CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently

Changchuan Ye,Mengjie Yang,Xiangfang Zeng,Shiyan Qiao,Xi Chen

doi:10.1186/s13036-021-00270-9

Abstract

T7 Expression System is a common method of ensuring tight control and high-level induced expression. However, this system can only work in some bacterial strains in which the T7 RNA Polymerase gene resides in the chromosome. In this study, we successfully introduced a chromosomal copy of the T7 RNA Polymerase gene under control of the lacUV5 promoter into Escherichia coli BW25113. The T7 Expression System worked efficiently in this mutant strain named BW25113-T7. We demonstrated that this mutant strain could satisfactorily produce 5-Aminolevulinic Acid via C5 pathway. A final study was designed to enhance the controllability of T7 Expression System in this mutant strain by constructing a T7 Promoter Variants Library. These efforts advanced E. coli BW25113-T7 to be a practical host for future metabolic engineering efforts.

Highlights

Metabolic engineering plays a critical part in bio-based production of fuels, chemicals and materials from biomass, and often involves integration of multiple genes to re-direct metabolic fluxes [1]
Growth rate was similar between E. coli BW25113 and BW25113-T7 in these three types of medium (Fig. 4). These results indicated that induction of a chromosomal copy of the T7 RNA Polymerase gene does not impact growth characteristics of E. coli BW25113-T7
The fluorescent signal in E. coli BW25113-T7 was detected as 5636, which was about 4 times greater than that in E. coli BL21(DE3) (Fig. 6 A). It means that expression strength of sYFP under control of T7-Lac promoter in BW25113-T7 was greater than BL21(DE3) (p < 0.001). These results revealed that the T7 expression system has an enhanced efficiency in E. coli BW25113-T7

Summary

Introduction

Metabolic engineering plays a critical part in bio-based production of fuels, chemicals and materials from biomass, and often involves integration of multiple genes to re-direct metabolic fluxes [1]. As synthetic biology applications have grown in complexity, so too has the sophistication of available genetic and biochemical tools. A dramatic change in the scope and complexity manufacturing processes within the space of synthetic biology has occurred. Thanks to development of genetic engineering, accomplishments have progressed from simple reconstitution of biosynthetic pathways in heterologous hosts to Synthetic biologists prefer two organisms for expression and optimization of heterologous biosynthetic pathways: Escherichia coli and Saccharomyces cerevisiae. E. coli is the most widely used prokaryotic system that produces heterologous proteins for industrial production of bacterial metabolites by batch and fed-batch operations [16, 17]. E. coli has been regarded as the workhorse of modern biotechnology in the microbial production of biofuels and biochemicals [19]

Methods

Results

Conclusion