formaldehyde, acetaldehyde, isovaleraldehyde, benzaldehyde, and phenylacetaldehyde were also observed in some SsADH proteins. Our results indicate that multiple alcohol dehydrogenases in S. stipitis are involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion.

  15. Fermentation strategy for second generation ethanol production from sugarcane bagasse hydrolyzate by Spathaspora passalidarum and Scheffersomyces stipitis.

    PubMed

    Nakanishi, Simone C; Soares, Lauren B; Biazi, Luiz Eduardo; Nascimento, Viviane M; Costa, Aline C; Rocha, George Jackson M; Ienczak, Jaciane L

    2017-10-01

    Alcoholic fermentation of released sugars in pretreatment and enzymatic hydrolysis of biomass is a central feature for second generation ethanol (E2G) production. Saccharomyces cerevisiae used industrially in the production of first generation ethanol (E1G) convert sucrose, fructose, and glucose into ethanol. However, these yeasts have no ability to ferment pentose (xylose). Therefore, the present work has focused on E2G production by Scheffersomyces stipitis and Spathaspora passalidarum. The fermentation strategy with high pitch, cell recycle, fed-batch mode, and temperature decrease for each batch were performed in a hydrolyzate obtained from a pretreatment at 130°C with NaOH solution (1.5% w/v) added with 0.15% (w/w) of anthraquinone (AQ) and followed by enzymatic hydrolysis. The process strategy has increased volumetric productivity from 0.35 to 0.38 g · L(-1)  · h(-1) (first to third batch) for S. stipitis and from 0.38 to 0.81 g · L(-1)  · h(-1) for S. passalidarum (first to fourth batch). Mass balance for the process proposed in this work showed the production of 177.33 kg ethanol/ton of sugar cane bagasse for S. passalidarum compared to 124.13 kg ethanol/ton of sugar cane bagasse for S. stipitis fermentation. The strategy proposed in this work can be considered as a promising strategy in the production of second generation ethanol. Biotechnol. Bioeng. 2017;114: 2211-2221. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  16. 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

  17. Overexpression of membrane proteins using Pichia pastoris.

    PubMed

    Bornert, Olivier; Alkhalfioui, Fatima; Logez, Christel; Wagner, Renaud

    2012-02-01

    Among the small number of expression systems validated for the mass production of eukaryotic membrane proteins (EMPs), the methylotrophic yeast Pichia pastoris stands as one of the most efficient hosts. This system has been used to produce crystallization-grade proteins for a variety of EMPs, from which high-resolution 3D structures have been determined. This unit describes a set of guidelines and instructions to overexpress membrane proteins using the P. pastoris system. Using a G protein-coupled receptor (GPCR) as a model EMP, these protocols illustrate the necessary steps, starting with the design of the DNA sequence to be expressed, through the preparation and analysis of samples containing the corresponding membrane protein of interest. In addition, recommendations are given on a series of experimental parameters that can be optimized to substantially improve the amount and/or the functionality of the expressed EMPs.

  18. Synthetic Core Promoters for Pichia pastoris

    PubMed Central

    2013-01-01

    Synthetic promoters are commonly used tools for circuit design or high level protein production. Promoter engineering efforts in yeasts, such as Saccharomyces cerevisiae and Pichia pastoris have mostly been focused on altering upstream regulatory sequences such as transcription factor binding sites. In higher eukaryotes synthetic core promoters, directly needed for transcription initiation by RNA Polymerase II, have been successfully designed. Here we report the first synthetic yeast core promoter for P. pastoris, based on natural yeast core promoters. Furthermore we used this synthetic core promoter sequence to engineer the core promoter of the natural AOX1 promoter, thereby creating a set of core promoters providing a range of different expression levels. As opposed to engineering strategies of the significantly longer entire promoter, such short core promoters can directly be added on a PCR primer facilitating library generation and are sufficient to obtain variable expression yields. PMID:24187969

  19. Native valve endocarditis due to Pichia ohmeri.

    PubMed

    João, Isabel; Duarte, José; Cotrim, Carlos; Rodrigues, Ana; Martins, Cristina; Fazendas, Paula; Oliveira, L Moura; Diogo, José; Carrageta, Manuel

    2002-09-01

    Candida species can cause clinical manifestations in various organs of the cardiovascular system, i.e., the pericardium, myocardium, and endocardium, with endocarditis being the best-known clinical entity. Endocarditis is seen primarily in intravenous drug users and in individuals with damaged native valves, especially in congenital heart disease or rheumatic valvular diseases, and in prosthetic heart valves. The authors present a case of Pichia ohmeri endocarditis in an intravenous drug user, with an unusual presentation form. This is a case of a 42-year-old man, an intravenous heroin user, who was admitted to our Vascular Surgery Department because of fever and acute serious ischemia of the left inferior limb. He presented with fever (39 degrees C), a pale and cold left limb, absence of the left popliteal pulse, and a pansystolic murmur at the cardiac apex. The transthoracic echocardiogram showed a large vegetation on the anterior leaflet of the mitral valve and severe mitral regurgitation with good left ventricular systolic function. Empirical antibiotic therapy was started. Six days after admission, embolectomy was performed with partial clinical recovery. Three blood cultures and the embolus showed a teleomorphic form of Candida guilliermondii - Pichia ohmeri. Therapy with intravenous liposomal amphotericin B, fluocitosin, imipenem, and aztreonam was started. Two weeks later, his clinical condition deteriorated with acute heart failure refractory to medical therapy, mandating mechanical ventilation and high-dose vasopressor and inotropic amine support. He underwent urgent mitral valve replacement with a biologic prosthetic valve. Rapid stabilization of the cardiac status occurred, but ischemic limb lesions required further vascular interventions.

  20. (13) C metabolic flux profiling of Pichia pastoris grown in aerobic batch cultures on glucose revealed high relative anabolic use of TCA cycle and limited incorporation of provided precursors of branched-chain amino acids.

    PubMed

    Zhang, Meng; Yu, Xiao-Wei; Xu, Yan; Jouhten, Paula; Swapna, Gurla V T; Glaser, Ralf W; Hunt, John F; Montelione, Gaetano T; Maaheimo, Hannu; Szyperski, Thomas

    2017-07-21

    Carbon metabolism of Crabtree-negative yeast Pichia pastoris was profiled using (13) C nuclear magnetic resonance (NMR) to delineate regulation during exponential growth and to study the import of two precursors for branched-chain amino acid biosynthesis, α-ketoisovalerate and α-ketobutyrate. Cells were grown in aerobic batch cultures containing (a) only glucose, (b) glucose along with the precursors, or (c) glucose and Val. The study provided the following new insights. First, (13) C flux ratio analyses of central metabolism reveal an unexpectedly high anaplerotic supply of the tricarboxylic acid cycle for a Crabtree-negative yeast, and show that a substantial fraction of glucose catabolism proceeds through the pentose phosphate pathway. A comparison with previous flux ratio analyses for batch cultures of Crabtree-negative Pichia stipitis and Crabtree-positive Saccharomyces cerevisiae indicate that the overall regulation of central carbon metabolism in P. pastoris is intermediate in between P. stipitis and S. cerevisiae. Second, excess α-ketoisovalerate in the medium is not transported into the cytoplasm indicating that P. pastoris lacks a suitable transporter. In contrast, excess Val is efficiently taken up and largely fulfills demands for both Val and Leu for protein synthesis. Third, excess α-ketobutyrate is transported into the mitochondria for Ile biosynthesis. However, the import does not efficiently inhibit the synthesis of α-ketobutyrate from pyruvate indicating that P. pastoris has not been optimized evolutionarily to take full advantage of this carbon source. These findings have direct implications for preparing uniformly (2) H,(13) C,(15) N-labeled proteins containing protonated Ile, Val, and Leu methyl groups in P. pastoris for NMR-based structural biology. Acetohydroxy acid isomeroreductase (EC 1.1.1.86), branched-chain amino acid aminotransferase (BCAT, EC 2.6.1.42), fumarase (EC 4.2.1.2), malic enzyme (EC 1.1.1.39/1.1.1.40), phosphoenolpyruvate

  1. Betaine supplementation is less effective than methionine restriction in correcting phenotypes of CBS deficient mice.

    PubMed

    Gupta, Sapna; Wang, Liqun; Kruger, Warren D

    2016-01-01

    Cystathionine beta synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by elevated serum total homocysteine (tHcy). Betaine supplementation, which can lower tHcy by stimulating homocysteine remethylation to methionine, is often given to CBS deficient patients in combination with other treatments such as methionine restriction and supplemental B-vitamins. However, the effectiveness of betaine supplementation by itself in the treatment of CBS deficiency has not been well explored. Here, we have examined the effect of a betaine supplemented diet on the Tg-I278T Cbs (-/-) mouse model of CBS deficiency and compared its effectiveness to our previously published data using a methionine restricted diet. Tg-I278T Cbs (-/-) mice on betaine, from the time of weaning until for 240 days of age, had a 40 % decrease in mean tHcy level and a 137 % increase in serum methionine levels. Betaine-treated Tg-I278T Cbs (-/-) mice also exhibited increased levels of betaine-dependent homocysteine methyl transferase (BHMT), increased levels of the lipogenic enzyme stearoyl-coenzyme A desaturase (SCD-1), and increased lipid droplet accumulation in the liver. Betaine supplementation largely reversed the hair loss phenotype in Tg-I278T Cbs (-/-) animals, but was far less effective than methionine restriction in reversing the weight-loss, fat-loss, and osteoporosis phenotypes. Surprisingly, betaine supplementation had several negative effects in control Tg-I278T Cbs (+/-) mice including decreased weight gain, lean mass, and bone mineral density. Our findings indicate that while betaine supplementation does have some beneficial effects, it is not as effective as methionine restriction for reversing the phenotypes associated with severe CBS deficiency in mice.

  2. Optimized fed-batch fermentation of Scheffersomyces stipitis for efficient production of ethanol from hexoses and pentoses.

    PubMed

    Unrean, Pornkamol; Nguyen, Nhung H A

    2013-03-01

    Scheffersomyces stipitis was cultivated in an optimized, controlled fed-batch fermentation for production of ethanol from glucose-xylose mixture. Effect of feed medium composition was investigated on sugar utilization and ethanol production. Studying influence of specific cell growth rate on ethanol fermentation performance showed the carbon flow towards ethanol synthesis decreased with increasing cell growth rate. The optimum specific growth rate to achieve efficient ethanol production performance from a glucose-xylose mixture existed at 0.1 h(-1). With these optimized feed medium and cell growth rate, a kinetic model has been utilized to avoid overflow metabolism as well as to ensure a balanced feeding of nutrient substrate in fed-batch system. Fed-batch culture with feeding profile designed based on the model resulted in high titer, yield, and productivity of ethanol compared with batch cultures. The maximal ethanol concentration was 40.7 g/L. The yield and productivity of ethanol production in the optimized fed-batch culture was 1.3 and 2 times higher than those in batch culture. Thus, higher efficiency ethanol production was achieved in this study through fed-batch process optimization. This strategy may contribute to an improvement of ethanol fermentation from lignocellulosic biomass by S. stipitis on the industrial scale.

  3. Bioethanol production by recycled Scheffersomyces stipitis in sequential batch fermentations with high cell density using xylose and glucose mixture.

    PubMed

    Santos, Samantha Christine; de Sousa, Amanda Silva; Dionísio, Suzane Rodrigues; Tramontina, Robson; Ruller, Roberto; Squina, Fabio Márcio; Vaz Rossell, Carlos Eduardo; da Costa, Aline Carvalho; Ienczak, Jaciane Lutz

    2016-11-01

    Here, it is shown three-step investigative procedures aiming to improve pentose-rich fermentations performance, involving a simple system for elevated mass production by Scheffersomyces stipitis (I), cellular recycle batch fermentations (CRBFs) at high cell density using two temperature strategies (fixed at 30°C; decreasing from 30 to 26°C) (II), and a short-term adaptation action seeking to acclimatize the microorganism in xylose rich-media (III). Cellular propagation provided 0.52gdrycellweightgRS(-1), resulting in an expressive value of 45.9gdrycellweightL(-1). The yeast robustness in CRBF was proven by effective ethanol production, reaching high xylose consumption (81%) and EtOH productivity (1.53gL(-1)h(-1)). Regarding the short-term adaptation, S. stipitis strengthened its robustness, as shown by a 6-fold increase in xylose reductase (XR) activity. The short fermentation time (20h for each batch) and the fermentation kinetics for ethanol production from xylose are quite promising.

  4. Rational optimization of culture conditions for the most efficient ethanol production in Scheffersomyces stipitis using design of experiments.

    PubMed

    Unrean, Pornkamol; Nguyen, Nhung H A

    2012-01-01

    Optimization of culture parameters for achieving the most efficient ethanol fermentation is challenging due to multiple variables involved. Here we presented a rationalized methodology for multi-variables optimization through the design of experiments DoE approach. Three critical parameters, pH, temperature, and agitation speed, affecting ethanol fermentation in S. stipitis was investigated. A predictive model showed that agitation speed significantly affected ethanol synthesis. Reducing pH and temperature also improved ethanol production. The model identified the optimum culture conditions for the most efficient ethanol production with the yield and productivity of 0.46 g/g and 0.28 g/l h, respectively, which is consistent with experimental observation. The results also indicated the scalability of the model from shake flask to bioreactor. Thus, DoE is a promising tool permitting the rapid establishment of culture conditions for the most efficient ethanol fermentation in S. stipitis. The approach could be useful to reduce process development time in lignocellulosic ethanol industry.

  5. In vivo unnatural amino acid expression in the methylotrophic yeast Pichia pastoris

    DOEpatents

    Young, Travis [San Diego, CA; Schultz, Peter G [La Jolla, CA

    2014-02-11

    The invention provides orthogonal translation systems for the production of polypeptides comprising unnatural amino acids in methyltrophic yeast such as Pichia pastoris. Methods for producing polypeptides comprising unnatural amino acids in methyltrophic yeast such as Pichia pastoris are also provided.

  6. In vivo unnatural amino acid expression in the methylotrophic yeast Pichia pastoris

    DOEpatents

    Young, Travis; Schultz, Peter G.

    2017-08-15

    The invention provides orthogonal translation systems for the production of polypeptides comprising unnatural amino acids in methylotrophic yeast such as Pichia pastoris. Methods for producing polypeptides comprising unnatural amino acids in methylotrophic yeast such as Pichia pastoris are also provided.

  7. Genome-Scale NAD(H/+) Availability Patterns as a Differentiating Feature between Saccharomyces cerevisiae and Scheffersomyces stipitis in Relation to Fermentative Metabolism

    PubMed Central

    Acevedo, Alejandro; Aroca, German; Conejeros, Raul

    2014-01-01

    Scheffersomyces stipitis is a yeast able to ferment pentoses to ethanol, unlike Saccharomyces cerevisiae, it does not present the so-called overflow phenomenon. Metabolic features characterizing the presence or not of this phenomenon have not been fully elucidated. This work proposes that genome-scale metabolic response to variations in NAD(H/+) availability characterizes fermentative behavior in both yeasts. Thus, differentiating features in S. stipitis and S. cerevisiae were determined analyzing growth sensitivity response to changes in available reducing capacity in relation to ethanol production capacity and overall metabolic flux span. Using genome-scale constraint-based metabolic models, phenotypic phase planes and shadow price analyses, an excess of available reducing capacity for growth was found in S. cerevisiae at every metabolic phenotype where growth is limited by oxygen uptake, while in S. stipitis this was observed only for a subset of those phenotypes. Moreover, by using flux variability analysis, an increased metabolic flux span was found in S. cerevisiae at growth limited by oxygen uptake, while in S. stipitis flux span was invariant. Therefore, each yeast can be characterized by a significantly different metabolic response and flux span when growth is limited by oxygen uptake, both features suggesting a higher metabolic flexibility in S. cerevisiae. By applying an optimization-based approach on the genome-scale models, three single reaction deletions were found to generate in S. stipitis the reducing capacity availability pattern found in S. cerevisiae, two of them correspond to reactions involved in the overflow phenomenon. These results show a close relationship between the growth sensitivity response given by the metabolic network and fermentative behavior. PMID:24489927

  8. The Coaching Behavior Scale for Sport (CBS-S): A psychometric evaluation of the Swedish version.

    PubMed

    Carlsson, A; Lundqvist, C

    2016-01-01

    The present study validated a Swedish version of the 47-item Coaching Behavior Scale for Sport (CBS-S). Sample 1 consisted of 506 team sport athletes [262 men and 244 women; mean age: 22.20, standard deviation (SD) = 3.90] distributed across 41 coaches at the two highest national levels of various sports. Athletes completed the CBS-S and established questionnaires of coaching behaviors (LSS), self-confidence (CSAI-2R), and coach-athlete relationship (CART-Q). An additional sample of 39 basketball players (21 men and 18 women; mean age = 17.40, SD = 2.39) completed the CBS-S twice, approximately 4 weeks apart. Confirmatory factor analysis showed an acceptable model fit for the seven-factor version of the CBS-S, although two items of the negative personal rapport subscale displayed insufficient factor loadings. Correlations between the subscales of the CBS-S and established instruments were in accordance with theoretical expectations, supporting the concurrent validity. Cronbach's alpha (> 0.82) for all dimensions provided support for the reliability of the CBS-S, and test-retest correlations indicated moderate stability over time. Cultural differences in the assessment of coaching behaviors and the usability of the CBS-S by coaches for self-reflection and development are discussed. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. A thermophilic endo-1,4-β-glucanase from Talaromyces emersonii CBS394.64 with broad substrate specificity and great application potentials.

    PubMed

    Wang, Kun; Luo, Huiying; Bai, Yingguo; Shi, Pengjun; Huang, Huoqing; Xue, Xianli; Yao, Bin

    2014-08-01

    Thermophilic cellulases are of significant interest to the efficient conversion of plant cell wall polysaccharides into simple sugars. In this study, a thermophilic and thermostable endo-1,4-β-glucanase, TeEgl5A, was identified in the thermophilic fungus Talaromyces emersonii CBS394.64 and functionally expressed in Pichia pastoris. Purified recombinant TeEgl5A exhibits optimal activity at pH 4.5 and 90 °C. It is highly stable at 70 °C and over a broad pH range of 1.0-10.0, and shows strong resistance to most metal ions, sodium dodecyl sulfate (SDS), and proteases. TeEgl5A has broad substrate specificity and exhibits high activity on substrates containing β-1,4-glycosidic bonds and β-1,3-glycosidic bonds (barley β-glucan, laminarin, lichenan, CMC-Na, carob bean gum, and birchwood xylan). Under simulated mashing conditions, addition of 60 U TeEgl5A reduced more viscosity (10.0 vs.7.6 %) than 80 U of Ultraflo XL from Novozymes. These properties make TeEgl5A a good candidate for extensive application in the detergent, textile, feed, and food industries.

  10. A tracer test at the Beowawe geothermal field, Nevada, using fluorescein and tinopal CBS

    SciTech Connect

    Rose, P.E.; Adams, M.C.; Benoit, D.

    1995-12-31

    An interwell tracer test using fluorescein and tinopal CBS was performed at the Beowawe geothermal field in north-central Nevada in order to assess the effects of recent changes to the injection strategy. Fluorescein return curves established injection-production flow patterns and verified that produced water is being reinjected into a region of the reservoir that is in excellent communication with the production wells. An analysis of the tinopal CBS return curves indicated that tinopal CBS was apparently strongly adsorbed onto the reservoir rock. The fluorescein return curves were used to estimate the overall (fractures and matrix) reservoir volume.

  11. Refined Pichia pastoris reference genome sequence.

    PubMed

    Sturmberger, Lukas; Chappell, Thomas; Geier, Martina; Krainer, Florian; Day, Kasey J; Vide, Ursa; Trstenjak, Sara; Schiefer, Anja; Richardson, Toby; Soriaga, Leah; Darnhofer, Barbara; Birner-Gruenberger, Ruth; Glick, Benjamin S; Tolstorukov, Ilya; Cregg, James; Madden, Knut; Glieder, Anton

    2016-10-10

    Strains of the species Komagataella phaffii are the most frequently used "Pichia pastoris" strains employed for recombinant protein production as well as studies on peroxisome biogenesis, autophagy and secretory pathway analyses. Genome sequencing of several different P. pastoris strains has provided the foundation for understanding these cellular functions in recent genomics, transcriptomics and proteomics experiments. This experimentation has identified mistakes, gaps and incorrectly annotated open reading frames in the previously published draft genome sequences. Here, a refined reference genome is presented, generated with genome and transcriptome sequencing data from multiple P. pastoris strains. Twelve major sequence gaps from 20 to 6000 base pairs were closed and 5111 out of 5256 putative open reading frames were manually curated and confirmed by RNA-seq and published LC-MS/MS data, including the addition of new open reading frames (ORFs) and a reduction in the number of spliced genes from 797 to 571. One chromosomal fragment of 76kbp between two previous gaps on chromosome 1 and another 134kbp fragment at the end of chromosome 4, as well as several shorter fragments needed re-orientation. In total more than 500 positions in the genome have been corrected. This reference genome is presented with new chromosomal numbering, positioning ribosomal repeats at the distal ends of the four chromosomes, and includes predicted chromosomal centromeres as well as the sequence of two linear cytoplasmic plasmids of 13.1 and 9.5kbp found in some strains of P. pastoris.

  12. Reduced response of Cystathionine Beta-Synthase (CBS) to S-Adenosylmethionine (SAM): Identification and functional analysis of CBS gene mutations in Homocystinuria patients.

    PubMed

    Mendes, Marisa I S; Colaço, Henrique G; Smith, Desirée E C; Ramos, Rúben J J F; Pop, Ana; van Dooren, Silvy J M; Tavares de Almeida, Isabel; Kluijtmans, Leo A J; Janssen, Mirian C H; Rivera, Isabel; Salomons, Gajja S; Leandro, Paula; Blom, Henk J

    2014-03-01

    A reduced response of cystathionine beta-synthase (CBS) to its allosteric activator S-adenosylmethionine (SAM) has been reported to be a cause of CBS dysfunction in homocystinuria patients. In this work we performed a retrospective analysis of fibroblast data from 62 homocystinuria patients and found that 13 of them presented a disturbed SAM activation. Their genotypic background was identified and the corresponding CBS mutant proteins were produced in E. coli. Nine distinct mutations were detected in 22 independent alleles: the novel mutations p.K269del, p.P427L, p.S500L and p.L540Q; and the previously described mutations p.P49L, p.C165Rfs*2, p.I278T, p.R336H and p.D444N. Expression levels and residual enzyme activities, determined in the soluble fraction of E. coli lysates, strongly correlated with the localization of the affected amino acid residue. C-terminal mutations lead to activities in the range of the wild-type CBS and to oligomeric forms migrating faster than tetramers, suggesting an abnormal conformation that might be responsible for the lack of SAM activation. Mutations in the catalytic core were associated with low protein expression levels, decreased enzyme activities and a higher content of high molecular mass forms. Furthermore, the absence of SAM activation found in the patients' fibroblasts was confirmed for all but one of the characterized recombinant proteins (p.P49L). Our study experimentally supports a deficient regulation of CBS by SAM as a frequently found mechanism in CBS deficiency, which should be considered not only as a valuable diagnostic tool but also as a potential target for the development of new therapeutic approaches in classical homocystinuria.

  13. Analysis of MTHFR, CBS, Glutathione, Taurine, and Hydrogen Sulfide Levels in Retinas of Hyperhomocysteinemic Mice.

    PubMed

    Cui, Xuezhi; Navneet, Soumya; Wang, Jing; Roon, Penny; Chen, Wei; Xian, Ming; Smith, Sylvia B

    2017-04-01

    Hyperhomocysteinemia (Hhcy) is implicated in certain retinal neurovascular diseases, although whether it is causative remains uncertain. In isolated ganglion cells (GCs), mild Hhcy induces profound death, whereas retinal phenotypes in Hhcy mice caused by mutations in remethylation (methylene tetrahydrofolatereductase [Mthfr+/-]) or transsulfuration pathways (cystathionine β-synthase [Cbs+/-]) demonstrate mild GC loss and mild vasculopathy. The current work investigated compensation in vivo of one pathway for the other, and, because the transsulfuration pathway yields cysteine necessary for formation of glutathione (GSH), taurine, and hydrogen sulfide (H2S), they were analyzed also. Retinas isolated from wild-type (WT), Mthfr+/-, and Cbs+/- mice (12 and 22 weeks) were analyzed for methylene tetrahydrofolate reductase (MTHFR), cystathionine-β-synthase (CBS), and cystathionase (CTH) RNA/protein levels. Retinas were evaluated for levels of reduced:oxidized GSH (GSH:GSSG), Slc7a11 (xCT), taurine, taurine transporter (TAUT), and H2S. Aside from decreased CBS RNA/protein levels in Cbs+/- retinas, there were minimal alterations in remethylation/transsulfuration pathways in the two mutant mice strains. Glutathione and taurine levels in Mthfr+/- and Cbs+/- retinas were similar to WT, which may be due to robust levels of xCT and TAUT in mutant retinas. Interestingly, levels of H2S were markedly increased in retinas of Mthfr+/- and Cbs+/- mice compared with WT. Ganglion cell loss and vasculopathy observed in Mthfr+/- and Cbs+/- mouse retinas may be milder than expected, not because of compensatory increases of enzymes in remethylation/transsulfuration pathways, but because downstream transsulfuration pathway products GSH, taurine, and H2S are maintained at robust levels. Elevation of H2S is particularly intriguing owing to neuroprotective properties reported for this gasotransmitter.

  14. Analysis of MTHFR, CBS, Glutathione, Taurine, and Hydrogen Sulfide Levels in Retinas of Hyperhomocysteinemic Mice

    PubMed Central

    Cui, Xuezhi; Navneet, Soumya; Wang, Jing; Roon, Penny; Chen, Wei; Xian, Ming; Smith, Sylvia B.

    2017-01-01

    Purpose Hyperhomocysteinemia (Hhcy) is implicated in certain retinal neurovascular diseases, although whether it is causative remains uncertain. In isolated ganglion cells (GCs), mild Hhcy induces profound death, whereas retinal phenotypes in Hhcy mice caused by mutations in remethylation (methylene tetrahydrofolatereductase [Mthfr+/−]) or transsulfuration pathways (cystathionine β-synthase [Cbs+/−]) demonstrate mild GC loss and mild vasculopathy. The current work investigated compensation in vivo of one pathway for the other, and, because the transsulfuration pathway yields cysteine necessary for formation of glutathione (GSH), taurine, and hydrogen sulfide (H2S), they were analyzed also. Methods Retinas isolated from wild-type (WT), Mthfr+/−, and Cbs+/− mice (12 and 22 weeks) were analyzed for methylene tetrahydrofolate reductase (MTHFR), cystathionine-β-synthase (CBS), and cystathionase (CTH) RNA/protein levels. Retinas were evaluated for levels of reduced:oxidized GSH (GSH:GSSG), Slc7a11 (xCT), taurine, taurine transporter (TAUT), and H2S. Results Aside from decreased CBS RNA/protein levels in Cbs+/− retinas, there were minimal alterations in remethylation/transsulfuration pathways in the two mutant mice strains. Glutathione and taurine levels in Mthfr+/− and Cbs+/− retinas were similar to WT, which may be due to robust levels of xCT and TAUT in mutant retinas. Interestingly, levels of H2S were markedly increased in retinas of Mthfr+/− and Cbs+/− mice compared with WT. Conclusions Ganglion cell loss and vasculopathy observed in Mthfr+/− and Cbs+/− mouse retinas may be milder than expected, not because of compensatory increases of enzymes in remethylation/transsulfuration pathways, but because downstream transsulfuration pathway products GSH, taurine, and H2S are maintained at robust levels. Elevation of H2S is particularly intriguing owing to neuroprotective properties reported for this gasotransmitter. PMID:28384716

  15. Novel Compound Heterozygous CBS Mutations Cause Homocystinuria in a Han Chinese Family.

    PubMed

    Gong, Bo; Liu, Liping; Li, Zhiwei; Ye, Zimeng; Xiao, Ying; Zeng, Guangqun; Shi, Yi; Wang, Yumeng; Feng, Xiaoyun; Li, Xiulan; Hao, Fang; Liu, Xiaoqi; Qu, Chao; Li, Yuanfeng; Mu, Guoying; Yang, Zhenglin

    2015-12-15

    The cystathionine β-synthase (CBS) gene has been shown to be related to homocystinuria. This study was aimed to detect the mutations in CBS in a Han Chinese family with homocystinuria. A four-generation family from Shandong Province of China was recruited in this study. All available members of the family underwent comprehensive medical examinations. Genomic DNA was collected from peripheral blood of all the participants. The coding sequence of CBS was amplified by polymerase chain reaction (PCR), followed by direct DNA sequencing. Among all the family members, three affected individuals showed typical clinical features of homocystinuria. Two novel compound heterozygous mutations in the CBS gene, c.407T > C (p. L136P) and c.473C > T (p.A158V), were identified by sequencing analysis in this family. Both of the two missense mutations were detected in the three patients. Other available normal individuals, including the patients' parents, grand parents, her younger sister and brother in this family either carried one of the two mutations, or none. In addition, the two mutations were not found in 600 ethnically matched normal controls. This study provides a mutation spectrum of CBS resulting in homocystinuriain a Chinese population, which may shed light on the molecular pathogenesis and clinical diagnosis of CBS-associated homocystinuria.

  16. Novel Compound Heterozygous CBS Mutations Cause Homocystinuria in a Han Chinese Family

    PubMed Central

    Gong, Bo; Liu, Liping; Li, Zhiwei; Ye, Zimeng; Xiao, Ying; Zeng, Guangqun; Shi, Yi; Wang, Yumeng; Feng, Xiaoyun; Li, Xiulan; Hao, Fang; Liu, Xiaoqi; Qu, Chao; Li, Yuanfeng; Mu, Guoying; Yang, Zhenglin

    2015-01-01

    The cystathionine β-synthase (CBS) gene has been shown to be related to homocystinuria. This study was aimed to detect the mutations in CBS in a Han Chinese family with homocystinuria. A four-generation family from Shandong Province of China was recruited in this study. All available members of the family underwent comprehensive medical examinations. Genomic DNA was collected from peripheral blood of all the participants. The coding sequence of CBS was amplified by polymerase chain reaction (PCR), followed by direct DNA sequencing. Among all the family members, three affected individuals showed typical clinical features of homocystinuria. Two novel compound heterozygous mutations in the CBS gene, c.407T > C (p. L136P) and c.473C > T (p.A158V), were identified by sequencing analysis in this family. Both of the two missense mutations were detected in the three patients. Other available normal individuals, including the patients’ parents, grand parents, her younger sister and brother in this family either carried one of the two mutations, or none. In addition, the two mutations were not found in 600 ethnically matched normal controls. This study provides a mutation spectrum of CBS resulting in homocystinuriain a Chinese population, which may shed light on the molecular pathogenesis and clinical diagnosis of CBS-associated homocystinuria. PMID:26667307

  17. The CBS/CSE system: a potential therapeutic target in NAFLD?

    PubMed

    Sarna, Lindsei K; Siow, Yaw L; O, Karmin

    2015-01-01

    Non-alcoholic fatty liver disease (NAFLD) is a broad spectrum liver disorder diagnosed in patients without a history of alcohol abuse. NAFLD is growing at alarming rates worldwide. Its pathogenesis is complex and incompletely understood. The cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) system regulates homocysteine and cysteine metabolism and contributes to endogenous hydrogen sulfide (H2S) biosynthesis. This review summarizes our current understanding of the hepatic CBS/CSE system, and for the first time, positions this system as a potential therapeutic target in NAFLD. As will be discussed, the CBS/CSE system is highly expressed and active in the liver. Its dysregulation, presenting as alterations in circulating homocysteine and (or) H2S levels, has been reported in NAFLD patients and in NAFLD-associated co-morbidities such as obesity and type 2 diabetes. Intricate links between the CBS/CSE system and a number of metabolic and stress related molecular mediators have also emerged. Various dysfunctions in the hepatic CBS/CSE system have been reported in animal models representative of each NAFLD spectrum. It is anticipated that a newfound appreciation for the hepatic CBS/CSE system will emerge that will improve our understanding of NAFLD pathogenesis, and give rise to new prospective targets for management of this disorder.

  18. Development of quantitative metabolomics for Pichia pastoris.

    PubMed

    Carnicer, Marc; Canelas, André B; Ten Pierick, Angela; Zeng, Zhen; van Dam, Jan; Albiol, Joan; Ferrer, Pau; Heijnen, Joseph J; van Gulik, Walter

    2012-04-01

    Accurate, reliable and reproducible measurement of intracellular metabolite levels has become important for metabolic studies of microbial cell factories. A first critical step for metabolomic studies is the establishment of an adequate quenching and washing protocol, which ensures effective arrest of all metabolic activity and removal of extracellular metabolites, without causing leakage of metabolites from the cells. Five different procedures based on cold methanol quenching and cell separation by filtration were tested for metabolomics of Pichia pastoris regarding methanol content and temperature of the quenching solution as key parameters. Quantitative evaluation of these protocols was carried out through mass balance analysis, based on metabolite measurements in all sample fractions, those are whole broth, quenched and washed cells, culture filtrate and quenching and washing solution. Finally, the optimal method was used to study the time profiles of free amino acid and central carbon metabolism intermediates in glucose-limited chemostat cultures. Acceptable recoveries (>90%) were obtained for all quenching procedures tested. However, quenching at -27°C in 60% v/v methanol performed slightly better in terms of leakage minimization. We could demonstrate that five residence times under glucose limitation are enough to reach stable intracellular metabolite pools. Moreover, when comparing P. pastoris and S. cerevisiae metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both yeasts, except for the lower glycolysis, where the levels of these metabolites in P. pastoris suggested an enzymatic capacity limitation in that part of the metabolism. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0308-1) contains supplementary material, which is available to authorized users.

  19. The Biology of Pichia membranifaciens Killer Toxins.

    PubMed

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

    2017-03-23

    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.).

  20. 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

  1. 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

  2. 21 CFR 573.750 - Pichia pastoris dried yeast.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Pichia pastoris dried yeast. 573.750 Section 573.750 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS...

  3. Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel.

    PubMed

    Yamada, Toshiki; Krzeminski, Mickael; Bozoky, Zoltan; Forman-Kay, Julie D; Strange, Kevin

    2016-11-01

    Eukaryotic CLC anion channels and transporters are homodimeric proteins composed of multiple α-helical membrane domains and large cytoplasmic C-termini containing two cystathionine-β-synthase domains (CBS1 and CBS2) that dimerize to form a Bateman domain. The Bateman domains of adjacent CLC subunits interact to form a Bateman domain dimer. The functions of CLC CBS and Bateman domains are poorly understood. We utilized the Caenorhabditis elegans CLC-1/2/Ka/Kb anion channel homolog CLH-3b to characterize the regulatory roles of CLC cytoplasmic domains. CLH-3b activity is reduced by phosphorylation or deletion of a 14-amino-acid activation domain (AD) located on the linker connecting CBS1 and CBS2. We demonstrate here that phosphorylation-dependent reductions in channel activity require an intact Bateman domain dimer and concomitant phosphorylation or deletion of both ADs. Regulation of a CLH-3b AD deletion mutant is reconstituted by intracellular perfusion with recombinant 14-amino-acid AD peptides. The sulfhydryl reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET) alters in a phosphorylation-dependent manner the activity of channels containing single cysteine residues that are engineered into the short intracellular loop connecting membrane α-helices H and I (H-I loop), the AD, CBS1, and CBS2. In contrast, MTSET has no effect on channels in which cysteine residues are engineered into intracellular regions that are dispensable for regulation. These studies together with our previous work suggest that binding and unbinding of the AD to the Bateman domain dimer induces conformational changes that are transduced to channel membrane domains via the H-I loop. Our findings provide new, to our knowledge, insights into the roles of CLC Bateman domains and the structure-function relationships that govern the regulation of CLC protein activity by diverse ligands and signaling pathways.

  4. A mutation in the COX5 gene of the yeast Scheffersomyces stipitis alters utilization of amino acids as carbon source, ethanol formation and activity of cyanide insensitive respiration.

    PubMed

    Freese, Stefan; Passoth, Volkmar; Klinner, Ulrich

    2011-04-01

    Scheffersomyces stipitis PJH was mutagenized by random integrative mutagenesis and the integrants were screened for lacking the ability to grow with glutamate as sole carbon source. One of the two isolated mutants was damaged in the COX5 gene, which encodes a subunit of the cytochrome c oxidase. BLAST searches in the genome of Sc. stipitis revealed that only one singular COX5 gene exists in Sc. stipitis, in contrast to Saccharomyces cerevisiae, where two homologous genes are present. Mutant cells had lost the ability to grow with the amino acids glutamate, proline or aspartate and other non-fermentable carbon sources, such as acetic acid and ethanol, as sole carbon sources. Biomass formation of the mutant cells in medium containing glucose or xylose as carbon source was lower compared with the wild-type cells. However, yields and specific ethanol formation of the mutant were much higher, especially under conditions of higher aeration. The mutant cells lacked both cytochrome c oxidase activity and cyanide-sensitive respiration, whereas ADH and PDC activities were distinctly enhanced. SHAM-sensitive respiration was obviously essential for the fermentative metabolism, because SHAM completely abolished growth of the mutant cells with both glucose or xylose as carbon source.

  5. Transcultural validation of the ALS-CBS Cognitive Section for the Brazilian population.

    PubMed

    Branco, Lucas M T; Zanao, Tamires; De Rezende, Thiago J; Casseb, Raphael F; Balthazar, Marcio F; Woolley, Susan C; França, Marcondes C

    2017-02-01

    Cognitive decline (CD) is common but often under-recognized in ALS due to the scarcity of adequate cognitive screening methods. In this scenario, the Amyotrophic Lateral Sclerosis Cognitive Behavioural Screen (ALS-CBS) is the most investigated instrument and presents high sensitivity to identify CD. Currently, there are no validated cognitive screening tools for ALS patients in the Brazilian population and little is known about the frequency of ALS related CD in the country. We assessed the accuracy of the Brazilian Portuguese version of ALS-CBS Cognitive Section (ALS-CBS-Br) for classifying the cognitive status of Brazilian patients compared to a standard neuropsychological battery, and estimated the prevalence of CD in the Brazilian ALS population. Among 73 initially recruited ALS patients, 49 were included. Twenty-four patients were excluded due to severe motor disability, FTD diagnosis or non-acceptance. Ten healthy controls were also included. Ten ALS patients (20%) were diagnosed with executive dysfunction (ALSci) based on the battery results. ALS-CBS-Br scores were significantly lower in the ALSci group (p < 0.001). The scale accuracy in detecting executive dysfunction was 0.906. Optimal cut-off score was 10/20 (specificity 0.872 and sensitivity 0.900). In conclusion, the ALS-CBS-Br may facilitate the recognition of CD in routine clinical care and complement future studies in our population.

  6. Endogenous CBS-H2S Pathway Contributes to the Development of CCI-Induced Neuropathic Pain.

    PubMed

    Gui, Yulong; Li, Aiyuan; Qiu, Bihui; Chen, Feng; Chen, Liang; Liu, Daming; Chen, Shuxian; Zhou, Wei; Zhou, Hong

    2016-06-01

    Studies showed a complex relationship between hydrogen sulfide (H2S) and neuropathic pain. In this study, the relationship between endogenous CBS-H2S pathway in L4-6 spinal cord and neuropathic pain was explored. A total of 163 adult Kunming mice were used in this study. CBS expression and H2S formation in L4-6 spinal cord were detected in the development of neuropathic pain firstly. Then, effect of AOAA, an CBS inhibitor, on treatment of neuropathic pain by chronic construction injury surgery (CCI) was detected. Pain thresholds and activation of NF-κB(p65), ERK1/2 and CREB were measured as biomarks of neuropathic pain. Results showed that CCI surgery significantly upregulated protein expression of CBS and H2S formation. Correlation analysis showed pain thresholds had negative relationships with protein expression of CBS and H2S formation. Treatment with AOAA, a CBS inhibitor, inhibited CCI-induced upregulation of CBS expression and H2S formation (P < 0.05). Further, AOAA significantly decreased activation of NF-κB(p65), ERK1/2 and CREB pathway, and reversed CCI-induced allodynia (P < 0.05). This indicated that CBS-H2S pathway promoted the development of neuropathic pain. CBS-H2S pathway could be a promising target for treatment of neuropathic pain.

  7. Endurance and failure characteristics of modified Vasco X-2, CBS 600 and AISI 9310 spur gears

    NASA Technical Reports Server (NTRS)

    Townsend, D. P.; Zaretsky, E. V.

    1980-01-01

    Gear endurance tests and rolling-element fatigue tests were conducted to compare the performance of spur gears made from AISI 9310, CBS 600 and modified Vasco X-2 and to compare the pitting fatigue lives of these three materials. Gears manufactured from CBS 600 exhibited lives longer than those manufactured from AISI 9310. However, rolling-element fatigue tests resulted in statistically equivalent lives. Modified Vasco X-2 exhibited statistically equivalent lives to AISI 9310. CBS 600 and modified Vasco X-2 gears exhibited the potential of tooth fracture occurring at a tooth surface fatigue pit. Case carburization of all gear surfaces for the modified Vasco X-2 gears results in fracture at the tips of the gears.

  8. Chaperone therapy for homocystinuria: the rescue of CBS mutations by heme arginate.

    PubMed

    Melenovská, Petra; Kopecká, Jana; Krijt, Jakub; Hnízda, Aleš; Raková, Kateřina; Janošík, Miroslav; Wilcken, Bridget; Kožich, Viktor

    2015-03-01

    Classical homocystinuria is caused by mutations in the cystathionine β-synthase (CBS) gene. Previous experiments in bacterial and yeast cells showed that many mutant CBS enzymes misfold and that chemical chaperones enable proper folding of a number of mutations. In the present study, we tested the extent of misfolding of 27 CBS mutations previously tested in E. coli under the more folding-permissive conditions of mammalian CHO-K1 cells and the ability of chaperones to rescue the conformation of these mutations. Expression of mutations in mammalian cells increased the median activity 16-fold and the amount of tetramers 3.2-fold compared with expression in bacteria. Subsequently, we tested the responses of seven selected mutations to three compounds with chaperone-like activity. Aminooxyacetic acid and 4-phenylbutyric acid exhibited only a weak effect. In contrast, heme arginate substantially increased the formation of mutant CBS protein tetramers (up to sixfold) and rescued catalytic activity (up to ninefold) of five out of seven mutations (p.A114V, p.K102N, p.R125Q, p.R266K, and p.R369C). The greatest effect of heme arginate was observed for the mutation p.R125Q, which is non-responsive to in vivo treatment with vitamin B(6). Moreover, the heme responsiveness of the p.R125Q mutation was confirmed in fibroblasts derived from a patient homozygous for this genetic variant. Based on these data, we propose that a distinct group of heme-responsive CBS mutations may exist and that the heme pocket of CBS may become an important target for designing novel therapies for homocystinuria.

  9. Betaine supplementation is less effective than methionine restriction in correcting phenotypes of CBS deficient mice

    PubMed Central

    Gupta, Sapna; Wang, Liqun; Kruger, Warren D.

    2015-01-01

    Cystathionine beta synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by elevated serum total homocysteine (tHcy). Betaine supplementation, which can lower tHcy by stimulating homocysteine remethylation to methionine, is often given to CBS deficient patients in combination with other treatments such as methionine restriction and supplemental B-vitamins. However, the effectiveness of betaine supplementation by itself in the treatment of CBS deficiency has not been well explored. Here, we have examined the effect of a betaine supplemented diet on the TgI278T Cbs−/− mouse model of CBS deficiency and compared its effectiveness to our previously published data using a methionine restricted diet. TgI278T Cbs−/− mice on betaine, from the time of weaning until for 240 days of age, had a 40% decrease in mean tHcy level and a 137% increase in serum methionine levels. Betaine-treated Tg-I278T Cbs−/− mice also exhibited increased levels of betaine-dependent homocysteine methyl transferase (BHMT), increased levels of the lipogenic enzyme stearoyl-coenzyme A desaturase (SCD-1), and increased lipid droplet accumulation in the liver. Betaine supplementation largely reversed the hair loss phenotype in Tg-I278T Cbs−/− animals, but was far less effective than methionine restriction in reversing the weight-loss, fat-loss, and osteoporosis phenotypes. Surprisingly, betaine supplementation had several negative effects in control Tg-I278T Cbs+/− mice including decreased weight gain, lean mass, and bone mineral density. Our findings indicate that while betaine supplementation does have some beneficial effects, it is not as effective as methionine restriction for reversing the phenotypes associated with severe CBS deficiency in mice. PMID:26231230

  10. Extension of the composite CBS-QB3 method to singlet diradical calculations

    NASA Astrophysics Data System (ADS)

    Sirjean, Baptiste; Fournet, René; Glaude, Pierre-Alexandre; Ruiz-López, Manuel F.

    2007-02-01

    The composite CBS-QB3 method is widely used to obtain accurate energies of molecules and radicals although its use in the case of singlet diradicals gives rise to some difficulties. The problem is related to the parameterized correction this method introduces to account for spin-contamination. We report a new term specifically designed to describe singlet diradicals separated by at least one CH 2 unit. As a test case, we have computed the formation enthalpy of a series of diradicals that includes hydrocarbons as well as systems involving heteroatoms (nitrogen, oxygen). The resulting CBS-QB3 energies are very close to experiment.

  11. Identification and Comparative Analysis of CBS Domain-Containing Proteins in Soybean (Glycine max) and the Primary Function of GmCBS21 in Enhanced Tolerance to Low Nitrogen Stress

    PubMed Central

    Hao, Qingnan; Shang, Weijuan; Zhang, Chanjuan; Chen, Haifeng; Chen, Limiao; Yuan, Songli; Chen, Shuilian; Zhang, Xiaojuan; Zhou, Xinan

    2016-01-01

    Nitrogen is an important macronutrient required for plant growth, and is a limiting factor for crop productivity. Improving the nitrogen use efficiency (NUE) is therefore crucial. At present, the NUE mechanism is unclear and information on the genes associated with NUE in soybeans is lacking. cystathionine beta synthase (CBS) domain-containing proteins (CDCPs) may be implicated in abiotic stress tolerance in plants. We identified and classified a CBS domain–containing protein superfamily in soybean. A candidate gene for NUE, GmCBS21, was identified. GmCBS21 gene characteristics, the temporal expression pattern of the GmCBS21 gene, and the phenotype of GmCBS21 overexpression in transgenic Arabidopsis thaliana under low nitrogen stress were analyzed. The phenotypes suggested that the transgenic Arabidopsis thaliana seedlings performed better under the nitrogen-deficient condition. GmCBS21-overexpressing transgenic plants exhibit higher low nitrogen stress tolerance than WT plants, and this suggests its role in low nitrogen stress tolerance in plants. We conclude that GmCBS21 may serve as an excellent candidate for breeding crops with enhanced NUE and better yield. PMID:27128900

  12. Identification and Comparative Analysis of CBS Domain-Containing Proteins in Soybean (Glycine max) and the Primary Function of GmCBS21 in Enhanced Tolerance to Low Nitrogen Stress.

    PubMed

    Hao, Qingnan; Shang, Weijuan; Zhang, Chanjuan; Chen, Haifeng; Chen, Limiao; Yuan, Songli; Chen, Shuilian; Zhang, Xiaojuan; Zhou, Xinan

    2016-04-26

    Nitrogen is an important macronutrient required for plant growth, and is a limiting factor for crop productivity. Improving the nitrogen use efficiency (NUE) is therefore crucial. At present, the NUE mechanism is unclear and information on the genes associated with NUE in soybeans is lacking. cystathionine beta synthase (CBS) domain-containing proteins (CDCPs) may be implicated in abiotic stress tolerance in plants. We identified and classified a CBS domain-containing protein superfamily in soybean. A candidate gene for NUE, GmCBS21, was identified. GmCBS21 gene characteristics, the temporal expression pattern of the GmCBS21 gene, and the phenotype of GmCBS21 overexpression in transgenic Arabidopsis thaliana under low nitrogen stress were analyzed. The phenotypes suggested that the transgenic Arabidopsis thaliana seedlings performed better under the nitrogen-deficient condition. GmCBS21-overexpressing transgenic plants exhibit higher low nitrogen stress tolerance than WT plants, and this suggests its role in low nitrogen stress tolerance in plants. We conclude that GmCBS21 may serve as an excellent candidate for breeding crops with enhanced NUE and better yield.

  13. 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.

  14. 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

  15. Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

    PubMed Central

    Pan, Heng-Yen; Whittaker, Mei M.; Bouveret, Romaric; Berna, Anne; Bernier, François; Whittaker, James W.

    2007-01-01

    High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an α-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4×104 U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions. PMID:17399681

  16. Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

    SciTech Connect

    Pan, Heng-Yen; Whittaker, Mei M.; Bouveret, Romaric; Berna, Anne; Bernier, Francois; Whittaker, James W. . E-mail: jim@ebs.ogi.edu

    2007-05-18

    High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an {alpha}-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4 x 10{sup 4} U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions.

