Short-chain alcohols fueled orthogonal conversion of pyruvate via a non-natural cofactor

As platform chemicals, short-chain alcohols can serve as energy donors for biocatalytic applications. Ubiquitous nicotinamide cofactors such as nicotinamide adenine dinucleotide mediate energy transfer in biological systems, yet they lack the specificity to channel exogenous reducing equivalents directionally toward targeted products. Herein, we constructed non-natural cofactor nicotinamide cytosine dinucleotide (NCD)-dependent multi-enzyme catalytic cascades that efficiently channeled the reducing power of short-chain alcohols to drive pyruvate conversion. Guided by thermodynamic analysis, NCD-preferring methanol dehydrogenase, formaldehyde dehydrogenase and formate dehydrogenase were coupled with either d -lactate dehydrogenase or malic enzyme. This design overcame the thermodynamic barrier of methanol oxidation and enabled pyruvate reduction and reductive carboxylation. Based on the stoichiometry of complete methanol oxidation, the energy conversion efficiencies reached 81% and 95%, respectively. Furthermore, whole-cell biocatalysis was established for the selective reduction of pyruvate to d -lactate with energy conversion efficiencies of 26% and 41%, respectively, using methanol and formaldehyde as exogenous energy donors. To demonstrate broader application scenarios, other short-chain alcohols were then tested as energy donors. Notably, isopropanol achieved a maximum energy yield of 100% to d -lactate, indicating the orthogonal delivery of exogenous chemical energy into the target product via NCD mediation. Overall, this work develops an efficient non-natural cofactor-mediated biocatalytic strategy that employs short-chain alcohols as efficient fuels for reductive biosynthesis, providing a new and reliable platform for the design and optimization of high-performance biocatalytic redox transformation.