Customized design of host-independent T7 expression system (HITES) for a broad host range

Efficient methods and universal DNA elements are eagerly required for the expression of proteins and the production of target chemicals in synthetic biology and metabolic engineering. This paper develops a customized-design approach by utilizing the host-independent T7 expression system (HITES), which facilitates the rational design and rapid construction of T7 expression systems. Firstly, the E_iL (Upper-limit value of initial enzyme activity) value is discovered to play a pivotal factor in the successful construction of the T7 expression system, different host strains exhibit varying E_iL values, and this study presents a method to measure the E_iL values. Secondly, E. coli DH5α is chosen as the host strain, and it demonstrates that various strategies to modulate T7 RNA polymerase activity can efficiently construct the HITES T7 expression system in E. coli DH5α under the guidance of E_iL. Lastly, the customized-design of HITES enables the efficient expression of sfGFP and D-MIase proteins across 13 host strains, guided by E_iL values. This customized-design method of HITES offers a streamlined pathway for T7 system construction across a broad range of hosts and serves as an enabling tool for synthetic biology, metabolic engineering, and enzyme engineering.