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Improved Yield of High Molecular Weight Hyaluronic Acid Production in a Stable Strain of Streptococcus zooepidemicus via the Elimination of the Hyaluronidase-Encoding Gene

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Abstract

Despite the significant potential of Streptococcus zooepidemicus for hyaluronic acid (HA) production with high molecular weight (MW), the HA degrading properties of hyaluronidase prevents the bacteria to achieve enhanced HA yield with high MW. In the present study, we aim to knockout the hyaluronidase enzyme and assess its effects on the yield and MW of the produced HA. The kanamycin resistance gene between the left and right arms of hyaluronidase gene was inserted into pUC18 plasmid to construct pUC18-L-kanar-R as a recombinant suicide plasmid. The construct was then transferred into S. zooepidemicus to induce the homologous recombination between the hyaluronidase gene and the kanamycin resistance gene. Gene deletion was confirmed by PCR and enzyme assay. The product was cultured on selectable medium in which the MW of HA was increased from 1.5 to 3.8 MDa. The yield of HA production using the mutant strain was higher in all different concentrations of glucose from 40 to 120 g/l. Moreover, glucose increase results in higher HA production within both wild-type and recombinant strains. However, the growth rate of HA concentration (the slope of the plot), as a consequence of increased glucose concentration, is always higher for the recombinant strain. Unlike the wild-type strain, there was no sharp HA production drop approaching the 6 g/l HA concentration. In conclusion, hyaluronidase activity and HA concentration and MW exhibited a mutual control on each other. Based on our results, deletion of the hyaluronidase gene positively affects the yield and MW of HA.

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References

  1. Meyer, K., & Palmer, J. W. (1934). The polysaccharide of the vitreous humor. Journal of Biological Chemistry, 107(3), 629–634.

    CAS  Google Scholar 

  2. Balazs, E. A., Leshchiner, E., Larsen, N. E., & Band, P. (1993). Application of hyaluronan and its derivatives. Biotechnological polymers. Technomic, Lancaster, 41–65.

  3. Burdick, J. A., & Prestwich, G. D. (2011). Hyaluronic acid hydrogels for biomedical applications. Advanced Materials, 23(12), 41–56. doi:10.1002/adma.201003963.

    Article  Google Scholar 

  4. Choh, S. Y., Cross, D., & Wang, C. (2011). Facile synthesis and characterization of disulfide-cross-linked hyaluronic acid hydrogels for protein delivery and cell encapsulation. Biomacromolecules, 12, 1126–1136. doi:10.1021/bm101451k.

    Article  CAS  Google Scholar 

  5. Kablik, J., Monheit, G. D., Yu, L., Chang, G., & Gershkovich, J. (2009). Comparative physical properties of hyaluronic acid dermal fillers. Dermatologic Surgery, 35(SUPPL. 1), 302–312. doi:10.1111/j.1524-4725.2008.01046.x.

    Article  CAS  Google Scholar 

  6. Taylor, P., Hahn, S. K., Jelacic, S., Maier, R. V, Patrick, S., & Hoffman, A. S. (2012). Anti-inflammatory drug delivery from hyaluronic acid hydrogels. Journal of Biomaterials Science, 15, 37–41.

    Google Scholar 

  7. Mero, A., & Campisi, M. (2014). Hyaluronic acid bioconjugates for the delivery of bioactive molecules. Polymers, 6(1), 346–369. doi:10.3390/polym6020346.

    Article  Google Scholar 

  8. Chong, B. F., Blank, L. M., Mclaughlin, R., & Nielsen, L. K. (2005). Microbial hyaluronic acid production. Applied Microbiology and Biotechnology, 66(4), 341–351. doi:10.1007/s00253-004-1774-4.

    Article  CAS  Google Scholar 

  9. D’Ayala, G. G., Malinconico, M., & Laurienzo, P. (2008). Marine derived polysaccharides for biomedical applications: Chemical modification approaches. Molecules, 13(9), 2069–2106. doi:10.3390/molecules13092069.

    Article  Google Scholar 

  10. Kim, J., Park, Y., Tae, G., Kyu, B. L., Chang, M. H., Soon, J. H., et al. (2009). Characterization of low-molecular-weight hyaluronic acid-based hydrogel and differential stem cell responses in the hydrogel microenvironments. Journal of Biomedical Materials Research—Part A, 88(4), 967–975. doi:10.1002/jbm.a.31947.

    Article  Google Scholar 

  11. Kim, J., Kim, I. S., Cho, T. H., Lee, K. B., Hwang, S. J., Tae, G., et al. (2007). Bone regeneration using hyaluronic acid-based hydrogel with bone morphogenic protein-2 and human mesenchymal stem cells. Biomaterials, 28(10), 1830–1837. doi:10.1016/j.biomaterials.2006.11.050.

    Article  CAS  Google Scholar 

  12. Marcellin, E., Steen, J. A., & Nielsen, L. K. (2014). Insight into hyaluronic acid molecular weight control. Applied Microbiology and Biotechnology, 98(16), 6947–6956. doi:10.1007/s00253-014-5853-x.

