Different construction strategies affected on the physiology of Pichia pastoris strains highly expressed lipase by transcriptional analysis of key genes

Jinjin Huang,Qing Wang,Wei Bu,Lingxiao Chen,Zhen Yang,Weifa Zheng,Ying Li,Jilun Li

doi:10.1080/21655979.2019.1614422

Abstract

ABSTRACT We demonstrated previously that expression of Rhizomucor miehei lipase (RML) in Pichia pastoris could be significantly increased by addition of gene propeptide, optimized signal peptide codons and manipulation of gene dosage. In this study, effects of various strategies on the protein synthesis and secretion pathways were analyzed. Using nine strains previously constructed, we evaluated cell culture properties, enzymatic activities, and analyzed transcriptional levels of nine genes involved in protein synthesis and secretion pathways by qPCR. We observed that (i) Addition of propeptide decreased lipase folding stress by down-regulated four UPR-related genes. (ii) Signal peptide codons optimization had no effect on host with no change in the nine detected genes. (iii) Folding stress and limited transport capacity produced when rml gene dosage exceed 2. Different limiting factors on lipase expression in strains with different construction strategies were identified. This study provides a theoretical basis for further improving RML by transforming host.

Highlights

The methylotrophic yeast Pichia pastoris is one of the most widely used foreign protein expression systems for heterologous protein production [1]
In order to better understand the changes that occurred in the host when foreign protein expression, in the present study, we examined the variation on the steps of protein synthesis and secretion pathway in recombinant strains constructed with different strategies previously
In order to answer the questions above, relative transcription levels of nine genes (HAC1, KAR2, protein disulfide isomerase (PDI), ERO1, SEC31, SSO2, MON2, VPS10, IMH1) participated in different process of protein synthesis and secretion pathway were analyzed in this study before and after adding propeptide when fermented at 48 h and 96 h

Summary

Introduction

The methylotrophic yeast Pichia pastoris is one of the most widely used foreign protein expression systems for heterologous protein production [1]. Numerous studies explore ways to increase protein yield, such as optimizing gene codons, gene dosage, culture conditions, change promoters and host transformation, etc [4,5]. Among these strategies, modification of host to enhance the yield of foreign proteins is getting more and more attention. Strategies for optimizing protein yield are random because of uncertain the limiting factors, and rarely study on the relationship between physiological of host and protein yields used various strategies when expression of heterologous protein

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Conclusion