Engineering Mixotrophy in the Chemolithoautotrophic Cupriavidus necator through Hydrogenase Induction.

Mixotrophy offers a promising strategy for biosynthesis by simultaneously utilizing organic carbon and CO2; however, mixotrophic microorganisms are rarely isolated outside of photoautotrophic microalgae. In this study, the chemoautotroph Cupriavidus necator H16 was found to preferentially consume fructose when coexisting with CO2 and H2, switching to utilization of only CO2 and H2 after fructose depletion. Transcriptomic analysis revealed significant differences in genes involved in energy metabolism, electron generation, and the respiratory chain. The molecular mechanism underlying the inability of C. necator H16 to simultaneously utilize carbohydrates and CO2 was identified as the suppression of hydrogenase expression in the presence of fructose. By inducing regulator hoxA to activate hydrogenase expression, an engineered C. necator strain capable of mixotrophic growth was developed. This engineered strain can simultaneously utilize fructose, CO2, and H2, maintain optimal growth, and approach carbon-neutral cultivation. This work provides insights for the mixotrophic cultivation of C. necator and serves as a reference for developing mixotrophic microorganisms in future studies.