Construction Of Engineered E.coli Based On Synthesis Of Xylitol Using Whole-cell Catalysis And Coenzyme Regeneration

This paper intends to clone and express the xylose reductase(AR)gene from Candida boidinii and the glucose dehydrogenase(GDH)gene from Bacillus subtilis168 to achieve coenzyme NADPH cycle regeneration and improve the catalytic potential of whole cell catalytic production of xylitol in recombinant Escherichia coli.The research contents and results are as follows:(1)Using pET28a(+)expression vector,xylose reductase(AR)gene and glucose dehydrogenase(GDH)gene were cloned into pET28a(+)to obtain recombinant expression vectors pET28a(+)-AR and pET28a(+)-GDH;Then the T7 promoter and glucose dehydrogenase(GDH)gene of pET28a(+)-GDH were co-cloned into pET28a(+)-AR,and the double gene expression vector pET28a(+)-AR-GDH with double T7promoter was obtained.The recombinant plasmid was verified by restriction enzyme digestion and sequencing,and the results showed that the fragment size of the digested product was consistent with its corresponding theoretical value,and there was no mutation in xylose reductase gene and glucose dehydrogenase gene.(2)The recombinant expression vector was separately transferred to E.coli BL21(DE3),IPTG was added to induce the expression of the recombinant protein,and the crude enzyme solution was extracted for molecular detection of enzyme protein,determination of enzyme activity,and study of related enzyme properties.The results showed that the molecular weight of AR was about 39 KDa,the activity of xylose reductase was 3.2 U/mL,the optimum temperature is 25?,the optimum pH is 7,the half-life at 30?is 37.2 h,the molecular weight of GDH is about 39 KDa,the activity of GDH is 1.3 U/mL,the optimum temperature is 40?,the optimum pH is 8,and the half-life at 40?is 12.5 h.(3)Using E.coli BL21(DE3)/pET28a(+)-AR-GDH as whole cell catalyst,the induced expression conditions of enzyme and the conditions of xylitol synthesis were optimized.The results showed that the optimum induction conditions were as follows:the biomass of Escherichia coli OD₆₀₀ was 0.6 mM,the induction temperature was27?and the induction time was 12 h.After the optimization of induction conditions,the yield of xylitol increased by 17.4%to 15.3 g/L,and the conversion rate was76.7%.The optimum whole-cell catalytic conditions were as follows:catalytic temperature 35?,cell wet weight 0.08 g/mL,cell permeable agent Triton X-100concentration 0.4%(v/v),rotational speed 150 r/min.After the optimization of whole-cell catalytic conditions,the yield of xylitol increased by 21.6%compared with that before optimization,reaching 18.6 g/L,and the conversion rate was 93.2%.(4)After the optimum induction conditions and whole-cell catalytic conditions were obtained,the concentration of xylose was increased to 50 g/L and 100 g/L to investigate the potential of the recombinant strain to produce xylitol.The results showed that the yield of xylitol reached 46.2 g/L and the conversion rate was 91.4%when the concentration of xylose was 50 g/L.when the concentration of xylose was100 g/L,the yield of xylitol reached 77.8 g/L,and the conversion rate was 78.2%.