Efficient biosynthesis of Pro-Xylane through semi-rational engineering of carbonyl reductase from Canariomyces notabilis

Abstract

Pro-Xylane (boseine), a key cosmetic ingredient renowned for its anti-wrinkle effects and bioaffinity, has drawn considerable attention for its efficient biosynthesis. In this study, a NADP(H)-dependent carbonyl reductase from Canariomyces notabilis (CnCR) was identified through in silico screening. Using alanine scanning and semi-saturation mutagenesis, the double mutant Y98K/N208Q was obtained, exhibiting a 15.8-fold increase in specific activity (60.11 U mg⁻¹) and an 11.5-fold improvement in catalytic efficiency (k_cat/K_m = 0.25 mM⁻¹ s⁻¹) compared to the wild-type enzyme. Mechanistic insights into the improved performance of Y98K/N208Q were revealed through molecular docking. In the enzyme-β-acetone xyloside complex, Y98K/N208Q showed enhanced hydrogen bonding and a refined active site, which improving substrate binding and anchoring. The shorter distance to NADPH and stronger interactions with the substrate boosted the mutant's catalytic efficiency compared to the wild-type. To enable efficient cofactor regeneration, glucose dehydrogenase from Bacillus amyloliquefaciens was integrated into the Y98K/N208Q-mediated Pro-Xylane biosynthetic pathway. Under optimized conditions (30 °C, pH 7.5, 40 g L⁻¹ cells, and a co-substrate-to-substrate ratio of 1.5 : 1), the coupled system produced Pro-Xylane at a titer of 160 g L⁻¹ within 10 h, achieving a conversion rate and diastereomeric excess (S-enantiomer) of ≥99 %. To our knowledge, this is the highest reported Pro-Xylane production to date. This study highlights the discovery and semi-rational engineering of a novel enzyme for efficient Pro-Xylane biosynthesis, offering a robust platform for industrial-scale production of this valuable compound.