Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris

Juhani Von Hellens,Xiaojing Chen,Robert Speight,Laura Navone,Thomas Vogl,Carlos Luna-Flores,Pawarisa Luangthongkam,Jo-Anne Blinco

doi:10.1186/s12934-020-01499-7

Abstract

BackgroundPichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments in synthetic biology have extended the toolbox for genetic engineering of P. pastoris to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critical to achieve cost-effective industrial manufacture. Several enzymes are commercially produced in P. pastoris, with phytases being one of the biggest on the global market. Phytases are ubiquitously used as a dietary supplement for swine and poultry to increase digestibility of phytic acid, the main form of phosphorous storage in grains.ResultsPotential bottlenecks for expression of E. coli AppA phytase in P. pastoris were explored by applying bidirectional promoters (BDPs) to express AppA together with folding chaperones, disulfide bond isomerases, trafficking proteins and a cytosolic redox metabolism protein. Additionally, transcriptional studies were used to provide insights into the expression profile of BDPs. A flavoprotein encoded by ERV2 that has not been characterised in P. pastoris was used to improve the expression of the phytase, indicating its role as an alternative pathway to ERO1. Subsequent AppA production increased by 2.90-fold compared to the expression from the state of the AOX1 promoter.DiscussionThe microbial production of important industrial enzymes in recombinant systems can be improved by applying newly available molecular tools. Overall, the work presented here on the optimisation of phytase production in P. pastoris contributes to the improved understanding of recombinant protein folding and secretion in this important yeast microbial production host.

Highlights

Pichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities
Several bidi‐ rectional promoters (BDPs) were tested for co-expression of the phytase together with folding and secretion chaperones, comparing phytase production yields with the commonly used PAOX1
Transcriptional optimization of AppA phytase expression The AppA gene from E. coli was expressed under the control of the commonly used methanol inducible P. pastoris Monodirectional promoter (MDP) PAOX1, PDAS1 and PCAT1, the methanol inducible PHpFMD from H. polymorpha [62] and the constitutive P

Summary

Introduction

Pichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Phytases can be found in animals and plants but microbial phytases have been the most extensively studied and constitute all commercial formulations available in the livestock feed supplement market [16, 38]. There has Navone et al Microb Cell Fact (2021) 20:8 been significant commercial work towards process optimisation to increase phytase production yields to achieve technical and economic manufacturing feasibility, but there is limited available information in the academic literature on P. pastoris strain engineering for phytase production. Previous investigations on the production of AppA phytase in P. pastoris include the expression under the strong constitutive promoter of the glyceraldehyde 3-phosphate dehydrogenase gene (PGAP) and methanol inducible alcohol oxidase 1 promoter (PAOX1) promoters [8, 9, 32, 38, 53, 56]

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