Establishment And Characteristics Of Reaction System For The Asymmetric Bioreduction Of COBE Into (S)-CHBE By Recombinant Escherichia Coli CCZU-K14

It is well-known that optically active alcohols are important building blocks for the production of agricultural and pharmaceutical chemicals. Ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), one of the optically active alcohols, is a key chiral synthon for the side-chain of cholesterol-lowering drug atorvastatin (Lipitor), which is the inhibitor of hydroxymethylglutaryl CoA (HMG-CoA) reductase. Compared to traditional chemical method, asymmetric bioreduction of ethyl 4-chloro-3-oxobutanoate (COBE) into optically pure (S)-CHBE is of great interest due to its mild reaction condition and high enantioselectivity with 100% of theoretical yield. Therefore, it is worthy of in-depth study. In this study, one high-throughput colorimetric screening strategy was built, and recombinant E. coli CCZU-T15, CCZU-Y10 and CCZU-K14 were used for the bioreduction of COBE into (S)CHBE. Furthermore, the hydrolyzates obtained from the enzymatic hydrolysis of sugarcane bagagge pretreated with combination pretreatments as reaction media were attempted to enhance the biosynthesis of (S)-CHBE (> 99.9% e.e.). Moreover, one highly stereoselective bioreduction of COBE (> 3000 mM) into (S)-CHBE by whole-cells was successfully demonstrated.Firstly, one modified high-throughput screening strategy was built for screening COBE-reducing biocatalysts with UV-Vis spectrophotometer in this study. It was found that the highest characteristic peak was observed at 510nm in the absorption spectrum, and the optimum color-generating conditions were obtained:ferric perchlorate concentration 80 mM, incubation temperature 30℃, incubation time 20 min. Moreover, this spectrophotometric method could be effectively used for the determination of aliphatic and aromatic β-carbonyl carboxylic esters.Secondly, the reaction media of E. coli CCZU-T15, E. coli CCZU-Y10 and E. coli CCZU-K14 were investigated in this study. It was found that E. coli CCZU-K14 had the highest COBE-reducing activity. After the optimization, the optimum reaction conditions were achieved as follows:1.5 mmol cosubstrate glucose/mmol COBE,0.1μmol NAD+/mmol COBE, and metal ion additive Mn2+(0.1 mM). Moreover, COBE at a high concentration of 3000 mM could be asymmetrically reduced to (S)-CHBE in the high yield (> 99.0%) and high enantiometric excess value (> 99.9% e.e.) after 14 h. Significantly, E. coli CCZU-K14 showed high potential for the synthesis of (S)-CHBE(>99.9%e.e.).Thirdly, to avoid to add additional cofactor and effectively biotransform COBE, L-glutamine and glycine were used to enhance the intracellular NADH content and the biocatalytic activity of E. coli CCZU-K14. Adding the mixture of 200 mM of L-glutamine and 500 mM of glycine to the reaction media, a 1.67-fold of biocatalytic activity was increased over the control without the addition of the two compounds. Moreover, β-cyclodextrin (0.4 mmol β-cyclodextrin/mmol COBE) was also added into this reaction media containing L-glutamine (200 mM) and glycine (500 mM), and the biocatalytic activity of E. coli CCZU-K14 whole-cell was increased by 1.34-fold than that without (3-cyclodextrin. In this β-cyclodextrin-water media containing L-glutamine (200 mM) plus glycine (500 mM), (S)-CHBE (> 99.9% e.e.) could be obtained from 3000 mM COBE in the yield of 98.4% after 8 h.Finally, hydrolyzates could be used as reaction media for the biotransformation of COBE. Sugarcane bagasse was pretreated with combination pretreatment (e.g., sequential KOH extraction and ionic liquid soaking, sequential KOH extraction and Fenton soaking, or sequential KOH extraction and glycerol soaking). After the enzymatic hydrolysis of pretreated sugarcane bagasses, three concentrated hydrolyzates could be used for asymmetrically bioreduction of COBE into (5)-CHBE. Compared with glucose, arabinose and cellobiose couldn’t promote the initial reaction rate, and xylose could increase the intracellular NADH content. Moreover, it was the first report that hydrolyzates could be used for effectively biosynthesizing (S)-CHBE (98.0% yield) from 3000 COBE by whole-cells of E. coli CCZU-K14 in the presence of β-cyclodextrin (0.4 mmol P-cyclodextrin /mmol COBE), L-glutamine (200 mM) and glycine (500 mM). Significantly, it is a new alternative to utilize bioresource for the synthesis of key chiral intermediate (S)-CHBE.In conclusion, high substrate COBE concentration (3000 mM) can be effectively biotransformed into (S)-CHBE (> 99.9%e.e.) by whole cells of recombinant E. coli CCZU-K14. Significantly, these results will lay the foundation for the industry production.