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Enhanced Production of Astaxanthin in Paracoccus haeundaensis Strain by Physical and Chemical Mutagenesis

물리·화학적 돌연변이 유도를 통한 Paracoccus haeundaensis의 astaxanthin 생산량 증대

  • Seo, Yong Bae (Department of Microbiology, College of Natural Sciences, Pukyong National University) ;
  • Jeong, Tae Hyug (Department of Marine and Fisheries Resources, College of Natural Sciences, Mokpo National University) ;
  • Choi, Seong Seok (Department of Microbiology, College of Natural Sciences, Pukyong National University) ;
  • Lim, Han Kyu (Department of Marine and Fisheries Resources, College of Natural Sciences, Mokpo National University) ;
  • Kim, Gun-Do (Department of Microbiology, College of Natural Sciences, Pukyong National University)
  • Received : 2017.01.31
  • Accepted : 2017.03.06
  • Published : 2017.03.30

Abstract

Carotenoids are natural lipid-soluble pigments, which are produced primarily by bacteria, algae, and plants. Many studies have focused on the identification, production, and utilization of natural sources of astaxanthin from algae, yeast, and crustacean byproducts as an alternative to the synthetic pigment, which is mostly used today. The aim of the present study was to identify a mutant of Paracoccus haeundaensis by exposure to UV and ethyl methanesulfonate (EMS). The mutant was then exposed to nutrient stress conditions to isolate an astaxanthin-hyperproducing strain, followed by characterization of the mutant. The survival rate decreased in accordance with an increase in the UV exposure time and an increase in the EMS concentration. A mutant of the original P. haeundaensis strain was identified that showed hyperproduction of astaxanthin following exposure to UV irradiation (20 min) and EMS treatment (0.4 M concentration). The optimal culture conditions for the PUE mutant were $25^{\circ}C$, pH 7-8, and 3% NaCl. The effects of various carbon and nitrogen sources on the growth and astaxanthin production of PUE were examined. The addition of 1% raffinose and 3% potassium nitrate influenced cell growth and astaxanthin production. The selected mutant exhibited an increase of 1.58 folds in astaxanthin content compared to initial wild type strain. A genetically stable mutant strain obtained using mutagen (UV irradiation and EMS treatment) may be a suitable candidate for further industrial scale production of astaxanthin.

Carotenoid는 천연 지용성 색소이며, 세균, 조류, 식물 등이 생산한다. 세계 시장의 대부분을 차지하는 합성 염료의 대안으로서 현재는 조류나 세균, 갑각류 등의 원료로부터 아스타잔틴의 생산, 정제, 이용이 주목 받고 있다. 이 연구는 UV와 EMS를 이용하여 P. haeundaensis의 돌연변이를 유도하고, 결과적으로 astaxanthin을 과잉 생산하는 돌연변이주를 선별하고 특성을 확인하기 위해 다양한 배양 및 영양 조건을 이용하여 astaxanthin 생산량을 확인하였다. 실험 결과 UV 조사 시간이 증가하거나, EMS 농도가 증가할수록 균주의 생존율이 감소하였다. Astaxanthin 과잉 생산 돌연변이 균주의 경우 400 mM EMS와 UV 20분을 순차적으로 처리한 방법에서 선별된 변이주가 가장 높은 astaxanthin 생산량을 보이는 것을 확인하였으며, 이 균주의 이름을 PUE로 명명하였다. PUE의 최적 배양 조건은 $25^{\circ}C$, pH 7-8, 3% NaCl이며, 1% raffinose, 3% potassium nitrate 첨가 시 astaxanthin 생산량이 증가하는 것으로 밝혀졌다. PUE에서는 wild type 균주에 비해 astaxanthin 생산량이 1.58배 증가함을 확인할 수 있었다. 본 연구의 실험 결과, 돌연변이 유도에 의해 선별된 변이주는 astaxanthin의 산업적 생산에 활용 가능한 후보가 될 수 있을 것으로 사료된다.

