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Multienzymatic cascade synthesis of fucosyloligosaccharide via a two-step fermentation strategy in Escherichia coli

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Abstract

Objectives

To achieve multienzymatic cascade synthesis of fucosyl oligosaccharide from d-mannose by two-step fermentation pathway in Escherichia coli.

Results

E. coli BL21(DE3) harboring pET-22b(+) vectors with six genes, i.e., glucokinase (Glk), phosphomannomutase (ManB), mannose-1-phosphate guanylytransferase (ManC), GDP-mannose 4,6-dehydratase (Gmd), GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase/4-reductase (WcaG), and α-1,2-fucosyltransferase (Fuct) were co-inoculated, and the multienzyme synthetic pathway was constructed to produce fucosyloligosaccharide using d-mannose as substrate. The product, analyzed by LC/MS, fucosyloligosaccharide was formed under the catalysis of Fuct using GDP-fucose as donor substrate and lactose as acceptor substrate. Fucosyloligosaccharides reached 22 mM by a two-step fermentation compared to 3.7 mM with a one-pot fermentation.

Conclusions

Fucosyloligosaccharide was produced by a two-step fermentation to avoid the inhibitory effect of GDP-fucose on Gmd. Two-step fermentation is a rational synthetic pathway for accumulating fucosyloligosaccharide.

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Acknowledgments

This work was supported by the National High-Tech Research and Development Plan (“863” Plan, 2012AA021502) and the Overseas High-level Talents Program of Tianjin University of Science and Technology, China. We acknowledge the excellent support of the Tianjin Food Safety & Low Carbon Manufacturing Collaborative Innovation Center 300457, Tianjin, China.

Supporting information

Supplementary Materials and methods—Enzyme activity assays.

Supplementary Materials and methods—Fermentation and product analysis of fucosyloligosaccharide.

Supplementary Table 1—Primer used for the construction of recombinant enzymes.

Supplementary Fig. 1—SDS-PAGE analyses of the proteins overexpressed in E. coli.

Supplementary Fig. 2—Cell growth curves of BL21(DE3) strains.

Author information

Authors and Affiliations

  1. Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China

    Hui-Min Qin, Songtao Li, Yu-Fu Zhang, Jian-Wen Wang, Jixuan Li, Shiyi Song, Fuping Lu & Yu Li

  2. Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China

    Hui-Min Qin, Songtao Li, Yu-Fu Zhang, Jian-Wen Wang, Jixuan Li, Shiyi Song, Fuping Lu & Yu Li

  3. National Engineering Laboratory for Industrial Enzymes, College of Biotechnology, Tianjin University of Science and Technology, No. 29 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China

    Hui-Min Qin, Songtao Li, Yu-Fu Zhang, Jian-Wen Wang, Jixuan Li, Shiyi Song, Fuping Lu & Yu Li

Authors
  1. Hui-Min Qin
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  2. Songtao Li
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  3. Yu-Fu Zhang
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  4. Jian-Wen Wang
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  5. Jixuan Li
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  7. Fuping Lu
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  8. Yu Li
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Corresponding author

Correspondence to Yu Li.

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Qin, HM., Li, S., Zhang, YF. et al. Multienzymatic cascade synthesis of fucosyloligosaccharide via a two-step fermentation strategy in Escherichia coli . Biotechnol Lett 38, 1747–1752 (2016). https://doi.org/10.1007/s10529-016-2151-y

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  • DOI: https://doi.org/10.1007/s10529-016-2151-y

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