Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin, Gyeonggi-Do, 449-791, Korea.
Can J Microbiol. 2012 Mar;58(3):278-86. doi: 10.1139/w11-132. Epub 2012 Feb 22.
The development of microbial strains for the enhanced production of α-ketoglutarate (α-KG) was investigated using a strain of Corynebacterium glutamicum that overproduces of l-glutamate, by disrupting three genes involved in the α-KG biosynthetic pathway. The pathways competing with the biosynthesis of α-KG were blocked by knocking out aceA (encoding isocitrate lyase, ICL), gdh (encoding glutamate dehydrogenase, l-gluDH), and gltB (encoding glutamate synthase or glutamate-2-oxoglutarate aminotransferase, GOGAT). The strain with aceA, gltB, and gdh disrupted showed reduced ICL activity and no GOGAT and l-gluDH activities, resulting in up to 16-fold more α-KG production than the control strain in flask culture. These results suggest that l-gluDH is the key enzyme in the conversion of α-KG to l-glutamate; therefore, prevention of this step could promote α-KG accumulation. The inactivation of ICL leads the carbon flow to α-KG by blocking the glyoxylate pathway. However, the disruption of gltB did not affect the biosynthesis of α-KG. Our results can be applied in the industrial production of α-KG by using C. glutamicum as producer.
本研究旨在利用一株谷氨酸棒杆菌（Corynebacterium glutamicum）高产 l-谷氨酸的特性，通过敲除参与 α-酮戊二酸（α-KG）生物合成途径的三个基因，开发用于增强 α-KG 生产的微生物菌株。通过敲除 aceA（编码异柠檬酸裂解酶，ICL）、gdh（编码谷氨酸脱氢酶，l-gluDH）和 gltB（编码谷氨酸合酶或谷氨酸-2-酮戊二酸氨基转移酶，GOGAT），阻断与 α-KG 生物合成竞争的途径。与对照菌株相比，敲除 aceA、gltB 和 gdh 的菌株 ICL 活性降低，GOGAT 和 l-gluDH 活性缺失，导致摇瓶培养中 α-KG 的产量增加了 16 倍。这些结果表明，l-gluDH 是将 α-KG 转化为 l-谷氨酸的关键酶；因此，阻止这一步骤可以促进 α-KG 的积累。ICL 的失活通过阻断乙醛酸途径使碳流流向 α-KG。然而，gltB 的敲除并不影响 α-KG 的生物合成。我们的研究结果可应用于利用谷氨酸棒杆菌作为生产菌株进行 α-KG 的工业生产。