Keywords

References

  1. An, G. H., Bielich J., Auerbach, R. and Johnson, E. A. 1991. Isolation and characterization of carotenoid hyperproducing mutants of yeast by flow cytometry and cell sorting. Bio. Tech. 9, 70-73.
  2. Castelblanco-Matiz, L. M., Barbachano-Torres, A., Ponce-Noyola, T., Ramos-Valdivia, A. C., Cerda Garcia-Rojas, C. M., Flores-Ortiz, C. M., Barahona-Crisostomo, S. K., Baeza-Cancino, M. E., Alcaino-Gorman, J. and Cifuentes-Guzman, V. H. 2015. Carotenoid production and gene expression in an astaxanthin-overproducing Xanthophyllomyces dendrorhous mutant strain. Arch. Microbiol. 197, 1129-39 https://doi.org/10.1007/s00203-015-1153-9
  3. Cheng, J., Li, K., Yang, Z., Zhou, J. and Cen, K. 2016. Enhancing the growth rate and astaxanthin yield of Haematococcus pluvialis by nuclear irradiation and high concentration of carbon dioxide stress. Bioresour. Technol. 204, 49-54 https://doi.org/10.1016/j.biortech.2015.12.076
  4. Choi, E. S. and An, G. H. 2003. Preparation of the red yeast, Xanthophyllomyces dendrorhous, as feed additive with increased availability of astaxanthin. Biotechnol. Lett. 25, 767-771. https://doi.org/10.1023/A:1023568319114
  5. Emmerstorfer-Augustin, A., Moser, S. and Pichler, H. 2016. Screening for improved isoprenoid biosynthesis in microorganisms. J. Biotechnol. 235, 112-120. https://doi.org/10.1016/j.jbiotec.2016.03.051
  6. Glaeser, J. and Klug, G. 2005. Photo-oxidative stress in Rhodobacter sphaeroides: protective role of carotenoids and expression of selected genes. Microbiology 151, 1927-1938. https://doi.org/10.1099/mic.0.27789-0
  7. Helmut, S. and Stahl, W. 2003. Antioxidant activity of carotenoids. Mol. Aspects Med. 24, 345-351. https://doi.org/10.1016/S0098-2997(03)00030-X
  8. Higuera-Ciapara, I., Felix-Valenzuela, L. and Goycoolea, F. M. 2006. Astaxanthin: A Review of its chemistry and applications. Crit. Rev. Food Sci. Nutr. 46, 185-196. https://doi.org/10.1080/10408690590957188
  9. Johnson, E. A. and Schroeder, W. A. 1996. Microbial carotenoids. Adv. Biochem. Eng. Biotechnol. 53, 119-178.
  10. Kobayashi, M., Kakizono, T. and Nagai, S. 1993. Enhanced carotenoid biosynthesis by oxidative stress in acetate-induced cyst cells of a green unicellular alga, Haematococcus pluvialis. Appl. Environ. Microbiol. 59, 867-873.
  11. Krubasik, P. and Sandmann, G. 2000. A carotenogenic gene cluster from Brevibacterium linens with novel lycopene cyclase genes involved in the synthesis of aromatic carotenoids. Mol. Gen. Genet. 263, 423-432. https://doi.org/10.1007/s004380051186
  12. Lee, J. H., Nam, S. W., Choi, T. J., Lee, W. J. and Kim, Y. T. 2004. Paracoccus haeundaensis sp. nov., a gram-negative, halophilic, astaxanthin-producing bacterium. Int. J. Syst. Evol. Microbiol. 54, 1699-1702. https://doi.org/10.1099/ijs.0.63146-0
  13. Lee, J. H., Seo, Y. B., Jeong, S. Y., Nam, S. W. and Kim, Y. T. 2007. Functional analysis of combinations in astaxanthin biosynthesis genes from Paracoccus haeundaensis. Biotechnol. Bioprocess Eng. 54, 1699-1702.
  14. Lee, P. C. and Schmidt-Dannert, C. 2002. Metabolic engineering towards biotechnological production of carotenoids in microorganisms. Appl. Microbiol. Biotechnol. 60, 1-11. https://doi.org/10.1007/s00253-002-1101-x
