Kinase Screening in Pichia pastoris Identified Promising Targets Involved in Cell Growth and Alcohol Oxidase 1 Promoter (PAOX1) Regulation.

Wei Shen,Menghao Cai,Chuixing Kong,Mian Zhou,Yiqi Liu,Ying Xue,Xiangshan Zhou,Yuanxing Zhang,Tianyi Jiang,Shihui Yang

doi:10.1371/journal.pone.0167766

Wei Shen, Menghao Cai + Show 8 more

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https://doi.org/10.1371/journal.pone.0167766

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Abstract

As one of the most commonly used eukaryotic recombinant protein expression systems, P. pastoris relies heavily on the AOX1 promoter (PAOX1), which is strongly induced by methanol but strictly repressed by glycerol and glucose. However, the complicated signaling pathways involved in PAOX1 regulation when supplemented with different carbon sources are poorly understood. Here we constructed a kinase deletion library in P. pastoris and identified 27 mutants which showed peculiar phenotypes in cell growth or PAOX1 regulation. We analyzed both annotations and possible functions of these 27 targets, and then focused on the MAP kinase Hog1. In order to locate its potential downstream components, we performed the phosphoproteome analysis on glycerol cultured WT and Δhog1 strains and identified 157 differentially phosphorylated proteins. Our results identified important kinases involved in P. pastoris cell growth and PAOX1 regulation, which could serve as valuable targets for further mechanistic studies.

Highlights

As one of the most commonly used expression systems, P. pastoris is highly efficient and cost effective for both secretive and intracellular protein expression
After examining cell growth and PAOX1 activity of these knockout strains on glucose, glycerol or methanol, we identified 27 kinases involved in cell growth or PAOX1 regulation
In order to exclude any possibilities of post-transcriptional and post-translational control, a PAOX1-GFP reporter was expressed in those mutants with interesting phenotypes to confirm the promoter activity.) The growth rates and alcohol oxidase (Aox) enzymatic activities of all of the 92 knockout strains on three carbon sources are shown in Fig A in S1 File

Summary

Introduction

As one of the most commonly used expression systems, P. pastoris is highly efficient and cost effective for both secretive and intracellular protein expression. Several important features of P. pastoris render it ideally suitable for large-scale production of recombinant proteins [1]. Over 5000 recombinant proteins have been successfully expressed in P. pastoris including insulin, α-interferon and hepatitis B antigen (http://www.pichia.com/). Recombinant protein expression in P. pastoris relies on the AOX1 gene promoter (PAOX1). AOX1 is the major gene encoding alcohol oxidase (Aox), which is substantially induced when cells are cultured in methanol and occupies 30% of total soluble proteins in the yeast cell [2]. P. pastoris belongs to the group of methylotrophic yeasts which are capable of utilizing methanol as the sole carbon and energy source for cell growth. While strongly induced by methanol, PAOX1 is strictly repressed by other carbon sources such as glucose, glycerol and ethanol [3]

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