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Metabolic engineering of Escherichia coli for the production of Lacto-N-neotetraose (LNnT)

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Abstract

Lacto-N-neotetraose (LNnT), one of the most important human milk oligosaccharides, can be used as infants’ food additives. Nowadays, extraction, chemical and biological synthesis were utilized to obtain LNnT, while these methods still face some problems such as low yield and high cost. The aim of current work is to construct a de novo biosynthesis pathway of LNnT in E. coli K12 MG1655. The lgtA and lgtB were first expressed by a plasmid, resulting in a LNnT titer of 0.04 g/L. To improve the yield of LNnT on substrate lactose, lacZ and lacI were knocked out, and lacY was over-expressed. As a result, the yield of LNnT on lactose increased from 0.01 to 0.09 mol/mol, and the titer of LNnT elevated to 0.41 g/L. In addition, the pathway was regulated using the titer of Lacto-N-triose II (LNTII) as a measure, and obtained a high titer strain of LNnT for 1.04 g/L. Finally, the gene expressions were fine-tuned, the titer of LNnT reached 1.2 g/L, which was 93% higher than the control strain, and the yield on lactose reached 0.28 mol/mol. The engineering strategy of pathway construction and modulation used in this study is applicable to facilitate the microbial production of other metabolites in E. coli.

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Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

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Funding

This work was supported by the National Natural Science Foundation of China (31930085, 32021005), and the key research and development program of China (2018YFA0900300, 2020YFA0908300).

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Authors and Affiliations

  1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Wei Zhang, Xueqin Lv, Xiaomin Dong, Yanfeng Liu, Jianghua Li, Guocheng Du & Long Liu

  2. State Key Laboratory of Dairy Biotechnology, Shanghai Engineering Research Center of Dairy Biotechnology, Dairy Research Institute, Bright Dairy & Food Co., Ltd, Shanghai, 200436, China

    Zhenmin Liu & Nan Li

  3. Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China

    Wei Zhang, Xueqin Lv, Xiaomin Dong, Yanfeng Liu, Jianghua Li, Guocheng Du & Long Liu

  4. School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China

    Mengyue Gong

Authors
  1. Wei Zhang
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  2. Zhenmin Liu
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  3. Mengyue Gong
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  4. Nan Li
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  6. Xiaomin Dong
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  10. Long Liu
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Contributions

WZ has a major contribution to the study, and writing of the manuscript. ZML, GMY, NL and XQL analyzed the experimental data. XMD, LYF, LJH and DGC revised the manuscript. LL conceived and supervised the project and contributed to the writing of the manuscript. All the authors read and approved the manuscript.

Corresponding authors

Correspondence to Zhenmin Liu or Long Liu.

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The authors declare that they have no conflicts of interest.

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Zhang, W., Liu, Z., Gong, M. et al. Metabolic engineering of Escherichia coli for the production of Lacto-N-neotetraose (LNnT). Syst Microbiol and Biomanuf 1, 291–301 (2021). https://doi.org/10.1007/s43393-021-00023-1

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  • DOI: https://doi.org/10.1007/s43393-021-00023-1

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