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Metabolic pathway of 3,6-anhydro-L-galactose in agar-degrading microorganisms

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Abstract

Recently, agarose-containing macroalgae have gained attention as possible renewable sources for bioethanol-production because of their high polysaccharide content. Complete hydrolysis of agarose produces two monomers, D-galactose (D-Gal) and 3,6-anhydro-L-galactose (L-AnG). However, at present, bioethanol yield from agarophyte macroalgae is low due to the inability of bioethanolproducing microorganisms to convert non-fermentable sugars, such as L-AnG, to bioethanol. Therefore, to increase the bioethanol productivity of agarophytes, it is necessary to determine how agar-degrading microorganisms metabolize L-AnG, and accordingly, construct recombinant microorganisms that can utilize both D-Gal and L-AnG. Previously, we isolated a novel microorganism belonging to a new genus, Postechiella marina M091, which hydrolyzes and metabolizes agar as the carbon and energy source. Here, we report a comparative genomic analysis of P. marina M091, Pseudoalteromonas atlantica T6c, and Streptomyces coelicolor A3(2), of the classes Flavobacteria, Gammaproteobacteria, and Actinobacteria, respectively. In this bioinformatic analysis of these agarolytic bacteria, we found candidate common genes that were believed to be involved in L-AnG metabolism. We then experimentally confirmed the enzymatic function of each gene product in the L-AnG cluster. The formation of two key intermediates, 2-keto-3-deoxy-L-galactonate and 2-keto-3-deoxy-D-gluconate, was also verified using enzymes that utilize these molecules as substrates. Combining bioinformatic analysis and experimental data, we showed that L-AnG is metabolized to pyruvate and D-glyceraldehyde-3-phosphate via six enzymecatalyzed reactions in the following reaction sequence: 3,6-anhydro-L-galactose → 3,6-anhydro-L-galactonate → 2-keto-3-deoxy-L-galactonate → 2,5-diketo-3-deoxy-L-galactonate → 2-keto-3-deoxy-D-gluconate → 2-keto-3-deoxy-6-phospho-D-gluconate → pyruvate + D-glyceraldehyde-3- phosphate. To our knowledge, this is the first report on the metabolic pathway of L-AnG degradation.

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References

  1. Roesijadi, G., S. B. Jones, L. J. Snowden-Swan, and Y. Zhu (2010) Macroalgae as A Biomass Feedstock: A Preliminary Analysis. Pacific Northwest National Laboratory.

    Book  Google Scholar 

  2. Meinita, M. D. N., B. Marhaeni, T. Winanto, G. T. Jeong, M. N. A. Khan, and Y. K. Hong (2013) Comparison of agarophytes (Gelidium, Gracilaria, and Gracilariopsis) as potential resources for bioethanol production. J. Appl. Phycol. 25: 1957–1961.

    Article  CAS  Google Scholar 

  3. Trivedi, N., V. Gupta, C. R. Reddy, and B. Jha (2013) Enzymatic hydrolysis and production of bioethanol from common macrophytic green alga Ulva fasciata Delile. Bioresour. Technol. 150: 106–112.

    Article  CAS  Google Scholar 

  4. Choi, W. Y., D.-H. Kang, and H.-Y. Lee (2013) Enhancement of the saccharification yields of Ulva pertusa Kjellmann and rape stems by the high-pressure steam pretreatment process. Biotechnol. Bioproc. Eng. 18: 728–735.

    Article  CAS  Google Scholar 

  5. Wargacki, A. J., E. Leonard, M. N. Win, D. D. Regitsky, C. N. Santos, P. B. Kim, S. R. Cooper, R. M. Raisner, A. Herman, A. B. Sivitz, A. Lakshmanaswamy, Y. Kashiyama, D. Baker, and Y. Yoshikuni (2012) An engineered microbial platform for direct biofuel production from brown macroalgae. Science 335: 308–313.

    Article  CAS  Google Scholar 

  6. Enquist-Newman, M., A. M. Faust, D. D. Bravo, C. N. Santos, R. M. Raisner, A. Hanel, P. Sarvabhowman, C. R. Le, D. D., S. R. Cooper, L. Peereboom, A. Clark, Y. Martinez, J. Goldsmith, M. Y. Cho, P. D. Donohoue, L. Luo, B. Lamberson, P. Tamrakar, E. J. Kim, J. L. Villari, A. Gill, S. A. Tripathi, P. Karamchedu, C. J. Paredes, V. Rajgarhia, H. K. Kotlar, R. B. Bailey, D. J. Miller, N. L. Ohler, C. Swimmer, and Y. Yoshikuni (2014) Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform. Nature 505: 239–243

    Article  CAS  Google Scholar 

  7. Ra, C. H. and S.-K. Kim (2013) Optimization of pretreatment conditions and use of a two-stage fermentation process for the production of ethanol from seaweed, Saccharina japonica. Biotechnol. Bioproc. Eng. 18: 715–720.

    Article  CAS  Google Scholar 

  8. Wei, N., J. Quarterman, and Y.-S. Jin (2013) Marine macroalgae: An untapped resource for producing fuels and chemicals. Trends Biotechnol. 31: 70–77.

