Applied Microbiology and Biotechnology

, Volume 66, Issue 4, pp 341–351 | Cite as

Microbial hyaluronic acid production

  • Barrie Fong Chong
  • Lars M. Blank
  • Richard Mclaughlin
  • Lars K. NielsenEmail author


Hyaluronic acid (HA) is a commercially valuable medical biopolymer increasingly produced through microbial fermentation. Viscosity limits product yield and the focus of research and development has been on improving the key quality parameters, purity and molecular weight. Traditional strain and process optimisation has yielded significant improvements, but appears to have reached a limit. Metabolic engineering is providing new opportunities and HA produced in a heterologous host is about to enter the market. In order to realise the full potential of metabolic engineering, however, greater understanding of the mechanisms underlying chain termination is required.


Hyaluronic Acid Lactic Acid Bacterium Stress Urinary Incontinence Metabolic Engineering Chemically Define Medium 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Abbe K, Takahashi S, Yamada T (1982) Involvement of oxygen-sensitive pyruvate-formate-lyase in mixed acid fermentation by Streptococcus mutans under strictly anaerobic conditions. J Bacteriol 152:175–182PubMedGoogle Scholar
  2. Armstrong DC (1997) The molecular weight properties of hyaluronic acid produced Streptococcus zooepidemicus. PhD. Thesis, University of QueenslandGoogle Scholar
  3. Armstrong DC, Johns MR (1997) Culture conditions affect the molecular weight properties of hyaluronic acid produced by Streptococcus zooepidemicus. Appl Environ Microbiol 63:2759–2764Google Scholar
  4. Asari A, Miyauchi S (2000) Medical applications of Hyaluronan. Glycoforum - Hyaluronan Today.
  5. Ashbaugh CD, Alberti S, Wessels MR (1998) Molecular analysis of the capsule gene region of group A streptococcus: the hasAB genes are sufficient for capsule expression. J Bacteriol 180:4955–4959PubMedGoogle Scholar
  6. Balazs EA, Leshchiner E, Larsen NE, Band P (1993) Application of hyaluronan and its derivatives. In: Gebelein CG (ed) Biotechnological polymers. Technomic, Lancaster, pp 41–65Google Scholar
  7. Cleary PP, Larkin A (1979) Hyaluronic acid capsule: strategy for oxygen resistance in group A streptococci. J Bacteriol 140:1090–1097PubMedGoogle Scholar
  8. Cooney MJ, Goh LT, Lee PL, Johns MR (1999) Structured model-based analysis and control of the hyaluronic acid fermentation by Streptococcus zooepidemicus: Physiological implications of glucose and complex nitrogen-limited growth. Biotechnol Prog 15:898–910PubMedGoogle Scholar
  9. Crater DL, Dougherty BA, van de Rijn I (1995) Molecular characterization of hasC from an operon required for hyaluronic acid synthesis in group A streptococci. Demonstration of UDP- glucose pyrophosphorylase activity. J Biol Chem 270:28676–28680PubMedGoogle Scholar
  10. Cywes C, Wessels MR (2001) Group A Streptococcus tissue invasion by CD44-mediated cell signalling. Nature 414:648–652CrossRefPubMedGoogle Scholar
  11. DeAngelis PL, Papaconstantinou J, Weigel PH (1993a) Isolation of a Streptococcus-Pyogenes gene locus that directs hyaluronan biosynthesis in acapsular mutants and in heterologous bacteria. J Biol Chem 268:14568–14571PubMedGoogle Scholar
  12. DeAngelis PL, Papaconstantinou J, Weigel PH (1993b) Molecular cloning, identification, and sequence of the hyaluronan synthase gene from group A Streptococcus pyogenes. J Biol Chem 268:19181–19184PubMedGoogle Scholar
  13. DeAngelis PL, Weigel PH (1994) Immunochemical confirmation of the primary structure of streptococcal hyaluronan synthase and synthesis of high molecular weight product by the recombinant enzyme. Biochemistry 33:9033–9039PubMedGoogle Scholar
  14. de Ruyter P, Kuipers OP, de Vos WM (1996) Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol 62:3662–3667PubMedGoogle Scholar
  15. Dougherty BA, van de Rijn I (1993) Molecular characterization of hasB from an operon required for hyaluronic acid synthesis in group A streptococci. Demonstration of UDP- glucose dehydrogenase activity. J Biol Chem 268:7118–7124PubMedGoogle Scholar
  16. Dougherty BA, van de Rijn I (1994) Molecular characterization of hasA from an operon required for hyaluronic acid synthesis in group A streptococci. J Biol Chem 269:169–175PubMedGoogle Scholar
  17. Ellwood DC, Evans CGT, Dunn GM, McInnes N et al (1995) Production of hyaluronic acid. US Patent 5411874Google Scholar
