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<p>Despite its prominent industrial use for L-proline production, microbial fermentation by <i>Corynebacterium glutamicum</i> achieves a suboptimal yield of glucose conversion, far below the theoretical maximum of 0.63&#xa0;g/g. This study aimed to develop an efficient engineered <i>Escherichia coli</i> chassis to produce L-proline from glucose by addressing the rate-limiting enzymatic step and high glucose conversion. The γ-glutamyl kinase encoding gene (<i>proB</i>) was screened by directed evolution, and the catalytic efficiency of the <i>Sc</i>ProB** variant toward L-glutamate increased 2.75-fold. After channeling 2-oxoglutarate flux and supplying sufficient NADPH, L-proline production reached 86.27 ± 2.58&#xa0;g/L, with a 0.33 ± 0.01&#xa0;g/g glucose yield in 10-L fed-batch fermentation. Further increasing the flux through central carbon metabolism by implementing a non-PTS glucose uptake system, amplifying the anaplerotic reaction, and redirecting the flux away from the glyoxylate cycle, the resulting strain BP23 achieved 119.89 ± 5.66&#xa0;g/L L-proline, with a productivity of 1.93 ± 0.03&#xa0;g/L/h. The glucose conversion yield improved to 0.49 ± 0.01&#xa0;g/g after 62&#xa0;h of fed-batch fermentation, indicating that the <i>E. coli</i> strain is a promising workhorse for producing L-proline with high glucose conversion.</p>

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Efficient Escherichia coli platform for L-proline biosynthesis via systematic metabolic engineering

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月29日

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  • Shumei Cui,
  • Xiaolu Hu,
  • Xinwei Yang,
  • Yongtao Chen,
  • Tianqi Bao,
  • Bingyan Wang,
  • Yong Tao,
  • Jianzhong Huang,
  • Chongrong Ke

摘要

Despite its prominent industrial use for L-proline production, microbial fermentation by Corynebacterium glutamicum achieves a suboptimal yield of glucose conversion, far below the theoretical maximum of 0.63 g/g. This study aimed to develop an efficient engineered Escherichia coli chassis to produce L-proline from glucose by addressing the rate-limiting enzymatic step and high glucose conversion. The γ-glutamyl kinase encoding gene (proB) was screened by directed evolution, and the catalytic efficiency of the ScProB** variant toward L-glutamate increased 2.75-fold. After channeling 2-oxoglutarate flux and supplying sufficient NADPH, L-proline production reached 86.27 ± 2.58 g/L, with a 0.33 ± 0.01 g/g glucose yield in 10-L fed-batch fermentation. Further increasing the flux through central carbon metabolism by implementing a non-PTS glucose uptake system, amplifying the anaplerotic reaction, and redirecting the flux away from the glyoxylate cycle, the resulting strain BP23 achieved 119.89 ± 5.66 g/L L-proline, with a productivity of 1.93 ± 0.03 g/L/h. The glucose conversion yield improved to 0.49 ± 0.01 g/g after 62 h of fed-batch fermentation, indicating that the E. coli strain is a promising workhorse for producing L-proline with high glucose conversion.