  17. 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

  18. 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

  19. Adhesion of Candida spp. and Pichia spp. to Wooden Surfaces

    PubMed Central

    Tomičić, Ružica; Tomičić, Zorica

    2017-01-01

    Summary Yeast adhesion to and biofilm formation on surfaces is present in many different environments. In food industry, biofilms may be a source of contaminations, causing food spoilage and reducing quality of products. Candida and Pichia are two common yeast genera involved in the spoilage of some food products. The aim of this study is to assess the potential of Candida and Pichia species to adhere to two types of wooden surfaces (smooth and rough), one of the materials typical for the food processing industry, and investigate the influence of surface roughness of wood on the degree of yeast adhesion. The adhesion of the cells to the wooden surfaces was determined by rinsing them from the surface, followed by methylene blue staining, and quantification after imaging under microscope by automatic counting of viable cells. The results showed that all Candida and Pichia strains were able to adhere to the wooden surfaces in a species- and strain-dependent manner. On the other hand, our data indicated that adhesion by these yeasts was not significantly affected by the roughness of the wood surfaces. PMID:28559743

  20. CBS-QB3 calculation of quantum chemical molecular descriptors of isomeric thiadiazoles.

    PubMed

    Glossman-Mitnik, Daniel

    2006-12-01

    The results of the calculation of several molecular descriptors of isomeric thiadiazoles through the CBS-QB3 model chemistry are presented in this work. The results could be useful in quantitative structure-activity relationship (QSAR) or quantitative structure-property relationship (QSPR) studies of derivatives of the nitrogen-containing analogs of thiophene.

  1. Complete Genome Sequences of the Xylose-Fermenting Candida intermedia Strains CBS 141442 and PYCC 4715

    PubMed Central

    Moreno, Antonio D.; Tellgren-Roth, Christian; Soler, Lucile; Dainat, Jacques; Olsson, Lisbeth

    2017-01-01

    ABSTRACT Sustainable biofuel production from lignocellulosic materials requires efficient and complete use of all abundant sugars in the biomass, including xylose. Here, we report on the de novo genome assemblies of two strains of the xylose-fermenting yeast Candida intermedia: CBS 141442 and PYCC 4715. PMID:28385851

  2. Draft Genome of Debaryomyces fabryi CBS 789T, Isolated from a Human Interdigital Mycotic Lesion

    PubMed Central

    Tafer, Hakim; Sterflinger, Katja

    2016-01-01

    The yeast genus Debaryomyces comprises species isolated from various natural habitats, man-made environments, and clinical materials. Here, the draft genome of D. fabryi CBS 789T, isolated from a human interdigital mycotic lesion, is presented. PMID:26847909

  3. Draft Genome Sequence of the Animal and Human Pathogen Malassezia pachydermatis Strain CBS 1879

    PubMed Central

    Triana, Sergio; González, Andrés; Ohm, Robin A.; Wösten, Han A. B.; de Cock, Hans; Restrepo, Silvia

    2015-01-01

    Malassezia pachydermatis is a basidiomycetous yeast that causes infections in humans and animals. Here, we report the genome sequence of Malassezia pachydermatis strain CBS 1879, which will facilitate the study of mechanisms underlying pathogenicity of the only non-lipid-dependent Malasezzia species. PMID:26472839

  4. Progression of brain atrophy in PSP and CBS over 6 months and 1 year.

    PubMed

    Dutt, Shubir; Binney, Richard J; Heuer, Hilary W; Luong, Phi; Attygalle, Suneth; Bhatt, Priyanka; Marx, Gabe A; Elofson, Jonathan; Tartaglia, Maria C; Litvan, Irene; McGinnis, Scott M; Dickerson, Bradford C; Kornak, John; Waltzman, Dana; Voltarelli, Lisa; Schuff, Norbert; Rabinovici, Gil D; Kramer, Joel H; Jack, Clifford R; Miller, Bruce L; Rosen, Howard J; Boxer, Adam L

    2016-11-08

    To examine the utility and reliability of volumetric MRI in measuring disease progression in the 4 repeat tauopathies, progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS), to support clinical development of new tau-directed therapeutic agents. Six- and 12-month changes in regional MRI volumes and PSP Rating Scale scores were examined in 55 patients with PSP and 33 patients with CBS (78% amyloid PET negative) compared to 30 normal controls from a multicenter natural history study. Longitudinal voxel-based morphometric analyses identified patterns of volume loss, and region-of-interest analyses examined rates of volume loss in brainstem (midbrain, pons, superior cerebellar peduncle), cortical, and subcortical regions based on previously validated atlases. Results were compared to those in a replication cohort of 226 patients with PSP with MRI data from the AL-108-231 clinical trial. Patients with CBS exhibited greater baseline atrophy and greater longitudinal atrophy rates in cortical and basal ganglia regions than patients with PSP; however, midbrain and pontine atrophy rates were similar. Voxel-wise analyses showed distinct patterns of regional longitudinal atrophy in each group as compared to normal controls. The midbrain/pons volumetric ratio differed between diagnoses but remained stable over time. In both patient groups, brainstem atrophy rates were correlated with disease progression measured using the PSP Rating Scale. Volume loss is quantifiable over a period of 6 months in CBS and PSP. Future clinical trials may be able to combine CBS and PSP to measure therapeutic effects. © 2016 American Academy of Neurology.

  5. Benchmarking Compound Methods (CBS-QB3, CBS-APNO, G3, G4, W1BD) against the Active Thermochemical Tables: Formation Enthalpies of Radicals.

    PubMed

    Somers, Kieran P; Simmie, John M

    2015-08-20

    The 298.15 K formation enthalpies of 38 radicals with molecular formula CxHyOz have been computed via the atomization procedure using the five title methods. The computed formation enthalpies are then benchmarked against the values recommended in the Active Thermochemical Tables (ATcT). The accuracy of the methods have been interpreted in terms of descriptive statistics, including the mean-signed error, mean-unsigned error, maximum average deviation, 2σ uncertainties, and 2×root-mean-square-deviations (2RMSD). The results highlight the following rank order of accuracy for the methods studied G4 > G3 > W1BD > CBS-APNO > CBS-QB3. The findings of this work are also considered in light of a recent companion study, which took an identical approach to quantifying the accuracies of these methods for 48 closed-shell singlet CxHyOz compounds. A similar order of accuracies and precisions were observed therein: G3 > G4 > W1BD > CBS-APNO > CBS-QB3. Both studies highlight systematic biases/deviations from the ATcT for the methods investigated, which are discussed in some detail, with methods having clear tendencies to over- or underpredict the recommended formation enthalpies for radical and/or closed-shell CxHyOz compounds. We show that one can improve the accuracy of their computation, and simultaneously reduce the uncertainty, by taking unweighted average formation enthalpies from various combinations of methods used. The reader should note that the statistical analyses preceding these conclusions also highlight that these error cancellation effects are unique for closed-shell and radical species. By extension, these error-cancellation effects can be expected to be different for various homologous series and chemical functionalities and their closed- and open-shell subgroups. Hence, further benchmarking studies are advised for other homologous series, such that the scientists and engineers (e.g., combustion/atmospheric/astrochemical) who frequently use these methods can

  6. Xylulose fermentation by Saccharomyces cerevisiae and xylose-fermenting yeast strains.

    PubMed

    Yu, S; Jeppsson, H; Hahn-Hägerdal, B

    1995-12-01

    Xylulose fermentation by four strains of Saccharomyces cerevisiae and two strains of xylose-fermenting yeasts, Pichia stipitis CBS 6054 and Candida shehatae NJ 23, was compared using a mineral medium at a cell concentration of 10 g (dry weight)/l. When xylulose was the sole carbon source and fermentation was anaerobic, S. cerevisiae ATCC 24860 and CBS 8066 showed a substrate consumption rate of 0.035 g g cells-1 h-1 compared with 0.833 gg cells-1 h-1 for glucose. Bakers' yeast and S. cerevisiae isolate 3 consumed xylulose at a much lower rate although they fermented glucose as rapidly as the ATCC and the CBS strains. While P. stipitis CBS 6054 consumed both xylulose and glucose very slowly under anaerobic conditions, C. shehatae NJ 23 fermented xylulose at a rate of 0.345 gg cells-1 h-1, compared with 0.575 gg cells-1 h-1 for glucose. For all six strains, the addition of glucose to the xylulose medium did not enhance the consumption of xylulose, but increased the cell biomass concentrations. When fermentation was performed under oxygen-limited conditions, less xylulose was consumed by S. cerevisiae ATCC 24860 and C. shehatae NJ 23, and 50%- 65% of the assimilated carbon could not be accounted for in the products determined.

  7. Biocontrol of mold growth in high-moisture wheat stored under airtight conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae.

    PubMed Central

    Petersson, S; Schnürer, J

    1995-01-01

    Pichia anomala inhibits the growth of Penicillium roqueforti and Aspergillus candidus on agar. In this investigation, antagonistic activity on agar against 17 mold species was determined. The abilities of Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae to inhibit the growth of the mold Penicillium roqueforti in nonsterile high-moisture wheat were compared by adding 10(3) Penicillium roqueforti spores and different amounts of yeast cells per gram of wheat. Inoculated grain was packed in glass tubes, incubated at 25 degrees C with a restricted air supply, and the numbers of yeast and mold CFU were determined on selective media after 7 and 14 days. Pichia anomala reduced growth on agar plates for all of the mold species tested in a dose-dependent manner. Aspergillus fumigatus and Eurotium amstelodami were the most sensitive, while Penicillium italicum and Penicillium digitatum were the most resistant. Pichia anomala had the strongest antagonistic activity in wheat, with 10(5) and 10(6) CFU/g completely inhibiting the growth of Penicillium roqueforti. Inhibition was least pronounced at the optimum temperature (21 degrees C) and water activity (0.95) for the growth of Penicillium roqueforti. Pichia guilliermondii slightly reduced the growth of Penicillium roqueforti in wheat inoculated with 10(5) and 10(6) yeast CFU/g. S. cerevisiae inhibited mold growth only weakly at the highest inoculum level. Pichia anomala grew from 10(3) to 10(7) CFU/g of wheat in 1 week. To reach the same level, Pichia guilliermondii had to be inoculated at 10(4) CFU while S. cerevisiae required an inoculum of 10(5) CFU to reach 10(7) CFU/g of wheat. PMID:7793907

  8. Isolation and characterization of acetic acid-tolerant galactose-fermenting strains of Saccharomyces cerevisiae from a spent sulfite liquor fermentation plant.

    PubMed Central

    Lindén, T; Peetre, J; Hahn-Hägerdal, B

    1992-01-01

    From a continuous spent sulfite liquor fermentation plant, two species of yeast were isolated, Saccharomyces cerevisiae and Pichia membranaefaciens. One of the isolates of S. cerevisiae, no. 3, was heavily flocculating and produced a higher ethanol yield from spent sulfite liquor than did commercial baker's yeast. The greatest difference between isolate 3 and baker's yeast was that of galactose fermentation, even when galactose utilization was induced, i.e., when they were grown in the presence of galactose, prior to fermentation. Without acetic acid present, both baker's yeast and isolate 3 fermented glucose and galactose sequentially. Galactose fermentation with baker's yeast was strongly inhibited by acetic acid at pH values below 6. Isolate 3 fermented galactose, glucose, and mannose without catabolite repression in the presence of acetic acid, even at pH 4.5. The xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) activities were determined in some of the isolates as well as in two strains of S. cerevisiae (ATCC 24860 and baker's yeast) and Pichia stipitis CBS 6054. The S. cerevisiae strains manifested xylose reductase activity that was 2 orders of magnitude less than the corresponding P. stipitis value of 890 nmol/min/mg of protein. The xylose dehydrogenase activity was 1 order of magnitude less than the corresponding activity of P. stipitis (330 nmol/min/mg of protein). Images PMID:1622236

  9. Simultaneous saccharification and ethanol fermentation of oxalic acid pretreated corncob assessed with response surface methodology.

    PubMed

    Lee, Jae-Won; Rodrigues, Rita C L B; Jeffries, Thomas W

    2009-12-01

    Response surface methodology was used to evaluate optimal time, temperature and oxalic acid concentration for simultaneous saccharification and fermentation (SSF) of corncob particles by Pichia stipitis CBS 6054. Fifteen different conditions for pretreatment were examined in a 2(3) full factorial design with six axial points. Temperatures ranged from 132 to 180 degrees C, time from 10 to 90 min and oxalic acid loadings from 0.01 to 0.038 g/g solids. Separate maxima were found for enzymatic saccharification and hemicellulose fermentation, respectively, with the condition for maximum saccharification being significantly more severe. Ethanol production was affected by reaction temperature more than by oxalic acid and reaction time over the ranges examined. The effect of reaction temperature was significant at a 95% confidence level in its effect on ethanol production. Oxalic acid and reaction time were statistically significant at the 90% level. The highest ethanol concentration (20 g/l) was obtained after 48 h with an ethanol volumetric production rate of 0.42 g ethanol l(-1) h(-1). The ethanol yield after SSF with P. stipitis was significantly higher than predicted by sequential saccharification and fermentation of substrate pretreated under the same condition. This was attributed to the secretion of beta-glucosidase by P. stipitis. During SSF, free extracellular beta-glucosidase activity was 1.30 pNPG U/g with P. stipitis, while saccharification without the yeast was 0.66 pNPG U/g.

  10. Development of yeasts for xylose fermentation

    SciTech Connect

    Jeffries, T.W.; Yang, V.; Marks, J.; Amartey, S.; Kenealy, W.R.; Cho, J.Y.; Dahn, K.; Davis, B.P.

    1993-12-31

    Xylose is an abundant sugar in hardwoods and agricultural residues. Its use is essential for any economical conversion of lignocellulose to ethanol. Only a few yeasts ferment xylose effectively. Our results show that the best strains are Candida shehatae ATCC 2984 and Pichia stipitis CBS 6054. Wild type strains of C. shehatae ATCC 22984 will produce 56 g/L of ethanol from xylose within 48 h in a fed batch fermentation. We have obtained improved mutants of P.stipitis by selecting for growth on L-xylose and L-arabinose. Mutant strains produce up to 55% more ethanol than the parent and exhibit higher specific fermentation rates. We have also developed an effective transformation system that enables the introduction and expression of heterologous DNA on integrating and autonomous vectors. The transformation system for P. stipitis is based on its URA3 gene as a selectable marker and an autonomous replication sequence (ARS) which we isolated from the parent. We are using integrating and ARS vectors to metabolically engineer P. stipitis by altering the regulation and expression of key enzymes. As model systems we are examining the expression of alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) that are present in limiting amounts or induced only under non-growth conditions.

  11. Molecular identification of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila

    PubMed Central

    Mota, Adolfo Jose; Back-Brito, Graziella Nuernberg; Nobrega, Francisco G.

    2012-01-01

    Traditional phenotypic methods and commercial kits based on carbohydrate assimilation patterns are unable to consistently distinguish among isolates of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila. As result, these species are often misidentified. In this work, we established a reliable method for the identification/differentiation of these species. Our assay was validated by DNA sequencing of the polymorphic region used in a real-time PCR assay driven by species-specific probes targeted to the fungal ITS 1 region. This assay provides a new tool for pathogen identification and for epidemiological, drug resistance and virulence studies of these organisms. PMID:22481884

  12. Molecular identification of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila.

    PubMed

    Mota, Adolfo Jose; Back-Brito, Graziella Nuernberg; Nobrega, Francisco G

    2012-01-01

    Traditional phenotypic methods and commercial kits based on carbohydrate assimilation patterns are unable to consistently distinguish among isolates of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila. As result, these species are often misidentified. In this work, we established a reliable method for the identification/differentiation of these species. Our assay was validated by DNA sequencing of the polymorphic region used in a real-time PCR assay driven by species-specific probes targeted to the fungal ITS 1 region. This assay provides a new tool for pathogen identification and for epidemiological, drug resistance and virulence studies of these organisms.

  13. Simplification of the CBS-QB3 method for predicting gas-phase deprotonation free energies

    NASA Astrophysics Data System (ADS)

    Casasnovas, Rodrigo; Frau, Juan; Ortega-Castro, Joaquín; Salvà, Antoni; Donoso, Josefa; Muñoz, Francisco

    Simplified versions of CBS-QB3 model chemistry were used to calculate the free energies of 36 deprotonation reactions in the gas phase. The best such version, S9, excluded coupled cluster calculation [CCSD(T)], and empirical (ΔEemp) and spin-orbit (ΔEint) correction terms. The mean absolute deviation and root mean square thus obtained (viz. 1.24 and 1.56 kcal/mol, respectively) were very-close to those provided by the original CBS-QB3 method (1.19 and 1.52 kcal/mol, respectively). The high-accuracy of the proposed simplification and its computational expeditiousness make it an excellent choice for energy calculations on gas-phase deprotonation reactions in complex systems.

  14. Optimized simultaneous saccharification and co-fermentation of rice straw for ethanol production by Saccharomyces cerevisiae and Scheffersomyces stipitis co-culture using design of experiments.

    PubMed

    Suriyachai, Nopparat; Weerasaia, Khatiya; Laosiripojana, Navadol; Champreda, Verawat; Unrean, Pornkamol

    2013-08-01

    Herein an ethanol production process from rice straw was optimized. Simultaneous saccharification and co-fermentation (SSCF) using Saccharomyces cerevisiae and Scheffersomyces stipitis co-culture was carried out to enhance ethanol production. The optimal saccharification solid loading was 5%. Key fermentation parameters for co-culture including cell ratio, agitation rate and temperature was rationally optimized using design of experiment (DoE). Optimized co-culture conditions for maximum ethanol production efficiency were at S. cerevisiae:S. stipitis cell ratio of 0.31, agitation rate of 116 rpm and temperature of 33.1°C. The optimized SSCF process reached ethanol titer of 15.2g/L and ethanol yield of 99% of theoretical yield, consistent with the DoE model prediction. Moreover, SSCF process under high biomass concentration resulted in high ethanol concentration of 28.6g/L. This work suggests the efficiency and scalability of the developed SSCF process which could provide an important basis for the economic feasibility of ethanol production from lignocelluloses. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Ethanol production by a new pentose-fermenting yeast strain, Scheffersomyces stipitis UFMG-IMH 43.2, isolated from the Brazilian forest.

    PubMed

    Ferreira, Adriana D; Mussatto, Solange I; Cadete, Raquel M; Rosa, Carlos A; Silva, Silvio S

    2011-07-01

    The ability of a recently isolated Scheffersomyces stipitis strain (UFMG-IMH 43.2) to produce ethanol from xylose was evaluated. For the assays, a hemicellulosic hydrolysate produced by dilute acid hydrolysis of sugarcane bagasse was used as the fermentation medium. Initially, the necessity of adding nutrients (MgSO(4)·7H(2)O, yeast extract and/or urea) to this medium was verified, and the yeast extract supplementation favoured ethanol production by the yeast. Then, in a second stage, assays under different initial xylose and cell concentrations, supplemented or not with yeast extract, were performed. All these three variables showed significant (p < 0.05) influence on ethanol production. The best results (ethanol yield and productivity of 0.19 g/g and 0.13 g/l/h, respectively) were obtained using the hydrolysate containing an initial xylose concentration of 30 g/l, supplemented with 5.0 g/l yeast extract and inoculated with an initial cell concentration of 2.0 g/l. S. stipitis UFMG-IMH 43.2 was demonstrated to be a yeast strain with potential for use in xylose conversion to ethanol. The establishment of the best fermentation conditions was also proved to be of great importance to increasing the product formation by this yeast strain. These findings open up new perspectives for the establishment of a feasible technology for ethanol production from hemicellulosic hydrolysates.

  16. Dynamic model-based analysis of furfural and HMF detoxification by pure and mixed batch cultures of S. cerevisiae and S. stipitis.

    PubMed

    Hanly, Timothy J; Henson, Michael A

    2014-02-01

    Inhibitory compounds that result from biomass hydrolysis are an obstacle to the efficient production of second-generation biofuels. Fermentative microorganisms can reduce compounds such as furfural and 5-hydroxymethyl furfural (HMF), but detoxification is accompanied by reduced growth rates and ethanol yields. In this study, we assess the effects of these furan aldehydes on pure and mixed yeast cultures consisting of a respiratory deficient mutant of Saccharomyces cerevisiae and wild-type Scheffersomyces stipitis using dynamic flux balance analysis. Uptake kinetics and stoichiometric equations for the intracellular reduction reactions associated with each inhibitor were added to genome-scale metabolic reconstructions of the two yeasts. Further modification of the S. cerevisiae metabolic network was necessary to satisfactorily predict the amount of acetate synthesized during HMF reduction. Inhibitory terms that captured the adverse effects of the furan aldehydes and their corresponding alcohols on cell growth and ethanol production were added to attain qualitative agreement with batch experiments conducted for model development and validation. When the two yeasts were co-cultured in the presence of the furan aldehydes, inoculums that reduced the synthesis of highly toxic acetate produced by S. cerevisiae yielded the highest ethanol productivities. The model described here can be used to generate optimal fermentation strategies for the simultaneous detoxification and fermentation of lignocellulosic hydrolysates by S. cerevisiae and/or S. stipitis.

  17. Draft Genome Sequence of the Mycoparasitic Oomycete Pythium oligandrum Strain CBS 530.74.

    PubMed

    Kushwaha, Sandeep K; Vetukuri, Ramesh R; Grenville-Briggs, Laura J

    2017-05-25

    The oomycete Pythium oligandrum is a mycoparasite and licenced biological control agent. Here, we report the draft genome sequence of P. oligandrum strain CBS 530.74, which is 36.80 Mb. It contains 341 scaffolds and 11,647 predicted protein-coding genes. As reported for plant-pathogenic Pythium species, RXLR-type effector sequences are absent. Copyright © 2017 Kushwaha et al.

  18. Draft genome sequence of CBS 2479, the standard type strain of Trichosporon asahii.

    PubMed

    Yang, Rong Ya; Li, Hai Tao; Zhu, He; Zhou, Guang Peng; Wang, Meng; Wang, Lei

    2012-11-01

    Trichosporon asahii is one of the important opportunistic pathogenic fungi. Here, we first report the draft nuclear chromosome genome sequence and mitochondrial genome sequence of T. asahii CBS 2479, which is a standard strain of T. asahii that was isolated from a progressive psoriatic lesion. COG analysis predicted that 3,131 genes were assigned to 23 functional categories and that 628 genes were predicted to have a general function.

  19. A case of homocystinuria due to CBS gene mutations revealed by cerebral venous thrombosis.

    PubMed

    Sarov, Mariana; Not, Adeline; de Baulny, Hélène Ogier; Masnou, Pascal; Vahedi, Katayoun; Bousser, Marie-Germaine; Denier, Christian

    2014-01-15

    Homocystinuria caused by cystathionine beta synthase (CBS) deficiency is most often diagnosed in childhood and has a variable expressivity. The most frequent abnormalities include intellectual disability, ectopia lentis, myopia, skeletal abnormalities or thromboembolism. To report a case of homocystinuria unraveled by cerebral venous thrombosis (CVT). A 17 year old female was admitted in our department of neurology for subacute headache and presented seizures in the emergency room. Cerebral imaging revealed CVT. Severe hyperhomocysteinemia was found and led to the diagnosis of homocystinuria due to composite heterozygous mutations in the CBS gene. Further investigations disclosed lens subluxation in association with myopia, mild scoliosis and osteopenia. The patient was treated by heparin followed by warfarin, vitamin therapy and dietary methionine restriction. Total homocysteine and methionine levels became normal in a few weeks and the patient had a complete recovery. In patients with CVT, plasma total homocysteine measurement as part of the etiologic work up may reveal severe hyperhomocysteinemia due to CBS or remethylation defects that require specific treatment and management including perhaps protein-restricted diet and/or vitamin therapy for life. © 2013.

  20. Genome Sequence of Type Strain Fonsecaea multimorphosa CBS 980.96T, a Causal Agent of Feline Cerebral Phaeohyphomycosis

    PubMed Central

    Leao, Aniele C. Ribas; Weiss, Vinicius Almir; Vicente, Vania Aparecida; Costa, Flavia; Bombassaro, Amanda; Raittz, Roberto Tadeu; Steffens, Maria Berenice R.; Pedrosa, Fabio Oliveira; Gomes, Renata R.; Baura, Valter; Faoro, Helisson; Sfeir, Michelle Zibetti Tadra; Balsanelli, Eduardo; Moreno, Leandro F.; Najafzadeh, M. Javad

    2017-01-01

    ABSTRACT A draft genome sequence of type strain Fonsecaea multimorphosa CBS 980.96T was obtained. This species was first isolated from a cat with cerebral phaeohyphomycosis in Queensland, Australia. PMID:28209838

  1. Boryl substitution of acetaldehyde makes it an enol: inconsistency between Gn/CBS and ab initio/DFT data.

    PubMed

    Balabin, Roman M

    2010-03-18

    Tautomerism, a particular case of isomerism, plays an important role in modern organic chemistry, biochemistry, medicinal chemistry, pharmacology, and molecular biology. Inconsistency between results of complex energy computation methods Gn/CBS (G2, G3, CBS-4M, and CBS-QB3) and high-level ab initio/DFT ones (CCSD(T)/CBS, MP2/CBS, and B3LYP/aug-cc-pVTZ) is found. Gn/CBS methods provide a qualitatively different description of tautomeric (keto-enol) equilibrium in 2-substituted acetaldehydes. According to valence focal point analysis (FPA) based on CCSD(T)/aug-cc-pVTZ, MP3/aug-cc-pVQZ, and MP2/aug-cc-pV5Z energies, boryl substitution of acetaldehyde makes it an enol. In other words, enol was found to be the global minimum on the potential energy surface (PES) of C(2)H(5)BO. Gn/CBS methods predict the keto form to be the minimum. The relative energy of alkenol, CH(BH(2))=CH(OH), is calculated to be -1.67 +/- 0.82 kcal mol(-1) at CCSD(T)/CBS level of theory. Hydrogen shift effects are also calculated in two other 2-substituted acetaldehydes, namely, 3-oxopropanenitrile (C(3)H(3)NO) and ethanal (C(2)H(4)O), with a general formula of XH(2)C-CHO (X = BH(2), CN, and H). Electron density (charge) transfer between the C=C double bond and the free p orbital of the boron atom (B) in a boryl group (BH(2)) greatly stabilizes enol with respect to ketone, CH(2)(BH(2))-CHO. The first known stabilization of enol in an acetaldehyde derivative, without an intramolecular hydrogen bond (H-bond), questions the accuracy of complex energy computation methods for boron-containing molecules. The possible reasons and consequences of this finding are discussed.

  2. Colloidal bismuth subcitrate (CBS) impedes proton entry into Helicobacter pylori and increases the efficacy of growth dependent antibiotics

    PubMed Central

    Marcus, Elizabeth A.; Sachs, George; Scott, David R.

    2015-01-01

    Background Successful eradication of Helicobacter pylori is becoming more difficult, mainly due to emerging antibiotic resistance. Treatment regimens containing bismuth have increased efficacy, but the mechanism is unknown. H. pylori is a neutralophile adapted to survive the acidic gastric environment via acid acclimation, but demonstrates more robust growth at neutral pH. Many antibiotics used to treat H. pylori rely on bacterial growth. Aim To investigate the mechanism of increased efficacy of bismuth-containing H. pylori treatment regimens. Methods RNAseq and qPCR, urease activity in permeabilized and intact bacteria, internal pH, and membrane potential were measured with and without colloidal bismuth subcitrate (CBS). Bacterial survival was assessed with CBS and/or ampicillin. Results Genes involved with metabolism and growth were upregulated in the presence of CBS at acidic pH. Urease activity of permeabilized H. pylori at pH 7.4 and 4.5 decreased in the presence of CBS, but intact urease activity only decreased at acidic pH. The fall in cytoplasmic pH with external acidification was diminished by CBS. The increase in membrane potential in response to urea addition at acidic medium pH was unaffected by CBS. The impact of CBS and ampicillin on H. pylori survival was greater than either agent alone. Conclusions Bismuth is not acting directly on urease or the urea channel. CBS impedes proton entry into the bacteria, leading to a decrease in the expected fall in cytoplasmic pH. With cytoplasmic pH remaining within range for increased metabolic activity of a neutralophile, the efficacy of growth dependent antibiotics is augmented. PMID:26238858

  3. CBS9106-induced CRM1 degradation is mediated by cullin ring ligase activity and the neddylation pathway.

    PubMed

    Saito, Naoya; Sakakibara, Keiichi; Sato, Takuji; Friedman, Jonathan M; Kufe, Donald W; VonHoff, Daniel D; Kawabe, Takumi

    2014-12-01

    Chromosome region maintenance 1 (CRM1) mediates the nuclear export of proteins and mRNAs, and is overexpressed in various cancers. Recent studies have also reported that CRM1 protein expression is a negative prognostic factor in patients with cancer. Therefore, CRM1 is considered a potential target for anticancer therapy. Our previous study demonstrated that CBS9106, a synthetic small-molecular inhibitor of CRM1, decreases CRM1 protein through proteasomal degradation without affecting CRM1 mRNA levels. However, the mechanism by which CRM1 is degraded is not well understood. Here, we demonstrate a novel signaling pathway that plays an important role in CBS9106-induced CRM1 degradation. We found that MLN4924, a selective inhibitor of NEDD8-activating enzyme (NAE), effectively inhibits cullin neddylation and attenuates CBS9106-induced CRM1 degradation in a time- and dose-dependent manner. MLN4924 also attenuated CBS9106-induced nuclear accumulation of Ran-binding protein 1 (RanBP1), cell growth inhibition, and apoptosis. Furthermore, RNAi-mediated knockdown of neddylation pathway proteins (NEDD8 and UBA3) or cullin ring ligase (CRL) component protein (Rbx1) attenuated CRM1 protein degradation and G1 phase cell-cycle arrest by CBS9106. Knockdown of CSN5 or CAND1 also partially inhibited CBS9106-induced CRM1 degradation. These findings demonstrate that CBS9106-induced CRM1 degradation is conferred by CRL activity involving the neddylation pathway, and that this response to CBS9106 leads to cell growth inhibition and apoptosis.

  4. DNA hypomethylation of CBS promoter induced by folate deficiency is a potential noninvasive circulating biomarker for colorectal adenocarcinomas.

    PubMed

    Xue, Geng; Lu, Chao-Jing; Pan, Shu-Jun; Zhang, Yin-Ling; Miao, Hui; Shan, Shi; Zhu, Xiao-Ting; Zhang, Yi

    2017-08-01

    Aberrant DNA methylation patterns, which induced by folate deficiency, play important roles in tumorigenesis of colorectal cancer (CRC). Some DNA methylation alterations can also be detected in cell-free DNA (cfDNA) of patients' plasma, making cfDNA an ideal noninvasive circulating biomarker. However, exact DNA methylation alterations induced by folate deficiency in tumorigenesis of CRC and exact potential circulating cfDNA methylation biomarker are still unclear. Therefore, DNA methylation patterns of the normal human colon mucosal epithelial cell line (NCM460), cultured with normal or low folate content, were screened and the DNA hypomethylation of cystathionine-beta-synthase (CBS) promoter was further validated in vitro and vivo. Then, the correlation analysis between folate level, DNA methylation alteration in promoter and expression of CBS was carried out in vitro and vivo. Further, the methylation patterns of CBS promoter in plasma cfDNA were detected and statistically correlated with pathological parameters and clinical outcome. Our study showed that DNA hypomethylation in CBS promoter, induced by folate deficiency, would lead to up-regulation of CBS both in vitro and vivo. Patients with cfDNA hypomethylation of CBS promoter in plasma were correlated with high tumor stage and poor clinical outcome. In addition, cfDNA hypomethylation of CBS promoter in plasma was shown to be an independent prognostic factor for recurrence and cancer-related death in CRC. Our results indicated that DNA hypomethylation of CBS promoter induced by folate deficiency could serve as a potential noninvasive circulating biomarker and may be helpful in developing more effective prognostic markers for CRC.

  5. 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.

  6. Improving cyanobacterail O2-tolerance using CBS hydrogenase for hydrogen production

    SciTech Connect

    Maness, Pin-Ching; Eckert, Carrie; Wawrousek, Karen; Noble, Scott; Pennington, Grant; Yu, Jianping

    2016-11-11

    Cyanobacterial H2 production is a viable path to renewable H2 with water serving as the electron donor and sunlight the energy source. A grand challenge is the sensitivity of the underlying hydrogenase to O2, the latter an inherent byproduct of oxygenic photosynthesis. This challenge has been identified as a technical barrier in the Fuel Cell Technologies Office (FCTO) Multi-year Research, Development and Deployment Plan. One solution is to express in cyanobacterium an O2-tolerant hydrogenase to circumvent this barrier. We have uncovered an O2-tolerant hydrogenase from a photosynthetic bacterium Rubrivivax gelatinosus CBS (Casa Bonita Strain; hereafter “CBS”) with a half-life near 21 h when exposed to ambient O2. We sequenced the CBS genome and identified two sets of maturation machineries hyp1 and hyp2. Transcripts expression analysis and mutagenesis revealed that hyp1 is responsible for the assembly of the O2-tolerant CO-oxidation (Coo) hydrogenase and hyp2 is involved in the maturation of a H2-uptake hydrogenase. The structural genes encoding the O2-tolerant hydrogenase (cooLXUH) and maturation genes hyp1FABCDE were therefore cloned and expressed in the model cyanobacterium Synechocystis sp. PCC 6803. We obtained several recombinants displaying hydrogenase activity in a Synechocystis host lacking background activity, suggesting that the CBS hydrogenase is active in Synechocystis. Yet the activity is extremely low. To ensure balanced protein expression, we systematically optimized heterologous expression of 10 CBS genes by using stronger promoters and better ribosome binding site. Moreover we attempted the expression of cooM and cooK genes, verified to be important in CBS to afford activity. CooM is a very large protein and both CooM and CooK are membrane-associated. These properties limited our success in expressing both genes in Synechocystis, although they

  7. A Database of Formation Enthalpies of Nitrogen Species by Compound Methods (CBS-QB3, CBS-APNO, G3, G4).

    PubMed

    Simmie, John M

    2015-10-22

    Accurate thermochemical data for compounds containing C/H/N/O are required to underpin kinetics simulation and modeling of the reactions of these species in different environments. There is a dearth of experimental data so computational quantum chemistry has stepped in to fill this breach and to verify whether particular experiments are in need of revision. A number of composite model chemistries (CBS-QB3, CBS-APNO, G3, and G4) are used to compute theoretical atomization energies and hence enthalpies of formation at 0 and 298.15 K, and these are benchmarked against the best available compendium of values, the Active Thermochemical Tables or ATcT. In general the agreement is very good for some 28 species with the only discrepancy being for hydrazine. It is shown that, although individually the methods do not perform that well, collectively the mean unsigned error is <1.7 kJ mol(-1); hence, this approach provides a useful tool to screen published values and validate new experimental results. Using multiple model chemistries does have some drawbacks but can produce good results even for challenging molecules like HOON and CN2O2. The results for these smaller validated molecules are then used as anchors for determining the formation enthalpies of larger species such as methylated hydrazines and diazenes, five- and six-membered heterocyclics via carefully chosen isodesmic working reactions with the aim of resolving some discrepancies in the literature and establishing a properly validated database. This expanded database could be useful in testing the performance of computationally less-demanding density function methods with newer functionals that have the capacity to treat much larger systems than those tested here.

  8. 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

    2017-09-18

    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 were 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 were no significant sequence matches of LegHb to known allergens or toxins. Eleven Pichia proteins had modest identity matches to minor environmental allergens and thirteen Pichia proteins had significant matches to proteins from toxic sources. Yet the matched allergens and toxins had 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. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  9. Physiological characteristics of the biocontrol yeast Pichia anomala J121.

    PubMed

    Fredlund, Elisabeth; Druvefors, Ulrika; Boysen, Marianne E; Lingsten, Karl-Johan; Schnürer, Johan

    2002-08-01

    The yeast Pichia anomala J121 prevents mold spoilage and enhances preservation of moist grain in malfunctioning storage systems. Development of P. anomala J121 as a biocontrol agent requires in-depth knowledge about its physiology. P. anomala J121 grew under strictly anaerobic conditions, at temperatures between 3 degrees C and 37 degrees C, at pH values between 2.0 and 12.4, and at a water activity of 0.92 (NaCl) and 0.85 (glycerol). It could assimilate a wide range of C- and N-sources and produce killer toxin. A selective medium containing starch, nitrate, acetic acid, and chloramphenicol was developed for P. anomala. P. anomala was equally sensitive as Candida albicans to common antifungal compounds. Growth ability at a range of environmental conditions contributes to the competitive ability of the biocontrol yeast P. anomala J121.

  10. Production and Analysis of Perdeuterated Lipids from Pichia pastoris Cells

    PubMed Central

    de Ghellinck, Alexis; Schaller, Hubert; Laux, Valérie; Haertlein, Michael; Sferrazza, Michele; Maréchal, Eric; Wacklin, Hanna; Jouhet, Juliette; Fragneto, Giovanna

    2014-01-01

    Probing molecules using perdeuteration (i.e deuteration in which all hydrogen atoms are replaced by deuterium) is extremely useful in a wide range of biophysical techniques. In the case of lipids, the synthesis of the biologically relevant unsaturated perdeuterated lipids is challenging and not usually pursued. In this work, perdeuterated phospholipids and sterols from the yeast Pichia pastoris grown in deuterated medium are extracted and analyzed as derivatives by gas chromatography and mass spectrometry respectively. When yeast cells are grown in a deuterated environment, the phospholipid homeostasis is maintained but the fatty acid unsaturation level is modified while the ergosterol synthesis is not affected by the deuterated culture medium. Our results confirm that the production of well defined natural unsaturated perdeuterated lipids is possible and gives also new insights about the process of desaturase enzymes. PMID:24747350

  11. Functional analysis of alcohol dehydrogenase (ADH) genes in Pichia pastoris.

    PubMed

    Karaoglan, Mert; Karaoglan, Fidan Erden; Inan, Mehmet

    2016-03-01

    To characterize the genes responsible for ethanol utilization in Pichia pastoris. ADH3 (XM_002491337) and ADH (FN392323) genes were disrupted in P. pastoris. The ADH3 mutant strain, MK115 (Δadh3), lost its ability to grow on minimal ethanol media but produced ethanol in minimal glucose medium. ADH3p was responsible for 92 % of total Adh enzyme activity in glucose media. The double knockout strain MK117 (Δadh3Δadh) also produced ethanol. The Adh activities of X33 and MK116 (Δadh) strains were not different. Thus, the ADH gene does not play a role in ethanol metabolism. The PpADH3 is the only gene responsible for consumption of ethanol in P. pastoris.

  12. Nicotine (NC)-induced "depressive" behavioral symptoms and effects of antidepressants including cannabinoids (CBs).

    PubMed

    Hayase, Tamaki

    2008-12-01

    Depression is one of the frequently-observed psychiatric symptoms associated with nicotine (NC) use. In the present study, considering the unique effects of NC (e.g. antidepressant effects have also been reported), the time course of the NC-induced depressive behavioral alterations in a mouse model was compared with a typical depression-inducing stressor. Furthermore, based on the involvement of cannabinoid (CB) receptors in the behavioral effects of NC, the effects of antidepressants including CB ligands (CBs) against the NC-induced behavioral alterations were also investigated. Repeated subcutaneous NC treatments (0.3 mg/kg, 4 days), like repeated immobilization stress (IM) treatments (10 min, 4 days), caused prolonged depressive effects (increased immobility time) at both 2 hr and 1 day time points after the last treatment in the tail suspension test. However, in the NC group, depressive effects (suppressed swimming behaviors) were observed only at the 2 hr time point in the forced swimming test. The antidepressants amitriptyline, clomipramine and fluvoxamine, the endogenous mixed CB agonist/antagonist virodhamine and the anandamide-like cannabimimetic O-2093 provided antagonistic effects against the depressive behaviors in the tail suspension test. However, in the forced swimming test, NC-induced depressive behaviors were antagonized only by the CBs virodhamine and O-2093. The present results demonstrated depressive effects of NC in two typical behavioral tests, which support the risk of repeated NC use. The shortened behavioral alterations in the forced swimming test, as compared to the IM group, seemed to reflect the neuronal modifications peculiar to NC, which are antagonized by some CBs.

  13. The association between the 844ins68 polymorphism in the CBS gene and breast cancer

    PubMed Central

    Figuera-Villanueva, Luis Eduardo; Ramos-Silva, Adriana; Salas-González, Efraín; Puebla-Pérez, Ana María; Peralta-Leal, Valeria; García-Ortiz, José Elías; Dávalos-Rodríguez, Ingrid Patricia; Zúñiga-González, Guillermo Moisés

    2014-01-01

    Introduction The cystathionine beta synthase (CBS) gene plays an important role in homocysteine metabolism because it catalyzes the first step of the transsulfuration pathway, during which homocysteine is converted to cystathionine. Polymorphisms of CBS have been associated with cancer. Material and methods We examined the role of the 844ins68 polymorphism by comparing the genotypes of 371 healthy Mexican women with the genotypes of 323 Mexican women with breast cancer (BC). Results The observed genotype frequencies for controls and BC patients were 1% and 2% for Ins/Ins, 13% and 26% for W/Ins, and 86% and 72% for W/W, respectively. We found that the odds ratio (OR) was 2.2, with a 95% confidence interval (95% CI) of 1.5–3.3, p = 0.0001. The association was also evident when comparing the distribution of the W/Ins-Ins/Ins genotypes in patients in the following categories: 1) menopause and high γ-glutamyltransferase (GGT) levels (OR of 2.17, 95% CI: 1.17–4.26, p = 0.02), 2) chemotherapy response and high lactate dehydrogenase (LDH) levels (OR 2.2, 95% CI: 1.08–4.4, p = 0.027), 3) chemotherapy response and high GGT levels (OR 2.46, 95% CI: 1.2–4.8, p = 0.007), and 4) body mass index (BMI) and III–IV tumor stage (OR 3.2, 95% CI: 1.2–8.3, p = 0.013). Conclusions We conclude that the genotypes W/Ins-Ins/Ins of the 844ins68 polymorphism in the CBS gene contribute significantly to BC susceptibility in the analyzed sample from the Mexican population. PMID:25624861

  14. The association between the 844ins68 polymorphism in the CBS gene and breast cancer.

    PubMed

    Gallegos-Arreola, Martha Patricia; Figuera-Villanueva, Luis Eduardo; Ramos-Silva, Adriana; Salas-González, Efraín; Puebla-Pérez, Ana María; Peralta-Leal, Valeria; García-Ortiz, José Elías; Dávalos-Rodríguez, Ingrid Patricia; Zúñiga-González, Guillermo Moisés

    2014-12-22

    The cystathionine beta synthase (CBS) gene plays an important role in homocysteine metabolism because it catalyzes the first step of the transsulfuration pathway, during which homocysteine is converted to cystathionine. Polymorphisms of CBS have been associated with cancer. We examined the role of the 844ins68 polymorphism by comparing the genotypes of 371 healthy Mexican women with the genotypes of 323 Mexican women with breast cancer (BC). The observed genotype frequencies for controls and BC patients were 1% and 2% for Ins/Ins, 13% and 26% for W/Ins, and 86% and 72% for W/W, respectively. We found that the odds ratio (OR) was 2.2, with a 95% confidence interval (95% CI) of 1.5-3.3, p = 0.0001. The association was also evident when comparing the distribution of the W/Ins-Ins/Ins genotypes in patients in the following categories: 1) menopause and high γ-glutamyltransferase (GGT) levels (OR of 2.17, 95% CI: 1.17-4.26, p = 0.02), 2) chemotherapy response and high lactate dehydrogenase (LDH) levels (OR 2.2, 95% CI: 1.08-4.4, p = 0.027), 3) chemotherapy response and high GGT levels (OR 2.46, 95% CI: 1.2-4.8, p = 0.007), and 4) body mass index (BMI) and III-IV tumor stage (OR 3.2, 95% CI: 1.2-8.3, p = 0.013). We conclude that the genotypes W/Ins-Ins/Ins of the 844ins68 polymorphism in the CBS gene contribute significantly to BC susceptibility in the analyzed sample from the Mexican population.

  15. Polymorphisms in MTHFR, MS and CBS Genes and Homocysteine Levels in a Pakistani Population

    PubMed Central

    Yakub, Mohsin; Moti, Naushad; Parveen, Siddiqa; Chaudhry, Bushra; Azam, Iqbal; Iqbal, Mohammad Perwaiz

    2012-01-01

    Background Hyperhomocysteinemia (>15 µmol/L) is highly prevalent in South Asian populations including Pakistan. In order to investigate the genetic determinants of this condition, we studied 6 polymorphisms in genes of 3 enzymes - methylenetetrahydrofolate reductase (MTHFR; C677T; A1298C), methionine synthase (MS; A2756G), cystathionine-β-synthase (CBS; T833C/844ins68, G919A) involved in homocysteine metabolism and investigated their interactions with nutritional and environmental factors in a Pakistani population. Methodology/Principal Findings In a cross-sectional survey, 872 healthy adults (355 males and 517 females; age 18–60 years) were recruited from a low-income urban population in Karachi. Fasting venous blood was obtained and assessed for plasma/serum homocysteine; folate, vitamin B12, pyridoxal phosphate and blood lead. DNA was isolated and genotyping was performed by PCR-RFLP (restriction-fragment-length- polymorphism) based assays. The average changes in homocysteine levels for MTHFR 677CT and TT genotypes were positive [β(SE β), 2.01(0.63) and 16.19(1.8) µmol/L, respectively]. Contrary to MTHFR C677T polymorphism, the average changes in plasma homocysteine levels for MS 2756AG and GG variants were negative [β(SE β), −0.56(0.58) and −0.83(0.99) µmol/L, respectively]. The average change occurring for CBS 844ins68 heterozygous genotype (ancestral/insertion) was −1.88(0.81) µmol/L. The combined effect of MTHFR C677T, MS A2756G and CBS 844ins68 genotypes for plasma homocysteine levels was additive (p value <0.001). Odds of having hyperhomocysteinemia with MTHFR 677TT genotype was 10-fold compared to MTHFR 677CC genotype [OR (95%CI); 10.17(3.6–28.67)]. Protective effect towards hyperhomocysteinemia was observed with heterozygous (ancestral/insertion) genotype of CBS 844ins68 compared to homozygous ancestral type [OR (95% CI); 0.58 (0.34–0.99)]. Individuals with MTHFR 677CT or TT genotypes were at a greater risk of hyperhomocysteinemia in

  16. The distribution of cystathionine beta-synthase (CBS) in the eye: implication of the presence of a trans-sulfuration pathway for oxidative stress defense.

    PubMed

    Persa, Cristina; Osmotherly, Kaila; Chao-Wei Chen, Kate; Moon, Sungchur; Lou, Marjorie F

    2006-10-01

    Clinical abnormalities in cystathionine-beta-synthase (CBS) deficiency, a key enzyme in the trans-sulfuration pathway, associate with many eye disorders. However, little is known about this enzyme in the eye. The goal of this study is to examine the distribution of CBS in the various regions of the eye, including conjunctiva, cornea, iris, lens, vitreous, retina and optic nerve using fresh eyes from both pigs (6 months) and humans (4-82 years). We have found that pig eye showed the highest CBS protein presence in cornea, conjunctiva and iris, followed by retina and optic nerve. The whole lens had a relatively lower amount and vitreous body had none. CBS protein distribution in the human eyes showed a similar pattern, with high level in the anterior segments but much lower amount in retina and optic nerve. CBS in anterior segments remained high throughout the lifespan, but retinal CBS showed a trend of age-dependent increase. The presence of CBS in human and pig eye tissues was further confirmed by RT-PCR, CBS activity assay, both showed similar distribution profiles as the Western blot analysis. This is the first evidence of the presence of CBS enzyme in the eye outside of the lens, which indicates that a functional trans-sulfuration pathway may be present in various eye tissues.

  17. Mechanisms of hyperhomocysteinemia induced skeletal muscle myopathy after ischemia in the CBS-/+ mouse model.

    PubMed

    Veeranki, Sudhakar; Tyagi, Suresh C

    2015-01-06

    Although hyperhomocysteinemia (HHcy) elicits lower than normal body weights and skeletal muscle weakness, the mechanisms remain unclear. Despite the fact that HHcy-mediated enhancement in ROS and consequent damage to regulators of different cellular processes is relatively well established in other organs, the nature of such events is unknown in skeletal muscles. Previously, we reported that HHcy attenuation of PGC-1α and HIF-1α levels enhanced the likelihood of muscle atrophy and declined function after ischemia. In the current study, we examined muscle levels of homocysteine (Hcy) metabolizing enzymes, anti-oxidant capacity and focused on protein modifications that might compromise PGC-1α function during ischemic angiogenesis. Although skeletal muscles express the key enzyme (MTHFR) that participates in re-methylation of Hcy into methionine, lack of trans-sulfuration enzymes (CBS and CSE) make skeletal muscles more susceptible to the HHcy-induced myopathy. Our study indicates that elevated Hcy levels in the CBS-/+ mouse skeletal muscles caused diminished anti-oxidant capacity and contributed to enhanced total protein as well as PGC-1α specific nitrotyrosylation after ischemia. Furthermore, in the presence of NO donor SNP, either homocysteine (Hcy) or its cyclized version, Hcy thiolactone, not only increased PGC-1α specific protein nitrotyrosylation but also reduced its association with PPARγ in C2C12 cells. Altogether these results suggest that HHcy exerts its myopathic effects via reduction of the PGC-1/PPARγ axis after ischemia.

