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Jens Nielsen - One of the best experts on this subject based on the ideXlab platform.
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heat shock response improves Heterologous Protein secretion in saccharomyces cerevisiae
Applied Microbiology and Biotechnology, 2013Co-Authors: Dina Petranovic, Jens Nielsen, Jin Hou, Zihe Liu, Tobias OsterlundAbstract:The yeast Saccharomyces cerevisiae is a widely used platform for the production of Heterologous Proteins of medical or industrial interest. However, Heterologous Protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote Protein folding and prevent the accumulation of mis-folded or aggregated Proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on Heterologous Protein secretion. We found that moderate and high level over-expression of HSF1-R206S increased Heterologous α-amylase yield 25 and 70 % when glucose was fully consumed, and 37 and 62 % at the end of the ethanol phase, respectively. Moderate and high level over-expression also improved endogenous invertase yield 118 and 94 %, respectively. However, human insulin precursor was only improved slightly and this only by high level over-expression of HSF1-R206S, supporting our previous findings that the production of this Protein in S. cerevisiae is not limited by secretion. Our results provide an effective strategy to improve Protein secretion and demonstrated an approach that can induce ER and cytosolic chaperones simultaneously.
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imbalance of Heterologous Protein folding and disulfide bond formation rates yields runaway oxidative stress
BMC Biology, 2012Co-Authors: Dina Petranovic, Jens NielsenAbstract:Background: The Protein secretory pathway must process a wide assortment of native Proteins for eukaryotic cells to function. As well, recombinant Protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from Heterologous production of a small Protein (insulin precursor) or a larger Protein (a-amylase). Results: HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of Protein/recycling amino acids, (b) overall transcription/ translation repression, and (c) oxidative stress were broadly associated with secretory stress. Conclusions: Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when Protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing Heterologous Protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant Proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.
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engineering of vesicle trafficking improves Heterologous Protein secretion in saccharomyces cerevisiae
Metabolic Engineering, 2012Co-Authors: Jin Hou, Dina Petranovic, Keith E J Tyo, Zihe Liu, Jens NielsenAbstract:The yeast Saccharomyces cerevisiae is a widely used platform for the production of Heterologous Proteins of medical or industrial interest. However, Heterologous Protein productivity is often restricted due to the limitations of the host strain. In the Protein secretory pathway, the Protein trafficking between different organelles is catalyzed by the soluble NSF (N-ethylmaleimide-sensitive factor) receptor (SNARE) complex and regulated by the Sec1/Munc18 (SM) Proteins. In this study, we report that over-expression of the SM Protein encoding genes SEC1 and SLY1, improves the Protein secretion in S. cerevisiae. Engineering Sec1p, the SM Protein that is involved in vesicle trafficking from Golgi to cell membrane, improves the secretion of Heterologous Proteins human insulin precursor and α-amylase, and also the secretion of an endogenous Protein invertase. Enhancing Sly1p, the SM Protein regulating the vesicle fusion from endoplasmic reticulum (ER) to Golgi, increases α-amylase production only. Our study demonstrates that strengthening the Protein trafficking in ER-to-Golgi and Golgi-to-plasma membrane process is a novel secretory engineering strategy for improving Heterologous Protein production in S. cerevisiae.
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imbalance of Heterologous Protein folding and disulfide bond formation rates yields runaway oxidative stress
BMC Biology, 2012Co-Authors: Dina Petranovic, Keith E J Tyo, Zihe Liu, Jens NielsenAbstract:The Protein secretory pathway must process a wide assortment of native Proteins for eukaryotic cells to function. As well, recombinant Protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from Heterologous production of a small Protein (insulin precursor) or a larger Protein (α-amylase). HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of Protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress. Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when Protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing Heterologous Protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant Proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.
Katsuhiko Kitamoto - One of the best experts on this subject based on the ideXlab platform.