    Article  CAS  Google Scholar 

  13. Lai, Z. W., Rahim, R. A., Ariff, A. B., & Mohamad, R. (2012). Biosynthesis of high molecular weight hyaluronic acid by Streptococcus zooepidemicus using oxygen vector and optimum impeller tip speed. Journal of Bioscience and Bioengineering, 114(3), 286–291. doi:10.1016/j.jbiosc.2012.04.011.

    Article  CAS  Google Scholar 

  14. Choi, S., Choi, W., Kim, S., Lee, S.-Y., Noh, I., & Kim, C.-W. (2014). Purification and biocompatibility of fermented hyaluronic acid for its applications to biomaterials. Biomaterials Research, 18(1), 6. doi:10.1186/2055-7124-18-6.

    Article  Google Scholar 

  15. O’Regan, M., Martini, I., Crescenzi, F., De Luca, C., & Lansing, M. (1994). Molecular mechanisms and genetics of hyaluronan biosynthesis. International Journal of Biological Macromolecules, 16(6), 283–286. doi:10.1016/0141-8130(94)90056-6.

    Article  Google Scholar 

  16. Wei, Z., Fu, Q., Chen, Y., Cong, P., Xiao, S., Mo, D., et al. (2012). The capsule of Streptococcus equi ssp. zooepidemicus is a target for attenuation in vaccine development. Vaccine, 30(31), 4670–4675. doi:10.1016/j.vaccine.2012.04.092.

    Article  CAS  Google Scholar 

  17. Schiraldi, C., Gatta, A. La, & De Rosa, M. (2010). Biotechnological production and application of hyaluronan. Biopolymers. doi:10.5772/10271.

  18. Mao, Z., Shin, H. D., & Chen, R. (2009). A recombinant E. coli bioprocess for hyaluronan synthesis. Applied Microbiology and Biotechnology, 84(1), 63–69. doi:10.1007/s00253-009-1963-2.

    Article  CAS  Google Scholar 

  19. Jin, P., Kang, Z., Yuan, P., Du, G., & Chen, J. (2016). Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168. Metabolic Engineering, 35, 21–30. doi:10.1016/j.ymben.2016.01.008.

    Article  CAS  Google Scholar 

  20. Jia, Y., Zhu, J., Chen, X., Tang, D., Su, D., Yao, W., et al. (2013). Metabolic engineering of Bacillus subtilis for the efficient biosynthesis of uniform hyaluronic acid with controlled molecular weights. Bioresource Technology, 132, 427–431. doi:10.1016/j.biortech.2012.12.150.

    Article  CAS  Google Scholar 

  21. Chauhan, A. S., Badle, S. S., Ramachandran, K. B., & Jayaraman, G. (2014). The P170 expression system enhances hyaluronan molecular weight and production in metabolically-engineered Lactococcus lactis. Biochemical Engineering Journal, 90, 73–78. doi:10.1016/j.bej.2014.05.012.

    Article  CAS  Google Scholar 

  22. Yu, H., & Stephanopoulos, G. (2008). Metabolic engineering of Escherichia coli for biosynthesis of hyaluronic acid. Metabolic Engineering, 10(1), 24–32. doi:10.1016/j.ymben.2007.09.001.

    Article  CAS  Google Scholar 

  23. Jeong, E., Shim, W. Y., & Kim, J. H. (2014). Metabolic engineering of Pichia pastoris for production of hyaluronic acid with high molecular weight. Journal of Biotechnology, 185, 28–36. doi:10.1016/j.jbiotec.2014.05.018.

    Article  CAS  Google Scholar 

  24. Huang, W. C., Chen, S. J., & Chen, T. L. (2008). Production of hyaluronic acid by repeated batch fermentation. Biochemical Engineering Journal, 40(3), 460–464. doi:10.1016/j.bej.2008.01.021.

    Article  CAS  Google Scholar 

  25. Patil, K. P., Kamalja, K. K., & Chaudhari, B. L. (2011). Optimization of medium components for hyaluronic acid production by Streptococcus zooepidemicus MTCC 3523 using a statistical approach. Carbohydrate Polymers, 86(4), 1573–1577. doi:10.1016/j.carbpol.2011.06.065.

    Article  CAS  Google Scholar 

  26. Badle, S. S., Jayaraman, G., & Ramachandran, K. B. (2014). Ratio of intracellular precursors concentration and their flux influences hyaluronic acid molecular weight in Streptococcus zooepidemicus and recombinant Lactococcus lactis. Bioresource Technology, 163, 222–227. doi:10.1016/j.biortech.2014.04.027.

    Article  CAS  Google Scholar 

  27. Tao, L., Song, F., Xu, N., Li, D., Linhardt, R. J., & Zhang, Z. (2017). New insights into the action of bacterial chondroitinase AC I and hyaluronidase on hyaluronic acid. Carbohydrate Polymers, 158, 85–92. doi:10.1016/j.carbpol.2016.12.010.

