Elsevier

Biotechnology Advances

Volume 36, Issue 1, January–February 2018, Pages 182-195
Biotechnology Advances

Research review paper
Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: A review

https://doi.org/10.1016/j.biotechadv.2017.11.002Get rights and content

Abstract

Pichia pastoris has been recognized as one of the most industrially important hosts for heterologous protein production. Despite its high protein productivity, the optimization of P. pastoris cultivation is still imperative due to strain- and product-specific challenges such as promoter strength, methanol utilization type and oxygen demand. To address the issues, strategies involving genetic and process engineering have been employed. Optimization of codon usage and gene dosage, as well as engineering of promoters, protein secretion pathways and methanol metabolic pathways have proved beneficial to innate protein expression levels. Large-scale production of proteins via high cell density fermentation additionally relies on the optimization of process parameters including methanol feed rate, induction temperature and specific growth rate. Recent progress related to the enhanced production of proteins in P. pastoris via various genetic engineering and cultivation strategies are reviewed. Insight into the regulation of the P. pastoris alcohol oxidase 1 (AOX1) promoter and the development of methanol-free systems are highlighted. Novel cultivation strategies such as mixed substrate feeding are discussed. Recent advances regarding substrate and product monitoring techniques are also summarized. Application of P. pastoris to the production of biodiesel and other value-added products via metabolic engineering are also reviewed. P. pastoris is becoming an indispensable platform through the use of these combined engineering strategies.

Introduction

The methylotrophic yeast Pichia pastoris has been established as a successful protein production platform, especially in the sector of industrial enzymes and the biopharmaceutical industry. As a “generally regarded as safe” (GRAS) microorganism, it has been used for the production of over 500 pharmaceutical proteins and more than 1000 recombinant proteins as of 2009 (Fickers, 2014). Driven by increasing demands in the food and feed industries, P. pastoris has also become an important host to produce enzymes such as xylanase and phytase, which are relevant to these sectors (Spohner et al., 2015). Recently, P. pastoris has also been favored in the expression of eukaryotic membrane proteins, facilitating advances in structural biology (Byrne, 2015, Goncalves, 2013). Using cell surface display techniques, P. pastoris has been used to synthesize biofuels and other chemicals (Tanaka et al., 2012). The success of P. pastoris as such a versatile system is mainly attributed to its ability to grow to a high biomass concentration on defined media, its capacity to perform complex post-translational modifications which include correct protein folding, disulfide bond formation as well as glycosylation, its high secretion efficiency and its repertoire of both inducible and constitutive promoters.

The successful development of high-yield yeast strains is imposed with strain- and product-specific challenges. To overcome these challenges, engineering strategies comprising genetic and process engineering approaches have been employed (Fig. 1). Extensive progress has been made for protein expression in P. pastoris. In this review, we will focus on recent progress related to the production of proteins and other by-products, aiming to update our previously published review (Potvin et al., 2012) from an engineering perspective. Strategies involving genetic and bioprocess engineering will be discussed. State-of-the-art monitoring techniques for substrates and products will also be briefly summarized.

Section snippets

Advances of AOX1 promoter regulation

The alcohol oxidase І (AOX1) promoter regulates the metabolism of methanol and catalyzes the first step of methanol assimilation, converting methanol to formaldehyde. It is widely used to drive heterologous protein expression due to its tight regulation and strong inducibility when methanol is used as the sole carbon source. Although it is the most widely studied promoter in P. pastoris, the mechanisms for the regulation of PAOX1 are still gaining great attention. This is because insights into

Substrate monitoring

Methanol monitoring is crucial for the success of the PAOX1 systems. Excessive methanol accumulation, particularly for the MUT strain, is cytotoxic while insufficient methanol may reduce protein yield. Monitoring techniques are generally classified as on-line and off-line methods. Compared with conventional time-consuming off-line methods such as gas chromatography (GC) or high performance liquid chromatography (HPLC), on-line methods eliminates manual sampling and offers rapid and efficient

Fed-batch cultivation

Fed-batch is an efficient operational strategy for high cell density fermentation. It is initiated with a batch phase, followed by a carbon source feeding phase to achieve high biomass accumulation for methanol induction and product formation. Carbon source depletion at the end of the batch phase is indicated by a DO spike. In P. pastoris fermentation, a carbon source such as glucose or glycerol is commonly used in the batch phase to develop a base of cell growth prior to feeding the

Production of other bio-products in P. pastoris

P. pastoris has been widely used as a factory for protein production. In recent years, P. pastoris has gained considerable interest as a host for metabolic engineering to produce value-added products. Despite the lack of autonomous plasmids and a limited choice of promoters, P. pastoris has been engineered into many different cellular factories, producing various products. Carotenoids such as lycopene have applications in animal feed supplements, cosmetics and pharmaceutical compounds. Bhataya

Conclusions and prospects

P. pastoris has received great attention as a powerful system for protein expression. Efficient production of recombinant proteins relies on multi-level optimization strategies incorporating promoters, codon bias, signal peptides, gene dosage and cultivation strategies. Systems biology methods such as GEMs and MFA are particularly effective tools for enhanced protein production. Although the engineering strategies (Fig. 1) discussed in this review can be applied in a combinatorial fashion to

Acknowledgements

Financial support for this work was provided by a discovery grant from the Natural Sciences and Engineering Research Council of Canada. Zhiliang Yang was the recipient of a doctoral scholarship from the China Scholarship Council for the duration of this work.

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