  18. Galactose utilization sheds new light on sugar metabolism in the sequenced strain Dekkera bruxellensis CBS 2499.

    PubMed

    Moktaduzzaman, Md; Galafassi, Silvia; Capusoni, Claudia; Vigentini, Ileana; Ling, Zhihao; Piškur, Jure; Compagno, Concetta

    2015-03-01

    Dekkera bruxellensis and Saccharomyces cerevisiae are considered two phylogenetically distant relatives, but they share several industrial relevant traits such as the ability to produce ethanol under aerobic conditions (Crabtree effect), high tolerance towards ethanol and acids, and ability to grow without oxygen. Beside a huge adaptability, D. bruxellensis exhibits a broader spectrum in utilization of carbon and nitrogen sources in comparison to S. cerevisiae. With the aim to better characterize its carbon source metabolism and regulation, the usage of galactose and the role that glucose plays on sugar metabolism were investigated in D. bruxellensis CBS 2499. The results indicate that in this yeast galactose is a non-fermentable carbon source, in contrast to S. cerevisiae that can ferment it. In particular, its metabolism is affected by the nitrogen source. Interestingly, D. bruxellensis CBS 2499 exhibits the 'short-term Crabtree effect', and the expression of genes involved in galactose utilization and in respiratory metabolism is repressed by glucose, similarly to what occurs in S. cerevisiae. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  19. Sterol glycosides and cerebrosides accumulate in Pichia pastoris, Rhynchosporium secalis and other fungi under normal conditions or under heat shock and ethanol stress.

    PubMed

    Sakaki, T; Zähringer, U; Warnecke, D C; Fahl, A; Knogge, W; Heinz, E

    2001-06-01

    The occurrence of glycolipids such as sterol glycosides, acylated sterol glycosides, cerebrosides and glycosyldiacylglycerols was examined in the three yeast species Candida albicans, Pichia pastoris and Pichia anomala, as well as in the six fungal species Sordaria macrospora, Pyrenophora teres, Ustilago maydis, Acremonium chrysogenum, Penicillium olsonii and Rhynchosporium secalis. Cerebroside was found in all organisms tested, whereas acylated sterol glycosides and glycosyldiacylglycerols were not found in any organism. Sterol glycosides were detected in P. pastoris strain GS115, U. maydis, S. macrospora and R. secalis. This glycolipid occurred in both yeast and filamentous forms of U. maydis but in neither form of C. albicans. This suggests that sterol glycoside is not correlated with the separately grown dimorphic forms of these organisms. Cerebrosides and sterol glycosides from P. pastoris and R. secalis were purified and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The cerebrosides are beta-glucosyl ceramides consisting of a saturated alpha-hydroxy or non-hydroxy fatty acid and a Delta4,8-diunsaturated, C9-methyl-branched sphingobase. Sterol glycoside from P. pastoris was identified as ergosterol-beta-D-glucopyranoside, whereas the sterol glucosides from R. secalis contain two derivatives of ergosterol. The biosynthesis of sterol glucoside in P. pastoris CBS7435 and GS115 depended on the culture conditions. The amount of sterol glucoside in cells grown in complete medium was much lower than in cells from minimal medium and a strong increase in the content of sterol glucoside was observed when cells were subjected to stress conditions such as heat shock or increased ethanol concentrations. From these data we suggest that, in addition to Saccharomyces cerevisiae, new yeast and fungal model organisms should be used to study the physiological functions of glycolipids in eukaryotic cells. This suggestion is based on the

  20. Benchmarking Compound Methods (CBS-QB3, CBS-APNO, G3, G4, W1BD) against the Active Thermochemical Tables: A Litmus Test for Cost-Effective Molecular Formation Enthalpies.

    PubMed

    Simmie, John M; Somers, Kieran P

    2015-07-16

    The theoretical atomization energies of some 45 CxHyOz molecules present in the Active Thermochemical Tables compilation and of particular interest to the combustion chemistry community have been computed using five composite model chemistries as titled. The species contain between 1-8 "heavy" atoms, and a few are conformationally diverse with up to nine conformers. The enthalpies of formation at 0 and 298.15 K are then derived via the atomization method and compared against the recommended values. In general, there is very good agreement between our averaged computed values and those in the ATcT; those for 1,3-cyclopentadiene exceptionally differ considerably, and we show from isodesmic reactions that the true value for 1,3-cyclopentadiene is closer to 134 kJ mol(-1) than the reported 101 kJ mol(-1). If one is restricted to using a single method, statistical measures indicate that the best methods are in the rank order G3 ≈ G4 > W1BD > CBS-APNO > CBS-QB3. The CBS-x methods do on average predict ΔfH(⊖)(298.15 K) within ≈5 kJ mol(-1) but are prone to occasional lapses. There are statistical advantages to be gained from using a number of methods in tandem, and all possible combinations have been tested. We find that the average formation enthalpy coming from using CBS-APNO/G4, CBS-APNO/G3, and G3/G4 show lower mean signed and mean unsigned errors, and lower standard and root-mean-squared deviations, than any of these methods in isolation. Combining these methods also leads to the added benefit of providing an uncertainty rooted in the chemical species under investigation. In general, CBS-APNO and W1BD tend to underestimate the formation enthalpies of target species, whereas CBS-QB3, G3, and G4 have a tendency to overestimate the same. Thus, combining CBS-APNO with a G3/G4 combination leads to an improvement in all statistical measures of accuracy and precision, predicting the ATcT values to within 0.14 ± 4.21 kJ mol(-1), thus rivalling "chemical accuracy" (

  1. [Expression of capsid gene of Chinese isolate of rabbit hemorrhagic disease virus in Pichia pastoris].

    PubMed

    Yan, Wei-Wei; Cui, Zhi-Zhong; Wang, Yong-Kun

    2005-01-01

    The capsid protein (VP60) gene of RHDV was subcloned into the Pichia expressin vector pPICZ B to express the VP60 protein intracellularly. The recombinant plasmid was initially transformed into a E. coli strain TOP10 F'. After verification of the construct by sequencing, the recombinant plasmid was linearized by Sac I in the 5' AOX1 region and then transformed into Pichia pastoris strain GS115 using the Pichia EasyComp Kit. After selecting and verifing for the insertion of VP60 gene in the genome, two clones of Pichia transformants were select for expression test. The recombinant clones were first inoculate with BMGY in baffled flask at 28-30 degrees C in a shaking incubator (250-300 r/min) until culture reaches an OD600 = 2-6, then resuspend the cell pellet to an OD6oo of 1.0 in BMMY medium to induce expression for 5 days by methanol at a concentration of 0.5% in a 1 liter baffled flask covered with 2 layers of sterile gauze. Collect the cell pellets and break it by acid-washed 0.5 mm glass beads. The expression of recombinant Pichia strains was detected by SDS-PAGE and Western analysis with a polyclonal serum which showed a specific protein band of 60kD. Theses results indicates that the recombinant VP60 produced in Pichia was antigenically similar to the viral polypeptide. Electron microscopic observation of the recombinant Pichia-derived protein revealed the presence of virus-like particles similar in size and appearance to native virus capsids. In the haemagglutination test, the recombinant VLPs, like the native RHDV, also agglutinated human blood type O erythrocytes and could be inhibited by the anti-RHDV polyclonal serum.

  2. Endurance and failure characteristics of modified Vasco X-2, CBS 600 and AISI 9310 spur gears. [aircraft construction materials

    NASA Technical Reports Server (NTRS)

    Townsend, D. P.; Zaretsky, E. V.

    1980-01-01

    Gear endurance tests and rolling-element fatigue tests were conducted to compare the performance of spur gears made from AISI 9310, CBS 600 and modified Vasco X-2 and to compare the pitting fatigue lives of these three materials. Gears manufactured from CBS 600 exhibited lives longer than those manufactured from AISI 9310. However, rolling-element fatigue tests resulted in statistically equivalent lives. Modified Vasco X-2 exhibited statistically equivalent lives to AISI 9310. CBS 600 and modified Vasco X-2 gears exhibited the potential of tooth fracture occurring at a tooth surface fatigue pit. Case carburization of all gear surfaces for the modified Vasco X-2 gears results in fracture at the tips of the gears.

  3. Crystallization and preliminary crystallographic analysis of merohedrally twinned crystals of MJ0729, a CBS-domain protein from Methanococcus jannaschii

    PubMed Central

    Fernández-Millán, Pablo; Kortazar, Danel; Lucas, María; Martínez-Chantar, María Luz; Astigarraga, Egoitz; Fernández, José Andrés; Sabas, Olatz; Albert, Armando; Mato, Jose M.; Martínez-Cruz, Luis Alfonso

    2008-01-01

    CBS domains are small protein motifs, usually associated in tandem, that are implicated in binding to adenosyl groups. Several genetic diseases in humans have been associated with mutations in CBS sequences, which has made them very promising targets for rational drug design. Trigonal crystals of the CBS-domain protein MJ0729 from Methanococcus jannaschii were grown by the vapour-diffusion method at acidic pH. Preliminary analysis of nine X-ray diffraction data sets using Yeates statistics and Britton plots showed that slight variation in the pH as well as in the buffer used in the crystallization experiments led to crystals with different degrees of merohedral twinning that may vary from perfect hemihedral twinning to perfect tetartohedral twinning. PMID:18607087

  4. Down-regulated CBS/H2S pathway is involved in high-salt-induced hypertension in Dahl rats.

    PubMed

    Huang, Pan; Chen, Siyao; Wang, Yuan; Liu, Jia; Yao, Qiuyu; Huang, Yaqian; Li, Hongxia; Zhu, Mingzhu; Wang, Suxia; Li, Lin; Tang, Chaoshu; Tao, Yinghong; Yang, Guosheng; Du, Junbao; Jin, Hongfang

    2015-04-30

    The study was designed to explore the significance of endogenous H2S in the development of high-salt-induced hypertension in rats. High-salt-induced hypertension rat model was made by feeding Dahl rat high-salt diet containing 8% NaCl for 8 weeks with SD rats as control. SBP and aorta structure in rats were observed. Endogenous H2S content and expression of cystathionine β-lyase (CBS), cystathionine γ-lyase and mercaptopyruvate sulfurtransferase in renal tissues were detected. Mechanisms for the impact of high-salt on CBS/H2S in renal tissues were studied, targeting HIF-1α pathway. The effect of H2S on RAS in serum and renal tissue of rats were tested. High-salt reduced endogenous H2S content and inhibited the expression of CBS in renal tissue in salt-sensitive Dahl rats. H2S donor, however, inhibited salt-sensitive hypertension, reversed aortic structural remodeling and inhibited activation of the RAS system in renal tissues in Dahl rats. Expression of HIF-1α was decreased but expression of PHD2 was increased in renal tissue of Dahl rats with high-salt diet, whereas they did not alter in renal tissue of SD rats with high-salt diet. Ex vivo experiment showed that inhibitor of HIF-1α degradation could rescue down-regulated CBS/H2S pathway in renal tissue of Dahl rats with high-salt. In contrast, inhibitor of HIF-1α activity decreased the CBS/H2S pathway in the renal tissue of SD rats treated with high-salt. Down-regulated CBS/H2S pathway in renal tissues under high-salt insult might be an important pathogenesis of salt-sensitive hypertension. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Crystallization and preliminary crystallographic studies of CbsA, a secretory exoglucanase from Xanthomonas oryzae pv. oryzae.

    PubMed

    Kumar, Sushil; Haque, Asfarul S; Jha, Gopaljee; Sonti, Ramesh V; Sankaranarayanan, Rajan

    2012-10-01

    The bacterial pathogen Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. The secreted exoglucanase CbsA is an important virulence factor of this pathogen. It belongs to the glycosyl hydrolase 6 family of proteins based on the carbohydrate-active enzyme (CAZY) classification. In this study, CbsA has been overexpressed, purified and crystallized. The crystal diffracted to a resolution of 1.86 Å and belonged to space group P2(1)2(1)2(1). It contained one monomer per asymmetric unit, with a solvent content of 45.8%.

  6. 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.

  7. Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System.

    PubMed

    Baeshen, Mohammed N; Bouback, Thamer A F; Alzubaidi, Mubarak A; Bora, Roop S; Alotaibi, Mohammed A T; Alabbas, Omar T O; Alshahrani, Sultan M; Aljohani, Ahmed A M; Munshi, Rayan A A; Al-Hejin, Ahmed; Ahmed, Mohamed M M; Redwan, Elrashdy M; Ramadan, Hassan A I; Saini, Kulvinder S; Baeshen, Nabih A

    2016-01-01

    Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan 5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system.

  8. Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System

    PubMed Central

    Baeshen, Mohammed N.; Bouback, Thamer A. F.; Alzubaidi, Mubarak A.; Alabbas, Omar T. O.; Alshahrani, Sultan M.; Aljohani, Ahmed A. M.; Munshi, Rayan A. A.; Al-Hejin, Ahmed; Redwan, Elrashdy M.; Ramadan, Hassan A. I.; Saini, Kulvinder S.; Baeshen, Nabih A.

    2016-01-01

    Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan 5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system. PMID:27579308

  9. 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 ...

  10. Inactivation of a GAL4-like transcription factor improves cell fitness and product yield in glycoengineered Pichia pastoris strains.

    PubMed

    Jiang, Bo; Argyros, Rebecca; Bukowski, John; Nelson, Stephanie; Sharkey, Nathan; Kim, Sehoon; Copeland, Victoria; Davidson, Robert C; Chen, Ronghua; Zhuang, Jun; Sethuraman, Natarajan; Stadheim, Terrance A

    2015-01-01

    With a completely reengineered and humanized glycosylation pathway, glycoengineered Pichia pastoris has emerged as a promising production host for the manufacture of therapeutic glycoproteins. However, the extensive genetic modifications have also negatively affected the overall fitness levels of the glycoengineered host cells. To make glycoengineered Pichia strains more compatible with a scalable industrial fermentation process, we sought to identify genetic solutions to broadly improve cell robustness during fermentation. In this study, we report that mutations within the Pichia pastoris ATT1 (PpATT1) gene (a homolog of the Saccharomyces cerevisiae GAL4 [ScGAL4] transcriptional activator) dramatically increased the cellular fitness levels of glycoengineered Pichia strains. We demonstrate that deletion of the PpATT1 gene enabled glycoengineered Pichia strains to improve their thermal tolerance levels, reduce their cell lysis defects, and greatly improve fermentation robustness. The extension of the duration of fermentation enabled the PpATT1-modified glycoengineered Pichia strains to increase their product yields significantly without any sacrifice in product quality. Because the ATT1 gene could be deleted from any Pichia strains, including empty hosts and protein-expressing production strains alike, we suggest that the findings described in this study are broadly applicable to any Pichia strains used for the production of therapeutic proteins, including monoclonal antibodies, Fc fusions, peptides, hormones, and growth factors.

  11. Inactivation of a GAL4-Like Transcription Factor Improves Cell Fitness and Product Yield in Glycoengineered Pichia pastoris Strains

    PubMed Central

    Argyros, Rebecca; Bukowski, John; Nelson, Stephanie; Sharkey, Nathan; Kim, Sehoon; Copeland, Victoria; Davidson, Robert C.; Chen, Ronghua; Zhuang, Jun; Sethuraman, Natarajan; Stadheim, Terrance A.

    2014-01-01

    With a completely reengineered and humanized glycosylation pathway, glycoengineered Pichia pastoris has emerged as a promising production host for the manufacture of therapeutic glycoproteins. However, the extensive genetic modifications have also negatively affected the overall fitness levels of the glycoengineered host cells. To make glycoengineered Pichia strains more compatible with a scalable industrial fermentation process, we sought to identify genetic solutions to broadly improve cell robustness during fermentation. In this study, we report that mutations within the Pichia pastoris ATT1 (PpATT1) gene (a homolog of the Saccharomyces cerevisiae GAL4 [ScGAL4] transcriptional activator) dramatically increased the cellular fitness levels of glycoengineered Pichia strains. We demonstrate that deletion of the PpATT1 gene enabled glycoengineered Pichia strains to improve their thermal tolerance levels, reduce their cell lysis defects, and greatly improve fermentation robustness. The extension of the duration of fermentation enabled the PpATT1-modified glycoengineered Pichia strains to increase their product yields significantly without any sacrifice in product quality. Because the ATT1 gene could be deleted from any Pichia strains, including empty hosts and protein-expressing production strains alike, we suggest that the findings described in this study are broadly applicable to any Pichia strains used for the production of therapeutic proteins, including monoclonal antibodies, Fc fusions, peptides, hormones, and growth factors. PMID:25344235

  12. Funding 3D-CBS:. Changing the Role of PET for Cancer Screening

    NASA Astrophysics Data System (ADS)

    Crosetto, Dario B.

    2010-04-01

    The role of Positron Emission Technology (PET) should be changed with use of the 30-CBS (Three Dimensional Complete Body screening) for maximizing the capture of signals that will detect minimum abnormal metabolism (or other biological processes), achievable by capturing simultaneously and accurately as many signals as possible from the tumor markers from all organs of the body in order to identify the smallest anomaly, at the lowest cost per signal captured and requiring the minimum radiation to the patient. This paper provides scientific arguments for setting new parameters for industry to establish the correct relation between the goal of obtaining substantial reduction in cancer deaths and the implementation of innovations and technology that will provide the expected results through early cancer detection.

  13. Geosmin and Related Volatiles in Bioreactor-Cultured Streptomyces citreus CBS 109.60

    PubMed Central

    Pollak, F. C.; Berger, R. G.

    1996-01-01

    Streptomyces citreus CBS 109.60 produced geosmin and a complex pattern of other volatile compounds during cultivation in a 2.5-liter laboratory bioreactor. Volatiles were isolated from disrupted cells, from the culture medium, and from the waste air of the bioreactor by adsorption on Lewatit OC 1064MD. Quantitative and qualitative analyses were carried out using capillary gas chromatography and coupled gas chromatography-mass spectroscopy. S. citreus produced 56 volatile compounds, which were mainly terpenoids but also included aliphatic ketones, alcohols, esters, pyrazines, furan(on)es, and aromatic types during the growth phase. The major components were geosmin and a germacradienol. A biosynthetic pathway for geosmin including eudesmanolides is proposed. PMID:16535293

  14. Polymorphisms in the folate-metabolizing genes MTR, MTRR, and CBS and breast cancer risk.

    PubMed

    Weiner, Alexandra S; Boyarskikh, Uljana A; Voronina, Elena N; Selezneva, Inna A; Sinkina, Tatiana V; Lazarev, Alexandr F; Petrova, Valentina D; Filipenko, Maxim L

    2012-04-01

    Alterations in the nucleotide sequences of folate-metabolizing genes can increase the risk of malignant transformation. The aim of our study was to investigate the association of three single-nucleotide polymorphisms (SNPs) in the folate-metabolizing genes - A2756G MTR, A66G MTRR, and 844ins68 CBS - which have putative functional significance in breast cancer risk. The allele and genotype frequencies of the SNPs were determined in a case group (840 women with sporadic breast cancer) and a control group (770 women). No statistically significant association of studied SNPs with breast cancer was revealed. A meta-analysis, which included data obtained from the literature and the present research, did not reveal any statistically significant associations of these SNPs with breast cancer. The results obtained provide evidence that these SNPs are not involved in the development of breast cancer.

  15. Novel progranulin mutation: screening for PGRN mutations in a Portuguese series of FTD/CBS cases.

    PubMed

    Guerreiro, Rita Joao; Santana, Isabel; Bras, Jose Miguel; Revesz, Tamas; Rebelo, Olinda; Ribeiro, Maria Helena; Santiago, Beatriz; Oliveira, Catarina Resende; Singleton, Andrew; Hardy, John

    2008-07-15

    Mutations in the progranulin (PGRN) gene were recently described as the cause of ubiquitin positive frontotemporal dementia (FTD) in many families. Different frequencies of these genetic changes have been reported in diverse populations leading us to determine if these mutations were a major cause of FTD in the Portuguese population. The entire coding sequence plus exon 0 of PGRN were sequenced in a consecutive series of 46 FTD/CBS Portuguese patients. Two mutations were found: a novel pathogenic insertion (p.Gln300GlnfsX61) and a previously described point variant (p.T182M) of unclear pathogenicity. Pathogenic mutations in the PGRN gene were found in one of the 36 probands studied (3% of the probands in our series) who had a corticobasal syndrome presentation, indicating that in the Portuguese population, mutations in this gene are not a major cause of FTD.

  16. Expression analysis for genes involved in arachidonic acid biosynthesis in Mortierella alpina CBS 754.68.

    PubMed

    Samadlouie, Hamid-Reza; Hamidi-Esfahani, Zohreh; Alavi, Seyed-Mehdi; Varastegani, Boshra

    2014-01-01

    The time courses for production of fungal biomass, lipid, phenolic and arachidonic acid (ARA) as well as expression of the genes involved in biosynthesis of ARA and lipid were examined in Mortierella alpina CBS 754.68. A significant increase in the arachidonic acid content in lipids that coincided with reduced levels of lipid was obtained. Reduced gene expression occurred presumably due to the steady reduction of carbon and nitrogen resources. However, these energy resources were inefficiently compensated by the breakdown of the accumulated lipids that in turn, induced up-regulated expression of the candidate genes. The results further indicated that the expression of the GLELO encoding gene is a rate-limiting step in the biosynthesis of ARA in the early growth phase.

  17. Open Science CBS Neuroimaging Repository: Sharing ultra-high-field MR images of the brain.

    PubMed

    Tardif, Christine Lucas; Schäfer, Andreas; Trampel, Robert; Villringer, Arno; Turner, Robert; Bazin, Pierre-Louis

    2016-01-01

    Magnetic resonance imaging at ultra high field opens the door to quantitative brain imaging at sub-millimeter isotropic resolutions. However, novel image processing tools to analyze these new rich datasets are lacking. In this article, we introduce the Open Science CBS Neuroimaging Repository: a unique repository of high-resolution and quantitative images acquired at 7 T. The motivation for this project is to increase interest for high-resolution and quantitative imaging and stimulate the development of image processing tools developed specifically for high-field data. Our growing repository currently includes datasets from MP2RAGE and multi-echo FLASH sequences from 28 and 20 healthy subjects respectively. These datasets represent the current state-of-the-art in in-vivo relaxometry at 7 T, and are now fully available to the entire neuroimaging community. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. 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.

  19. Electrochemical studies of a truncated laccase produced in Pichia pastoris

    SciTech Connect

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

    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 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 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.

  20. 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.

  1. 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.

  2. Biocontrol of postharvest Rhizopus decay of peaches with Pichia caribbica.

    PubMed

    Xu, Baitian; Zhang, Hongyin; Chen, Keping; Xu, Qin; Yao, Yao; Gao, Hui

    2013-08-01

    A new yeast antagonist, Pichia caribbica, isolated in our laboratory from the soil collected from unsprayed orchards, was evaluated for its biocontrol capability against Rhizopus stolonifer on peaches and the possible mechanisms involved. The decay incidence and lesion diameter of Rhizopus decay of peaches treated by P. caribbica were significantly reduced compared with the control fruits, and the higher the concentration of P. caribbica, the better the efficacy of the biocontrol. Rapid colonization of the yeast in peach wounds stored at 25 °C was observed. In peaches, the activities of peroxidase (POD), catalase (CAT), and phenylalanine ammonia-lyase (PAL) were significantly induced by P. caribbica treatment compared to those of the control fruits. All these results indicated that P. caribbica has a great potential for the development of commercial formulations to control postharvest Rhizopus decay of peaches. Its modes of action were based on competition for space and nutrients with pathogens, inducement of activities of defense-related enzymes such as POD, CAT, and PAL of peaches.

  3. Expression and characterization of Aspergillus thermostable phytases in Pichia pastoris.

    PubMed

    Promdonkoy, Patcharee; Tang, Kittapong; Sornlake, Warasirin; Harnpicharnchai, Piyanun; Kobayashi, Rutchadaporn Sriprang; Ruanglek, Vasimon; Upathanpreecha, Tewa; Vesaratchavest, Mongkol; Eurwilaichitr, Lily; Tanapongpipat, Sutipa

    2009-01-01

    Two thermostable phytases were identified from Thai isolates of Aspergillus japonicus BCC18313 (TR86) and Aspergillus niger BCC18081 (TR170). Both genes of 1404 bp length, coding for putative phytases of 468 amino acid residues, were cloned and transferred into Pichia pastoris. The recombinant phytases, r-PhyA86 and r-PhyA170, were expressed as active extracellular, glycosylated proteins with activities of 140 and 100 U mL(-1), respectively. Both recombinant phytases exhibited high affinity for phytate but not for p-nitrophenyl phosphate. Optimal phytase activity was observed at 50 degrees C and pH 5.5. High thermostability, which is partly dependent on glycosylation, was demonstrated for both enzymes, as >50% activity was retained after heating at 100 degrees C for 10 min. The recombinant phytases also exhibited broad pH stability from 2.0 to 8.0 and are resistant to pepsin. In vitro digestibility tests suggested that r-PhyA86 and r-PhyA170 are at least as efficient as commercial phytase for hydrolyzing phytate in corn-based animal feed and are therefore suitable sources of phytase supplement.

  4. Crystal Structure of Alcohol Oxidase from Pichia pastoris

    PubMed Central

    Valerius, Oliver; Feussner, Ivo; Ficner, Ralf

    2016-01-01

    FAD-dependent alcohol oxidases (AOX) are key enzymes of methylotrophic organisms that can utilize lower primary alcohols as sole source of carbon and energy. Here we report the crystal structure analysis of the methanol oxidase AOX1 from Pichia pastoris. The crystallographic phase problem was solved by means of Molecular Replacement in combination with initial structure rebuilding using Rosetta model completion and relaxation against an averaged electron density map. The subunit arrangement of the homo-octameric AOX1 differs from that of octameric vanillyl alcohol oxidase and other dimeric or tetrameric alcohol oxidases, due to the insertion of two large protruding loop regions and an additional C-terminal extension in AOX1. In comparison to other alcohol oxidases, the active site cavity of AOX1 is significantly reduced in size, which could explain the observed preference for methanol as substrate. All AOX1 subunits of the structure reported here harbor a modified flavin adenine dinucleotide, which contains an arabityl chain instead of a ribityl chain attached to the isoalloxazine ring. PMID:26905908

  5. 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

  6. Constitutive expression of Botrytis aclada laccase in Pichia pastoris

    PubMed Central

    Kittl, Roman; Gonaus, Christoph; Pillei, Christian; Haltrich, Dietmar; Ludwig, Roland

    2012-01-01

    The heterologous expression of laccases is important for their large-scale production and genetic engineering—a prerequisite for industrial application. Pichia pastoris is the preferred expression host for fungal laccases. The recently cloned laccase from the ascomycete Botrytis aclada (BaLac) has been efficiently expressed in P. pastoris under the control of the inducible alcohol oxidase (AOX1) promoter. In this study, we compare these results to the constitutive expression in the same organism using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The results show that the amounts of BaLac produced with the GAP system (517 mgL-1) and the AOX1 system (495 mgL-1) are comparable. The constitutive expression is, however, faster, and the specific activity of BaLac in the culture supernatant is higher (41.3 Umg-1 GAP, 14.2 Umg-1 AOX1). In microtiter plates, the constitutive expression provides a clear advantage due to easy manipulation (simple medium, no methanol feeding) and fast enzyme production (high-throughput screening assays can already be performed after 48 h). PMID:22705842

  7. [Expression of Arabidopsis thaliana thioesterase gene in Pichia pastoris].

    PubMed

    Hao, Zhaocheng; Wang, Tengfei; Li, Zhongkui; Hao, Zikai; Dai, Kun; Wang, Ruiming

    2015-01-01

    Thioesterase catalyzes the hydrolysis of acyl-ACP and saturated fatty acyl chain. It plays a key role in the accumulation of medium chain fatty acids in vivo. In this study, to construct an engineering strain to produce MCFAs, the Arabidopsis acyl-ACP thioesterase gene AtFatA was amplified by PCR from cDNA of arabidopsis and double digested by EcoR I/Xba I, then linked to the plasmid digested with same enzymes to get the recombinant plasmid pPICZaA-AtFatA. We transformed the gene into Pichia pastoris GS115 by electroporation and screened positive colonies by YPD medium with Zeocin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the recombinant enzyme had a molecular of 45 kDa band which was consistent with the predicted molecular mass and we constructed the expression system of gene AtFatA in fungus for the first time. Under shake-flask conditions, Gas Chromatograph-Mass Spectrometer-computer results indicated that recombinant strain produced 51% more extracellular free MCFAs than the wild and its yield reached 28.7% of all extracellular fatty acids. This figure is 10% higher than the control group. The result provides a new way to produce MCFAs.

  8. 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

  9. Long-term performance of thermoplastic composite material with cotton burr and stem (CBS) as a partial filler

    USDA-ARS?s Scientific Manuscript database

    Rationale: Cotton burr and stem (CBS) fraction of cotton gin byproducts has shown promise as a fiber filler in thermoplastic composites, with physical and mechanical properties comparable to that made with wood fiber fillers. However, the long-term performance of this composite material is not known...

  10. Surface fatigue life and failure characteristics of EX-53, CBS 1000M, and AISI 9310 gear materials

    NASA Technical Reports Server (NTRS)

    Townsend, D. P.

    1985-01-01

    Spur gear endurance tests and rolling-element surface fatigue tests are conducted to investigate EX-53 and CBS 1000M steels for use as advanced application gear materials, to determine their endurance characteristics, and to compare the results with the standard AISI 9310 gear material. The gear pitch diameter is 8.89 cm (3.50 in). Gear test conditions are an oil inlet temperature of 320 K (116 F), an oil outlet temperature of 350 K (170 F), a maximum Hertz stress of 1.71 GPa (248 ksi), and a speed of 10,000 rpm. Bench-type rolling-element fatigue tests are conducted at ambient temperature with a bar specimen speed of 12,500 rpm and a maximum Hertz stress of 4.83 GPa (700 ksi). The EX-53 test gears have a surface fatigue life of twice that of the AISI 9310 spur gears. The CBS 1000M test gears have a surface fatigue life of more than twice that of the AISI 9310 spur gears. However, the CBS 1000M gears experience a 30-percent tooth fracture failure which limits its use as a gear material. The rolling-contact fatigue lines of RC bar specimens of EX-53 and ASISI 9310 are approximately equal. However, the CBS 1000M RC specimens have a surface fatigue life of about 50 percent that of the AISI 9310.

  11. An assessment of selected properties of the fluorescent tracer, Tinopal CBS-X related to conservative behavior, and suggested improvements

    NASA Astrophysics Data System (ADS)

    Licha, Tobias; Niedbala, Anne; Bozau, Elke; Geyer, Tobias

    2013-03-01

    SummaryA conservative or well known reactive behavior of water tracers is a prerequisite in the quantitative evaluation of their tracer breakthrough curves. The fluorescent dye, Tinopal CBS-X, is one of the few licensed fluorescent dyes for water tracing with safe use. Its main advantage is its blue fluorescence, which is barely visible to the human eye and thus can be used when coloring water must be avoided. However, scientists have described the recovery of this dye as poor to very poor in field tracer experiments. Hence, this study focuses on examining the interaction of the main water chemistry with Tinopal CBS-X by determining the solubility products of the dye with most common cations. The findings of this investigation reveal that the tracer forms precipitates of very low aqueous solubility with di- and trivalent cations (pKsp 6.4-16.8). As a consequence, Tinopal CBS-X is not a conservative tracer and respective tracer breakthrough curves will exhibit strong tailings at least in part, as result of precipitations formed. The addition of a chemical modifier, EDTA, is suggested to enhance the solubility of Tinopal CBS-X in order to overcome its non-conservative behavior. Equations for estimating the amount of EDTA necessary are provided. In the light of these results, earlier reported tracer breakthrough curves are revisited and re-interpreted.

  12. The Zygosaccharomyces rouxii strain CBS732 contains only one copy of the HOG1 and the SOD2 genes.

    PubMed

    Kinclová, O; Potier, S; Sychrová, H

    2001-06-15

    The osmotolerant yeast Zygosaccharomyces rouxii CBS732 contains only one copy of the ZrHOG1 and ZrSOD2-22 genes. Both genes were cloned and sequenced (Acc. Nos. AJ132606 and AJ252273, respectively) and their sequences were compared to homologous pairs of genes from Z. rouxii ATCC42981 (genes Z-HOG1, Z-HOG2, Z-SOD2, Z-SOD22). The CBS732 ZrHog1p is shorter than its ATCC42981 counterparts (380 aa residues vs. 407 and 420 aa, respectively) and is more similar to ATCC42981 Z-Hog2p than to Z-Hog1p. Also its promoter region corresponds to that one of Z-HOG2. The CBS732 ZrHOG1 promoter region is recognised by Saccharomyces cerevisiae, and the gene product (MAP kinase ZrHog1p) presence fully complements the osmosensitivity of a S. cerevisiae hog1 mutant strain. The CBS ZrSOD2-22 gene is highly similar to ATCC42981 Z-SOD2 but it contains also a segment of 15 aa residues specific for Z-SOD22. Z. rouxii ZrSod2-22 Na(+)/H(+) antiporter expressed in S. cerevisiae shows better activity toward toxic Na(+) and Li(+) cations than does S. cerevisiae's own Nha1 antiporter, and is efficient in improving the halotolerance of some S. cerevisiae wild types.

  13. Toxicological Evaluation of Lactase Derived from Recombinant Pichia pastoris

    PubMed Central

    Liu, Yifei; Chen, Delong; Luo, Yunbo; Huang, Kunlun; Zhang, Wei; Xu, Wentao

    2014-01-01

    A recombinant lactase was expressed in Pichia pastoris, resulting in enzymatic activity of 3600 U/mL in a 5 L fermenter. The lactase product was subjected to a series of toxicological tests to determine its safety for use as an enzyme preparation in the dairy industry. This recombinant lactase had the highest activity of all recombinant strains reported thus far. Acute oral toxicity, mutagenicity, genotoxic, and subchronic toxicity tests performed in rats and mice showed no death in any groups. The lethal dose 50% (LD50) based on the acute oral toxicity study is greater than 30 mL/kg body weight, which is in accordance with the 1500 L milk consumption of a 50 kg human daily. The lactase showed no mutagenic activity in the Ames test or a mouse sperm abnormality test at levels of up to 5 mg/plate and 1250 mg/kg body weight, respectively. It also showed no genetic toxicology in a bone marrow cell micronucleus test at levels of up to 1250 mg/kg body weight. A 90-day subchronic repeated toxicity study via the diet with lactase levels up to 1646 mg/kg (1000-fold greater than the mean human exposure) did not show any treatment-related significant toxicological effects on body weight, food consumption, organ weights, hematological and clinical chemistry, or histopathology compared to the control groups. This toxicological evaluation system is comprehensive and can be used in the safety evaluation of other enzyme preparations. The lactase showed no acute, mutagenic, genetic, or subchronic toxicity under our evaluation system. PMID:25184300

  14. Production of sialylated O-linked glycans in Pichia pastoris.

    PubMed

    Hamilton, Stephen R; Cook, W James; Gomathinayagam, Sujatha; Burnina, Irina; Bukowski, John; Hopkins, Daniel; Schwartz, Shaina; Du, Min; Sharkey, Nathan J; Bobrowicz, Piotr; Wildt, Stefan; Li, Huijuan; Stadheim, Terrance A; Nett, Juergen H

    2013-10-01

    The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. Previously, we have reported the glycoengineering of this organism to produce human-like N-linked glycans but up to now no one has addressed engineering the O-linked glycosylation pathway. Typically, O-linked glycans produced by wild-type P. pastoris are linear chains of four to five α-linked mannose residues, which may be capped with β- or phospho-mannose. Previous genetic engineering of the N-linked glycosylation pathway of P. pastoris has eliminated both of these two latter modifications, resulting in O-linked glycans which are linear α-linked mannose structures. Here, we describe a method for the co-expression of an α-1,2-mannosidase, which reduces these glycans to primarily a single O-linked mannose residue. In doing so, we have reduced the potential of these glycans to interact with carbohydrate-binding proteins, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin. Furthermore, the introduction of the enzyme protein-O-linked-mannose β-1,2-N-acetylglucosaminyltransferase 1, resulted in the capping of the single O-linked mannose residues with N-acetylglucosamine. Subsequently, this glycoform was extended into human-like sialylated glycans, similar in structure to α-dystroglycan-type glycoforms. As such, this represents the first example of sialylated O-linked glycans being produced in yeast and extends the utility of the P. pastoris production platform beyond N-linked glycosylated biotherapeutics to include molecules possessing O-linked glycans.

  15. Toxicological evaluation of lactase derived from recombinant Pichia pastoris.

    PubMed

    Zou, Shiying; He, Xiaoyun; Liu, Yifei; Chen, Delong; Luo, Yunbo; Huang, Kunlun; Zhang, Wei; Xu, Wentao

    2014-01-01

    A recombinant lactase was expressed in Pichia pastoris, resulting in enzymatic activity of 3600 U/mL in a 5 L fermenter. The lactase product was subjected to a series of toxicological tests to determine its safety for use as an enzyme preparation in the dairy industry. This recombinant lactase had the highest activity of all recombinant strains reported thus far. Acute oral toxicity, mutagenicity, genotoxic, and subchronic toxicity tests performed in rats and mice showed no death in any groups. The lethal dose 50% (LD50) based on the acute oral toxicity study is greater than 30 mL/kg body weight, which is in accordance with the 1500 L milk consumption of a 50 kg human daily. The lactase showed no mutagenic activity in the Ames test or a mouse sperm abnormality test at levels of up to 5 mg/plate and 1250 mg/kg body weight, respectively. It also showed no genetic toxicology in a bone marrow cell micronucleus test at levels of up to 1250 mg/kg body weight. A 90-day subchronic repeated toxicity study via the diet with lactase levels up to 1646 mg/kg (1000-fold greater than the mean human exposure) did not show any treatment-related significant toxicological effects on body weight, food consumption, organ weights, hematological and clinical chemistry, or histopathology compared to the control groups. This toxicological evaluation system is comprehensive and can be used in the safety evaluation of other enzyme preparations. The lactase showed no acute, mutagenic, genetic, or subchronic toxicity under our evaluation system.

  16. 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.

  17. 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

  18. Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris.

    PubMed

    Guerrero-Olazarán, Martha; Escamilla-Treviño, Luis L; Castillo-Galván, Mauricio; Gallegos-López, Juan A; Viader-Salvadó, José M

    2009-01-01

    Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS-PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the induction step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of induction with alanine supplemented medium, a 29-kDa band from the cell-free culture medium was clearly observed by SDS-PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 microg/mL. Our results demonstrate that high density cell cultures with alanine in the induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen.

  19. How reliable are gas-phase proton affinity values of small carbanions? A comparison of experimental data with values calculated using Gaussian-3 and CBS compound methods

    NASA Astrophysics Data System (ADS)

    Danikiewicz, Witold

    2009-08-01

    Gas-phase proton affinities (PA) of a series of 25 small, aliphatic carbanions were computed using different Gaussian-3 methods: G3, G3(B3LYP), G3(MP2) and G3(MP2, B3LYP) and Complete Basis Set Extrapolation methods: CBS-4M, CBS-Q, CBS-QB3, and CBS-APNO. The results were compared with critically selected experimental data. The analysis of the results shows that for the majority of the studied molecules all compound methods (Gaussian-3 and CBS), except for CBS-4M, give comparable results, which differ no more than +/-2 kcal mol-1 from the experimental data. Taking into account the calculation time, G3(MP2) and G3(MP2, B3LYP) methods offer the best compromise between accuracy and computational cost. As an additional proof, the results obtained by these two methods were compared with the values obtained using CCSD(T) ab initio method with large basis set. It was found also that some of the published experimental data are erroneous and should be corrected. The results described in this work show that for the majority of the studied compounds PA values calculated using compound methods can be used with the same or even higher confidence as the experimental ones because even the largest differences between Gaussian-3 and CBS methods listed above are still comparable with the accuracy of the typical PA measurements.

  20. Expression of Cellobiose Dehydrogenase from Neurospora crassa in Pichia pastoris and its purification and characterization

    USDA-ARS?s Scientific Manuscript database

    A gene encoding cellobiose dehydrogenase (CDH) from Neurospora crassa strain FGSC 2489 has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter. Recombinant CDH without the native signal sequence and fused with a his6-tag (rNC-...

  1. Genome Sequence of Pichia kudriavzevii M12, a Potential Producer of Bioethanol and Phytase

    PubMed Central

    Gan, Han Ming; Ling, How Lie; Rashid, Noor Aini Abdul

    2012-01-01

    A draft genome sequence of Pichia kudriavzevii M12 is presented here. The genome reveals the presence of genes encoding enzymes involved in xylose utilization and the pentose phosphate pathway for bioethanol production. Strain M12 is also a potential producer of phytases, enzymes useful in food processing and agriculture. PMID:23027839

  2. 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...

  3. 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...

  4. Efficacy of Pichia anomala WLR-076 to control aflatoxin on corn in Texas, 2005

    USDA-ARS?s Scientific Manuscript database

    The experiments were conducted at three Texas Agricultural Experiment Stations on yellow corn hybrids to test the biocontrol yeast, Pichia anomala WRL-076. There were five replicates per treatment arranged in a randomized complete block design. The treatments were: (1) P. anomala WLR-076 applied ...

  5. A high-throughput protein expression system in Pichia pastoris using a newly developed episomal vector

    USDA-ARS?s Scientific Manuscript database

    We describe here the construction of a Gateway-compatible vector, pBGP1-DEST, for rapid and convenient preparation of expression plasmids for production of secretory proteins in Pichia pastoris. pBGP1-DEST directs the synthesis of a fusion protein consisting of the N-terminal signal and pro-sequence...

  6. Detailed kinetic study of the ring opening of cycloalkanes by CBS-QB3 calculations.

    PubMed

    Sirjean, B; Glaude, P A; Ruiz-Lopez, M F; Fournet, R

    2006-11-23

    This work reports a theoretical study of the gas-phase unimolecular decomposition of cyclobutane, cyclopentane and cyclohexane by means of quantum chemical calculations. A biradical mechanism has been envisaged for each cycloalkane, and the main routes for the decomposition of the biradicals formed have been investigated at the CBS-QB3 level of theory. Thermochemical data(DeltaHf(o), S(o), Cp(o)) for all the involved species have been obtained by means of isodesmic reactions. The contribution of hindered rotors has also been included. Activation barriers of each reaction have been analyzed to assess the energetically most favorable pathways for the decomposition of biradicals. Rate constants have been derived for all elementary reactions using transition-state theory at 1 atm and temperatures ranging from 600 to 2000 K. Global rate constant for the decomposition of the cyclic alkanes in molecular products have been calculated. Comparison between calculated and experimental results allowed us to validate the theoretical approach. An important result is that the rotational barriers between the conformers, which are usually neglected, are of importance in decomposition rate of the largest biradicals. Ring strain energies (RSE) in transition states for ring opening have been estimated and show that the main part of RSE contained in the cyclic reactants is removed upon the activation process.

  7. Development of AN Innovative Three-Dimensional Complete Body Screening Device - 3D-CBS

    NASA Astrophysics Data System (ADS)

    Crosetto, D. B.

    2004-07-01

    This article describes an innovative technological approach that increases the efficiency with which a large number of particles (photons) can be detected and analyzed. The three-dimensional complete body screening (3D-CBS) combines the functional imaging capability of the Positron Emission Tomography (PET) with those of the anatomical imaging capability of Computed Tomography (CT). The novel techniques provide better images in a shorter time with less radiation to the patient. A primary means of accomplishing this is the use of a larger solid angle, but this requires a new electronic technique capable of handling the increased data rate. This technique, combined with an improved and simplified detector assembly, enables executing complex real-time algorithms and allows more efficiently use of economical crystals. These are the principal features of this invention. A good synergy of advanced techniques in particle detection, together with technological progress in industry (latest FPGA technology) and simple, but cost-effective ideas provide a revolutionary invention. This technology enables over 400 times PET efficiency improvement at once compared to two to three times improvements achieved every five years during the past decades. Details of the electronics are provided, including an IBM PC board with a parallel-processing architecture implemented in FPGA, enabling the execution of a programmable complex real-time algorithm for best detection of photons.

  8. Ethanol production using whole plant biomass of Jerusalem artichoke by Kluyveromyces marxianus CBS1555.

    PubMed

    Kim, Seonghun; Park, Jang Min; Kim, Chul Ho

    2013-03-01

    Jerusalem artichoke is a low-requirement sugar crop containing cellulose and hemicellulose in the stalk and a high content of inulin in the tuber. However, the lignocellulosic component in Jerusalem artichoke stalk reduces the fermentability of the whole plant for efficient bioethanol production. In this study, Jerusalem artichoke stalk was pretreated sequentially with dilute acid and alkali, and then hydrolyzed enzymatically. During enzymatic hydrolysis, approximately 88 % of the glucan and xylan were converted to glucose and xylose, respectively. Batch and fed-batch simultaneous saccharification and fermentation of both pretreated stalk and tuber by Kluyveromyces marxianus CBS1555 were effectively performed, yielding 29.1 and 70.2 g/L ethanol, respectively. In fed-batch fermentation, ethanol productivity was 0.255 g ethanol per gram of dry Jerusalem artichoke biomass, or 0.361 g ethanol per gram of glucose, with a 0.924 g/L/h ethanol productivity. These results show that combining the tuber and the stalk hydrolysate is a useful strategy for whole biomass utilization in effective bioethanol fermentation from Jerusalem artichoke.

  9. Utilisation of aromatic organosulfur compounds as sulfur sources by Lipomyces starkeyi CBS 1807.

    PubMed

    Linder, Tomas

    2016-10-01

    Aromatic organosulfur compounds that contain aryl carbon-sulfur bonds are rare in nature but occur frequently in industrial processes and products. The utilisation of aryl sulfur compounds as a sulfur source has not been described previously among the budding yeasts (phylum Ascomycota, sub-phylum Saccharomycotina). A total of 31 strains representing 30 species and 28 genera of the budding yeasts were assayed for utilisation of benzene sulfonate as source of sulfur. Lipomyces starkeyi CBS 1807 was the only strain in this screen that was able to utilise benzene sulfonate as a sulfur source. Further study of L. starkeyi demonstrated that it could also utilise methyl phenyl sulfoxide, 1-naphthalene sulfonate, 2-naphthalene sulfonate, 1-anthraquinone sulfonate and 2-anthraquinone sulfonate as sulfur sources but not diphenylsulfone. This study represents the first reported case of the utilisation of sulfonated naphthalenes and anthraquinones as sulfur sources by a fungus. However, it should be stressed that the remaining 30 strains in this study were not assayed for the utilisation of these additional aryl sulfur compounds as sulfur sources. The present study highlights the potential of L. starkeyi and related species as a source of novel enzyme activities with possible applications in chemical manufacturing, disposal of xenobiotics and bioremediation.

  10. Investigation of CBS, MTR, RFC-1 and TC polymorphisms as maternal risk factors for Down syndrome.

    PubMed

    Fintelman-Rodrigues, N; Corrêa, J C; Santos, J M; Pimentel, M M G; Santos-Rebouças, C B

    2009-01-01

    Recent evidence shows that almost 92% of the DS children are born from young mothers, suggesting that other risk factors than advanced maternal age must be involved. In this context, some studies demonstrated a possible link between DS and maternal polymorphisms in genes involved in folate metabolism. These polymorphisms, as well as low intake of folate could generate genomic instability, DNA hypomethylation and abnormal segregation, leading to trisomy 21. We compared the frequency of CBS 844ins68, MTR 2756A>G, RFC-1 80G> A and TC 776C>G polymorphisms among 114 case mothers and 110 matched controls, in order to observe whether these variants act as risk factors for DS. The genotype distributions revealed that there were not significant differences between both samples. However, when we proceed the multiplicative interaction analyses between the four polymorphisms described above together with the previously studied MTHFR 677C>T, MTHFR 1298A>C and MTRR 66A>G polymorphisms, our results show that the combined genotype TC 776CC / MTHFR 677TT and TC 776CC / MTR 2756AG were significantly higher in the control sample. Nevertheless, there was no significant association after Bonferroni correction. Our results suggest that maternal folate-related polymorphisms studied here have no influence on trisomy 21 susceptibility in subjects of Brazilian population.

  11. Changes in bone mineral density and body composition of children with well-controlled homocystinuria caused by CBS deficiency.

    PubMed

    Lim, J S; Lee, D H

    2013-09-01

    Homocystinuria due to cystathionine β-synthase (CBS) deficiency is an inherited disorder of the metabolism of methionine. Clinical manifestations include mental retardation, dislocation of the optic lens, vascular lesions, arterial and venous thromboembolism, skeletal abnormalities, and osteoporosis. Most homocystinuria patients diagnosed in adulthood have severe osteoporosis, and homocystinuria is frequently mentioned as a cause of osteoporosis. Good control of plasma homocysteine may prevent or delay some of these complications. However, the effectiveness of bone mineral density (BMD) gain or fracture prevention has not been addressed. Here, we describe changes in BMD and body composition in 5 CBS deficiency patients who were diagnosed at young age and were managed with good metabolic control. We found that the BMD of each region was within the normal range. BMD gain was adequate and the patients had no significant change in skeletal morphology.

