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<p>Amino acids are important bio-based products with a multi-billion-dollar market. The development of efficient high-throughput screening technologies utilizing biosensors is essential for the rapid identification of high-performance amino acid producers. However, there remains a pressing need for biosensors that specifically target certain critical amino acids, such as <span>l</span>-threonine and <span>l</span>-proline. In this study, a novel transcriptional regulator-based biosensor for <span>l</span>-threonine and <span>l</span>-proline was successfully developed, inspired by our new finding that SerE can export <span>l</span>-proline in addition to the previously known <span>l</span>-threonine and <span>l</span>-serine. Through directed evolution of SerR (the corresponding transcriptional regulator of SerE), the mutant SerR<sup>F104I</sup> which can recognize both <span>l</span>-threonine and <span>l</span>-proline as effectors and effectively distinguish strains with varying production levels was identified. Subsequently, the SerR<sup>F104I</sup>-based biosensor was employed for high-throughput screening of the superior enzyme mutants of <span>l</span>-homoserine dehydrogenase and γ-glutamyl kinase, which are critical enzymes in the biosynthesis of <span>l</span>-threonine and <span>l</span>-proline, respectively. A total of 25 and 13 novel mutants that increased the titers of <span>l</span>-threonine and <span>l</span>-proline by over 10% were successfully identified. Notably, six of the newly identified mutants exhibited similarities to the most effective mutants reported to date, indicating the promising application potential of the SerR<sup>F104I</sup>-based biosensor. This study illustrates an effective strategy for the development of transcriptional regulator-based biosensors for amino acids and other chemical compounds.</p>

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Engineering of l-threonine and l-proline biosensors by directed evolution of transcriptional regulator SerR and application for high-throughput screening

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年1月19日

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  • Wei Pu,
  • Jinhui Feng,
  • Jiuzhou Chen,
  • Jiao Liu,
  • Xuan Guo,
  • Lixian Wang,
  • Xiaojia Zhao,
  • Ningyun Cai,
  • Wenjuan Zhou,
  • Yu Wang,
  • Ping Zheng,
  • Jibin Sun

摘要

Amino acids are important bio-based products with a multi-billion-dollar market. The development of efficient high-throughput screening technologies utilizing biosensors is essential for the rapid identification of high-performance amino acid producers. However, there remains a pressing need for biosensors that specifically target certain critical amino acids, such as l-threonine and l-proline. In this study, a novel transcriptional regulator-based biosensor for l-threonine and l-proline was successfully developed, inspired by our new finding that SerE can export l-proline in addition to the previously known l-threonine and l-serine. Through directed evolution of SerR (the corresponding transcriptional regulator of SerE), the mutant SerRF104I which can recognize both l-threonine and l-proline as effectors and effectively distinguish strains with varying production levels was identified. Subsequently, the SerRF104I-based biosensor was employed for high-throughput screening of the superior enzyme mutants of l-homoserine dehydrogenase and γ-glutamyl kinase, which are critical enzymes in the biosynthesis of l-threonine and l-proline, respectively. A total of 25 and 13 novel mutants that increased the titers of l-threonine and l-proline by over 10% were successfully identified. Notably, six of the newly identified mutants exhibited similarities to the most effective mutants reported to date, indicating the promising application potential of the SerRF104I-based biosensor. This study illustrates an effective strategy for the development of transcriptional regulator-based biosensors for amino acids and other chemical compounds.