Effect of nitrogen source on gene expression of first steps of methanol utilization pathway in Pichia pastoris

Abstract Background Methanol expression regulator 1 (Mxr1p) is a key transcription factor that plays a vital role in the methanol utilization pathway in Pichia pastoris ( P. pastoris ). Most genes referred to the methanol utilization pathway were regulated by Mxr1p. However, some genes did not show a significant difference between methanol and glycerol even though they play an important role in the methanol utilization pathway. So far, the regulation mechanism about these genes and the relationship with Mxr1p are still unknown. Results Methanol metabolic pathway analysis revealed that most of the methanol-induced genes were upregulated in transcriptional level when cultured in methanol. Whereas some genes like tkl1 (transketolase 1) did not show significant up-regulation in methanol even though it plays a very important role in Xu5P recycle, the reason is still not clear. To clarify this point, firstly, pull-down and MS experiments were performed. The result shows that Tkl1p protein combined with Mxr1p in vitro . Subsequently, this result was further confirmed by yeast two-hybrid in vivo , and the binding region mainly located from 150AA to 400AA. Moreover, Ser215 phosphorylation did not affect this interaction. In addition, Mxr1p-400AA integration into Δmxr1 could rescue cell growth in methanol. All the above results proved that Mxr1p played a post-translational role in the methanol utilization pathway and Mxr1p-400AA may achieved most of Mxr1p functions. Secondly, the function of Mxr1p-Tkl1p complex was expounded by detecting formaldehyde consumption and xylulose production in cell-free systems. Results showed that Mxr1p-Tkl1p mixture could promote formaldehyde consumption and xylulose production in vitro . Conclusion Mxr1p promotes methanol utilization via combining with Tkl1p to accelerate Xu5P recycle and this interaction was not affected by Ser215 phosphorylation.

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Production of foreign proteins in the yeast Pichia pastoris

Canadian Journal of Botany ◽

10.1139/b95-336 ◽

1995 ◽

Vol 73 (S1) ◽

pp. 891-897 ◽

Cited By ~ 46

Author(s):

James M. Cregg ◽

David R. Higgins

Keyword(s):

Gene Expression ◽

Pichia Pastoris ◽

Heterologous Gene Expression ◽

Expression System ◽

Methylotrophic Yeast ◽

Culture Broth ◽

Heterologous Gene ◽

Foreign Gene ◽

Heterologous Proteins ◽

Yeast Pichia Pastoris

The methanol-utilizing yeast Pichia pastoris has been developed as a host system for the production of heterologous proteins of commercial interest. An industrial yeast selected for efficient growth on methanol for biomass generation, P. pastoris is readily grown on defined medium in continuous culture at high volume and density. A unique feature of the expression system is the promoter employed to drive heterologous gene expression, which is derived from the methanol-regulated alcohol oxidase I gene (AOX1) of P. pastoris, one of the most efficient and tightly regulated promoters known. The strength of the AOX1 promoter results in high expression levels in strains harboring only a single integrated copy of a foreign-gene expression cassette. Levels may often be further enhanced through the integration of multiple cassette copies into the P. pastoris genome and strategies to construct and select multicopy cassette strains have been devised. The system is particularly attractive for the secretion of foreign-gene products. Because P. pastoris endogenous protein secretion levels are low, foreign secreted proteins often appear to be virtually the only proteins in the culture broth, a major advantage in processing and purification. Key words: heterologous gene expression, methylotrophic yeast, Pichia pastoris, secretion, glycosylation.

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The nature of the nitrogen source added to nitrogen depleted vinifications conducted by a Saccharomyces cerevisiae strain in synthetic must affects gene expression and the levels of several volatile compounds

Antonie van Leeuwenhoek ◽

10.1007/s10482-006-9135-1 ◽

2007 ◽

Vol 92 (1) ◽

pp. 61-75 ◽

Cited By ~ 42

Author(s):

Elena Jiménez-Martí ◽

Agustín Aranda ◽

Alexandra Mendes-Ferreira ◽

Arlete Mendes-Faia ◽

Marcel lí del Olmo

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Nitrogen Source ◽

Volatile Compounds

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

Applied and Environmental Microbiology ◽

10.1128/aem.00218-12 ◽

2012 ◽

Vol 78 (16) ◽

pp. 5845-5854 ◽

Cited By ~ 45

Author(s):

Yang Yang ◽

Fangfang Fan ◽

Rui Zhuo ◽

Fuying Ma ◽

Yangmin Gong ◽

...

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Pichia Pastoris ◽

Oxidative Damage ◽

White Rot ◽

Antioxidative System ◽

White Rot Fungus ◽

Laccase Gene ◽

Content Type ◽

Glutamylcysteine Synthetase

ABSTRACTLaccase is a copper-containing polyphenol oxidase that has great potential in industrial and biotechnological applications. Previous research has suggested that fungal laccase may be involved in the defense against oxidative stress, but there is little direct evidence supporting this hypothesis, and the mechanism by which laccase protects cells from oxidative stress also remains unclear. Here, we report that the expression of the laccase gene from white rot fungus inPichia pastoriscan significantly enhance the resistance of yeast to H2O2-mediated oxidative stress. The expression of laccase in yeast was found to confer a strong ability to scavenge intracellular H2O2and to protect cells from lipid oxidative damage. The mechanism by which laccase gene expression increases resistance to oxidative stress was then investigated further. We found that laccase gene expression inPichia pastoriscould increase the level of glutathione-based antioxidative activity, including the intracellular glutathione levels and the enzymatic activity of glutathione peroxidase, glutathione reductase, and γ-glutamylcysteine synthetase. The transcription of the laccase gene inPichia pastoriswas found to be enhanced by the oxidative stress caused by exogenous H2O2. The stimulation of laccase gene expression in response to exogenous H2O2stress further contributed to the transcriptional induction of the genes involved in the glutathione-dependent antioxidative system, includingPpYAP1,PpGPX1,PpPMP20,PpGLR1, andPpGSH1. Taken together, these results suggest that the expression of the laccase gene inPichia pastoriscan enhance the resistance of yeast to H2O2-mediated oxidative stress by stimulating the glutathione-based antioxidative system to protect the cell from oxidative damage.

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