Cultivation strategies to enhance productivity of Pichia pastoris: A review

Abstract

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.

Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules

“…It shows low viscosity morphology, allowing easy, large-scale, high-cell-density growth in fermenters using inexpensive substrates. Furthermore, this fungus supports broad ranges of pH (4.5-7.0) and temperature (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44) C) for growth. A C1 genetic toolbox is available, and the 3eight megabase pair genome has been sequenced, enabling the development of various gene expression strategies.…”

“…One such non-conventional strain is Pichia pastoris, a yeast originally introduced in the 1960s as an additive for animal foods, and later, thanks to the industrial high cell density fermentation processes already developed for its initial application and the use of strong and tightly regulated promoters, as a new heterologous expression system. Over the years, P. pastoris has become a widely used expression system, [24][25][26][27][28][29] and has been investigated for the production of recombinant protein antigens for human vaccines. 30,31 Several examples of recombinant protein antigen expression in P. pastoris show how this system can offer the benefit of higher eukaryotic host cells, with glycosylated and well-folded proteins, while having the advantage of being easy and inexpensive to cultivate.…”

Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing

“…1000 bp), a high variance in targeting efficiency is observed, indicating the prevalence of the non-homologous end joining (NHEJ) pathway in P. pastoris 181920. As a result, a high clonal variability is found after transformation, necessitating a time and labour-intensive screening process for the clone with the desired characteristics2122. Concerning productivity characteristics, the different expression levels of clones typically originate from varying gene copy numbers2324.…”

High Expression of Human Cathepsin S by Recombinant <b><i>Pichia pastoris</i></b> with Cod Skin as an Organic Co-Nitrogen Source

“…However, a recent trend has been to move away from standard protocols (i.e., fixed recipes for growth-dependent and growth-independent production kinetics) towards a conceptual approach that allows for the development of a specific process strategy that is tailored to a particular product, multilevel carbon sources, alternative nitrogen sources, even trace elements and salts, or the characteristics of specific bioreactor equipment [Looser et al, 2015].…”

“…After the depletion of residual glycerol, the methanol induction phase is initiated to express recombinant proteins [Looser et al, 2015]. As is well known, heterologous protein expression by methylotrophic P. pastoris controlled by the AOX promotor takes place in the presence of methanol as the sole carbon source.…”