Xylose Reductase Gene Clone And Expression Strain Of Metabolic Engineering Transform

Xylitol as a five carbon sugar alcohol and low caloric content, has been widely used in medicine, food and chemical fields. Xylitol’s chemical name is1,2,3,4,5-pentanol, the formula is C5H12O5. It is a white crystalline, its solubility, solution density and other physical and chemical properties and refraction coefficients sucrose is basically the same as sugar substitutes, and the nutritional supplement for people with diabetes and people’s attention.Firstly obtained the target xr gene by PCR from Neurospora crassa cell bacteria genome, constructed the expression plasmid pET30a-xr, and analysis of recombinant plasmids expression in the E.coli BL21(DE3). The results showed that the molecular weight of38kDa xylose reductase (XR) can be effectively expressed, enzyme tests showed a specific activity of recombinant strains XR is7.25U/mg.Secondly, xylose reductase is cofactor NADPH-dependent enzyme, this paper obtained by PCR from the genome of E.coli K-12target genes, respectively gnd and zwf, construct expression plasmids pCDFDuet-gnd, pCDFDuet-zwf, pCDFDuet-gnd-zwf, and analyze the recombinant plasmid expression in E.coli BL21(DE3). The results showed that the molecular weight of51kDa6-phosphogluconate dehydrogenase (6-PGDH),54kDa glucose6-phosphate dehydrogenase enzyme (G6PDH) were effectively expressed, the enzyme tests showed that the recombinant strains6-PGDH activity ratio was2.26U/mg, G6PDH activity ratio was1.31U/mg. By coexpression pET30a(+) and pCDFDuet-1series of vectors to produce xylitol.On this basis, mutations in the xylB gene can reduce phosphorylation of xylulose, xylose reducted into xylitol instead of HMP pathway. The xylB gene was knocked out successfully by Red-recombination systems, xylulose by xylose weakened the phosphorylated saccharide to from a5-phosphate-D-xylulose enters the pentose phosphate pathway. By comparison the after xylB knockout conversion of xylose to xylitol, clear knock out xylitol to improve the efficiency and yield.Finally, the paper through the medium composition and culture conditions (seed age、initial sugar concentration、temperature、pH and inoculum size) to optimize conditions to obtain the optimum conditions for the production of xylitol bioconversion engineered bacteria:the kind of age is12h, initial xylose concentration is60g/L, the temperature is28℃, pH7.0, inoculum is10%. Fermentation showed that the recombinant strain produced before optimization of xylitol production rate is0.88g/(L·h), after optimization of xylitol production rate is1.04g/(L·h), improved24.32%.