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Production of Hyaluronic Acid by Mutant Strains of Group C Streptococcus

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Abstract

This study addresses the influence of upstream region sequence on the strength of has operon promoter in highly encapsulated S. equi subsp. zooepidemicus (SEZ). For this purpose, seven different strains were constructed. Each strain carries a point mutation in one of the following positions upstream of the has promoter: −43, −44, −49, and −50 bp. To facilitate measuring of the recombinant promoter relative strength, ß-glucuronidase gene was used as a reporter gene. Three mutations located in positions −49 and −50: AT, GT, and AG, positively impacted has promoter strength when compared to the wild type sequence GG. Conversely, two other mutations: TG and TT, exhibited a slight inhibitory effect. Further, three different strains carrying chromosomal mutations in the has promoter region were constructed. In two cases, the has operon is under the control of a stronger promoter and in the third strain the has operon is controlled by a weaker promoter. The laboratory fermenter scale cultivations confirmed the increase of hyaluronan yields for SEZPhasAG and SEZPhas2G, resulting 116 and 105 %, respectively. As expected, the yield of the hyaluronic acid of SEZPhas2B strain fell to 41 %.

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Abbreviations

SEZ:

Streptococcus equi subsp. zooepidemicus

HA:

Hyaluronic acid

GlcUA:

Glucuronic acid

GlcNAc:

N-acetylglucosamine

GAS:

Group A streptococci

TT:

Transcriptional terminator

References

  1. Alberti, S., Ashbaugh, C. D., & Wessels, M. R. (1998). Structure of the has operon promoter and regulation of hyaluronic acid capsule expression in group A Streptococcus. Molecular Microbiology, 28, 343–353.

    Article  CAS  Google Scholar 

  2. Ausubel, F., Brent, M. R., Kingston, R. E., Moore, D. D., Siedman, J. G., Smith, J. A., et al. (1987). Current protocols in molecular biology. New York: Wiley.

    Google Scholar 

  3. Blank, L. M., Hugenholtz, P., & Nielsen, L. K. (2008). Evolution of the hyaluronic acid synthesis (has) operon in Streptococcus zooepidemicus and other pathogenic streptococci. Journal of Molecular Evolution, 67, 13–22.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Chen, W. Y., Marcellin, E., Hung, J., & Nielsen, L. K. (2009). Hyaluronan molecular weight is controlled by UDP-N-acetylglucosamine concentration in Streptococcus zooepidemicus. Journal of Biological Chemistry, 284(27), 18007–18014.

    Article  CAS  Google Scholar 

  6. Chien, L. J., & Lee, C. K. (2007). Enhanced hyaluronic acid production in Bacillus subtilis by coexpressing bacterial hemoglobin. Biotechnology Progress, 23(5), 1017–1022.

    CAS  Google Scholar 

  7. Chong, B. F., Blank, L. M., McLaughlin, R., & Nielsen, L. K. (2005). Microbial hyaluronic acid production. Applied Microbiology and Biotechnology, 66, 341–351.

    Article  CAS  Google Scholar 

  8. Crater, D. L., & van de Rijn, I. (1995). Hyaluronic acid synthesis operon (has) expression in group A streptococci. Journal of Biological Chemistry, 270, 18452–18458.

    Article  CAS  Google Scholar 

  9. DeAngelis, P. L., Jing, W., Graves, M. V., Burbank, D. E., & Van Etten, J. L. (1997). Hyaluronan synthase of Chlorella virus PBCV-1. Science, 278, 1800–1803.

    Article  CAS  Google Scholar 

  10. DeAngelis, P. L., Papaconstantinou, J., & Weigel, P. H. (1993). Molecular cloning, identification, and sequence of the hyaluronan synthase gene from group A Streptococcus pyogenes. Journal of Biological Chemistry, 268, 19181–19184.

    CAS  Google Scholar 

  11. Dougherty, B. A., & van de Rijn, I. (1993). Molecular characterization of hasB from an operon required for hyaluronic acid synthesis in group A streptococci. Journal of Biological Chemistry, 10, 7118–7124.

    Google Scholar 

  12. Dougherty, B. A., & van de Rijn, I. (1994). Molecular characterization of hasA from an operon required for hyaluronic acid synthesis in group A streptococci. Journal of Biological Chemistry, 269, 169–175.

    CAS  Google Scholar 

  13. Grafe, S., Ellinger, T., & Malke, H. (1996). Structural dissection and functional analysis of the complex promoter of the streptokinase gene from Streptococcus equisimilis H46A. Medical Microbiology and Immunology, 185, 11–17.

    Article  CAS  Google Scholar 

  14. Graves, M. V., Burbank, D. E., Roth, R., Heuser, J., DeAngelis, P. L., & Van Etten, J. L. (1999). Hyaluronan synthesis in virus PBCV-1 infected Chlorella-like green algae. Virology, 257, 15–23.

