Synthesis of 2′-fucosyllactose using multi-enzyme cascade with cofactor regeneration

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Synthesis of 2′-fucosyllactose using multi-enzyme cascade with cofactor regeneration

References

  1. Zhu, Y., Cao, H., Wang, H. & Mu, W. Biosynthesis of human milk oligosaccharides via metabolic engineering approaches: current advances and challenges. Curr. Opin. Biotechnol. 78, 102841. https://doi.org/10.1016/j.copbio.2022.102841 (2022).

    Google Scholar 

  2. Castanys-Muñoz, E., Martin, M. J. & Prieto, P. A. 2′-Fucosyllactose: an abundant, genetically determined soluble glycan present in human milk. Nutr. Rev. 71(12), 773–789. https://doi.org/10.1111/nure.12079 (2013).

    Google Scholar 

  3. Salminen, S., Stahl, B., Vinderola, G. & Szajewska, H. Infant formula supplemented with biotics: current knowledge and future perspectives. Nutrients 12(7), 1–20. https://doi.org/10.3390/nu12071952 (2020).

    Google Scholar 

  4. Anderson, A. & Donald, A. S. R. Improved method for the isolation of 2′-fucosyllactose from human milk. J. Chromatogr. A. 211, 170–174 (1981).

    Google Scholar 

  5. Zhu, Y. et al. Recent advances on 2′-fucosyllactose: physiological properties, applications, and production approaches. Crit. Rev. Food Sci. Nutr. 62(8), 2083–2092. https://doi.org/10.1080/10408398.2020.1850413 (2022).

    Google Scholar 

  6. Bych, K. et al. Production of HMOs using microbial hosts — from cell engineering to large scale production. Curr. Opin. Biotechnol. 56, 130–137. https://doi.org/10.1016/j.copbio.2018.11.003 (2019).

    Google Scholar 

  7. Zeuner, B., Teze, D., Muschiol, J. & Meyer, A. S. Synthesis of human milk oligosaccharides: protein engineering strategies for improved enzymatic transglycosylation. Molecules 24(11). https://doi.org/10.3390/molecules24112033 (2019).

  8. Li, M. et al. Semi-rationally designed site-saturation mutation of Helicobacter pylori α-1,2-fucosyltransferase for improved catalytic activity and thermostability. Int. J. Biol. Macromol. 259(P2), 129316. https://doi.org/10.1016/j.ijbiomac.2024.129316 (2024).

    Google Scholar 

  9. Li, C. et al. Efficient biosynthesis of 2′-fucosyllactose using an in vitro multienzyme cascade. J. Agric. Food Chem. 68(39), 10763–10771. https://doi.org/10.1021/acs.jafc.0c04221 (2020).

    Google Scholar 

  10. Agoston, K., Hederos, M. J., Bajza, I. & Dekany, G. Kilogram scale chemical synthesis of 2′-fucosyllactose. Carbohydr. Res. 476, 71–77. https://doi.org/10.1016/j.carres.2019.03.006 (2019).

    Google Scholar 

  11. Deng, J. et al. Engineering the substrate transport and cofactor regeneration systems for enhancing 2′-fucosyllactose synthesis in Bacillus subtilis. ACS Synth. Biol. 8(10), 2418–2427. https://doi.org/10.1021/acssynbio.9b00314 (2019).

    Google Scholar 

  12. You, R., Wang, L., Hu, M. & Tao, Y. Efficient production of 2′-fucosyllactose from fructose through metabolically engineered recombinant Escherichia coli. Microb. Cell. Fact. 23(1), 1–14. https://doi.org/10.1186/s12934-024-02312-5 (2024).

    Google Scholar 

  13. Hollands, K. et al. Engineering two species of yeast as cell factories for 2′-fucosyllactose. Metab. Eng. No. 52, 232–242. https://doi.org/10.1016/j.ymben.2018.12.005 (2018).

    Google Scholar 

  14. Lee, J. W. et al. Enhanced 2′-fucosyllactose production by engineered Saccharomyces cerevisiae using xylose as a co-substrate. Metab. Eng. 62, 322–329. https://doi.org/10.1016/j.ymben.2020.10.003 (2020).

    Google Scholar 

  15. Parschat, K., Schreiber, S., Wartenberg, D., Engels, B. & Jennewein, S. High-titer de novo biosynthesis of the predominant human milk oligosaccharide 2′-fucosyllactose from sucrose in Escherichia coli. ACS Synth. Biol. 9(10), 2784–2796. https://doi.org/10.1021/acssynbio.0c00304 (2020).

    Google Scholar 

  16. Chin, Y. W., Kim, J. Y., Lee, W. H. & Seo, J. H. Enhanced production of 2′-fucosyllactose in engineered Escherichia coli BL21star(DE3) by modulation of lactose metabolism and fucosyltransferase. J. Biotechnol. 210, 107–115. https://doi.org/10.1016/j.jbiotec.2015.06.431 (2015).