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enhanced production of bovine chymosin by autophagy deficiency in the filamentous fungus aspergillus oryzae
PLOS ONE, 2013Co-Authors: Jaewoo Yoon, Jun-ichi Maruyama, Takashi Kikuma, Katsuhiko KitamotoAbstract:Aspergillus oryzae has been utilized as a host for Heterologous Protein production because of its high Protein secretory capacity and food-safety properties. However, A. oryzae often produces lower-than-expected yields of target Heterologous Proteins due to various underlying mechanisms, including degradation processes such as autophagy, which may be a significant bottleneck for Protein production. In the present study, we examined the production of Heterologous Protein in several autophagy (Aoatg) gene disruptants of A. oryzae. We transformed A. oryzae gene disruptants of Aoatg1, Aoatg13, Aoatg4, Aoatg8, or Aoatg15, with a bovine chymosin (CHY) expression construct and found that the production levels of CHY increased up to three fold compared to the control strain. Notably, however, conidia formation by the Aoatg gene disruptants was significantly reduced. As large amounts of conidia are necessary for inoculating large-scale cultures, we also constructed Aoatg gene-conditional expression strains in which the promoter region of the Aoatg gene was replaced with the thiamine-controllable thiA promoter. Conidiation by the resultant transformants was clearly enhanced in the absence of thiamine, while autophagy remained repressed in the presence of thiamine. Moreover, these transformants displayed increased CHY productivity, which was comparable to that of the Aoatg gene disruptants. Consequently, we succeeded in the construction of A. oryzae strains capable of producing high levels of CHY due to defects in autophagy. Our finding suggests that the conditional regulation of autophagy is an effective method for increasing Heterologous Protein production in A. oryzae.
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Improved Heterologous Protein production by a tripeptidyl peptidase gene (AosedD) disruptant of the filamentous fungus Aspergillus oryzae
The Journal of general and applied microbiology, 2012Co-Authors: Lin Zhu, Jun-ichi Maruyama, Jaewoo Yoon, Nemoto Takeshi, Katsuhiko KitamotoAbstract:Proteolytic degradation is one of the serious bottlenecks limiting the yields of Heterologous Protein production by Aspergillus oryzae. In this study, we selected a tripeptidyl peptidase gene AosedD (AO090166000084) as a candidate potentially degrading the Heterologous Protein, and performed localization analysis of the fusion Protein AoSedD-EGFP in A. oryzae. As a result, the AoSedD-EGFP was observed in the septa and cell walls as well as in the culture medium, suggesting that AoSedD is a secretory enzyme. An AosedD disruptant was constructed to investigate an effect of AoSedD on the production level of Heterologous Proteins and protease activity. Both of the total protease and tripeptidyl peptidase activities in the culture medium of the AosedD disruptant were decreased as compared to those of the control strain. The maximum yields of recombinant bovine chymosin (CHY) and human lysozyme (HLY) produced by the AosedD disruptants showed approximately 2.9- and 1.7-fold increases, respectively, as compared to their control strains. These results suggest that AoSedD is one of the major proteases involved in the proteolytic degradation of recombinant Proteins in A. oryzae.
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enhanced production and secretion of Heterologous Proteins by the filamentous fungus aspergillus oryzae via disruption of vacuolar Protein sorting receptor gene aovps10
Applied and Environmental Microbiology, 2010Co-Authors: Jaewoo Yoon, Jun-ichi Maruyama, Tuerxun Aishan, Katsuhiko KitamotoAbstract:Filamentous fungi have received attention as hosts for Heterologous Protein production because of their high secretion capability and eukaryotic posttranslational modifications. However, despite these positive attributes, a bottleneck in posttranscriptional processing limits Protein yields. The vacuolar Protein sorting gene VPS10 encodes a sorting receptor for the recognition and delivery of several yeast vacuolar Proteins. Although it can also target recombinant and aberrant Proteins for vacuolar degradation, there is limited knowledge of the effect of its disruption on Heterologous Protein production. In this study, cDNA encoding AoVps10 from the filamentous fungus Aspergillus oryzae was cloned and sequenced. Microscopic observation of the transformant expressing AoVps10 fused with enhanced green fluorescent Protein showed that the fusion Protein localized at the Golgi and prevacuolar compartments. Moreover, disruption of the Aovps10 gene resulted in missorting and secretion of vacuolar carboxypeptidase AoCpyA into the medium, indicating that AoVps10 is required for sorting of vacuolar Proteins to vacuoles. To investigate the extracellular production levels of Heterologous Proteins, ΔAovps10 mutants expressing either bovine chymosin (CHY) or human lysozyme (HLY) were constructed. Interestingly, the ΔAovps10 mutation increased the maximum extracellular production levels of CHY and HLY by 3- and 2.2-fold, respectively. Western blot analysis of extracellular Heterologous Proteins also demonstrated an improvement in productivity. These results suggest that AoVps10 plays a role in the regulation of Heterologous Protein secretion in A. oryzae and may be involved in the vacuolar Protein degradation through the Golgi apparatus.