    Article  CAS  Google Scholar 

  15. Heldermon, C. D., Tlapak-Simmons, V. L., Baggenstoss, B. A., & Weigel, P. H. (2001). Site-direct mutation of conserved cysteine residues does not inactivate the Streptococcus pyogenes hyaluronan synthase. Glycobiology, 11, 1017–1024.

    Article  CAS  Google Scholar 

  16. Husman, L. K., Yung, D. L., Hollingshead, S. K., & Scott, J. R. (1997). Role of putative virulence of Streptococcus pyogenes in mouse models of long-term throat colonization and pneumonia. Infection and Immunity, 65, 1422–1430.

    Google Scholar 

  17. Johnson, D. R., Stevens, D. L., & Kaplan, E. L. (1992). Epidemiologic analysis of group A streptococcal serotypes associated with severe systemic infections, rheumatic fever, or uncomplicated pharyngitis. Journal of Infectious Diseases, 166, 374–382.

    Article  CAS  Google Scholar 

  18. Kendall, F., Heidelberger, M., & Dawson, M. (1937). A serologically inactive polysaccharide elaborated by mucoid strains of group A hemolytic Streptococcus. Journal of Biological Chemistry, 118, 61–69.

    CAS  Google Scholar 

  19. Kovacic, R. T. (1987). The 0°C closed complexes between Escherichia coli RNA polymerase and two promoters, T7–A3 and lacUV. Biological Chemistry, 262, 13654–13661.

    CAS  Google Scholar 

  20. Krahulec, J., & Krahulcová, J. (2006). Increase in hyaluronic acid production by Streptococcus equi subspecies zooepidemicus strain deficient in β-glucuronidase in laboratory conditions. Applied Microbiology and Biotechnology, 71(4), 415–422.

    Article  CAS  Google Scholar 

  21. Krahulec, J., & Krahulcová, J. (2007). Characterization of the new β-glucuronidase from Streptococcus equi subsp. zooepidemicus. Applied Microbiology and Biotechnology, 74(5), 1016–1022.

    Article  CAS  Google Scholar 

  22. Krahulec, J., Tlustá, M., Stuchlík, S., & Turňa, J. (2011). Structure of the has operon promoter and the effect of mutations on the has promoter strength in Streptococcus equi subsp. zooepidemicus. Molecular Biotechnology, 49(2), 166–175.

    Article  CAS  Google Scholar 

  23. Kumari, K., Baggenstoss, B. A., Parker, A. L., & Weigel, P. H. (2006). Mutation of two intramembrane polar residues conserved within the hyaluronan synthase family alters hyaluronan product size. Journal of Biological Chemistry, 281, 11755–11760.

    Article  CAS  Google Scholar 

  24. Kumari, K., Tlapak-Simmons, V. L., Baggenstoss, B. A., & Weigel, P. H. (2002). The streptococcal hyaluronan synthases are inhibited by sulfhydryl-modifying reagents, but conserved cysteine residues are not essential for enzyme function. Journal of Biological Chemistry, 277, 13943–13951.

    Article  CAS  Google Scholar 

  25. Landstein, D., Graves, M. V., Burbank, D. E., DeAngelis, P. L., & Van Etten, J. L. (1998). Chlorella virus PBCV-1 encodes functional glutamine: Fructose-6-phosphate amidotransferase and UDP-glucose dehydrogenase enzymes. Virology, 250, 388–396.

    Article  CAS  Google Scholar 

  26. Laurent, T. C., & Fraser, J. R. E. (1992). Hyaluronan. FASEB Journal, 6, 2397–2404.

    CAS  Google Scholar 

  27. Lee, J. Y., & Spicer, A. P. (2000). Hyaluronan: A multifunctional, megaDalton, stealth molecule. Current Opinion in Cell Biology, 12, 581–586.

    Article  CAS  Google Scholar 

  28. Marcellin, E., Chen, W. Y., & Nielsen, K. L. (2010). Understanding effect on hyaluronic acid molecular weight produced by Streptococcus equi subsp. zooepidemicus. Metabolic Engineering, 12, 62–69.

    Article  CAS  Google Scholar 

  29. McCure, P. M., & Wilson, W. D. (1989). Equine mastitis: A review of 28 cases. Equine Veterinary Journal, 21, 351–353.

    Article  Google Scholar 

  30. McIver, K. S., Heath, A. S., Green, B. D., & Scott, J. R. (1995). Specific binding of the activator Mga to promoter sequences of the emm and scpA genes in the group A streptococcus. Journal of Bacteriology, 177, 6619–6624.

    CAS  Google Scholar 

  31. Meyer, K. (1934). The polysaccharide of the vitreous humor. Journal of Biological Chemistry, 107, 629–634.

    CAS  Google Scholar 

  32. Morrison, D. A., & Jaurin, R. (1990). Streptococcus pneumoniae possesses canonical Escherichia coli (sigma 70) promoters. Molecular Microbiology, 4, 1143–1152.

