(326g) Modification of the T7 Expression System By Genetic Engineering to Increase Recombinant Protein Production in BL21(DE3)

The combination of E. coli BL21(DE3) and pET expression system is currently the most widely used recombinant protein expression system and plays a prominent role in experimental research and industrial production. However, not every protein can be highly expressed in BL21(DE3). On the one hand, the Lac UV5 promoter is more prone to leaky expression than the wild-type Lac promoter (P_LacWT), resulting in stronger Lac UV5 promoter driving large amounts of T7 RNA polymerase transcription during both non-induction and induction phases, leading to more resources for cell growth devoted to recombinant protein production, causing a restricted allocation of resources for cell growth and protein production. On the other hand, the activity of certain enzymes can interfere with the host metabolism. Constructing more expression hosts is a good choice to enhance the yield of recombinant proteins, however, most of the previous studies implemented separate optimization strategies for different proteins, which is time-consuming and difficult to apply quickly.The expression level of T7 RNA polymerase is the core control node of the pET expression system, and regulating its expression level is an effective way to increase the yield of difficult-to-express proteins, most commonly at the transcription level and translation level. Based on this, we first used CRISPR-Cas9 technology to replace the original promoters with other inducible promoters: P_araBAD, P_rhaBAD, and P_tet, respectively, to regulate the transcriptional level and leakage level of the T7 RNA polymerase and optimize the production of labile autolysins and membrane proteins. Secondly, a BL21(DE3)-derived variant strain library with different RBS sequences of T7 RNAP was constructed using Base Editor and CRISPR-Cas9 technology. Notably, the CRISPR-Cas9 technology combined with degenerate primers enabled the construction of an RBS library with 87.5% of the theoretical coverage in 3 rounds of editing, which is more convenient and efficient than the use of Base Editor, and the expression level of a target gene in the variant strain library ranged from 28 to 220% of the parental strain. Furthermore, a high-throughput host-screening platform for recombinant protein production was constructed upon the variant strain library, which could obtain the best expression hosts of different recombinant target proteins, including autolytic protein, membrane proteins, antimicrobial peptides, and hardly soluble proteins, in only 3 days. Among them, the expression of glucose dehydrogenase in the best host exhibited a 298-fold increase compared to the parental strain. In summary, the engineered strains constructed in this study as well as the library of variant strains provide more host options for the production of recombinant proteins.