    Article  CAS  Google Scholar 

  28. Hynes, W. L., & Walton, S. L. (2000). Hyaluronidases of gram-positive bacteria. FEMS Microbiology Letters, 183(2), 201–207. doi:10.1016/S0378-1097(99)00669-2.

    Article  CAS  Google Scholar 

  29. Maniatis, T., Fritsch, E. F., & Sambrook, J. (1982). Molecular cloning: A laboratory manual (Vol. 545). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

    Google Scholar 

  30. 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. doi:10.1074/jbc.M109.011999.

    Article  CAS  Google Scholar 

  31. Bitter, T., & Muir, H. M. (1962). A modified uronic acid carbazole reaction. Analytical Biochemistry, 4, 330–334. doi:10.1016/0003-2697(62)90095-7.

    Article  CAS  Google Scholar 

  32. Hynes, W. L., Ferretti, J. J., & Hynes, W. L. (1994). Assays for hyaluronidase activity. Methods in Enzymology, 235, 606–616.

    Article  CAS  Google Scholar 

  33. Holden, M. T. G., Heather, Z., Paillot, R., Steward, K. F., Webb, K., Ainslie, F., et al. (2009). Genomic evidence for the evolution of Streptococcus equi host restriction, increased virulence, and genetic exchange with human pathogens. PLoS Pathogens. doi:10.1371/journal.ppat.1000346.

    Google Scholar 

  34. Hong-jie, F., Fu-yu, T., Ying, M., & Cheng-ping, L. (2009). Virulence and antigenicity of the szp-gene deleted Streptococcus equi ssp. zooepidemicus mutant in mice. Vaccine, 27(1), 56–61. doi:10.1016/j.vaccine.2008.10.037.

    Article  Google Scholar 

  35. Liu, L., Du, G., Chen, J., Wang, M., & Sun, J. (2008). Influence of hyaluronidase addition on the production of hyaluronic acid by batch culture of Streptococcus zooepidemicus. Food Chemistry, 110(4), 923–926. doi:10.1016/j.foodchem.2008.02.082.

    Article  CAS  Google Scholar 

  36. Park, M. G., Jang, J. D., & Kang, W. K. (1996). Streptococcus zooepidemicus medium and process for preparing hyaluronic acid. Google Patents.

  37. Starr, C. R., & Engleberg, N. C. (2006). Role of hyaluronidase in subcutaneous spread and growth of group A streptococcus. Infection and Immunity, 74(1), 40–48. doi:10.1128/IAI.74.1.40-48.2006.

    Article  CAS  Google Scholar 

  38. Schanté, C. E., Zuber, G., Herlin, C., & Vandamme, T. F. (2011). Chemical modifications of hyaluronic acid for the synthesis of derivatives for a broad range of biomedical applications. Carbohydrate Polymers, 85(3), 469–489. doi:10.1016/j.carbpol.2011.03.019.

    Article  Google Scholar 

  39. Im, J. H., Song, J. M., Kang, J. H., & Kang, D. J. (2009). Optimization of medium components for high-molecular-weight hyaluronic acid production by Streptococcus sp. ID9102 via a statistical approach. Journal of Industrial Microbiology and Biotechnology, 36(11), 1337–1344. doi:10.1007/s10295-009-0618-8.

    Article  CAS  Google Scholar 

  40. Zakeri, A., Rasaee, M. J., & Pourzardosht, N. (2017). Enhanced hyluronic acid production in Streptococcus zooepidemicus by over expressing HasA and molecular weight control with Niscin and glucose. Biotechnology Reports. doi:10.1016/j.btre.2017.02.007.

    Google Scholar 

  41. Armstrong, D. C., & Johns, M. R. (1997). Culture conditions affect the molecular weight properties of hyaluronic acid produced by Streptococcus zooepidemicus. Applied and Environmental Microbiology, 63(7), 2759–2764.

    CAS  Google Scholar 

  42. Tian, X., Azpurua, J., Hine, C., Vaidya, A., Myakishev-Rempel, M., Ablaeva, J., et al. (2013). High-molecularmass hyaluronan mediates the cancer resistance of the naked mole rat. Nature, 499(7458), 346–349. doi:10.1038/nature12234.

    Article  CAS  Google Scholar 

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Acknowledgement

The authors wish to thank Tarbiat Modares University of Tehran for supporting the conduct of this research.

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  1. Department of Clinical Biochemistry, School of Medical Sciences, Tarbiat Modares University, Chamran -Jalal Crossing, P.O. Box 14115-331, Tehran, Iran

    Navid Pourzardosht & Mohammad Javad Rasaee

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  1. Navid Pourzardosht
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  2. Mohammad Javad Rasaee
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Correspondence to Mohammad Javad Rasaee.

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Pourzardosht, N., Rasaee, M.J. Improved Yield of High Molecular Weight Hyaluronic Acid Production in a Stable Strain of Streptococcus zooepidemicus via the Elimination of the Hyaluronidase-Encoding Gene. Mol Biotechnol 59, 192–199 (2017). https://doi.org/10.1007/s12033-017-0005-z

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