Asymmetric Reduction Of Prochiral Carbonyl Compounds By Recombinant Oxidoreductases Expressed In Escherichia Coli

We collected some stereoselective oxidoreductases from various microorganisms and constructed several recombinant Escherichia coli BL21(DE3) overexpressing the above oxidoreductases respectively. Several prochiral carbonyl compounds were applied as substrate to conduct asymmetric reduction to investigate the principle of the biocatalysis. Meanwhile, we adopted cell-free extract as biocatalyst to construct a cell-free system and a biphasic cell-free system for asymmetric carbonyl reduction. We also built a self-assembled cofactor system by investigating the endogenous enzymes of the cell-free extract which could be used for asymmetric conversion efficiently.(1) We constructed 13 recombinant E. coli overexpressing different oxidoreductases from 7 different microorganisms by using pET21c as expression plasmid and E. coli BL21(DE3) as expression host. After the induction of IPTG, the recombinants were applied to conduct asymmetric reduction of 2-hydroxyacetophenone. The results indicated that the optimal induction temperature and IPTG concentration were 17℃and 1 mM respectively. 2-Hydroxyacetophenone, acetophenone and ethyl 4-chloroacetoacetate (COBE) were used as substrates. Nine of the recombinants showed ability of asymmetric reduction of 2-hydroxyacetophenone to produce (R)- or (S)-enantiomers with >90% ee. RCR, SCR, SCR1,SCR2 and SCR3 that come form Candida parapsilosis CCTCC M203011 can achieve asymmetric reduction of 2-hydroxyacetophenone at higher substrate concentration with >90% ee and >85% yield; S1, ADHR and SCR1 exhibited ability of asymmetric reduction acetophenone. S1 and ADHR showed better performance on reduction acetophenone to afford (R)-1-phenylethanol with >97% ee; KRD, CR2, S1, C1, C2 and SCR1 could asymmetric reduce COBE. KRD, CR2 and S1 exhibited better performance to produce (S)- or (R)-enantiomers with >98% ee and >50% yield.(2) We applied SCR1 as model enzyme and 2-hydroxyacetophenone as model substrate to construct a cell-free system involving a self-assembled cofactor recycling system. This cell-free system is characterized by using glucose and trace NADP+ to conduct asymmetric reduction of 40 mM 2-hydroxyacetophenone to give the (S)-enantiomer with >99.9% ee and 93.9% yield in 6 h in an aqueous environment. Besides, after screening several organic solvents, ethyl laurate was adopted as organic phase to form a cell-free biphasic system.In order to examine the generality of the cell-free biphasic system, various aromatic ketones were tested as substrates. The range of substrates included a-substituted acetophenone derivatives, acetophenone and ortho-, meta-, and para-substituted acetophenones. Among all the aromatic ketones,2-hydroxyacetophenone was more suitable for this cell-free system mediated asymmetric reduction, giving corresponding chiral alcohol of 87.3% yield in high substrate concentration (80 mM). Besides, a clear trend was observed that the cell-free system performance was significantly influenced by the group substituted at a-position. In addition, for a-substituted acetophenone derivatives, a-bromo-substitution showed lower yield than a-hydroxy-substitution, suggesting that hydroxyl group was more suitable for SCR1. Although substrates varied, the corresponding alcohols were synthesized with >99% ee in all cases, even on the poor substrates. The high ee value indicated that the cell-free extract, though complex, produced no side reaction. Besides, the biphasic cell-free system showed much higher yield in comparison with aqueous whole-cell system.(3) After screening of the 13 recombinants for asymmetric reduction of COBE, CR2 was chosen to synthesis (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) and S1 for (R)-CHBE. S1 could asymmetric reduce 125 mM COBE to give (R)-CHBE with 99.9% ee and 61.2% yield and CR2 could produce (S)-CHBE with 99.9% ee and 68.6% yield.