    Article  CAS  Google Scholar 

  33. Moses, A. E., Wessels, M. R., Zalcman, K., Alberti, S., Natanson-Yaron, S., Menes, T., et al. (1997). Relative contributions of hyaluronic acid capsule and M protein to virulence in a mucoid strain of group A Streptococcus. Infection and Immunity, 65, 64–71.

    CAS  Google Scholar 

  34. Nimrod, A. (1998). Method of producing high molecular weight sodium hyaluronate by fermentation of streptococcus. US Patent, 4(780), 414.

    Google Scholar 

  35. O’Regan, M., Martini, I., Crescenzi, F., DeLuka, C., et al. (1994). Molecular mechanisms and genetics of hyaluronan biosynthesis. International Journal of Biological Macromolecules, 16, 283–286.

    Article  Google Scholar 

  36. Schickor, P., Metzger, W., Werel, W., Lederer, H., & Heumann, H. (1990). Topography of intermediates in transcription initiation in E. coli. EMBO Journal, 9, 2215–2220.

    CAS  Google Scholar 

  37. Schrager, H. M., Rheinwald, J. G., & Wessels, M. R. (1996). Hyaluronic acid capsule and the role of streptococcal entry into keratinocytes in invasive skin infection. Journal of Clinical Investigation, 98, 1954–1958.

    Article  CAS  Google Scholar 

  38. Stocks, S. M. & Brown, S. (2007). Production of low molecular weight hyaluronic acid. Patent WO2007/093179.

  39. Weigel, P. H. (2002). Functional characteristics and catalytic mechanisms of the bacterial hyaluronan synthases. IUBMB Life, 54, 201–211.

    Article  CAS  Google Scholar 

  40. Weigel, P. H., & DeAngelis, P. L. (2007). Hyaluronan synthases: A decade-plus of novel glycosyltransferases. Journal of Biological Chemistry, 282(51), 36777–36781.

    Article  CAS  Google Scholar 

  41. Weigel, P. H., Hascall, V. C., & Tammi, M. (1997). Hyaluronan synthases. Journal of Biological Chemistry, 272, 13997–14000.

    Article  CAS  Google Scholar 

  42. Weissman, B., & Meyer, K. (1954). The structure of hyalobiuronic acid and of hyaluronic acid from umbilical cord. Journal of the American Chemical Society, 76, 1753–1757.

    Article  Google Scholar 

  43. Wessels, M. R., & Bronze, M. S. (1994). Critical role of the group A streptococcal capsule in pharyngeal colonization and infection in mice. Proceedings of the National Academy of Sciences USA, 91, 12238–12242.

    Article  CAS  Google Scholar 

  44. Wessels, M. R., Moses, A. E., Goldberg, J. B., & DiCesare, T. J. (1991). Hyaluronic acid capsule is a virulence factor for mucoid group A streptococci. Proceedings of the National Academy of Sciences USA, 88, 8317–8321.

    Article  CAS  Google Scholar 

  45. Wibawan, I. W., Pasaribu, F. H., Utama, I. H., Abdulmawjood, A., & Lammler, C. (1999). The role of hyaluronic acid capsular material of Streptococcus equi subsp. zooepidemicus in mediating adherence to HeLa cells and in resisting phagocytosis. Research in Veterinary Science, 67, 131–135.

    Article  CAS  Google Scholar 

  46. Widner, B., Behr, R., Von Dollen, S., Tang, M., Heu, T., Sloma, A., et al. (2005). Hyaluronic acid production in Bacillus subtilis. Applied and Environment Microbiology, 71(7), 3747–3752.

    Article  CAS  Google Scholar 

  47. Widner, B., Thomas, M., Sternberg, D., Lammon, D., Behr, R., & Sloma, A. (2000). Development of marker-free strains of Bacillus subtilis capable of secreting high levels of industrial enzymes. Journal of Industrial Microbiology and Biotechnology, 25(2), 04–212.

    Google Scholar 

  48. Yamada, T., & Kawasaki, T. (2005). Microbial synthesis of hyaluronan and chitin: New approaches. Journal of Bioscience and Bioengineering, 99(6), 521–528.

    Article  CAS  Google Scholar 

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Acknowledgments

This study was supported by Contipro Biotech s r.o.

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Authors and Affiliations

  1. Contipro Biotech s r.o., Dolní Dobrouč 401, Czech Republic

    Marcela Tlustá, Stanislav Pepeliaev, Lukáš Franke, Zbyněk Černý & Jana Jílková

  2. Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina, 842 15, Bratislava 4, Slovak Republic

    Ján Krahulec

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  1. Marcela Tlustá
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Correspondence to Marcela Tlustá.

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Tlustá, M., Krahulec, J., Pepeliaev, S. et al. Production of Hyaluronic Acid by Mutant Strains of Group C Streptococcus . Mol Biotechnol 54, 747–755 (2013). https://doi.org/10.1007/s12033-012-9622-8

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