전분 분해성 산업용 Saccharomyces cerevisiae에서 Achlya bisexualis $\beta$-Amylase의 발현 특성 규명

Characterization of Achlya bisexualis $\beta$-Amylase Expression in an Amylolytic Industrial Strain of Saccharomyces cerevisiae

  • 이옥희 (전남대학교 자연과학대학 생물학과) ;
  • 임미현 (전남대학교 자연과학대학 생물학과) ;
  • 김지혜 (전남대학교 자연과학대학 생물학과) ;
  • 유은혜 (전남대학교 자연과학대학 생물학과) ;
  • 고현미 (전남대학교 자연과학대학 생물학과) ;
  • 진종언 (동강대학 피부미용계열) ;
  • 배석 (전남대학교 자연과학대학 생물학과)
  • Lee, Ok-Hee (Department of Biological Sciences, College of Natural Sciences, Chonnam National University) ;
  • Lim, Mi-Hyeon (Department of Biological Sciences, College of Natural Sciences, Chonnam National University) ;
  • Kim, Ji-Hye (Department of Biological Sciences, College of Natural Sciences, Chonnam National University) ;
  • Ryu, Eun-Hye (Department of Biological Sciences, College of Natural Sciences, Chonnam National University) ;
  • Ko, Hyun-Mi (Department of Biological Sciences, College of Natural Sciences, Chonnam National University) ;
  • Chin, Jong-Eon (Department of Cosmetology, Dongkang College University) ;
  • Bai, Suk (Department of Biological Sciences, College of Natural Sciences, Chonnam National University)
  • 발행 : 2008.09.30

초록

$\beta$-Amylase를 생산하여 전분 분해능을 갖는 산업용 효모를 개발하기 위해 산업용 Saccharomyces cerevisiae에서 Achlya bisexualis $\beta$-amylase (BAMY)유전자를 ADC1 promoter에 연결하여 구성적으로 발현시켰다. 효모의 형질전환은 $\delta$-서열을 재조합 부위로 하는integration 시스템을 이용하였다. Integration 시스템의 세균 유전자 부분은 제거되고 BAMY 유전자와 $\delta$-서열을 갖고 있는 짧은integrative cassette를 제조하였다. BAMY 유전자를 발현하는 재조합 S. cerevisiae 형질전환체는 세포외 배지로 45 kDa의 $\beta$-amylase를 분비하였고, $\beta$-amylase 활성은 A. bisexualis에 비해 약 18.5배 높았다. 형질전환체에 다중도입된 BAMY 유전자는 비선택배지에서 100세대 생장 후에도 안정되게 유지되었다. 각종전분을 기질로 했을 매 $\beta$-amylase의 활성은soluble starch를 기질로 했을 경우와 유사하게 높았고, 가수분해산물 분석 결과 maltose가 주 분해산물이었다.

To develop an amylolytic industrial yeast strain producing $\beta$-amylase, the BAMY gene encoding Achlya bisexualis $\beta$-amylase was constitutively expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p) in an industrial strain of Saccharomyces cerevisiae. Yeast transformation was carried out by an integration system containing $\delta$-sequences as the recombination site. The integrative cassette devoid of bacterial DNA sequences was constructed that contains the BAMY gene and $\delta$-sequences. Industrial S. cerevisiae transformed with this integrative cassette secreted 45 kDa $\beta$-amylase into the culture medium. The $\beta$-amylase activity of the transformant was approximately 18.5-times higher than that of A. bisexualis. The multi-integrated BAMY genes in the transform ant were stable after 100 generations of growth in nonselective medium. Hydrolysis of soluble starch and various starches with the enzyme released maltose but not glucose or oligosaccharides.

