Sustainable chemo-enzymatic NADP(H) synthesis from biomass-derived xylose, polyphosphate, and nicotinamide†

Nicotinamide adenine dinucleotide phosphate (NADP(H)) is a universally conserved redox cofactor used in a variety of redox reactions. NADP(H)-dependent enzymes have become valuable biocatalysts for synthesizing specialty and commodity chemicals due to their broad catalytic capabilities. However, the high NADP(H) cost makes it unaffordable for industrial-scale biomanufacturing. Despite multiple efforts to modify the cofactor specificity of NADP(H)-dependent enzymes via protein engineering, the number of successfully engineered enzymes remains limited. As such, developing a cost-effective NADP(H) synthesis method using a sustainable and economical feedstock would greatly expand the industrial applications of NADP(H)-dependent enzymes. Here, we present a chemo-enzymatic cascade for NADP(H) synthesis from crude xylose derived from sugarcane bagasse, a major biomass byproduct of the sugar industry, and purified recombinant enzymes from probiotic Escherichia coli Nissle (endotoxin-free). We successfully streamlined an 11-enzyme cascade in one pot to minimize waste generation and avoid intermediate purification steps. In this cascade, a polyphosphate-based ATP regeneration system activates bagasse-derived crude xylose and nicotinamide to generate NADP⁺, which is further reduced using phosphite, a decarbonized reducing equivalent, by phosphite dehydrogenase, yielding NADPH at titers up to 2.0 g L⁻¹ (∼2.6 mM). To demonstrate practical application, we utilized the sugarcane bagasse- and polyphosphate-derived NADPH to synthesize tetrahydrofolate, an essential micronutrient to maintain the health of nervous system, from folic acid using NADPH-dependent dihydrofolate reductase, showcasing the potential and applicability of our sustainable NADP(H) biomanufacturing method in driving diverse NADP(H)-dependent reactions for industrial biocatalysis.