Extensive gene mining and facile engineering of a novel carbonyl reductase for asymmetric synthesis of anti-aging (S)-Pro-Xylane from d-xylose

Carbonyl reductases (CRs) are promising biological macromolecules for the asymmetric synthesis of chiral secondary alcohols. A novel CR was evolved for efficient conversion of d-xylose into Anti-aging (S)-Pro-Xylane. Through extensive gene mining, an active carbonyl reductase ZmCR was identified from Zygofabospora marxiana with high reductive activity towards d-xylose-derived 1-C-(β-d-xylopyranosyl)-acetone (XPSA) and in situ cofactor regeneration capability. Using a three-round of concise engineering strategy, a hextuple mutant C85V/V128C/S129A/V161S/Y166H/T217A (M6) was identified from merely 157 mutants with specific activity of 46.00 U·mg^-1, about 306 folds of WT. The Shannon-Wiener index of M6 was 2.21, much higher than WT, indicating its extended substrate scope. M6 could efficiently catalyze the asymmetric reduction of 190 g·L^-1 XPSA into (S)-XPSP. The k_cat/K_M^XPSA of M6 was 203.84 s^-1·mM^-1, about 910 folds of WT. More non-bonded interactions were monitored in M6, contributing to the stabilization of reactive catalytic conformation. This study provides a promising biocatalyst for the chemoenzymatic synthesis of value-added Anti-aging (S)-Pro-Xylane from lignocellulosic d-xylose.