Creating a System of Dual Regulation of Translation and Transcription to Enhance the Production of Recombinant Protein.

When constructing cell factories, it is crucial to reallocate intracellular resources towards the synthesis of target compounds. However, imbalanced resource allocation can lead to a tradeoff between cell growth and production, reducing overall efficiency. Reliable gene expression regulation tools are needed to coordinate cell growth and production effectively. The orthogonal translation system, developed based on genetic code expansion (GCE), incorporates non-canonical amino acids (ncAAs) into proteins by assigning them to expanded codons, which enables the control of target protein expression at the translational level in an ncAA-dependent manner. However, the stringency of this regulatory tool remains inadequate. This study achieved strict translational-level control of the orthogonal translation system by addressing the abnormal leakage caused by the arabinose-inducible promoter. Further validation was conducted on the relationship between ncAA concentration and expression level, as well as the host's adaptability to the system. Subsequently, the system's applicability across multiple Escherichia coli hosts was verified by examining the roles of RF1 (peptide chain release factor 1) and endogenous TAG codons. By combining this strategy with inducible promoters, dual-level regulation of target gene expression at both transcriptional and translational levels was achieved and the dynamic range was further increased to over 20-fold. When using ncAA to control the expression of T7 RNA polymerase (T7 RNAP), the leakage expression was reduced by 82.7%, mitigating the low production efficiency caused by extensive leakage in the T7 system. As proof of concept, the strategy enhanced the production of alcohol dehydrogenase (ADH) by 9.82-fold, demonstrating its excellent capability in controlling gene expression in developing cell factories.