Enhanced production of recombinant proteins in Corynebacterium glutamicum by constructing a bicistronic gene expression system

Manman Sun,Xiuxia Liu,Yankun Yang,An Li,Zihao Zhao,Yali Wang,Xiong Gao,Zhonghu Bai

doi:10.1186/s12934-020-01370-9

Abstract

BackgroundCorynebacterium glutamicum is a traditional food-grade industrial microorganism, in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is still difficult to be solved. Here, according to the bacteria-specific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum.ResultsIn this research, the Escherichia coli (E. coli) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic expression vectors were isolated and exhibited high efficiency under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-α (BoIFN-α), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type Ι N-terminal peptide (PΙNP). All examined proteins were highly expressed compared with the original vector with tac promoter. Large-scale production of PΙNP was also performed in fed-batch cultivation, and the highest PΙNP production level was 1.2 g/L.ConclusionIn this study, the strength of the inducible tac promoter for C. glutamicum was improved by screening and inserting fore-cistron sequences in front of the target genes. Those vectors with bicistronic expression patterns have strong compatibility for expressing various heterogeneous proteins in high yield. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.

Highlights

Corynebacterium glutamicum has been used as an important industrial microorganism to produce amino acid since the 1950s, during which people have gained abundant knowledge about its growing condition andSun et al Microb Cell Fact (2020) 19:113 cellular mechanism [1,2,3]
The N-terminal 62 bp of the open reading frame (ORF) from 24 genes were taken as the forecistron sequences embedded between Tac promoter (Ptac) and target gene, these 24 genes were originated from 12 highly transcribed genes (GEO accession number: GSE77502) and 12 highly expressed genes in C. glutamicum [25, 26] (Additional file 1: Table S3)
The results showed that the expression intensities were varying among different bicistronic design (BCD) vectors (Fig. 2a), with 15 of 24 constructs having higher Enhanced Green Fluorescent Protein (EGFP) expression levels than the Ptac plasmid

Summary

Introduction

Corynebacterium glutamicum has been used as an important industrial microorganism to produce amino acid since the 1950s, during which people have gained abundant knowledge about its growing condition andSun et al Microb Cell Fact (2020) 19:113 cellular mechanism [1,2,3]. Trials of developing a recombinant protein expression system in this microorganism have been made in recent years given its characteristics of endotoxin-free, low extracellular protease abundance and the ability of protein secretion [4]. To enhance the protein yield in C. glutamicum expression system, various strategies including screening promoters and other genetic parts, optimizing culture conditions and engineering host cells [6, 8, 9] have been carried out. Corynebacterium glutamicum is a traditional food-grade industrial microorganism, in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. According to the bacteriaspecific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum

Methods

Results

Discussion

Conclusion