  18. Ellwood DC, Evans CGT, Dunn GM, McInnes N et al (1996) Production of hyaluronic acid. US Patent 5411874Google Scholar
  19. Fong Chong B (2002) Improving the cellular economy of Streptococcus zooepidemicus through metabolic engineering. PhD Thesis, The University of QueenslandGoogle Scholar
  20. Fong Chong B, Nielsen LK (2003a) Aerobic cultivation of Streptococcus zooepidemicus and the role of NADH oxidase. Biochem Eng J 16:153–162CrossRefGoogle Scholar
  21. Fong Chong B, Nielsen LK (2003b) Amplifying the cellular reduction potential of Streptococcus zooepidemicus. J Biotechnol 100:33–41CrossRefPubMedGoogle Scholar
  22. Forsee WT, Cartee RT, Yother J (2000) Biosynthesis of type 3 capsular polysaccharide in Streptococcus pneumoniae—enzymatic chain release by an abortive translocation process. J Biol Chem 275:25972–25978CrossRefPubMedGoogle Scholar
  23. Fouissac E, Milas M, Rinaudo M (1993) Shear rate, concentration, molecular weight and temperature viscosity dependences of hyaluronate, a wormlike polyelectrolytes. Macromolecules 26:6945–6951Google Scholar
  24. Goh L-T (1998) Effect of culture conditions on rates of intrinsic hyaluronic acid production by Streptococcusequi subsp. zooepidemicus. PhD Thesis. University of QueenslandGoogle Scholar
  25. Heldermon C, DeAngelis PL, Weigel PH (2001) Topological organization of the hyaluronan synthase from Streptococcus pyogenes. J Biol Chem 276:2037–2046CrossRefPubMedGoogle Scholar
  26. Heldermon C, Kumari K, Tlapak-Simmons V, Weigel PH (2000) Streptococcal hyaluronan synthases and the synthesis of “designer” hyaluronan. In: Abatangelo G, Weigel PH (eds) New frontiers in medical sciences: redefining hyaluronan. Elsevier, New YorkGoogle Scholar
  27. Jacques M, Graham L (1989) Improved preservation of bacterial capsule for electron microscopy. J Electron Microsc Tech 11:167–169PubMedGoogle Scholar
  28. Johns MR, Goh LT, Oeggerli A (1994) Effect of pH, agitation and aeration on hyaluronic-acid production by Streptococcus-Zooepidemicus. Biotechnol Lett 16:507–512Google Scholar
  29. Kim JH, Yoo SJ, Oh DK, Kweon YG et al. (1996) Selection of a Streptococcus equi mutant and optimization of culture conditions for the production of high molecular weight hyaluronic acid. Enzyme Microb Technol 19:440–445CrossRefGoogle Scholar
  30. Kitchen JR, Cysyk RL (1995) Synthesis and release of hyaluronic acid by Swiss 3T3 fibroblasts. Biochem J 309:649–656PubMedGoogle Scholar
  31. Kumari K, Weigel PH (1997) Molecular cloning, expression, and characterization of the authentic hyaluronan synthase from group C Streptococcus equisimilis. J Biol Chem 272:32539–32546CrossRefPubMedGoogle Scholar
  32. Lander ES, Linton LM, Birren B, Nusbaum C et al. (2001) Initial sequencing and analysis of the human genome. Nature 409:860–921CrossRefPubMedGoogle Scholar
  33. Leonard BA, Woischnik M, Podbielski A (1998) Production of stabilized virulence factor-negative variants by group A streptococci during stationary phase. Infect Immun 66:3841–3847PubMedGoogle Scholar
  34. Lertwerawat Y (1993) Hyaluronic acid production and its instability in Streptococcus zooepidemicus. PhD Thesis. University of QueenslandGoogle Scholar
  35. Maguin E, Prevost H, Ehrlich SD, Gruss A (1996) Efficient insertional mutagenesis in lactococci and other Gram-positive bacteria. J Bacteriol 178:931–935PubMedGoogle Scholar
  36. Matsubara C, Kajiwara M, Akasaka H, Haze S (1991) Carbon-13 nuclear magnetic resonance studies on the biosynthesis of hyaluronic acid. Chem Pharm Bull 39:2446–2448Google Scholar
  37. Mausolf A, Jungmann J, Robenek H, Prehm P (1990) Shedding of hyaluronate synthase from streptococci. Biochem J 267:191–196PubMedGoogle Scholar
  38. Meyer K, Palmer JW (1934) The polysaccharide of the vitreous humor. J Biol Chem 107:629–634Google Scholar
  39. O’Regan M, Martini I, Crescenzi F, De Luca C et al (1994) Molecular Mechanisms and genetics of hyaluron Biosynthesis. Int J Biol Macromol 16:283–286CrossRefPubMedGoogle Scholar
  40. Praest BM, Helmut G, Rudiger K (1997) Effects of oxygen-derived free radicals on the molecular weight and the polydispersity of hyaluronan solutions. Carbohydr Res 303:153–157CrossRefGoogle Scholar
  41. Prehm P (1984) Hyaluronate is synthesised at plasma membrane. Biochem J 220:597–600PubMedGoogle Scholar
  42. Saettone MF, Giannaccini B, Chetoni P, Torracca MT et al (1991) Evaluation of high-molecular-weight and low-molecular-weight fractions of sodium hyaluronate and an ionic complex as adjuvants for topical ophthalmic vehicles containing pilocarpine. Int J Pharm 72:131–139Google Scholar
