Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032

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• 作者：Masaru Wada¹ ; ^{wada@chem.agr.hokudai.ac.jp" class="auth_mail" title="E-mail the corresponding author} ; Kazunori Sawada¹ ; ^{kazunori.sawada3@gmail.com" class="auth_mail" title="E-mail the corresponding author} ; Kotaro Ogura¹ ; ^{k-ogura@frontier.hokudai.ac.jp" class="auth_mail" title="E-mail the corresponding author} ; Yuta Shimono¹ ; ^{shimono-yuta@frontier.hokudai.ac.jp" class="auth_mail" title="E-mail the corresponding author} ; Takuya Hagiwara¹ ; ^{t-hagiwara@frontier.hokudai.ac.jp" class="auth_mail" title="E-mail the corresponding author} ; Masakazu Sugimoto² ; ^{masakazu_sugimoto@ajinomoto.com" class="auth_mail" title="E-mail the corresponding author} ; Akiko Onuki² ; ^{akiko_oonuki@ajinomoto.com" class="auth_mail" title="E-mail the corresponding author} ; Atsushi Yokota¹ ; ^{yokota@chem.agr.hokudai.ac.jp" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Corynebacterium glutamicum ; Glutamic acid ; Aspartic acid ; Phosphoenolpyruvate carboxylase ; Pyruvate kinase ; Anaplerotic pathway ; Feed-back inhibition
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：2016
• 出版时间：February 2016
• 年：2016
• 卷：121
• 期：2
• 页码：172-177
• 全文大小：353 K

文摘

Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum.