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<p>Glucosamine (GlcN) and GlcN-based supplements such as N-acetyl-glucosamine (GlcNAc) are widely used by osteoarthritis patients to support joint health. However, current methods for producing GlcN-based products are not environmentally friendly and pose risks to individuals allergic to shrimp. Microbial cell-based systems offer a sustainable alternative for GlcN and GlcNAc production. This study focused on developing an <i>Escherichia coli</i> strain for GlcNAc fermentation. <i>E. coli</i> naturally synthesizes N-acetyl-glucosamine-1-phosphate (GlcNAc-1-P) as part of its peptidoglycan biosynthesis pathway. To enhance GlcNAc production, the <i>glmS</i> gene (encoding for glucosamine-6-P synthase) from <i>E. coli</i> and the <i>GNA1</i> gene (encoding for N-acetylglucosamine-6-P N-acetyltransferase) from <i>Saccharomyces cerevisiae</i> were cloned and combined into an artificial operon (<i>glmS-GNA1</i>) to establish an alternative GlcNAc pathway. The qPCR analysis of the resulting strain revealed a substantial increase of <i>glmS</i> and <i>GNA1</i> expression. This artificial operon was then placed under the control of a salicylate-inducible promoter (Pm<i>-glmS-GNA1</i>), which enabled the cell growth and GlcNAc production in two phases. The <i>E. coli</i> AP520 strain containing the salicylate inducible cassette produced 6.2&#xa0;g/L of GlcNAc in shaking flask fermentation, validating the strain design and engineering strategy for constructing a functional GlcNAc fermentation pathway in <i>E. coli</i>.</p>

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Metabolic engineering of Escherichia coli for N-acetyl glucosamine fermentation

  • 期刊论文

  • 发表日期：

    2025年11月11日

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  • Anica Tasnim Protity,
  • Shengde Zhou

摘要

Glucosamine (GlcN) and GlcN-based supplements such as N-acetyl-glucosamine (GlcNAc) are widely used by osteoarthritis patients to support joint health. However, current methods for producing GlcN-based products are not environmentally friendly and pose risks to individuals allergic to shrimp. Microbial cell-based systems offer a sustainable alternative for GlcN and GlcNAc production. This study focused on developing an Escherichia coli strain for GlcNAc fermentation. E. coli naturally synthesizes N-acetyl-glucosamine-1-phosphate (GlcNAc-1-P) as part of its peptidoglycan biosynthesis pathway. To enhance GlcNAc production, the glmS gene (encoding for glucosamine-6-P synthase) from E. coli and the GNA1 gene (encoding for N-acetylglucosamine-6-P N-acetyltransferase) from Saccharomyces cerevisiae were cloned and combined into an artificial operon (glmS-GNA1) to establish an alternative GlcNAc pathway. The qPCR analysis of the resulting strain revealed a substantial increase of glmS and GNA1 expression. This artificial operon was then placed under the control of a salicylate-inducible promoter (Pm-glmS-GNA1), which enabled the cell growth and GlcNAc production in two phases. The E. coli AP520 strain containing the salicylate inducible cassette produced 6.2 g/L of GlcNAc in shaking flask fermentation, validating the strain design and engineering strategy for constructing a functional GlcNAc fermentation pathway in E. coli.