Identification and two-step tunnel engineering of a carbonyl reductase for biosynthesis of an (R)-α-lipoic acid intermediate

As a "universal antioxidant", α-lipoic acid (LA) is beneficial for health and therapy, combatting Alzheimer's disease, diabetes. The main active form of LA has the R-configuration. Biocatalytic synthesis of (R)-ethyl 8‑chloro-6-hydroxycaprylate ((R)-ECHO) from ethyl 8‑chloro-6-oxocrylate (ECOO) coupled with subsequent chemical synthesis is a competitive route to (R)-LA. However, very few enzymes have been confirmed to prepare the key intermediate (R)-ECHO with high efficiency and absolute stereoselectivity. In this work, carbonyl reductase CoCR13 from Candida orthopsilosis Co 90–125 was mined, and then engineered by a stepwise strategy with the aim of expanding the substrate entry tunnel. This led to the optimal variant M3 (R129L/D210T/S126A), which exhibited a 93.42-fold higher catalytic efficiency (kcat/Km = 85.95 mm−1·s−1) towards ECOO than the wild type, an ee value greater than 99.5 % (R) from initial 86 % (R), and a space time yield of 99.9 g·L−1·h−1. This study expands the scarce enzyme inventory for the biosynthesis of (R)-ECHO and offers a new choice for the industrial preparation of (R)-LA through a synergistic enzymatic-chemical approach.