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Metabolic engineering of Corynebacterium glutamicum to produce GDP-l-fucose from glucose and mannose

Abstract

Wild-type Corynebacterium glutamicum was metabolically engineered to convert glucose and mannose into guanosine 5′-diphosphate (GDP)-l-fucose, a precursor of fucosyl-oligosaccharides, which are involved in various biological and pathological functions. This was done by introducing the gmd and wcaG genes of Escherichia coli encoding GDP-d-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase-4-reductase, respectively, which are known as key enzymes in the production of GDP-l-fucose from GDP-d-mannose. Coexpression of the genes allowed the recombinant C. glutamicum cells to produce GDP-l-fucose in a minimal medium containing glucose and mannose as carbon sources. The specific product formation rate was much higher during growth on mannose than on glucose. In addition, the specific product formation rate was further increased by coexpressing the endogenous phosphomanno-mutase gene (manB) and GTP-mannose-1-phosphate guanylyl-transferase gene (manC), which are involved in the conversion of mannose-6-phosphate into GDP-d-mannose. However, the overexpression of manA encoding mannose-6-phosphate isomerase, catalyzing interconversion of mannose-6-phosphate and fructose-6-phosphate showed a negative effect on formation of the target product. Overall, coexpression of gmd, wcaG, manB and manC in C. glutamicum enabled production of GDP-l-fucose at the specific rate of 0.11 mg g cell−1 h−1. The specific GDP-l-fucose content reached 5.5 mg g cell−1, which is a 2.4-fold higher than that of the recombinant E. coli overexpressing gmd, wcaG, manB and manC under comparable conditions. Well-established metabolic engineering tools may permit optimization of the carbon and cofactor metabolisms of C. glutamicum to further improve their production capacity.

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References

  1. 1.

    Kunz C, Rudloff S (1993) Biological functions of oligosaccharides in human milk. Acta Paediatr 82:903

  2. 2.

    Bode L (2006) Recent advances on structure, metabolism, and function of human milk oligosaccharides. J Nutr 136(8):2127

  3. 3.

    Boehm G, Stahl B (2007) Oligosaccharides from milk. J Nutr 137(3):847S–849S

  4. 4.

    Han NS, Kim TJ, Park YC, Kim J, Seo JH (2012) Biotechnological production of human milk oligosaccharides. Biotechnol Adv 30(6):1268–1278

  5. 5.

    Albermann C, Distler J, Piepersberg W (2000) Preparative synthesis of GDP-β-l-fucose by recombinant enzymes from enterobacterial sources. Glycobiology 10(9):875–881

  6. 6.

    Bulter T, Elling L (1999) Enzymatic synthesis of nucleotide sugars. Glycoconjugate J 16(2):147–159

  7. 7.

    Lee W, Han N, Park Y, Seo J (2009) Modulation of guanosine 5′-diphosphate-d-mannose metabolism in recombinant Escherichia coli for production of guanosine 5′-diphosphate-l-fucose. Bioresource Technol 100(24):6143–6148

  8. 8.

    Lee WH, Shin SY, Kim MD, Han NS, Seo JH (2011) Modulation of guanosine nucleotides biosynthetic pathways enhanced GDP-l-fucose production in recombinant Escherichia coli. Appl Microbiol Biotechnol 93:2327–2334

  9. 9.

    Koizumi S, Endo T, Tabata K, Nagano H, Ohnishi J, Ozaki A (2000) Large-scale production of GDP-fucose and Lewis X by bacterial coupling. J Industrial Microbiol Biotechnol 25(4):213–217

  10. 10.

    Demain A, Jackson M, Vitali R, Hendlin D, Jacob T (1966) Production of guanosine-5′-monophosphate and inosine-5′-monophosphate by fermentation. Appl Environ Microbiol 14(5):821–825

  11. 11.

    Furuya A, Okachi R, Takayama K, Abe S (1973) Accumulation of 5′-guanine nucleotides by mutants of Brevibacterium ammoniagenes. Biotechnol Bioeng 15(4):795–803

  12. 12.

    Eggeling L, Bott M (2005) Handbook of Corynebacterium glutamicum. CRC press, Boca Raton

  13. 13.

    Doo EH, Lee WH, Seo HS, Seo JH, Park JB (2009) Productivity of cyclohexanone oxidation of the recombinant Corynebacterium glutamicum expressing chnB of Acinetobacter calcoaceticus. J Biotechnol 142(2):164–169

  14. 14.

    Marx A, De Graaf A, Wiechert W, Eggeling L, Sahm H (1996) Determination of the fluxes in central metabolism of Corynebacterium glutamicum by NMR spectroscopy combined with metabolite balancing. Biotechnol Bioeng 49:111–129

  15. 15.