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Isolation of Aspergillus oryzae mutants for Heterologous Protein production from a double Proteinase gene disruptant.
Applied microbiology and biotechnology, 2009Co-Authors: Takashi Nemoto, Jun-ichi Maruyama, Taisuke Watanabe, Yutaka Mizogami, Katsuhiko KitamotoAbstract:Aspergillus oryzae has attracted much attention as a host for Heterologous Protein production because of its high secretion ability and safety. However, there have been only a few reports on construction of this organism to improve its properties as a production host. We previously reported that the double disruptant of the Proteinase gene (tppA, pepE) improved human lysozyme (HLY) production. In this double disruptant, however, the HLY expression plasmid cannot be removed due to its random integration into the genome. In this study, we re-constructed the tppA pepE disruptant as a host for Heterologous Protein production. By the use of the tppA pepE disruptant, bovine chymosin (CHY) production was enhanced by 1.9-fold. Moreover, we generated HLY-producing strain from the tppA pepE disruptant by curable niaD marker, and then isolated HLY-hyperproducing mutants using the halo assay based on HLY activity. Subsequently, the niaD-based plasmid for HLY production was cured from the mutants by positive selection. The cured strains (named AUT strains) showed production levels of HLY and CHY that were 2.6- and 3.2-fold higher than those of the wild-type strain, respectively. Thus, the AUT strains are expected to be good hosts for obtaining higher production levels of various Heterologous Proteins.
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Construction of quintuple protease gene disruptant for Heterologous Protein production in Aspergillus oryzae.
Applied microbiology and biotechnology, 2008Co-Authors: Jaewoo Yoon, Jun-ichi Maruyama, Shinya Kimura, Katsuhiko KitamotoAbstract:Aspergillus oryzae has received attention as a host for Heterologous Protein production. However, A. oryzae has 134 protease genes, which is recognized to be one of the major reasons for the proteolytic degradation of Heterologously produced Proteins. We previously reported that double disruption of the protease genes (tppA and pepE) improved Heterologous Protein (human lysozyme) production by A. oryzae. In this study, we performed successive round of five protease genes (tppA, pepE, nptB, dppIV, and dppV) disruption in A. oryzae by pyrG marker recycling with highly efficient gene-targeting background (ΔligD). The multiple disruption of protease genes were confirmed by Southern blot analysis. Furthermore, the quintuple protease gene disruptants showed the maximum production level of bovine chymosin (CHY) that was 34% higher than those of the double protease gene disruptant (ΔtppA ΔpepE). Consequently, we successfully constructed a multiple protease gene disruptant bearing enhanced levels of CHY productivity. We presented the first evidence that the quintuple disruption of the protease genes improved the production level of a Heterologous Protein by A. oryzae.
Dina Petranovic - One of the best experts on this subject based on the ideXlab platform.
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heat shock response improves Heterologous Protein secretion in saccharomyces cerevisiae
Applied Microbiology and Biotechnology, 2013Co-Authors: Dina Petranovic, Jens Nielsen, Jin Hou, Zihe Liu, Tobias OsterlundAbstract:The yeast Saccharomyces cerevisiae is a widely used platform for the production of Heterologous Proteins of medical or industrial interest. However, Heterologous Protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote Protein folding and prevent the accumulation of mis-folded or aggregated Proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on Heterologous Protein secretion. We found that moderate and high level over-expression of HSF1-R206S increased Heterologous α-amylase yield 25 and 70 % when glucose was fully consumed, and 37 and 62 % at the end of the ethanol phase, respectively. Moderate and high level over-expression also improved endogenous invertase yield 118 and 94 %, respectively. However, human insulin precursor was only improved slightly and this only by high level over-expression of HSF1-R206S, supporting our previous findings that the production of this Protein in S. cerevisiae is not limited by secretion. Our results provide an effective strategy to improve Protein secretion and demonstrated an approach that can induce ER and cytosolic chaperones simultaneously.
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imbalance of Heterologous Protein folding and disulfide bond formation rates yields runaway oxidative stress
BMC Biology, 2012Co-Authors: Dina Petranovic, Jens NielsenAbstract:Background: The Protein secretory pathway must process a wide assortment of native Proteins for eukaryotic cells to function. As well, recombinant Protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from Heterologous production of a small Protein (insulin precursor) or a larger Protein (a-amylase). Results: HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of Protein/recycling amino acids, (b) overall transcription/ translation repression, and (c) oxidative stress were broadly associated with secretory stress. Conclusions: Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when Protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing Heterologous Protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant Proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.
