Corynebacterium glutamicum Tailored for Efficient Isobutanol Production
Corynebacterium glutamicum Tailored for Efficient Isobutanol Production
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DOI：
10.1128/aem.02972-10
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发表时间：
2011-05-01
影响因子：
4.4
通讯作者：
Eikmanns, Bernhard J.
中科院分区：
文献类型：
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作者：
Blombach, Bastian;Riester, Tanja;Eikmanns, Bernhard J.
我们最近改造了谷氨酸棒杆菌，通过灭活丙酮酸脱氢酶复合体丙酮酸：醌氧化还原酶，转氨酶B，以及编码乙酰羟酸合成酶、乙酰羟酸异构酶和二羟基脱水酶的ilvBNCD基因的额外过表达，来有氧生产2-酮基异戊酸酯。在此基础上，我们通过对L乳酸和苹果酸脱氢酶的失活、乳酸乳球菌的酮酸脱羧酶的实现、酿酒酵母的酒精脱氢酶2(Adh2)的实现以及在大肠杆菌中表达pntAB转氢酶基因的方法，使谷氨酸杆菌在缺氧条件下从葡萄糖中生产异丁醇。产生的菌株产生异丁醇，底物比产量(Y-P/S)为每摩尔葡萄糖0.60+/-0.02摩尔。有趣的是，染色体上编码的乙醇脱氢酶而不是来自酿酒酵母的质粒编码的adh2参与了谷氨酸链霉菌异丁醇的合成，相应的adha基因的过表达使Y-P/S增加到每摩尔葡萄糖0.77+/-0.01mol异丁醇。苹果酸酶的失活显著降低了Y-P/S，表明由丙酮酸和/或磷酸烯醇式丙酮酸羧化酶、苹果酸脱氢酶和苹果酸酶组成的代谢循环负责NADH+H+向NADPH+H+的转化。在由好氧生长阶段和缺氧生产阶段组成的补料分批发酵过程中，最有希望的菌株是谷氨酸菌Delta Acee Delta pqo Delta Ilve Delta LDHA Delta mdh(pJC4ilvBNCD-pntAB)(pBB1kivd-Adha)，其异丁醇产量约为175 mm，体积产率为4.4 mm h(-1)，在生产阶段的Y-P/S约为每摩尔葡萄糖0.48摩尔。
We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate: quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of L-lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis, alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae, and expression of the pntAB transhydrogenase genes from Escherichia coli. The resulting strain produced isobutanol with a substrate-specific yield (Y-P/S) of 0.60 +/- 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum, and overexpression of the corresponding adhA gene increased the Y-P/S to 0.77 +/- 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the Y-P/S, indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH+H+ to NADPH+H+. In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum Delta aceE Delta pqo Delta ilvE Delta ldhA Delta mdh (pJC4ilvBNCD-pntAB)(pBB1kivd-adhA), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h(-1), and showed an overall Y-P/S of about 0.48 mol per mol of glucose in the production phase.