  43. Sakamoto M, Komagata K (1996) Aerobic growth of and activities of NADH oxidase and NADH peroxidase in lactic acid bacteria. J Ferment Bioeng 82:210–216Google Scholar
  44. Salzberg SL, White O, Peterson J, Eisen JA (2001) Microbial genes in the human genome: lateral transfer or gene loss? Science 292:1903–1906PubMedGoogle Scholar
  45. Saso L, Bonanni G, Grippa E, Gatto MT et al (1999) Interaction of hyaluronic acid with mucin, evaluated by gel permeation chromatography. Res Commun Mol Pathol Pharmacol 104:277–284PubMedGoogle Scholar
  46. Schmidt KH, Gunther E, Courtney HS (1996) Expression of both M protein and hyaluronic acid capsule by group A streptococcal strains results in a high virulence for chicken embryos. Med Microbiol Immunol 184:169–173PubMedGoogle Scholar
  47. Scott JE, Heatley F (1999) Hyaluronan forms specific stable tertiary structures in aqueous solution: a C-13 NMR study. Proc Natl Acad Sci USA 96:4850–4855PubMedGoogle Scholar
  48. Snoep JL, de Graef MR, de Mattos MJT, Neijssel OM (1992) Pyruvate catabolism during transient state conditions in chemostat cultures of Enterococcus faecalis NCTC 775: importance of internal pyruvate concentrations and NADH/NAD+ ratios. J Gen Microbiol 138:2015–2020PubMedGoogle Scholar
  49. Spurlock SL, Spurlock GH, Bernstad S, Michanek P et al. (1999) Treatment of acute superficial flexor tendon injuries in performance horses with high molecular weight sodium hyaluronate. J Equine Vet Sci 19:338–344Google Scholar
  50. Stangohl S (2000) Methods and means for the production of hyaluronic acid. US Patent 6090596Google Scholar
  51. Stangohl S (2003) Methods and means for the production of hyaluronic acid. US Patent 6537795Google Scholar
  52. Suzuki Y, Yamaguchi T (1993) Effect of hyaluronic acid on macrophage phagacytosis and active oxygen release. Agents Actions 38:32–37PubMedGoogle Scholar
  53. Thomas EL, Pera KA (1983) Oxygen metabolism of Streptococcus mutans: uptake of oxygen and release of superoxide and hydrogen peroxide. J Bacteriol 154:1236–1244PubMedGoogle Scholar
  54. Thomas TD, Ellwood DC, Longyear VMC (1979) Change from homo- to heterolactic fermentation by Streptococcus lactis resulting from glucose limitation in anaerobic chemostat cultures. J Bacteriol 138:109–117PubMedGoogle Scholar
  55. Tlapak-Simmons VL, Baggenstoss BA, Clyne T, Weigel PH (1999a) Purification and lipid dependence of the recombinant hyaluronan synthases from Streptococcus pyogenes and Streptococcus equisimilis. J Biol Chem 274:4239–4245CrossRefPubMedGoogle Scholar
  56. Tlapak-Simmons VL, Baggenstoss BA, Kumari K, Heldermon C et al. (1999b) Kinetic characterization of the recombinant hyaluronan synthases from Streptococcus pyogenes and Streptococcus equisimilis. J Biol Chem 274:4246–4253CrossRefPubMedGoogle Scholar
  57. Tlapak-Simmons VL, Kempner ES, Baggenstoss BA, Weigel PH (1998) The active streptococcal hyaluronan synthases (HASs) contain a single HAS monomer and multiple cardiolipin molecules. J Biol Chem 273:26100–26109CrossRefPubMedGoogle Scholar
  58. van de Rijn I, Kessler RE (1980) Growth characteristics of group A streptococci in a new chemically defined medium. Infect Immun 27:444–448PubMedGoogle Scholar
  59. Ward PN, Field TR, Ditcham WGF, Maguin E et al (2001) Identification and disruption of two discrete loci encoding hyaluronic acid capsule biosynthesis genes hasA, hasB, and hasC in Streptococcus uberis. Infect Immun 69:392–399CrossRefPubMedGoogle Scholar
  60. Weigel PH (2002) Functional characteristics and catalytic mechanisms of the bacterial hyaluronan synthases. IUBMB Life 54:201–211PubMedGoogle Scholar
  61. Weigel PH, Hascall VC, Tammi M (1997) Hyaluronan synthases. J Biol Chem 272:13997–14000CrossRefPubMedGoogle Scholar
  62. Weissman B, Meyer K (1954) The structure of hyalobiuronic acid and of hyaluronic acid from umbilical cord. J Am Chem Soc 76:1753–1757Google Scholar
  63. Wessels MR, Moses A, Goldberg JB, DiCesare TJ (1991) Hyaluronic acid capsule is a virulence factor for mucoid group A streptococci. Proc Natl Acad Sci USA 88:8317–8321PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Barrie Fong Chong
    • 1
  • Lars M. Blank
    • 1
  • Richard Mclaughlin
    • 1
  • Lars K. Nielsen
    • 1
    Email author
  1. 1.Department of Chemical EngineeringThe University of QueenslandBrisbaneAustralia

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