    Baumchen C, Krings E, Bringer S, Eggeling L, Sahm H (2008) Myo-inositol facilitators IolT1 and IolT2 enhance d-mannitol formation from d-fructose in Corynebacterium glutamicum. FEMS Microbiol Lett 290(2):227–235

  16. 16.

    Keilhauer C, Eggeling L, Sahm H (1993) Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon. J Bacteriol 175(17):5595–5603

  17. 17.

    Gerstmeir R, Cramer A, Dangel P, Schaffer S, Eikmanns B (2004) RamB, a novel transcriptional regulator of genes involved in acetate metabolism of Corynebacterium glutamicum. J Bacteriol 186(9):2798–2809

  18. 18.

    Sigal N, Gorzalczany Y, Sarfstein R, Weinbaum C, Zheng Y, Pick E (2003) The guanine nucleotide exchange factor trio activates the phagocyte NADPH oxidase in the absence of GDP to GTP exchange on Rac. “THE EMPEROR’S NEW CLOTHES”. J Biol Chem 278(7):4854–4861

  19. 19.

    Sasaki M, Teramoto H, Inui M, Yukawa H (2011) Identification of mannose uptake and catabolism genes in Corynebacterium glutamicum and genetic engineering for simultaneous utilization of mannose and glucose. Appl Microbiol Biotechnol 89:1905–1916

  20. 20.

    Padgett PJ, Phibbs PV (1986) Phosphomannomutase activity in wild-type and alginate-producing strains of Pseudomonas aeruginosa. Curr Microbiol 14(4):187–192

  21. 21.

    Dover LG, Cerdeño-Tárraga AM, Pallen MJ, Parkhill J, Besra GS (2004) Comparative cell wall core biosynthesis in the mycolated pathogens, Mycobacterium tuberculosis and Corynebacterium diphtheriae. FEMS Microbiol Rev 28(2):225–250

  22. 22.

    Mishra A, Alderwick L, Rittmann D, Wang C, Bhatt A, Jacobs W Jr, Takayama K, Eggeling L, Besra G (2008) Identification of a novel α(1 → 6) mannopyranosyltransferase MptB from Corynebacterium glutamicum by deletion of a conserved gene, NCgl1505, affords a lipomannan-and lipoarabinomannan-deficient mutant. Mol Microbiol 68(6):1595–1613

  23. 23.

    Mishra A, Klein C, Gurcha S, Alderwick L, Babu P, Hitchen P, Morris H, Dell A, Besra G, Eggeling L (2008) Structural characterization and functional properties of a novel lipomannan variant isolated from a Corynebacterium glutamicum pimB’ mutant. Anton Leeuw Int J G 94(2):277–287

  24. 24.

    Korduláková J, Gilleron M, Mikus͂ová K, Puzo G, Brennan PJ, Gicquel B, Jackson M (2002) Definition of the first mannosylation step in phosphatidylinositol mannoside synthesis. J Biol Chem 277(35):31335–31344

  25. 25.

    Schaeffer ML, Khoo KH, Besra GS, Chatterjee D, Brennan PJ, Belisle JT, Inamine JM (1999) The pimB gene of Mycobacterium tuberculosis encodes a mannosyltransferase involved in lipoarabinomannan biosynthesis. J Biol Chem 274(44):31625–31631

  26. 26.

    Kim S, Yun J, Kim S, Seo J, Park J (2010) Production of xylitol from d-xylose and glucose with recombinant Corynebacterium glutamicum. Enzyme Microb Tech 46(5):366–371

  27. 27.

    Eikmanns B, Kleinertz E, Liebl W, Sahm H (1991) A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing. Gene 102(1):93–98

  28. 28.

    Wendisch V (1997) Physiologische und NMR-spektroskopische Untersuchungen zur in vivo-Aktivität zentraler Stoffwechselwege im Wildstamm und in rekombinanten Stämmen von Corynebacterium glutamicum. PhD thesis, Düsseldorf University and Research Centre, Jülich, Germany

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Acknowledgments

This work was supported by the Advanced Biomass R and D Center (ABC) of Korea Grant funded by the Ministry of Education, Science and Technology (2011-0031359). This work was also supported by a grant from Marine Biotechnology Program Funded by Ministry of Land, Transport and Maritime Affairs of Korean Government.

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Correspondence to Jin-Ho Seo.

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Chin, Y., Park, J., Park, Y. et al. Metabolic engineering of Corynebacterium glutamicum to produce GDP-l-fucose from glucose and mannose. Bioprocess Biosyst Eng 36, 749–756 (2013). https://doi.org/10.1007/s00449-013-0900-z

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Keywords

  • GDP-l-fucose
  • GDP-d-mannose
  • Corynebacterium glutamicum
  • Guanosine nucleotide