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.

Fermentation development of recombinant Pichia pastoris expressing the heterologous gene: bovine lysozyme.

Abstract: The methylotrophic yeast, Pichia pastoris, has been shown to be an outstanding host for high-level heterologous gene expression.'+ The alcohol oxidase promoter was isolated and cloned by Ellis et a/.' and transformation of Pichia pastoris was first reported in 1985.6 The success of the Pichia expression system is linked to the strong, tightly regulated alcohol oxidase (AOXl ) promoter. The strength of the promoter is demonstrated by the observation that AOX comprises up to 30% of the soluble protein in extracts of Pichia pastoris grown on methanol (MeOH) in a chemostat.' Another key feature of the system is that high cell densities can be achieved using a simple MeOH-salts medium.' The strong promoter coupled with the high-cell-density fermentations have allowed production of recombinant products a t high intercellular (e.g., 400 mg/L hepatitis surface antigen)' and extracellular (e.g., 2.5 g /L invertase and 250 mg/L bovine l y ~ o z y m e ) ~ ~ ~ concentrations. The recombinant Pichia fermentations referenced above'.234 employed an MeOH utilization mutant (Mut-) created by integration of the recombinant expression cassette into the host genome a t the AOXl locus, resulting in disruption of the AOXl structural gene. The Mutstrains were grown in a repressed, batch regime for 24 hours, followed by an MeOH-induced, fed-batch regime for 100-200 hours. The long induction period is a consequence of the slow growth rate of the Mutstrain on MeOH ( p = 0.01-0.04 h' ) . The Mutstrains are still able to utilize MeOH due to the presence of the AOX2 gene, which yields 10-20 times less AOX activity than the AOXI gene.' The original Mutprotocols offer several desirable features. They are extremely easy to scale up to large volumes and they consistently give maximal levels of expression.' Additionally, low growth rates may be desirable for production of certain recombinant product^.'*^^'^ Furthermore, the Mutstrains are not as sensitive to transient high residual MeOH concentrations, which can cause as much as a 99% loss of AOX activity and cell death following certain culture perturbations of wild-type met hylotrophic yeast. 'J I-'

Reliable high‐throughput screening with Pichia pastoris by limiting yeast cell death phenomena

Abstract: Comparative screening of gene expression libraries employing the potent industrial host Pichia pastoris for improving recombinant eukaryotic enzymes by protein engineering was an unsolved task. We simplified the protocol for protein expression by P. pastoris and scaled it down to 0.5-ml cultures. Optimising standard growth conditions and procedures, programmed cell death and necrosis of P. pastoris in microscale cultures were diminished. Uniform cell growth in 96-deep-well plates now allows for high-throughput protein expression and screening for improved enzyme variants. Furthermore, the change from one host for protein engineering to another host for enzyme production becomes dispensable, and this accelerates the protein breeding cycles and makes predictions for large-scale production more accurate.

Laccase from the white-rot fungus Trametes versicolor : cDNA cloning of lcc1 and expression in Pichia pastoris

Abstract: A cDNA coding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene lcc1, which encodes a laccase isoenzyme of 498 amino-acid residues preceded by a 22-residue signal peptide. The lcc1 cDNA was cloned into the vector pHIL-D2 for expression in Pichia pastoris under the control of the AOX1 promoter. Transformants were found to secrete active recombinant enzyme after induction with methanol. The use of growth medium buffered to pH 6.0 and control of pH during cultivation were found to be important, or even necessary, for obtaining activity in liquid cultures. The effect of exchanging the native secretion signal for the Saccharomyces cerevisiae α-factor pre-pro secretion signal was studied by cloning the portion encoding the mature enzyme into the vector pPIC9. The activity obtained for the construct encoding the native laccase signal sequence was found to be seven-fold higher than for the construct encoding the α-factor secretion signal. Utilisation of the P. pastoris pep4 mutant strain SMD1168 was found to provide a two-fold higher level of activity compared with P. pastoris GS115.

Modeling of growth and energy metabolism of Pichia pastoris producing a fusion protein

Abstract: A fusion protein composed of a cellulose binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B (CBD-lipase) was produced by Pichia pastoris methanol utilization plus phenotype in high cell-density cultures. The genes expressing CBD-lipase were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. To control the repression and induction of AOX1 and oxygen demand at high cell density, a four-stage process was used. Batch growth on glycerol was used in the first step to provide biomass (28 g L–1) while product formation was prevented due to repression of the AOX1. The second stage was exponential fed-batch growth on glycerol, which caused a slight increase of the enzyme alcohol oxidase activity due to derepression of the AOX1. This procedure resulted in smooth transition to exponential fed-batch growth on methanol, the third stage, in which the AOX1 was strongly induced. The fourth stage was constant fed-batch growth on methanol used to control the oxygen demand at the high cell density. A kinetic model was developed that could predict biomass growth and oxygen consumption in processes with and without oxygen-enriched air. With oxygen enrichment to 34% O2 in the inlet air the methanol feed rate could be increased by 50% and this resulted in 14% higher final cell density (from 140 to 160 g L–1 cell dry weight). The increased methanol feed rate resulted in a proportionally increased specific rate of product secretion to the medium. After an initial decrease, the synthesis capacity of the cell was kept constant throughout the cultivation, which made the product concentration increase almost constantly during the process. The kinetic model also describes how the low maintenance demand of P. pastoris compared with E. coli enables this organism to grow to such high cell densities.