Abstract
Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.
Highlights
The methylotrophic yeast Pichia pastoris, currently reclassified as Komagataella pastoris, has become a substantial workhorse for biotechnology, especially for heterologous protein production (Kurtzman 2009). It was introduced more than 40 years ago by Phillips Petroleum for commercial production of single cell protein (SCP) as animal feed additive based on a high cell density fermentation process utilizing methanol as carbon source
We focus on recent developments for heterologous protein production and describe examples for the commercial use of this expression system
We describe specific strategies for obtaining high-level expression strains and summarize important applications of P. pastoris for production of biopharmaceuticals, membrane proteins and industrial proteins
Summary
The methylotrophic yeast Pichia pastoris, currently reclassified as Komagataella pastoris, has become a substantial workhorse for biotechnology, especially for heterologous protein production (Kurtzman 2009) It was introduced more than 40 years ago by Phillips Petroleum for commercial production of single cell protein (SCP) as animal feed additive based on a high cell density fermentation process utilizing methanol as carbon source. In the 1980s, P. pastoris was developed as a heterologous protein expression system using the strong and tightly regulated AOX1 promoter (Cregg et al 1985). In combination with the already developed fermentation process for SCP production, the AOX1 promoter provided exceptionally high levels of heterologous proteins. We describe specific strategies for obtaining high-level expression strains and summarize important applications of P. pastoris for production of biopharmaceuticals, membrane proteins and industrial proteins.
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