한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)

  • 제47권4호
  • /
  • Pages.662-666
  • /
  • 2019
  • /
  • 1598-642X(pISSN)
  • /
  • 2234-7305(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지
DOI QR코드

DOI QR Code

출아효모에서 재조합 neoagarobiose hydrolyase의 생산을 위한 최적 발현시스템

Optimal Expression System for Production of Recombinant Neoagarobiose Hydrolyase in Saccharomyces cerevisiae

  • 정혜원 (동의대학교 바이오응용공학부 의생명공학전공) ;
  • 김연희 (동의대학교 바이오응용공학부 의생명공학전공)
  • Jung, Hye-Won (Biomedical Engineering and Biotechnology Major, Division of Applied Bioengineering, Dong-Eui University) ;
  • Kim, Yeon-Hee (Biomedical Engineering and Biotechnology Major, Division of Applied Bioengineering, Dong-Eui University)
  • 투고 : 2019.06.17
  • 심사 : 2019.07.04
  • 발행 : 2019.12.28
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

본 연구에서는 Saccharomyces cerevisiae를 이용해서 neoagarobiose hydrolase (NABH)를 효율적으로 생산하기 위한 NABH558 유전자 발현시스템을 구축하였다. ADH1 promoter와 GAL10 promoter 하류에 NABH558 유전자를 가진 pAMFα-NABH plasmid와 pGMFα-NABH plasmid는 S. cerevisiae 2805 균주에 형질전환되었다. 2805/pAMFα-NABH 균주는 YPD (2% dextrose) 배지에서 가장 높은 NABH 효소 활성(0.069 unit/ml/DCW)을 보였고, 2805/pGMFα-NABH 균주의 경우는 배지의 조성과 상관없이 비슷한 수준의 NABH 활성(0.02-0.027 unit/ml/DCW)을 보였다. RT-PCR을 통한 NABH558 유전자의 transcription level은 NABH 활성 증가에 따라 비슷한 수준으로 증가되었음을 확인할 수 있었다. 또한 재조합균주에서 생산된 NABH는 agarose를 galactose와 AHG로 분해하였다. 따라서 NABH558 유전자의 발현에는 ADH1 promoter를 사용하는 것이 더 효율적이며 GAL10 promoter와 비교해서 최대 3배정도 높은 활성의 재조합 NABH를 생산할 수 있음을 알 수 있었다.

In this study, the NABH558 gene expression system was constructed to efficiently produce neoagarobiose hydrolase (NABH) in Saccharomyces cerevisiae strain. The ADH1 and GAL10 promoters of the pAMFα-NABH and pGMFα-NABH plasmids were examined to determine the suitable promoter for the NABH558 gene expression, respectively. The effect of promoter and carbon sources on NABH558 gene expression was investigated by transforming each plasmid into the S. cerevisiae 2805 strain. The NABH activity in the 2805/pAMFα-NABH strain was 0.069 unit/ml/DCW in YPD medium, whereas that in the 2805/pGMFα-NABH strain was similar (0.02-0.027 unit/ml/DCW) irrespective of the medium composition. The higher NABH activity in the YPD medium was due to the increased NABH558 gene transcription. NABH produced in the recombinant strains could degrade agarose to galactose and AHG. This indicated that ADH1 promoter was a more optimal promoter for the expression of NABH558 gene than the GAL10 promoter. The NABH activity induced by the ADH1 promoter was about 3-fold higher than that induced by the GAL10 promoter.

