돌연변이를 통한 미세조류 Haematococcus pluvialis의 Astaxanthin 생산성의 향상

Enhancement of Astaxanthin Production of Haematococcus pluvialis by Mutation

  • 박복준 (인하대학교 공과대학 생물공학과/생물산업기술연구소) ;
  • 김법민 (인하대학교 공과대학 생물공학과/생물산업기술연구소) ;
  • 심수현 (인하대학교 공과대학 생물공학과/생물산업기술연구소) ;
  • 김정동 (인하대학교 공과대학 생물공학과/생물산업기술연구소) ;
  • 이철균 (인하대학교 공과대학 생물공학과/생물산업기술연구소)
  • Park Bok-Jun (Institute of Industrial Biotechnology, Department of Biological Engineering Inha University) ;
  • Kim Beob-Min (Institute of Industrial Biotechnology, Department of Biological Engineering Inha University) ;
  • Shim Su-Hyun (Institute of Industrial Biotechnology, Department of Biological Engineering Inha University) ;
  • Kim Jeong-Dong (Institute of Industrial Biotechnology, Department of Biological Engineering Inha University) ;
  • Lee Choul-Gyun (Institute of Industrial Biotechnology, Department of Biological Engineering Inha University)
  • 발행 : 2006.06.01

초록

Haematococcus pluvialis는 astaxanthin을 많이 생산하는 미세조류로써 astaxanthin은 항산화제로 면역반응을 강화시켜주며 항암효과 등을 가지고 있다. 그런데 야생종의 낮은 생장속도와 astaxanthin의 생산에는 한계가 있기 때문에 이를 극복하기위해 돌연변이 방법을 사용하였다. 돌연변이 방법으로는 자외선 조사와 EMS와 colchicines 처리를 사용하여 야생종보다 colony가 크고 더 붉은 것은 선택하였다. 선별된 돌연변이들은 carotenoid 생합성과정을 억제하는 nicotine 과 diphenylamine을 이용하여 다시 선별하였다. 그때 생존율은 40-50%이었고 여기서 선별된 균주들을 다시 규모를 키워 배양한 결과 자외선 처리한 돌연변이인 U15-5가 야생종보다 세포당 total carotenold 생산량이 1.68배 증가하였고, colchicine 처리한 DS와 M4-3은 생장속도가 $20\sim30%$ 증가하였다.

Haematococcus pluvialis is a great producer of astaxanthin (3,3'-dihydroxy-$\beta$,$\beta$-carotene-4,4'-dione). The activities of astaxanthin include potential cancer prevention, immune response enhancement, antioxidant activity, and so on. Nevertheless, it tried to manipulate by mutation for overcoming low growth rate of wild type and limited production of astaxanthin. Mutated colony that is lager and more reddish one than wild type was selected by attempting to expose strains to UV irradiation and to treat chemical such as EMS and colchicines as mutagen. Selected mutants were further screened using inhibitors of the carotenoid biosynthetic pathway. Inhibitors used were nicotine and diphenylamine and both had decreased the survival rate by 40-50%. Among over 50,000 mutant colonies screened, two strains were selected. One selected mutant strain (U15-5) from UV treatment showed 1.68-fold higher total carotenoid contents per cell than that of the wild type strain. On the other hand, the other selected mutant strains (DS, M4-3) from colchicine treatment showed 20$\sim$30% faster cell growth than the wild type strain.

