Deciphering Transcription-Translation-Folding (TX-TL-FD) for Enhancing Cutinase Production in T7 System and Genetic Chaperone-Equipped Strains.

T7 RNA polymerase (T7RNAP), orthogonal to the T7 promoter, is a powerful tool in engineered that enables the production of many different harsh enzymes. Still, it requires precise control, particularly when expressing toxic proteins. The optimized strategy for the interconnected processes of transcription (TX), translation (TL), and protein folding (FD) in the T7 system is still not well understood. Therefore, we developed a quantitative adjustment index (AI) to evaluate all regulatory factors within the "tri-synergistic TX-TL-FD" pathway to obtain high-level production of leaf-branch compost cutinase mutant (ICCM), an enzyme challenging to express in soluble form. Among six chassis (BD, B7G, BKJ, C43, C7G, and CKJ), and considering the effect of replication origin, ribosome binding site (RBS), and chaperones, we identified T7RNAP level and translation initiation region (TIR) as the primary determinants of expression efficiency. Coordinated regulation of TX-TL proved the most effective performance, thus enhancing ICCM expression by 90%. In contrast, FD optimization through temperature modulation yielded only 10% enhancement. Notably, molecular chaperones of GroELS and DnaK/J showed benefits only after achieving optimal TX-TL balance. This hierarchical framework of trisynergistic regulation in the T7 system provides a universal strategy to express complex proteins in engineered .