中文摘要：

枯草芽孢杆菌Bacillus subtilis 168是一株安全生产菌株,首次通过弱化B.subtilis 168磷酸戊糖途径（PPP）中的关键酶葡萄糖-6-磷酸脱氢酶（G6PDH）基因zwf,研究了其对胞内NADH水平的影响,进而研究其对2,3-丁二醇（2,3-BD）及副产物合成的影响。弱化菌株B.subtilis168△zwf进行摇瓶发酵实验,与出发菌株相比,胞内辅酶NADH水平得到了增强,2,3-BD产量提高了15.0%,主要副产物AC积累量下降了10.6%,但乙酸、乳酸等有机酸的积累量提高。为了进一步提高2,3-BD生产效率,在B.subtilis168中克隆表达了不同来源的ACR基因,研究发现克雷伯氏菌来源的ACR酶活力最高,将此来源的ACR的基因kphs克隆到B.subtilis168△zwf中加强表达,对重组菌株B.subtilis168△zwf/p MA5-kphs进行摇瓶发酵实验,与出发菌相比,2,3-BD产量提高了37.3%,主要副产物AC积累量下降了28.1%,同时,乙酸等分支路径的其他副产物也有不同程度的降低。

英文摘要：

Acetoin is the main by-product during the 2,3-butanediol（ 2,3-BD） fermentation.Acetoin reductase（ ACR） catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD＋.Therefore,intracellular 2,3-BD production is likely governed by the quantities of rate-limiting factor（ s） ACR and/or NADH.In bacterial metabolism,glucose is first converted to pyruvate before generation of major products,then pyruvate produced from glucose is channeled into 2,3-BD fermentation pathway.While,glucose can be converted to pyruvate through two main metabolism pathway PPP and EMP.However,there are NADPH generated via the former pathway,which was not necessary for 2,3-BD production.Glucose-6-phosphate dehydrogenase（ G6PDH） is the key enzyme in PPP pathway and to enhance the level of intracellular NADH,metabolic flux of EMP pathway was increased by disrupting G6 PDH,an key enzyme in PPP pathway.Flask fermentation experiments indicates that the level of intracellular NADH was enhanced in the recombinant strain B.subtilis168△zwf,and the yield of 2,3-BD was increased by 15.0%,and the titer of main by-products AC was decreased by 10.6%,however,the accumulation of acetic acid,lactic acid and other organic acid were enhanced.In order to improve the yield and production efficiency of 2,3-BD further,a recombinant strain B.subtilis168 △zwf/p MA5-kphs was constructed by overexpressing ACR in B.subtilis168 △zwf.Compared with parental strain,the yield of 2,3-BD was increased by 37.3 %,AC was decreased by 28.1%,and acetate acid,lactate acid,succinic acid and other by-products also were decreased.