产1,3-丙二醇新型基因工程菌的构建

Construction of Novel Recombinant Strains Capable of Producing 1,3-propanediol

以甘油为底物转化生产1,3-丙二醇的生物合成途径中,往往由于还原力NADH的不足,限制了1,3-丙二醇的合成,引起中间代谢产物3-羟基丙醛累积,进而抑制甘油脱水酶的活性,阻碍菌体的生长,严重影响1,3-丙二醇的合成途径.为了解决合成途径中还原力不足这一主要矛盾,本文以大肠杆菌和克雷伯氏菌染色体DNA为模板克隆得到yqhD和dhaB、dhaT基因,构建双启动子表达载体pEtac-dhaB-tac-yqhD及表达载体pUC-tac-dhaT,成功共转入E.coliJM109,得到可利用两种辅酶(NADH、NADPH)将甘油转化为1,3-丙二醇且传代稳定的重组大肠杆菌双质粒系统,发酵结果表明,1,3-丙二醇产量提高了28.6%.

Biosynthesis of 1,3-propanediol （1,3- PD） from glycerol needs NADH as a coenzyme to provide H^＋. Deficiency of NADH causes the accumulation of intermediate metabolite 3-hydroxypropionaldehyde and inhibits the activity of glycerol dehydratase, which has the further negative effect on biosynthesis of 1,3-propanediol and cell growth. To resolve this problem, two recombinant plasmids, pEtac-dhaB-tac-yqhD harboring the genes encoding glycerol dehydratase （DHAB） and 1,3-propanediol oxidoreductase isoenzyme （YQHD） from Klebsiella pneumoniae and Escherichia coli, and pUC-tac-dhaT harboring the genes encoding 1,3-propanediol oxidoreductase, were constructed and coexpressed by polycistronic expression and coexistence of incompatible plasmids transformed into E. coli JM109 （pEtac-dhaB-tac-yqhD/pUC-tac-dhaT）. A stable two- plasmid expression system was obtained, and it could convert glycerol to 1,3- PD by using both NADH and NADPH, so as to eliminate the accumulation of 3-hydroxypropionaldehyde and raise the yield of 1,3- PD. The fermentation results demonstrated that the yield of 1,3-PD of recombinant strain E. coil JM109 was increased by 28.6 %. Fig 6, Tab 1, Ref 17