Green Process: Improved Semi-Continuous Fermentation of Pichia pastoris Based on the Principle of Vitality Cell Separation

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.

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.

Pichia pastoris, a methylotrophic yeast, is an established system for the production of heterologous proteins, particularly biopharmaceuticals and industrial enzymes. To maximise and optimise the production of recombinant products, recent molecular research has focused on numerous issues including the design of expression vectors, optimisation of gene copy number, co-expression of secretory proteins such as chaperones, engineering of glycosylation and secretory pathways, etc. However, the physiological effects of different cultivation strategies are often difficult to separate from the molecular effects of the gene construct (e.g., cellular stress through over-expression or incorrect post-translational processing). Hence, overall system optimisation is difficult, even though it is urgently required in order to describe and understand the behaviour of new molecular constructs. This review focuses on particular aspects of recombinant protein production related to variations in biomass growth and their implications for strain design and screening, as well as on the concept of rational comparisons between cultivation systems for the development of specific production processes in bioreactors. The relationship between specific formation rates of secreted recombinant proteins, qp, and specific growth rates, μ, has been analysed in a conceptual attempt to compare different systems, particularly those based on AOX1/methanol and GAP/glucose, and this has now evolved into a pivotal concept for bioprocess engineering of P. pastoris.