On the basis of our previous studies of microbial L-valine production under oxygen deprivation, we developed isobutanol-producing Corynebacterium glutamicum strains. The artificial isobutanol synthesis pathway was composed of the first three steps of the L-valine synthesis pathway; and the subsequent Ehrlich Pathway: pyruvate was converted to 2-ketoisovalerate in the former reactions; and the 2-keto acid was decarboxylated into isobutyraldehyde, and subsequently reduced into isobutanol in the latter reactions. Although there exists redox cofactor imbalance in the overall reactions, i.e., NADH is generated via glycolysis whereas NADPH is required to synthesize isobutanol, it was resolved by taking advantage of the NAD-preferring mutant acetohydroxy acid isomeroreductase encoded by ilvC^TM and the NAD-specific alcohol dehydrogenase encoded by adhA. Each enzyme activity to synthesize isobutanol was finely tuned by using two kinds of lac promoter derivatives. Efficient suppression of succinate by-production and improvement of isobutanol yield resulted from inactivation of pckA, which encodes phosphoenolpyruvate carboxykinase, whereas glucose consumption and isobutanol production rates decreased because of the elevated intracellular NADH/NAD⁺ ratio. On the other hand, introduction of the exogenous Entner–Doudoroff pathway effectively enhanced glucose consumption and productivity. Overexpression of phosphoenolpyruvate:carbohydrate phosphotransferase system specific to glucose and deletion of ilvE, which encodes branched-chain amino acid transaminase, further suppressed by-products and improved isobutanol productivity. Finally, the produced isobutanol concentration reached 280 mM at a yield of 84% (mol/mol glucose) in 24 h.

根据我们以前在缺氧条件下生产微生物L-缬氨酸的研究，我们开发了异丁醇生产谷氨酸棒杆菌菌株。人工异丁醇合成途径由L-缬氨酸合成途径的前三个步骤组成。以及随后的Ehrlich途径：在先前的反应中，丙酮酸转化为2-酮异戊酸；然后将2-酮酸脱羧成异丁醛，然后在后面的反应中还原成异丁醇。尽管在整个反应中存在氧化还原辅因子失衡，即NADH是通过糖酵解产生的，而NADPH是合成异丁醇所必需的，但它可以通过利用NAD优选的突变体乙酰羟酸异构还原酶来解决ilvC ^TM和NAD特异性醇脱氢酶由编码ADHA。通过使用两种lac启动子衍生物对合成异丁醇的每种酶活性进行了微调。有效抑制琥珀酸副产物和提高异丁醇产量的原因是pckA失活，该磷酸化编码磷酸烯醇丙酮酸羧激酶，而葡萄糖消耗和异丁醇生产率却由于细胞内NADH / NAD ⁺升高而降低。 比。另一方面，引入外源性Entner-Doudoroff途径可有效提高葡萄糖的消耗和生产率。对葡萄糖特异的磷酸烯醇丙酮酸：碳水化合物磷酸转移酶系统的过表达和编码支链氨基酸转氨酶的ilvE的缺失，进一步抑制了副产物并提高了异丁醇的生产率。最后，在24小时内产生的异丁醇浓度达到280 mM，产率为84％（摩尔/摩尔葡萄糖）。