Tuning an efficient Escherichia coli whole-cell catalyst expressing l-pantolactone dehydrogenase for the biosynthesis of d-(−)-pantolactone

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.