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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。
Bacillus subtilis 168, a safe strain, was the first to attenuate the gene zwf, a key enzyme in the pentose phosphate (B.subtilis 168) phosphate pentose pathway (PPP) NADH levels, and then study its 2,3-butanediol (2,3-BD) and by-product synthesis. Weak strain B.subtilis168 △ zwf shake flask fermentation experiments, compared with the starting strain, intracellular coenzyme NADH levels have been enhanced, 2,3-BD increased by 15.0%, the main byproduct of AC accumulation decreased by 10.6% However, the accumulation of organic acids such as acetic acid and lactic acid is increased. In order to further improve the production efficiency of 2,3-BD, different sources of ACR genes were cloned and expressed in B.subtilis168. It was found that ACR activity derived from Klebsiella was the highest. The kphs gene of this ACR was cloned into B .subtilis168 △ zwf, and the shake flask fermentation of recombinant strain B.subtilis168 △ zwf / p MA5-kphs was carried out. Compared with the starting strain, the yield of 2,3-BD increased by 37.3%, the accumulation of major by-products AC A decrease of 28.1%. At the same time, other by-products such as acetic acid branch pathways also decreased to some extent.