DOI QR코드

DOI QR Code

Secretory Expression System of Xylose Reductase (GRE3) for Optimal Production of Xylitol

Xylitol 생산에 최적화된 xylose reductase (GRE3)의 분비발현 시스템

  • Jung, Hoe-Myung (Department of Smart-Biohealth, Dong-Eui University) ;
  • Kim, Jae-Woon (Department of Biotechnology and Bioengineering, Dong-Eui University) ;
  • Kim, Yeon-Hee (Department of Smart-Biohealth, Dong-Eui University)
  • 정회명 (동의대학교 스마트바이오헬스학과) ;
  • 김재운 (동의대학교 생명공학과) ;
  • 김연희 (동의대학교 스마트바이오헬스학과)
  • Received : 2016.10.04
  • Accepted : 2016.11.11
  • Published : 2016.12.30

Abstract

Xylitol is widely used in the food and medical industry. It is produced by the reduction of xylose (lignocellulosic biomass) in the Saccharomyces cerevisiae strain, which is considered genetically safe. In this study, the expression system of the GRE3 (YHR104W) gene that encodes xylose reductase was constructed to efficiently produce xylitol in the S. cerevisiae strain, and the secretory production of xylose reductase was investigated. To select a suitable promoter for the expression of the GRE3 gene, pGMF-GRE3 and pAMF-GRE3 plasmid with GAL10 promoter and ADH1 promoter, respectively, were constructed. The mating factor ${\alpha}$ ($MF{\alpha}$) signal sequence was also connected to each promoter for secretory production. Each plasmid was transformed into S. cerevisiae $SEY2102{\Delta}trp1$, and $SEY2102{\Delta}trp1$/pGMF- GRE3 and $SEY2102{\Delta}trp1$/pAMF-GRE3 transformants were selected. In the $SEY2102{\Delta}trp1$/pGMF-GRE3 strain, the total activity of xylose reductase reached 0.34 unit/mg-protein when NADPH was used as a cofactor; this activity was 1.5 fold higher than that in $SEY2102{\Delta}trp1$/pAMF-GRE3 with ADH1 as the promoter. The secretion efficiency was 91% in both strains, indicating that most of the recombinant xylose reductase was efficiently secreted in the extracellular fraction. In a baffled flask culture of the $SEY2102{\Delta}trp1$/pGMF-GRE3 strain, 12.1 g/l of xylitol was produced from 20 g/l of xylose, and ~83% of the consumed xylose was reduced to xylitol.

Xylitol은 식품 및 의료산업에서 이용가치가 높은 물질로, lignocellulosic biomass인 xylose의 환원으로부터 생산되며, 대부분 유전적으로 안전한 Saccharomyces cerevisiae 균주를 사용하여 생산되고 있다. 따라서 본 연구에서는 S. cerevisiae에서 xylitol을 효율적으로 생산하기 위해 xylose reductase를 code하는 GRE3 (YHT104W)유전자의 발현시스템을 구축하여, xylose reductase의 분비생산 및 xylitol 생산성을 조사하고자 하였다. 먼저 GRE3 유전자의 발현에 적합한 promoter의 선별을 위해 GAL10 promoter와 ADH1 promoter 하류에 각각 mating factor ${\alpha}$ ($MF{\alpha}$) signal sequence와 GRE3 유전자를 가진 pGMF-GRE3와 pAMF-GRE3 plasmid를 구축하였다. 각각의 plasmid는 S. cerevisiae $SEY2102{\Delta}trp1$균주에 형질전환되었고, $SEY2102{\Delta}trp1$/pGMF-GRE3와 $SEY2102{\Delta}trp1$/pAMF-GRE3 형질전환주가 선별되었다. 그 중 $SEY2102{\Delta}trp1$/pGMF-GRE3 균주에서 NADPH를 cofactor로 사용했을 때 0.34 unit/mg-protein의 xylose reductase 활성(total activity)을 보였고, ADH1 promoter를 가진 $SEY2102{\Delta}trp1$/pAMF-GRE3 균주에 비해 1.5배 높은 활성증가를 확인 할 수 있었다. 또한 두 균주에서 모두 91%의 분비효율을 보여 대부분의 재조합 xylose reductase가 세포 밖으로 효율적으로 발현 분비되었음을 알 수 있었다. $SEY2102{\Delta}trp1$/pGMF-GRE3 균주를 사용한 baffled flask 배양에서 xylitol 생산량을 조사해 본 결과, 20 g/l의 xylose로부터 12.1 g/l의 xylitol을 생산하였고, 소모된 xylose의 약 83%정도가 xylitol로 환원되었음을 알 수 있었다.

