High Efficiently Asymmetric Reduction Of Methyl Acetoacetate Catalyzed By Engineered E. Coli Whole Cells

Asymmetric synthesis of chiralβ-hydroxy esters,the key building blocks for many functional materials,is currently of great interest.Generally,enantiomerically pureβ-hydroxy esters is prepared by asymmetric catalytic reduction with isolated enzymes as well as whole cells.Compared with isolated enzymes,the asymmetric bio-reduction of 3-oxo esters using resting whole cells,especially for engineered E.coli cells,is well accepted to be a safe,economical,and mild method to prepare for chiralβ-hydroxy ester.The strategy of adding carbohydrate during the reaction can be adopted for in situ regeneration of co-enzymes with whole cells.Additionaly,the engineered E.coli is preferable to the wild strain for its higher expression level,cheaper medium components,sample enzyme systems and purification procedures.To date,the biocatalytic anti-Prelog stereoselectivity reduction of methyl acetoacetate（MAA）to methyl 3-hydroxybutyrate（HBME）using engineered E.coli cells has remained largely untapped.Accordingly,the bioreduction of MAA to（R）-HBME was successfully carried out in this study with high enantioselectivity using the whole cell of engineered E.coli,which harbored an AcCR gene from Acetobacter sp.CCTCC M209061and a GDH gene from Bacillus Subtilis168 for the in situ regeneration of the coenzyme.The key gene of the AcCR of Acetobacter sp.CCTCC M209061 was successfully obtained through genetic engineering.This gene sequence is a complete reading frame which composed of 762 bp bases,encoding 253 amino acids,with a molecular weight of about 27kDa.In addition,the gene of glucose dehydrogenase（GDH）from Bacillus subtilis 168 was successfully amplificated,and it composed an complete reading frame of 786 bp bases,encoding 261 amino acids,the molecular weight was 28.7 kDa.By co-expression of AcCR and GDH in E.coli,an co-expressed E.coli of BL21（DE3）（pETDuet-gaccr-gdh）containing the recombinant carbonyl reductase AcCR gene（with increased protein soluble tag）and GDH gene was obtained.It catalyzes the asymmetric transformation of 4’-chloroacetophenone and can achieve the in situ regeneration of coenzyme NADH.The specific activity of intracellular enzymes reaches 304.9±4.57 U/g-dw,which is 9 times higher than that of wild-type strain.Compared with the corresponding wild strain,the engineered E.coli cells were proved to be more effective for the bio-reduction of MAA,and afforded much higher productivity.The optimal buffer pH,co-substrate concentration,reaction temperature,substrate concentration and shaking rate were 7.0,800 mM,40℃,350 mM and 200 rpm,respectively.Under the optimized conditions,the product e.e.was>99.9%and the maximum yield was 85.3%after a reaction time of 7 h,which were much higher than those reported previously.Besides,the effects of adding Na₂CO₃,C₆H₁₁NaO₇,Arg,Lys,His,and His-Na2CO3 in a fed-batch mode to tune pH were performed,and it was found that adding six different alkaline substances to reaction solution promoted the efficiency of the reaction obviously.The production of 747m M of（R）-HBME was realized within 8 h at a substrate concentration of 1000 mM by using His-Na₂CO₃ to tuning pH,with a space-time yield of approximately 2.241 mol/L·d,thus the issue in previous research of low substrate concentrations appears to be solved.Additionally,the use of immobilized cells as bicatalysts can not only facilitate product separation,but also make cells recyclable and reusable,which can simplify the production process and lower production cost greatly.The study adopts the method of calcium alginate embedding recombinant E.coli cells,and it was found that the immobilized cells on storage stability,thermal stability and operating stability are better than that of free cells.Besides,the optimum substrate concentration is 600 mM,which is much better than that of free cells.The established bio-catalytic system in this study was proved to be efficient for the bio-reduction of MAA to（R）-HBME with relatively high substrate concentration and selectivity.For further industrial application,these results open a way to use of whole cells of engineered E.coli for challenging higher substrate concentrations ofβ-ketone esters enantioselective reduction reactions.