Isobutanol is a valuable chemical and is considered a new generation biofuel. Construction of isobutanol synthesis pathways in bacteria is a hot topic in isobutanol production. Here, we show that an isobutanol synthesis pathway exists naturally in Klebsiella pneumoniae; however, this pathway is dormant in the wild-type bacterium. K. pneumoniae is a 2,3-butanediol producer, and the synthesis pathways of 2,3-butanediol, valine and isobutanol all start from condensation of two pyruvate molecules to yield α-acetolactate. Inactivation of α-acetolactate decarboxylase (encoded by budA) resulted in α-acetolactate flowing into the valine pathway, which led to synthesis of isobutanol and 2-ketoisovalerate (a precursor of isobutanol). ldhA (lactate dehydrogenase) deletion further increased the isobutanol and 2-ketoisovalerate production. In the first step of this pathway, BudB (α-acetolactate synthase) was identified as responsible for most of the α-acetolactate synthesis. Complementation of ilvBN or ilvIH (isoenzymes of budB) both resulted in a remarkable increase in 2-ketoisovalerate production. Thus, α-acetolactate formation is the rate-limiting step of 2-ketoisovalerate production. ilvC (acetohydroxy acid isomeroreductase) and ilvD (dihydroxy acid dehydratase) were identified responsible for 2-ketoisovalerate synthesis from α-acetolactate. ipdC, which encodes an indole-3-pyruvate decarboxylase, was identified responsible for most of the isobutyraldehyde formation from 2-ketoisovalerate, and isobutanol production was increased 15.7 fold in the ipdC complementation strain, with a final titer of 2.45g/L. Isobutanol dehydrogenase activity is distributed across multiple alcohol dehydrogenase enzymes expressed by K. pneumoniae. BudC, DhaT, DhaD and YqhD all had isobutanol dehydrogenase activity in vitro. YqhD uses NADPH as the coenzyme, while the other dehydrogenases use NADH. However, inactivating one or two of these dehydrogenases had no effect on isobutanol production in vivo with isobutyraldehyde as the substrate. These results reveal a novel method for biological production of isobutanol and 2-ketoisovalerate.

中文翻译：

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肺炎克雷伯氏菌通过天然途径生产异丁醇和2-酮异戊酸。

异丁醇是一种有价值的化学品，被认为是新一代生物燃料。细菌中异丁醇合成途径的构建是异丁醇生产中的热门话题。在这里，我们表明异丁醇合成途径在肺炎克雷伯菌中自然存在。但是，该途径在野生型细菌中处于休眠状态。肺炎克雷伯菌是2,3-丁二醇的生产者，而2,3-丁二醇，缬氨酸和异丁醇的合成途径均始于两个丙酮酸分子的缩合以生成α-乙酰乳酸。α-乙酰乳酸脱羧酶（由budA编码）的失活导致α-乙酰乳酸流入缬氨酸途径，从而导致异丁醇和2-酮异戊酸（异丁醇的前体）的合成。ldhA（乳酸脱氢酶）的缺失进一步增加了异丁醇和2-酮异戊酸酯的产量。在该途径的第一步，已确定BudB（α-乙酰乳酸合酶）是大多数α-乙酰乳酸合成的原因。ilvBN或ilvIH（budB的同功酶）的互补均导致2-酮异戊酸产量的显着提高。因此，α-乙酰乳酸的形成是2-酮异戊酸酯生产的限速步骤。确定了ilvC（乙酰羟酸异构还原酶）和ilvD（二羟酸脱水酶）负责从α-乙酰乳酸合成2-酮异戊酸酯。编码吲哚-3-丙酮酸脱羧酶的ipdC被认为是由2-酮异戊酸形成大部分异丁醛的原因，ipdC互补菌株中异丁醇的产量增加了15.7倍，最终滴度为2.45g / L。异丁醇脱氢酶活性分布在肺炎克雷伯菌表达的多种醇脱氢酶中。BudC，DhaT，DhaD和YqhD在体外均具有异丁醇脱氢酶活性。YqhD使用NADPH作为辅酶，而其他脱氢酶则使用NADH。然而，以异丁醛为底物使这些脱氢酶中的一种或两种失活对体内异丁醇生产没有影响。这些结果揭示了生物生产异丁醇和2-酮异戊酸酯的新方法。