    Google Scholar 

  17. Chen, Q. et al. Engineering a colanic acid biosynthesis pathway in E. coli for manufacturing 2′-fucosyllactose. Process. Biochem. 94(99), 79–85. https://doi.org/10.1016/j.procbio.2020.04.017 (2020).

    Google Scholar 

  18. Zhu, Y. et al. Elimination of byproduct generation and enhancement of 2′-fucosyllactose synthesis by expressing a novel Α1,2-fucosyltransferase in engineered Escherichia coli. J. Agric. Food Chem. 71(12), 4915–4923. https://doi.org/10.1021/acs.jafc.3c00139 (2023).

    Google Scholar 

  19. Ebel, W. & Trempy, J. E. Escherichia coli RcsA, a positive activator of colanic acid capsular polysaccharide synthesis, functions to activate its own expression. J. Bacteriol. 181(2), 577–584. https://doi.org/10.1128/jb.181.2.577-584.1999 (1999).

    Google Scholar 

  20. Drouillard, S., Driguez, H. & Samain, E. Large-scale synthesis of H-antigen oligosaccharides by expressinghelicobacter pylori Α1,2-fucosyltransferase in metabolically engineered Escherichia coli cells. Angew Chemie. 118(11), 1810–1812. https://doi.org/10.1002/ange.200503427 (2006).

    Google Scholar 

  21. Liu, Y. et al. High-level de novo biosynthesis of 2′-fucosyllactose by metabolically engineered Escherichia coli. J. Agric. Food Chem. 70(29), 9017–9025. https://doi.org/10.1021/acs.jafc.2c02484 (2022).

    Google Scholar 

  22. Zhang, K., Gao, M., Cao, C. & Zhang, M. Intensification of pathway by using a novel fucosyltransferase from Bacillus cereus. No April. 1–9 https://doi.org/10.3389/fbioe.2025.1569597 (2025).

  23. Bornscheuer, U. T. et al. Engineering the third wave of biocatalysis. Nature 485(7397), 185–194. https://doi.org/10.1038/nature11117 (2012).

    Google Scholar 

  24. Zou, Z., Ji, Y. & Schwaneberg, U. Empowering Site-Specific bioconjugations in vitro and in vivo: advances in sortase engineering and sortase-Mediated ligation. Angew Chemie – Int. Ed. 63(12). https://doi.org/10.1002/anie.202310910 (2024).

  25. France, S. P., Hepworth, L. J., Turner, N. J. & Flitsch, S. L. Constructing biocatalytic cascades: in vitro and in vivo approaches to de novo multi-enzyme pathways. ACS Catal. 7(1), 710–724. https://doi.org/10.1021/acscatal.6b02979 (2017).

    Google Scholar 

  26. Li, M. et al. Multi-level metabolic engineering of Escherichia coli for high-titer biosynthesis of 2′-fucosyllactose and 3-fucosyllactose. Microb. Biotechnol. 15(12), 2970–2981. https://doi.org/10.1111/1751-7915.14152 (2022).

    Google Scholar 

  27. Wan, L. et al. Efficient production of 2′-fucosyllactose from l -fucose via self-assembling multienzyme complexes in engineered Escherichia coli. ACS Synth. Biol. 10(10), 2488–2498. https://doi.org/10.1021/acssynbio.1c00102 (2021).

    Google Scholar 

  28. Chen, Y. et al. De Novo biosynthesis of 2′-Fucosyllactose in a metabolically engineered Escherichia coli using a novel α1,2-Fucosyltransferase from azospirillum lipoferum. Bioresour Technol. 374(1800), 128818. https://doi.org/10.1016/j.biortech.2023.128818 (2023).

    Google Scholar 

  29. Ni, Z. et al. Multi-path optimization for efficient production of 2′-fucosyllactose in an engineered Escherichia coli C41 (DE3) derivative. Front. Bioeng. Biotechnol. 8, 1–13. https://doi.org/10.3389/fbioe.2020.611900 (2020).

    Google Scholar 

  30. Lee, W. H., Chin, Y. W., Han, N. S., Kim, M. D. & Seo, J. H. Enhanced production of GDP-l-fucose by overexpression of NADPH regenerator in recombinant Escherichia coli. Appl. Microbiol. Biotechnol. 91(4), 967–976. https://doi.org/10.1007/s00253-011-3271-x (2011).

    Google Scholar 

  31. Motomura, K. et al. New subfamily of polyphosphate kinase 2 (Class III PPK2) catalyzes both nucleoside monophosphate phosphorylation and nucleoside diphosphate phosphorylation Kei. Appl. Environ. Microbiol. 80(8), 2602–2608. https://doi.org/10.1128/AEM.03971-13 (2014).

    Google Scholar 

  32. Zhang, H., Ishige, K. & Kornberg, A. A. Polyphosphate kinase (PPK2) widely conserved in bacteria. Proc. Natl. Acad. Sci. 99(26), 16678–16683. https://doi.org/10.1073/pnas.262655199 (2002).