  12. Production of recombinant human antithrombin by Pichia pastoris.

    PubMed

    Kuwae, Shinobu; Ohyama, Masao; Ohya, Tomoshi; Ohi, Hideyuki; Kobayashi, Kaoru

    2005-03-01

    This paper deals with the production of recombinant human antithrombin (rAT) by the methylotrophic yeast Pichia pastoris. In preliminary methanol-limited fed-batch fermentation, the rAT concentration reached 324 mg/l at 192 h of cultivation, but the specific heparin cofactor (HC) activity of rAT in the culture supernatant was 10% of that of plasma-derived antithrombin (pAT). To improve the specific HC activity of rAT, effort was first focused on the optimization of culture pH and media composition, resulting in protection of rAT against pH-dependent instability and proteolytic degradation. However, even in the optimized methanol-limited fed-batch fermentation, the specific HC activity of rAT in the culture supernatant was still 20% that of pAT. To investigate the unknown mechanisms involved in the decreased specific HC activity of rAT, the culture supernatant of mock-transfected cells was prepared by methanol-limited fed-batch fermentation. When pAT was added to this supernatant, a rapid decrease in HC activity was observed; the residual HC activity was 26% after 24 h of incubation at 25 degrees C. The loss of pAT activity was prevented by addition of a formaldehyde scavenger, amino urea, to the supernatant. In addition, alcohol oxidase activity was observed in the supernatant, resulting in the accumulation of formaldehyde in the culture broth. These results suggest that the formaldehyde produced by methanol oxidation in the culture broth of P. pastoris might decrease the HC activity of rAT during fermentation. Replacing the methanol with glycerol as the carbon source improved the specific HC activity of rAT from 20% to above 40% of that of pAT. In the glycerol-limited fed-batch fermentation, rAT is expressed at 100 mg/l under the control of the truncated mutated AOX2 promoter.

  13. Cognitive impairment in ALS patients and validation of the Spanish version of the ALS-CBS test.

    PubMed

    Turon-Sans, Janina; Gascon-Bayarri, Jordi; Reñé, Ramon; Rico, Imma; Gámez, Cristina; Paipa, Andres; Povedano, Monica

    2016-01-01

    Our aim was to develop and validate the Spanish version of the Amyotrophic Lateral Sclerosis Cognitive Behavioural Screen (ALS-CBS) and investigate behavioural/cognitive impairment in our ALS patients. We enrolled 50 patients with definite or probable ALS, evaluated by the Motor Neuron Disease Unit (using El Escorial criteria) and Dementia Unit, and assessed with the Spanish ALS-CBS. The patients' cognitive/behavioural status was classified according to current criteria. Patients were classified into each diagnostic category: ALS-no impairment, 36%; ALS-mild cognitive impairment, 34%; ALS-mild behavioural impairment, 6%; ALS-mild cognitive/behavioural impairment, 12%; ALS-frontotemporal dementia, 12%. Cognitive impairment was more common in bulbar (90.9%) than spinal (48.7%) forms (p < 0.012). The Spanish ALS-CBS was validated. Performance to differentiate normal vs. impaired individuals was: 1) cognition (cut-off 15; AUC, 84.7%): sensitivity 86.2%, specificity 62%, positive predictive value 75.8%, negative predictive value 76.5%; 2) behaviour (cut-off 36; AUC, 83.3%): sensitivity 93.3%, specificity 74.3%, positive predictive value 61%, negative predictive value 96.3%. Performance to differentiate between patients with and without dementia: 1) cognition (cut-off 8; AUC, 87.3%): sensitivity 83.3%, specificity 75%, positive predictive value 31.3%, negative predictive value 97.1%; 2) behaviour (cut-off 35; AUC, 80.9%): sensitivity 83.3%, specificity 69%, positive predictive value 25%, negative predictive value 96.7%. In conclusion, cognitive impairment is common in ALS patients, particularly in bulbar forms. The Spanish version of the ALS-CBS is useful for screening cognitive/behavioural impairment in this population.

  14. Ionization potential and electron affinity for six common explosive compounds by DFT, MP2, and CBS-QB3

    SciTech Connect

    Cooper, Jason K.; Grant, Christian D.; Zhang, Jin Z.

    2012-07-20

    The vertical and adiabatic ionization potential (IPV and IPA) and vertical electron affinity (EAV) for six explosives (RDX, HMX, TNT, PETN, HMTD, and TATP) have been studied by ab initio computational methods. The IPV was calculated using MP2 and CBS-QB3 while the IPA was calculated with B3LYP, CAM-B3LYP, ω B97XD, B2PLYP, and MP2. RDX and TNT IPA’s were also reported using CBS -QB3. Excluding results by CBS-QB3, B3LYP and B2PLYPD provided superior and more consistent results for calculating the IP. The EAV were calculated using the same methods however B3LYP performed the worst in this case with MP2 and B2PLYPD predicting values closest to those made by CBS-QB3, which was used a reference due to lacking experimental data. Basis set effects were evaluated using 6- 31+G(d,p), 6-311+G(d,p), and 6-311+(3df,2p) for both IP and EA. 6-31+G(d,p) gave satisfactory results for calculating both IP however 6-311+G(3df,2p) had improved results in calculating the EA. The four nitro containing compounds had exothermic reduction potentials while the peroxides were unfavorable. Additionally, it was seen that RDX, HMX, TATP and HMTD were unstable in their reduced forms. Results are aimed to assist detection and screening methods.

  15. Ionization potential and electron affinity for six common explosive compounds by DFT, MP2, and CBS-QB3

    DOE PAGES

    Cooper, Jason K.; Grant, Christian D.; Zhang, Jin Z.

    2012-07-20

    The vertical and adiabatic ionization potential (IPV and IPA) and vertical electron affinity (EAV) for six explosives (RDX, HMX, TNT, PETN, HMTD, and TATP) have been studied by ab initio computational methods. The IPV was calculated using MP2 and CBS-QB3 while the IPA was calculated with B3LYP, CAM-B3LYP, ω B97XD, B2PLYP, and MP2. RDX and TNT IPA’s were also reported using CBS -QB3. Excluding results by CBS-QB3, B3LYP and B2PLYPD provided superior and more consistent results for calculating the IP. The EAV were calculated using the same methods however B3LYP performed the worst in this case with MP2 and B2PLYPDmore » predicting values closest to those made by CBS-QB3, which was used a reference due to lacking experimental data. Basis set effects were evaluated using 6- 31+G(d,p), 6-311+G(d,p), and 6-311+(3df,2p) for both IP and EA. 6-31+G(d,p) gave satisfactory results for calculating both IP however 6-311+G(3df,2p) had improved results in calculating the EA. The four nitro containing compounds had exothermic reduction potentials while the peroxides were unfavorable. Additionally, it was seen that RDX, HMX, TATP and HMTD were unstable in their reduced forms. Results are aimed to assist detection and screening methods.« less

  16. Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine [beta]-synthase

    SciTech Connect

    Koutmos, Markos; Kabil, Omer; Smith, Janet L.; Banerjee, Ruma

    2011-08-17

    The catalytic potential for H{sub 2}S biogenesis and homocysteine clearance converge at the active site of cystathionine {beta}-synthase (CBS), a pyridoxal phosphate-dependent enzyme. CBS catalyzes {beta}-replacement reactions of either serine or cysteine by homocysteine to give cystathionine and water or H{sub 2}S, respectively. In this study, high-resolution structures of the full-length enzyme from Drosophila in which a carbanion (1.70 {angstrom}) and an aminoacrylate intermediate (1.55 {angstrom}) have been captured are reported. Electrostatic stabilization of the zwitterionic carbanion intermediate is afforded by the close positioning of an active site lysine residue that is initially used for Schiff base formation in the internal aldimine and later as a general base. Additional stabilizing interactions between active site residues and the catalytic intermediates are observed. Furthermore, the structure of the regulatory 'energy-sensing' CBS domains, named after this protein, suggests a mechanism for allosteric activation by S-adenosylmethionine.

  17. Purification, crystallization and preliminary crystallographic analysis of the CBS-domain pair of cyclin M2 (CNNM2)

    PubMed Central

    Gómez-García, Inmaculada; Stuiver, Marchel; Ereño, June; Oyenarte, Iker; Corral-Rodríguez, María Angeles; Müller, Dominik; Martínez-Cruz, Luis Alfonso

    2012-01-01

    This work describes the purification and preliminary crystallographic analysis of the CBS-domain pair of the murine CNNM2 magnesium transporter (formerly known as ancient domain protein 2; ACDP2), which consists of a pair of cystathionine β-synthase (CBS) motifs and has 100% sequence identity to its human homologue. CNNM proteins represent the least-studied members of the eight different types of magnesium transporters identified to date in mammals. In humans, the CNNM family is encoded by four genes: CNNM1–4. CNNM1 acts as a cytosolic copper chaperone, whereas CNNM2 and CNNM4 have been associated with magnesium handling. Interestingly, mutations in the CNNM2 gene cause familial dominant hypomagnesaemia (MIM:607803), a rare human disorder characterized by renal and intestinal magnesium (Mg2+) wasting, which may lead to symptoms of Mg2+ depletion such as tetany, seizures and cardiac arrhythmias. This manuscript describes the preliminary crystallographic analysis of two different crystal habits of a truncated form of the protein containing its regulatory CBS-domain pair, which has been reported to host the pathological mutation T568I in humans. The crystals belonged to space groups P21212 and I222 (or I212121) and diffracted X-­rays to 2.0 and 3.6 Å resolution, respectively, using synchrotron radiation. PMID:23027747

  18. A novel transgenic mouse model of CBS-deficient homocystinuria does not incur hepatic steatosis or fibrosis and exhibits a hypercoagulative phenotype that is ameliorated by betaine treatment.

    PubMed

    Maclean, Kenneth N; Sikora, Jakub; Kožich, Viktor; Jiang, Hua; Greiner, Lori S; Kraus, Eva; Krijt, Jakub; Overdier, Katherine H; Collard, Renata; Brodsky, Gary L; Meltesen, Lynne; Crnic, Linda S; Allen, Robert H; Stabler, Sally P; Elleder, Milan; Rozen, Rima; Patterson, David; Kraus, Jan P

    2010-01-01

    Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine (Hcy) and serine to cystathionine, which is then hydrolyzed to cysteine by cystathionine gamma-lyase. Inactivation of CBS results in CBS-deficient homocystinuria more commonly referred to as classical homocystinuria, which, if untreated, results in mental retardation, thromboembolic complications, and a range of connective tissue disorders. The molecular mechanisms that underlie the pathology of this disease are poorly understood. We report here the generation of a new mouse model of classical homocystinuria in which the mouse cbs gene is inactivated and that exhibits low-level expression of the human CBS transgene under the control of the human CBS promoter. This mouse model, designated "human only" (HO), exhibits severe elevations in both plasma and tissue levels of Hcy, methionine, S-adenosylmethionine, and S-adenosylhomocysteine and a concomitant decrease in plasma and hepatic levels of cysteine. HO mice exhibit mild hepatopathy but, in contrast to previous models of classical homocystinuria, do not incur hepatic steatosis, fibrosis, or neonatal death with approximately 90% of HO mice living for at least 6months. Tail bleeding determinations indicate that HO mice are in a hypercoagulative state that is significantly ameliorated by betaine treatment in a manner that recapitulates the disease as it occurs in humans. Our findings indicate that this mouse model will be a valuable tool in the study of pathogenesis in classical homocystinuria and the rational design of novel treatments. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. Lewis Acids as Activators in CBS-Catalysed Diels-Alder Reactions: Distortion Induced Lewis Acidity Enhancement of SnCl4.

    PubMed

    Nödling, Alexander R; Möckel, Robert; Tonner, Ralf; Hilt, Gerhard

    2016-09-05

    The effect of several Lewis acids on the CBS catalyst (named after Corey, Bakshi and Shibata) was investigated in this study. While (2) H NMR spectroscopic measurements served as gauge for the activation capability of the Lewis acids, in situ FT-IR spectroscopy was employed to assess the catalytic activity of the Lewis acid oxazaborolidine complexes. A correlation was found between the Δδ((2) H) values and rate constants kDA , which indicates a direct translation of Lewis acidity into reactivity of the Lewis acid-CBS complexes. Unexpectedly, a significant deviation was found for SnCl4 as Lewis acid. The SnCl4 -CBS adduct was much more reactive than the Δδ((2) H) values predicted and gave similar reaction rates to those observed for the prominent AlBr3 -CBS adduct. To rationalize these results, quantum mechanical calculations were performed. The frontier molecular orbital approach was applied and a good correlation between the LUMO energies of the Lewis acid-CBS-naphthoquinone adducts and kDA could be found. For the SnCl4 -CBS-naphthoquinone adduct an unusual distortion was observed leading to an enhanced Lewis acidity. Energy decomposition analysis with natural orbitals for chemical valence (EDA-NOCV) calculations revealed the relevant interactions and activation mode of SnCl4 as Lewis acid in Diels-Alder reactions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Effect of S-adenosyl-L-methionine (SAM), an allosteric activator of cystathionine-β-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro

    PubMed Central

    Módis, Katalin; Coletta, Ciro; Asimakopoulou, Antonia; Szczesny, Bartosz; Chao, Celia; Papapetropoulos, Andreas; Hellmich, Mark R.; Szabo, Csaba

    2014-01-01

    Recent data show that colon cancer cells selectively overexpress cystathionine-β-synthase (CBS), which produces hydrogen sulfide (H2S), to maintain cellular bioenergetics, support tumor growth and stimulate angiogenesis and vasorelaxation in the tumor microenvironment. The purpose of the current study was to investigate the effect of the allosteric CBS activator S-adenosyl-L-methionine (SAM) on the proliferation and bioenergetics of the CBS-expressing colon cancer cell line HCT116. The non-transformed, non-tumorigenic colon epithelial cell line NCM356 was used as control. For assessment of cell proliferation, the xCELLigence system was used. Bioenergetic function was measured by Extracellular Flux Analysis. Experiments using human recombinant CBS or HCT116 homogenates complemented the cell-based studies. SAM markedly enhanced CBS-mediated H2S production in vitro, especially when a combination of cysteine and homocysteine was used as substrates. Addition of SAM (0.1 – 3 mM) to HCT116 cells induced a concentration-dependent increase H2S production. SAM exerted time-and concentration-dependent modulatory effects on cell proliferation. At 0.1–1 mM SAM increased HCT116 proliferation between 0–12 h, while the highest SAM concentration (3 mM) inhibited proliferation. Over a longer time period (12–24 h), only the lowest concentration of SAM used (0.1 mM) stimulated cell proliferation; higher SAM concentrations produced a concentration-dependent inhibition. The short-term stimulatory effects of SAM were attenuated by the CBS inhibitor aminooxyacetic acid (AOAA) or by stable silencing of CBS. In contrast, the inhibitory effects of SAM on cell proliferation was unaffected by CBS inhibition or CBS silencing. In contrast to HCT116 cells, the lower rate of proliferation of the low-CBS expressor NCM356 cells was unaffected by SAM. Short-term (1h) exposure of HCT116 cells to SAM induced a concentration-dependent increase in oxygen consumption and bioenergetic function at

  1. Effect of S-adenosyl-L-methionine (SAM), an allosteric activator of cystathionine-β-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro.

    PubMed

    Módis, Katalin; Coletta, Ciro; Asimakopoulou, Antonia; Szczesny, Bartosz; Chao, Celia; Papapetropoulos, Andreas; Hellmich, Mark R; Szabo, Csaba

    2014-09-15

    Recent data show that colon cancer cells selectively overexpress cystathionine-β-synthase (CBS), which produces hydrogen sulfide (H2S), to maintain cellular bioenergetics, support tumor growth and stimulate angiogenesis and vasorelaxation in the tumor microenvironment. The purpose of the current study was to investigate the effect of the allosteric CBS activator S-adenosyl-L-methionine (SAM) on the proliferation and bioenergetics of the CBS-expressing colon cancer cell line HCT116. The non-transformed, non-tumorigenic colon epithelial cell line NCM356 was used as control. For assessment of cell proliferation, the xCELLigence system was used. Bioenergetic function was measured by Extracellular Flux Analysis. Experiments using human recombinant CBS or HCT116 homogenates complemented the cell-based studies. SAM markedly enhanced CBS-mediated H2S production in vitro, especially when a combination of cysteine and homocysteine was used as substrates. Addition of SAM (0.1-3 mM) to HCT116 cells induced a concentration-dependent increase H2S production. SAM exerted time- and concentration-dependent modulatory effects on cell proliferation. At 0.1-1 mM SAM increased HCT116 proliferation between 0 and 12 h, while the highest SAM concentration (3 mM) inhibited proliferation. Over a longer time period (12-24 h), only the lowest concentration of SAM used (0.1 mM) stimulated cell proliferation; higher SAM concentrations produced a concentration-dependent inhibition. The short-term stimulatory effects of SAM were attenuated by the CBS inhibitor aminooxyacetic acid (AOAA) or by stable silencing of CBS. In contrast, the inhibitory effects of SAM on cell proliferation was unaffected by CBS inhibition or CBS silencing. In contrast to HCT116 cells, the lower rate of proliferation of the low-CBS expressor NCM356 cells was unaffected by SAM. Short-term (1 h) exposure of HCT116 cells to SAM induced a concentration-dependent increase in oxygen consumption and bioenergetic function at 0

  2. [Comparative study of the protein makeup in diploid and haploid forms of Saccharomyces and Pichia].

    PubMed

    Temina, A V; Tolstorukov, I I; Korogodin, V I; Gololobov, A D

    1979-01-01

    The rates of growth, biomass accumulation, and electrophoretic spectra of mobile cytoplasmic proteins were studied with nonisogenous haploid and diploid cultures of Saccharomyces cerevisiae and Pichia guilliermondii as well as with isogenous haploid-diploid pairs of Saccharomyces cerevisiae and Pichia pinus. On a mineral medium with glucose, differences in these parameters in various yeast strains were found to be due to the genotype of a strain rather than to ploidy: nonisogenous haploid and diploid cultures displayed considerable and random variability of these properties while no differences were found in isogenous haploid-diploid pairs. Studies on solubility of protein fractions in various solvents made it possible to reveal differences connected with ploidy, namely: both in nonisogenous and isogenous haploid-diploid systems, the content of the water-soluble fraction decreased in diploid cultures.

  3. Secretory Expression and Characterization of Chinese Narcissus GNA-Like Lectin in Pichia pastoris.

    PubMed

    Li, Xinyu; Zhang, Pengpeng; Zhang, Ning; Liang, Xiao; Wang, Wenya; Yuan, Qipeng; Li, Qiang

    2017-08-01

    Narcissus tazetta lectin (NTL) is a GNA-like lectin, which has a wide potential application in medicine, agriculture, and glycobiology. In the present paper, the codon-optimized ntl gene was transformed into the yeast Pichia pastoris; SDS-PAGE gel and western blotting analysis revealed that the recombinant lectin was expressed successfully in Pichia yeast. The similarity between the recombinant NTL and the native NTL was confirmed by circular dichroism (CD) and hemagglutination assay further. In the 5-L scale fermentator, the protein yield was as high as 1.2 g/L after fermentation for 96 h. In addition, the effect of metal ions (K(+), Mg(2+), Ca(2+), and Cu(2+)), acid, and alkaline on hemagglutinating activity of NTL was tested, which provided biochemical characterizations of the mannose-binding lectin from Chinese Narcissus.

  4. Expression of the human tumor suppressor p53 induces cell death in Pichia pastoris.

    PubMed

    Abdelmoula-Souissi, Salma; Mabrouk, Imed; Gargouri, Ali; Mokdad-Gargouri, Raja

    2012-02-01

    The human tumor suppressor p53 is known as guardian of genome because of its involvement in many signals related to cell life or death. In this work, we report that human p53 induces cell death in the yeast Pichia pastoris. We showed a growth inhibition effect, which increased with the p53 protein expression level in recombinant Mut(s) (methanol utilization slow) strain of Pichia. However, no effect of p53 was observed in recombinant strain of Mut(+) (methanol utilization plus) phenotype. Interestingly, human p53 induces cell death in recombinant strains Mut(s) with characteristic markers of apoptosis such as DNA fragmentation, exposure of phosphatidylserine, and reactive oxygen species generation. Taken together, our results strongly suggest that human p53 is biologically active in this heterologous context. Thus, we propose that P. pastoris could be a useful tool to better understand the biological function of human p53.

  5. [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.

  6. Recovery of fermented inulin fiber by lactic acid bacteria (LAB) from inulin hydrolysate using fungi inulinase enzymes of Scopulariopsis sp.-CBS1 and class of Deuteromycetes-CBS4 as cholesterol binder

    NASA Astrophysics Data System (ADS)

    Susilowati, Agustine; Melanie, Hakiki; Maryati, Yati; Aspiyanto

    2017-01-01

    Fermentation of Lactobacillus Acid Bacteria (LAB) which are mixtures of Lactobacillus acidophilus, Bifidobacteriumbifidum, Lactobacillus bulgaricus and Streptococcus thermophillus on hydrolysate as a result of inulin hydrolysis using inulinase enzymes obtained from endophytic fungi ofScopulariopsis sp.-CBS1 (inulin hydrolysate of S) and Class of Deuteromycetes-CBS4 (inulin hydrolysate of D) generate potential fermented inulin fiber as cholesterol binder. Fermentation process was conducted under concentrations of inulin hydrolysate 50% (w/v), LAB 15% (v/v) and skim milk 12.5% (w/v) at room temperature and 40°C for 0, 12, 24, 36 and 48 hours, respectively. Result of experimental work showed that longer time of LAB fermentation increased total acids, TPC and CBC at pH 2, but decreased total sugar, reducing, IDF, SDF, CBC pH 2 and CBC pH 7. Based on Cholesterol Binding Capacity (CBC), optimization of fermentation process on inulin hydrolysate of S was achieved by combining treatment at 40°C for 24 hours resulted in CBC pH 2 of 19.11 mg/g TDF and inulin hydrolysate of D was achieved by fermentation at 40 °C for 48 hours resulted in CBC pH 2 of 24.28 mg/g TDF. Inulin hydrolysate of class of Deutrymecetes CBS4 fermented by LAB had better functional property as cholesterol binder than that inulin hydrolysate of S fermented by LAB. This is due to cholesterol binder and cholesterol derivatives as a result of degradation of LAB on digestive system (stomach) when compared to higher colon under optimal process condition.

  7. 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

  8. Biological production of acetaldehyde from ethanol using non-growing Pichia pastoris whole cells

    SciTech Connect

    Chiang, Heien-Kun; Foutch, G.L.; Fish, W.W.

    1991-12-31

    Acetaldehyde has been produced biologically using whole-cell Pichia Pass in a semibatch fermentor. Ethanol and air were fed continuously, and the product, acetaldehyde, was removed by the air stream. Operation of the reactor exceeded 100 h, maintaining high alcohol oxidase activity. Low cell-mass concentration (9.9 g/L) minimized product inhibition. Ethanol concentration in the broth, oxygen concentration in the air, and pH were evaluated for their effects on the fermentation process.

  9. Integrated single-cell analysis shows Pichia pastoris secretes protein stochastically.

    PubMed

    Love, Kerry Routenberg; Panagiotou, Vasiliki; Jiang, Bo; Stadheim, Terrance A; Love, J Christopher

    2010-06-01

    The production of heterologous proteins by secretion from cellular hosts is an important determinant for the cost of biotherapeutics. A single-cell analytical method called microengraving was used to examine the heterogeneity in secretion by the methylotrophic yeast Pichia pastoris. We show that constitutive secretion of a human Fc fragment by P. pastoris is not cell-cycle dependent, but rather fluctuates between states of high and low productivity in a stochastic manner.

  10. Enrichment and identification of Δ(9)-Tetrahydrocannabinolic acid synthase from Pichia pastoris culture supernatants.

    PubMed

    Lange, Kerstin; Poetsch, Ansgar; Schmid, Andreas; Julsing, Mattijs K

    2015-09-01

    This data article refers to the report Δ(9)-Tetrahydrocannabinolic acid synthase (THCAS) production in Pichia pastoris enables chemical synthesis of cannabinoids (Lange et. al. 2015) [2]. THCAS was produced on a 2 L lab scale using recombinant P. pastoris KM71 KE1. Enrichment of THCAS as a technically pure enzyme was realized using dialysis and cationic exchange chromatography. nLC-ESI-MS/MS analysis identified THCAS in different fractions obtained by cationic exchange chromatography.

  11. Isolation and characterization of Pichia heedii mutants defective in xylose uptake

    SciTech Connect

    Does, A.L.; Bisson, L.F. )

    1990-11-01

    To investigate the role of xylose uptake in xylose metabolism in yeasts, we isolated a series of mutated strains of the yeast Pichia heedii which are defective in xylose utilization. Four of these demonstrated defects in xylose uptake. Overlaps between the functional or regulatory mechanisms for glucose and xylose uptake may exist in this yeast since some of the mutants defective in xylose uptake were also defective in glucose transport. None of the mutants were defective in xylose reductase or xylitol dehydrogenase activities.

  12. 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

  13. Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology.

    PubMed

    Jacobs, Pieter P; Geysens, Steven; Vervecken, Wouter; Contreras, Roland; Callewaert, Nico

    2009-01-01

    Here we provide a protocol for engineering the N-glycosylation pathway of the yeast Pichia pastoris. The general strategy consists of the disruption of an endogenous glycosyltransferase gene (OCH1) and the stepwise introduction of heterologous glycosylation enzymes. Each engineering step results in the introduction of one glycosidase or glycosyltransferase activity into the Pichia endoplasmic reticulum or Golgi complex and consists of a number of stages: transformation with the appropriate GlycoSwitch vector, small-scale cultivation of a number of transformants, sugar analysis and heterologous protein expression analysis. If desired, the resulting clone can be further engineered by repeating the procedure with the next GlycoSwitch vector. Each engineering step takes approximately 3 weeks. The conversion of any wild-type Pichia strain into a strain that modifies its glycoproteins with Gal(2)GlcNAc(2)Man(3)GlcNAc(2)N-glycans requires the introduction of five GlycoSwitch vectors. Three examples of the full engineering procedure are provided to illustrate the results that can be expected.

  14. A functional Kv1.2-hERG chimaeric channel expressed in Pichia pastoris

    NASA Astrophysics Data System (ADS)

    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

    2014-02-01

    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.

  15. 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.

  16. A Rapid Method for Determining the Concentration of Recombinant Protein Secreted from Pichia pastoris

    NASA Astrophysics Data System (ADS)

    Sun, L. W.; Zhao, Y.; Niu, L. P.; Jiang, R.; Song, Y.; Feng, H.; feng, K.; Qi, C.

    2011-02-01

    Pichia secretive expression system is one of powerful eukaryotic expression systems in genetic engineering, which is especially suitable for industrial utilization. Because of the low concentration of the target protein in initial experiment, the methods and conditions for expression of the target protein should be optimized according to the protein yield repetitively. It is necessary to set up a rapid, simple and convenient analysis method for protein expression levels instead of the generally used method such as ultrafiltration, purification, dialysis, lyophilization and so on. In this paper, acetone precipitation method was chosen to concentrate the recombinant protein firstly after comparing with four different protein precipitation methods systematically, and then the protein was analyzed by SDS-Polyacrylamide Gel Electrophoresis. The recombinant protein was determined with the feature of protein band by the Automated Image Capture and 1-D Analysis Software directly. With this method, the optimized expression conditions of basic fibroblast growth factor secreted from pichia were obtained, which is as the same as using traditional methods. Hence, a convenient tool to determine the optimized conditions for the expression of recombinant proteins in Pichia was established.

  17. The association between CBS 844ins68 polymorphism and head and neck squamous cell carcinoma risk – a case-control analysis

    PubMed Central

    Galbiatti, Ana L.S; Ruiz, Mariangela T; Raposo, Luis S; Maniglia, José V; Pavarino-Bertelli, Erika C

    2010-01-01

    Introduction Susceptibility to head and neck squamous cell carcinoma may be modified by functional polymorphisms in genes involved in the folate pathway, such as cystathionine beta-synthase (CBS). The CBS 844ins68 polymorphism is associated with DNA methylation changes and cancer development. Material and methods A case-control retrospective study was conducted in 322 patients with head and neck squamous cell carcinoma and in 531 control subjects without cancer. The polymerase chain reaction-restriction fragment length polymorphism technique was used to genotype the polymorphism. For statistical analysis, χ2 test was conducted to examine whether the genotypic frequency of CBS 844ins68 was in Hardy-Weinberg equilibrium and multiple logistic regression was used for comparisons between groups, and for interactions between the polymorphism and risk factors and clinical histopathological parameters. Results No significant difference in CBS 844ins68 genotypic distribution was observed between the groups. Age > 50 years, male gender and tobacco consumption were predictors of the disease with increased risk of 7.89 (95% CI: 5.56-11.21), 2.49 (95% CI: 1.72-3.62), 6.44 (95% CI: 4.63-8.96) and 2.29 times (95% CI: 1.71-3.06) respectively. There was no association between the distribution of the CBS 844ins68 genotype and risk factors for this disease. According to clinical histopathological parameters, CBS 884ins68 polymorphism presented high frequency in oral cavity (p < 0.05) and patients with the polymorphism presented less survival time (p < 0.05). Conclusions We concluded that the CBS 844ins68 polymorphism is not associated with HNSCC risk and there is increased risk of this disease in male gender individuals smokers aged over 50 years. In adittion, the polymorphism is more frequent in patients with oral cavity as primary site and in patients with less survival time. PMID:22419938

  18. Determination of Tinopal CBS-X in rice papers and rice noodles using HPLC with fluorescence detection and LC-MS/MS.

    PubMed

    Ko, Kyung Yuk; Lee, Chae A; Choi, Jae Chon; Kim, Meehye

    2014-01-01

    To date there have been no reports of methods to determine Tinopal CBS-X. We developed a rapid and simple method to determine the Tinopal CBS-X content in rice noodles and rice papers using HPLC equipped with fluorescence detection. Heating the rice noodles and rice papers to 80°C after adding 75% methanol solution induced the release of Tinopal CBS-X from processed rice products. Tinopal CBS-X was separated using an isocratic mobile phase comprising 50% acetonitrile/water containing 0.4% tetrabutyl ammonium hydrogen sulphate at pH 8.0. The samples suspected to be positive by HPLC analysis were then confirmed by LC-MS/MS analysis. This study also investigated the Tinopal CBS-X content of three rice noodle products and two rice papers. The limits of quantification for rice papers and rice noodles were 1.58 and 1.51 µg kg(-1), respectively, and their correlation curves showed good linearity with r(2) ≥ 0.9997 and ≥ 0.9998, respectively. Moreover, rice papers had recoveries of 70.3-83.3% with precision ranging from 5.0% to 7.9%, whereas rice noodles had slightly lower recoveries of 63.4-78.7% and precisions of 8.5-11.5%. Only one rice noodle product contained Tinopal CBS-X, at around 2.1 mg kg(-1), whereas it was not detected in four other samples. Consequently, Tinopal CBS-X from rice noodles and rice papers can be successfully detected using the developed pre-treatment and ion-pairing HPLC system coupled with fluorescence detection.

  19. Mechanisms of HNO and NO production from Angeli's salt: density functional and CBS-QB3 theory predictions.

    PubMed

    Dutton, Andrew S; Fukuto, Jon M; Houk, K N

    2004-03-31

    The mechanism of decomposition of Angeli's salt, Na(2)N(2)O(3), was explored with B3LYP and CBS-QB3 computational methods. Angeli's salt produces both nitroxyl (HNO) and nitric oxide (NO), depending upon the pH of the solution. These calculations show that protonation on N(2), while less favorable than O protonation, leads spontaneously to HNO production, while diprotonation at O(3) leads to NO generation. K(a) values for protonation at different centers and rate constants have been found which reproduce experimental data satisfactorily.

  20. The mechanism of NO formation from the decomposition of dialkylamino diazeniumdiolates: density functional theory and CBS-QB3 predictions.

    PubMed

    Dutton, Andrew S; Fukuto, Jon M; Houk, K N

    2004-02-09

    The mechanism of decomposition of dialkylamino diazeniumdiolate salts, R(2)N[NONO](-)M(+), to form nitric oxide, NO, was explored using theoretical methods. B3LYP/6-311+G(d) density functional theory calculations gave the optimized geometries, and energetics were further evaluated with the high accuracy CBS-QB3 method, when feasible. Relative pK(a) values were estimated using the PCM model for aqueous solvation. The terminal oxygen is the most basic site, followed closely by the internal oxygen. Protonation of these sites does not lead to decomposition. However, protonation of the weakly basic amino nitrogen leads to very rapid decomposition and NO generation.

  1. A 15-year review of ABC, CBS, and NBC news coverage of organ donation: implications for organ donation campaigns.

    PubMed

    Quick, Brian L; Kim, Do Kyun; Meyer, Kevin

    2009-03-01

    This content analysis represents news coverage of organ donation from January 1990 to December 2005. Specifically, ABC, CBS, and NBC news broadcasts were examined to gain a greater understanding of organ donation coverage on TV. Overall this investigation revealed that organ donation received modest coverage (N = 1,507). Although the majority of coverage was positive, attention to the need for organs and the process of becoming a potential organ donor received modest exposure. In addition, non-living donor and living-donor donations received approximately equal coverage. Results are discussed with a focus on message design for practitioners and advocates of organ donation.

  2. Comparative genomics of citric-acid producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88

    SciTech Connect

    Grigoriev, Igor V.; Baker, Scott E.; Andersen, Mikael R.; Salazar, Margarita P.; Schaap, Peter J.; Vondervoot, Peter J.I. van de; Culley, David; Thykaer, Jette; Frisvad, Jens C.; Nielsen, Kristen F.; Albang, Richard; Albermann, Kaj; Berka, Randy M.; Braus, Gerhard H.; Braus-Stromeyer, Susanna A.; Corrochano, Luis M.; Dai, Ziyu; Dijck, Piet W.M. van; Hofmann, Gerald; Lasure, Linda L.; Magnusson, Jon K.; Meijer, Susan L.; Nielsen, Jakob B.; Nielsen, Michael L.; Ooyen, Albert J.J. van; Panther, Kathyrn S.; Pel, Herman J.; Poulsen, Lars; Samson, Rob A.; Stam, Hen; Tsang, Adrian; Brink, Johannes M. van den; Atkins, Alex; Aerts, Andrea; Shapiro, Harris; Pangilinan, Jasmyn; Salamov, Asaf; Lou, Yigong; Lindquist, Erika; Lucas, Susan; Grimwood, Jane; Kubicek, Christian P.; Martinez, Diego; Peij, Noel N.M.E. van; Roubos, Johannes A.; Nielsen, Jens

    2011-04-28

    The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compels additional exploration. We therefore undertook whole genome sequencing of the acidogenic A. niger wild type strain (ATCC 1015), and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was utilized to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 megabase of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis revealed up-regulation of the electron transport chain, specifically the alternative oxidative pathway in ATCC 1015, while CBS 513.88 showed significant up-regulation of genes relevant to glucoamylase A production, such as tRNA-synthases and protein transporters. Our results and datasets from this integrative systems biology analysis resulted in a snapshot of fungal evolution and will support further optimization of cell factories based on filamentous fungi.[Supplemental materials (10 figures, three text documents and 16 tables) have been made available

  3. Quantitative description of the growth of Saccharomyces cerevisiae CBS 426 on a mixed substrate of glucose and ethanol

    SciTech Connect

    Geurts, T.G.E.; De Kok, H.E.; Roels, J.A.

    1980-01-01

    S. cerevisiae CBS 426 was grown aerobically in continuous culture with a mixture of glucose and EtOH as the C source. The flows of biomass, glucose, EtOH, O, and CO/sub 2/ were measured. A model for growth with 2 substrates was derived. Application of this model to the above-mentioned system yielded values for YATP and P/O. The joint confidence regions for these parameters were calculated. The relevance to industrial production of bakers' yeast is discussed.

  4. Polymorphisms in the CBS gene and homocysteine, folate and vitamin B12 levels: association with polymorphisms in the MTHFR and MTRR genes in Brazilian children.

    PubMed

    Aléssio, Ana C M; Siqueira, Lúcia H; Bydlowski, Sérgio P; Höehr, Nelci F; Annichino-Bizzacchi, Joyce M

    2008-10-15

    Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR) and cystathionine beta-synthase (CBS) genes, involved in the intracellular metabolism of homocysteine (Hcy), can result in hyperhomocysteinemia. The objective of this study was to evaluate prevalence estimates of CBS T833C, G919A and the insertion of 68-bp (844ins68) polymorphisms and their correlation with Hcy, folate and B(12) in 220 children previously genotyped for MTHFR C677T, A1298C, and MTRR A66G. The prevalence of heterozygote children for 844ins68 was 19.5%. The T833C CBS mutation was identified in association with 844ins68 in all the carriers of the insertion. Genotyping for CBS G919A mutation showed that all the children presented the GG genotype. Analysis of Hcy, B(12) and folate, according to the combination of the different genotypes of the C677T and A1298C MTHFR, A66G MTRR, and 844ins68 CBS showed that the 677TT/1298AA/68WW genotype is associated with an increase in Hcy, when compared to the 677CC/1298AC/68WW (P = 0.033) and the 677CT/1298AA/68WW genotypes (P = 0.034). Since B(12) and folate were not different between these groups, a genetic interaction between diverse polymorphisms probably influences Hcy. Our results emphasize the role of genetic interactions in Hcy levels. 2008 Wiley-Liss, Inc.

  5. Efficient expression and secretion of recombinant human growth hormone in the methylotrophic yeast Pichia pastoris: potential applications for other proteins.

    PubMed

    Apte-Deshpande, Anjali; Rewanwar, Sachin; Kotwal, Prakash; Raiker, Veena A; Padmanabhan, Sriram

    2009-11-13

    A simple high yielding process for the production of rhGH (recombinant human growth hormone) in the Pichia pastoris system is described. The approach adopted the addition of surfactants during fermentation along with methanol induction. A Pichia integrant harbouring multiple-copy, non-codon-optimized hGH showed poor expression in complex and defined media. Inclusion of the surfactants Tween 20 or Tween 80 during induction enhanced the expression levels significantly in shake flask studies. The combination of 2 litres of basal salt medium and Tween 20 in a bioreactor culminated in 3 x 10(4)-fold elevated expression of the protein (approximately 500 mg/l) as estimated by ELISA. SDS/PAGE and Western-blot analyses revealed that the Pichia-derived rhGH migrated as a single band with a molecular mass of approximately 22 kDa and had the same immunoreactivity as native commercial rhGH. Analysis of Pichia-derived purified rhGH and commercial rhGH on an Agilent 2100 Bioanalyzer revealed overlapping peaks displaying authentic N-terminal processing of Pichia-derived rhGH, which was further confirmed by N-terminal sequencing. In addition, matrix-assisted laser-desorption ionization-time of flight analysis of the protein confirmed its authenticity. These results indicate that the P. pastoris expression system can be effective in the production of rhGH at commercially relevant levels.

  6. Codon optimization, promoter and expression system selection that achieved high-level production of Yarrowia lipolytica lipase in Pichia pastoris.

    PubMed

    Zhou, Wen-Jing; Yang, Jiang-Ke; Mao, Lin; Miao, Li-Hong

    2015-04-01

    Lipase (EC 3.1.1.3) stands amongst the most important and promising biocatalysts for industrial applications. In this study, in order to realize a high-level expression of the Yarrowia lipolytica lipase gene in Pichia pastoris, we optimized the codon of LIP2 by de novo gene design and synthesis, which significantly improved the lipase expression when compared to the native lip2 gene. We also comparatively analyzed the effects of the promoter types (PAOX1 and PFLD1) and the Pichia expression systems, including the newly developed PichiaPink system, on lipase production and obtained the optimal recombinants. Bench-top scale fermentation studies indicated that the recombinant carrying the codon-optimized lipase gene syn-lip under the control of promoter PAOX1 has a significantly higher lipase production capacity in the fermenter than other types of recombinants. After undergoing methanol inducible expression for 96h, the wet cell weight of Pichia, the lipase activity and the protein content in the fermentation broth reached their highest values of 262g/L, 38,500U/mL and 2.82g/L, respectively. This study has not only greatly facilitated the bioapplication of lipase in industrial fields but the strategies utilized, such as de novo gene design and synthesis, the comparative analysis among promoters and different generations of Pichia expression systems will also be useful as references for future work in this field. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Effects of B2O3 content and sintering temperature on crystallization and microstructure of CBS glass-ceramic coatings

    NASA Astrophysics Data System (ADS)

    Li, Pengyang; Wang, Shubin; Liu, Jianggao; Feng, Mengjie; Yang, Xinwang

    2015-11-01

    Borosilicate glass-ceramics precursors with varying compositional ratios in the CaO-SiO2-B2O3 (CBS) system were synthesized by sol-gel method. The precursors were calcined at 1200 °C for 2 h to form glass powders. The glass-ceramics were prepared by overlaying glass slurries on the substrates before sintering at different temperatures. The as-prepared glasses and glass-ceramics were characterized by differential scanning calorimetry and X-ray diffraction. The crystallization activation energies (Ec) were calculated using the Kissinger method from DSC results. The morphology and crystallization behavior of the glass-ceramics were monitored by scanning electron microscopy. Both glass transition and crystallization temperatures decreased, however, the metastable zone increased. The Ec values of CBS glasses and glass-ceramics were 254.1, 173.2 and 164.4 kJ/mol with increasing B2O3 content, whereas that of the calcined G3 glass was 104.9 kJ/mol. Finally, the coatings were prepared at a low temperature (700 °C). The crystals that grew on the surface of multilayer coatings demonstrated heterogeneous surface nucleation and crystallization after heat-treatment from 700 °C to 850 °C for 4 h.

  8. Unique gating properties of C. elegans ClC anion channel splice variants are determined by altered CBS domain conformation and the R-helix linker

    PubMed Central

    Dave, Sonya; Sheehan, Jonathan H; Meiler, Jens

    2010-01-01

    All eukaryotic and some prokaryotic ClC anion transport proteins have extensive cytoplasmic C-termini containing two cystathionine-β-synthase (CBS) domains. CBS domain secondary structure is highly conserved and consists of two α-helices and three β-strands arranged as β1-α1-β2-β3-α2. ClC CBS domain mutations cause muscle and bone disease and alter ClC gating. However, the precise functional roles of CBS domains and the structural bases by which they regulate ClC function are poorly understood. CLH-3a and CLH-3b are C. elegans ClC anion channel splice variants with strikingly different biophysical properties. Splice variation occurs at cytoplasmic N- and C-termini and includes several amino acids that form α2 of the second CBS domain (CBS2). We demonstrate that interchanging α2 between CLH-3a and CLH-3b interchanges their gating properties. The “R-helix” of ClC proteins forms part of the ion-conducting pore and selectivity filter and is connected to the cytoplasmic C-terminus via a short stretch of cytoplasmic amino acids termed the “R-helix linker”. C-terminus conformation changes could cause R-helix structural rearrangements via this linker. X-ray structures of three ClC protein cytoplasmic C-termini suggest that α2 of CBS2 and the R-helix linker could be closely apposed and may therefore interact. We found that mutating apposing amino acids in α2 and the R-helix linker of CLH-3b was sufficient to give rise to CLH-3a-LIKE gating. We postulate that the R-helix linker interacts with CBS2 α2, and that this putative interaction provides a pathway by which cytoplasmic C-terminus conformational changes induce conformational changes in membrane domains that in turn modulate ClC function. PMID:20581474

  9. Cell culture using centrifugal microfluidic platform with demonstration on Pichia pastoris.

    PubMed

    Ren, Yong; Chow, Larry Ming-Cheung; Leung, Wallace Woon-Fong

    2013-04-01

    This paper discusses the vortical flow, mixing and cell culture of Pichia pastoris using a centrifugal microfluidic (CM) chamber. The resultant "spiral toroidal vortex" in the chamber is made up of a primary vortex induced from inertial acceleration/deceleration of the chamber superposed by a secondary toroidal vortex due to Coriolis acceleration acting on the primary vortex. A validated numerical fluid-flow model with minimized numerical diffusion effect has been developed to investigate the flow and consequently mixing of two-color liquids through cyclic constant acceleration-and-deceleration in the same rotation direction until homogeneous mixing of the two liquids in the CM chamber has been established. The specific mixing time is found to improve with increase in acceleration/deceleration and angular span of the chamber. An experimental CM platform with three cell-culture chambers of different angular spans has been built and Pichia pastoris cell culture has been successfully demonstrated. Cell growth can be monitored over time on the extracted samples by measuring the optical density at 600-nm wave-length. Comparing with conventional cell culture, Pichia pastoris cultured on CM platform exhibits a very short lag (cell preparation/budding) phase prior to the log phase (cell growth). While it takes 8 to 12 h for the conventional shake flask in the lag phase, it takes only 2 h for the CM platform irrespective of initial cell concentration (8.1 × 10(4) to 8.1 × 10(5)/ml), acceleration/deceleration (10 to 32/s(2)) and angular span of the culture chamber (π/12 to π/4), representing significant time reduction. This is largely attributed to better growth conditions due to enhanced mixing and appropriate shear-stress stimulation from the efficient spiral toroidal vortex.

  10. [Construction of Pichia pastoris strain expressing salivary plasminogen activator from vampire bat (Desmodus rotundus)].

    PubMed

    Liu, Yan; Su, Chang; Song, Xiaoshuang; Tang, Yalan; Bao, Zhenhong

    2009-04-01

    Vampire bat saliva contains a plasminogen activator that presumably assists these hematophagous animals during feeding. Bat-PA (H), the full-length form of Vampire Bat Salivary Plasminogen Activator (DSPAalpha1), is homologous and similar efficacy to tissue-type plasminogen activator (t-PA). The strict fibrin dependence of activity is a characteristic which could be desirable in the fibrinolytic therapy. It is a unique fibrinolytic enzyme that does not promote neurodegeneration. In this study, according to the reported gene sequence (GenBank Accession No. J05082) of Vampire bat (D. rotundus) plasminogen activator. It was the first time to synthesize the full sequence of DSPAalpha1 in vitro and clone it into the expression vector pPIC9K, the recombinant plasmid was linearized and transformed into Pichia pastoris GS115 strain. Secreted expression of recombinant DSPAalpha1 was attained by methanol induction and its molecular mass is 47 kD. To get recombinant GS115 with high amount of protein, hundreds of His+ transformants had been screened to isolate clones resistant to high levels G418 (2-4 mg/mL), the selected clones mini-expressed in Pichia pastoris, and tested their fibrinolytic activities and expressed protein bands by fibrin plate assay and SDS-PAGE. DSPAalpha1 was determined by optical density after SDS-PAGE, the yield is about 30 mg per liter of fermentation culture. DSPAalpha1 derived often from mammalian cells: Chinese hamster ovary (CHO) cells, Baby hamster kidney (BHK) cells, COS cells, which might be produced at high cost. In Pichia pastoris, it is expected to higher yield and lower cost, thus it might be able to serve as new thrombolytic candidate.

  11. Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.

    PubMed

    Clark, Lindsay; Zahm, Jacob A; Ali, Rustam; Kukula, Maciej; Bian, Liangqiao; Patrie, Steven M; Gardner, Kevin H; Rosen, Michael K; Rosenbaum, Daniel M

    2015-07-01

    (13)C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific (13)C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient (13)C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.

  12. 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.

  13. New opportunities by synthetic biology for biopharmaceutical production in Pichia pastoris

    PubMed Central

    Vogl, Thomas; Hartner, Franz S; Glieder, Anton

    2013-01-01

    Biopharmaceuticals are an integral part of modern medicine and pharmacy. Both, the development and the biotechnological production of biopharmaceuticals are highly cost-intensive and require suitable expression systems. In this review we discuss established and emerging tools for reengineering the methylotrophic yeast Pichia pastoris for biopharmaceutical production. Recent advancements of this industrial expression system through synthetic biology include synthetic promoters to avoid methanol induction and to fine-tune protein production. New platform strains and molecular cloning tools as well as in vivo glycoengineering to produce humanized glycoforms have made P. pastoris an important host for biopharmaceutical production. PMID:23522654

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

    PubMed

    Wang, Man; Jiang, Shuai; Wang, Yefu

    2016-04-01

    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.

  15. [Cloning of mMR-1 gene and expression in Pichia pastoris systems].

