Highly stereoselective reduction of prochiral ketones by a bacterial reductase coupled with cofactor regeneration

Abstract

A carbonyl reductase gene (yueD) from Bacillus sp. ECU0013 was heterologously overexpressed in Escherichia coli, and the encoded protein (BYueD) was purified to homogeneity and characterized. The NADPH-dependent reductase showed a broad substrate spectrum towards different aromatic ketones, and α- and β-ketoesters. Although the enantioselectivity was high to moderate for the reduction of α-ketoesters, all the tested β-ketoesters and aromatic ketones were reduced to the corresponding chiral alcohols in enantiomerically pure forms. Furthermore, the practical applicability of this enzyme was evaluated for the reduction of ethyl 4-chloro-3-oxobutanoate (1a). Using Escherichia coli cells coexpressing BYueD and glucose dehydrogenase, 215 g L(-1) (1.3 M) of 1a was stoichiometrically converted to ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-1b) in an aqueous-toluene biphasic system by using a substrate fed-batch strategy, resulting in an overall hydroxyl product yield of 91.7% with enantiomeric purity of 99.6% ee.

Efficient biosynthesis of ethyl (R)-4-chloro-3-hydroxybutyrate using a stereoselective carbonyl reductase from Burkholderia gladioli

“… a Calculated using Advanced Chemistry Development (ACD/Labs) Software V11.02 b Partition coefficient is the molar ratio of each compound found in the organic phase to the same compound in the aqueous phase (data from ref. [ 18 , 46 , 47 ]) c COBE (73.9 μmol) and CHBE (71.2 μmol) in 0.7 mL phosphate buffer (100 mM, 6.5) were shaken with organic solvents (0.7 mL) at 30 °C in an eppendorf tube. The concentrations of COBE and CHBE in each phase were determined by GC analysis …”

“…Since then, several microorganisms and enzymes capable of converting ethyl 4-chloro-3-oxobutanoate (COBE) to ( R )-CHBE have been documented, including gox2036 from Gluconobacter oxydans [ 14 ], AKRs from Sporobolomyces salmonicolor and Lodderomyces elongisporus [ 15 – 17 ], and a reductase from Bacillus sp. ECU0013 [ 18 ]. However, all of them suffer from impediment such as low substrate concentration, unsatisfactory stereoselectivity, or high substrate/catalyst (S/C) ratio.…”

Efficient production of l-menthol in a two-phase system with SDS using an immobilized Bacillus subtilis esterase

“…The application of organic-aqueous biphasic systems has proven to be an efficient approach to solve the problem of inhibition [30,31]. In order to eliminate the inhibition of product l-menthol and realize the complete bioconversion of l-menthyl acetate at high loads, an organic-aqueous biphasic system was adopted as the reaction medium.…”

A genomic search approach to identify carbonyl reductases in Gluconobacter oxydans for enantioselective reduction of ketones

“…The apparent Km value of Gox2036 for COBE was 0.77 mM, which is lowest in the test enzymes. The Km value is similar to that of BYueD (0.70 mM) from Bacillus sp.ECU001327) 33) and much lower than those of ARII (1.49 mM) from Sporobolomyces salmonicolor, S1 (4.6 mM) from Candida magnolia, and PsCRI (4.9 mM) and PsCRII (3.3 mM) from Pichia stipitis. 34) These results demonstrated that the Gox2036 could reach its maximal reactive velocity at a lower substrate concentration, which is important because high concentrations of substrate can destabilize the enzyme.…”