Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Republic of Korea.
School of Chemical and Biological Engineering, Institute of Bioengineering, Seoul National University, Seoul, Republic of Korea.
Appl Microbiol Biotechnol. 2016 Jun;100(12):5527-36. doi: 10.1007/s00253-016-7433-8. Epub 2016 Mar 12.
While glutamate and glutamate-rich compounds are widely used for culturing Streptomyces sp., little is known regarding glutamate catabolism at molecular level. Noting the presence of two distinct putative glutamate dehydrogenases (GDH), we constructed knockout mutants of each gene with Streptomyces coelicolor M145 and examined the functionality related to antibiotic production. Out of the two, the sco2999 knockout (ΔgdhB, NAD(+)-specific) showed outstanding effects; it decreased the growth sevenfold but initiated the undecylprodigiosin (RED) production in complex Difco nutrient media which otherwise does not support the production from M145. With glucose supplementation, the growth difference by ΔgdhB disappeared but we could obtain significantly increased actinorhodin (ACT) and RED biosynthesis with the mutant by limiting the glucose content (0.5∼1.0 %, w/v). Complementing the gene to the knockout mutant inhibited the production, confirming its gene specificity. Along with the extended impacts on overall nitrogen metabolism based on the intracellular metabolite analysis and enzyme assays, GdhB and glutamate utilization were shown to interfere with N-acetylglucosamine metabolism and the activity of its associated global transcriptional regulator (DasR). Taken together, GdhB-subjected to the nutritional context-dependent regulation-is proposed as a key member of central nitrogen metabolism to control the secondary metabolism initiation in exploiting the organic nitrogen sources.
虽然谷氨酸和富含谷氨酸的化合物被广泛用于培养链霉菌属，但对于谷氨酸在分子水平上的代谢知之甚少。鉴于存在两种不同的推定谷氨酸脱氢酶（GDH），我们构建了链霉菌属橙色 M145 中每个基因的敲除突变体，并研究了与抗生素生产相关的功能。在这两种酶中， sco2999 敲除（ΔgdhB，NAD(+)-特异性）表现出显著的效果；它使生长减少了七倍，但在复杂的 Difco 营养培养基中启动了十一烷丙基灵菌红素（RED）的产生，而在该培养基中，M145 本身并不支持 RED 的产生。在葡萄糖补充的情况下，ΔgdhB 引起的生长差异消失了，但通过限制葡萄糖含量（0.5∼1.0%，w/v），我们可以从突变体中获得显著增加的放线紫红素（ACT）和 RED 生物合成。将基因互补到敲除突变体中抑制了产物的产生，证实了其基因特异性。通过对细胞内代谢物分析和酶活性测定，扩展了对整体氮代谢的影响，表明 GdhB 和谷氨酸的利用会干扰 N-乙酰葡萄糖胺代谢及其相关全局转录调节剂（DasR）的活性。总之，受营养条件调节的 GdhB 被提议作为中央氮代谢的关键成员，以控制在利用有机氮源时次级代谢的启动。
