Scholarlink

Abstract <p>The fermentation of nitrogen-containing compounds by biological nitrogen fixation is a sustainable strategy that is independent of the Haber–Bosch process. We previously reported that the nitrogen-fixing bacterium <i>Klebsiella pasteurii</i> (formerly <i>K. oxytoca</i>) NG13 synthesized and excreted large amounts of ʟ-glutamate using gaseous nitrogen when citrate synthase (CS) and citrate transporter (CitS) were overproduced; however, the majority of carbon atoms in ʟ-glutamate were derived from citrate, not glucose, in the glucose and citrate-containing medium. To examine biased carbon flux to ʟ-glutamate, <i>K. pasteurii</i> overproducing CS and a 2-oxoglutarate (2-OG) transporter (KgtP) was constructed, and its carbon origin was investigated. This strain produced 2-OG-derived ʟ-glutamate in a culture medium containing glucose and 2-OG as the carbon sources. Since CS was inhibited by 2-OG competitively with oxaloacetate, a cognate substrate of CS, the deviated carbon flux from citrate/2-OG to ʟ-glutamate was attributed to the suppression of CS by 2-OG. Based on the structural model of CS from <i>K. pasteurii</i> (KpCS), H227 and V362 were selected as candidates to detect 2-OG binding, and KpCS variants (KpCS*) with H227L, H227Q, and V362L substitutions were confirmed to have inhibition constants that increased by 2.5- to 12.5-fold. As expected, the strains co-overproducing each of the KpCS variants and CitS generated larger amounts of ʟ-glutamate from glucose than the wild-type KpCS + CitS strain. When the KpCS(H227Q) + CitS strain was cultured under continuous glucose-fed conditions, maximum ʟ-glutamate production reached 2.35&#xa0;g L<sup>−1</sup>. These results suggest the potential of the Haber–Bosch process-independent strategy as a technological basis for the sustainable and eco-friendly utilization of nitrogen.</p> Key points <p>• <i>CS was inhibited by 2-OG in K. pasteurii</i></p> <p>• <i>CS variants with increased K</i><sub>i</sub><sup>2−OG</sup><i> allowed glucose-derived ʟ-glutamate production</i></p> <p>• <i>Under glucose-fed culture, ʟ-glutamate production finally reached 2.35 g L</i><sup>−1</sup></p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Increased ʟ-glutamate production from gaseous nitrogen using Klebsiella pasteurii NG13 with modified citrate synthase

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年12月19日

查看全文
  • Daisuke Yoshidome,
  • Keitaro Kuze,
  • Atsushi Ichiyanagi,
  • Ayako Yoshida,
  • Saori Kosono,
  • Makoto Nishiyama

摘要

Abstract

The fermentation of nitrogen-containing compounds by biological nitrogen fixation is a sustainable strategy that is independent of the Haber–Bosch process. We previously reported that the nitrogen-fixing bacterium Klebsiella pasteurii (formerly K. oxytoca) NG13 synthesized and excreted large amounts of ʟ-glutamate using gaseous nitrogen when citrate synthase (CS) and citrate transporter (CitS) were overproduced; however, the majority of carbon atoms in ʟ-glutamate were derived from citrate, not glucose, in the glucose and citrate-containing medium. To examine biased carbon flux to ʟ-glutamate, K. pasteurii overproducing CS and a 2-oxoglutarate (2-OG) transporter (KgtP) was constructed, and its carbon origin was investigated. This strain produced 2-OG-derived ʟ-glutamate in a culture medium containing glucose and 2-OG as the carbon sources. Since CS was inhibited by 2-OG competitively with oxaloacetate, a cognate substrate of CS, the deviated carbon flux from citrate/2-OG to ʟ-glutamate was attributed to the suppression of CS by 2-OG. Based on the structural model of CS from K. pasteurii (KpCS), H227 and V362 were selected as candidates to detect 2-OG binding, and KpCS variants (KpCS*) with H227L, H227Q, and V362L substitutions were confirmed to have inhibition constants that increased by 2.5- to 12.5-fold. As expected, the strains co-overproducing each of the KpCS variants and CitS generated larger amounts of ʟ-glutamate from glucose than the wild-type KpCS + CitS strain. When the KpCS(H227Q) + CitS strain was cultured under continuous glucose-fed conditions, maximum ʟ-glutamate production reached 2.35 g L−1. These results suggest the potential of the Haber–Bosch process-independent strategy as a technological basis for the sustainable and eco-friendly utilization of nitrogen.

Key points

• CS was inhibited by 2-OG in K. pasteurii

• CS variants with increased Ki2−OG allowed glucose-derived ʟ-glutamate production

• Under glucose-fed culture, ʟ-glutamate production finally reached 2.35 g L−1