키워드

참고문헌

  1. Bamabas, B., B. Obert, and G. Kovacs. 1999. Colchicine, an efficient genome-doubling agent for maize (Zea mays L.) microspores cultured in anthero. Plant Cell Rep. 18: 858-862 https://doi.org/10.1007/s002990050674
  2. Becker, E. W. 1994. Biomechanical model of the P-type ion pumps of the cell. Naturwissenschaflen 81: 21-27 https://doi.org/10.1007/BF01138556
  3. Borowitzka, M. A. 1997. Microalgae for aquaculture: Opportunities and constraints. J. Appl. Phycol. 9:393-401 https://doi.org/10.1023/A:1007921728300
  4. Borowitzka, M. A. 1999. Commercial production of microalgae: ponds, tanks, tubes and fermenters. J. Biotechnol. 70: 313-321 https://doi.org/10.1016/S0168-1656(99)00083-8
  5. Borowitzka., M. A. and L. J. Borowitzka. 1988. Micro-algal biotechnology. Cambridge, UK: Cambridge University Press
  6. Chumpolkulwong, N., T. Kakizono, T. Handa, and N. Nishio. 1997. Isolation and characterization of compactin resistant mutants of an astaxanthin synthesizing green alga Haematococcus pluvialis. Biotechnol. Lett. 19: 299-302 https://doi.org/10.1023/A:1018330329357
  7. Chumpolkulwong, N., T. Kakizono, H. Ishii, and N. Nishio. 1997. Enzymatic conversion of beta-carotene to astaxanthin by cell-extracts of a green alga Haematococcus pluvialis. Biotechnol. Lett. 19: 443-446 https://doi.org/10.1023/A:1018392010218
  8. Chumpolkulwong, N., T. Kakizono, S. Nagai, and N. Nishio. 1997. Increased astaxanthin production by Phaffza rhodozyma mutants isolated as resistant to diphenylamine. J. Ferment. Bioeng. 83: 429-434 https://doi.org/10.1016/S0922-338X(97)82996-0
  9. Harker, M., A. J. Tsavalos, and A. J. Young. 1996. Factors responsible for astaxanthin formation in the chlorophyte Haematococcus pluvialis. Bioresour. Technol. 55: 207-214 https://doi.org/10.1016/0960-8524(95)00002-X
  10. Hasegawa., H., S. Takashima., and A. Nakamura. 1995. Effect of gamma-ray irradiation on cultured anther of tobacco (Nicotiana tobacum L.). Radioactivity and morphological variants appearing in the haplod plants. Plant Tiss. Cult. Lett. 12: 281-287
  11. Herrera, J. C., L. G. Moreno, J. R. Acuna, M. De Pena, and D. Osorio. 2002. Colchicine-induced microspore embryogenesis in coffee. Plant Cell Tiss. Org. 71: 89-92 https://doi.org/10.1023/A:1016564816602
  12. Hong, S. P., M. H. Kim., and J. K. Hwang. 1998. Biological functions and production technology of carotenoids. J. Korean Soc. Food Sci. Nutr. 27: 1297-1306
  13. Ishikawa, T., T. Takayama, and H. Ishizaka. 1999. Amphidiploids between Alstroemeria ligtu L. hybrid and A. pelegrina L, var. rosea induced through colchicine treatment and their reproductive characteristics. Sci. Hortic. 80: 235-246 https://doi.org/10.1016/S0304-4238(98)00246-5
  14. Kobayashi, M. 2003. Astaxanthin biosynthesis enhanced by reactive oxygen species in the green alga Haematococcus pluvialis. Biotechnol. Bioprocess Eng. 8: 322-330. https://doi.org/10.1007/BF02949275
  15. Lee, Y. K. 2001. Microalgal mass culture systems and methods: Their limitation and potential. J. Appl. Phycol. 13: 307-315 https://doi.org/10.1023/A:1017560006941
  16. Margalith, P. Z. 1999. Production of ketocarotenoids by microalgae. Appli. Microbiol. Biotechnol. 51: 431-438 https://doi.org/10.1007/s002530051413
  17. Palozza, P. and N. I. Krinsky. 1992. Antioxidant effects of carotenoids invivo and invitro - an overview. Methods Enzymol. 213: 403-420 https://doi.org/10.1016/0076-6879(92)13142-K
  18. Park, E. K. and C. G. Lee. 2001. Astaxanthin production by Haematococcus pluvialis under various light intensities and wavelengths. J. Microbiol. Biotech. 11: 1024-1030
  19. Tripathi, U., G. Venkateshwaran, R. Sarada, and G. A. Ravishankar. 2001. Studies on Haematococcus pluvialis for improved production of astaxanthin by mutagenesis. World J. Microb. Biotechnol. 17: 143-148 https://doi.org/10.1023/A:1016609815405
  20. Wang., A. S., D. S. K. Chang., J. B. Milcic., and T. C. Yang. 1988. Effect of X-ray irradiation on maize inbred line B73 tissue cultures and regenerated plants. Crop. Sci. 28: 358-362
  21. Zhang, D. H. and Y. K. Lee. 1997. Enhanced accumulation of secondary carotenoids in a mutant of the green alga, Chlorococcum sp. J. Appl. Phycol. 9: 459-463 https://doi.org/10.1023/A:1007902103419