    Article  CAS  Google Scholar 

  9. Hwang, H. J., S. Y. Lee, S. M. Kim, and S. B. Lee (2011) Fermentation of seaweed sugars by Lactobacillus species and the potential of seaweed as a biomass feedstock. Biotechnol. Bioproc. Eng. 16: 1231–1239.

    Article  CAS  Google Scholar 

  10. Cole, K. M. and R. G. Sheath (1990) Biology of the Red Algae. Cambridge University Press, NY, USA.

    Google Scholar 

  11. Araki, C. (1956) Structure of the agarose constituent of agar-agar. Bull. Chem. Soc. Japan 29: 543–544.

    Article  CAS  Google Scholar 

  12. Hamer, G. K., S. S. Bhattacharjee, and W. Yaphe (1977) Analysis of the enzymic hydrolysis products of agarose by 13C-n.m.r. spectroscopy. Carbohydr. Res. 54: C7–C10.

    Article  CAS  Google Scholar 

  13. Chi, W. J., Y. K. Chang, and S. K. Hong (2012) Agar degradation by microorganisms and agar-degrading enzymes. Appl. Microbiol. Biotechnol. 94: 917–930.

    Article  CAS  Google Scholar 

  14. Day, D. F. and W. Yaphe (1975) Enzymatic hydrolysis of agar: purification and characterization of neoagarobiose hydrolase and p-nitrophenyl α-galactoside hydrolase. Can. J. Microbiol. 21: 1512–1518.

    Article  CAS  Google Scholar 

  15. O’Nell, A. N. and D. K. R. Stewart (1956) On the structure of agar from Gelidium cartilaginium. Can. J. Chem. 34: 1700–1703.

    Article  Google Scholar 

  16. Siegel, B. Z. and S. M. Siegel (1973) The chemical composition of algal cell walls. Crit. Rev. Microbiol. 3: 1–26.

    Article  CAS  Google Scholar 

  17. Park, J. H., J. Y. Hong, H. C. Jang, S. G. Oh, S. H. Kim, J. J. Yoon, and Y. J. Kim (2012) Use of Gelidium amansii as a promising resource for bioethanol: a practical approach for continuous diluteacid hydrolysis and fermentation. Bioresour. Technol. 108: 83–88.

    Article  CAS  Google Scholar 

  18. Kumar, S., R. Gupta, G. Kumar, D. Sahoo, and R. C. Kuhad (2013) Bioethanol production from Gracilaria verrucosa, a red alga, in a biorefinery approach. Bioresour. Technol. 135: 150–156.

    Article  CAS  Google Scholar 

  19. Lee, D. H., S. J. Cho, S. M. Kim, and S. B. Lee (2012) Postechiella marina gen. nov., sp. nov., isolated from seawater. Int. J. Syst. Evol. Microbiol. 62: 1528–1535.

    Article  CAS  Google Scholar 

  20. Yaphe, W. (1957) The use of agarase from Pseudomonas atlantica in the identification of agar in marine algae (Rhodophyceae) Can. J. Microbiol. 3: 987–993.

    Article  CAS  Google Scholar 

  21. Morrice, L. M., M. W. McLean, F. B. Williamson, and W. F. Long (1983) β-Agarases I and II from Pseudomonas atlantica: Purifications and some properties. Eur. J. Biochem. 135: 553–558.

    Article  CAS  Google Scholar 

  22. Stanier, R. Y. (1942) Agar-decomposing strains of the Actinomyces coelicolor species-group. J. Bacteriol. 44: 555–570.

    CAS  Google Scholar 

  23. Kim, S. M., K. H. Paek, and S. B. Lee (2012) Characterization of NADP+-specific L-rhamnose dehydrogenase from the thermoacidophilic Archaeon Thermoplasma acidophilum. Extremophiles 16: 447–454

    Article  CAS  Google Scholar 

  24. Sambrook, J. and D. W. Russell (2001) Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

    Google Scholar 

  25. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.

    Article  CAS  Google Scholar 

  26. Sugano, Y., H. Kodama, I. Terada, Y. Yamazaki, and M. Noma (1994) Purification and characterization of a novel enzyme, a-neoagarooligosaccharide hydrolase (α-NAOS hydrolase), from a marine bacterium, Vibrio sp. strain JT0107. J. Bacteriol. 176: 6812–6818.

    CAS  Google Scholar 

  27. Duckworth, M. and W. Yaphe (1970) Thin-layer chromatographic analysis of enzymic hydrolysates of agar. J. Chromatogr. 49: 482–487.

    Article  CAS  Google Scholar 

  28. Skoza, L. and S. Mohos (1976) Stable thiobarbituric acid chromophore with dimethyl sulphoxide. Application to sialic acid assay in analytical de-O-acetylation. Biochem. J. 159: 457–462.

    CAS  Google Scholar 

  29. Kim, S. and S. B. Lee (2006) Characterization of Sulfolobus solfataricus 2-keto-3-deoxy-D-gluconate kinase in the modified Entner- Doudoroff pathway. Biosci. Biotechnol. Biochem. 70: 1308–1316.

