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Background <p>1,5-Pentanediol (1,5-PDO) is a high-value chemical with broad uses in polymer, cosmetic, and pharmaceutical industries. Although diverse biosynthetic pathways have been constructed, current recombinant strains typically rely on plasmid-based overexpression, which necessitates antibiotics and hinders industrial-scale production.</p> Results <p>We developed a robust, plasmid-free Escherichia coli platform for de novo 1,5-PDO synthesis by integrating pathway genes (<i>davB</i>, <i>davA</i>, <i>gabT</i>, <i>yahK</i>, <i>car</i>, <i>sfp</i> and <i>yqhD</i>) into the chromosome of a lysine-hyperproducing strain via CRISPR/Cas9. Screening of carboxylic acid reductases identified <i>Nocardia iowensis</i> CAR-Ni as the most effective, yielding a base strain (D13) that produced 0.672&#xa0;g/L 1,5-PDO. Integrated analysis confirmed the alcohol dehydrogenase (ADH)-mediated reduction of 5-hydroxypentanal (5-HP) as an underappreciated bottleneck. We subsequently screened ten endogenous ADHs and selected YjgB for computational optimization. Docking-guided saturation mutagenesis at position E205 yielded the variant YjgB(E205C), which exhibited a 3.34-fold increase in in vitro activity, reduced 5-HP accumulation, and elevated the titer to 0.935&#xa0;g/L. Enhancing NADPH supply by integrating <i>pntAB</i> further raised the shake-flask titer to 1.5&#xa0;g/L. In a 5-L fed-batch bioreactor, the final strain (D91) achieved 12.1&#xa0;g/L 1,5-PDO (yield of 0.225&#xa0;mol/mol glucose) without antibiotics or inducers. To our knowledge, this is the highest reported 1,5-PDO titer in <i>E. coli</i>.</p> Conclusion <p>This study establishes a scalable, sustainable biosynthetic platform through synergistic metabolic engineering and computational enzyme optimization.</p>

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Combining computer-aided enzyme design and chromosomal integration for plasmid-free biosynthesis of 1,5-pentanediol in Escherichia coli

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月25日

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  • Hongyu Deng,
  • Jing Wang,
  • Fei Meng,
  • Chunling Ma,
  • Junlin Li,
  • Junqing Wang,
  • Xiaonan Wang,
  • Chunhui Zhao,
  • Yan Zhang,
  • Ruiming Wang,
  • Ning Chen

摘要

Background

1,5-Pentanediol (1,5-PDO) is a high-value chemical with broad uses in polymer, cosmetic, and pharmaceutical industries. Although diverse biosynthetic pathways have been constructed, current recombinant strains typically rely on plasmid-based overexpression, which necessitates antibiotics and hinders industrial-scale production.

Results

We developed a robust, plasmid-free Escherichia coli platform for de novo 1,5-PDO synthesis by integrating pathway genes (davB, davA, gabT, yahK, car, sfp and yqhD) into the chromosome of a lysine-hyperproducing strain via CRISPR/Cas9. Screening of carboxylic acid reductases identified Nocardia iowensis CAR-Ni as the most effective, yielding a base strain (D13) that produced 0.672 g/L 1,5-PDO. Integrated analysis confirmed the alcohol dehydrogenase (ADH)-mediated reduction of 5-hydroxypentanal (5-HP) as an underappreciated bottleneck. We subsequently screened ten endogenous ADHs and selected YjgB for computational optimization. Docking-guided saturation mutagenesis at position E205 yielded the variant YjgB(E205C), which exhibited a 3.34-fold increase in in vitro activity, reduced 5-HP accumulation, and elevated the titer to 0.935 g/L. Enhancing NADPH supply by integrating pntAB further raised the shake-flask titer to 1.5 g/L. In a 5-L fed-batch bioreactor, the final strain (D91) achieved 12.1 g/L 1,5-PDO (yield of 0.225 mol/mol glucose) without antibiotics or inducers. To our knowledge, this is the highest reported 1,5-PDO titer in E. coli.

Conclusion

This study establishes a scalable, sustainable biosynthetic platform through synergistic metabolic engineering and computational enzyme optimization.