Selective Electron Transfer from Formate Mediated by Nicotinamide Cytosine Dinucleotide for d -Lactate Synthesis in Escherichia coli

Abstract As a renewable C1 feedstock, formate can be produced via CO2 reduction and is widely utilized as an electron donor for NAD(P)-dependent biocatalysis in biomanufacturing. However, the ubiquitous involvement of NAD(P) in native metabolic networks hinders selective energy channeling from formate to target reactions. In this study, we constructed a synthetic system by coupling formate oxidation and carbon metabolism with the non-natural cofactor nicotinamide cytosine dinucleotide (NCD). An orthogonal cascade reaction was first established for d-lactate synthesis using NCD-preferred formate dehydrogenase (FDH*) and d-lactate dehydrogenase (DLDH*), achieving a stoichiometric yield of d-lactate from formate. Subsequently, engineered Escherichia coli strains harboring different NCD synthesis modules were constructed, and selective d-lactate production was realized upon formate supplementation. To enhance pyruvate formation and rebalance intracellular redox chemistry, an NADH oxidase was introduced, leading to nearly stoichiometric production of d-lactate from glucose, namely 2 mol of d-lactate per mol of glucose. Further analysis of carbon distribution confirmed that carbon flux was redirected to d-lactate and energy from formate was selectively transferred. This work demonstrates that NCD and FDH* function as a module for selective electron transfer from formate, which drives carbon flux to a target metabolite, thereby offering a practical route for precise energy regulation and C1-driven cell factories for sustainable bioproduction.