Carbonyl Reduction of Cyclohexanone Derivatives Catalyzed by Recombinant Strain <i>Escherichia coli</i> BL21 (pET-<i>eryKR1</i>)2

Abstract

In order to find substrate specificity and enantioselectivity of ketoreductase domain (EryKR1) in the first module of polyketide synthase from Saccharopolyspora erythraea, the whole cells of recombinant Escherichia coli BL21 (pET-eryKR1)2 harboring eryKR1 gene fermented with different kinds of cyclohexanone derivatives such as methylcyclohexanone and cyclohexanedione. Gas Chromatography (GC) or Gas Chromatography Mass Spectrum (GC-MS) analyses of these ferments indicate that when the recombinant cells were employed as biocatalysts, only one carbonyl of 1,2-cyclohexanedione and 1,4-cyclohexanedione could be reduced with the conversion rate 20.44% and 6.03%, respectively. In addition, 2-methylcyclohexanone and 3-methylcyclohexanone except 4-methylcyclohexanone could be significantly turned over by the recombinant cells to their corresponding cis-methylcyclohexanols, of which the conversion rate of 40 mM 2-methylcyclohexanone was 12.2% and enantiomeric excess of cis-2-methylcyclohexanol was 57.98%, and the conversion rate of 10 mM 3-methylcyclohexanone was 38.26% and enantiomeric excess of cis-3-methylcyclohexanol was 53.9%. Furthermore, the unexpected reduction of 1,2-cyclohexanedione and 2-chlorocyclohexanone by the control E.coli BL21 (pET-28a) reveals that the attracting electron effect induced by the carbonyl or chlorin group in their ortho position could make some NAD(P)H-dependent oxo-reductase except EryKR1 domain within the recombinants reduce the carbonyl of 1,2-cyclohexanedione and 2-chlorocyclohexanone. Therefore, it is concluded that 1,4-cyclohexanedione, 2-methylcyclohexanone and 3-methylcyclohexanone except 1,3-cyclohexanedione and 4-methylcyclohexanone are accepted substrates of EryKR1 domain which takes on a certain biocatalyst activity and stereospecificity.