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<p><span>l</span>-threonine is an integral nutrient for mammals, often used in animal feeds to enhance growth and reduce breeding costs. Developing <span>l</span>-threonine engineered strains that meet industrial production specifications has significant economic value. Here, we developed a biosensor that monitors <span>l</span>-threonine concentration to assist in high-throughput screening to capture high-yielding <span>l</span>-threonine mutants. Among them, the P<sub><i>cysK</i></sub> promoter and CysB protein were used to construct a primary <span>l</span>-threonine biosensor, and then the CysB<sup>T102A</sup> mutant was obtained through directed evolution resulting in a 5.6-fold increase in the fluorescence responsiveness of biosensor over the 0–4&#xa0;g/L <span>l</span>-threonine concentration range. In addition, the metabolic network of mutant was further optimized through multi-omics analysis and in silico simulation. Ultimately, the THRM13 strain produced 163.2&#xa0;g/L <span>l</span>-threonine, with a yield of 0.603&#xa0;g/g glucose in a 5&#xa0;L bioreactor. The biosensor constructed here could be employed for iterative upgrading of subsequent strains, and these engineering strategies described provide guidance for other chemical overproducers.</p> Graphical Abstract <p></p>

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Combining biosensor and metabolic network optimization strategies for enhanced l-threonine production in Escherichia coli

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年3月26日

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  • Zhenqiang Zhao,
  • Rongshuai Zhu,
  • Xuanping Shi,
  • Fengyu Yang,
  • Meijuan Xu,
  • Minglong Shao,
  • Rongzhen Zhang,
  • Youxi Zhao,
  • Jiajia You,
  • Zhiming Rao

摘要

l-threonine is an integral nutrient for mammals, often used in animal feeds to enhance growth and reduce breeding costs. Developing l-threonine engineered strains that meet industrial production specifications has significant economic value. Here, we developed a biosensor that monitors l-threonine concentration to assist in high-throughput screening to capture high-yielding l-threonine mutants. Among them, the PcysK promoter and CysB protein were used to construct a primary l-threonine biosensor, and then the CysBT102A mutant was obtained through directed evolution resulting in a 5.6-fold increase in the fluorescence responsiveness of biosensor over the 0–4 g/L l-threonine concentration range. In addition, the metabolic network of mutant was further optimized through multi-omics analysis and in silico simulation. Ultimately, the THRM13 strain produced 163.2 g/L l-threonine, with a yield of 0.603 g/g glucose in a 5 L bioreactor. The biosensor constructed here could be employed for iterative upgrading of subsequent strains, and these engineering strategies described provide guidance for other chemical overproducers.

Graphical Abstract