Different from NAD(P)H regeneration approaches mediated by a single enzyme or a whole-cell microorganism, we demonstrate high-yield generation of NAD(P)H from a renewable biomass sugar--cellobiose through in vitro synthetic enzymatic pathways consisting of 12 purified enzymes and coenzymes. When the NAD(P)H generation system was coupled with its consumption reaction mediated by xylose reductase, the NADPH yield was as high as 11.4 mol NADPH per cellobiose (i.e., 95% of theoretical yield--12 NADPH per glucose unit) in a batch reaction. Consolidation of endothermic reactions and exothermic reactions in one pot results in a very high energy-retaining efficiency of 99.6% from xylose and cellobiose to xylitol. The combination of this high-yield and projected low-cost biohydrogenation and aqueous phase reforming may be important for the production of sulfur-free liquid jet fuel in the future.

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