키워드

참고문헌

  1. Choi, E.Y., J.N. Park, H.O. Kim, D.J. Shin, Y.H. Chun, S.Y. Im, S.B. Chun, and S. Bai. 2002. Construction of an industrial polyploid strain of Saccharomyces cerevisiae containing Saprolegnia ferax $\beta$-amylase gene and secreting $\beta$-amylase. Biotechnol. Lett. 24, 1785-1790 https://doi.org/10.1023/A:1020613306127
  2. Cho, K.M., Y.J. Yoo, and H.S. Kang. 1999. $\delta$-Integration of endo/exo-glucanase and $\beta$-glucosidase genes into the yeast chromosomes for direct conversion of cellulose to ethanol. Enzyme Microbiol. Technol. 25, 23-30 https://doi.org/10.1016/S0141-0229(99)00011-3
  3. Dohmen, R.J., A.W.M. Strasser, U.M. Dahlemsand, and C.P. Hollenberg. 1990. Cloning of the Schwanniomyces occidentalis glucoamylase gene (GAM1) and its expression in Saccharomyces cerevisiae. Gene 95, 111-121 https://doi.org/10.1016/0378-1119(90)90421-M
  4. Eksteen, J.M., P. Van Renseburg, R.R. Cordero Otero, and I.S. Pretorius. 2003. Starch fermentation by recombinant Saccharomyces cerevisiae strains expressing the $\alpha$-amylase and glucoamylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera. Biotechnol. Bioeng. 84, 639-646 https://doi.org/10.1002/bit.10797
  5. Ghang, D.M., L. Yu, M.H. Lim, H.M. Ko, S.Y. Im, H.B. Lee, and S. Bai. 2007. Efficient one-step starch utilization by industrial strains of Saccharomyces cerevisiae expressing the glucoamylase and $\alpha$-amylase genes from Debaryomyces occidentalis. Biotechnol. Lett. 29, 1203-1208 https://doi.org/10.1007/s10529-007-9371-0
  6. Gietz, D., A. St. Jean, R.A. Woods, and R.H. Schiestl. 1992. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20, 1425 https://doi.org/10.1093/nar/20.6.1425
  7. Janse, B.J.H. and I.S. Pretorius. 1995. One-step enzymatic hydrolysis of starch using a recombinant strain of Saccharomyces cerevisiae producing $\alpha$-amylase, glucoamylase and pullulanase. Appl. Microbiol. Biotechnol. 42, 876-883
  8. Jeong, T.H., H.O. Kim, J.N. Park, H.J. Lee, D.J. Shin, H.B. Lee, S.B. Chun, and S. Bai. 2001. Cloning and sequencing of the $\beta$-amylase gene from Paenibacillus sp. and its expression in Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 11, 65-71
  9. Kang, H.A. and J.W.B. Hershey. 1994. Effect of initiation factor elF-5A depletion on protein synthesis and proliferation of Saccharomyces cerevisiae. J. Biol. Chem. 269, 3934-3940
  10. Kang, N.Y., J.N. Park, J.E. Chin, H.B. Lee, S.Y. Im, and S. Bai. 2003. Construction of an amylolytic industrial strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis $\alpha$-amylase gene. Biotechnol. Lett. 25, 1847-1851 https://doi.org/10.1023/A:1026281627466
  11. Kim, H.O., J.N. Park, H.J. Shon, D.J. Shin, C. Choi, S.Y. Im, H.B. Lee, S.B. Chun, and S. Bai. 2000. Cloning and expression in Saccharomyces cerevisiae of a $\beta$-amylase gene from the oomycete Saprolegnia ferax. Biotechnol. Lett. 22, 1493-1498 https://doi.org/10.1023/A:1005646013313
  12. Kim, H.O., J.N. Park, D.J. Shin, H.B. Lee, S.B. Chun, and S. Bai. 2001. A gene encoding $\beta$-amylase from Saprolegnia parasitica and its expression in Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 11, 529-533
  13. Kim, H.O., J.N. Park, D.J. Shin, H.B. Lee, S.B. Chun, and S. Bai. 2001. A gene encoding Achlya bisexualis $\beta$-amylase and its expression in Saccharomyces cerevisiae. Biotechnol. Lett. 23, 1101-1107 https://doi.org/10.1023/A:1010503731486
  14. Kim, K., C.S. Park, and J.R. Mattoon. 1988. High-efficiency, onestep utilization by transformed Saccharomyces cells which secrete both yeast glucoamylase and mouse $\alpha$-amylase. Appl. Environ. Microbiol. 54, 966-971
  15. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage $T_4$. Nature (London) 227, 680-685 https://doi.org/10.1038/227680a0
  16. Lee, F.W.F. and N.A. Da Silva. 1997. Improved efficiency and stability of multiple cloned gene insertions at the $\delta$ sequences of Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 48, 339-345 https://doi.org/10.1007/s002530051059
  17. Marin, D., A. Jimenez, and M.F. Lobato. 2001. Construction of an efficient amylolytic industrial yeast strain containing DNA exclusively derived from yeast. FEMS Microbiol. Lett. 201, 249-253 https://doi.org/10.1111/j.1574-6968.2001.tb10764.x
  18. Monroe, J.D., M.D. Salminen, and J. Preiss. 1991. Nucleotide sequence of a cDNA clone encoding a $\beta$-amylase from Arabidopsis thaliana. Plant Physiol. 97, 1599-1601 https://doi.org/10.1104/pp.97.4.1599
  19. Nanmori, T., M. Nagai, Y. Shimizu, R. Shinke, and B. Mikami. 1993. Cloning of the $\beta$-amylase gene from Bacillus cereus and characteristics of the primary structure of the enzyme. Appl. Environ. Microbiol. 59, 623-627
  20. Ness, F., F. Lavallee, D. Dubourdieu, M. Aigle, and L. Dulau. 1993. Identification of yeast strains using the polymerase chain reaction. J. Sci. Food Agric. 62, 89-94 https://doi.org/10.1002/jsfa.2740620113
  21. Nieto, A., J.A. Prieto, and P. Sanz. 1999. Stable high-copy number integration of Aspergillus orizae $\alpha$-amylase cDNA in an industrial baker's yeast strain. Biotechnol. Prog. 15, 459-466 https://doi.org/10.1021/bp9900256
  22. Park, J.N., K.H. Lee, H.M. Ko, K.H. Seo, J.E. Chin, H.B. Lee, and S. Bai. 2004. Expression of ethionine resistance conferring gene in an industrial strains of Saccharomyces cerevisiae. Kor. J. Microbiol. Biotechnol. 32, 356-361
  23. Sambrook, J. and D.W. Russell. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, N.Y., USA
  24. Steyn, A.J.C. and I.S. Pretorius. 1991. Co-expression of a Saccharomyces diastaticus glucoamylase-encoding gene and a Bacillus amyloliquefaciens $\alpha$-amylase-encoding gene in Saccharomyces cerevisiae. Gene 100, 85-93 https://doi.org/10.1016/0378-1119(91)90353-D
  25. Wang, X., Z. Wang, and N.A. Da Silva. 1996. G418 selection and stability of cloned genes integrated at chromosomal $\delta$ sequences of Saccharomyces cerevisiae. Biotechnol. Bioeng. 49, 45-51 https://doi.org/10.1002/(SICI)1097-0290(19960105)49:1<45::AID-BIT6>3.0.CO;2-T
  26. Zhu, H., F. Qu, and L.H. Zhu. 1993. Isolation of genomic DNAs from plant, fungi and bacteria using benzyl chloride. Nucleic Acids Res. 21, 5279-5280 https://doi.org/10.1093/nar/21.22.5279