Frontiers in Bioengineering and Biotechnology (Jan 2024)

Engineering allosteric inhibition of homoserine dehydrogenase by semi-rational saturation mutagenesis screening

  • Xinyang Liu,
  • Xinyang Liu,
  • Xinyang Liu,
  • Xinyang Liu,
  • Jiao Liu,
  • Jiao Liu,
  • Zhemin Liu,
  • Zhemin Liu,
  • Qianqian Qiao,
  • Qianqian Qiao,
  • Qianqian Qiao,
  • Qianqian Qiao,
  • Xiaomeng Ni,
  • Xiaomeng Ni,
  • Xiaomeng Ni,
  • Jinxing Yang,
  • Jinxing Yang,
  • Guannan Sun,
  • Guannan Sun,
  • Fanghe Li,
  • Fanghe Li,
  • Wenjuan Zhou,
  • Wenjuan Zhou,
  • Xuan Guo,
  • Xuan Guo,
  • Jiuzhou Chen,
  • Jiuzhou Chen,
  • Shiru Jia,
  • Shiru Jia,
  • Yu Zheng,
  • Yu Zheng,
  • Ping Zheng,
  • Ping Zheng,
  • Ping Zheng,
  • Jibin Sun,
  • Jibin Sun,
  • Jibin Sun

DOI
https://doi.org/10.3389/fbioe.2023.1336215
Journal volume & issue
Vol. 11

Abstract

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Allosteric regulation by pathway products plays a vital role in amino acid metabolism. Homoserine dehydrogenase (HSD), the key enzyme for the biosynthesis of various aspartate family amino acids, is subject to feedback inhibition by l-threonine and l-isoleucine. The desensitized mutants with the potential for amino acid production remain limited. Herein, a semi-rational approach was proposed to relieve the feedback inhibition. HSD from Corynebacterium glutamicum (CgHSD) was first characterized as a homotetramer, and nine conservative sites at the tetramer interface were selected for saturation mutagenesis by structural simulations and sequence analysis. Then, we established a high-throughput screening (HTS) method based on resistance to l-threonine analog and successfully acquired two dominant mutants (I397V and A384D). Compared with the best-ever reported desensitized mutant G378E, both new mutants qualified the engineered strains with higher production of CgHSD-dependent amino acids. The mutant and wild-type enzymes were purified and assessed in the presence or absence of inhibitors. Both purified mutants maintained >90% activity with 10 mM l-threonine or 25 mM l-isoleucine. Moreover, they showed >50% higher specific activities than G378E without inhibitors. This work provides two competitive alternatives for constructing cell factories of CgHSD-related amino acids and derivatives. Moreover, the proposed approach can be applied to engineering other allosteric enzymes in the amino acid synthesis pathway.

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