    PubMed

    Li, Tian-Bo; Hu, Yang; Wang, Yi-Guang; Xia, Huan-Zhang

    2005-01-01

    hMR-1 (Homo Myofibrillogenesis Regulator 1, AF417001) is a novel homo gene, which was firstly cloned in our laboratory. The former studies revealed that hMR-1 is a transmembrane protein which shows protein interaction with sarcomeric proteins like myomesin I, myosin regulatory light chain, alpha-enolase and some cell regulator proteins such as eukaryotic translation initiation factor3 subunit 5 (eIF3S5) and etc. In this work, we focused on cloning the homologous gene of hMR-1 from mouse C57BL/6J and exploring its expression using Pichia pastoris yeast system. Two pairs of primers were synthesized according to the hMR-1 gene homologous sequence on mouse genome chromosome 1. The mouse MR-1 gene (mMR-1) was cloned by PCR following the first round RT-PCR from mouse C57BL/6J spleen total RNA. Sequence analysis verified that mMR-1 gene and amino acids sequence showed 90.4% and 90.1% identity with hMR-1, respectively. The prediction of hydrophobic transmembrane structure of mMR-1 suggested it is also a transmembrane protein. The mMR-1 Pichia pastoris expression vector pPIC9-mMR-1 was constructed by fusion of the flanking mMR-1 ORF in the pPIC9 plasmid. After linearization of pPIC9-mMR-1 with Sal I, the 8.5kb DNA fragment was transformed into Pichia pastoris GS115 strain by electroporation. GS115/Mut+ pPIC9-mMR-1 transformants were selected on minimal methanol medium. Integration of mMR-1 gene into the yeast genome in the recombinants was verified by PCR from the transformants total DNA. The mMR-1 protein was expressed by induction under the concentration of 0.5 % methanol. The specific induced protein of 25 kD molecular mass in SDS-PAGE was confirmed to be the mMR-1 protein by Western blot rsing hMR-1 polyclonal antibody. The expression level of this recombinant mMR-1 protein was about 50 mg/L. The successful expression of mMR-1 in the Pichia pastoris GS115 will facilitate the further functional analysis of the novel gene MR-1 in animal model.

  16. High-level expression of Rhodotorula gracilis D-amino acid oxidase in Pichia pastoris.

    PubMed

    Abad, Sandra; Nahalka, Jozef; Winkler, Margit; Bergler, Gabriele; Speight, Robert; Glieder, Anton; Nidetzky, Bernd

    2011-03-01

    By combining gene design and heterologous over-expression of Rhodotorula gracilis D-amino acid oxidase (RgDAO) in Pichia pastoris, enzyme production was enhanced by one order of magnitude compared to literature benchmarks, giving 350 kUnits/l of fed-batch bioreactor culture with a productivity of 3.1 kUnits/l h. P. pastoris cells permeabilized by freeze-drying and incubation in 2-propanol (10% v/v) produce a highly active (1.6 kUnits/g dry matter) and stable oxidase preparation. Critical bottlenecks in the development of an RgDAO catalyst for industrial applications have been eliminated.

  17. Involvement of carnitine acyltransferases in peroxisomal fatty acid metabolism by the yeast Pichia guilliermondii.

    PubMed Central

    Pagot, Y; Belin, J M

    1996-01-01

    This article provides information about peroxisomal fatty acid metabolism in the yeast Pichia guilliermondii. The existence of inducible mitochondrial carnitine palmitoyltransferase and peroxisomal carnitine octanoyl-transferase activities was demonstrated after culture of this yeast in a medium containing methyl oleate. The subcellular sites and induction patterns were studied. The inhibition of carnitine octanoyl- and palmitoyl-transferases by chlorpromazine to a large extent prevented the otherwise observed metabolism-dependent inactivation of thiolase by 2-bromofatty acids in vivo. We concluded that the metabolism of long- and medium-chain fatty acids in the peroxisome of this yeast involved carnitine intermediates. PMID:8837442

  18. Improving functional annotation for industrial microbes: a case study with Pichia pastoris

    PubMed Central

    Dikicioglu, Duygu; Wood, Valerie; Rutherford, Kim M.; McDowall, Mark D.; Oliver, Stephen G.

    2014-01-01

    The research communities studying microbial model organisms, such as Escherichia coli or Saccharomyces cerevisiae, are well served by model organism databases that have extensive functional annotation. However, this is not true of many industrial microbes that are used widely in biotechnology. In this Opinion piece, we use Pichia (Komagataella) pastoris to illustrate the limitations of the available annotation. We consider the resources that can be implemented in the short term both to improve Gene Ontology (GO) annotation coverage based on annotation transfer, and to establish curation pipelines for the literature corpus of this organism. PMID:24929579

  19. Response Surface Optimization of Bioethanol Production from Sugarcane Molasses by Pichia veronae Strain HSC-22

    PubMed Central

    Hamouda, Hamed I.; Nassar, Hussein N.; Madian, Hekmat R.; Abu Amr, Salem S.; El-Gendy, Nour Sh.

    2015-01-01

    Pichia veronae strain HSC-22 (accession number KP012558) showed a good tolerance to relatively high temperature, ethanol and sugar concentrations. Response surface optimization based on central composite design of experiments predicted the optimal values of the influencing parameters that affect the production of bioethanol from sugarcane molasses to be as follows: initial pH 5, 25% (w : v) initial molasses concentration, 35°C, 116 rpm, and 60 h. Under these optimum operating conditions the maximum bioethanol production on a batch fermenter scale was recorded as 32.32 g/L with 44% bioethanol yield. PMID:26779347

  20. Nitrile bioconversion by Microbacterium imperiale CBS 498-74 resting cells in batch and ultrafiltration membrane bioreactors.

    PubMed

    Cantarella, M; Cantarella, L; Gallifuoco, A; Spera, A

    2006-03-01

    The biohydration of acrylonitrile, propionitrile and benzonitrile catalysed by the NHase activity contained in resting cells of Microbacterium imperiale CBS 498-74 was operated at 5, 10 and 20 degrees C in laboratory-scale batch and membrane bioreactors. The bioreactions were conducted in buffered medium (50 mM Na(2)HPO(4)/NaH(2)PO(4), pH 7.0) in the presence of distilled water or tap-water, to simulate a possible end-pipe biotreatment process. The integral bioreactor performances were studied with a cell loading (dry cell weight; DCW) varying from 0.1 mg(DCW) per reactor to 16 mg(DCW) per reactor, in order to realize near 100% bioconversion of acrylonitrile, propionitrile and benzonitrile without consistent loss of NHase activity.

  1. Outlines for the definition of halotolerance/halophily in yeasts: Candida versatilis (halophila) CBS4019 as the archetype?

    PubMed

    Silva-Graça, Magda; Neves, Luisa; Lucas, Cândida

    2003-06-01

    Candida versatilis (halophila) CBS4019 was chosen to study the physiological reactions of long-term exposure to extremely high salt concentrations. In general, our results show a significant increase in enzyme expression during growth under stress conditions. Although glycerol and mannitol pathways are not under glucose repression, they were found to be metabolically regulated. Glycerol-3P-dehydrogenase used either of its cofactors NADPH or NADH, being in favor of NADPH during growth with high salt concentrations. This ability of interchanging cofactors, an increased fermentation rate, and the observed mannitol pathway activity are suggested to contribute to the yeasts' redox stability. Enzymes per se were not salt-tolerant in vitro. Consistently, intracellular sodium was low and intracellular potassium, a requirement for growth, was high. The concept of halophily and its applicability to yeasts is discussed.

  2. The gene for cystathionine beta-synthase (CBS) maps to the subtelomeric region on human chromosome 21q and to proximal mouse chromosome 17.

    PubMed Central

    Münke, M; Kraus, J P; Ohura, T; Francke, U

    1988-01-01

    The human gene for cystathionine beta-synthase (CBS), the enzyme deficient in classical homocystinuria, has been assigned to the subtelomeric region of band 21q22.3 by in situ hybridization of a rat cDNA probe to structurally rearranged chromosomes 21. The homologous locus in the mouse (Cbs) was mapped to the proximal half of mouse chromosome 17 by Southern analysis of Chinese hamster X mouse somatic cell hybrid DNA. Thus, CBS/Cbs and the gene for alpha A-crystalline (CRYA1/Crya-1 or Acry-1) form a conserved linkage group on human (HSA) chromosome region 21q22.3 and mouse (MMU) chromosome 17 region A-C. Features of Down syndrome (DS) caused by three copies of these genes should not be present in mice trisomic for MMU 16 that have been proposed as animal models for DS. Mice partially trisomic for MMU 16 or MMU 17 should allow gene-specific dissection of the trisomy 21 phenotype. Images Figure 1 Figure 2 Figure 4 PMID:2894761

  3. Deciphering the Hybridisation History Leading to the Lager Lineage Based on the Mosaic Genomes of Saccharomyces bayanus Strains NBRC1948 and CBS380T

    PubMed Central

    Nguyen, Huu-Vang; Legras, Jean-Luc; Neuvéglise, Cécile; Gaillardin, Claude

    2011-01-01

    Saccharomyces bayanus is a yeast species described as one of the two parents of the hybrid brewing yeast S. pastorianus. Strains CBS380T and NBRC1948 have been retained successively as pure-line representatives of S. bayanus. In the present study, sequence analyses confirmed and upgraded our previous finding: S. bayanus type strain CBS380T harbours a mosaic genome. The genome of strain NBRC1948 was also revealed to be mosaic. Both genomes were characterized by amplification and sequencing of different markers, including genes involved in maltotriose utilization or genes detected by array-CGH mapping. Sequence comparisons with public Saccharomyces spp. nucleotide sequences revealed that the CBS380T and NBRC1948 genomes are composed of: a predominant non-cerevisiae genetic background belonging to S. uvarum, a second unidentified species provisionally named S. lagerae, and several introgressed S. cerevisiae fragments. The largest cerevisiae-introgressed DNA common to both genomes totals 70kb in length and is distributed in three contigs, cA, cB and cC. These vary in terms of length and presence of MAL31 or MTY1 (maltotriose-transporter gene). In NBRC1948, two additional cerevisiae-contigs, cD and cE, totaling 12kb in length, as well as several smaller cerevisiae fragments were identified. All of these contigs were partially detected in the genomes of S. pastorianus lager strains CBS1503 (S. monacensis) and CBS1513 (S. carlsbergensis) explaining the noticeable common ability of S. bayanus and S. pastorianus to metabolize maltotriose. NBRC1948 was shown to be inter-fertile with S. uvarum CBS7001. The cross involving these two strains produced F1 segregants resembling the strains CBS380T or NRRLY-1551. This demonstrates that these S. bayanus strains were the offspring of a cross between S. uvarum and a strain similar to NBRC1948. Phylogenies established with selected cerevisiae and non-cerevisiae genes allowed us to decipher the complex hybridisation events linking S

  4. 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

  5. Cloning, expression, and purification of lipoprotein-associated phospholipase A(2) in Pichia pastoris.

    PubMed

    Zhang, Fujun; Wang, Yiping

    2006-05-01

    Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) has been shown to play a crucial role in atherosclerosis, and has been proposed as a promising target for drug discovery. Here, we cloned the Lp-PLA(2) gene from differentiated THP-1 cells, and inserted a carboxy-terminal His(6)-tagged version of the gene into the pPIC9 Pichia expression vector. The Lp-PLA(2) fusion protein was successfully expressed in Pichia pastoris expression system and could be rapidly purified to apparent homogeneity using a single-step purification method. The activity of our recombinant Lp-PLA(2) was strong when [3H] PAF was used as a substrate, and the Lp-PLA(2) inhibitor SB435495 exhibited an inhibitory curve against the recombinant Lp-PLA2 (IC50 = 15.93 +/- 1 microM). This novel recombinant Lp-PLA(2) could prove useful as a screening model for Lp-PLA(2) inhibitors, and may facilitate further investigation of this protein in atherosclerosis.

  6. Overexpression and biochemical characterization of a thermostable phytase from Bacillus subtilis US417 in Pichia pastoris.

    PubMed

    Hmida-Sayari, Aïda; Elgharbi, Fatma; Farhat, Ameny; Rekik, Hatem; Blondeau, Karine; Bejar, Samir

    2014-09-01

    The overexpression of the native gene encoding the thermostable Bacillus subtilis US417 phytase using Pichia pastoris system is described. The phytase gene, in which the sequence encoding the signal peptide was replaced by that of the α-factor of Saccharomyces cerevisiae, was placed under the control of the methanol-inducible promoter of the alcohol oxidase 1 gene and expressed in Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. A recombinant strain was selected and produces 43 and 227 U/mL of phytase activity in shake flasks and in high-cell-density fermentation, respectively. The purified phytase was glycosylated protein and varied in size (50-65 kDa). It has a molecular mass of 43 kDa when it was deglycosylated. The purified r-PHY maintains 100% of its activity after 10 min incubation at 75 °C and pH 7.5. This thermostable phytase, which is also active over broad pH ranges, may be useful as feed additives, since it can resist the temperature used in the feed-pelleting process.

  7. Codon Optimization Significantly Improves the Expression Level of a Keratinase Gene in Pichia pastoris

    PubMed Central

    Hu, Hong; Gao, Jie; He, Jun; Yu, Bing; Zheng, Ping; Huang, Zhiqing; Mao, Xiangbing; Yu, Jie; Han, Guoquan; Chen, Daiwen

    2013-01-01

    The main keratinase (kerA) gene from the Bacillus licheniformis S90 was optimized by two codon optimization strategies and expressed in Pichia pastoris in order to improve the enzyme production compared to the preparations with the native kerA gene. The results showed that the corresponding mutations (synonymous codons) according to the codon bias in Pichia pastoris were successfully introduced into keratinase gene. The highest keratinase activity produced by P. pastoris pPICZαA-kerAwt, pPICZαA-kerAopti1 and pPICZαA-kerAopti2 was 195 U/ml, 324 U/ml and 293 U/ml respectively. In addition, there was no significant difference in biomass concentration, target gene copy numbers and relative mRNA expression levels of every positive strain. The molecular weight of keratinase secreted by recombinant P. pastori was approx. 39 kDa. It was optimally active at pH 7.5 and 50°C. The recombinant keratinase could efficiently degrade both α-keratin (keratin azure) and β-keratin (chicken feather meal). These properties make the P. pastoris pPICZαA-kerAopti1 a suitable candidate for industrial production of keratinases. PMID:23472192

  8. 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.

  9. 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.

  10. Heterologous, Expression, and Characterization of Thermostable Glucoamylase Derived from Aspergillus flavus NSH9 in Pichia pastoris

    PubMed Central

    Karim, Kazi Muhammad Rezaul; Hossain, Md. Anowar; Sing, Ngieng Ngui; Mohd Sinang, Fazia; Hussain, Mohd Hasnain Md.; Roslan, Hairul Azman

    2016-01-01

    A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfully identified, isolated, and overexpressed in Pichia pastoris GS115. The complete open reading frame of glucoamylase from Aspergillus flavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned into Pichia pastoris and the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg++, Fe++, Zn++, Cu++, and Pb++) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries. PMID:27504454

  11. Expression of a cold-adapted fish trypsin in Pichia pastoris.

    PubMed

    Macouzet, Martin; Simpson, Benjamin K; Lee, Byong H

    2005-06-01

    Trypsin is a highly valuable protease that has many industrial and biomedical applications. The growing demand for non-animal sources of the enzyme and for trypsins with special properties has driven the interest to clone and express this protease in microorganisms. Reports about expression of recombinant trypsins show wide differences in the degree of success and are contained mainly in patent applications, which disregard the difficulties associated with the developments. Although the yeast Pichia pastoris appears to be the microbial host with the greatest potential for the production of trypsin, it has shown problems when expressing cold-adapted fish trypsins (CAFTs). CAFTs are considered of immense value for their comparative advantage over other trypsins in a number of food-processing and biotechnological applications. Thus, to investigate potential obstacles related to the production of CAFTs in P. pastoris, the cunner fish trypsin (CFT) was cloned in different Pichia expression vectors. The vectors were constructed targeting both internal and secreted expression and keeping the CFT native signal peptide. Western-blotting analysis confirmed the expression with evident differences for each construct, observing a major effect of the leader peptide sequence on the expression patterns. Immobilized nickel affinity chromatography yielded a partially purified recombinant CFT, which exhibited trypsin-specific activity after activation with bovine enterokinase.

  12. Genome Sequences of Cyberlindnera fabianii 65, Pichia kudriavzevii 129, and Saccharomyces cerevisiae 131 Isolated from Fermented Masau Fruits in Zimbabwe

    PubMed Central

    van Rijswijck, Irma M. H.; Derks, Martijn F. L.; Abee, Tjakko; de Ridder, Dick

    2017-01-01

    ABSTRACT Cyberlindnera fabianii 65, Pichia kudriavzevii 129, and Saccharomyces cerevisiae 131 have been isolated from the microbiota of fermented masau fruits. C. fabianii and P. kudriavzevii especially harbor promising features for biotechnology and food applications. Here, we present the draft annotated genome sequences of these isolates. PMID:28385833

  13. 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.

  14. 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...

  15. Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris

    USDA-ARS?s Scientific Manuscript database

    To improve expression efficiency of the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse in the methylotrophic yeast Pichia pastoris (P. pastoris) GS115, the DNA sequence encoding for CBL-scFv was designed and synthesized based on the codon bias of P. p...

  16. Differential response of Pichia guilliermondii spoilage isolates to biological and physico-chemical factors prevailing in Patagonian wine fermentations.

    PubMed

    Lopes, Christian A; Sáez, Julieta S; Sangorrín, Marcela P

    2009-07-01

    Pichia guilliermondii can produce volatile phenols in the initial stages of wine fermentation; however, its response to different wine stress conditions has been poorly studied. In this work, we present an analysis of the response of 23 P. guilliermondii indigenous isolates to physical and chemical wine stress factors and to indigenous wine killer yeasts. Principal coordinates analysis (PCoA), based on data obtained from response patterns, was carried out to evaluate the relationships among the isolates. Major differences among the isolates were detected in media plates containing 8% ethanol and in those containing 280 g/L glucose. PCoA identified 3 clusters of isolates with different stress response patterns, indicating a relationship between the tolerance to these compounds and the origin of the isolates. Pichia guilliermondii isolates were sensitive to the toxins produced by the species Hanseniaspora uvarum, Metschnikowia pulcherrima, Wickerhamomyces anomala (ex Pichia anomala), and Pichia kluyveri, with a maximum level of sensitivity against W. anomala (91% on average). Those isolates obtained from fermenting must proved to be more resistant to killer yeasts than those obtained from grape surfaces. The combined evaluation of the response to physico-chemical and biological factors presented in this work could be a useful standard protocol for the evaluation of the potential spoilage capacity of yeasts in winemaking.

  17. 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...

  18. Accurate reaction barrier heights of pericyclic reactions: Surprisingly large deviations for the CBS-QB3 composite method and their consequences in DFT benchmark studies.

    PubMed

    Karton, Amir; Goerigk, Lars

    2015-04-05

    Accurate barrier heights are obtained for the 26 pericyclic reactions in the BHPERI dataset by means of the high-level Wn-F12 thermochemical protocols. Very often, the complete basis set (CBS)-type composite methods are used in similar situations, but herein it is shown that they in fact result in surprisingly large errors with root mean square deviations (RMSDs) of about 2.5 kcal mol(-1). In comparison, other composite methods, particularly G4-type and estimated coupled cluster with singles, doubles, and quasiperturbative triple excitations [CCSD(T)/CBS] approaches, show deviations well below the chemical-accuracy threshold of 1 kcal mol(-1). With the exception of SCS-MP2 and the herein newly introduced MP3.5 approach, all other tested Møller-Plesset perturbative procedures give poor performance with RMSDs of up to 8.0 kcal mol(-1). The finding that CBS-type methods fail for barrier heights of these reactions is unexpected and it is particularly troublesome given that they are often used to obtain reference values for benchmark studies. Significant differences are identified in the interpretation and final ranking of density functional theory (DFT) methods when using the original CBS-QB3 rather than the new Wn-F12 reference values for BHPERI. In particular, it is observed that the more accurate Wn-F12 benchmark results in lower statistical errors for those methods that are generally considered to be robust and accurate. Two examples are the PW6B95-D3(BJ) hybrid-meta-general-gradient approximation and the PWPB95-D3(BJ) double-hybrid functionals, which result in the lowest RMSDs of the entire DFT study (1.3 and 1.0 kcal mol(-1), respectively). These results indicate that CBS-QB3 should be applied with caution in computational modeling and benchmark studies involving related systems.

  19. [Expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity analysis].

    PubMed

    Ma, Dong-Mei; Bai, Jun-Jie; Jian, Qing; Lao, Hai-Hua; Ye, Xing; Luo, Jian-Ren

    2003-09-01

    Cystatin, which widely distributed in both tissues and body fluids of animal and plant, was a superfamily of cysteine proteinase inhibitors. It could form activity-inhibitor complexes with cysteine proteinases to inhibit the hydrolytic activity of proteinases. Cystatin played important roles not only in the inhibition of the proteolytic degradation of fish muscle, but also in biological defense systems against invaders. To explore the functions of fish cystatin and the potential values in fish disease prevention and cure, as well as seafood processing, the recombinant yeast strains which could express Chinese sturgeon cystatin were constructed. First, the cystatin cDNA of Chinese sturgeon, which had been PCR modified, was subcloned into yeast integrated vector pPICZaA. After extracted and purified, the recombinant plasmids were linearized by Sac I. The yeast Pichia pastoris GS115 strain was transformed by use of the Lithium Chloride transformation method, and the recombinant cystatin yeast strains got. After 0.5% methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215mg x L(-1) with the percentage about 73.6%. The recombinant cystatin was purified through Q-Sepharose anion-exchange chromatography, and the purity reached about 94.2%. The inhibitory activity of recombinant cystatin was measured by inhibiting the proteinase activity of papain. The results showed that about 1 microg recombinant cystatin could inhibit the activity of 15 microg papain. Heat stability assay results showed that there was a decrease in inhibitory activity of cystatin with the increasing of temperature. When solution of recombinant cystatin was kept at 70 degrees C for 5min, the inhibitory activity reduced fast. While the recombinant cystatin was heated to 90 degrees C for 5min, the inhibitory activity of recombinant cystatin was undetected. The inhibitory activity for recombinant Chinese sturgeon cystatin was higher

  20. Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency.

    PubMed

    Jaouadi, Bassem; Ellouz-Chaabouni, Semia; Rhimi, Moez; Bejar, Samir

    2008-09-01

    We have described previously the potential use of an alkaline protease from Bacillus pumilus CBS as an effective additive in laundry detergent formulations [B. Jaouadi, S. Ellouz-Chaabouni, M. Ben Ali, E. Ben Messaoud, B. Naili, A. Dhouib, S. Bejar, A novel alkaline protease from Bacillus pumilus CBS having a high compatibility with laundry detergent and a high feather-degrading activity, Process Biochem, submitted for publication]. Here, we purified this enzyme (named SAPB) and we cloned, sequenced and over-expressed the corresponding gene. The enzyme was purified to homogeneity using salt precipitation and gel filtration HPLC. The pure protease was found to be monomeric protein with a molecular mass of 34598.19Da as determined by MALDI-TOF mass spectrometry. The NH2-terminal sequence of first 21 amino acids (aa) of the purified SAPB was AQTVPYGIPQIKAPAVHAQGY and was completely identical to proteases from other Bacillus pumilus species. This protease is strongly inhibited by PMSF and DFP, showing that it belongs to the serine proteases superfamily. Interestingly, the optimum pH is 10.6 while the optimum temperature was determined to be 65 degrees C. The enzyme was completely stable within a wide range of pH (7.0-10.6) and temperature (30-55 degrees C). One of the distinguishing properties is its catalytic efficiency (kcat/Km) calculated to be 45,265min(-1)mM(-1) and 147,000min(-1)mM(-1) using casein and AAPF as substrates, respectively, which is higher than that of Subtilisin Carlsberg, Subtilisin BPN' and Subtilisin 309 determined under the same conditions. In addition, SAPB showed remarkable stability, for 24h at 40 degrees C, in the presence of 5% Tween-80, 1% SDS, 15% urea and 10% H2O2, which comprise the common bleach-based detergent formulation. The sapB gene encoding SAPB was cloned, sequenced and over-expressed in Escherichia coli. The purified recombinant enzyme (rSAPB) has the same physicochemical and kinetic properties as the native one. SapB gene had

  1. Calcitriol prevents peripheral RSC96 Schwann neural cells from high glucose & methylglyoxal-induced injury through restoration of CBS/H2S expression.

    PubMed

    Zhang, Hui; Zhuang, Xiao-dong; Meng, Fu-hui; Chen, Li; Dong, Xiao-bian; Liu, Guo-Hui; Li, Jian-hua; Dong, Qi; Xu, Ji-de; Yang, Chun-tao

    2016-01-01

    A meta-analysis has suggested that vitamin D deficiency is involved in diabetic peripheral neuropathy (DPN) and the levels of hydrogen sulfide (H2S) are also decreased in type 2 diabetes. The injection of vitamin D induces cystathionine-β-synthase (CBS) expression and H2S generation. However, it remains unclear whether the supplementation of vitamin D prevents DPN through improvement of CBS/H2S expression. In the present study, RSC96 cells, a rat Schwann cell line, were exposed to high glucose and methylglyoxal (HG&MG) to simulate diabetic peripheral nerve injury in vivo. Before the exposure to HG&MG, the cells were preconditioned with calcitriol (CCT), an active form of vitamin D, and then CCT-mediated neuroprotection was investigated in respect of cellular viability, superoxide anion (O2(-)) generation, inducible nitric oxide (NO) synthase (iNOS)/NO expression, mitochondrial membrane potential (MMP), as well as CBS expression and activity. It was found that both high glucose and MGO decreased cell viability and co-treatment with the two induced a more serious injury in RSC96 cells. Therefore, the exposure to HG&MG was used in the present study. The exposure to HG&MG markedly induced iNOS expression, NO and O2(-) generation, as well as MMP loss. In addition, the exposure to HG&MG depressed CBS expression and activity in RSC96 cells. However, the preconditioning with CCT significantly antagonized HG&MG-induced cell injury including the decreased viability, iNOS overexpression, NO and O2(-) accumulation, as well as MMP loss. CCT also partially restored the decreased CBS expression and activity triggered by HG&MG, while the inhibition of CBS with hydroxylamine attenuated CCT-mediated neuroprotection. Moreover, the exogenous donation of H2S produced similar cellular protective effects to CCT. The data indicate that the supplementation of vitamin D prevents HG&MG-induced peripheral nerve injury involving the restoration of endogenous H2S system, which may provide a

  2. Comparative Production and Characterization of a Catalytically Inactive BoNT/C1 Holoprotein in Two Production Strains of Pichia Pastoris

    DTIC Science & Technology

    2008-01-01

    phosphoribosylaminoimidazole carboxylase gene AOX1 alcohol oxidase 1 gene AEBSF 4-(2-Aminomethyl)benzenesulfonyl fluoride hydrochloride BabyBIG botulism...PichiaPink A synthetic ciBoNT/C1 HP open reading frame (ORF) containing a P. pastoris alcohol oxidase 1 (AOX1) gene codon bias was manufactured by DNA...COMPARATIVE PRODUCTION AND CHARACTERIZATION OF A CATALYTICALLY INACTIVE BoNT/C1 HOLOPROTEIN IN TWO PRODUCTION STRAINS OF PICHIA PASTORIS by

  3. Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system.

    PubMed

    Rabert, Claudia; Weinacker, Daniel; Pessoa, Adalberto; Farías, Jorge G

    2013-01-01

    The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed.

  4. Optimization of a glycoengineered Pichia pastoris cultivation process for commercial antibody production.

    PubMed

    Ye, Jianxin; Ly, Jeffrey; Watts, Kathryn; Hsu, Amy; Walker, Andre; McLaughlin, Kathleen; Berdichevsky, Marina; Prinz, Bianka; Sean Kersey, D; d'Anjou, Marc; Pollard, David; Potgieter, Thomas

    2011-01-01

    Glycoengineering enabled the production of proteins with human N-linked glycans by Pichia pastoris. This study used a glycoengineered P. pastoris strain which is capable of producing humanized glycoprotein with terminal galactose for monoclonal antibody production. A design of experiments approach was used to optimize the process parameters. Followed by further optimization of the specific methanol feed rate, induction duration, and the initial induction biomass, the resulting process yielded up to 1.6 g/L of monoclonal antibody. This process was also scaled-up to 1,200-L scale, and the process profiles, productivity, and product quality were comparable with 30-L scale. The successful scale-up demonstrated that this glycoengineered P. pastoris fermentation process is a robust and commercially viable process.

  5. Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris

    PubMed Central

    Clark, Lindsay; Zahm, Jacob A.; Ali, Rustam; Kukula, Maciej; Bian, Liangqiao; Patrie, Steven M.; Gardner, Kevin H.; Rosen, Michael K.; Rosenbaum, Daniel M.

    2015-01-01

    13C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific 13C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient 13C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets. PMID:26025061

  6. Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris.

    PubMed

    Shah, Kartik A; Clark, John J; Goods, Brittany A; Politano, Timothy J; Mozdzierz, Nicholas J; Zimnisky, Ross M; Leeson, Rachel L; Love, J Christopher; Love, Kerry R

    2015-12-01

    Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria, or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, "hit-to-lead identification" remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigen-specific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV).

  7. [Expression Of DNA-Encoded Antidote to Organophosphorus Toxins in the Methylotrophic Yeast Pichia Pastoris].

    PubMed

    Terekhov, S S; Bobik, T V; Mokrushina, Yu A; Stepanova, A V; Aleksandrova, N M; Smirnov, I V; Belogurov, A A; Ponomarenko, N A; Gabibov, A G

    2016-01-01

    A platform for the cloning and expression of active human butyrylcholinesterase (BuChE) in the yeast Pichia pastoris is first presented. Genetic constructs for BuChE gene expression, separately and in conjunction with a proline-rich peptide called proline-rich attachment domain (PRAD), are based on the vector pPICZαA. It is shown that the highest level of production is achieved in the expression of a BuChE gene without PRAD pPICZαA. It is found that one can obtain up to 125 mg of active enzyme from 1 L of culture medium at an optimal pH environment (pH 7.6), an optical seed culture density of 3 o.u., and an optimum methanol addition mode of (0.5% methanol in the first day and 0.2% thereafter from the second day).

  8. Production in Pichia pastoris of protein-based polymers with small heterodimer-forming blocks.

    PubMed

    Domeradzka, Natalia E; Werten, Marc W T; de Vries, Renko; de Wolf, Frits A

    2016-05-01

    Some combinations of leucine zipper peptides are capable of forming α-helical heterodimeric coiled coils with very high affinity. These can be used as physical cross-linkers in the design of protein-based polymers that form supramolecular structures, for example hydrogels, upon mixing solutions containing the complementary blocks. Such two-component physical networks are of interest for many applications in biomedicine, pharmaceutics, and diagnostics. This article describes the efficient secretory production of A and B type leucine zipper peptides fused to protein-based polymers in Pichia pastoris. By adjusting the fermentation conditions, we were able to significantly reduce undesirable proteolytic degradation. The formation of A-B heterodimers in mixtures of the purified products was confirmed by size exclusion chromatography. Our results demonstrate that protein-based polymers incorporating functional heterodimer-forming blocks can be produced with P. pastoris in sufficient quantities for use in future supramolecular self-assembly studies and in various applications.

  9. Expression and purification of a recombinant avidin with a lowered isoelectric point in Pichia pastoris.

    PubMed

    Zocchi, Andrea; Jobé, Anna Marya; Neuhaus, Jean-Marc; Ward, Thomas R

    2003-12-01

    A recombinant glycosylated avidin (recGAvi) with an acidic isoelectric point was expressed and secreted by the methylotrophic yeast Pichia pastoris. The coding sequence for recGAvi was de novo synthesized based on the codon usage of P. pastoris. RecGAvi is secreted at approximately 330mg/L of culture supernatant. RecGAvi monomer displays a molecular weight of 16.5kDa, as assessed by ESI mass spectrometry. N-terminal amino acid sequencing indicates the presence of three additional amino acids (E-A-E), which contribute to further lowering the isoelectric point to 5.4. The data presented here demonstrate that the P. pastoris system is suitable for the production of recGAvi and that the recombinant avidin displays biotin-binding properties similar to those of the hen-egg white protein.

  10. Sterol glucosyltransferases have different functional roles in Pichia pastoris and Yarrowia lipolytica.

    PubMed

    Stasyk, Oleh V; Nazarko, Taras Y; Stasyk, Olena G; Krasovska, Olena S; Warnecke, Dirk; Nicaud, Jean-Marc; Cregg, James M; Sibirny, Andrei A

    2003-01-01

    Mutants of the methanol-utilizing yeast Pichia pastoris and the alkane-utilizing yeast Yarrowia lipolytica defective in the orthologue of UGT51 (encoding sterol glucosyltransferase) were isolated and compared. These mutants do not contain the specific ergosterol derivate, ergosterol glucoside. We observed that the P. pastoris UGT51 gene is required for pexophagy, the process by which peroxisomes containing methanol-metabolizing enzymes are selectively shipped to and degraded in the vacuole upon shifting methanol-grown cells of this yeast to glucose or ethanol. PpUGT51 is also required for other vacuole related processes. In contrast, the Y. lipolytica UGT51 gene is required for utilization of decane, but not for pexophagy. Thus, sterol glucosyltransferases play different functional roles in P. pastoris and Y. lipolytica.

  11. Physiological Functionality and Enzyme Activity of Biomass from Pichia anomala Grown on Ginseng-Steaming Effluent

    PubMed Central

    Kim, Na-Mi; So, Seung-Ho; Lee, Sung-Gae; Song, Jung-Eun; Seo, Dong-Soo

    2008-01-01

    A novel biomass was prepared from Pichia anomala KCCM 11473, which grew well in ginseng-steaming effluent (GSE), and its physiological functionalities and enzyme activities were determined. When the strain was cultured in the GSE (pH 6.0) at 30℃ for 48 h, 1.6 mg of biomass per ml-cultures was produced. The cell-free extract of the biomass showed high antihypertensive angiotensin I-converting enzyme inhibitory activity of 72.0% and anticholesteromia HMG-CoA reductase inhibitory activity of 46.5%. The cell-free extract also showed 13.0 U per ml and 8.5 U per ml of neutral protease activity and alkaline protease, respectively. PMID:23997615

  12. Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system

    PubMed Central

    Rabert, Claudia; Weinacker, Daniel; Pessoa, Adalberto; Farías, Jorge G.

    2013-01-01

    The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed. PMID:24294221

  13. Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris.

    PubMed

    Mansur, Manuel; Cabello, Cecilia; Hernández, Lester; País, José; Varas, Laura; Valdés, Jorge; Terrero, Yanet; Hidalgo, Abdel; Plana, Liuba; Besada, Vladimir; García, Liudys; Lamazares, Emilio; Castellanos, Lila; Martínez, Eduardo

    2005-03-01

    We have found a direct relationship between protein production in Pichia pastoris and the number of introduced synthetic genes of miniproinsulin (MPI), fused to the Saccharomyces cerevisiae pre-pro alpha factor used as secretion signal, and inserted between the alcohol oxidase 1 (AOX1) promoter and terminator sequences. Two consecutive approaches were followed to increase the number of integrated cassettes: the head-to-tail expression cassette multimerization procedure and re-transformation with a dominant selection marker. This increased expression from 19 to 250 mg l(-1) when about 11 copies have been integrated. Further, the correct position of one of the disulphide bridges of the purified molecule was verified by digestion with Glu-C endoprotease, followed by mass spectrometry of the isolated fragments.

  14. Purification of Candida guilliermondii and Pichia ohmeri killer toxin as an active agent against Penicillium expansum.

    PubMed

    Coelho, Alexandre Rodrigo; Tachi, Masahico; Pagnocca, Fernando Carlos; Nobrega, Gisele Maria Andrade; Hoffmann, Fernando Leite; Harada, Ken-Ichi; Hirooka, Elisa Yoko

    2009-01-01

    An antifungal assay with cell-free culture supernatant of Pichia ohmeri 158 and Candida guilliermondii P3 was tested against Penicillium expansum strain #2 at 25 degrees C by measuring hyphal length and percentage conidia germination. C. guilliermondii was more effective against P. expansum conidia germination (58.15% inhibition), while P. ohmeri showed higher inhibition of mycelial growth (66.17%), indicating a probable mechanism associated with killer activity. This killer toxin (molecular mass <3 kDa) was partially purified by normal phase HPLC, using TSKgel Amide-80 analytical and preparative columns. Compared with crude extract, the killer toxin eluted from the post analytical column significantly inhibited P. expansum:% inhibition rose from 42.16 to 90.93% (C. guilliermondii) and 39.32 to 91.12% (P. ohmeri) (p < 0.05). The one-step purification process was adequate in isolating killer toxin from culture supernatant and also increased anti-Penicillium activity.

  15. Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris.

    PubMed

    Mizutani, Kimihiko; Toyoda, Mayuko; Otake, Yuichiro; Yoshioka, Soshi; Takahashi, Nobuyuki; Mikami, Bunzo

    2012-08-01

    The medaka fish α-amylase was expressed and purified. The expression systems were constructed using methylotrophic yeast Pichia pastoris, and the recombinant proteins were secreted into the culture medium. Purified recombinant α-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and K(M) and V(max) values were determined; chloride ions were essential for enzyme activity. The purified protein was also crystallized and examined by X-ray crystallography. The structure has the (α/β)(8) barrel fold, as do other known α-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) α-amylases. A novel expression plasmid was developed. Using this plasmid, high-throughput construction of an expression system by homologous recombination in P. pastoris cells, previously reported for membrane proteins, was successfully applied to the secretory protein.

  16. Expression, Purification, and Characteristic of Tibetan Sheep Breast Lysozyme Using Pichia pastoris Expression System

    PubMed Central

    Li, Jianbo; Jiang, Mingfeng; Wang, Yong

    2014-01-01

    A lysozyme gene from breast of Tibetan sheep was successfully expressed by secretion using a-factor signal sequence in the methylotrophic yeast, Pichia pastoris GS115. An expression yield and specific activity greater than 500 mg/L and 4,000 U/mg was obtained. Results at optimal pH and temperature showed recombinant lysozyme has higher lytic activity at pH 6.5 and 45°C. This study demonstrates the successful expression of recombinant lysozyme using the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, this study shows the feasibility of subsequent industrial manufacture of the enzyme with this expression system together with a high purity scheme for easy high-yield purification. PMID:25049990

  17. 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.

  18. 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.

  19. High-throughput recombinant gene expression systems in Pichia pastoris using newly developed plasmid vectors.

    PubMed

    Sasagawa, Takahiro; Matsui, Makoto; Kobayashi, Yuki; Otagiri, Masato; Moriya, Shigeharu; Sakamoto, Yasuharu; Ito, Yukishige; Lee, Charles C; Kitamoto, Katsuhiko; Arioka, Manabu

    2011-01-01

    We describe here the construction of Gateway-compatible vectors, pBGP1-DEST and pPICZα-DEST, for rapid and convenient preparation of expression plasmids for production of secretory proteins in Pichia pastoris. Both vectors direct the synthesis of fusion proteins consisting of the N-terminal signal and pro-sequences of Saccharomyces cerevisiae α-factor, the recognition sites for Kex2 and Ste13 processing proteases, the mature region of a foreign protein flanked by attB1- and attB2-derived sequences at N- and C-termini, respectively, and myc plus hexahistidine tags added at the extreme C-terminus. To test the usefulness of these vectors, production of endo-glucanases and xylanases from termite symbionts, as well as a fungal glucuronoyl esterase, was performed. Enzyme activities were detected in the culture supernatants, indicating that the chimeric proteins were synthesized and secreted as designed.

  20. An episomal expression vector for screening mutant gene libraries in Pichia pastoris.

    PubMed

    Lee, Charles C; Williams, Tina G; Wong, Dominic W S; Robertson, George H

    2005-07-01

    Screening mutant gene libraries for isolating improved enzyme variants is a powerful technique that benefits from effective and reliable biological expression systems. Pichia pastoris is a very useful organism to express proteins that are inactive in other hosts such as Escherichia coli and Saccharomyces cerevisiae. However, most P. pastoris expression plasmids are designed to integrate into the host chromosome and hence are not as amenable to high-throughput screening projects. We have designed a P. pastoris expression vector, pBGP1, incorporating an autonomous replication sequence that allows the plasmid to exist as an episomal element. This vector contains the alpha-factor signal sequence to direct secretion of the mutant enzymes. Expression of the genes is driven by the constitutive GAP promoter, thus eliminating the need for timed or cell density-specific inductions. The pBGP1 plasmid was used to screen a xylanase gene library to isolate higher activity mutants.

  1. Metabolomics sampling of Pichia pastoris revisited: rapid filtration prevents metabolite loss during quenching.

    PubMed

    Russmayer, Hannes; Troyer, Christina; Neubauer, Stefan; Steiger, Matthias G; Gasser, Brigitte; Hann, Stephan; Koellensperger, Gunda; Sauer, Michael; Mattanovich, Diethard

    2015-09-01

    Metabolomics can be defined as the quantitative assessment of a large number of metabolites of a biological system. A prerequisite for the accurate determination of intracellular metabolite concentrations is a reliable and reproducible sample preparation method, which needs to be optimized for each organism individually. Here, we compare the performance of rapid filtration and centrifugation after quenching of Pichia pastoris cells in cold methanol. During incubation in the quenching solution, metabolites are lost from the cells with a half-life of 70-180 min. Metabolites with lower molecular weights showed lower half-lifes compared to metabolites with higher molecular weight. Rapid filtration within 2 min after quenching leads to only minor losses below 2%, and is thus the preferred method for cell separation.

  2. Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in Pichia pastoris.

    PubMed

    Ruth, C; Zuellig, T; Mellitzer, A; Weis, R; Looser, V; Kovar, K; Glieder, A

    2010-09-01

    Natural tools for recombinant protein production show technological limitations. Available natural promoters for gene expression in Pichia pastoris are either constitutive, weak or require the use of undesirable substances or procedures for induction. Here we show the application of deletion variants based on the well known methanol inducible AOX1 promoter and small synthetic promoters, where cis-acting elements were fused to core promoter fragments. They enable differently regulated target protein expression and at the same time to replace methanol induction by a glucose or glycerol feeding strategy. Trypsinogen, the precursor of the serine protease trypsin, was expressed using these different promoters. Depending on the applied promoter the production window (i.e. the time of increasing product concentration) changed significantly. In fedbatch processes trypsinogen yields before induction with methanol were up to 10 times higher if variants of the AOX1 promoter were applied. In addition, the starting point of autoproteolytic product degradation can be predetermined by the promoter choice.

  3. Systematic single-cell analysis of Pichia pastoris reveals secretory capacity limits productivity.

    PubMed

    Love, Kerry Routenberg; Politano, Timothy J; Panagiotou, Vasiliki; Jiang, Bo; Stadheim, Terrance A; Love, J Christopher

    2012-01-01

    Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity.

  4. Inulinase production by a marine yeast Pichia guilliermondii and inulin hydrolysis by the crude inulinase.

    PubMed

    Gong, Fang; Sheng, Jun; Chi, Zhenming; Li, Jing

    2007-03-01

    Marine yeast strain 1, isolated from the surface of a marine alga, was found to secrete a large amount of inulinase into the medium. This marine yeast was identified as a strain of Pichia guilliermondii according to the results of routine yeast identification and molecular methods. The crude inulinase produced by this marine yeast worked optimally at pH 6.0 and 60 degrees C. The optimal medium for inulinase production was seawater containing 4.0% (w/v) inulin and 0.5% (w/v) yeast extract, while the optimal cultivation conditions for inulinase production were pH 8.0, 28 degrees C and 170 rpm. Under the optimal conditions, over 60 U ml(-1) of inulinase activity was produced within 48 h of fermentation in shake flasks. A large amount of monosaccharides and a trace amount of oligosaccharides were detected after the hydrolysis, indicating that the crude inulinase had a high exoinulinase activity.

  5. 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.

  6. Expression of a Deschampsia antarctica Desv. Polypeptide with Lipase Activity in a Pichia pastoris Vector

    PubMed Central

    Rabert, Claudia; Gutiérrez-Moraga, Ana; Navarrete-Gallegos, Alejandro; Navarrete-Campos, Darío; Bravo, León A.; Gidekel, Manuel

    2014-01-01

    The current study isolated and characterized the Lip3F9 polypeptide sequence of Deschampsia antarctica Desv. (GeneBank Accession Number JX846628), which was found to be comprised of 291 base pairs and was, moreover, expressed in Pichia pastoris X-33 cells. The enzyme was secreted after 24 h of P. pastoris culture incubation and through induction with methanol. The expressed protein showed maximum lipase activity (35 U/L) with an optimal temperature of 37 °C. The lipase-expressed enzyme lost 50% of its specific activity at 25 °C, a behavior characteristic of a psychrotolerant enzyme. Recombinant enzyme activity was measured in the presence of ionic and non-ionic detergents, and a decrease in enzyme activity was detected for all concentrations of ionic and non-ionic detergents assessed. PMID:24514564

  7. Bioproduction of benzaldehyde in a solid-liquid two-phase partitioning bioreactor using Pichia pastoris.

    PubMed

    Jain, Ashu N; Khan, Tanya R; Daugulis, Andrew J

    2010-11-01

    The bioproduction of benzaldehyde from benzyl alcohol using Pichia pastoris was examined in a solid-liquid two-phase partitioning bioreactor (TPPB) to reduce substrate and product inhibition. Rational polymer selection identified Elvax 40W as an effective sequestering phase, possessing partition coefficients for benzyl alcohol and benzaldehyde of 3.5 and 35.4, respectively. The use of Elvax 40W increased the overall mass of benzaldehyde produced by approx. 300% in a 5 l bioreactor, relative to a single phase biotransformation. The two-phase system had a molar yield of 0.99, indicating that only minor losses occurred. These results provide a promising starting point for solid-liquid TPPBs to enhance benzaldehyde production, and suggest that multiple, targeted polymers may provide relief for transformations characterized by multiple inhibitory substrates/product/by-products.

  8. Expression of a Deschampsia antarctica Desv. polypeptide with lipase activity in a Pichia pastoris vector.

    PubMed

    Rabert, Claudia; Gutiérrez-Moraga, Ana; Navarrete, Alejandro; Navarrete-Campos, Darío; Bravo, León; Gidekel, Manuel

    2014-02-07

    The current study isolated and characterized the Lip3F9 polypeptide sequence of Deschampsia antarctica Desv. (GeneBank Accession Number JX846628), which was found to be comprised of 291 base pairs and was, moreover, expressed in Pichia pastoris X-33 cells. The enzyme was secreted after 24 h of P. pastoris culture incubation and through induction with methanol. The expressed protein showed maximum lipase activity (35 U/L) with an optimal temperature of 37 °C. The lipase-expressed enzyme lost 50% of its specific activity at 25 °C, a behavior characteristic of a psychrotolerant enzyme. Recombinant enzyme activity was measured in the presence of ionic and non-ionic detergents, and a decrease in enzyme activity was detected for all concentrations of ionic and non-ionic detergents assessed.

  9. Bioremediation of Parboiled Rice Effluent Supplemented with Biodiesel-Derived Glycerol Using Pichia pastoris X-33

    PubMed Central

    Gil de los Santos, Diego; Gil Turnes, Carlos; Rochedo Conceição, Fabricio

    2012-01-01

    This paper describes the use of Pichia pastoris X-33 as a bioremediator to reduce the chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN), and phosphorus (P-PO4   3−), after culture in parboiled rice effluent supplemented with p.a. glycerol or a glycerol by-product of the biodiesel industry. The greatest reduction in the COD (55%), TKN (45%), and P-PO4   3− (52%) of the effluent was observed in cultures of P. pastoris X-33 supplemented with 15 g ·L−1 of biodiesel-derived glycerol. Furthermore, the overall biomass yield was 2.1 g ·L−1. These data suggest that biodiesel-derived glycerol is an efficient carbon source for the bioremediation of parboiled rice effluent and biomass production. PMID:22919327

  10. 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.

  11. [Synthesis of diisooctyl adipate catalyzed by lipase-displaying Pichia pastoris whole-cell biocatalysts].

    PubMed

    Zhang, Na; Jin, Zi; Lin, Ying; Zheng, Suiping; Han, Shuangyan

    2013-07-01

    An enzyme-displaying yeast as a whole-cell biocatalyst is an alternative to immobilized enzyme, due to its low-cost preparation and simple recycle course. Here, lipase-displaying Pichia pastoris whole-cell was used as a biocatalyst to synthesize diisooctyl adipate in the non-aqueous system. The maximum productivity of diisooctyl adipate was obtained as 85.0% in a 10 mL reaction system. The yield could be reached as high as 97.8% when the reaction system was scaled up to 200 mL. The purity obtained is 98.2% after vacuum distillation. Thus, the lipase-displaying P. pastoris whole-cell biocatalyst was promising in commercial application for diisooctyl adipate synthesis in non-aqueous phase.

  12. Characterization of the oligosaccharides assembled on the Pichia pastoris-expressed recombinant aspartic protease.

    PubMed

    Montesino, R; Nimtz, M; Quintero, O; García, R; Falcón, V; Cremata, J A

    1999-10-01

    Aspartic protease, widely used as a milk-coagulating agent in industrial cheese production, contains three potential N-glycosylation sites. In this study, we report the characterization of N-linked oligosaccharides on recombinant aspartic protease secreted from the methylotrophic yeast Pichia pastoris using a combination of mass spectrometric, 2D chromatographic, chemical and enzymatic methods. The carbohydrates from site I (Asn79) were found to range from Man6-17GlcNAc2 with 50% bearing a phospho-diester-motif, site II (Asn113) was not occupied and site III (Asn188) contained mostly uncharged species ranging from Man-13GlcNAc2. These charged groups are not affecting the transport through the secretion pathway of the recombinant glycoprotein. Changes from a molasses-based medium to a minimal salts-based medium led to a clear reduction of the degree of phosphorylation of the N-glycan population.

