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Metabolic Engineering of Bacillus subtilis for Ethanol Production: Lactate Dehydrogenase Plays a Key Role in Fermentative Metabolism

机译:枯草芽孢杆菌用于乙醇生产的代谢工程:乳酸脱氢酶在发酵代谢中起关键作用

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Wild-type Bacillus subtilis ferments 20 g/liter glucose in 48 h, producing lactate and butanediol, but not ethanol or acetate. To construct an ethanologenic B. subtilis strain, homologous recombination was used to disrupt the native lactate dehydrogenase (LDH) gene (ldh) by chromosomal insertion of the Zymomonas mobilis pyruvate decarboxylase gene (pdc) and alcohol dehydrogenase II gene (adhB) under the control of the ldh native promoter. The values of the intracellular PDC and ADHII enzymatic activities of the engineered B. subtilis BS35 strain were similar to those found in an ethanologenic Escherichia coli strain. BS35 produced ethanol and butanediol; however, the cell growth and glucose consumption rates were reduced by 70 and 65%, respectively, in comparison to those in the progenitor strain. To eliminate butanediol production, the acetolactate synthase gene (alsS) was inactivated. In the BS36 strain (BS35 ΔalsS), ethanol production was enhanced, with a high yield (89% of the theoretical); however, the cell growth and glucose consumption rates remained low. Interestingly, kinetic characterization of LDH from B. subtilis showed that it is able to oxidize NADH and NADPH. The expression of the transhydrogenase encoded by udhA from E. coli allowed a partial recovery of the cell growth rate and an early onset of ethanol production. Beyond pyruvate-to-lactate conversion and NADH oxidation, an additional key physiological role of LDH for glucose consumption under fermentative conditions is suggested. Long-term cultivation showed that 8.9 g/liter of ethanol can be obtained using strain BS37 (BS35 ΔalsS udhA+). As far as we know, this is the highest ethanol titer and yield reported with a B. subtilis strain.
机译:野生型枯草芽孢杆菌在48小时内发酵20克/升葡萄糖,产生乳酸和丁二醇,但不产生乙醇或乙酸。为了构建产乙醇的枯草芽孢杆菌菌株,使用同源重组通过在控制下的运动发酵单胞菌丙酮酸丙酮酸脱羧酶基因(pdc)和酒精脱氢酶II基因(adhB)的染色体插入来破坏天然乳酸脱氢酶(LDH)基因(ldh)。 ldh本地启动子。工程枯草芽孢杆菌BS35菌株的细胞内PDC和ADHII酶促活性值与产乙醇的大肠杆菌菌株相似。 BS35产生乙醇和丁二醇;然而,与祖先菌株相比,其细胞生长和葡萄糖消耗率分别降低了70%和65%。为了消除丁二醇的产生,乙酰乳酸合酶基因(alsS)被灭活。在BS36菌株(BS35ΔalsS)中,乙醇的产量提高了,产率很高(理论值的89%)。然而,细胞生长和葡萄糖消耗率仍然很低。有趣的是,来自枯草芽孢杆菌的LDH的动力学表征表明它能够氧化NADH和NADPH。大肠杆菌中udhA编码的转氢酶的表达可部分恢复细胞的生长速度,并提早产生乙醇。除了丙酮酸到乳酸的转化和NADH氧化以外,还提出了LDH在发酵条件下对葡萄糖消耗的另外的关键生理作用。长期培养表明,使用菌株BS37(BS35ΔalsSudhA +)可获得8.9 g /升的乙醇。据我们所知,这是枯草芽孢杆菌菌株报道的最高乙醇滴度和产量。

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