Yeast expression systems have been successfully used for over 20 years for the production of recombinant proteins. With the growing interest in recombinant protein expression for various uses, yeast expression systems, such as the popular Pichia pastoris, are becoming increasingly important. Although P. pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, there is still room for improvement of this expression system. In particular, secretion of recombinant proteins is still one of the main reasons for using P. pastoris. Therefore, endoplasmic reticulum protein folding, correct glycosylation, vesicular transport to the plasma membrane, gene dosage, secretion signal sequences, and secretome studies are important considerations for improved recombinant protein production.

Protein secretion in Pichia pastoris and advances in protein production.

The rapid increase in the number of diabetic patients globally and exploration of alternate insulin delivery methods such as inhalation or oral route that rely on higher doses, is bound to escalate the demand for recombinant insulin in near future. Current manufacturing technologies would be unable to meet the growing demand of affordable insulin due to limitation in production capacity and high production cost. Manufacturing of therapeutic recombinant proteins require an appropriate host organism with efficient machinery for posttranslational modifications and protein refolding. Recombinant human insulin has been produced predominantly using E. coli and Saccharomyces cerevisiae for therapeutic use in human. We would focus in this review, on various approaches that can be exploited to increase the production of a biologically active insulin and its analogues in E. coli and yeast. Transgenic plants are also very attractive expression system, which can be exploited to produce insulin in large quantities for therapeutic use in human. Plant-based expression system hold tremendous potential for high-capacity production of insulin in very cost-effective manner. Very high level of expression of biologically active proinsulin in seeds or leaves with long-term stability, offers a low-cost technology for both injectable as well as oral delivery of proinsulin.

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Cell factories for insulin production.

Pichia pastoris is an efficient host for the expression and secretion of heterologous proteins and the most important feature of P. pastoris is the existence of a strong and tightly regulated promoter from the alcohol oxidase I (AOX1) gene. The AOX1 promoter (pAOX1) has been used to express foreign genes and to produce a variety of recombinant proteins in P. pastoris. However, some efforts have been made to develop new alternative promoters to pAOX1 to avoid the use of methanol. The glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) has been used for constitutive expression of many heterologous proteins. The pGAP-based expression system is more suitable for large-scale production because the hazard and cost associated with the storage and delivery of large volume of methanol are eliminated. Some important developments and features of this expression system will be summarized in this review.

Recent advances on the GAP promoter derived expression system of Pichia pastoris

The success of Pichia pastoris as a heterologous expression system lies predominantly in the impressive yields that can be achieved due to high volumetric productivity. However, low specific productivity still inhibits the potential success of this platform. Multi-(gene) copy clones are potentially a quick and convenient method to increase recombinant protein titer, yet they are not without their pitfalls. It has been more than twenty years since the first reported use of multi-copy clones and it is still an active area of research to find the fastest and most efficient method for generating these strains. It has also become apparent that there is not always a linear correlation between copy number and protein titer, leading to in-depth investigations into how to minimize the negative impact of secretory stress and achieve clonal stability.

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Can too many copies spoil the broth

Aims: This study attempted to fully explore the expression potentials of Pichia pastoris for producing porcine insulin precursor (PIP) through PIP copy number optimization.



Methods and Results:  Multi-copy strains were screened employing a highly efficient improved in vivo method and their copy numbers were quantified by real-time qPCR. A range of Mut+P. pastoris strains harbouring 0, 1, 3, 6, 12, 18, 29, 52 copies of PIP were obtained. After 96 h methanol induction, a bell-shaped correlation curve was observed between gene dosage and protein yield, and the maximum PIP expression level of 181 mg l−1 was achieved by a 12-copy strain. Specific growth rate and methanol utilization capacity were found to decrease remarkably for high copy strains (>12 copies). Transcriptional analysis of KAR2 suggested higher copy strains were suffering more from ER stress.



Conclusions:  A copy number around 12 is optimal for secretory expression of PIP in P. pastoris. Excess PIP gene dosage (>12 copies) significantly impaired the growth of P. pastoris hosts.



Significance and Impact of the Study:  The methods developed and the discoveries made by this systematical investigation will be helpful to the application and understanding of Pichia pastoris expression system for heterologous overexpression.

https://sfamjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2672.2009.04279.x

Efficient generation of multi-copy strains for optimizing secretory expression of porcine insulin precursor in yeast Pichia pastoris

We have found a direct relationship between protein production in Pichia pastoris and the number of introduced synthetic genes of miniproinsulin (MPI), fused to the Saccharomyces cerevisiae pre-pro alpha factor used as secretion signal, and inserted between the alcohol oxidase 1 (AOX1) promoter and terminator sequences. Two consecutive approaches were followed to increase the number of integrated cassettes: the head-to-tail expression cassette multimerization procedure and re-transformation with a dominant selection marker. This increased expression from 19 to 250 mg l(-1) when about 11 copies have been integrated. Further, the correct position of one of the disulphide bridges of the purified molecule was verified by digestion with Glu-C endoprotease, followed by mass spectrometry of the isolated fragments.

Liudys García

Papers

Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris.