Construction of a host-independent T7 expression system with small RNA regulation

• We integrate T7 RNA Polymerase gene and T7 Promoter into a single plasmid.
• We use small antisense RNA to regulate T7 RNA Polymerase expression on plasmid.
• We construct a novel T7 expression system independent on DE3 lysogenic hosts.
• We prove the expression ability of this system in 4 non-DE3 E. coli strains.
• We enhance the protein expression in an industrial wild-type strain by this system.

It is desirable to build a universal and efficient protein expression system for wild-type prokaryotic strains in biotechnology industry and the outstanding T7 expression system could be a good candidate. However, the current utilization of T7 system depends on the specific DE3 lysogenic hosts, which severely limits its application in wild-type strains. In this study, a host-independent T7 expression system without relying on DE3 lysogenic hosts to provide T7 RNA Polymerase was developed. T7 RNA Polymerase gene (Gene1) and T7 Promoter were successfully integrated into a single plasmid with the regulation of proper antisense RNA to limit T7 RNA Polymerase expression at a non-lethal level. This host-independent T7 expression system realized efficient protein expression in 4 non-DE3 Escherichia coli strains and a wild-type Sinorhizobium strain TH572.