The Construction Of Coenzyme Regeneration Improved In E.Coli With Mulit-copy Integrated Expression Of Xylose Reductase

Hemicellulose resource is one of the most abundant renewable resource in nature,and it is an effective way to produce xylitol by microbial transformation.In this study,we improved NADPH regeneration of Escherichia coli W3110 through the CRISPR/Cas9 system and obtained a strain which can produce high-yield of xylitol based on the previous researches in our lab.A double plasmid CRISPR system was used in this work.We constructed different pTargetF plasmids and repairing templates,to replace the ptsG,ptsF and xylAB genes by xylose reductase(xr),and obtained the WZ04 with integrated 3 xr copies in genome.On this basis,we analyzed the glucose metabolism pathway of E.coli,constructed the strains and detected the effects of deleting the phosphofructokinase(pfkA and pfkB)gene(s),phosphoglucose isomerase(pgi)gene and transhydrogenase(sthA)gene by measuring both the ratio of NADPH/NADP+ and concentration of xylitol after fermentation in flask.Finally,we got the strain WZ41 with knocked-out pfkAB.The ratio of intracellular coenzyme NADPH/NADP+ of WZ41 was 1.68 times of the starting strain WZ04,showing that the yield of xylitol to glucose was improved.After that,we confirmed the best copies of xr was 5 through RT-qPCR and flask fermentation.After 30 h mixed sugar fermentation,the xylitol concentration was 16.76 g/L,and the residual amount of glucose and xylose was 0.44 g/L and 10.50 g/L,respectively.In the end,we got the recombinant strain IS5-d ?pfkA with the application of coenzyme improvement in strain IS5-d.The strain having the same growth rate with IS5-d in the flask fermentation,can transform all xylose to xylitol without any glucose and xylose remained.We obtained 126.62 g/L xylitol after 63 h fermentation using cheap substrate and simple process in 15L fermenter,and the productivity of xylitol was 2.01 g/L/h.