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.