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engineering of vesicle trafficking improves Heterologous Protein secretion in saccharomyces cerevisiae
Metabolic Engineering, 2012Co-Authors: Jin Hou, Dina Petranovic, Keith E J Tyo, Zihe Liu, Jens NielsenAbstract:The yeast Saccharomyces cerevisiae is a widely used platform for the production of Heterologous Proteins of medical or industrial interest. However, Heterologous Protein productivity is often restricted due to the limitations of the host strain. In the Protein secretory pathway, the Protein trafficking between different organelles is catalyzed by the soluble NSF (N-ethylmaleimide-sensitive factor) receptor (SNARE) complex and regulated by the Sec1/Munc18 (SM) Proteins. In this study, we report that over-expression of the SM Protein encoding genes SEC1 and SLY1, improves the Protein secretion in S. cerevisiae. Engineering Sec1p, the SM Protein that is involved in vesicle trafficking from Golgi to cell membrane, improves the secretion of Heterologous Proteins human insulin precursor and α-amylase, and also the secretion of an endogenous Protein invertase. Enhancing Sly1p, the SM Protein regulating the vesicle fusion from endoplasmic reticulum (ER) to Golgi, increases α-amylase production only. Our study demonstrates that strengthening the Protein trafficking in ER-to-Golgi and Golgi-to-plasma membrane process is a novel secretory engineering strategy for improving Heterologous Protein production in S. cerevisiae.
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imbalance of Heterologous Protein folding and disulfide bond formation rates yields runaway oxidative stress
BMC Biology, 2012Co-Authors: Dina Petranovic, Keith E J Tyo, Zihe Liu, Jens NielsenAbstract:The Protein secretory pathway must process a wide assortment of native Proteins for eukaryotic cells to function. As well, recombinant Protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from Heterologous production of a small Protein (insulin precursor) or a larger Protein (α-amylase). HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of Protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress. Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when Protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing Heterologous Protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant Proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.
Keith E J Tyo - One of the best experts on this subject based on the ideXlab platform.
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engineering of vesicle trafficking improves Heterologous Protein secretion in saccharomyces cerevisiae
Metabolic Engineering, 2012Co-Authors: Jin Hou, Dina Petranovic, Keith E J Tyo, Zihe Liu, Jens NielsenAbstract:The yeast Saccharomyces cerevisiae is a widely used platform for the production of Heterologous Proteins of medical or industrial interest. However, Heterologous Protein productivity is often restricted due to the limitations of the host strain. In the Protein secretory pathway, the Protein trafficking between different organelles is catalyzed by the soluble NSF (N-ethylmaleimide-sensitive factor) receptor (SNARE) complex and regulated by the Sec1/Munc18 (SM) Proteins. In this study, we report that over-expression of the SM Protein encoding genes SEC1 and SLY1, improves the Protein secretion in S. cerevisiae. Engineering Sec1p, the SM Protein that is involved in vesicle trafficking from Golgi to cell membrane, improves the secretion of Heterologous Proteins human insulin precursor and α-amylase, and also the secretion of an endogenous Protein invertase. Enhancing Sly1p, the SM Protein regulating the vesicle fusion from endoplasmic reticulum (ER) to Golgi, increases α-amylase production only. Our study demonstrates that strengthening the Protein trafficking in ER-to-Golgi and Golgi-to-plasma membrane process is a novel secretory engineering strategy for improving Heterologous Protein production in S. cerevisiae.
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imbalance of Heterologous Protein folding and disulfide bond formation rates yields runaway oxidative stress
BMC Biology, 2012Co-Authors: Dina Petranovic, Keith E J Tyo, Zihe Liu, Jens NielsenAbstract:The Protein secretory pathway must process a wide assortment of native Proteins for eukaryotic cells to function. As well, recombinant Protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from Heterologous production of a small Protein (insulin precursor) or a larger Protein (α-amylase). HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of Protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress. Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when Protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing Heterologous Protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant Proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.
Joan Lin Cereghino - One of the best experts on this subject based on the ideXlab platform.
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Heterologous Protein expression in the methylotrophic yeast Pichia pastoris
FEMS Microbiology Reviews, 2000Co-Authors: Joan Lin CereghinoAbstract:During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of Heterologous Proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign Proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit. In this paper, we review the P. pastoris expression system: how it was developed, how it works, and what Proteins have been produced. We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.