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Pichia pastoris

About: Pichia pastoris is a research topic. Over the lifetime, 7937 publications have been published within this topic receiving 162645 citations. The topic is also known as: Komagataella pastoris.


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Journal ArticleDOI
TL;DR: P. pastoris is a potential host to express high levels of A. fumigatus phytase and the thermostability of the recombinant enzyme is modulated by the specificity of buffer used in the heat treatment.

126 citations

Journal ArticleDOI
TL;DR: It is proposed that PFK1 protein directly modulates glucose-induced microautophagy independent of its ability to metabolize glucose intermediates and therefore does not require a catalytically active phosphofructokinase.
Abstract: We have characterized biochemically, morphologically, and genetically two distinct pathways for the selective degradation of peroxisomes in Pichia pastoris. These pathways are independently regulated and analogous to microautophagy and macroautophagy that have been defined in mammalian cells. When P. pastoris is grown in methanol, cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized. During adaptation from methanol to glucose, these enzymes are rapidly and selectively degraded within the yeast vacuole by microautophagy. We have isolated gsa mutants that are defective in glucose-induced selective autophagy of peroxisomes. In this study, we have shown that gsa1 is unable to sequester peroxisomes into the yeast vacuole. In addition, we provide evidence that the glucose-induced selective autophagy 1 (GSA1) protein is the alpha subunit of the phosphofructokinase enzyme complex encoded by PFK1. First, we can rescue the gsa1 mutant by transformation with a vector containing PFK1. Second, cellular levels of both PFK1 mRNA and phosphofructokinase activity are dramatically reduced in gsa1 when compared to the parental GS115. Third, a PFK1 knockout (delta pfk1) is unable to degrade alcohol oxidase during glucose adaptation. As observed in gsa1, the peroxisomes in delta pfk1 remain outside the vacuole during adaptation. Our data are consistent with the concept that PFK1 protein is required for an event upstream of vacuole degradation (i.e. signaling, selection, or sequestration). However, the degradation of peroxisomes does not require a catalytically active phosphofructokinase. The inability of delta pfk1 cells to degrade alcohol oxidase can be rescued by transformation with either normal PFK1 or mutant pfk1 whose catalytic site had been inactivated by a single amino acid mutation. We propose that PFK1 protein directly modulates glucose-induced microautophagy independent of its ability to metabolize glucose intermediates.

126 citations

Journal ArticleDOI
TL;DR: Results show that glycerol inhibits expression driven by the AOX1 promoter even at extremely limited availability and demonstrate the benefits of on-line methanol control in Pichia fermentation research.
Abstract: In the last few years the Pichia pastoris expres- sion system has been gaining more and more interest for the expression of recombinant proteins. Many groups have employed fermentation technology in their investi- gations because the system is fairly easy to scale up and suitable for the production in the milligram to gram range. A large number of heterologous proteins from different sources has been expressed, but the fermenta- tion process technology has been investigated to a lesser extent. A large number of fermentations are carried out in standard bioreactors that may be insufficiently equipped to meet the demands of high-cell-density fer- mentations of methylotrophic yeasts. In particular, the lack of on-line methanol analysis leads to fermentation protocols that may impair the optimal expression of the desired products. We have used a commercially avail- able methanol sensor to investigate in detail the effects of supplementary glycerol feeding while maintaining a constant methanol concentration during the induction of aM ut + strain of Pichia pastoris. Specific glycerol feed rates in the range of 38-4.2 mg ? g ˛1 ? h ˛1 (mg glycerol per gram fresh weight per hour) were investigated. Ex- pression of the recombinant scFv antibody fragment was only observed at specific feed rates below 6 mg ? g ˛1 ? h ˛1 . At low specific feed rates, growth was even lower than with methanol as the sole carbon source and the harvest expression level of the scFv was only half of that found in the control fermentation. These results show that glycerol inhibits expression driven by the AOX1 pro- moter even at extremely limited availability and demon- strate the benefits of on-line methanol control in Pichia fermentation research. © 2001 John Wiley & Sons, Inc. Bio- technol Bioeng 74: 344-352, 2001.

126 citations

Journal ArticleDOI
TL;DR: The pas2 mutant of the methylotrophic yeast Pichia pastoris is characterized by a deficiency in peroxisome biogenesis and cloned the PpPAS2 gene by functional complementation and it is shown that it encodes a protein of 455 amino acids with a molecular mass of 52 kDa.

126 citations

Journal ArticleDOI
TL;DR: This work has developed a cultivation and induction protocol amendable to automation to increase the number of clones screened for protein expression and is able to screen for and identify expression clones which produce heterologous protein with a yield of 5 mg l(-1) culture volume or higher.

126 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
2023150
2022340
2021255
2020303
2019374
2018401