  13. Pichia kudriavzevii as a representative yeast of North Patagonian winemaking terroir.

    PubMed

    Del Mónaco, Silvana M; Rodríguez, María E; Lopes, Christian A

    2016-08-02

    Terroir concept includes specific soil, topography, climate, landscape characteristics and biodiversity features. In reference to the last aspect, recent studies investigating the microbial biogeography (lately called 'microbial terroir') have revealed that different wine-growing regions maintain different microbial communities. The aim of the present work was to identify potential autochthonous fermentative yeasts isolated from native plants in North Patagonia, Schinus johnstonii, Ephedra ochreata and Lycium chilense, that could be associated to the specific vitivinicultural terroir of this region. Different Pichia kudriavzevii isolates were recovered from these plants and physiologically and genetically compared to regional wine isolates and foreign reference strains of the same species. All isolates were subjected to molecular characterization including mtDNA-RFLP, RAPD-PCR and sequence analysis. Both wine and native P. kudriavzevii isolates from Patagonia showed similar features, different from those showed by foreign strains, suggesting that this species could be part of a specific regional terroir from North Patagonia.

  14. Improving 3'-Hydroxygenistein Production in Recombinant Pichia pastoris Using Periodic Hydrogen Peroxide-Shocking Strategy.

    PubMed

    Wang, Tzi-Yuan; Tsai, Yi-Hsuan; Yu, I-Zen; Chang, Te-Sheng

    2016-03-01

    3'-Hydroxygenistein can be obtained from the biotransformation of genistein by the engineered Pichia pastoris X-33 strain, which harbors a fusion gene composed of CYP57B3 from Aspergillus oryzae and a cytochrome P450 oxidoreductase gene (sCPR) from Saccharomyces cerevisiae. P. pastoris X-33 mutants with higher 3'-hydroxygenistein production were selected using a periodic hydrogen peroxide-shocking strategy. One mutant (P2-D14-5) produced 23.0 mg/l of 3'-hydroxygenistein, representing 1.87-fold more than that produced by the recombinant X-33. When using a 5 L fermenter, the P2-D14-5 mutant produced 20.3 mg/l of 3'- hydroxygenistein, indicating a high potential for industrial-scale 3'-hydroxygenistein production.

  15. Different mosquito species host Wickerhamomyces anomalus (Pichia anomala): perspectives on vector-borne diseases symbiotic control.

    PubMed

    Ricci, Irene; Mosca, Michela; Valzano, Matteo; Damiani, Claudia; Scuppa, Patrizia; Rossi, Paolo; Crotti, Elena; Cappelli, Alessia; Ulissi, Ulisse; Capone, Aida; Esposito, Fulvio; Alma, Alberto; Mandrioli, Mauro; Sacchi, Luciano; Bandi, Claudio; Daffonchio, Daniele; Favia, Guido

    2011-01-01

    The genetic manipulation of the microbial community associated with hematophagus insects is particularly relevant for public health applications. Within mosquito populations, this relationship has been overlooked until recently. New advances in molecular biotechnology propose the genetic manipulation of mosquito symbionts to prevent the transmission of pathogens to humans by interfering with the obligatory life cycle stages within the insect through the use of effector molecules. This approach, defined as 'paratransgenesis', has opened the way for the investigation and characterization of microbes residing in the mosquito body, particularly those localised within the gut. Some interesting bacteria have been identified as candidates for genetic modification, however, endosymbiotic yeasts remain largely unexplored with little information on the symbiotic relationships to date. Here we review the recent report of symbiotic relationship between Wickerhamomyces anomalus (Pichia anomala) and several mosquito vector species as promising methods to implement control of mosquito-borne diseases.

  16. Synthesis and high expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris GS115.

    PubMed

    Kang, Lixin; Chen, Xiaomei; Zhai, Chao; Ma, Lixin

    2012-09-01

    A gene, ClCDA, encoding chitin deacetylase from Colletotrichum lindemuthianum, was optimized according to the codon usage bias of Pichia pastoris and synthesized in vitro by overlap extension PCR. It was secretorily expressed in P. pastoris GS115 using the constitutive expression vector pHMB905A. The expression level reached the highest with 110 mg/l culture supernatant after 72 h of methanol induction, which comprised 77.27 U/mg chitin deacetylase activity. SDS-PAGE, mass spectrometry, and deglycosylation assays demonstrated that partial recombinant protein was glycosylated with an apparent molecular mass of 33 kDa. The amino acid sequences of recombinant proteins were confirmed by mass spectrometry.

  17. [Heterologous expression and characterization of Yarrowia lipolytica lipase 4 and lipase 5 in Pichia pastoris].

    PubMed

    Zhao, Heyun; Xiao, Xiao; Xu, Li; Liu, Yun; Yan, Yunjun

    2011-10-01

    To clone cDNA sequences of lipase 4 (LIP4) and lipase 5 (LIPS), analyze gene structures and express them in Pichia pastoris so as to investigate their enzymatic characteristics. We first cloned cDNA sequences of LIP4 and LIP5 by reverse transcription PCR and analyzed their gene structures by SignalP 3.0. Then, intracellular expression vectors pPIC3. 5K-Lip4, pPIC3. 5K-Lip5 and inducible secretion vectors pPIC9K-Lip4, pPIC9K-Lip5 were constructed. All vectors were transformed into Pichia pastoris GS115 by electroporation, resulting in a series of engineered strains. After fermentation and NTA-Ni resin purification, the enzymatic properties of LIP4 and LIP5 were examined. The cloned cDNA sequences revealed that there was no intron in both of Lip4 and Lip5. The two lipases both contained catalytic triads and conserved GHSLG motifs. Their optimal substrate, pH, temperature were respectively pNP-caprylate (C8), 7.0 and 40 degrees C. The activities of LIP4 and LIPS were 10.16 U/mg and 5.1 U/mg, respectively. It was found that LIP4 was more sensitive to the variations of pH and temperature than LIP5. LIP4 and LIP5 could both be stimulated by Ca2+, besides LIPS could also be activated by Mg2+. They were both strongly inhibited by Hg2+, Phenylmethanesulfonyl fluoride (PMSF) and Dithiothreitol (DTT). The cloning of Lip4 and Lip5, expression in P. pastoris and characterization of their properties would offer a solid basis for their large-scale production and future application. In addition, the results also enriched the data for a systematic research on the lipase gene family of Y. lipolytica.

  18. Metabolic engineering of Pichia pastoris for the production of dammarenediol-II.

    PubMed

    Liu, Xin-Bin; Liu, Min; Tao, Xin-Yi; Zhang, Zhong-Xi; Wang, Feng-Qing; Wei, Dong-Zhi

    2015-12-20

    Dammarenediol-II is the nucleus of dammarane-type ginsenosides, which are a group of active triterpenoids exhibiting various pharmacological activities. Based on the native triterpene synthetic pathway, a dammarenediol-II synthetic pathway was established in Pichia pastoris by introducing a dammarenediol-II synthase gene (PgDDS) from Panax ginseng, which is responsible for the cyclization of 2,3-oxidosqualene to dammarenediol-II in this study. To enhance productivity, a strategy of "increasing supply and reducing competitive consumption of 2,3-oxidosqualene" was used. To increase the supply of 2,3-oxidosqualene, we augmented expression of the ERG1 gene, which is responsible for 2,3-oxidosqualene synthesis. This significantly improved the yield of dammarenediol-II over 6.7-fold, from 0.030mg/g dry cell weight (DCW) to 0.203mg/g DCW. Subsequently, to reduce competition for 2,3-oxidosqualene from ergosterol biosynthesis without affecting the normal growth of P. pastoris, we targeted the ERG7gene, which is responsible for conversion of 2,3-oxidosqualene to lanosterol. This gene was downregulated by replacing its native promoter with a thiamine-repressible promoter, using a marker-recycling and gene-targeting Cre- lox71/66 system developed for P. pastoris herein. The yield of dammarenediol-II was further increased more than 3.6-fold, to 0.736mg/g DCW. Furthermore, the direct addition of 0.5g/L squalene into the culture medium further enhanced the yield of dammarenediol-II to 1.073mg/g DCW, which was 37.5-fold higher than the yield from the strain with the PgDDS gene introduction only. The P. pastoris strains engineered in this study constitute a good platform for further production of ginsenosides in Pichia species.

  19. High yield production of a mutant Nippostrongylus brasiliensis acetylcholinesterase in Pichia pastoris and its purification.

    PubMed

    Richter, Sven; Nieveler, Jens; Schulze, Holger; Bachmann, Till T; Schmid, Rolf D

    2006-04-05

    The mutant M301A of the acetylcholinesterase B from Nippostrongylus brasiliensis (NbAChE) was produced in a high-cell-density fermentation of a recombinant methylotrophic yeast Pichia pastoris. Dissolved oxygen (DO) spikes were used as an indicator for feeding the carbon source. Wet cell weight (WCW) reached after 8 days a maximum value of 316 g/L and the OD600 at this time was 280. The acetylcholinesterase activity increased up to 6,600 U/mL corresponding to an expression rate of 2 g of NbAChE per liter supernatant. The specific activity of the mutant NbAChE was determined after purification as 3,300 U/mg. Active site titration with chlorpyrifos, a strong AChE inhibitor, yielded in a specific activity of 3,400 U/mg. The enzyme was secreted by Pichia pastoris. Therefore, it could be concentrated from culture broth by cross-flow-filtration (50 kDa cut-off membrane). It was further purified in one-step anion-exchange chromatography, using a XK 50/20 column filled with 125 mL Q Sepharose HP. Mutant NbAChE was purified 1.9-fold up to a purity of 97% and a yield of 87%. The isolated enzyme was nearly homogenous, as seen on the silver stained SDS-PAGE as well as by a single peak after gel filtration. This extraordinary high expression rate and the ease of purification is an important prerequisite for their practical application, for example in biosensors for the detection of neurotoxic insecticides.

  20. Production of human pro-relaxin H2 in the yeast Pichia pastoris.

    PubMed

    Cimini, D; Corte, K Della; Finamore, R; Andreozzi, L; Stellavato, A; Pirozzi, A V A; Ferrara, F; Formisano, R; De Rosa, M; Chino, M; Lista, L; Lombardi, A; Pavone, V; Schiraldi, C

    2017-01-14

    Initially known as the reproductive hormone, relaxin was shown to possess other therapeutically useful properties that include extracellular matrix remodeling, anti-inflammatory, anti-ischemic and angiogenic effects. All these findings make relaxin a potential drug for diverse medical applications. Its precursor, pro-relaxin, is an 18 kDa protein, that shows activity in in vitro assays. Since extraction of relaxin from animal tissues raises several issues, prokaryotes and eukaryotes were both used as expression systems for recombinant relaxin production. Most productive results were obtained when using Escherichia coli as a host for human relaxin expression. However, in such host, relaxin precipitated in the form of inclusion bodies and, therefore, required several expensive recovery steps as cell lysis, refolding and reduction. To overcome the issues related to prokaryotic expression here we report the production and purification of secreted human pro-relaxin H2 by using the methylotrophic yeast Pichia pastoris as expression host. The methanol inducible promoter AOX1 was used to drive expression of the native and histidine tagged forms of pro-relaxin H2 in dual phase fed-batch experiments on the 22 L scale. Both protein forms presented the correct structure, as determined by mass spectrometry and western blotting analyses, and demonstrated to be biologically active in immune enzymatic assays. The presence of the tag allowed to simplify pro-relaxin purification obtaining higher purity. This work presents a strategy for microbial production of recombinant human pro-relaxin H2 in Pichia pastoris that allowed the obtainment of biologically active pro-hormone, with a final concentration in the fermentation broth ranging between 10 and 14 mg/L of product, as determined by densitometric analyses.

  1. Cloning and expression of recombinant human platelet-derived growth factor-BB in Pichia Pink.

    PubMed

    Babavalian, H; Latifi, A M; Shokrgozar, M A; Bonakdar, S; Tebyanian, H; Shakeri, F

    2016-07-31

    The PDGF-BB plays a key role in several pathogenesis diseases and it is believed to be an important mediator for wound healing. The recombinant human PDGF-BB is safe and effective to stimulate the healing of chronic, full thickness and lower extremity diabetic neurotrophic ulcers. In the present study, we attempted to produce a PDGF-BB growth factor and also, evaluate its functionality in cell proliferation in yeast host Pichia pink. Pichia pink yeast was used as a host for evaluation of the rhPDGF-BB expression. The coding sequence of PDGF-BB protein was synthesized after optimization and packed into the pGEM. Recombinant proteins were produced and purified. The construct of pPinkα-HC-pdgf was confirmed by sequence, the PDGF-BB protein was expressed and purified with using a nickel affinity chromatography column and then characterized by SDS-PAGE electrophoresis. The biological activity of PDGF-BB was estimated with using human fibroblast cell line. The measurement of protein concentration was determined by Bradford and human PDGF-BB ELISA kit. Purified rhPDGF-BB showed similar biological activity (as the standard PDGF-BB) and suggested that the recombinant protein has a successful protein expression (as well as considerable biological activity in P. pink host). The exact amount of recombinant PDGF-BB concentrations were measured by specific ELISA test which it was about 30 μg/ml. Our study suggested that efficiency of biological activity of PDGF-BB protein may be related to its conformational similarity with standard type and also, it practically may be important in wound healing and tissue regeneration.

  2. Purification and characterization of the first recombinant bird pancreatic lipase expressed in Pichia pastoris: the turkey.

    PubMed

    Bou Ali, Madiha; Ben Ali, Yassine; Karray, Aida; Fendri, Ahmed; Gargouri, Youssef

    2011-01-27

    The turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Some biochemical properties and kinetic studies were determined using emulsified system and monomolecular film techniques. Those studies have shown that despite the accumulation of free fatty acids at the olive oil/water interface, TPL continues to hydrolyse efficiently the olive oil and the TC4 in the absence of colipase and bile salts, contrary to most classical digestive lipases which denaturate rapidly under the same conditions. The aim of the present study was to express TPL in the methylotrophic yeast Pichia pastoris in order to get a large amount of this enzyme exhibiting interesting biochemical properties, to purify and characterize the recombinant enzyme. The recombinant TPL was secreted into the culture medium and the expression level reached about 15 mg/l after 4 days of culture. Using Q-PCR, the number of expression cassette integrated on Pichia genomic DNA was estimated to 5. The purified rTPL, with molecular mass of 50 kDa, has a specific activity of 5300 U/mg on emulsified olive oil and 9500 U/mg on tributyrin. The optimal temperature and pH of rTPL were 37°C and pH 8.5. The stability, reaction kinetics and effects of calcium ions and bile salts were also determined. Our results show that the expressed TPL have the same properties as the native TPL previously purified. This result allows us the use of the recombinant enzyme to investigate the TPL structure-function relationships.

  3. Purification and characterization of the first recombinant bird pancreatic lipase expressed in Pichia pastoris: The turkey

    PubMed Central

    2011-01-01

    Background The turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Some biochemical properties and kinetic studies were determined using emulsified system and monomolecular film techniques. Those studies have shown that despite the accumulation of free fatty acids at the olive oil/water interface, TPL continues to hydrolyse efficiently the olive oil and the TC4 in the absence of colipase and bile salts, contrary to most classical digestive lipases which denaturate rapidly under the same conditions. The aim of the present study was to express TPL in the methylotrophic yeast Pichia pastoris in order to get a large amount of this enzyme exhibiting interesting biochemical properties, to purify and characterize the recombinant enzyme. Results The recombinant TPL was secreted into the culture medium and the expression level reached about 15 mg/l after 4 days of culture. Using Q-PCR, the number of expression cassette integrated on Pichia genomic DNA was estimated to 5. The purified rTPL, with molecular mass of 50 kDa, has a specific activity of 5300 U/mg on emulsified olive oil and 9500 U/mg on tributyrin. The optimal temperature and pH of rTPL were 37°C and pH 8.5. The stability, reaction kinetics and effects of calcium ions and bile salts were also determined. Conclusions Our results show that the expressed TPL have the same properties as the native TPL previously purified. This result allows us the use of the recombinant enzyme to investigate the TPL structure-function relationships. PMID:21272342

  4. Transcriptome of the Alternative Ethanol Production Strain Dekkera bruxellensis CBS 11270 in Sugar Limited, Low Oxygen Cultivation

    PubMed Central

    Tiukova, Ievgeniia A.; Petterson, Mats E.; Tellgren-Roth, Christian; Bunikis, Ignas; Eberhard, Thomas; Pettersson, Olga Vinnere; Passoth, Volkmar

    2013-01-01

    Dekkera bruxellensis can outcompete Saccharomyces cerevisiae in environments with low sugar concentrations. It is usually regarded as a spoilage yeast but has lately been identified as an alternative ethanol production organism. In this study, global gene expression in the industrial isolate D. bruxellensis CBS 11270 under oxygen and glucose limitation was investigated by whole transcriptome sequencing using the AB SOLiD technology. Among other observations, we noted expression of respiratory complex I NADH-ubiquinone reductase although D. bruxellensis is a Crabtree positive yeast. The observed higher expression of NADH-generating enzymes compared to NAD+-generating enzymes might be the reason for the previously observed NADH imbalance and resulting Custer effect in D. bruxellensis. Low expression of genes involved in glycerol production is probably the molecular basis for high efficiency of D. bruxellensis metabolism under nutrient limitation. No D. bruxellensis homologs to the genes involved in the final reactions of glycerol biosynthesis were detected. A high number of expressed sugar transporter genes is consistent with the hypothesis that the competitiveness of D. bruxellensis is due to a higher affinity for the limiting substrate. PMID:23516483

  5. Genome Sequencing of the Pyruvate-producing Strain Candida glabrata CCTCC M202019 and Genomic Comparison with Strain CBS138

    PubMed Central

    Xu, Nan; Ye, Chao; Chen, Xiulai; Liu, Jia; Liu, Liming; Chen, Jian

    2016-01-01

    Candida glabrata CCTCC M202019 as an industrial yeast strain that is widely used to produce α-oxocarboxylic acid. Strain M202019 has been proven to have a higher pyruvate-producing capacity than the reference strain CBS138. To characterize the genotype of the M202019 strain, we generated a draft sequence of its genome, which has a size of 12.1 Mbp and a GC content of 38.47%. Evidence accumulated during genome annotation suggests that strain M202019 has strong capacities for glucose transport and pyruvate biosynthesis, defects in pyruvate catabolism, as well as variations in genes involved in nutrient and dicarboxylic acid transport, oxidative phosphorylation, and other relevant aspects of carbon metabolism, which might promote pyruvate accumulation. In addition to differences in its central carbon metabolism, a genomic analysis revealed genetic differences in adhesion metabolism. Forty-nine adhesin-like proteins of strain M202019 were identified classified into seven subfamilies. Decreased amounts of adhesive proteins, and deletions or changes of low-complexity repeats and functional domains might lead to lower adhesion and reduced pathogenicity. Further virulence experiments validated the biological safety of strain M202019. Analysis of the C. glabrata CCTCC M202019 genome sequence provides useful insights into its genetic context, physical characteristics, and potential metabolic capacity. PMID:27713500

  6. Cold exposure affects carbohydrates and lipid metabolism, and induces Hog1p phosphorylation in Dekkera bruxellensis strain CBS 2499.

    PubMed

    Galafassi, Silvia; Toscano, Marco; Vigentini, Ileana; Zambelli, Paolo; Simonetti, Paolo; Foschino, Roberto; Compagno, Concetta

    2015-05-01

    Dekkera bruxellensis is a yeast known to affect the quality of wine and beer. This species, due to its high ethanol and acid tolerance, has been reported also to compete with Saccharomyces cerevisiae in distilleries producing fuel ethanol. In order to understand how this species responds when exposed to low temperatures, some mechanisms like synthesis and accumulation of intracellular metabolites, changes in lipid composition and activation of the HOG-MAPK pathway were investigated in the genome sequenced strain CBS 2499. We show that cold stress caused intracellular accumulation of glycogen, but did not induce accumulation of trehalose and glycerol. The cellular fatty acid composition changed after the temperature downshift, and a significant increase of palmitoleic acid was observed. RT-PCR analysis revealed that OLE1 encoding for Δ9-fatty acid desaturase was up-regulated, whereas TPS1 and INO1 didn't show changes in their expression. In D. bruxellensis Hog1p was activated by phosphorylation, as described in S. cerevisiae, highlighting a conserved role of the HOG-MAP kinase signaling pathway in cold stress response.

  7. Transcriptome of the alternative ethanol production strain Dekkera bruxellensis CBS 11270 in sugar limited, low oxygen cultivation.

    PubMed

    Tiukova, Ievgeniia A; Petterson, Mats E; Tellgren-Roth, Christian; Bunikis, Ignas; Eberhard, Thomas; Pettersson, Olga Vinnere; Passoth, Volkmar

    2013-01-01

    Dekkera bruxellensis can outcompete Saccharomyces cerevisiae in environments with low sugar concentrations. It is usually regarded as a spoilage yeast but has lately been identified as an alternative ethanol production organism. In this study, global gene expression in the industrial isolate D. bruxellensis CBS 11270 under oxygen and glucose limitation was investigated by whole transcriptome sequencing using the AB SOLiD technology. Among other observations, we noted expression of respiratory complex I NADH-ubiquinone reductase although D. bruxellensis is a Crabtree positive yeast. The observed higher expression of NADH-generating enzymes compared to NAD(+)-generating enzymes might be the reason for the previously observed NADH imbalance and resulting Custer effect in D. bruxellensis. Low expression of genes involved in glycerol production is probably the molecular basis for high efficiency of D. bruxellensis metabolism under nutrient limitation. No D. bruxellensis homologs to the genes involved in the final reactions of glycerol biosynthesis were detected. A high number of expressed sugar transporter genes is consistent with the hypothesis that the competitiveness of D. bruxellensis is due to a higher affinity for the limiting substrate.

  8. Depicting the Discrepancy between Tri Genotype and Chemotype on the Basis of Strain CBS 139514 from a Field Population of F. graminearum Sensu Stricto from Argentina

    PubMed Central

    Kulik, Tomasz; Buśko, Maciej; Bilska, Katarzyna; Ostrowska-Kołodziejczak, Anna; van Diepeningen, Anne D.; Perkowski, Juliusz; Stenglein, Sebastian

    2016-01-01

    Recent studies on a field population of F. graminearum sensu stricto from Argentina revealed an atypical panel of strains identified through PCR genotyping as 15ADON genotypes, but producing high levels of 3ADON. Based on representative strain CBS 139514, we asked if the discrepancy between the trichothecene genotype and chemotype might result from an inter-chemotype recombination of the chemotype-determining genes. To answer this, we sequenced the complete core Tri gene cluster (around 30,200 bp) from this strain and compared its sequence to sequence data of typical type B trichothecene genotypes/chemotypes. Sequence alignment showed that CBS 139514 has an identical sequence within the entire core Tri cluster to the 15ADON genotype. The revealed discrepancy underlines the need for using both molecular and chemical methods for reliable characterization of toxigenic strains of Fusarium. PMID:27845742

  9. TLR4 upregulates CBS expression through NF-κB activation in a rat model of irritable bowel syndrome with chronic visceral hypersensitivity.

    PubMed

    Yuan, Bo; Tang, Wei-Hong; Lu, Li-Juan; Zhou, Yuan; Zhu, Hong-Yan; Zhou, You-Lang; Zhang, Hong-Hong; Hu, Chuang-Ying; Xu, Guang-Yin

    2015-07-28

    To investigate the roles of toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB on cystathionine β synthetase (CBS) expression and visceral hypersensitivity in rats. This study used 1-7-wk-old male Sprague-Dawley rats. Western blot analysis was employed to measure the expression of TLR4, NF-κB and the endogenous hydrogen sulfide-producing enzyme CBS in colon dorsal root ganglia (DRG) from control and "irritable bowel syndrome" rats induced by neonatal colonic inflammation (NCI). Colon-specific DRG neurons were labeled with Dil and acutely dissociated to measure excitability with patch-clamp techniques. Immunofluorescence was employed to determine the co-expression of TLR4, NF-κB and CBS in DiI-labeled DRG neurons. NCI significantly upregulated the expression of TLR4 in colon-related DRGs (0.34 ± 0.12 vs 0.72 ± 0.02 for the control and NCI groups, respectively, P < 0.05). Intrathecal administration of the TLR4-selective inhibitor CLI-095 significantly enhanced the colorectal distention threshold of NCI rats. CLI-095 treatment also markedly reversed the hyperexcitability of colon-specific DRG neurons and reduced the expression of CBS (1.7 ± 0.1 vs 1.1 ± 0.04, P < 0.05) and of the NF-κB subunit p65 (0.8 ± 0.1 vs 0.5 ± 0.1, P < 0.05). Furthermore, the NF-κB-selective inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the upregulation of CBS (1.0 ± 0.1 vs 0.6 ± 0.1, P < 0.05) and attenuated visceral hypersensitivity in the NCI rats. In vitro, incubation of cultured DRG neurons with the TLR4 agonist lipopolysaccharide significantly enhanced the expression of p65 (control vs 8 h: 0.9 ± 0.1 vs 1.3 ± 0.1; control vs 12 h: 0.9 ± 0.1 vs 1.3 ± 0.1, P < 0.05; control vs 24 h: 0.9 ± 0.1 vs 1.6 ± 0.1, P < 0.01) and CBS (control vs 12 h: 1.0 ± 0.1 vs 2.2 ± 0.4; control vs 24 h: 1.0 ± 0.1 vs 2.6 ± 0.1, P < 0.05), whereas the inhibition of p65 via pre-incubation with PDTC significantly reversed the upregulation of CBS expression (1.2 ± 0

  10. Neuroprotective Roles of l-Cysteine in Attenuating Early Brain Injury and Improving Synaptic Density via the CBS/H2S Pathway Following Subarachnoid Hemorrhage in Rats

    PubMed Central

    Li, Tong; Wang, Lingxiao; Hu, Quan; Liu, Song; Bai, Xuemei; Xie, Yunkai; Zhang, Tiantian; Bo, Shishi; Gao, Xiangqian; Wu, Shuhua; Li, Gang; Wang, Zhen

    2017-01-01

    l-Cysteine is a semi-essential amino acid and substrate for cystathionine-β-synthase (CBS) in the central nervous system. We previously reported that NaHS, an H2S donor, significantly alleviated brain damage after subarachnoid hemorrhage (SAH) in rats. However, the potential therapeutic value of l-cysteine and the molecular mechanism supporting these beneficial effects have not been determined. This study was designed to investigate whether l-cysteine could attenuate early brain injury following SAH and improve synaptic function by releasing endogenous H2S. Male Wistar rats were subjected to SAH induced by cisterna magna blood injection, and l-cysteine was intracerebroventricularly administered 30 min after SAH induction. Treatment with l-cysteine stimulated CBS activity in the prefrontal cortex (PFC) and H2S production. Moreover, l-cysteine treatment significantly ameliorated brain edema, improved neurobehavioral function, and attenuated neuronal cell death in the PFC; these effects were associated with a decrease in the Bax/Bcl-2 ratio and the suppression of caspase-3 activation 48 h after SAH. Furthermore, l-cysteine treatment activated the CREB–brain-derived neurotrophic factor (BDNF) pathway and intensified synaptic density by regulating synapse proteins 48 h after SAH. Importantly, all the beneficial effects of l-cysteine in SAH were abrogated by amino-oxyacetic acid, a CBS inhibitor. Based on these findings, l-cysteine may play a neuroprotective role in SAH by inhibiting cell apoptosis, upregulating CREB–BDNF expression, and promoting synaptic structure via the CBS/H2S pathway. PMID:28512446

  11. Neuroprotective Roles of l-Cysteine in Attenuating Early Brain Injury and Improving Synaptic Density via the CBS/H2S Pathway Following Subarachnoid Hemorrhage in Rats.

    PubMed

    Li, Tong; Wang, Lingxiao; Hu, Quan; Liu, Song; Bai, Xuemei; Xie, Yunkai; Zhang, Tiantian; Bo, Shishi; Gao, Xiangqian; Wu, Shuhua; Li, Gang; Wang, Zhen

    2017-01-01

    l-Cysteine is a semi-essential amino acid and substrate for cystathionine-β-synthase (CBS) in the central nervous system. We previously reported that NaHS, an H2S donor, significantly alleviated brain damage after subarachnoid hemorrhage (SAH) in rats. However, the potential therapeutic value of l-cysteine and the molecular mechanism supporting these beneficial effects have not been determined. This study was designed to investigate whether l-cysteine could attenuate early brain injury following SAH and improve synaptic function by releasing endogenous H2S. Male Wistar rats were subjected to SAH induced by cisterna magna blood injection, and l-cysteine was intracerebroventricularly administered 30 min after SAH induction. Treatment with l-cysteine stimulated CBS activity in the prefrontal cortex (PFC) and H2S production. Moreover, l-cysteine treatment significantly ameliorated brain edema, improved neurobehavioral function, and attenuated neuronal cell death in the PFC; these effects were associated with a decrease in the Bax/Bcl-2 ratio and the suppression of caspase-3 activation 48 h after SAH. Furthermore, l-cysteine treatment activated the CREB-brain-derived neurotrophic factor (BDNF) pathway and intensified synaptic density by regulating synapse proteins 48 h after SAH. Importantly, all the beneficial effects of l-cysteine in SAH were abrogated by amino-oxyacetic acid, a CBS inhibitor. Based on these findings, l-cysteine may play a neuroprotective role in SAH by inhibiting cell apoptosis, upregulating CREB-BDNF expression, and promoting synaptic structure via the CBS/H2S pathway.

  12. Assessment of CCSD(T), MP2, DFT-D, CBS-QB3, and G4(MP2) methods for conformational study of alanine and proline dipeptides

    NASA Astrophysics Data System (ADS)

    Kang, Young Kee; Park, Hae Sook

    2014-04-01

    The CCSD(T), MP2, dispersion-corrected DFT, CBS-QB3, and G4(MP2) levels of theory with various basis sets are assessed for their ability to describe the conformational preferences of the Ala and Pro dipeptides. The ωB97X-D/6-311++G(d,p) level of theory provided the rotational constants of the most stable conformer of the Ala dipeptide consistent with the values from microwave experiments. The double-hybrid DSD-PBEP86-D3BJ DFT method provided the best performance for relative energies of both dipeptides consistent with CCSD(T)/CBS limit values. The DSD-PBEP86-D3BJ/cc-pVTZ//M06-2X/6-31+G(d), B2PLYPD-D3BJ/cc-pVTZ//M06-2X/6-31+G(d), and M06-2X/cc-pVTZ//M06-2X/6-31+G(d) levels of theory may be an alternative to the CCSD(T)/CBS limit//ωB97X-D/6-311++G(d,p) level of theory with marginal deviations for conformational study of peptides.

  13. Sex-specific association of sequence variants in CBS and MTRR with risk for promoter hypermethylation in the lung epithelium of smokers.

    PubMed

    Flores, Kristina G; Stidley, Christine A; Mackey, Amanda J; Picchi, Maria A; Stabler, Sally P; Siegfried, Jill M; Byers, Tim; Berwick, Marianne; Belinsky, Steven A; Leng, Shuguang

    2012-08-01

    Gene promoter hypermethylation is now regarded as a promising biomarker for the risk and progression of lung cancer. The one-carbon metabolism pathway is postulated to affect deoxyribonucleic acid (DNA) methylation because it is responsible for the generation of S-adenosylmethionine (SAM), the methyl donor for cellular methylation reactions. This study investigated the association of single nucleotide polymorphisms (SNPs) in six one-carbon metabolism-related genes with promoter hypermethylation in sputum DNA from non-Hispanic white smokers in the Lovelace Smokers Cohort (LSC) (n = 907). Logistic regression was used to assess the association of SNPs with hypermethylation using a high/low methylation cutoff. SNPs in the cystathionine beta synthase (CBS) and 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) genes were significantly associated with high methylation in males [CBS rs2850146 (-8283G > C), OR = 4.9; 95% CI: 1.98, 12.2, P = 0.0006] and low methylation in females [MTRR rs3776467 (7068A > G), OR = 0.57, 95% CI: 0.42, 0.77, P = 0.0003]. The variant allele of rs2850146 was associated with reduced gene expression and increased plasma homocysteine (Hcy) concentrations. Three plasma metabolites, Hcy, methionine and dimethylglycine, were associated with increased risk for gene methylation. These studies suggest that SNPs in CBS and MTRR have sex-specific associations with aberrant methylation in the lung epithelium of smokers that could be mediated by the affected one-carbon metabolism and transsulfuration in the cells.

  14. rpL3 promotes the apoptosis of p53 mutated lung cancer cells by down-regulating CBS and NFκB upon 5-FU treatment

    PubMed Central

    Russo, Annapina; Saide, Assunta; Cagliani, Roberta; Cantile, Monica; Botti, Gerardo; Russo, Giulia

    2016-01-01

    5-FU is a chemotherapy drug commonly used for the treatment of human cancers; however drug resistance represents a major challenge for its clinical application. In the present study, we reporte that rpL3 induced by 5-FU treatment in Calu-6 cells represses CBS transcription and reduces CBS protein stability leading to a decrease of CBS protein levels. rpL3 also regulates negatively the activation of NFκB by preventing NFκB nuclear translocation through IκB-α up-regulation. Furthermore, we demonstrate that rpL3 significantly enhances the apoptosis of 5-FU treated Calu-6 cells promoting the overexpression of the pro-apoptotic proteins Bax and the inhibition of the anti-apoptotic protein Bcl-2. We finally demonstrate that rpL3 potentiates 5-FU efficacy inhibiting cell migration and invasion. Our results suggest that combination of rpL3 and 5-FU is a promising strategy for chemotherapy of lung cancers lacking functional p53 that are resistant to 5-FU. PMID:27924828

  15. rpL3 promotes the apoptosis of p53 mutated lung cancer cells by down-regulating CBS and NFκB upon 5-FU treatment.

    PubMed

    Russo, Annapina; Saide, Assunta; Cagliani, Roberta; Cantile, Monica; Botti, Gerardo; Russo, Giulia

    2016-12-07

    5-FU is a chemotherapy drug commonly used for the treatment of human cancers; however drug resistance represents a major challenge for its clinical application. In the present study, we reporte that rpL3 induced by 5-FU treatment in Calu-6 cells represses CBS transcription and reduces CBS protein stability leading to a decrease of CBS protein levels. rpL3 also regulates negatively the activation of NFκB by preventing NFκB nuclear translocation through IκB-α up-regulation. Furthermore, we demonstrate that rpL3 significantly enhances the apoptosis of 5-FU treated Calu-6 cells promoting the overexpression of the pro-apoptotic proteins Bax and the inhibition of the anti-apoptotic protein Bcl-2. We finally demonstrate that rpL3 potentiates 5-FU efficacy inhibiting cell migration and invasion. Our results suggest that combination of rpL3 and 5-FU is a promising strategy for chemotherapy of lung cancers lacking functional p53 that are resistant to 5-FU.

  16. Urothelium muscarinic activation phosphorylates CBS(Ser227) via cGMP/PKG pathway causing human bladder relaxation through H2S production.

    PubMed

    d'Emmanuele di Villa Bianca, Roberta; Mitidieri, Emma; Fusco, Ferdinando; Russo, Annapina; Pagliara, Valentina; Tramontano, Teresa; Donnarumma, Erminia; Mirone, Vincenzo; Cirino, Giuseppe; Russo, Giulia; Sorrentino, Raffaella

    2016-08-11

    The urothelium modulates detrusor activity through releasing factors whose nature has not been clearly defined. Here we have investigated the involvement of H2S as possible mediator released downstream following muscarinic (M) activation, by using human bladder and urothelial T24 cell line. Carbachol stimulation enhances H2S production and in turn cGMP in human urothelium or in T24 cells. This effect is reversed by cysthationine-β-synthase (CBS) inhibition. The blockade of M1 and M3 receptors reverses the increase in H2S production in human urothelium. In T24 cells, the blockade of M1 receptor significantly reduces carbachol-induced H2S production. In the functional studies, the urothelium removal from human bladder strips leads to an increase in carbachol-induced contraction that is mimicked by CBS inhibition. Instead, the CSE blockade does not significantly affect carbachol-induced contraction. The increase in H2S production and in turn of cGMP is driven by CBS-cGMP/PKG-dependent phosphorylation at Ser(227) following carbachol stimulation. The finding of the presence of this crosstalk between the cGMP/PKG and H2S pathway downstream to the M1/M3 receptor in the human urothelium further implies a key role for H2S in bladder physiopathology. Thus, the modulation of the H2S pathway can represent a feasible therapeutic target to develop drugs for bladder disorders.

  17. Optimization of the expression of surface antigen SAG1/2 of Toxoplasma gondii in the yeast Pichia pastoris.

    PubMed

    Thiruvengadam, G; Init, I; Fong, M Y; Lau, Y L

    2011-12-01

    Surface antigens are the most abundant proteins found on the surface of the parasite Toxoplasma gondii. Surface antigen 1 (SAG1) and Surface antigen 2 (SAG2) remain the most important and extensively studied surface proteins. These antigens have been identified to play a role in host cell invasion, immune modulation, virulence attenuation. Recombinant SAG1/2 was cloned and expressed in yeast Pichia pastoris. We describe here optimization of critical parameters involved in high yield expression of the recombinant SAG1/2. Our results suggest that recombinant SAG1/2 were best expressed at 30ºC, pH 6 and 1% methanol as the carbon source by X33 Pichia cells. Additional optimizations included the downstream process such as ammonium sulphate precipitation and dialysis. The fusion protein was purified using Ni-NTA purification system with 80% recovery. The purified protein was 100% specific and sensitive in detection of toxoplasmosis.

  18. 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

  19. Functional identification of a rice ent-kaurene oxidase, OsKO2, using the Pichia pastoris expression system.

    PubMed

    Ko, Kwang-Wook; Lin, Fengqiu; Katsumata, Takumi; Sugai, Yoshinori; Miyazaki, Sho; Kawaide, Hiroshi; Okada, Kazunori; Nojiri, Hideaki; Yamane, Hisakazu

    2008-12-01

    Rice ent-kaurene oxidase 2 (OsKO2) perhaps functions in the early steps of gibberellin biosynthesis. We found that microsomes from the methylotropic yeast Pichia pastoris expressing both OsKO2 and a fungal cytochrome P450 monooxygenase (P450) reductase converted ent-kaurene to ent-kaurenoic acid. This is direct evidence that OsKO2 is involved in the sequential oxidation of ent-kaurene to ent-kaurenoic acid in gibberellin biosynthesis in rice.

  20. 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.

  1. A simple Pichia pastoris fermentation and downstream processing strategy for making recombinant pandemic Swine Origin Influenza a virus Hemagglutinin protein.

    PubMed

    Athmaram, T N; Singh, Anil Kumar; Saraswat, Shweta; Srivastava, Saurabh; Misra, Princi; Kameswara Rao, M; Gopalan, N; Rao, P V L

    2013-02-01

    The present Influenza vaccine manufacturing process has posed a clear impediment to initiation of rapid mass vaccination against spreading pandemic influenza. New vaccine strategies are therefore needed that can accelerate the vaccine production. Pichia offers several advantages for rapid and economical bulk production of recombinant proteins and, hence, can be attractive alternative for producing an effective influenza HA based subunit vaccine. The recombinant Pichia harboring the transgene was subjected to fed-batch fermentation at 10 L scale. A simple fermentation and downstream processing strategy is developed for high-yield secretory expression of the recombinant Hemagglutinin protein of pandemic Swine Origin Influenza A virus using Pichia pastoris via fed-batch fermentation. Expression and purification were optimized and the expressed recombinant Hemagglutinin protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot and MALDI-TOF analysis. In this paper, we describe a fed-batch fermentation protocol for the secreted production of Swine Influenza A Hemagglutinin protein in the P. pastoris GS115 strain. We have shown that there is a clear relationship between product yield and specific growth rate. The fed-batch fermentation and downstream processing methods optimized in the present study have immense practical application for high-level production of the recombinant H1N1 HA protein in a cost effective way using P. pastoris.

  2. A dynamic method based on the specific substrate uptake rate to set up a feeding strategy for Pichia pastoris

    PubMed Central

    2011-01-01

    Background Pichia pastoris is one of the most important host organisms for the recombinant production of proteins in industrial biotechnology. To date, strain specific parameters, which are needed to set up feeding profiles for fed batch cultivations, are determined by time-consuming continuous cultures or consecutive fed batch cultivations, operated at different parameter sets. Results Here, we developed a novel approach based on fast and easy to do batch cultivations with methanol pulses enabling a more rapid determination of the strain specific parameters specific substrate uptake rate qs, specific productivity qp and the adaption time (Δtimeadapt) of the culture to methanol. Based on qs, an innovative feeding strategy to increase the productivity of a recombinant Pichia pastoris strain was developed. Higher specific substrate uptake rates resulted in increased specific productivity, which also showed a time dependent trajectory. A dynamic feeding strategy, where the setpoints for qs were increased stepwise until a qs max of 2.0 mmol·g-1·h-1 resulted in the highest specific productivity of 11 U·g-1·h-1. Conclusions Our strategy describes a novel and fast approach to determine strain specific parameters of a recombinant Pichia pastoris strain to set up feeding profiles solely based on the specific substrate uptake rate. This approach is generic and will allow application to other products and other hosts. PMID:21371310

  3. Binding studies using Pichia pastoris expressed human aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins.

    PubMed

    Zheng, Yujuan; Xie, Jinghang; Huang, Xin; Dong, Jin; Park, Miki S; Chan, William K

    2016-06-01

    The aryl hydrocarbon receptor (AHR) is a transcription factor which activates gene transcription by binding to its corresponding enhancer as the heterodimer, which is consisted of AHR and the aryl hydrocarbon receptor nuclear translocator (ARNT). Human AHR can be rather difficult to study, when compared among the AHR of other species, since it is relatively unstable and less sensitive to some ligands in vitro. Overexpression of human AHR has been limited to the baculovirus expression, which is costly and tedious due to the need of repetitive baculovirus production. Here we explored whether we could generate abundant amounts of human AHR and ARNT in a better overexpression system for functional study. We observed that human AHR and ARNT can be expressed in Pichia pastoris with yields that are comparable to the baculovirus system only if their cDNAs are optimized for Pichia expression. Fusion with a c-myc tag at their C-termini seems to increase the expression yield. These Pichia expressed proteins can effectively heterodimerize and form the ternary AHR/ARNT/enhancer complex in the presence of β-naphthoflavone or kynurenine. Limited proteolysis using thermolysin can be used to study the heterodimerization of these human AHR and ARNT proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Theoretical reduction potentials for nitrogen oxides from CBS-QB3 energetics and (C)PCM solvation calculations.

    PubMed

    Dutton, Andrew S; Fukuto, Jon M; Houk, Kendall N

    2005-05-30

    The complete basis set method, CBS-QB3, is used in combination with two continuum solvation models for aqueous solvation to compute reduction potentials previously determined experimentally for 36 nitrogen oxides and related species of the general formula H(V)C(W)N(X)O(Y)Cl(Z). The PCM model led to the correlation E(o)exp (vs NHE) = 0.84E(o)calc + 0.03 V with an average error of 0.12 V (2.8 kcal/mol) and a maximum error of 0.32 V (7.4 kcal/mol). The CPCM/UAKS model gave E(o)exp (vs NHE) = 0.83E(o)calc + 0.11 V with the same average error. This general method was used to predict reduction potentials (+/-0.3 V) for nitrogen oxides for which reduction potentials are not known with certainty: NO2/NO2- (0.6 V), NO3/NO3- (1.9 V), N2O3-/N2O3(2-) (0.5 V), HN2O3/HN2O3- (0.9 V), HONNO,H+/HONNOH (1.6 V), 2NO,H+/HONNO (0.0 V), 2NO/ONNO- (-0.1 V), ONNO-/ONNO(2-) (-0.4 V), HNO,H+/H2NO (0.6 V), H2NO,H+/H2NOH (0.9 V), HNO,2H+/H2NOH (0.8 V), and HNO/HNO- (-0.7 V).

  5. Correlation of Biological Activity and Reactor Performance in Biofiltration of Toluene with the Fungus Paecilomyces variotii CBS115145

    PubMed Central

    García-Peña, Inés; Hernández, Sergio; Auria, Richard; Revah, Sergio

    2005-01-01

    A biofiltration system inoculated with the mold Paecilomyces variotii CBS115145 showed a toluene elimination capacity (EC) of around 250 g/m3 of biofilter/h, which was higher than the values usually reported for bacteria. P. variotii assimilated m- and p-cresols but not the o isomer. Initial toluene hydroxylation occurred both on the methyl group and through the p-cresol pathway. These results were corroborated by detecting benzyl alcohol, benzaldehyde, and p-cresol as volatile intermediates. In liquid cultures with toluene as a substrate, the activity of toluene oxygenase (TO) was 5.6 nmol of O2/min/mg of biomass, and that of benzyl alcohol dehydrogenase was 16.2 nmol of NADH/min/mg of protein. Toluene biodegradation determined from the TO activity in the biofilter depended on the biomass distribution and the substrate concentration. The specific enzymatic activity decreased from 6.3 to 1.9 nmol of O2/min/mg of biomass along the reactor. Good agreement was found between the EC calculated from the TO activity and the EC measured on the biofilter. The results were confirmed by short-time biofiltration experiments. Average EC measured in different biofiltration experiments and EC calculated from the TO activity showed a linear relation, suggesting that in the biofilters, EC was limited by biological reaction. As the enzymatic activities of P. variotii were similar to those reported for bacteria, the high performance of the fungal biofilters can possibly be explained by the increased transfer of the hydrophobic compounds, including oxygen, from the gas phase to the mycelia, overcoming the transfer problems associated with the flat bacterial biofilms. PMID:16085815

  6. Purification and characterization of extracellular alpha-amylase and glucoamylase from the yeast Candida antarctica CBS 6678.

    PubMed

    De Mot, R; Verachtert, H

    1987-05-04

    An alpha-amylase and a glucoamylase were purified to homogeneity from the culture fluid of beta-cyclodextrin-grown Candida antarctica CBS 6678 by protamine sulfate treatment, ammonium sulfate precipitation, gel filtration (Sephadex G-75 sf, Ultrogel AcA 54), DEAE-Sephacel chromatography, hydroxyapatite chromatography and affinity chromatography on acarbose--AH-Sepharose 4B. Both enzymes were monomeric glycoproteins with fairly different amino acid compositions. Their apparent relative molecular mass, sedimentation coefficient (Szero20,w), isoelectric point, absorption coefficient (280 nm), pH and temperature optima were estimated as 48,500, 4.7 S, 10.1, 1.74 cm2 mg-1, 4.2 and 57 degrees C, respectively, for glucoamylase and as 50,000, 4.9 S, 10.3, 1.53 cm2 mg-1, 4.2 and 62 degrees C, respectively, for alpha-amylase. Kinetic analyses indicated that both enzymes preferentially hydrolyzed high-molecular-mass substrates, including some raw starches. alpha-Amylase was active on cyclodextrins, whereas debranching activity was demonstrated for glucoamylase. Trestatins were potent inhibitors of both alpha-amylase (Ki less than 1 microM) and glucoamylase (Ki less than 0.1 microM), being more effective than Bay e 4609 (Ki less than 10 microM). Glucoamylase was selectivity and strongly inhibited by acarbose (Ki less than 0.1 microM). Activity of the latter enzyme was also affected by 1-deoxynojirimycin (Ki less than 1 mM), maltitol and amino alcohols (Ki less than 10 mM). Unlike alpha-amylase, glucoamylase adsorbed strongly onto raw starch, the adsorption site being non-identical with the active site.

  7. Chemical Composition and Medicinal Value of the New Ganoderma tsugae var. jannieae CBS-120304 Medicinal Higher Basidiomycete Mushroom.

    PubMed

    Chan, Jannie Siew Lee; Asatiani, Mikheil D; Sharvit, Lital E; Trabelcy, Beny; Barseghyan, Gayane S; Wasser, Solomon P

    2015-01-01

    In this research, the chemical composition and anticancer and antioxidant activity of the new medicinal mushroom Ganoderma tsugae var. jannieae CBS-120304 were evaluated. The chemical composition assay includes amounts of total carbohydrates and proteins, amino acids, fatty acids, micro- and macroelements, and vitamins. The investigated medicinal mushroom seemed to be a rich source of nutritional components. Mycelium accumulated more than 2-fold more total protein compared with the fruiting body and reached 37% and 16% of dry weight, respectively. Carbohydrate content in the fruiting body seemed to be conspicuously higher than in the mycelium (50% of dry weight) and reached 80% of dry weight. Quantification of the identified fatty acids indicated that, in general, palmitic acid, oleic acid, and linoleic acid were the major fatty acids. Toxic elements, such as silver, arsenic, cadmium, and mercury, were found only in trace amounts in mycelium and were not detected in the fruiting body. Furthermore, the 1,1-diphenyl-2-picrylhydrazyl free radical scavenging assay was used to evaluate antioxidant activity. The highest radical scavenging activity was 9.0 mg/mL (65.9%) by ethanol extract. In addition, mycelial extracts were tested to inhibit MCF7 breast cancer cells. Ganoderma tsugae var. jannieae ethyl acetate extract (GTEAE) extract showed high potential by inhibiting reporter activity by more than 70%. Results demonstrated that GTEAE had a strong effect on inhibitory protein κΒα level in the higher concentration used (200 gg/mL), which could be compared with the effect of parthenolide. Furthermore, GTEAE demonstrated strong inhibition of IκΒα phosphorylation.