  15. Misawa, N., Satomi, Y., Kondo, K., Yokoyama, A., Kajiwara, S., Saito, T., Ohtani, T. and Miki, W. 1995. Structure and functional analysis of marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level. J. Bacteriol. 177, 6575-6584. https://doi.org/10.1128/jb.177.22.6575-6584.1995
  16. Nelis, H. J. and De Leenheer, A. P. 1991. Microbial sources of carotenoid pig pigments used in foods and feeds. J. Appl. Bacteriol. 70, 181-191. https://doi.org/10.1111/j.1365-2672.1991.tb02922.x
  17. Nishino, H., Murakosh, M., Ii, T., Takemura, M., Kuchide, M., Kanazawa, M., Mou, X.Y., Wada, S., Masuda, M., Ohsaka, Y., Yogosawa, S., Satomi, Y. and Jinno, K. 2002. Carotenoids in cancer chemoprevention. Cancer Metastasis Rev. 21, 257-264 https://doi.org/10.1023/A:1021206826750
  18. Pollmann, H., Breitenbach, J. and Sandmann, G. 2017. Engineering of the carotenoid pathway in Xanthophyllomyces dendrorhous leading to the synthesis of zeaxanthin. Appl. Microbiol. Biotechnol. 101. 103-111. https://doi.org/10.1007/s00253-016-7769-0
  19. Sandesh Kamath, B., Vidhyavathi, R., Sarada, R. and Ravishankar, G. A. 2008. Enhancement of carotenoids by mutation and stress induced carotenogenic genes in Haematococcus pluvialis mutants. Bioresour. Technol. 99, 8667-8673. https://doi.org/10.1016/j.biortech.2008.04.013
  20. Smith, T. A. D. 1998. Carotenoids and cancer: prevention and potential therapy. Br. J. Biomed. Sci. 55, 268-275.
  21. Sun, N. K., Lee, S. H. and Song, K. B. 2004. Characterization of a carotenoid-hyperproducing yeast mutant isolated by low-dose gamma irradiation. Int. J. Food Microbiol. 94, 263-267. https://doi.org/10.1016/S0168-1605(03)00311-8
  22. Vershinin, A. 1999. Biological functions of carotenoids-diversity and evolution. Biofactors 10, 99-104. https://doi.org/10.1002/biof.5520100203
  23. Wachi, Y., Burgess, J. G., Iwamoto, K., Yamada, N., Nakamura, N. and Matsunga, T. 1995. Effect of Ultraviolet A (UV-A) light on growth, photosynthetic acitivity and production of biopterin glucoside by the marine UV-A resistant cyanobacterium Oscillatoria sp. Biochemica. Biophysica. Acta. 124, 165-168.
  24. Wang, C. C., Ding, S., Chiu, K. H., Liu, W. S., Lin, T. J. and Wen, Z. H. 2016. Extract from a mutant Rhodobacter sphaeroides as an enriched carotenoid source. Food Nutr. Res. 60, 29580. https://doi.org/10.3402/fnr.v60.29580
  25. Wang, N., Guan, B., Kong, Q., Sun, H., Geng, Z. and Duan, L. 2016. Enhancement of astaxanthin production from Haematococcus pluvialis mutants by three-stage mutagenesis breeding. J. Biotechnol. 236, 71-77. https://doi.org/10.1016/j.jbiotec.2016.08.009
  26. Zhang, Y., He, M., Zou, S., Fei, C., Yan, Y., Zheng, H., Rajper, A. A. and Wang, C. 2016. Breeding of high biomass and lipid producing Desmodesmus sp. by Ethylmethane sulfonate-induced mutation. Bio. Tech. 207, 268-275. https://doi.org/10.1016/j.biortech.2016.01.120
  27. Zhao, Y., Shang, M., Xu, J. W., Zhao, P., Li, T. and Yu, X. 2015. Enhanced astaxanthin production from a novel strain of Haematococcus pluvialis using fulvic acid. Process. Biochem. 50, 2072-2077. https://doi.org/10.1016/j.procbio.2015.09.004