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<p>Lacto-<i>N</i>-neotetraose is an abundant human milk oligosaccharide with multifaceted physiological functions. In this study, <i>Escherichia coli</i> MG1655 has been used as the chassis strain for lacto-<i>N</i>-neotetraose production. First, the genes <i>lacz</i>, <i>ugd</i>, <i>gcd</i>, <i>nagB</i>, <i>ushA</i>, <i>setA</i> were deleted in MG1655, and the <i>Neisseria meningitidis</i> genes <i>lgtA</i> and <i>lgtB</i> encoding <i>β</i>-1,3-<i>N</i>-acetylglucosaminyltransferase and <i>β</i>-1,4-galactosyltransferase, respectively, were overexpressed, and the resulting strain WY011/pC-A/pR-B could produce 1.102&#xa0;g/L lacto-<i>N</i>-neotetraose. Next, the rate-limiting gene overexpression, expression vector screening, and ribosome-binding site strength were optimized, and the resulting strain WY011/pR-BEA1R could produce 2.634&#xa0;g/L lacto-<i>N</i>-neotetraose. Further optimization of fermentation parameters enabled the engineered strain WY011/pR-BEA1R to produce 3.533&#xa0;g/L lacto-<i>N</i>-neotetraose, and a whole-cell catalysis process developed by using this strain with the mixed substrates of glycerol, lactose, and galactose boosted the titer to 10.845&#xa0;g/L, which is the highest reported titer in small-scale systems so far. Ultimately, fed-batch fermentation of the strain WY011/pR-BEA1R in a 2-L bioreactor achieved an LNnT titer of 16.202&#xa0;g/L.</p>

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Metabolic engineering of Escherichia coli for production of lacto-N-neotetraose

  • 期刊论文

  • 发表日期：

    2025年11月24日

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  • Hongchen Yin,
  • Yuanming Wu,
  • Miaomiao Zhuang,
  • Lingyan Chen,
  • Xiaoqing Hu,
  • Xiaoyuan Wang

摘要

Lacto-N-neotetraose is an abundant human milk oligosaccharide with multifaceted physiological functions. In this study, Escherichia coli MG1655 has been used as the chassis strain for lacto-N-neotetraose production. First, the genes lacz, ugd, gcd, nagB, ushA, setA were deleted in MG1655, and the Neisseria meningitidis genes lgtA and lgtB encoding β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase, respectively, were overexpressed, and the resulting strain WY011/pC-A/pR-B could produce 1.102 g/L lacto-N-neotetraose. Next, the rate-limiting gene overexpression, expression vector screening, and ribosome-binding site strength were optimized, and the resulting strain WY011/pR-BEA1R could produce 2.634 g/L lacto-N-neotetraose. Further optimization of fermentation parameters enabled the engineered strain WY011/pR-BEA1R to produce 3.533 g/L lacto-N-neotetraose, and a whole-cell catalysis process developed by using this strain with the mixed substrates of glycerol, lactose, and galactose boosted the titer to 10.845 g/L, which is the highest reported titer in small-scale systems so far. Ultimately, fed-batch fermentation of the strain WY011/pR-BEA1R in a 2-L bioreactor achieved an LNnT titer of 16.202 g/L.