Biocatalytic stereoinversion of d-ribose to enantiopure l-ribose via a kinetic model-guided redox cascade

초록

l-Ribose (l-Rib) is a key chiral building block for antiviral and anticancer l-nucleoside drugs, yet its scarcity in nature necessitates efficient synthesis from abundant sugars. Existing chemical routes are multi-step, demand stoichiometric chiral auxiliaries, and generate substantial organic waste, while enzymatic alternatives often rely on rare or costly substrates and can suffer from low conversion. Here, we report a multi-enzyme redox cascade that converts bulk d-ribose (d-Rib) directly into enantiopure l-ribose through the achiral intermediate ribitol, a symmetrization-desymmetrization route not previously demonstrated from a d-ribose feedstock. Whereas established enzymatic routes reach l-ribose from l-arabinose or l-ribulose, or from ribitol by microbial oxidation and l-ribose isomerase, the cascade reported here installs a d-ribose-to-ribitol reduction as the front-end step, enabling enantiopure l-ribose to be produced directly from an abundant, low-cost feedstock. The redox enzyme cascade leverages the distinct cofactor specificities of NADPH-dependent aldo-keto reductase (ScGRE3) and NAD(+)-dependent mannitol dehydrogenase (MBP-AgMDH), coupled with cofactor regeneration systems using formate dehydrogenase (PsFdhV9) and NADH oxidase (LpNoxV), to drive the reactions and offset cofactor costs. Importantly, rather than empirical screening, we employed a mechanistic kinetic model coupled with constrained numerical optimization to identify operating conditions that maximize l-Rib yield while preserving high ee. This model-guided strategy delivered l-Rib with >98% yield and >99% enantiomeric excess (ee) at d-Rib loadings of 20-60 mM, decreasing to 96.4% and 97.5%, respectively, at the highest 70 mM loading evaluated, and, because the cascade runs in a single aqueous phase without downstream chromatographic resolution, achieves a simple E-factor as low as 1.97 g g(-1) and atom economy of 64.1%. Beyond l-Rib synthesis, this work demonstrates a kinetic model-guided design strategy for an orthogonal NADPH/NAD(+) redox cascade that produces an enantiopure l-sugar from an abundant d-sugar feedstock with minimal waste and without stoichiometric chiral auxiliaries, and that could in principle be extended to other polyol-mediated sugar interconversions.

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