Sustainable production of β-caryophyllene from fructose and carbon dioxide in Cupriavidus necator H16 through pathway enhancement and global regulatory optimization.

Cupriavidus necator H16, a hydrogen-oxidizing bacterium, is capable of chemolithoautotrophic growth and biosynthesis using carbon dioxide (CO2) as the sole carbon source. This organism has been engineered to convert CO2 into various value-added compounds; however, its application in autotrophic terpenoid biosynthesis remains limited due to low metabolic efficiency and poorly understood regulatory mechanisms. In this study, we developed an efficient C. necator H16 cell factory to autotrophically produce β-caryophyllene, a sesquiterpene with significant potential in the food, cosmetic, and biofuel sectors. Initially, a β-caryophyllene-producing strain was constructed by introducing the β-caryophyllene synthase gene and disrupting the polyhydroxybutyrate (PHB) biosynthetic pathway. Subsequently, production was further enhanced through three engineering modules: strengthening MEP pathway flux, improving cofactor availability, and optimizing global regulatory. Notably, fnr, a global transcriptional regulator, was identified as a crucial contributor to improved production in engineered C. necator H16. Transcriptomic analysis revealed that fnr overexpression facilitated enzyme maturation and improved electron transfer efficiency within the biosynthetic pathway. Ultimately, through integrated metabolic engineering strategies, β-caryophyllene production from CO2 was enhanced 42.3-fold, reaching a final titer of 48.67 mg/L. This represents the highest reported titer and productivity for β-caryophyllene produced from CO2. These results underscore the industrial potential of non-photosynthetic microbial platforms for β-caryophyllene production from CO2.