    Article  CAS  Google Scholar 

  30. Noh, M., J. H. Jung, and S. B. Lee (2006) Purification and characterization of glycerate kinase from the thermoacidophilic archaeon Thermoplasma acidophium: an enzyme belonging to the second glycerate kinase family. Biotechnol. Bioproc. Eng. 11: 344–350.

    Article  CAS  Google Scholar 

  31. Buchanan, C. L., H. Connaris, M. J. Danson, C. D. Reeve, and D. W. Hough (1999) An extremely thermostable aldolase from Sulfolobus solfataricus with specificity for non-phosphorylated substrates. Biochem. J. 343: 563–570.

    Article  CAS  Google Scholar 

  32. Lim, S. H. (2008) Thermostable aldolases from Thermoplasma acidophilum. Master Thesis, POSTECH, Korea.

    Google Scholar 

  33. Karlin, S., J. Mrazek, A. Campbell, and D. Kaiser (2001) Characterizations of highly expressed genes of four fast-growing bacteria. J. Bacteriol. 183: 5025–5040.

    Article  CAS  Google Scholar 

  34. Kumar, S., M. Nei, J. Dudley, and K. Tamura (2008) MEGA: A biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform. 9: 299–306.

    Article  CAS  Google Scholar 

  35. Ducatti, D. R., A. Massi, M. D. Noseda, M. E. Duarte, and A. Dondoni (2009) Production of carbohydrate building blocks from red seaweed polysaccharides. Efficient conversion of galactans into C-glycosyl aldehydes. Org. Biomol. Chem. 7: 576–588.

    Article  CAS  Google Scholar 

  36. Kim, S. and S. B. Lee (2005) Identification and characterization of Sulfolobus solfataricus D-gluconate dehydratase: A key enzyme in the non-phosphorylated Entner-Doudoroff pathway. Biochem. J. 387: 271–280.

    Article  CAS  Google Scholar 

  37. Jung, J. H. and S. B. Lee (2006) Identification and characterization of Thermoplasma acidophilum glyceraldehyde dehydrogenase: A new class of NADP+-specific aldehyde dehydrogenase. Biochem. J. 397: 131–138.

    Article  CAS  Google Scholar 

  38. Jung, J. H. and S. B. Lee (2005) Identification and characterization of Thermoplasma acidophilum 2-keto-deoxy-D-gluconate kinase: a new class of sugar kinases. Biotechnol. Bioproc. Eng. 10: 535–539.

    Article  CAS  Google Scholar 

  39. Condemine, G., N. Hugouvieuxcottepattat, and J. Robert-Baudouy (1984) An enzyme in the pectinolytic pathway of Erwinia chrysanthemi: 2-keto-3-deoxygluconate oxidoreductase. J. Gen. Microbiol. 130: 2839–2844.

    CAS  Google Scholar 

  40. Petsko, G. A. and D. Ringe (2004) Protein Structure and Function. p. 136. New Science Press, London.

    Google Scholar 

  41. Entner, E. and M. Doudoroff (1952) Glucose and gluconic acid oxidation of Pseudomonas saccharophila. J. Biol. Chem. 196: 853–862.

    CAS  Google Scholar 

  42. Peekhaus N. and T. Conway (1998) What’s for dinner?: Entner- Doudoroff metabolism in Escherichia coli. J. Bacteriol. 180: 3495–3502.

    CAS  Google Scholar 

  43. Kim, S. and S. B. Lee (2008) Identification and characterization of the bacterial D-gluconate dehydratase in Achromobacter xylosoxidans. Biotechnol. Bioproc. Eng. 13: 436–444.

    Article  CAS  Google Scholar 

  44. Andberg, M., H. Maaheimo, H. Boer, M. Penttilä, A. Koivula, and P. Richard (2012) Characterization of a novel Agrobacterium tumefaciens galactarolactone cycloisomerase enzyme for direct conversion of D-galactarolactone to 3-deoxy-2-keto-L-threohexarate. J. Biol. Chem. 287: 17662–17671.

    Article  CAS  Google Scholar 

  45. Hilditch, S., S. Berghall, N. Kalkkinen, M. Penttila, and P. Richard (2007) The missing link in the fungal D-galacturonate pathway: Identification of the L-threo-3-deoxy-hexulosonate aldolase. J. Biol. Chem. 282: 26195–26201.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, 790-784, Korea

    Sun Bok Lee, Sun Ja Cho, Jeong Ah Kim, Shin Youp Lee, Suk Min Kim & Hyun Seung Lim

  2. Graduate School of Engineering Mastership, Pohang University of Science and Technology, Pohang, 790-784, Korea

    Sun Bok Lee

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  1. Sun Bok Lee
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Correspondence to Sun Bok Lee.

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Lee, S.B., Cho, S.J., Kim, J.A. et al. Metabolic pathway of 3,6-anhydro-L-galactose in agar-degrading microorganisms. Biotechnol Bioproc E 19, 866–878 (2014). https://doi.org/10.1007/s12257-014-0622-3

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  • DOI: https://doi.org/10.1007/s12257-014-0622-3

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