Regulating The Expression Of Glk And Zwf In The PPP To Promote The Supply Of Intracellular NADPH In E.coli And Its Application In Asymmetric Reduction Reactions

NADPH is involved in the biosynthesis of many important products in cells,such as asymmetric reduction.The insufficiency of its availability restricts the industrialized production of industrial biotechnology products such as alcohol chemicals,bio-oils,organic acids,amino acids.In this work,in order to enhance intracellular NADPH availability,exogenous genes glk and zwf were introduced to regulate the PPP of recombinant E.coli through genetic engineering technology,thus promoting whole-cell catalytic asymmetric reduction reaction of recombinant E.coli to the synthetization of chiral alcohols.Firstly,the gene glk [encoding glucokinase(GLK)] from E.coli(strain K12 MG1655)and the gene zwf [encoding glucose-6-phosphate dehydrogenase(G6PDH)] from B.subtilis(strain 168)were recombined by genetic engineering technology into dual promoter expression vector p ETDuet-1,and then transformed into E.coli BL21(DE3)by electrotransformation method to successfully construct a recombinant E.coli BL21(DE3)/p ETDuet-1-glk-zwf.The activity of GLK or G6 PDH after induced expression in the recombinant strain was 2.98 and 2.99 times than that of the control strain E.coli BL21(DE3)/p ETDuet-1,respectively,indicating that the target genes glk and zwf were successfully expressed in the recombinant strain,and the expression effect was well.Secondly,in order to increase the supply of intracellular NADPH,the metabolic regulation strategy of overexpression of GLK and G6 PDH was applied to enhance the regeneration efficiency and bioavailability of intracellular NADPH in E.coli.When overexpressing intracellular GLK and G6 PDH,the intracellular NADPH amount increased by 1.98 and 2.61 times,respectively,compared with the control strain E.coli BL21(DE3)/p ETDuet-1.Further simultaneous expression of GLK and G6 PDH increased the amount of intracellular NADPH by 3.54 times.These results showed that regulating the expression of GLK and G6 PDH in PPP pathway can effectively promote intracellular NADPH regeneration and significantly improve intracellular NADPH supply.Finally,in order to study the effect of intracellular NADPH regeneration on chiral alcohol synthesis,E.coli BL21(DE3)/p ET28a-Bc CR&p ETDuet-1-glk-zwf was constructed,in which Bc CR,a gene from B.cereus,encodes a carbonyl reductase.When Bc CR,glk and zwf genes were simultaneously overexpressed in recombinant E.coli,the intracellular NADPH regeneration efficiency still increased,and the NADPH amount was2.1 times that of the control strain E.coli BL21(DE3)/p ET28a-Bc CR&p ETDuet-1.As a whole-cell catalyst,the engineered strain participated in the conversion of substrate COBE,and the yield of S-CHBE after reaction for 20 hours was 62.49% higher than that of the control strain E.coli BL21(DE3)/p ET28a-Bc CR&p ETDuet-1.In addition,the metabolic regulation strategy of overexpressed Bc CR,glk and zwf genes would promote the growth efficiency of the strain.These results indicated that the overexpression of GLK and G6 PDH in the recombinant strain which can synthesize chiral alcohols can increase the amount of intracellular NADPH and the yield of S-CHBE without affecting the growth of the strain.This research work showed that the introduction of glk and zwf genes to regulate the PPP metabolic pathway can enhance the intracellular NADPH regeneration,thereby providing a cell factory for the efficient synthetization of chiral alcohols.