Strategies for efficient production of recombinant proteins in Escherichia coli: alleviating the host burden and enhancing protein activity

Abstract

Escherichia coli, one of the most efficient expression hosts for recombinant proteins (RPs), is widely used in chemical, medical, food and other industries. However, conventional expression strains are unable to effectively express proteins with complex structures or toxicity. The key to solving this problem is to alleviate the host burden associated with protein overproduction and to enhance the ability to accurately fold and modify RPs at high expression levels. Here, we summarize the recently developed optimization strategies for the high-level production of RPs from the two aspects of host burden and protein activity. The aim is to maximize the ability of researchers to quickly select an appropriate optimization strategy for improving the production of RPs.

Keywords: Escherichia coli; Host burden; Inclusion bodies; Post-translational modification; Recombinant protein.

Fig. 1

The optimization expression strategies for T7 RNAP and pET plasmids. A Illustration of protein expression of recombinant protein genes on pET plasmids. B Optimization of T7 RNAP transcription and translation level, including substitutions of different promoters, and mutations in promoter functional region and RBS sequence. C regulation of T7 RNAP activity. The conventional approach is to utilize lysozyme or light-induction to regulate. D Optimization of pET plasmids based on expression intensity and copy numbers. Among them, the expression intensity was optimized by constructing an ITR library to screen for optimal expression results. The degree of binding of RNA-i to RNA-p determines the replication intensity of the plasmid to control the copy numbers. By constructing a promoter library for RNA-p, replacing the inducible promoter, and using dCas9 to regulate expression intensity, the copy numbers can be controlled

Fig. 2

The optimization expression strategies for decoupling the cell growth and RP production. A Manipulating the expression of RNAP subunits (β and β') or inhibiting RNAP activity by RNA polymerase inhibitor GP2 to prevent transcription of endogenous growth genes. B Inhibition of growth-related gene expression using CRISPRi. C Reducing competition for host ribosome using orthologous ribosome (O-ribosome) to specifically translate target proteins. D The uncoupling strategy allows to clearly divide an RP production process into two phases, namely the growth phase and the production phase. This allows resources to be used for RP production during fermentation

Fig. 3

The optimization strategies to enhance PTMs. A Principle of disulfide bond formation in the cytoplasm using the CyDisCo system. B Modification process of phosphorylation and acetylation. P: phosphonate; AC: acetyl. C Modification process of glycosylation by overexpression of a heterologous N/O-glycosylase. D Introduction of PTMs via ncAA. The figure shows the principle of phosphoserine introduction

Fig. 4

The routine workflow for expression optimization based on protein properties