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

• Published in: Frontiers in bioengineering and biotechnology 2024-01, Vol.11, p.1336215-1336215
• Main Authors: Liu, Xinyang, Liu, Jiao, Liu, Zhemin, Qiao, Qianqian, Ni, Xiaomeng, Yang, Jinxing, Sun, Guannan, Li, Fanghe, Zhou, Wenjuan, Guo, Xuan, Chen, Jiuzhou, Jia, Shiru, Zheng, Yu, Zheng, Ping, Sun, Jibin
• Format: Article
• Language: English
• Subjects: allosteric inhibition; Bioengineering and Biotechnology; Corynebacterium glutamicum; high-throughput screening (HTS); homoserine dehydrogenase; semi-rational design
• ISSN: 2296-4185; 2296-4185
• DOI: 10.3389/fbioe.2023.1336215

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 ( HSD) 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 HSD-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 HSD-related amino acids and derivatives. Moreover, the proposed approach can be applied to engineering other allosteric enzymes in the amino acid synthesis pathway.