Copper-inducible expression system for metabolic engineering of Escherichia coli

Abstract

Aims: The inducible expression system plays an important role in engineering Escherichia coli for chemical production. However, it still heavily relies on expensive chemical inducers, like IPTG. There is a pressing need to develop alternative expression systems with more affordable inducers.

Materials and results: We herein report a copper-inducible expression system in E. coli based on the two-component Cus system and T7 RNA polymerase (RNAP). By integrating the gene encoding T7 RNAP at the CusC locus, we managed to program eGFP expression under the T7 promoter in response to different concentrations of Cu2+ (0-20 μM). Subsequently, we demonstrated that the copper-inducible expression system was suitable for the metabolic engineering of E. coli toward protocatechuic acid overproduction, and the resulting strain with combined manipulation of the central metabolism via CRISPRi produced 4.12 g L-1 PCA under the optimal copper concentration and induction time.

Conclusions: We have established a copper-inducible T7 RNAP expression system in E. coli. The copper-inducible expression system could rationally control metabolic pathways in a temporal and dose-dependent manner. The gradient expression system based on copper inducer could be widely used in E. coli cell factories, and the design principle reported here would also be applicable in other prokaryotes.

Keywords: CusC promoter; Escherichia coli; CRISPRi; T7 RNAP; copper-regulated system; protocatechuic acid.