키워드

참고문헌

  1. Naik SN, Goud VV, Rout PK, Dalai AK. 2010. Production of first and second generation biofuels: a comprehensive review. Renew Sustainable Energy Rev. 14: 578-597. https://doi.org/10.1016/j.rser.2009.10.003
  2. John RP, Anisha GS, Nampoothiri KM, Pandey M. 2011. Micro and macroalgal biomass: a renewable source for bioethanol. Bioresour. Technol. 102: 186-193. https://doi.org/10.1016/j.biortech.2010.06.139
  3. Lee SM, Kim JH, Cho HY, Joo H, Lee JH. 2009. Production of bio-ethanol from brown algae by physicochemical hydrolysis. J. Korean Ind. Eng. Chem. 20: 517-521.
  4. Yanagisawa M, Kawai S, Murata K. 2013. Strategies for the production of high concentrations of bioethanol from seaweeds: production of high concentrations of bioethanol from seaweeds. Bioengineered 4: 224-235. https://doi.org/10.4161/bioe.23396
  5. Duckworth M, Yaphe W. 1971. The structure of agar. I. Fractionation of a complex mixture of polysaccharides. Carbohydr. Res. 16: 189-197. https://doi.org/10.1016/S0008-6215(00)86113-3
  6. Lee JS, Hong SK, Lee CR, Nam SW, Jeon SJ, Kim YH. 2019. Production of ethanol (agaro-bioethanol) from agarose by unified enzymatic saccharification and fermentation in recombinant yeast. J. Microbiol. Biotechol. 29: 625-632. https://doi.org/10.4014/jmb.1902.02012
  7. Potin P, Richard C, Rochas C, Kloareg B. 1993. Purification and characterization of the ${\alpha}$-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B. Eur. J. Biochem. 214: 599-607. https://doi.org/10.1111/j.1432-1033.1993.tb17959.x
  8. Kirimura K, Masuda N, Iwasaki Y, Nakagawa H, Kobayashi R, Usami S. 1999. Purification and characterization of a novel ${\beta}$-agarase from an alkalophilic bacterium, Alteromonas sp. E-1. J. Biosci. Bioeng. 87: 436-441. https://doi.org/10.1016/S1389-1723(99)80091-7
  9. Ha SC, Lee S, Lee J, Kim HT, Ko HJ, Kim KH, et al. 2011. Crystal structure of a key enzyme in the agarolytic pathway, ${\alpha}$-neoagarobiose hydrolase from Saccharophagus degradans 2-40. Biochem. Biophys. Res. Commun. 412: 238-244. https://doi.org/10.1016/j.bbrc.2011.07.073
  10. Lee S, Lee JY, Ha SC, Jung J, Shin DH, Kim KH, et al. 2009. Crystallization and preliminary X-ray analysis of neoagarobiose hydrolase from Saccharophagus degradans 2-40. Acta. Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 65: 1299-1301. https://doi.org/10.1107/S174430910904603X
  11. Seok JH, Kim HS, Hatada Y, Nam SW, Kim YH. 2012. Construction of an expression system for the secretory production of recombinant ${\alpha}$-agarase in yeast. Biotechnol. Lett. 34: 1041-1049. https://doi.org/10.1007/s10529-012-0864-0
  12. Park HS, Kim HC, Shin DH, Kim JK, Nam SW. 2005. Expression of Thermomonoepora fusca exoglucanase in Saccharomyces cerevisiae and its application to cellulose hydrolysis. Korean J. Microbiol. Biotechnol. 33: 267-273.
  13. Asghar S, Lee CR, Chi WJ, Kang DK, Hong SK. 2019.Molecular cloning and characterization of a novel cold-adapted alkaline 1,3-${\alpha}$-3,6-anhydro-L-galactosidase, Ahg558, from Gayadomonas joobiniege G7. Appl. Biochem. Biotechnol. 188: 1077-1095. https://doi.org/10.1007/s12010-019-02963-w
  14. Choi HJ, Kim YH. 2018. Simultaneous and sequential integration by cre/loxP site-specific recombination in Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 28: 826-830. https://doi.org/10.4014/jmb.1802.02004
  15. Kim MJ, Nam SW, Tamono K, Machida M, Kim SK, Kim YH. 2011. Optimization for production of exo-${\beta}$-1,3-glucanase (laminarinase) from Aspergillus oryzae in Saccharomyces cerevisiae. Korean Soc. Biotech. Bioeng. J. 26: 427-432.
  16. Latchinian-Sadek L, Thomas DY. 1993. Expression, purification, and characterization of the yeast KEX1 gene product, a polypeptide precursor processing carboxypeptidase. J. Biol. Chem. 268: 534-540. https://doi.org/10.1016/S0021-9258(18)54184-3
  17. Jo JH, Oh SY, Lee HS, Park YC, Seo JH. 2015. Dual utilization of NADPH and NADH cofactors enhances xylitol production in engineered Saccharomyces cerevisiae. Biotechnol. J. 10: 1935-1943. https://doi.org/10.1002/biot.201500068
  18. Jung HM, Kim JW, Kim YH. 2016. Secretory expression system of xylose reductase (GRE3) for optimal production of xylitol. J. Life Sci. 26: 1376-1382. https://doi.org/10.5352/JLS.2016.26.12.1376
  19. Hummon AB, Lim SR, Difilippantonio MJ, Ried T. 2007. Isolation and solubilization of proteins after TRIZOL extraction of RNA and DNA from patient material following prolonged storage. Biotechniques 42: 467-472. https://doi.org/10.2144/000112401