Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, Japan.
Appl Microbiol Biotechnol. 2011 Jul;91(1):143-51. doi: 10.1007/s00253-011-3275-6. Epub 2011 Apr 19.
Glutamate overproduction by Corynebacterium glutamicum is triggered by treatment with penicillin or Tween 40 and is accompanied by a decrease in 2-oxoglutarate dehydrogenase complex (ODHC) activity. We have reported that de novo synthesis of OdhI, which inhibits ODHC activity by interacting specifically with the E1o subunit of ODHC (OdhA), is induced by penicillin, and that odhI overexpression induces glutamate overproduction in the absence of any triggers for glutamate overproduction. In this study, to determine the function of OdhI in glutamate overproduction by C. glutamicum, changes in OdhI levels and phosphorylation status during penicillin- and Tween 40-induced glutamate overproduction were examined by western blot. The synthesis of both unphosphorylated and phosphorylated OdhI was increased by addition of Tween 40 or penicillin and the levels of unphosphorylated OdhI, which can inhibit ODHC activity, was significantly higher than those of phosphorylated OdhI, which is unable to inhibit ODHC activity. Meanwhile, the OdhA levels were maintained throughout the culture. These results indicate that OdhI synthesis is induced by additions of penicillin and Tween 40 and most synthesized OdhI is unphosphorylated, resulting in the decrease in ODHC activity and glutamate overproduction. Similarly, in the odhI-overexpressing strain, both unphosphorylated and phosphorylated OdhI were synthesized, while the levels of OdhA were nearly constant throughout culture. Our results suggest that high level of unphosphorylated OdhI regulates glutamate overproduction by C. glutamicum.
谷氨酸棒杆菌中的谷氨酸过度产生是由青霉素或吐温 40 处理引发的，并伴随着 2-氧戊二酸脱氢酶复合物（ODHC）活性的降低。我们已经报道过，新合成的 OdhI 通过与 ODHC 的 E1o 亚基（OdhA）特异性相互作用来抑制 ODHC 活性，青霉素诱导 OdhI 的合成，并且 odhI 的过表达在没有任何引发谷氨酸过度产生的情况下诱导谷氨酸过度产生。在这项研究中，为了确定 OdhI 在谷氨酸棒杆菌谷氨酸过度产生中的作用，通过 Western blot 检查了青霉素和吐温 40 诱导谷氨酸过度产生过程中 OdhI 水平和磷酸化状态的变化。添加吐温 40 或青霉素可增加未磷酸化和磷酸化 OdhI 的合成，可抑制 ODHC 活性的未磷酸化 OdhI 的水平明显高于不能抑制 ODHC 活性的磷酸化 OdhI 的水平。同时，OdhA 的水平在整个培养过程中保持不变。这些结果表明，青霉素和吐温 40 的添加诱导了 OdhI 的合成，并且大多数合成的 OdhI 是未磷酸化的，导致 ODHC 活性降低和谷氨酸过度产生。同样，在 odhI 过表达菌株中，合成了未磷酸化和磷酸化的 OdhI，而 OdhA 的水平在整个培养过程中几乎保持不变。我们的结果表明，高水平的未磷酸化 OdhI 调节谷氨酸棒杆菌的谷氨酸过度产生。
