D-pantolactone (D-PL) is the crucial chiral intermediate for the synthesis of D-pantothenic acid. The asymmetric synthesis of D-PL from ketopantothenic acid lactone (KPL) using conjugated polyketide reductases (CPRs) exhibited high catalytic efficiency and high enantioselectivity. A novel conjugated polyketide reductase KbCPR was isolated from Kazachstania barnettii and the mutant KbCPR Y29K/R65N/K215R was developed with the improved activity by 9.0 folds and the enhanced k cat / K m value by 2.6 folds than the wild-type based on integrated substrate pocket alteration strategy, to alter the substrate channel, to regulate the pocket size, and to facilitate proton transfer. An organic-aqueous biphase biosystem containing 40% dibutyl phthalate (v/v) was constructed to inhibit the hydrolysis of KPL and improve the catalytic efficiency. The time-space yield of 892.9 g L -1 d -1 was achieved, which was higher than ever reported. Furthermore, the organic-aqueous biphase system was employed for the first time in the multi-enzymatic system for D-PL preparation from L-pantolactone (L-PL), without any accumulation of KPL intermediate, high concentration of 1 M L-PL was 95.5% converted to D-PL, with the final production e.e. of >99.9%.
d -(−)-Pantolactone (DPL) is a key intermediate for the production of d -(+)-pantothenate (vitamin B5). Deracemization of d , l -pantolactone (D, L -PL) through oxidizing l -(+)-pantolactone (LPL) to ketopantoyl lactone (KPL) and subsequently reducing KPL to DPL is a promising route for synthesizing DPL. Herein, a newly mined l -pantolactone dehydrogenase from Rhodococcus hoagie ( Rho LPLDH) was used for the oxidative dehydrogenation of LPL. To alleviate inclusion bodies formed by membrane-bound Rho LPLDH intracellular expression in E. coli , strategies involving chaperone assistance and decreasing induction temperature were used to achieve Rho LPLDH soluble expression. To enhance its activity, directed evolution and hydrophilicity-based engineering yielded increased catalytic activity and thermostability . 1 M LPL was efficiently converted to KPL by engineering strain CM5 co-expressing Rho LPLDH L254I/V241I/I156L/F224Q/N164K and chaperone. A “two stages in one-pot” method was employed in deracemization of 1 M D, L -PL with 91.2% yield. These results demonstrated that CM5 catalyst exhibits great potential in enzyme cascade deracemization for the production of DPL.
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