Integrated chaperone engineering and process optimization enable enhanced production of ChCODH-II A559W in Escherichia coli.

Carbon monoxide dehydrogenase (ChCODH) from Carboxydothermus hydrogenoformans is a high-value metalloenzyme, however, its recombinant production in Escherichia coli BL21(DE3) is hindered by protein misfolding and challenges associated with functional maturation. In this study, we addressed these bottlenecks for the ChCODH-II A559W through the integrated optimization of chaperone assisted folding, cofactor assembly, and fermentation parameters. Co-expression of the ISC system significantly improved functional enzyme activity, highlighting the importance of proper cofactor maturation. A systematic screening of auxiliary chaperones identified the DnaK-DnaJ-GrpE (KJE) system as the most effective in enhancing soluble expression. The induction strategy was further simplified to single isopropyl β-D-1-thiogalactopyranoside (IPTG)-based system to improve process robustness and industrial feasibility. Fermentation conditions were subsequently optimized in a 5 L scale fermenter, where induction cell density was found to significantly influence enzyme productivity. Semi-quantitative RT-PCR analysis revealed that the endogenous GroEL-ES chaperone system was upregulated under elevated induction density, reflecting a cellular response to increased cultivation stress. Under optimized conditions, KJE co-expression with controlled induction timing, ChCODH-II A559W production achieved a 70.2-fold increase in activity per biomass (U/g WCW) and 14.4-fold increase in protein yield (mg/g WCW) relative to initial flask-scale CODH-only expression system. These results demonstrate a robust and scalable bioprocess engineering platform for the mass production of complex, oxygen-sensitive metalloenzymes, offering a viable path for their industrial application.