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Background <p>Rapid advancements in complex engineered metabolic system design have not been matched by corresponding screening and identification capabilities for high-performing microbial variants, creating considerable pacing constraints in strain development. Therefore, advancing high-throughput phenotyping methods is essential for propelling synthetic biology and metabolic engineering towards scalable biomanufacturing.</p> Results <p>Accordingly, we developed a rare codon-dependent fluorescent biosensor that enables real-time, high-content monitoring of intracellular L-arginine accumulation. This system employed L-arginine-rich peptide modules that are engineered with AGG rare codons fused to the StayGold fluorescent protein, developing a stringent link between fluorescence intensity and cytoplasmic L-arginine levels. By integrating ultraviolet mutagenesis with fluorescence-activated cell sorting, we efficiently isolated superior producers from an engineered <i>Escherichia coli</i> ARG library, achieving a screening efficiency of 55.12%. The top-performing isolate, <i>E. coli</i> ARG-B10, exhibited a 94.8% enhancement in L-arginine production. Its plasmid-cured derivative, <i>E. coli</i> B10, was used for scale-up fermentation, attaining a 120.5&#xa0;g/L titer and 0.45&#xa0;g/g glucose yield under industrially relevant conditions. Genomic analysis revealed missense mutations in key metabolic genes (<i>coaBC</i>, <i>gst</i>, <i>yihU</i>, and <i>fruB</i>), indicating improvements in the precursor supply and redox management.</p> Conclusion <p>This biosensor platform operates independently of orthogonal translation systems and is readily applicable to wild-type strains, offering a powerful and generalizable tool for accelerating microbial strain optimization.</p>

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Rare-codon-tuned fluorescent biosensor for high-throughput selection of Escherichia coli strain capable of L-arginine overproduction

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月12日

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  • Daixue Kou,
  • Guanhua Jiao,
  • Yinuo Li,
  • Dan Li,
  • Chuanzhuang Guo,
  • Jianbin Wang,
  • Junlin Li,
  • Yanwei Wei,
  • Ting Wang,
  • Piwu Li,
  • Jing Su,
  • Ruiming Wang,
  • Nan Li,
  • Junqing Wang,
  • Han Fan

摘要

Background

Rapid advancements in complex engineered metabolic system design have not been matched by corresponding screening and identification capabilities for high-performing microbial variants, creating considerable pacing constraints in strain development. Therefore, advancing high-throughput phenotyping methods is essential for propelling synthetic biology and metabolic engineering towards scalable biomanufacturing.

Results

Accordingly, we developed a rare codon-dependent fluorescent biosensor that enables real-time, high-content monitoring of intracellular L-arginine accumulation. This system employed L-arginine-rich peptide modules that are engineered with AGG rare codons fused to the StayGold fluorescent protein, developing a stringent link between fluorescence intensity and cytoplasmic L-arginine levels. By integrating ultraviolet mutagenesis with fluorescence-activated cell sorting, we efficiently isolated superior producers from an engineered Escherichia coli ARG library, achieving a screening efficiency of 55.12%. The top-performing isolate, E. coli ARG-B10, exhibited a 94.8% enhancement in L-arginine production. Its plasmid-cured derivative, E. coli B10, was used for scale-up fermentation, attaining a 120.5 g/L titer and 0.45 g/g glucose yield under industrially relevant conditions. Genomic analysis revealed missense mutations in key metabolic genes (coaBC, gst, yihU, and fruB), indicating improvements in the precursor supply and redox management.

Conclusion

This biosensor platform operates independently of orthogonal translation systems and is readily applicable to wild-type strains, offering a powerful and generalizable tool for accelerating microbial strain optimization.