  8. Cloning, sequence analysis, expression of Cyathus bulleri laccase in Pichia pastoris and characterization of recombinant laccase

    PubMed Central

    2012-01-01

    Background Laccases are blue multi-copper oxidases and catalyze the oxidation of phenolic and non-phenolic compounds. There is considerable interest in using these enzymes for dye degradation as well as for synthesis of aromatic compounds. Laccases are produced at relatively low levels and, sometimes, as isozymes in the native fungi. The investigation of properties of individual enzymes therefore becomes difficult. The goal of this study was to over-produce a previously reported laccase from Cyathus bulleri using the well-established expression system of Pichia pastoris and examine and compare the properties of the recombinant enzyme with that of the native laccase. Results In this study, complete cDNA encoding laccase (Lac) from white rot fungus Cyathus bulleri was amplified by RACE-PCR, cloned and expressed in the culture supernatant of Pichia pastoris under the control of the alcohol oxidase (AOX)1 promoter. The coding region consisted of 1,542 bp and encodes a protein of 513 amino acids with a signal peptide of 16 amino acids. The deduced amino acid sequence of the matured protein displayed high homology with laccases from Trametes versicolor and Coprinus cinereus. The sequence analysis indicated the presence of Glu 460 and Ser 113 and LEL tripeptide at the position known to influence redox potential of laccases placing this enzyme as a high redox enzyme. Addition of copper sulfate to the production medium enhanced the level of laccase by about 12-fold to a final activity of 7200 U L-1. The recombinant laccase (rLac) was purified by ~4-fold to a specific activity of ~85 U mg-1 protein. A detailed study of thermostability, chloride and solvent tolerance of the rLac indicated improvement in the first two properties when compared to the native laccase (nLac). Altered glycosylation pattern, identified by peptide mass finger printing, was proposed to contribute to altered properties of the rLac. Conclusion Laccase of C. bulleri was successfully produced extra

  9. Purification and biochemical characterization of simplified eukaryotic nitrate reductase expressed in Pichia pastoris.

    PubMed

    Barbier, Guillaume G; Joshi, Rama C; Campbell, Ellen R; Campbell, Wilbur H

    2004-09-01

    NAD(P)H:nitrate reductase (NaR, EC 1.7.1.1-3) is a useful enzyme in biotechnological applications, but it is very complex in structure and contains three cofactors-flavin adenine dinucleotide, heme-Fe, and molybdenum-molybdopterin (Mo-MPT). A simplified nitrate reductase (S-NaR1) consisting of Mo-MPT-binding site and nitrate-reducing active site was engineered from yeast Pichia angusta NaR cDNA (YNaR1). S-NaR1 was cytosolically expressed in high-density fermenter culture of methylotrophic yeast Pichia pastoris. Total amount of S-NaR1 protein produced was approximately 0.5 g per 10 L fermenter run, and methanol phase productivity was 5 microg protein/g wet cell weight/h. Gene copy number in genomic DNA of different clones showed direct correlation with the expression level. S-NaR1 was purified to homogeneity in one step by immobilized metal affinity chromatography (IMAC) and total amount of purified protein per run of fermentation was approximately 180 mg. Polypeptide size was approximately 55 kDa from electrophoretic analysis, and S-NaR1 was mainly homo-tetrameric in its active form, as shown by gel filtration. S-NaR1 accepted electrons efficiently from reduced bromphenol blue (kcat = 2081 s(-1)) and less so from reduced methyl viologen (kcat = 159 s(-1)). The nitrate KM for S-NaR1 was 30 +/- 3 microM, which is very similar to YNaR1. S-NaR1 is capable of specific nitrate reduction, and direct electric current, as shown by catalytic nitrate reduction using protein film cyclic voltammetry, can drive this reaction. Thus, S-NaR1 is an ideal form of this enzyme for commercial applications, such as an enzymatic nitrate biosensor formulated with S-NaR1 interfaced to an electrode system.

  10. Recombinant production of bacterial toxins and their derivatives in the methylotrophic yeast Pichia pastoris

    PubMed Central

    Gurkan, Cemal; Ellar, David J

    2005-01-01

    The methylotrophic yeast Pichia pastoris is a popular heterologous expression host for the recombinant production of a variety of prokaryotic and eukaryotic proteins. The rapid emergence of P. pastoris as a robust heterologous expression host was facilitated by the ease with which it can be manipulated and propagated, which is comparable to that of Escherichia coli and Saccharomyces cerevisiae. P. pastoris offers further advantages such as the tightly-regulated alcohol oxidase promoter that is particularly suitable for heterologous expression of foreign genes. While recombinant production of bacterial toxins and their derivatives is highly desirable, attempts at their heterologous expression using the traditional E. coli expression system can be problematic due to the formation of inclusion bodies that often severely limit the final yields of biologically active products. However, recent literature now suggests that P. pastoris may be an attractive alternative host for the heterologous production of bacterial toxins, such as those from the genera Bacillus, Clostridium, and Corynebacterium, as well as their more complex derivatives. Here, we review the recombinant production of bacterial toxins and their derivatives in P. pastoris with special emphasis on their potential clinical applications. Considering that de novo design and construction of synthetic toxin genes have often been necessary to achieve optimal heterologous expression in P. pastoris, we also present general guidelines to this end based on our experience with the P. pastoris expression of the Bacillus thuringiensis Cyt2Aa1 toxin. PMID:16336647

  11. Expression and characterization of recombinant Locusta migratoria manilensis acetylcholinesterase 1 in Pichia pastoris.

    PubMed

    Zhou, Xiaoxia; Xia, Yuxian

    2011-05-01

    The acetylcholinesterase 1 from Locusta migratoria manilensis (LmAChE1) was successfully expressed in methylotrophic yeast Pichia pastoris KM71. The maximum expression of recombinant LmAChE1 (reLmAChE1) was achieved after 9 days of induction at 2.5% methanol. The reLmAChE1 was first precipitated with ammonium sulfate (50% saturation) and then was purified with nickel affinity chromatography. The enzyme was purified 3.2×10(3)-fold with a yield of 68% and a specific activity of 8.1 U/mg. The purified reLmAChE1 exhibited highest activity at 30°C in 100 mM phosphate buffer (pH 7.4), and its activity could be inhibited by eserine sulfate and pentan-3-one-dibromide (BW284c51). Substrate specificity analysis showed that the purified reLmAChE1 preferred acetylthiocholine (ATC) and propionylthiocholine (BTC) rather than butyrylthiocholine (BTC). When ATC was used as substrate, the K(m) and V(max) values for the reLmAChE1 were 24.8 μM and 9.5 μmol/min/mg, respectively.

  12. Efficient microbial production of stylopine using a Pichia pastoris expression system

    PubMed Central

    Hori, Kentaro; Okano, Shunsuke; Sato, Fumihiko

    2016-01-01

    Stylopine is a protoberberine-type alkaloid that has potential biological activities. Based on the successful microbial production of (S)-reticuline, we attempted to produce stylopine from (S)-reticuline by the reaction of berberine bridge enzyme, cheilanthifoline synthase (CYP719A5), and stylopine synthase (CYP719A2). Biosynthetic enzyme expression was examined in a methanol-utilizing yeast (Pichia pastoris), and both a “consolidated” system with all genes expressed in one cell and a “co-culture” system with three cell lines that each express a single gene were examined. Although both systems efficiently converted reticuline to stylopine, the consolidated system was more rapid and efficient than the co-culture system. However, substrate-feeding experiments revealed a decrease in the conversion efficiency in the consolidated system during successive cultures, whereas the conversion efficiency in the co-culture system remained constant. Thus, the final amount of stylopine produced from reticuline after successive feedings in the co-culture system was more than 150 nmoles from 750 nmoles of (R, S)-reticuline (375 nmoles of (S)-reticuline). The advantages and drawbacks of the “consolidated” system and the “co-culture” system are discussed. PMID:26923560

  13. Characterization of the enterovirus 71 P1 polyprotein expressed in Pichia pastor as a candidate vaccine

    PubMed Central

    Han, Xue; Ying, Xiao-ling; Zhou, Shi-li; Han, Tao; Huang, Hao; Jin, Qi; Yang, Fan; Sun, Qi-ying; Sun, Xian-xun

    2014-01-01

    Human enterovirus 71 (EV71) plays an important role in hand, foot, and mouth disease (HFMD), which recently caused the death of hundreds of children in the Asia-Pacific region. However, there are no specific treatments available for EV71 infections; thus, a safe and effective vaccine is needed urgently. In this study, we developed an effective and economical method for producing EV71 polyprotein (P1 protein) in Pichia pastoris. Furthermore, we evaluated the potential of P1 protein as a candidate vaccine against EV71 virus. The data revealed that P1 protein induced persistent high cross-neutralization antibodies for different EV71 subtypes, and elicited significant splenocyte proliferation. The high levels of interleukin-10(IL-10) and interferon-gamma (IFN-γ) showed that P1 protein induced Th1 and Th2 immune responses. Interestingly, vaccinating female mice with the P1 protein conferred cross-protection against different EV71 subtypes to their neonatal offspring.Compared with heat-inactivated EV71, the P1 protein elicited improved humoral and cellular immune responses and showed good cross-protection with different EV71 subtypes. Therefore, the EV71-P1 protein produced by P. pastoris is a promising candidate vaccine against EV71. PMID:25424925

  14. Engineering Pichia pastoris for improved NADH regeneration: A novel chassis strain for whole-cell catalysis.

    PubMed

    Geier, Martina; Brandner, Christoph; Strohmeier, Gernot A; Hall, Mélanie; Hartner, Franz S; Glieder, Anton

    2015-01-01

    Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO2 represents an efficient and easy-to-use strategy for NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regeneration pathway expression and source of energy.

  15. Engineering Pichia pastoris for improved NADH regeneration: A novel chassis strain for whole-cell catalysis

    PubMed Central

    Geier, Martina; Brandner, Christoph; Strohmeier, Gernot A; Hall, Mélanie; Hartner, Franz S

    2015-01-01

    Summary Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO2 represents an efficient and easy-to-use strategy for NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regeneration pathway expression and source of energy. PMID:26664594

  16. Synergistic modular promoter and gene optimization to push cellulase secretion by Pichia pastoris beyond existing benchmarks.

    PubMed

    Mellitzer, Andrea; Ruth, Claudia; Gustafsson, Claes; Welch, Mark; Birner-Grünberger, Ruth; Weis, Roland; Purkarthofer, Thomas; Glieder, Anton

    2014-12-10

    Although successfully used for heterologous gene expression for more than twenty years, general knowledge about all factors influencing protein expression by Pichia pastoris is still lacking. For high titers of protein clones are optimized individually for each target protein. Optimization efforts in this study were focused on the DNA level, evaluating a set of 48 different individual synthetic genes (TrCBH2) coding for the same protein sequence of a Trichoderma reesei cellulase in combination with three different promoter sequences: PGAP (constitutive) and the synthetic AOX1 promoter variants PDeS (derepressed) and PEn (enhanced, inducible). Expression of active secreted enzyme varied from undetectable to ∼300% of the best known gene, as determined by secreted enzyme activity analyses of supernatants from 96 well plate and bioreactor cultivations. Finally, the best optimized gene and new promoters were combined to engineer highly productive P. pastoris CBH2 expression strains. Although no methanol was used for induction a final titer of more than 18g/l of secreted protein was produced under controlled conditions in small scale bioreactor cultivations after 60-70h of growth limiting glycerol feed. This is the highest concentration of secreted enzyme in P. pastoris published so far and single parts of the expression cassette could be independently optimized showing additive effects for improvements in protein production by P. pastoris.

  17. Expression and purification of non-N-glycosylated porcine interleukin 3 in yeast Pichia pastoris.

    PubMed

    Hermanrud, Christina E; Pathiraja, Vimukthi; Matar, Abraham; Duran-Struuck, Raimon; Crepeau, Rebecca L; Srinivasan, Srimathi; Sachs, David H; Huang, Christene A; Wang, Zhirui

    2012-03-01

    Yeast Pichia pastoris has been widely utilized to express heterologous recombinant proteins. P. pastoris expressed recombinant porcine interleukin 3 (IL3) has been used for porcine stem cell mobilization in allo-hematopoietic cell transplantation models and pig-to-primate xeno-hematopoietic cell transplantation models in our lab for many years. Since the yeast glycosylation mechanism is not exactly the same as those of other mammalian cells, P. pastoris expressed high-mannose glycoprotein porcine IL3 has been shown to result in a decreased serum half-life. Previously this was avoided by separation of the non-glycosylated porcine IL3 from the mixture of expressed glycosylated and non-glycosylated porcine IL3. However, this process was very inefficient and lead to a poor yield following purification. To overcome this problem, we engineered a non-N-glycosylated version of porcine IL3 by replacing the four potential N-glycosylation sites with four alanines. The codon-optimized non-N-glycosylated porcine IL3 gene was synthesized and expressed in P. pastoris. The expressed non-N-glycosylated porcine IL3 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50 HQ. In vivo mobilization studies performed in our research facility demonstrated that the non-N-glycosylated porcine IL3 still keeps the original stem cell mobilization function. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Comparison of two codon optimization strategies enhancing recombinant Sus scrofa lysozyme production in Pichia pastoris.

    PubMed

    Zhu, D; Cai, G; Wu, D; Lu, J

    2015-05-16

    Lysozyme has played an important role in animal feed additive industry, food additive industry and biological engineering. For improving expression efficiency of recombinant lysozyme from Sus scrofa, two genes respectively designed by the most used codon optimization strategies, "one amino acid one codon" and "codon randomization", were synthesized and expressed in Pichia pastoris X—33. At shaking flask level, Sus scrofa lysozyme (SSL) under two conditions had a highest activity of 153.33±10.41 and 538.33±15.18 U/mL after a 5 days induction of 1% methanol, with secreted protein concentration 80.03±1.94 and 239.60±4.16 mg/L, respectively. Compared with the original SSL gene, the expression of optimized SSL gene by the second strategy showed a 2.6 fold higher level, while the first method had no obvious improvement in production. In total secreted protein, the proportions of recombinant SSL encoded by the original gene, first method optimized gene and the second—strategy optimized one were 75.06±0.25%, 74.56±0.14% and 79.00±0.14%, respectively, with the same molecular weight about 18 kDa, optimum acidity pH 6.0 and optimum temperature 35degC.

  19. 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.

  20. Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris.

    PubMed

    Wriessnegger, Tamara; Augustin, Peter; Engleder, Matthias; Leitner, Erich; Müller, Monika; Kaluzna, Iwona; Schürmann, Martin; Mink, Daniel; Zellnig, Günther; Schwab, Helmut; Pichler, Harald

    2014-07-01

    The sesquiterpenoid (+)-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or Nootka cypress tree. Extraction of (+)-nootkatone from plant material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. Lately, major attention has been paid to biotechnological approaches, using cell extracts or whole-cell systems for the production of (+)-nootkatone. In our study, the yeast Pichia pastoris initially was applied as whole-cell biocatalyst for the production of (+)-nootkatone from (+)-valencene, the abundant aroma compound of oranges. Therefore, we generated a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylated extracellularly added (+)-valencene. Intracellular production of (+)-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolved the phase-transfer issues of (+)-valencene. Bi-phasic cultivations of P. pastoris resulted in the production of trans-nootkatol, which was oxidized to (+)-nootkatone by an intrinsic P. pastoris activity. Additional overexpression of a P. pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhanced the (+)-nootkatone yield to 208mg L(-1) cell culture in bioreactor cultivations. Thus, metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-nootkatone from simple carbon sources. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  1. A toolbox of endogenous and heterologous nuclear localization sequences for the methylotrophic yeast Pichia pastoris.

    PubMed

    Weninger, Astrid; Glieder, Anton; Vogl, Thomas

    2015-11-01

    Nuclear localization sequences (NLSs) are required for the import of proteins in the nucleus of eukaryotes. However many proteins from bacteria or bacteriophages are used for basic studies in molecular biology, to generate synthetic genetic circuits or for genome editing applications. Prokaryotic recombinases, CRISPR-associated proteins such as Cas9 or bacterial and viral polymerases require efficient NLSs to function in eukaryotes. The yeast Pichia pastoris is a widely used expression platform for heterologous protein production, but molecular tools such as NLSs are limited. Here we have characterized a set of 10 NLSs for P. pastoris, including the first endogenous NLSs (derived from P. pastoris proteins) and commonly used heterologous NLSs. The NLSs were evaluated by fusing them in N- and C-terminal position to an enhanced green fluorescent protein showing pronounced differences in fluorescence levels and nuclear targeting. Thereby we provide a set of different NLSs that can be applied to optimize the nuclear import of heterologous proteins in P. pastoris, paving the way for the establishment of intricate synthetic biology applications.

  2. Modular Integrated Secretory System Engineering in Pichia pastoris To Enhance G-Protein Coupled Receptor Expression.

    PubMed

    Claes, Katrien; Vandewalle, Kristof; Laukens, Bram; Laeremans, Toon; Vosters, Olivier; Langer, Ingrid; Parmentier, Marc; Steyaert, Jan; Callewaert, Nico

    2016-10-21

    Membrane protein research is still hampered by the generally very low levels at which these proteins are naturally expressed, necessitating heterologous expression. Protein degradation, folding problems, and undesired post-translational modifications often occur, together resulting in low expression levels of heterogeneous protein products that are unsuitable for structural studies. We here demonstrate how the integration of multiple engineering modules in Pichia pastoris can be used to increase both the quality and the quantity of overexpressed integral membrane proteins, with the human CXCR4 G-protein coupled receptor as an example. The combination of reduced proteolysis, enhanced ER folding capacity, GlycoDelete-based N-Glycan trimming, and nanobody-based fold stabilization improved the expression of this GPCR in P. pastoris from a low expression level of a heterogeneously glycosylated, proteolyzed product to substantial quantities (2-3 mg/L shake flask culture) of a nonproteolyzed, homogeneously glycosylated proteoform. We expect that this set of tools will contribute to successful expression of more membrane proteins in a quantity and quality suitable for functional and structural studies.

  3. Cloning and expression in Pichia pastoris of a blue mussel (Mytilus edulis) beta-mannanase gene.

    PubMed

    Xu, Bingze; Sellos, Daniel; Janson, Jan-Christer

    2002-03-01

    Using PCR, cloning and sequencing techniques, a 1.1-kb complementary DNA fragment encoding for a beta-mannanase (mannan endo-1,4-beta-mannosidase, EC 3.2.1.78) has been identified in the digestive gland of blue mussel, Mytilus edulis. The cDNA sequence shows significant sequence identity to several beta-mannanases in glycoside hydrolase family 5. The beta-mannanase gene has been isolated and sequenced from gill tissue of blue mussel and contains five introns. The beta-mannanase has been expressed extracellularly in Pichia pastoris using the Saccharomyces cerevisiae alpha-factor signal sequence. The beta-mannanase was produced in a 14-L fermenter with an expression level of 900 mg.L-1. The expression level is strongly affected by the induction temperature. A two-step purification procedure, composed of a combination of immobilized metal ion affinity chromatography and ion exchange chromatography, is required to give a pure beta-mannanase. However, due to post-translational modifications, structural varieties regarding molecular mass and isoelectric point were obtained. The specific activity of the purified recombinant M. edulis beta-mannanase was close to that of the wild-type enzyme. Also pH and temperature optima were the same as for the native protein. In conclusion, P. pastoris is regarded as a suitable host strain for the production of blue mussel beta-mannanase. This is the first time a mollusc beta-mannanase has been characterized at the DNA level.

  4. Heterologous expression of codon optimized Trichoderma reesei Cel6A in Pichia pastoris.

    PubMed

    Sun, Fubao Fuelbiol; Bai, Renhui; Yang, Huimin; Wang, Fei; He, Jing; Wang, Chundi; Tu, Maobing

    2016-10-01

    The Cel6A deficiency has become one of the limiting factors for cellulose saccharification in biochemical conversion of cellulosic biomass to fuels and chemicals. The work attempted to use codon optimization to enhance Trichoderma reesei Cel6A expression in Pichia pastoris. Two recombinants P. pastoris GS115 containing AOX1 and GAP promotors were successfully constructed, respectively. The optimal temperatures and pHs of the expressed Cel6A from two recombinants were consistent with each other, were also in the extremely similar range to that reported on the native Cel6A from T. reesei. Based on the shake flask fermentation, AOX1 promotor enabled the recombinant to produce 265U/L and 300mg/L of the Cel6A enzyme, and the GAP promotor resulted in 145U/L and 200mg/L. High cell density fed batch (HCDFB) fermentation significantly improved the enzyme titer (1100U/L) and protein yield (2.0g/L) for the recombinant with AOX1 promotor. Results have showed that the AOX1 promotor is more suitable than the GAP for the Cel6A expression in P. pastoris. And the HCDFB cultivation is a favorable way to express the Cel6A highly in the methanol inducible yeast.

  5. Cloning and expression of Trichoderma reesei cellobiohydrolase I in Pichia pastoris

    SciTech Connect

    Godbole, S.; Decker, S.R.; Nieves, R.A.; Adney, W.S.; Vinzant, T.B.; Baker, J.O.; Thomas, S.R.; Himmel, M.E.

    1999-10-01

    Pichia pastoris was transformed with the Trichoderma reesei cbh1 gene, and the recombinant enzyme was purified and analyzed kinetically and by circular dichroism. The P. pastoris rCBH I was recognized by MoAb raised to T. reesei CBH I but was found in multiple molecular weight species on SDS-PAGE gels. Carbohydrate content determination and SDS-PAGE western analysis indicated that the recombinant protein was hyperglycosylated, although a species very similar in molecular weight to the T. reesei enzyme could be isolated chromatographically. The P. pastoris rCBH I also demonstrated activity toward soluble and insoluble substrates (i.e., pNPL and Sigmacell), although at a level significantly lower than the wild-type enzyme. More seriously, the yeast-expressed enzyme showed non-wild-type secondary structure by circular dichroism. The authors conclude that P. pastoris may not serve as an adequate host for the site-directed mutagenesis of T. reesei CBH I.

  6. Codon optimization and expression of irisin in Pichia pastoris GS115.

    PubMed

    Duan, Huikun; Wang, Haisong; Ma, Baicheng; Jiang, Pingzhe; Tu, Peipei; Ni, Zaizhong; Li, Xiaodan; Li, Miao; Ma, Xiaofeng; Wang, Bin; Wu, Ri; Li, Minggang

    2015-08-01

    Irisin is a novel hormone which is related to many metabolic diseases. In order to illuminate the function and therapeutic effect of irisin, gaining active irisin is necessary. In this work, a codon-optimized irisin gene was designed according to Pichia pastoris synonymous codon usage bias and cloned into the pPIC9K expression vector. Sequencing result indicating that the sequence of irisin was consistent with the modified irisin and the irisin was in frame with α-factor secretion signal ATG. The plasmid pPIC9K-irisin was transformed into GS115 P. pastoris cells through electroporation. The positive transformants were screened on MD medium and analyzed by PCR. Five recombinant GS115/pPIC9K-irisin strains were obtained, but only one strain expressed irisin successfully. SDS-PAGE and Western blot were used to assess the expression level and purity of irisin. The irisin was also simply purified and the effect of pH value, methanol concentration and induction time on the production of irisin was investigated. The results showed that the best conditions of irisin expression were as follows: pH 6.0, 2.0% methanol and induction for 96 h. This work laid the basis for further investigation into the therapeutic and pharmacological effects of irisin, as well as development of irisin-based therapy.

  7. Artificial synthesis of swine hepcidin gene and expression in Pichia pastoris.

    PubMed

    Di, Yuanran; Cheng, Wei; Chang, Juan; Yin, Qingqiang; Lu, Min; Yuan, Lin; Dang, Xiaowei

    2014-01-01

    In order to express swine hepcidin gene in Pichia pastoris, a DNA fragment coding hepcidin gene was synthesized with adaptation to yeast codon usage of highly expressed genes. A Kex2 signal cleavage site was fused in the 5' end of the DNA fragment for getting a peptide with the same N-end as native hepcidin. The 96-bp DNA fragment was ligated into the expression plasmid of pGAPZaA to construct pGAPZaA-hepcidin vector, which was transferred into P. pastoris (X33) to express hepcidin gene for extracellular secretion of protein at 86 µg/mL. A band of 2.76 kD molecular mass was detected by Tricine sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) analysis. Through antibacterial assay, the expressed hepcidin displayed obvious antibacterial activity. The minimal inhibitory concentration (MIC) was 5.38 and 2.69 µg/mL for Staphylococcus aureus and Bacillus subtilis prolification inhibitions, respectively.

  8. 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.

  9. 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.

  10. 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.

  11. Purification and basic biochemical characterization of 19 recombinant plant peroxidase isoenzymes produced in Pichia pastoris☆

    PubMed Central

    Krainer, Florian W.; Pletzenauer, Robert; Rossetti, Laura; Herwig, Christoph; Glieder, Anton; Spadiut, Oliver

    2014-01-01

    The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity. PMID:24342173

  12. 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.

  13. Expression of a Bacillus phytase C gene in Pichia pastoris and properties of the recombinant enzyme.

    PubMed

    Guerrero-Olazarán, Martha; Rodríguez-Blanco, Lilí; Carreon-Treviño, J Gerardo; Gallegos-López, Juan A; Viader-Salvadó, José M

    2010-08-01

    The cloning and expression of a native gene encoding a Bacillus subtilis phytase using Pichia pastoris as the host is described. In addition, the influence of N-glycosylation on the biochemical properties of the B. subtilis phytase, the influence of pH on the thermostability of the recombinant and native B. subtilis phytases, and the resistance of both phytases to shrimp digestive enzymes and porcine trypsin are also described. After 48 h of methanol induction in shake flasks, a selected recombinant strain produced and secreted 0.82 U/ml (71 mg/liter) recombinant phytase. This phytase was N-glycosylated, had a molecular mass of 39 kDa after N-deglycosylation, exhibited activity within a pH range of 2.5 to 9 and at temperatures of 25 to 70 degrees C, had high residual activity (85% +/- 2%) after 10 min of heat treatment at 80 degrees C and pH 5.5 in the presence of 5 mM CaCl(2), and was resistant to shrimp digestive enzymes and porcine trypsin. Although the recombinant Bacillus phytase had pH and temperature activity profiles that were similar to those of the corresponding nonglycosylated native phytase, the thermal stabilities of the recombinant and native phytases were different, although both were calcium concentration and pH dependent.

  14. A novel methanol-free Pichia pastoris system for recombinant protein expression.

    PubMed

    Shen, Wei; Xue, Ying; Liu, Yiqi; Kong, Chuixing; Wang, Xiaolong; Huang, Mengmeng; Cai, Menghao; Zhou, Xiangshan; Zhang, Yuanxing; Zhou, Mian

    2016-10-21

    As one of the most popular expression systems, recombinant protein expression in Pichia pastoris relies on the AOX1 promoter (P AOX1 ) which is strongly induced by methanol. However, the toxic and inflammatory nature of methanol restricts its application, especially in edible and medical products. Therefore, constructing a novel methanol-free system becomes necessary. The kinases involved in P AOX1 activation or repression by different carbon sources may be promising targets. We identified two kinase mutants: Δgut1 and Δdak, both of which showed strong alcohol oxidase activity under non-methanol carbon sources. Based on these two kinases, we constructed two methanol-free expression systems: Δgut1-HpGCY1-glycerol (P AOX1 induced by glycerol) and Δdak-DHA (P AOX1 induced by DHA). By comparing their GFP expression efficiencies, the latter one showed better potential. To further test the Δdak-DHA system, three more recombinant proteins were expressed as examples. We found that the expression ability of our novel methanol-free Δdak-DHA system was generally better than the constitutive GAP promoter, and reached 50-60 % of the traditional methanol induced system. We successfully constructed a novel methanol-free expression system Δdak-DHA. This modified expression platform preserved the favorable regulatable nature of P AOX1 , providing a potential alternative to the traditional system.

  15. Metabolic engineering of Pichia pastoris to produce ricinoleic acid, a hydroxy fatty acid of industrial importance.

    PubMed

    Meesapyodsuk, Dauenpen; Chen, Yan; Ng, Siew Hon; Chen, Jianan; Qiu, Xiao

    2015-11-01

    Ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) has many specialized uses in bioproduct industries, while castor bean is currently the only commercial source for the fatty acid. This report describes metabolic engineering of a microbial system (Pichia pastoris) to produce ricinoleic acid using a "push" (synthesis) and "pull" (assembly) strategy. CpFAH, a fatty acid hydroxylase from Claviceps purpurea, was used for synthesis of ricinoleic acid, and CpDGAT1, a diacylglycerol acyl transferase for the triacylglycerol synthesis from the same species, was used for assembly of the fatty acid. Coexpression of CpFAH and CpDGAT1 produced higher lipid contents and ricinoleic acid levels than expression of CpFAH alone. Coexpression in a mutant haploid strain defective in the Δ12 desaturase activity resulted in a higher level of ricinoleic acid than that in the diploid strain. Intriguingly, the ricinoleic acid produced was mainly distributed in the neutral lipid fractions, particularly the free fatty acid form, but with little in the polar lipids. This work demonstrates the effectiveness of the metabolic engineering strategy and excellent capacity of the microbial system for production of ricinoleic acid as an alternative to plant sources for industrial uses.

  16. The effect of hypoxia on the lipidome of recombinant Pichia pastoris.

    PubMed

    Adelantado, Núria; Tarazona, Pablo; Grillitsch, Karlheinz; García-Ortega, Xavier; Monforte, Sergi; Valero, Francisco; Feussner, Ivo; Daum, Günther; Ferrer, Pau

    2017-05-19

    Cultivation of recombinant Pichia pastoris (Komagataella sp.) under hypoxic conditions has a strong positive effect on specific productivity when the glycolytic GAP promoter is used for recombinant protein expression, mainly due to upregulation of glycolytic conditions. In addition, transcriptomic analyses of hypoxic P. pastoris pointed out important regulation of lipid metabolism and unfolded protein response (UPR). Notably, UPR that plays a role in the regulation of lipid metabolism, amino acid metabolism and protein secretion, was found to be upregulated under hypoxia. To improve our understanding of the interplay between lipid metabolism, UPR and protein secretion, the lipidome of a P. pastoris strain producing an antibody fragment was studied under hypoxic conditions. Furthermore, lipid composition analyses were combined with previously available transcriptomic datasets to further understand the impact of hypoxia on lipid metabolism. Chemostat cultures operated under glucose-limiting conditions under normoxic and hypoxic conditions were analyzed in terms of intra/extracellular product distribution and lipid composition. Integrated analysis of lipidome and transcriptome datasets allowed us to demonstrate an important remodeling of the lipid metabolism under limited oxygen availability. Additionally, cells with reduced amounts of ergosterol through fluconazole treatment were also included in the study to observe the impact on protein secretion and its lipid composition. Our results show that cells adjust their membrane composition in response to oxygen limitation mainly by changing their sterol and sphingolipid composition. Although fluconazole treatment results a different lipidome profile than hypoxia, both conditions result in higher recombinant protein secretion levels.

  17. Purification of ß-glucosidases from Pichia etchellsii using CIM monolith columns.

    PubMed

    Gaonkar, Roopa K; Mishra, Saroj; Vijayalakshmi, M A

    2011-05-01

    β-Glucosidases (EC 3.2.1.21) are industrially important glycosyl hydrolases used for cellulose saccharification as well as for synthesis of glyco-conjugates. Crystal structure of only one β-glucosidase of family 3 of the glycosyl hydrolase families is available due to difficulty in purification of these closely related enzymes from a given source. Multiple steps used during purification result in low yield, making it difficult to study their properties. Conditions for purification of two closely related β-glucosidases (BGL I and BGL II) of family 3 from Pichia etchellsii were investigated in this study. Two weak anion exchange columns convective interaction media-diethyl amino ethyl (CIM-DEAE) and CIM-ethylenediamine (CIM-EDA) were used for this purpose. The results obtained at 0.34 ml disk (CIM-DEAE) level were scaled up to 8 ml CIM-DEAE tube column wherein BGL I and BGL II were separated from the major contaminants in the cell-free extract. The recovered enzymes were completely resolved in the second step using CIM-EDA. A final specific activity of 9,180 IU/mg and 2,345.3 IU/mg was achieved for BGL I and BGL II respectively with an overall yield of 33%. The system should be applicable to resolution of other closely related enzymes from this family.

  18. Optimization of the fermentation and downstream processes for human enterokinase production in Pichia pastoris.

    PubMed

    Melicherová, Kristína; Krahulec, Ján; Šafránek, Martin; Lišková, Veronika; Hopková, Diana; Széliová, Diana; Turňa, Ján

    2017-03-01

    Enterokinase is one of the most frequently used enzymes for the removal of affinity tags from target recombinant proteins. In this study, several fermentation strategies were assayed for the production of human enterokinase in Pichia pastoris under constitutive GAP promoter. Two of them with controlled specific growth rate during whole cultivation showed a very low enterokinase activity, under 1 U/ml, of the fermentation medium. On the contrary, the combined fermentation with a maximum specific growth rate at the initial phase of the fermentation and stationary-like phase during the rest of the fermentation showed a significant accumulation of the enterokinase in the medium, which counted up to 1400 U/ml. Lower cultivation temperature had a negative impact on the enzyme accumulation during this fermentation strategy. Downstream processes were focused on buffer environment optimization directly after cultivation, as at this time, the most amount of the activity is eliminated by endogenous proteases. Slightly positive effect on enzyme activity in the medium had an addition of liquid storage solution of EDTA and KOH to adjust pH to 8 and molarity of the EDTA to 50 mM. During the purification process, a significant amount of the enzyme was detected to be lost, which counted up to 90%. The purified enzyme, enterokinase, kept quality standard of the published enzymes.

  19. Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris.

    PubMed

    Hong, Feng; Meinander, Nina Q; Jönsson, Leif J

    2002-08-20

    Improved expression of recombinant laccase by Pichia pastoris carrying the lcc1 cDNA isolated from Trametes versicolor was achieved by optimization of the cultivation conditions in a fermentor equipped with a methanol sensor system. The results indicated that the activity obtained in fermentor cultivations was at least 7 times higher than in shake-flask cultures. Three different strategies for fermentor cultivations were compared: A (30 degrees C, 1.0% methanol), B (20 degrees C, 1.0% methanol), and C (20 degrees C, 0.5% methanol). The laccase activity, particularly the specific activity, could be improved by decreasing the cultivation temperature. The mechanisms behind the temperature effect on the laccase activity may be ascribed to poor stability, release of more proteases from dead cells, and folding problems at higher temperature. The results showed that the methanol concentration had a marked effect on the production of active heterologous laccase. A fivefold higher volumetric laccase activity was obtained when the methanol concentration was kept at 0.5% instead of 1.0%. The detrimental effect of methanol on the production of recombinant laccase may be attributed to lower laccase stability, a higher proteolytic activity, and folding problems due to higher growth rate at 1.0% methanol.

  20. Expression of Recombinant Human Mast Cell Chymase with Asn-linked Glycans in Glycoengineered Pichia pastoris

    PubMed Central

    Smith, Eliot T.; Perry, Evan T.; Sears, Megan B.; Johnson, David A.

    2014-01-01

    Recombinant human mast cell chymase (rhChymase) was expressed in secreted form as an active enzyme in the SuperMan5 strain of GlycoSwitch® Pichia pastoris, which is engineered to produce proteins with (Man)5(GlcNAc)2 Asn-linked glycans. Cation exchange and heparin affinity chromatography yielded 5 mg of active rhChymase per liter of fermentation medium. Purified rhChymase migrated on SDSPAGE as a single band of 30 kDa and treatment with peptide N-glycosidase F decreased this to 25 kDa, consistent with the established properties of native human chymase (hChymase). Polyclonal antibodies against hChymase detected rhChymase by Western blot. Active site titration with Eglin C, a potent chymase inhibitor, quantified the concentration of purified active enzyme. Kinetic analyses with succinyl-Ala-Ala-Pro-Phe (suc-AAPF) p-nitroanilide and thiobenzyl ester synthetic substrates showed that heparin significantly reduced Km, whereas heparin effects on kcat were minor. Pure rhChymase with Asn-linked glycans closely resembles hChymase. This bioengineering approach avoided hyperglycosylation and provides a source of active rhChymase for other studies as well as a foundation for production of recombinant enzyme with human glycosylation patterns. PMID:25131858

  1. Modulation of cadmium bioaccumulation and enhancing cadmium tolerance in Pichia kudriavzevii by sodium chloride preincubation.

    PubMed

    Ma, Ning; Li, Chunsheng; Zhang, Dandan; Yu, Jinzhi; Xu, Ying

    2016-07-01

    Application of growing microorganisms for cadmium removal is limited by the sensitivity of living cells to cadmium. The effects of sodium chloride (NaCl) preincubation on the cadmium bioaccumulation and tolerance of Pichia kudriavzevii and Saccharomyces cerevisiae were investigated in this study. NaCl preincubation significantly reduced the intracellular and cell-surface cadmium bioaccumulation of P. kudriavzevii at both 6 and 20 mg L(-1) cadmium, while no obvious effect was observed in S. cerevisiae except that the intracellular cadmium bioaccumulation at 20 mg L(-1) cadmium was reduced obviously by 20-60 g L(-1)  NaCl. For both yeasts, the improved contents of protein and proline after NaCl preincubation contributed to the cadmium tolerance. The thiol contents in P. kudriavzevii under cadmium stress were alleviated by NaCl preincubation, which might be due to the decrease of intracellular cadmium bioaccumulation. NaCl preincubation enhanced the contents of glycerol and trehalose in P. kudriavzevii under cadmium stress, while no acceleration was observed in S. cerevisiae. The results suggested that NaCl preincubation could be applied in cadmium removal by growing P. kudriavzevii to increase the cadmium tolerance of the yeast.

  2. Debaryomyces hansenii (Candida famata), a rare human fungal pathogen often misidentified as Pichia guilliermondii (Candida guilliermondii).

    PubMed

    Desnos-Ollivier, Marie; Ragon, Marie; Robert, Vincent; Raoux, Dorothée; Gantier, Jean-Charles; Dromer, Françoise

    2008-10-01

    Debaryomyces hansenii is a hemiascomycetous yeast commonly found in natural substrates and in various types of cheese. Pichia guilliermondii is widely distributed in nature and is a common constituent of the normal human microflora. Both species have been described in human infections but are extremely difficult to differentiate phenotypically. Thus, frequent errors in identification occur. The 62 clinical and environmental isolates sent between 2000 and 2007 to the French National Reference Center for Mycoses and Antifungals as D. hansenii or P. guilliermondii were analyzed by using the carbon assimilation pattern, the presence of pseudohyphae, and sequencing of the ITS and D1/D2 regions of the rRNA gene. The objective of this study was to assess using nucleotide sequences whether phenotypic identification was accurate and whether phenotypic characteristics could be used to differentiate the two species when sequencing was not available. We found that 58% of the isolates were misidentified and belong to seven different species: P. guilliermondii, P. caribbica, P. jadinii, D. hansenii, Candida palmioleophila, C. haemulonii type II, and Clavispora lusitaniae. In conclusion, D. hansenii may not be as common a human pathogen as previously thought. Sequencing of either ITS or D1/D2 regions is a good tool for differentiating the species more frequently confused with D. hansenii, keeping in mind that reliable databases should be used.

  3. Production of a soluble and functional recombinant apolipoproteinD in the Pichia pastoris expression system.

    PubMed

    Armanmehr, Shiva; Kalhor, Hamid Reza; Tabarraei, Alijan

    2016-05-01

    ApolipoproteinD (ApoD) is a human glycoprotein from the lipocalin family. ApoD contains a conserved central motif of an 8-stranded antiparallel β-sheet, which forms a beta-barrel that can be used for transport and storage of diverse hydrophobic ligands. Due to hydrophobic nature of ApoD, it has been difficult to generate a recombinant version of this protein. In the present work, we aimed at the production of ApoD in the robust Pichia pastoris expression system. To this end, the ApoD gene sequence was synthesized and subcloned for expression in the yeast host cells. Following integration of the ApoD gene into the yeast genomic region using homologous recombination, the ApoD recombinant protein was induced using methanol, reaching its maximum induction at 96 h. Having purified the ApoD recombinant protein by affinity chromatography, we measured the dissociation constant (KD) using its natural ligands: progesterone and arachidonic acid. Our results provide a viable solution to the production of recombinant ApoD protein in lieu of previous obstacles in generating soluble and functional ApoD protein. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Glutaminase-producing Meyerozyma (Pichia) guilliermondii isolated from Thai soy sauce fermentation.

    PubMed

    Aryuman, Phichayaphorn; Lertsiri, Sittiwat; Visessanguan, Wonnop; Niamsiri, Nuttawee; Bhumiratana, Amaret; Assavanig, Apinya

    2015-01-02

    In this study, 34 yeast isolates were obtained from koji and moromi samples of Thai soy sauce fermentation. However, the most interesting yeast strain was isolated from the enriched 2 month-old (M2) moromi sample and identified as Meyerozyma (Pichia) guilliermondii EM2Y61. This strain is a salt-tolerant yeast that could tolerate up to 20% (w/v) NaCl and produce extracellular and cell-bound glutaminases. Interestingly, its glutaminases were more active in 18% (w/v) NaCl which is a salt concentration in moromi. The extracellular glutaminase's activity was found to be much higher than that of cell-bound glutaminase. The highest specific activity and stability of the extracellular glutaminase were found in 18% (w/v) NaCl at pH4.5 and 37°C. A challenge test by adding partially-purified extracellular glutaminase from M. guilliermondii EM2Y61 into 1 month-old (M1) moromi sample showed an increased conversion of L-glutamine to L-glutamic acid. This is the first report of glutaminase producing M. guilliermondii isolated from the moromi of Thai soy sauce fermentation. The results suggested the potential application of M. guilliermondii EM2Y61 as starter yeast culture to increase l-glutamic acid during soy sauce fermentation.

  5. 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.

  6. A system for dual protein expression in Pichia pastoris and Escherichia coli.

    PubMed

    Lueking, A; Holz, C; Gotthold, C; Lehrach, H; Cahill, D

    2000-12-01

    We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning.

  7. An approach toward optimization of the influential growth determinants of opportunistic yeast isolate Pichia guilliermondii.

    PubMed

    Mukherjee, Suprabhat; Mukherjee, Niladri; Roy, Priya; Saini, Prasanta; Sinha Babu, Santi P

    2016-07-03

    The present study reports statistical optimization of growth conditions of an opportunistic fungal strain Pichia guilliermondii, isolated from the blood of patients suffering from bancroftian filariasis. Seven key determinants, namely, primary inoculums size (%), volume (mL) and pH of media, serum proportion, temperature (°C), incubation time (hr), and agitation speed (rpm) that influence in vitro growth of the pathogen were optimized statistically using response surface methodology (RSM). RSM with seven factors and two-level Box-Behnken design was employed for designing experimental run, prediction of case statistics, suitable exploration of quadratic response surfaces, and constructing a second-order polynomial equation. Analysis of variance (ANOVA) showed that primary inoculums size, volume of culture media, temperature, incubation time, and agitation speed exert most significant influence over fungal growth. The RSM study predicted that optimum fungal growth can be obtained using 10% primary inoculums size in 100 mL culture media with pH 6.0, 6.28% serum, 32.5°C temperature, and 24 hr of incubation, alongside agitation speed at 400 rpm. The desirability of the optimized growth model for P. guilliermondii is 99.123%, which indicated its accuracy and acceptability. Finally, the optimized growth module illustrated in the study could be useful in improving in vitro growth of clinically important P. guilliermondii.

  8. Efficient production and characterization of the sweet-tasting brazzein secreted by the yeast Pichia pastoris.

    PubMed

    Poirier, Nicolas; Roudnitzky, Natacha; Brockhoff, Anne; Belloir, Christine; Maison, Marie; Thomas-Danguin, Thierry; Meyerhof, Wolfgang; Briand, Loïc

    2012-10-03

    Brazzein is a small, heat-, and pH-stable sweet protein present in the fruits of the West African plant Pentadiplandra brazzeana Baillon. It exists in two forms differing in sweetness intensity. The major form, called pyrE-bra, contains a pyroglutamic acid at its N-terminus, while the minor form, called des-pyrE-bra, lacks this residue. Here we describe the heterologous expression in the methylotrophic yeast Pichia pastoris of two natural forms of brazzein, pyrE-bra and des-pyrE-bra, and an additional form, called Q1-bra, which is not naturally occurring in the fruit. Q1-bra differs from pyrE-bra in having a glutamine residue instead of pyrE at its N-terminus. Over an expression period of 6 days, we obtained approximately 90, 30, and 90 mg/L of purified recombinant pyrE-bra, Q1-bra, and des-pyrE-bra brazzein forms, respectively. Recombinant proteins were purified and submitted to mass spectrometry and (1)H NMR spectroscopy. The data indicate that the recombinant brazzein forms were properly folded. Moreover, they activated the human sweet receptor in vitro and evoked sweetness in vivo with properties similar to those of the two natural brazzein forms.

  9. Cloning and expression of an active aspartic proteinase from Mucor circinelloides in Pichia pastoris.

    PubMed

    Gama Salgado, Jose Antonio; Kangwa, Martin; Fernandez-Lahore, Marcelo

    2013-11-09

    Extracellular aspartic proteinase (MCAP) produced by Mucor circinelloides in solid state fermentations has been shown to possess milk clotting activity and represents a potential replacement for bovine chymosin in cheese manufacturing. Despite its prospects in the dairy industry, the molecular characteristics of this enzyme remain unknown. This work focuses on MCAP cloning and optimization of heterologous expression in Pichia pastoris, and characterization of the enzyme. The cloning of cDNA sequence encoding MCAP from M. circinelloides was performed using a fragment of approximately 1 kbp as a probe. The fragment was amplified using non-specific primers designed from the NDIEYYG and KNNYVVFN consensus motifs from aspartic proteinases of different fungi. Gene specific primers were designed to amplify a full-length cDNA using SMART™ RACE PCR. MCAP was expressed in P. pastoris under the control of the constitutive GAP promoter. It was shown that P. pastoris secreted non-glycosylated and glycosylated MCAPs with molecular weights of 33 and 37 kDa, respectively. A novel MCAP was expressed in P. pastoris and efficiently secreted into the culture medium. The expression of the heterologous proteins was significantly increased due to advantages in codon usage as compared to other expression systems. The results suggest that P. pastoris could be exploited as a safe production platform for the milk clotting enzyme.

  10. Cloning and expression of an active aspartic proteinase from Mucor circinelloides in Pichia pastoris

    PubMed Central

    2013-01-01

    Background Extracellular aspartic proteinase (MCAP) produced by Mucor circinelloides in solid state fermentations has been shown to possess milk clotting activity and represents a potential replacement for bovine chymosin in cheese manufacturing. Despite its prospects in the dairy industry, the molecular characteristics of this enzyme remain unknown. This work focuses on MCAP cloning and optimization of heterologous expression in Pichia pastoris, and characterization of the enzyme. Results The cloning of cDNA sequence encoding MCAP from M. circinelloides was performed using a fragment of approximately 1 kbp as a probe. The fragment was amplified using non-specific primers designed from the NDIEYYG and KNNYVVFN consensus motifs from aspartic proteinases of different fungi. Gene specific primers were designed to amplify a full-length cDNA using SMART™ RACE PCR. MCAP was expressed in P. pastoris under the control of the constitutive GAP promoter. It was shown that P. pastoris secreted non-glycosylated and glycosylated MCAPs with molecular weights of 33 and 37 kDa, respectively. Conclusion A novel MCAP was expressed in P. pastoris and efficiently secreted into the culture medium. The expression of the heterologous proteins was significantly increased due to advantages in codon usage as compared to other expression systems. The results suggest that P. pastoris could be exploited as a safe production platform for the milk clotting enzyme. PMID:24206750

  11. A Simplified and Efficient Process for Insulin Production in Pichia pastoris

    PubMed Central

    Polez, Sulena; Origi, Domenico; Zahariev, Sotir; Guarnaccia, Corrado; Tisminetzky, Sergio G.; Skoko, Nataša

    2016-01-01

    A significant barrier to insulin is affordability. In this manuscript we describe improvements to key steps in the insulin production process in Pichia pastoris that reduce cost and time. The strategy for recovery and processing of human insulin precursor has been streamlined to two steps from bioreactor to the transpeptidation reaction. In the first step the insulin precursor secreted during the methanol induction phase is recovered directly from the culture broth using Tangential Flow Filtration with a Prostak™ module eliminating the laborious and time-consuming multi-step clarification, including centrifugation. In the second step the protein is applied at very high loadings on a cation exchange resin and eluted in a mixture of water and ethanol to obtain a concentrated insulin precursor, suitable for use directly in the transpeptidation reaction. Overall the yield from insulin precursor to human insulin was 51% and consisted of three purification chromatography steps. In addition we describe a method for recovery of the excess of H-Thr(tBu)-OtBu from the transpeptidation reaction mixture, one of the more costly reagents in the process, along with its successful reuse. PMID:27907132

  12. 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.

