Feedstock-Efficient Conversion through Hydrogen and Formate-Driven Metabolism in Escherichia coli

ABSTRACT Product yields for biomanufacturing processes are often constrained by the tight coupling of cellular energy generation and carbon metabolism in sugar-based fermentation systems. To overcome this limitation, we engineered Escherichia coli to utilize hydrogen gas (H₂) and formate (HCOO⁻) as alternative sources of energy and reducing equivalents, thereby decoupling energy generation from carbon metabolism. This approach enabled precise suppression of decarboxylative oxidation during acetate growth, with 86.6 ± 1.6% of electrons from hydrogen gas (via soluble hydrogenase from Cupriavidus necator H16) and 98.4 ± 3.6% of electrons from formate (via formate dehydrogenase from Pseudomonas sp. 101) offsetting acetate oxidation. Hydrogen gas supplementation led to a titratable and stoichiometric reduction in CO₂ evolution in acetate-fed cultures. Metabolomic analysis suggests that this metabolic decoupling redirects carbon flux through the glyoxylate shunt, partially bypassing two decarboxylative steps in the TCA cycle. We demonstrated the utility of this strategy by applying it to mevalonate biosynthesis, where formate supplementation during glucose fermentation increased titers by 57.6% in our best-performing strain. Flux balance analysis further estimated that 99.0 ± 2.8% of electrons from formate were used to enhance mevalonate production. These findings highlight a broadly applicable strategy for enhancing biomanufacturing efficiency by leveraging external reducing power to optimize feedstock and energy use. Graphical AbstractCreated in BioRender. Panich, J. (2025) Competing Interest Statement RLB declares a financial interest in Redoxify. JBR declares a financial interest in AlkaLi Labs. JDK declares a financial interest in Ansa Biotechnologies, Apertor Pharma, Berkeley Yeast, BioMia, Cyklos Materials, Demetrix, Lygos, Napigen, ResVita Bio, and Zero Acre Farms. All other authors declare no competing interests. Footnotes Additional kinetic data for formate/FDH metabolism provided, expanded conclusions.