Strengthening NADPH Bioavailability Through Metabolic Regulation In Recombinant E.coli And Its Application In The Synthesis Of Chiral Alcohols

Chiral alcohols are widely used as one of the most critical building blocks for the synthesis of chiral pharmaceuticals,agrochemicals,and fine chemicals.The synthesis of chiral alcohols by whole-cell biocatalysts has the advantages of low pollution,remarkable enantioselectivity,and mild reaction conditions.Biocatalytic synthesis of chiral alcohols requires addressing two key issues:efficient carbonyl reductase and sufficient NADPH supply.In this work,an alkali-tolerant carbonyl reductase（BsCR,encoded by yueD）from Bacillus subtilis（strain 168）was obtained through gene mining.On the other hand,the supply of nicotinamide adenine dinucleotide phosphate（NADPH）is the crucial limit factor for the asymmetric synthesis of chiral alcohols.Regenerating NADPH in the glycolytic pathway（EMP）by the introduction of NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenases（GAPDH,encoded by gapB）and NAD kinase（NADK,encoded by yfjB）is a novel and promising route.Herein,a regeneration NADPH system was constructed and coupled with BsCR for the highly efficient and enantioselective synthesis of chiral alcohols.Firstly,an alkali-tolerant carbonyl reductase BsCR from Bacillus subtilis（strain168）was obtained through gene mining,and successfully heterologously expressed in E.coli with pET-32a.BsCR exhibited a broad substrate and preferred the cofactor NADPH to NADH as the electron donor.The optimal reaction pH and temperature for BsCR are 6.3 and 45°C.Besides,BsCR showed excellent alkali resistance and even kept more than 70%of its peak activity after incubation in Tris-HCl buffer at pH 9.0for 40 h.The Michaelis constants and maximal velocity of the BsCR to NADPH（A）and ethyl 4-chloroacetoacetate（B）are₈8))=5.390×10^-2 mmol/L,₈8))=1.855 mmol/L,and(1(1₈8)8)8)8)8))=147.3μmol·min^-1·mg^-1,respectively.Secondly,in order to increase the supply of intracellular NADPH,two different metabolic regulation systems were constructed in this study:（1）the NADP+-dependent GAPDH from Bacillus subtilis（strain 168）,which could catalyze the reaction:D-G3P+phosphate+NADP⁺→1,3-BPG+NADPH,was introduced into the EMP of recombinant E.coli as an additional mechanism to improve the regeneration of cellular NADPH.The results showed that the intracellular GAPDH activity of recombinant E.coli increased,which caused the intracellular NADPH amount increased by 53%;（2）NAD kinase,which acted as the only enzyme responsible for de nova NADP⁺biosynthesis,was overexpressed in the recombinant E.coli to catalyze the conversion of NAD⁺to NADP⁺which enhanced the cellular NADPH&NADP⁺pool.As we expected,the amount of intracellular NADPH increased by 74%.Thirdly,recombinant E.coli BL21（DE3）/pETDuet-1-gapB-yueD&pET-28a-yfjB and E.coli BL21（DE3）/pETDuet-1-yfjB-yueD&pET-28a-gapB were constructed.The relative normalized expression levels of the genes yueD,gapB and yfjB in the recombinant E.coli BL21（DE3）/pETDuet-1-gapB-yueD&pET-28a-yfjB were increased88.00-fold,150.00-fold and 157.82-fold,respectively;The relative normalized expression levels of the genes yueD,gapB and yfjB in E.coli BL21（DE3）/pETDuet-1-yfjB-yueD&pET-28a-gapB were increased 61.53-fold,172.24-fold and 168.40-fold,respectively.Further,the intracellular NADPH content in the two recombinant E.coli increased 2.40-fold and 2.34-fold,respectively.Finally,asymmetric reduction reactions of prochiral ketones by recombinant E.coli were performed.Applying the E.coli BL21（DE3）/pETDuet-1-yueD to catalyze the asymmetric reduction of ethyl 4-chloroacetoacetate（COBE）,and acetophenone,the yield of ethyl S-4-chloro-3-hydroxybutyrate（S-CHBE）reached 89.9%,and S-1-phenyl ethanol reached 66.7%,and e.e.（enantiomeric excess,%）of both products reached more than 99%.To examine the promoting efficiency of the NADPH strengthening regeneration system on asymmetric reduction reaction of chiral alcohols,whole-cell biocatalysts were performed and reaction results were evaluated.the result showed that recombinant E.coli BL21（DE3）/pETDuet-1-gapB-yueD&pET-28a-yfjB showed the best performance:the yield of S-CHBE reached 99.1%after 3 h of reaction,and the yield of S-phenyl ethanol reached 99.2%after 8 h.Further,the optimal reaction conditions for the catalytic reduction of acetophenone by recombinant E.coli BL21（DE3）/pETDuet-1-gapB-yueD&pET-28a-yfjB are:T=40°C,pH 8.0（PBS）,optimum substrate concentration is 70 mmol/L.This study not only obtained a high-performance carbonyl reductase through gene mining,enriched the chiral alcohol biosynthesis enzyme library,but also enhanced the supply of intracellular NADPH from intracellular EMP metabolic pathway and NADP⁺synthesis pathway.Thereby promoting the efficient synthesis of whole-cells catalyzed the asymmetric reduction of chiral alcohols.This research work provides a theoretical and practical basis for the metabolic regulation of NADPH regeneration and the biosynthesis of chiral alcohol,and has pushed the pace of biosynthesis industry forward.