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). Dera-cemization 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 (RhoLPLDH) was used for the oxidative dehydrogenation of LPL. To alleviate inclusion bodies formed by membrane-bound RhoLPLDH intracellular expression in E. coli, strategies involving chaperone assistance and decreasing induction temperature were used to achieve RhoLPLDH 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 RhoLPLDHL254I/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.

Automated summary

D-(-)-Pantolactone (DPL) is an important intermediate for producing D-(+)-pantothenate (vitamin B5). In this study, a newly discovered L-pantolactone dehydrogenase (RhoLPLDH) was used for the oxidation of L-(+)-pantolactone (LPL) to ketopantoyl lactone (KPL). Strategies involving chaperone assistance and decreasing induction temperature were employed to achieve soluble expression of RhoLPLDH in E. coli. Directed evolution and hydrophilicity-based engineering were used to enhance the activity and thermostability of RhoLPLDH. A two stages in one-pot method was successfully used to deracemize 1 M D,L-PL with a yield of 91.2%. These results demonstrate the potential of the CM5 catalyst for enzyme cascade deracemization in the production of DPL.