Engineering thermophilic Cupriavidus cauae PHS1 for formate-driven C1 biorefinery and high-value PHA production

Abstract

The emerging C1 economy can now tap three mutually reinforcing carbon-neutral streams: (i) electroreduced formate from captured CO₂, (ii) renewable H₂ generated by water electrolysis, and (iii) biocatalytic formate produced by tandem CO-dehydrogenase and formate-dehydrogenase conversion of industrial CO off-gas. Cupriavidus cauae PHS1, a 45°C hot-spring isolate with two circular chromosomes, emerges as a uniquely suitable chassis for high-temperature C1 biorefineries. Genomic and physiological analyses reveal four pillars that this project will exploit: 1) Native formate metabolism. PHS1 encodes both a soluble NAD-dependent and a membrane-bound—experimentally shown to be functionally expressed—an engineered tungsten(W)-dependent formate dehydrogenase (MeFDH) set, enabling efficient formate production or oxidation at thermophilic conditions. 2) Hydrogen as auxiliary energy source. Complete NiFe-hydrogenase loci (HoxFUYH and Hyp accessory genes) allow the strain to tap renewable H₂ for extra reducing power, facilitating redox-neutral product synthesis. 3) Calvin-BensonBassham (CBB) cycle. A fully functional RuBisCO operon coupled to two CO₂-concentrating transporters provides an inherent CO₂-fixation route, opening the door to mixotrophic or even lithoautotrophic operation. 4) Endogenous PHA machinery. A chromosomal β-ketothiolase/acetyl-CoA reductase/PHB synthase operon already supports high poly-3-hydroxy-butyrate titers at 45 °C. By combining thermotolerance, inherent PHA capacity, formate oxidation, hydrogen utilization, and dual CO₂-fixation routes in a single chassis, C. cauae PHS1 can serve as a plug-and-play platform for next-generation, C1 biorefineries—converting CO₂, formate, and H₂ directly into high-value biodegradable polymers and other green-chemistry building blocks.