    Google Scholar 

  33. Ishige, K., Zhang, H. & Kornberg, A. Polyphosphate kinase (PPK2), a potent, polyphosphate-driven generator of GTP. Proc. Natl. Acad. Sci. 99(26), 16684–16688. https://doi.org/10.1073/pnas.262655299 (2002).

    Google Scholar 

  34. Cao, H. et al. Rational design of substrate binding pockets in polyphosphate kinase for use in cost-effective ATP-dependent cascade reactions. Appl. Microbiol. Biotechnol. 101(13), 5325–5332. https://doi.org/10.1007/s00253-017-8268-7 (2017).

    Google Scholar 

  35. Cao, H. et al. Enzymatic production of glutathione coupling with an ATP regeneration system based on polyphosphate kinase. Appl. Biochem. Biotechnol. 185(2), 385–395. https://doi.org/10.1007/s12010-017-2664-4 (2018).

    Google Scholar 

  36. Zhu, W. et al. The synthesis of mannose-6-Phosphate using Polyphosphate-Dependent mannose kinase. Catalysts 9 (3). https://doi.org/10.3390/catal9030250 (2019).

  37. Sun, H. et al. Enhanced thermal stability of polyphosphate-dependent glucomannokinase by directed evolution. Catalysts 12(10), 1112. https://doi.org/10.3390/catal12101112 (2022).

    Google Scholar 

  38. Liu, W. et al. Enhancing lactose recognition of a key enzyme in 2′-fucosyllactose synthesis: α-1,2-fucosyltransferase. J. Sci. Food Agric. 103(3), 1303–1314. https://doi.org/10.1002/jsfa.12224 (2023).

    Google Scholar 

  39. Shin, J. et al. Directed evolution of soluble α-1,2-fucosyltransferase using kanamycin resistance protein as a phenotypic reporter for efficient production of 2′-fucosyllactose. J. Microbiol. Biotechnol. 32(11), 1471–1478. https://doi.org/10.4014/jmb.2209.09018 (2022).

    Google Scholar 

  40. Peng, L. et al. Directed evolution of Α1,2-fucosyltransferase via a scalable high-throughput screening platform. Int. J. Biol. Macromol. 331(P1), 148408. https://doi.org/10.1016/j.ijbiomac.2025.148408 (2025).

    Google Scholar 

  41. Sturla, L. et al. Purification and characterization of GDP-D-mannose 4,6-dehydratase from Escherichia coli. FEBS Lett. 412(1), 126–130. https://doi.org/10.1016/S0014-5793(97)00762-X (1997).

    Google Scholar 

  42. Somoza, J. R. et al. Structural and kinetic analysis of Escherichia coli GDP-mannose 4, 6 dehydratase provides insights into the enzyme’s catalytic mechanism and regulation by GDP-fucose. Structure 8(2), 123–135. https://doi.org/10.1016/S0969-2126(00)00088-5 (2000).

    Google Scholar 

  43. Batten, L. E. et al. Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis. Biosci. Rep. 36(1), e00294. https://doi.org/10.1042/BSR20150203 (2015).

    Google Scholar 

  44. Sureka, K., Sanyal, S., Basu, J. & Kundu, M. P. Kinase 2: A modulator of nucleoside diphosphate kinase activity in mycobacteria. Mol. Microbiol. 74(5), 1187–1197. https://doi.org/10.1111/j.1365-2958.2009.06925.x (2009).

    Google Scholar 

  45. Chuang, Y., Belchis, D. A. & Karakousis, C. The polyphosphate kinase gene Ppk2 is required for Mycobacterium tuberculosis inorganic polyphosphate regulation and virulence. MBio. 4(3), e00039-13. https://doi.org/10.1128/mBio.00039-13.Invited (2013).

  46. Engels, L. & Elling, L. WbgL: A novel bacterial Α1,2-fucosyltransferase for the synthesis of 2′-fucosyllactose. Glycobiology 24(2), 170–178. https://doi.org/10.1093/glycob/cwt096 (2014).

    Google Scholar 

  47. Liu, J. J. et al. L-Fucose production by engineered Escherichia coli. Biotechnol. Bioeng. 116(4), 904–911. https://doi.org/10.1002/bit.26907 (2019).

    Google Scholar 

  48. Albermann, C., Piepersberg, W. & Wehmeier, U. F. Synthesis of the milk oligosaccharide 2′-fucosyllactose using recombinant bacterial enzymes. Carbohydr. Res. 334(2), 97–103. https://doi.org/10.1016/S0008-6215(01)00177-X (2001).

    Google Scholar 

  49. Mao, G., Valliere, M., Wu, J., Yu, O. & Johnson, S. R. U S Patent Application No 18/170,576. https://patents.google.com/patent/US20240093255A1/en (2024).

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