Intercellular whole-cell cascade strategy for robust CO-to-formate bioconversion

Biocatalytic conversion of CO-containing C1 gas to formate provides a route for C1 gas valorization, but purified carbon monoxide dehydrogenase (CODH)-formate dehydrogenase (FDH) enzyme cascades require enzyme purification and immobilization steps. As a practical alternative, this study developed an ethyl viologen (EV)-mediated intercellular whole-cell cascade by coupling CODH-expressing Escherichia coli with FDH-expressing Methylobacterium extorquens AM1. In this cascade, the two whole-cell biocatalysts performed the oxidative and reductive half-reactions, respectively, and were functionally coupled through EV redox cycling. The native whole-cell cascade enabled CO-to-formate conversion but showed lower initial productivity than the immobilized enzyme benchmark under low-EV conditions, while maintaining activity and productivity more effectively during multi-day operation. Module-level activity analysis, EV-dependent FDH kinetics, and apparent Damköhler number analysis indicated that the initial productivity penalty was mainly associated with restricted mediator accessibility in AM1 whole cells. Mild Triton X-100 permeabilization produced envelope-associated morphological changes, increased FDH activity at 0.1 mM EV by 7.9-fold, and enhanced cascade productivity by approximately 7-fold while maintaining multi-day productivity retention. In a pH-controlled gas-fed bubble-column reactor, the permeabilized cascade outperformed the native cascade under both low- and high-cell-loading conditions and approached the theoretical CO-transfer upper bound when sufficient catalyst loading was supplied. These results demonstrate that pre-established enzyme-producing hosts can be assembled into a robust EV-mediated intercellular whole-cell cascade, providing a practical catalyst format for CO-to-formate bioconversion without constructing a single multifunctional strain or relying on purified and immobilized enzyme catalysts.