Asymmetric reduction of ketopantolactone using a strictly (R)-stereoselective carbonyl reductase through efficient NADPH regeneration and the substrate constant-feeding strategy.

OBJECTIVES: To characterize a recombinant carbonyl reductase from Saccharomyces cerevisiae (SceCPR1) and explore its use in asymmetric synthesis of (R)-pantolactone [(R)-PL]. RESULTS: The NADPH-dependent SceCPR1 exhibited strict (R)-enantioselectivity and high activity in the asymmetric reduction of ketopantolactone (KPL) to (R)-PL. Escherichia coli, coexpressing SceCPR1 and glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), was constructed to fulfill efficient NADPH regeneration. During the whole-cell catalyzed asymmetric reduction of KPL, the spontaneous hydrolysis of KPL significantly affected the yield of (R)-PL, which was effectively alleviated by the employment of the substrate constant-feeding strategy. The established whole-cell bioreduction for 6 h afforded 458 mM (R)-PL with the enantiomeric excess value of >99.9% and the yield of 91.6%. CONCLUSIONS: Escherichia coli coexpressing SceCPR1 and EsGDH efficiently catalyzed the asymmetric synthesis of (R)-PL through the substrate constant-feeding strategy.

Increasing catalytic efficiency of SceCPR by semi-rational engineering towards the asymmetric reduction of D-pantolactone.

D-pantolactone (D-PL) is one of the important chiral intermediates in the synthesis of D-pantothenic acid. Our previous study has revealed that ketopantolactone (KPL) reductase in Saccharomyces cerevisiae (SceCPR) could asymmetrically reduce KPL to D-PL with a relatively weak activity. In this study, engineering of SceCPR was performed using a semi-rational design to enhance its catalytic activity. Based on the computer-aided design including phylogenetic analysis and molecular dynamics simulation, Ser158, Asn159, Gln180, Tyr208, Tyr298 and Trp299 were identified as the potential sites. Semi-saturation, single and combined-site mutagenesis was performed on all six residues, and several mutants with improved enzymatic activities were obtained. Among them, the mutant SceCPRS158A/Y298H exhibited the highest catalytic efficiency in which the kcat/Km value is 2466.22 s-1·mM-1, 18.5 times higher than that of SceCPR. The 3D structural analysis showed that the mutant SceCPRS158A/Y298H had an expanded and increased hydrophilicity catalytic pocket, and an enhanced π-π interaction which could contribute to faster conversion efficiency and higher catalytic rate. The whole cell system containing SceCPRS158A/Y298H and glucose dehydrogenase (GDH), under the optimized condition, could reduce 490.21 mM D-PL with e.e.≧ 99%, conversion rate = 98%, and the space-time yield = 382.80 g·L-1·d-1, which is the highest level reported so far.

Recombinant expression and molecular insights into the catalytic mechanism of an NADPH-dependent conjugated polyketone reductase for the asymmetric synthesis of (R)-pantolactone.

(R)-pantolactone is a key chiral intermediate for synthesizing calcium (R)-pantothenate. The commercial synthesis of (R)-pantolactone is performed through the resolution of racemic pantolactone using lactonase-catalyzed enantioselective hydrolysis. The process needs highly toxic hydrogen cyanide and a tedious dynamic kinetic resolution. In this study, we investigated an alternative method to prepare (R)-pantolactone through asymmetric reduction of ketopantolactone (KPL). An NADPH-dependent conjugated polyketone reductase gene from Candida dubliniensis CD36 (CduCPR) was functionally overexpressed in Escherichia coli BL21 (DE3). Recombinant CduCPR belonged to the aldo-keto reductase superfamily, and showed high catalytic activity and stereoselectivity using KPL as the substrate. In a continuous feeding reaction, 200 mM ketopantolactone was reduced to (R)-pantolactone with 98% conversion and 99% enantiomeric excess (e.e.) within 2.0 h. The catalytic mechanism was further investigated. Tyr66 functions as a proton donor following hydrogen transfer from NADPH. Thr30 and His128 are critical residues to bind and orient KPL. Therefore, the recombinant CduCPR from C. dubliniensis exhibited potential application in the asymmetric synthesis of (R)-pantolactone.