  13. Heterologous expression of a Penicillium purpurogenum exo-arabinanase in Pichia pastoris and its biochemical characterization.

    PubMed

    Mardones, Wladimir; Callegari, Eduardo; Eyzaguirre, Jaime

    2015-12-01

    Arabinan is a component of pectin, which is one of the polysaccharides present in lignocelluose. The enzymes degrading the main chain of arabinan are the endo- (EC 3.2.1.99) and exo-arabinanases (3.2.1.-). Only three exo-arabinanases have been biochemically characterized; they belong to glycosyl hydrolase family 93. In this work, the cDNA of an exo-arabinanase (Arap2) from Penicillium purpurogenum has been heterologously expressed in Pichia pastoris. The gene is 1310 bp long, has three introns and codes for a protein of 380 amino acid residues; the mature protein has a calculated molecular mass of 39 823 Da. The heterologously expressed Arap2 has a molecular mass in the range of 60-80 kDa due to heterogeneous glycosylation. The enzyme is active on debranched arabinan with optimum pH of 4-5.5 and optimal temperature of 40 °C, and has an exo-type action mode, releasing arabinobiose from its substrates. The expression profile of arap2 in corncob and sugar beet pulp follows a different pattern and is not related to the presence of arabinan. This is the first exo-arabinanase studied from P. purpurogenum and the first expressed in yeast. The availability of heterologous Arap2 may be useful for biotechnological applications requiring acidic conditions. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  14. The Possible Mechanisms Involved in Degradation of Patulin by Pichia caribbica

    PubMed Central

    Zheng, Xiangfeng; Yang, Qiya; Zhang, Hongyin; Cao, Jing; Zhang, Xiaoyun; Apaliya, Maurice Tibiru

    2016-01-01

    In this work, we examined the mechanisms involved in the degradation of patulin by Pichia caribbica. Our results indicate that cell-free filtrate of P. caribbica reduced patutlin content. The heat-killed cells could not degrade patulin. However, the live cells significantly reduced the concentration of the patulin. In furtherance to this, it was observed that patulin was not detected in the broken yeast cells and cell wall. The addition of cycloheximide to the P. caribbica cells decreased the capacity of degradation of patulin. Proteomics analyses revealed that patulin treatment resulted in an upregulated protein which was involved in metabolism and stress response processes. Our results suggested that the mechanism of degradation of patulin by P. caribbica was not absorption; the presence of patulin can induce P. caribbica to produce associated intracellular and extracellular enzymes, both of which have the ability to degrade patulin. The result provides a new possible method that used the enzymes produced by yeast to detoxify patulin in food and feed. PMID:27735830

  15. Codon optimisation improves the expression of Trichoderma viride sp. endochitinase in Pichia pastoris

    PubMed Central

    Yu, Ping; Yan, Yuan; Gu, Qing; Wang, Xiangyang

    2013-01-01

    The mature cDNA of endochitinase from Trichoderma viride sp. was optimised based on the codon bias of Pichia pastoris GS115 and synthesised by successive PCR; the sequence was then transformed into P. pastoris GS115 via electroporation. The transformant with the fastest growth rate on YPD plates containing 4 mg/mL G418 was screened and identified. This transformant produced 23.09 U/mL of the recombinant endochitinase, a 35% increase compared to the original strain bearing the wild-type endochitinase cDNA. The recombinant endochitinase was sequentially purified by ammonia sulphate precipitation, DE-52 anion-exchange chromatography and Sephadex G-100 size-exclusion chromatography. Thin-layer chromatography indicated that the purified endochitinase could hydrolyse chito-oligomers or colloidal chitin to generate diacetyl-chitobiose (GlcNAc)2 as the main product. This study demonstrates (1) a means for high expression of Trichoderma viride sp. endochitinase in P. pastoris using codon optimisation and (2) the preparation of chito-oligomers using endochitinase. PMID:24154717

  16. Metabolite profiles of the biocontrol yeast Pichia anomala J121 grown under oxygen limitation.

    PubMed

    Fredlund, E; Broberg, A; Boysen, M E; Kenne, L; Schnürer, J

    2004-04-01

    The biocontrol yeast Pichia anomala J121 prevents mould growth during the storage of moist grain under low oxygen/high carbon dioxide conditions. Growth and metabolite formation of P. anomala was analyzed under two conditions of oxygen limitation: (a) initial aerobic conditions with restricted oxygen access during the growth period and (b) initial microaerobic conditions followed by anaerobiosis. Major intra- and extracellular metabolites were analyzed by high-resolution magic-angle spinning (HR-MAS) NMR and HPLC, respectively. HR-MAS NMR allows the analysis of major soluble compounds inside intact cells, without the need for an extraction step. Biomass production was higher in treatment (A), whereas the specific ethanol production rate during growth on glucose was similar in both treatments. This implies that oxygen availability affected the respiration and not the fermentation of the yeast. Following glucose depletion, ethanol was oxidized to acetate in treatment (A), but continued to be produced in (B). Arabitol accumulated in the culture substrate of both treatments, whereas glycerol only accumulated in treatment (B). Trehalose, arabitol, and glycerol accumulated inside the cells in both treatments. The levels of these metabolites were generally significantly higher in treatment (B) than in (A), indicating their importance for P. anomala during severe oxygen limitation/anaerobic conditions.

  17. rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization

    PubMed Central

    Ciaccio, Chiara; Gambacurta, Alessandra; Sanctis, Giampiero DE; Spagnolo, Domenico; Sakarikou, Christina; Petrella, Giovanni; Coletta, Massimo

    2006-01-01

    A Pichia pastoris expression system has for the first time been successfully developed to produce rhEPO (recombinant human eosinophil peroxidase). The full-length rhEPO coding sequence was cloned into the pPIC9 vector in frame with the yeast α-Factor secretion signal under the transcriptional control of the AOX (acyl-CoA oxidase) promoter, and transformed into P. pastoris strain GS115. Evidence for the production of rhEPO by P. pastoris as a glycosylated dimer precursor of approx. 80 kDa was determined by SDS/PAGE and gel filtration chromatography. Recombinant hEPO undergoes proteolytic processing, similar to that in the native host, to generate two chains of approx. 50 and 20 kDa. A preliminary biochemical characterization of purified rhEPO demonstrated that the spectral and kinetic properties of the recombinant wild-type EPO are comparable with those of the native enzyme and are accompanied by oxidizing activity towards several physiological anionic substrates such as SCN−, Br− and Cl−. On the basis of the estimated Km and kcat values it is evident that the pseudohalide SCN− is the most specific substrate for rhEPO, consistent with the catalytic properties of other mammalian EPOs purified from blood. PMID:16396635

  18. Expression and Characterization of the RKOD DNA Polymerase in Pichia pastoris

    PubMed Central

    Wang, Fei; Li, Shuntang; Zhao, Hui; Bian, Lu; Chen, Liang; Zhang, Zhen; Zhong, Xing; Ma, Lixin; Yu, Xiaolan

    2015-01-01

    The present study assessed high-level expression of the KOD DNA polymerase in Pichia pastoris. Thermococcus kodakaraensis KOD1 is a DNA polymerase that is widely used in PCR. The DNA coding sequence of KOD was optimized based on the codon usage bias of P. pastoris and synthesized by overlapping PCR, and the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus was fused to the C-terminus of KOD. The resulting novel gene was cloned into a pHBM905A vector and introduced into P. pastoris GS115 for secretory expression. The yield of the target protein reached approximately 250 mg/l after a 6-d induction with 1% (v/v) methanol in shake flasks. This yield is much higher than those of other DNA polymerases expressed heterologously in Escherichia coli. The recombinant enzyme was purified, and its enzymatic features were studied. Its specific activity was 19,384 U/mg. The recombinant KOD expressed in P. pastoris exhibited excellent thermostability, extension rate and fidelity. Thus, this report provides a simple, efficient and economic approach to realize the production of a high-performance thermostable DNA polymerase on a large scale. This is the first report of the expression in yeast of a DNA polymerase for use in PCR. PMID:26134129

  19. 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.

  20. Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris.

    PubMed

    Choi, Byung-Kwon; Warburton, Shannon; Lin, Heping; Patel, Rohan; Boldogh, Istvan; Meehl, Michael; Meehl, Meehl; d'Anjou, Marc; Pon, Liza; Stadheim, Terrance A; Sethuraman, Natarajan

    2012-08-01

    Yeast is capable of performing posttranslational modifications, such as N- or O-glycosylation. It has been demonstrated that N-glycans play critical biological roles in therapeutic glycoproteins by modulating pharmacokinetics and pharmacodynamics. However, N-glycan sites on recombinant glycoproteins produced in yeast can be underglycosylated, and hence, not completely occupied. Genomic homology analysis indicates that the Pichia pastoris oligosaccharyltransferase (OST) complex consists of multiple subunits, including OST1, OST2, OST3, OST4, OST5, OST6, STT3, SWP1, and WBP1. Monoclonal antibodies produced in P. pastoris show that N-glycan site occupancy ranges from 75-85 % and is affected mainly by the OST function, and in part, by process conditions. In this study, we demonstrate that N-glycan site occupancy of antibodies can be improved to greater than 99 %, comparable to that of antibodies produced in mammalian cells (CHO), by overexpressing Leishmania major STT3D (LmSTT3D) under the control of an inducible alcohol oxidase 1 (AOX1) promoter. N-glycan site occupancy of non-antibody glycoproteins such as recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was also significantly improved, suggesting that LmSTT3D has broad substrate specificity. These results suggest that the glycosylation status of recombinant proteins can be improved by heterologous STT3 expression, which will allow for the customization of therapeutic protein profiles.

  1. Recombinant expression of human cathelicidin (hCAP18/LL-37) in Pichia pastoris.

    PubMed

    Hong, In-Pyo; Lee, Sung-Jae; Kim, Yong-Seok; Choi, Shin-Geon

    2007-01-01

    The constitutive expression of human cathelicidin LL-37 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris. An LL-37 cDNA clone was amplified by PCR using human fetal cDNA library as template. The 111 bp fragment encoding mature LL-37 gene was subcloned into pGAPZ-E, an episomal form of the pGAPZB vector incorporating PARS1. It was then transformed into the P. pastoris X-33 strain for intracellular expression. A small peptide with a molecular mass of about 5 kDa was detected by 17% peptide-PAGE analysis. The recombinant LL-37 peptide was purified from the gel and its amino acid sequence was determined by LC-ESI-MS/MS analysis. The initiating amino acid, methionine, was still attached to the N-terminal region of recombinant LL-37. LL-37 crude extract from P. pastoris showed an antimicrobial activity against Micrococcus luteus as the test strain. The successful expression of human LL-37 indicates that the system may be applicable to the expression of other human defensins without resorting to fusion protein constructions.

  2. Dissociation and purification of the endogenous membrane-bound Vo complex from Pichia pastoris.

    PubMed

    Li, Sumei; Hong, Tao; Wang, Kun; Lu, Yinghong; Zhou, Min

    2017-10-01

    Most proteins occur and function in complexes rather than as isolated entities in membranes. In most cases macromolecules with multiple subunits are purified from endogenous sources. In this study, an endogenous membrane-protein complex was obtained from Pichia pastoris, which can be grown at high densities to significantly improve the membrane protein yield. We successfully isolated the membrane-bound Vo complex of V-ATPase from P. pastoris using a fusion FLAG tag attached to the C-terminus of subunit a to generate the vph-tag strain, which was used for dissociation and purification. After FLAG affinity and size exclusion chromatography purification, the production quantity and purity of the membrane-bound Vo complex was 20 μg l(-1) and >98%, respectively. The subunits of the endogenous membrane-bound Vo complex observed in P. pastoris were similar to those obtained from S. cerevisiae, as demonstrated by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Therefore, successful dissociation and purification of the membrane-bound Vo complex at a high purity and sufficient quantity was achieved via a rapid and simple procedure that can be used to obtain the endogenous membrane-protein complexes from P. pastoris. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production

    PubMed Central

    Nocon, Justyna; Steiger, Matthias G.; Pfeffer, Martin; Sohn, Seung Bum; Kim, Tae Yong; Maurer, Michael; Rußmayer, Hannes; Pflügl, Stefan; Ask, Magnus; Haberhauer-Troyer, Christina; Ortmayr, Karin; Hann, Stephan; Koellensperger, Gunda; Gasser, Brigitte; Lee, Sang Yup; Mattanovich, Diethard

    2014-01-01

    The production of recombinant proteins is frequently enhanced at the levels of transcription, codon usage, protein folding and secretion. Overproduction of heterologous proteins, however, also directly affects the primary metabolism of the producing cells. By incorporation of the production of a heterologous protein into a genome scale metabolic model of the yeast Pichia pastoris, the effects of overproduction were simulated and gene targets for deletion or overexpression for enhanced productivity were predicted. Overexpression targets were localized in the pentose phosphate pathway and the TCA cycle, while knockout targets were found in several branch points of glycolysis. Five out of 9 tested targets led to an enhanced production of cytosolic human superoxide dismutase (hSOD). Expression of bacterial β-glucuronidase could be enhanced as well by most of the same genetic modifications. Beneficial mutations were mainly related to reduction of the NADP/H pool and the deletion of fermentative pathways. Overexpression of the hSOD gene itself had a strong impact on intracellular fluxes, most of which changed in the same direction as predicted by the model. In vivo fluxes changed in the same direction as predicted to improve hSOD production. Genome scale metabolic modeling is shown to predict overexpression and deletion mutants which enhance recombinant protein production with high accuracy. PMID:24853352

  4. Expression and Characterization of Windmill Palm Tree (Trachycarpus fortunei) Peroxidase by Pichia pastoris.

    PubMed

    Wen, Boting; Baker, Margaret R; Zhao, Hongwei; Cui, Zongjun; Li, Qing X

    2017-06-14

    Currently, commercial plant peroxidases are all native and are isolated from plants such as horseradish and soybean. No recombinant plant peroxidase products have been available on the commercial market. The gene encoding peroxidase was cloned from windmill palm tree leaves. The codon-optimized gene was transformed into Pichia pastoris for expression. The recombinant windmill palm tree peroxidase (rWPTP) expressed by P. pastoris showed high stability under pH 2-10 and temperatures up to 70 °C to many metallic salts and organic solvents. The substrate specificity of WPTP was determined, and among the substrates tested, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was most suitable for WPTP. The Michaelis constants with the substrates H2O2 and ABTS were 4.6 × 10(-4) and 1.6 × 10(-4) M, respectively. The rWPTP expressed in P. pastoris may be a suitable enzyme for the biosynthesis of polymers because of its high stability and activity under acidic conditions.

  5. Production of Equine Infectious Anemia Virus (EIAV) antigen in Pichia pastoris.

    PubMed

    de Arruda Coutinho, Luciana Cavalcanti; de Jesus, André Luiz Santos; de Paiva Fontes, Karin Florêncio Lins; Coimbra, Eliane Campos; Mariz, Filipe Colaço; de Freitas, Antonio Carlos; de Cássia Carvalho Maia, Rita; de Castro, Roberto Soares

    2013-08-01

    Equine Infectious Anemia (EIA) is a persistent lentivirus infection of horses which causes a chronic clinical condition with worldwide importance in veterinary medicine. The p26 protein is usually prepared for use as an antigen in serological tests for EIA diagnosis since it is a well-conserved gene sequence and very immunogenic. In view of the ability of yeast to make post-translational modifications of proteins, this study was carried out to allow Pichia pastoris to be used for the expression of a synthetic codon-optimized EIAV p26 gene. The gene was cloned into pPICZαA vector after appropriate enzymatic digestion. P. pastoris clones transformed with the pPICZαAp26 construction were induced to produce the recombinant p26 protein (rp26) under the regulation of alcohol oxidase 1 promoter by adding methanol to the culture medium. The p26 gene expression was detected by RT-PCR and the production of rp26 was confirmed by dot blotting, Western blotting, ELISA and AGID. The P. pastoris expression system was capable of producing a functional EIAV p26 protein that can be used directly in the functionality tests without requiring laborious purification or recovery steps. This is the first reported study of EIAV p26 protein production in yeast cells. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Δ(9)-Tetrahydrocannabinolic acid synthase production in Pichia pastoris enables chemical synthesis of cannabinoids.

    PubMed

    Lange, Kerstin; Schmid, Andreas; Julsing, Mattijs K

    2015-10-10

    Δ(9)-Tetrahydrocannabinol (THC) is of increasing interest as a pharmaceutical and bioactive compound. Chemical synthesis of THC uses a laborious procedure and does not satisfy the market demand. The implementation of biocatalysts for specific synthesis steps might be beneficial for making natural product availability independent from the plant. Δ(9)-Tetrahydrocannabinolic acid synthase (THCAS) from C. sativa L. catalyzes the cyclization of cannabigerolic acid (CBGA) to Δ(9)-tetrahydrocannabinolic acid (THCA), which is non-enzymatically decarboxylated to THC. We report the preparation of THCAS in amounts sufficient for the biocatalytic production of THC(A). Active THCAS was most efficiently obtained from Pichia pastoris. THCAS was produced on a 2L bioreactor scale and the enzyme was isolated by single-step chromatography with a specific activity of 73Ug(-1)total protein. An organic/aqueous two-liquid phase setup for continuous substrate delivery facilitated in situ product removal. In addition, THCAS activity in aqueous environments lasted for only 20min whereas the presence of hexane stabilized the activity over 3h. In conclusion, production of THCAS in P. pastoris Mut(S) KM71 KE1, subsequent isolation, and its application in a two-liquid phase setup enables the synthesis of THCA on a mg scale.

  7. 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.

  8. NMR monitoring of accumulation and folding of 15N-labeled protein overexpressed in Pichia pastoris.

    PubMed

    de Lamotte, F; Boze, H; Blanchard, C; Klein, C; Moulin, G; Gautier, M F; Delsuc, M A

    2001-07-01

    Postgenomic studies have led to an increasing demand for isotope-labeled proteins. We present a method for producing large quantities of truly native (15)N-labeled protein. Based on the secretion capabilities of the yeast Pichia pastoris, the recombinant protein is easily purified in a single step as it is secreted. Control of all nitrogen sources permits very high labeling yields. As a result, accumulation and folding of the recombinant protein can be monitored by heteronuclear NMR without purification. Comparison of sample spectra with the spectrum of the purified recombinant protein allows detection of the secreted protein in the culture and monitoring of its folding, from the start of the induction phase. The detection limit for a (15)N-labeled protein is estimated as 20 microM and corresponds, for a 10-kDa protein, to a load of 40 mg/liter in the fermentor. This concentration is reached by most reported preparations in P. pastoris. Further concentration by ultrafiltration would compensate for lower production. This procedure may be useful in many structural genomics and combinatorial chemistry screening projects where most protein productions meet the requirements for this method. Copyright 2001 Academic Press.

  9. A toolbox of endogenous and heterologous nuclear localization sequences for the methylotrophic yeast Pichia pastoris

    PubMed Central

    Weninger, Astrid; Glieder, Anton; Vogl, Thomas

    2015-01-01

    Nuclear localization sequences (NLSs) are required for the import of proteins in the nucleus of eukaryotes. However many proteins from bacteria or bacteriophages are used for basic studies in molecular biology, to generate synthetic genetic circuits or for genome editing applications. Prokaryotic recombinases, CRISPR-associated proteins such as Cas9 or bacterial and viral polymerases require efficient NLSs to function in eukaryotes. The yeast Pichia pastoris is a widely used expression platform for heterologous protein production, but molecular tools such as NLSs are limited. Here we have characterized a set of 10 NLSs for P. pastoris, including the first endogenous NLSs (derived from P. pastoris proteins) and commonly used heterologous NLSs. The NLSs were evaluated by fusing them in N- and C-terminal position to an enhanced green fluorescent protein showing pronounced differences in fluorescence levels and nuclear targeting. Thereby we provide a set of different NLSs that can be applied to optimize the nuclear import of heterologous proteins in P. pastoris, paving the way for the establishment of intricate synthetic biology applications. PMID:26347503

  10. Expression of soluble recombinant transglutaminase from Zea mays in Pichia pastoris.

    PubMed

    Li, Hongbo; Zhang, Lanwei; Cui, Yanhua; Luo, Xue; Xue, Chaohui; Wang, Shumei

    2013-05-01

    Transglutaminases (TGases) catalyze post-translational protein modifications by ε-(γ-glutamyl) links and covalent amide bonds. In plant, this enzyme is poorly studied and only the Zea mays TGase gene (tgz) has been cloned. The tgz had been expressed in Escherichia coli, but the recombinant protein was mainly present in inclusion bodies. Therefore, to obtain active, soluble protein, we optimized its coding sequence according to the codon bias of Pichia pastoris and synthesized the sequence with SOEing-PCR. The optimized fragment was successfully transformed into P. pastoris GS115 by electroporation. The optimal conditions for expression were under a final concentration of 0.5 % methanol and a time-course of 96 h. The synthesized recombinant Zea mays transglutaminase (TGZs) was purified by affinity method, its production was 4.4 mg/L, and the specific activity was 0.889 U/mg under optimal expression condition. Optimal activity for TGZs was observed at 37 °C and a pH of 8.0, respectively. The cross-linking reaction of TGZs to the casein was studied, and the result was same as the reaction of casein by microbial transglutaminase. These results indicated that an effective procedure for expressing and purifying TGZs in P. pastoris GS115 was established.

  11. Antioxidant and Neuroprotective Effects of Doenjang Prepared with Rhizopus, Pichia, and Bacillus

    PubMed Central

    Kang, Su Jin; Seo, Ji Yeon; Cho, Kye Man; Lee, Chang Kwon; Kim, Jeong Hwan; Kim, Jong-Sang

    2016-01-01

    A new type of doenjang was manufactured by mixing soaked soybean, koji (Rhizopus oryzae), cheonggukjang (Bacillus amyloliquefaciens MJ1-4 and B. amyloliquefaciens EMD17), and Pichia farinosa SY80 as a yeast, salt, and water, followed by fermentation with koji that was made by fermenting whole wheat with R. oryzae. The mixed culture doenjang was designed to have a more palatable flavor and stronger biological activities than the conventional product. The extract of mixed culture doenjang showed higher antioxidant activity than the commercial doenjang as evaluated by the ferric reducing antioxidant power assay although it was not significantly different from the commercial product in 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activities. Further, the mixed culture doenjang reduced intracellular reactive oxygen species levels and protected cells from glutamate-induced cytotoxicity more efficiently in human hippocampal HT22 neuroblastoma cells than the commercial doenjang. In conclusion, a newly-developed mixed culture doenjang had a strong antioxidant activity in vitro and cultured cell model systems, exhibited a potential to prevent oxidative stress-associated disorders although animal and clinical studies are needed to confirm its in vivo efficacy. PMID:27752498

  12. 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.

  13. Macromolecular antimicrobial glycoprotein, achacin, expressed in a methylotrophic yeast Pichia pastoris.

    PubMed

    Ogawa, M; Nakamura, S; Atsuchi, T; Tamiya, T; Tsuchiya, T; Nakai, S

    1999-04-01

    A cDNA encoding achacin, an antimicrobial glycoprotein from the body surface mucus of giant African snail Achacina fulica Férussac, was expressed in a methylotrophic yeast, Pichia pastoris, and recombinant achacin (rAch) was secreted in yeast minimal medium in a polyglycosylated form with 80 kDa. Carbohydrate analysis revealed that the glycosylated moiety of rAch was composed of 50 mol mannose and 2 mol N-acetylglucosamine residues. Antimicrobial activity using Escherichia coli and Staphylococcus aureus showed that the rAch had a behavior similar to its native counterpart. The rAch showed so wide an antimicrobial spectrum that 0.1 mg/ml rAch inhibited the growth of Pseudomonas fluorescens, Staphylococcus epidermidis, and Streptococcus faecalis in addition to E. coli and S. aureus, whereas it did not appreciably affect the growth of Proteus mirabilis, Bacillus cereus and Micrococcus luteus. The rAch was also effective in preventing growth of Vibrio anguillarum and Vibrio parahaemolyticus. The results suggested that the rAch had great potential of using as an antimicrobial agent.

  14. Surface Display and Bioactivity of Bombyx mori Acetylcholinesterase on Pichia pastoris

    PubMed Central

    He, Yong-Sheng; Beier, Ross C.; Sun, Yuan-Ming; Xu, Zhen-Lin; Wu, Wei-Jian; Shen, Yu-Dong; Xiao, Zhi-Li; Lai, Li-Na; Wang, Hong; Yang, Jin-Yi

    2013-01-01

    A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC50 of 4.17×10−8 M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity. PMID:23940577

  15. Surface display and bioactivity of Bombyx mori acetylcholinesterase on Pichia pastoris.

    PubMed

    Dong, Jie-Xian; Xie, Xi; He, Yong-Sheng; Beier, Ross C; Sun, Yuan-Ming; Xu, Zhen-Lin; Wu, Wei-Jian; Shen, Yu-Dong; Xiao, Zhi-Li; Lai, Li-Na; Wang, Hong; Yang, Jin-Yi

    2013-01-01

    A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC50 of 4.17×10(-8) M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity.

  16. 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

  17. Binding of DC-SIGN to glycoproteins expressed in glycoengineered Pichia pastoris.

    PubMed

    Cukan, Michael C; Hopkins, Daniel; Burnina, Irina; Button, Michelle; Giaccone, Erin; Houston-Cummings, Nga Rewa; Jiang, Youwei; Li, Fang; Mallem, Muralidhar; Mitchell, Teresa; Moore, Renée; Nylen, Adam; Prinz, Bianka; Rios, Sandra; Sharkey, Nathan; Zha, Dongxing; Hamilton, Stephen; Li, Huijuan; Stadheim, Terrance A

    2012-12-14

    Previous studies have shown that glycoproteins expressed in wild-type Pichia pastoris bind to Dendritic cell-SIGN (DC-Specific Intercellular adhesion molecule-3 Grabbing Nonintegrin), a mannose-binding receptor found on dendritic cells in peripheral tissues which is involved in antigen presentation and the initiation of an immune response. However, the binding of DC-SIGN to glycoproteins purified from P. pastoris strains engineered to express humanized N- and O-linked glycans has not been tested to date. In this study, the binding of glycoproteins with specific high-mannose or human N- and O-linked glycan structures to DC-SIGN was tested. Proteins with humanized N-glycans including Man5 structures and O-glycans (up to as many as 24) with single mannose chain length showed DC-SIGN binding that was comparable to that measured for a CHO-produced IgG1 which lacks O-linked mannose. Glycoproteins with wild-type N-glycans and mannotriose and higher O-glycans bound to DC-SIGN in a manner that was strongly inhibited by either the use of enzymatic N-deglycosylation or sodium meta-periodate oxidation. Mannan purified from humanized P. pastoris also showed lower ability to inhibit DC-SIGN binding to glycoproteins with wild type fungal glycosylation than mannan purified from wild type strains. This study shows that humanized P. pastoris can produce glycoproteins that do not bind to DC-SIGN.

  18. 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

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

    PubMed Central

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

  20. Peroxisome Degradation by Microautophagy in Pichia pastoris: Identification of Specific Steps and Morphological Intermediates

    PubMed Central

    Sakai, Yasuyoshi; Koller, Antonius; Rangell, Linda K.; Keller, Gilbert A.; Subramani, Suresh

    1998-01-01

    We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor–product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version [GFP])-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis. PMID:9566964

  1. Heterologous expression of Pycnoporus cinnabarinus cellobiose dehydrogenase in Pichia pastoris and involvement in saccharification processes

    PubMed Central

    2011-01-01

    Background Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by lignocellulose-degrading fungi including Pycnoporus cinnabarinus. We investigated the cellulolytic system of P. cinnabarinus, focusing on the involvement of CDH in the deconstruction of lignocellulosic biomass. Results First, P. cinnabarinus growth conditions were optimized for CDH production. Following growth under cellulolytic conditions, the main components secreted were cellulases, xylanases and CDH. To investigate the contribution of P. cinnabarinus secretome in saccharification processes, the Trichoderma reesei enzymatic cocktail was supplemented with the P. cinnabarinus secretome. A significant enhancement of the degradation of wheat straw was observed with (i) the production of a large amount of gluconic acid, (ii) increased hemicellulose degradation, and (iii) increased overall degradation of the lignocellulosic material. P. cinnabarinus CDH was heterologously expressed in Pichia pastoris to obtain large amounts of pure enzyme. In a bioreactor, the recombinant CDH (rCDH) expression level reached 7800 U/L. rCDH exhibited values of biochemical parameters similar to those of the natural enzyme, and was able to bind cellulose despite the absence of a carbohydrate-binding module (CBM). Following supplementation of purified rCDH to T. reesei enzymatic cocktail, formation of gluconic acid and increased hemicellulose degradation were observed, thus confirming the previous results observed with P. cinnabarinus secretome. Conclusions We demonstrate that CDH offers an attractive tool for saccharification process enhancement due to gluconic acid production from raw lignocellulosic material. PMID:22204630

  2. The Membrane Dynamics of Pexophagy Are Influenced by Sar1p in Pichia pastoris

    PubMed Central

    Schroder, Laura A.; Ortiz, Michael V.

    2008-01-01

    Several Sec proteins including a guanosine diphosphate/guanosine triphosphate exchange factor for Sar1p have been implicated in autophagy. In this study, we investigated the role of Sar1p in pexophagy by expressing dominant-negative mutant forms of Sar1p in Pichia pastoris. When expressing sar1pT34N or sar1pH79G, starvation-induced autophagy, glucose-induced micropexophagy, and ethanol-induced macropexophagy are dramatically suppressed. These Sar1p mutants did not affect the initiation or expansion of the sequestering membranes nor the trafficking of Atg11p and Atg9p to these membranes during micropexophagy. However, the lipidation of Atg8p and assembly of the micropexophagic membrane apparatus, which are essential to complete the incorporation of the peroxisomes into the degradative vacuole, were inhibited when either Sar1p mutant protein was expressed. During macropexophagy, the expression of sar1pT34N inhibited the formation of the pexophagosome, whereas sar1pH79G suppressed the delivery of the peroxisome from the pexophagosome to the vacuole. The pexophagosome contained Atg8p in wild-type cells, but in cells expressing sar1pH79G these organelles contain both Atg8p and endoplasmic reticulum components as visualized by DsRFP-HDEL. Our results demonstrate key roles for Sar1p in both micro- and macropexophagy. PMID:18768759

  3. Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris.

    PubMed Central

    Cregg, J M; Madden, K R; Barringer, K J; Thill, G P; Stillman, C A

    1989-01-01

    In Pichia pastoris, alcohol oxidase (AOX) is the first enzyme in the methanol utilization pathway and is encoded by two genes, AOX1 and AOX2. The DNA and predicted amino acid sequences of the protein-coding portions of the genes are closely homologous, whereas flanking sequences share no homology. The functional roles of AOX1 and AOX2 in the metabolism of methanol were examined. Studies of strains with disrupted AOX genes revealed that AOX1 was the major source of methanol-oxidizing activity in methanol-grown P. pastoris. The results of two types of experiments each suggested that the difference in AOX activity contributed by the two genes was a consequence of sequences located 5' of the protein-coding portions of the genes. First, the coding portion of AOX2 was able to functionally substitute for that of AOX1 when placed under the control of AOX1 regulatory sequences. Second, when labeled oligonucleotide probes specific for the 5' nontranslated region of each gene were used, it was apparent that the steady-state level of AOX1 mRNA was much higher than that of AOX2. Except for the difference in the amount of mRNA present, the two genes appeared to be regulated in the same manner. A physiological reason for the existence of AOX2 was sought but was not apparent. Images PMID:2657390

  4. Heterologous expression of the plant cysteine protease bromelain and its inhibitor in Pichia pastoris.

    PubMed

    Luniak, Nora; Meiser, Peter; Burkart, Sonja; Müller, Rolf

    2017-01-01

    Expression of proteases in heterologous hosts remains an ambitious challenge due to severe problems associated with digestion of host proteins. On the other hand, proteases are broadly used in industrial applications and resemble promising drug candidates. Bromelain is an herbal drug that is medicinally used for treatment of oedematous swellings and inflammatory conditions and consists in large part of proteolytic enzymes. Even though various experiments underline the requirement of active cysteine proteases for biological activity, so far no investigation succeeded to clearly clarify the pharmacological mode of action of bromelain. The potential role of proteases themselves and other molecules of this multi-component extract currently remain largely unknown or ill defined. Here, we set out to express several bromelain cysteine proteases as well as a bromelain inhibitor molecule in order to gain defined molecular entities for subsequent studies. After cloning the genes from its natural source Ananas comosus (pineapple plant) into Pichia pastoris and subsequent fermentation and purification, we obtained active protease and inhibitor molecules which were subsequently biochemically characterized. Employing purified bromelain fractions paves the way for further elucidation of pharmacological activities of this natural product. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:54-65, 2017. © 2016 American Institute of Chemical Engineers.

  5. Differential gene expression in recombinant Pichia pastoris analysed by heterologous DNA microarray hybridisation

    PubMed Central

    Sauer, Michael; Branduardi, Paola; Gasser, Brigitte; Valli, Minoska; Maurer, Michael; Porro, Danilo; Mattanovich, Diethard

    2004-01-01

    Background Pichia pastoris is a well established yeast host for heterologous protein expression, however, the physiological and genetic information about this yeast remains scanty. The lack of a published genome sequence renders DNA arrays unavailable, thereby hampering more global investigations of P. pastoris from the beginning. Here, we examine the suitability of Saccharomyces cerevisiae DNA microarrays for heterologous hybridisation with P. pastoris cDNA. Results We could show that it is possible to obtain new and valuable information about transcriptomic regulation in P. pastoris by probing S. cerevisiae DNA microarrays. The number of positive signals was about 66 % as compared to homologous S. cerevisiae hybridisation, and both the signal intensities and gene regulations correlated with high significance between data obtained from P. pastoris and S. cerevisiae samples. The differential gene expression patterns upon shift from glycerol to methanol as carbon source were investigated in more detail. Downregulation of TCA cycle genes and a decrease of genes related to ribonucleotide and ribosome synthesis were among the major effects identified. Conclusions We could successfully demonstrate that heterologous microarray hybridisations allow deep insights into the transcriptomic regulation processes of P. pastoris. The observed downregulation of TCA cycle and ribosomal synthesis genes correlates to a significantly lower specific growth rate during the methanol feed phase. PMID:15610561

  6. Purification and immunogenicity study of human papillomavirus 58 virus-like particles expressed in Pichia pastoris.

    PubMed

    Jiang, Zijun; Tong, Guangjie; Cai, Beibei; Xu, Yihan; Lou, Jueren

    2011-12-01

    Two human papillomavirus (HPV) prophylactic vaccines are currently available in the market: Gardasil and Cervarix. These two vaccines work against tumor high-risk subtypes HPV 16 and HPV 18. However, they do not include other high-risk subtypes such as HPV 58. Epidemiological research in China shows that HPV 58 is a prevalent high-risk subtype, second only to HPV 16 and HPV 18. Thus, for cervical cancer prevention in China, developing a vaccine against HPV 58 is necessary. In this study, HPV 58 virus-like particles (VLPs) were expressed in the Pichia pastoris, and subsequently purified through pretreatment and a three-step purification process consisting of strong cation exchange chromatography, size-exclusion chromatography, and hydroxyapatite chromatography. The highly purified HPV 58 VLPs were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, electron microscopy, dynamic laser scattering, and ultracentrifugation. The purified VLPs were used to immunize mice to test their ability to induce humoral immunity. Enzyme-linked immunosorbent assays were performed on the sera of the immunized mice and significantly high anti-HPV 58 VLP antibody titers were observed. The immunogenicity study demonstrates that the purified HPV 58 VLPs are HPV vaccine candidates.

  7. Characterization of Trichoderma reesei endoglucanase II expressed heterologously in Pichia pastoris for better biofinishing and biostoning.

    PubMed

    Samanta, Sutanu; Basu, Asitava; Halder, Umesh Chandra; Sen, Soumitra Kumar

    2012-06-01

    The endoglucanase II of Trichoderma reesei is considered the most effective enzyme for biofinishing cotton fabrics and biostoning denim garments. However, the commercially available preparation of endoglucanase II is usually mixed with other cellulase components, especially endoglucanase I, resulting in hydrolysis and weight loss of garments during biofinishing and biostoning. We thus isolated the endoglucanase II gene from T. reesei to express this in Pichia pastoris, under the control of a methanol-inducible AOX1 promoter, to avoid the presence of other cellulase components. A highly expressible Mut(+) transformant was selected and its expression in BMMH medium was found most suitable for the production of large amounts of the recombinant protein. Recombinant endoglucanase II was purified to electrophoretic homogeneity, and functionally characterized by activity staining. The specific activity of recombinant endoglucanase II was found to be 220.57 EU/mg of protein. Purified recombinant endoglucanase II was estimated to have a molecular mass of 52.8 kDa. The increase in molecular mass was likely due to hyperglycosylation. Hyperglycosylation of recombinant endoglucanase II secreted by P. pastoris did not change the temperature or pH optima as compared to the native protein, but did result in increased thermostability. Kinetic analysis showed that recombinant endoglucanase was most active against amorphous cellulose, such as carboxymethyl cellulose, for which it also had a high affinity.

  8. Overexpression of Escherichia coli phytase in Pichia pastoris and its biochemical properties.

    PubMed

    Tai, Hsueh-Ming; Yin, Li-Jung; Chen, Wei-Chuan; Jiang, Shann-Tzong

    2013-06-26

    To obtain a Pichia pastoris mutant with an Escherichia coli phytase gene, which was synthesized according to P. pastoris codon preference, a mature phytase cDNA of E. coli being altered according to the codons usage preference of P. pastoris was artificially synthesized and cloned into an expression vector of pGAPZαC. The final extracellular phytase activity was 112.5 U/mL after 72 h of cultivation. The phytase, with a molecular mass of 46 kDa, was purified to electrophoretical homogeneity after Ni Sepharose 6 Fast Flow chromatography. The yield, purification fold, and specific activity were 63.97%, 26.17, and 1.57 kU/mg, respectively. It had an optimal pH and temperature of 4.0-6.0 and 50 °C, respectively, and was stable at pH 3.0-8.0 and 25-40 °C. The purified recombinant phytase was resistant to trypsin, highly inhibited by Cu(2+), Zn(2+), Hg(2+), Fe(2+), Fe(3+), phenylmethylsulfonyl fluoride, and N-tosyl-l-lysine chloromethyl ketone, but activated by Mg(2+), Ca(2+), Sr(2+), Ba(2+), glutathione, ethylenediaminetetraacetic acid, and N-ethylmaleimide. It revealed higher affinity to calcium phytate than to other phosphate conjugates.

  9. Influence of the metabolic state on the tolerance of Pichia kudriavzevii to heavy metals.

    PubMed

    Mesquita, Vanessa A; Machado, Manuela D; Silva, Cristina F; Soares, Eduardo V

    2016-11-01

    This work aims to examine the influence of the metabolic state of the yeast Pichia kudriavzevii on the susceptibility to a metals mixture (5 mg L(-1) Cd, 10 mg L(-1) Pb, and 5 mg L(-1) Zn). Cells exposed to the metals mixture in the presence of 25 mmol L(-1) glucose displayed a higher loss of membrane integrity and proliferation capacity, compared to cells incubated in the absence of glucose. The analysis of the effect of individual metals revealed that glucose increased the toxic effect of Cd marginally, and of Pb significantly. The increased susceptibility to heavy metals due to glucose was attenuated in the simultaneous presence of a mitochondrial respiration inhibitor such as sodium azide (NaN3 ). ATP-depleted yeast cells, resulting from treatment with the non-metabolizable glucose analogue 2-deoxy-d-glucose, showed an increased susceptibility to heavy metals mixture. Pre-incubation of yeast cells with 1 or 1.5 mmol L(-1) Ca(2+) reduced significantly (P < 0.05) the loss of membrane integrity induced by the metals mixture. These findings contribute to the understanding of metals mechanisms of toxicity in the non-conventional yeast P. kudriavzevii. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Characterization of Bovine Interferon α1: Expression in Yeast Pichia pastoris, Biological Activities, and Physicochemical Characteristics

    PubMed Central

    Shao, Jianwei; Cao, Chong; Bao, Jun; Liu, Hongtao; Peng, Tongquan

    2015-01-01

    A bovine interferon α (BoIFNα) gene that included signal sequence was amplified from bovine liver genomic DNA. The gene was named BoIFN-α1 according to the position at which the encoded gene of the bovine IFN was located in the bovine genome. The sequence included a 23-amino-acid signal peptide and a 166-amino-acid mature peptide. The structural characteristics and phylogenetic relationships of the BoIFN-α1 gene were analyzed. A recombinant mature BoIFN-α1 (rBoIFN-α1) was expressed in the yeast Pichia pastoris. Physicochemical characteristics and antiviral activity were determined in vitro. Recombinant BoIFN-α1 was found to be highly sensitive to trypsin and stable at pH 2.0 or 65°C. It also exhibited antiviral activity, which was neutralized by a rabbit anti-rBoIFNα polyclonal antibody. This study revealed that rBoIFN-α1 has the typical characteristics of IFNα and can be used for both research and industrial application. PMID:25343404

  11. High level expression of human enteropeptidase light chain in Pichia pastoris.

    PubMed

    Pepeliaev, Stanislav; Krahulec, Ján; Černý, Zbyněk; Jílková, Jana; Tlustá, Marcela; Dostálová, Jana

    2011-10-20

    Human enterokinase (enteropeptidase, rhEP), a serine protease expressed in the proximal part of the small intestine, converts the inactive form of trypsinogen to active trypsin by endoproteolytic cleavage. The high specificity of the target site makes enterokinase an ideal tool for cleaving fusion proteins at defined cleavage sites. The mature active enzyme is comprised of two disulfide-linked polypeptide chains. The heavy chain anchors the enzyme in the intestinal brush border membrane, whereas the light chain represents the catalytic enzyme subunit. The synthetic gene encoding human enteropeptidase light chain with His-tag added at the C-terminus to facilitate protein purification was cloned into Pichia pastoris expression plasmids under the control of an inducible AOX1 or constitutive promoters GAP and AAC. Cultivation media and conditions were optimized as well as isolation and purification of the target protein. Up to 4 mg/L of rhEP was obtained in shake-flask experiments and the expression level of about 60-70 mg/L was achieved when cultivating in lab-scale fermentors. The constitutively expressing strains proved more efficient and less labor-demanding than the inducible ones. The rhEP was immobilized on AV 100 sorbent (Iontosorb) to allow repeated use of enterokinase, showing specific activity of 4U/mL of wet matrix.

  12. High-level secretory production of recombinant bovine enterokinase light chain by Pichia pastoris.

    PubMed

    Peng, Lisheng; Zhong, Xiaofen; Ou, Jingxing; Zheng, Suilan; Liao, Jian; Wang, Lei; Xu, Anlong

    2004-03-04

    Enterokinase (EC 3.4.21.9) is a serine proteinase with a specific digest sequence (Asp)4-Lys in the duodenum. Its high specificity for the recognition site makes enterokinase (EK) a useful tool for an in vitro cleavage of fusion proteins. In this work, an active bovine enterokinase light chain (EK(L)) was produced in secretory form by a recombinant strain of the methylotrophic yeast Pichia pastoris. The influences of methanol utilization phenotype of the host strain, induction pH, and carbon source on the recombinant production were studied. The production of recombinant EK(L) by Mut(s) strain was much higher than that by Mut+ strain. When inducted at pH 6.0, on a glycerol/methanol medium, the concentration of recombinant EK(L) (rEK(L)) reached 350 mg l(-1), which was 20-fold higher than that reported previously. The recombinant EK(L) was purified in a simple procedure on the anion exchange chromatography and 15 mg pure active EK(L) were obtained from 100 ml culture broth supernatant. The specific activity of purified rEK(L) was approximately 9000 u mg(-1). To facilitate purification and removal of rEKL after cleavage of fusion protein, the C-terminal His-tagged EK(L) (EK(L)/His) was also expressed in P. pastoris, and this His-tagged EK(L) exhibited a similar enzymatic activity to the untagged EK(L).

  13. Recombinant expression and biological characterization of the antimicrobial peptide fowlicidin-2 in Pichia pastoris

    PubMed Central

    Xing, Li-Wei; Tian, Shi-Xun; Gao, Wei; Yang, Na; Qu, Pei; Liu, Di; Jiao, Jian; Wang, Jue; Feng, Xing-Jun

    2016-01-01

    Fowlicidins are a group of cathelicidin antimicrobial peptides that were initially identified in chickens. Fowlicidin-2, which is composed of 31 amino acids, is widely expressed in the majority of tissues in chickens and has an important role in innate immunity. In the present study, a recombinant expression system for fowlicidin-2 was successfully constructed using Pichia pastoris X-33 and the expression vector pPICZα-A. Under the optimized fermentation conditions, 85.6 mg fowlicidin-2 with >95% purity was obtained from 1 liter culture medium following purification by ion exchange chromatography and reversed phase high performance liquid chromatography. The recombinant fowlicidin-2 exhibited broad spectrum antimicrobial activity and had a minimum inhibitory concentration ranging from 1 to 4 µM. Furthermore, recombinant fowlicidin-2 exhibited hemolytic activity, promoting 50% human erythrocyte hemolysis in the concentration range of 128–256 µM, and anticancer activity, resulting in the death of 50% of A375 human malignant melanoma cells in the concentration range of 2–4 µM. The results of the present study suggest that recombinant fowlicidin-2 may be a promising candidate for therapeutic applications. PMID:27698732

  14. Improving the Secretory Expression of an α-Galactosidase from Aspergillus niger in Pichia pastoris

    PubMed Central

    Zheng, Xianliang; Fang, Bo; Han, Dongfei; Yang, Wenxia; Qi, Feifei; Chen, Hui; Li, Shengying

    2016-01-01

    α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1’ residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Muts) strain of AGA-I with the specific P1’ site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application. PMID:27548309

  15. Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing

    PubMed Central

    Schwarzhans, Jan-Philipp; Wibberg, Daniel; Winkler, Anika; Luttermann, Tobias; Kalinowski, Jörn; Friehs, Karl

    2016-01-01

    The non-conventional yeast Pichia pastoris is a popular host for recombinant protein production in scientific research and industry. Typically, the expression cassette is integrated into the genome via homologous recombination. Due to unknown integration events, a large clonal variability is often encountered consisting of clones with different productivities as well as aberrant morphological or growth characteristics. In this study, we analysed several clones with abnormal colony morphology and discovered unpredicted integration events via whole genome sequencing. These include (i) the relocation of the locus targeted for replacement to another chromosome (ii) co-integration of DNA from the E. coli plasmid host and (iii) the disruption of untargeted genes affecting colony morphology. Most of these events have not been reported so far in literature and present challenges for genetic engineering approaches in this yeast. Especially, the presence and independent activity of E. coli DNA elements in P. pastoris is of concern. In our study, we provide a deeper insight into these events and their potential origins. Steps preventing or reducing the risk for these phenomena are proposed and will help scientists working on genetic engineering of P. pastoris or similar non-conventional yeast to better understand and control clonal variability. PMID:27958335

  16. 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.

  17. Heterologous Expression of Tulip Petal Plasma Membrane Aquaporins in Pichia pastoris for Water Channel Analysis▿

    PubMed Central

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2009-01-01

    Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs. PMID:19251885

  18. Heterologous expression of tulip petal plasma membrane aquaporins in Pichia pastoris for water channel analysis.

    PubMed

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2009-05-01

    Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.

  19. Effects of pre- and pro-sequence of thaumatin on the secretion by Pichia pastoris.

    PubMed

    Ide, Nobuyuki; Masuda, Tetsuya; Kitabatake, Naofumi

    2007-11-23

    Thaumatin is a 22-kDa sweet-tasting protein containing eight disulfide bonds. When thaumatin is expressed in Pichia pastoris using the thaumatin cDNA fused with both the alpha-factor signal sequence and the Kex2 protease cleavage site from Saccharomyces cerevisiae, the N-terminal sequence of the secreted thaumatin molecule is not processed correctly. To examine the role of the thaumatin cDNA-encoded N-terminal pre-sequence and C-terminal pro-sequence on the processing of thaumatin and efficiency of thaumatin production in P. pastoris, four expression plasmids with different pre-sequence and pro-sequence were constructed and transformed into P. pastoris. The transformants containing pre-thaumatin gene that has the native plant signal, secreted thaumatin molecules in the medium. The N-terminal amino acid sequence of the secreted thaumatin molecule was processed correctly. The production yield of thaumatin was not affected by the C-terminal pro-sequence, and the pro-sequence was not processed in P. pastoris, indicating that pro-sequence is not necessary for thaumatin synthesis.

  20. 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.