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.

Role of the PAS1 gene of Pichia pastoris in peroxisome biogenesis.

Abstract: Several groups have reported the cloning and sequencing of genes involved in the biogenesis of yeast peroxisomes. Yeast strains bearing mutations in these genes are unable to grow on carbon sources whose metabolism requires peroxisomes, and these strains lack morphologically normal peroxisomes. We report the cloning of Pichia pastoris PAS1, the homologue (based on a high level of protein sequence similarity) of the Saccharomyces cerevisiae PAS1. We also describe the creation and characterization of P. pastoris pas1 strains. Electron microscopy on the P. pastoris pas1 cells revealed that they lack morphologically normal peroxisomes, and instead contain membrane-bound structures that appear to be small, mutant peroxisomes, or "peroxisome ghosts." These "ghosts" proliferated in response to induction on peroxisome-requiring carbon sources (oleic acid and methanol), and they were distributed to daughter cells. Biochemical analysis of cell lysates revealed that peroxisomal proteins are induced normally in pas1 cells. Peroxisome ghosts from pas1 cells were purified on sucrose gradients, and biochemical analysis showed that these ghosts, while lacking several peroxisomal proteins, did import varying amounts of several other peroxisomal proteins. The existence of detectable peroxisome ghosts in P. pastoris pas1 cells, and their ability to import some proteins, stands in contrast with the results reported by Erdmann et al. (1991) for the S. cerevisiae pas1 mutant, in which they were unable to detect peroxisome-like structures. We discuss the role of PAS1 in peroxisome biogenesis in light of the new information regarding peroxisome ghosts in pas1 cells.

Human kallikrein 6 activity is regulated via an autoproteolytic mechanism of activation/inactivation.

Abstract: Human kallikrein 6 (protease M/zyme/neurosin) is a serine protease that has been suggested to be a serum biomarker for ovarian cancer and may also be involved in pathologies of the CNS. The precursor form of human kallikrein 6 (pro-hK6) was overexpressed in Pichia pastoris and found to be autoprocessed to an active but unstable mature enzyme that subsequently yielded the inactive, self-cleavage product, hK6 (D81-K244). Site-directed mutagenesis was used to investigate the basis for the intrinsic catalytic activity and the activation mechanism of pro-hK6. A single substitution R80 --> Q stabilized the activity of the mature enzyme, while substitution of the active site serine (S197 --> A) resulted in complete loss of hK6 proteolytic activity and facilitated protein production. Our data suggest that the enzymatic activity of hK6 is regulated by an autoactivation/autoinactivation mechanism. Mature hK6 displayed a trypsin-like activity against synthetic substrates and human plasminogen was identified as a putative physiological substrate for hK6, as specific cleavage at the plasminogen internal bond S460-V461 resulted in the generation of angiostatin, an endogenous inhibitor of angiogenesis and metastatic growth.

Developing high cell density fed-batch cultivation strategies for heterologous protein production in Pichia pastoris using the nitrogen source-regulated FLD1 Promoter.

Abstract: A Pichia pastoris strain expressing a Rhizopus oryzae lipase gene under the transcriptional control of the promoter from the P. pastoris formaldehyde dehydrogenase 1 gene (PFLD) was utilized to study the feasibility of this expression system for recombinant protein production using methanol-free fed-batch high cell density cultivations. We have developed a simple and reliable fed-batch strategy using the PFLD system based on the use of methylamine and sorbitol as nitrogen and carbon sources, respectively, for the induction phase. Three different fed-batch fermentations were performed at three different constant growth rates, i.e., at a low growth rate (0.005/h), at an intermediate growth rate of (0.01/h), and at a constant residual sorbitol concentration of 8 g/L, i.e., allowing cells to grow at high (near micro(max)) growth rate (0.02/h). Important differences were observed between the lower and higher growth rate cultivation phases in terms of specific production rate (q(p)) profiles. In all three cases, maximum q(p) were reached soon after the start of the induction phase; after that maximum, an exponential decrease reaching final values close to zero were observed, except for the cells growing at near micro(max). The best results in terms of Y(P/X), productivity and specific productivity were obtained when the microorganism was growing at the highest growth rate. Furthermore, such results were significantly better in relation to those obtained with the PAOX-based system expressing the same protein.

High level expression of a recombinant acid phytase gene in Pichia pastoris.

Abstract: Aims: To achieve high phytase yield with improved enzymatic activity in Pichia pastoris. Methods and Results: The 1347-bp phytase gene of Aspergillus niger SK-57 was synthesized using a successive polymerase chain reaction and was altered by deleting intronic sequences, optimizing codon usage and replacing its original signal sequence with a synthetic signal peptide (designated MF4I) that is a codon-modified Saccharomyces cerevisiae mating factor α-prepro-leader sequence. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene promoter with the MF4I- or wild type α-signal sequence were used to transform Pichia pastoris. The P. pastoris strain that expressed the modified phytase gene (phyA-sh) with MF4I sequence produced 6·1 g purified phytase per litre of culture fluid, with the phytase activity of 865 U ml−1. The expressed phytase varied in size (64, 67, 87, 110 and 120 kDa), but could be deglycosylated to produce a homogeneous 64 kDa protein. The recombinant phytase had two pH optima (pH 2·5 and pH 5·5) and an optimum temperature of 60°C. Conclusions: The P. pastoris strain with the genetically engineered phytase gene produced 6·1 g l−1 of phytase or 865 U ml−1 phytase activity, a 14·5-fold increase compared with the P. pastoris strain with the wild type phytase gene. Significance and Impact of the Study: The P. pastoris strain expressing the modified phytase gene with the MF4I signal peptide showed great potential as a commercial phytase production system.