Engineering Mixotrophy in the Chemolithoautotrophic <i>Cupriavidus necator</i> 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 <i>Cupriavidus necator</i> 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 <i>C. necator</i> H16 to simultaneously utilize carbohydrates and CO₂ was identified as the suppression of hydrogenase expression in the presence of fructose. By inducing regulator <i>hoxA</i> to activate hydrogenase expression, an engineered <i>C. necator</i> 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 <i>C. necator</i> and serves as a reference for developing mixotrophic microorganisms in future studies.