Keywords

References

  1. Bradford, M. M. 1976. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  2. Clark, J. H. and Deswarte, F. 2011. Introduction to Chemicals from Biomass. pp. 344, John Wiley & Sons.
  3. Chung, B. H., Nam, S. W., Kim, B. M. and Park, Y. H. 1996. Highly-dfficient secretion of heterologous protein from Saccharomyces cerevisiae using inulinase signal peptide. Biotechnol. Bioeng. 49, 473-479.
  4. Ford, G. and Ellis, E. M. 2001. Three aldo-keto reductases of the yeast Saccharomyces cerevisiae. Chem. Biol. Interact 130, 685-698.
  5. Garay-Arroyo, A. and Covarrubias, A. A. 1999. Three genes whose expression is induced by stress in Saccharomyces cerevisiae. Yeast 15, 879-892. https://doi.org/10.1002/(SICI)1097-0061(199907)15:10A<879::AID-YEA428>3.0.CO;2-Q
  6. Gietz, R. D. and Schiestl, R. H. 1995. Transforming yeast with DNA. Methods Mol. Cell Biol. 5, 225-269.
  7. Hacker, B., Habenicht, A., Kiess, M. and Mattes, R. 1999. Xylose utilization: Cloning and characterization of the xylose reductase from Candida tenuis. Biological. Chem. 380, 1395-1403.
  8. Halborn, J., Walfridsson, M., Airaksinen, U., Ojamo, H., Hahnhagerdal, B., Penttila, M. and Keranen, S. 1991. Xylitol production by recombinant Saccharomyces cerevisiae. Bio Technol. 9, 1090-1095. https://doi.org/10.1038/nbt1191-1090
  9. Hummon, A. B., Lim, S. R., Difilippantonio, M. J. and 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
  10. Jeong, E. Y., Sopher, C., Kim, I. S. and Lee, H. 2001. Mutational study of the role of tyrosine-49 in the Saccharomyces cerevisiae xylose reductase. Yeast 18, 1081-1089. https://doi.org/10.1002/yea.758
  11. Jo, J. H., Oh, S. Y., Lee, H. S., Park, Y. C. and Seo, J. H. 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
  12. Kang, H. A., Nam, S. W., Kwon, K. S., Chung, B. H. and Yu, M. H. 1996. High-level secretion of human ${\alpha}1$-antitrypsin form Saccharomyces cerevisiae using inulinase signal sequence. J. Biotechnol. 48, 15-24. https://doi.org/10.1016/0168-1656(96)01391-0
  13. Kang, M. H., Ni, H. Y. and Jeffries, T. W. 2003. Molecular characterization of a gene for aldose reductase (CbXYL1) from Candida boidinii and its expression in Saccharomyces cerevisiae. Appl. Biochem. Biotechnol. 105. 265-276.
  14. Kim, M. J., Kim, B. H., Nam, S. W., Choi, E. S., Shin, D. H., Cho, H. Y., Son, K. H., Park, H. Y. and Kim, Y. H. 2013. Efficient secretory expression of recombinant endoxylanase from Bacillus sp. HY-20 in Saccharomyces cerevisiae. J. Life Sci. 23, 863-868. https://doi.org/10.5352/JLS.2013.23.7.863
  15. Kim, M. J., Nam, S. W., Tamano, K., Machida, M., Kim, S. K. and Kim, Y. H. 2011. Optimization for production of exo-${\beta}$-1,3-glucanase (laminarinase) from Aspergillus oryzae in Saccharomyces cerevisiae. Kor. J. Microbiol. Biotechnol. 26, 427-432.
  16. Kim, S. R., Kwee, N. R., Kim, H. J. and Jin, Y. S. 2013. Feasibility of xylose fermentation by engineered Saccharomyces cerevisiae overexpressing endogenous aldose reductase (GRE3), xylitol dehydrogenase (XYL2), and xylulokinase (XYL3) from Scheffersomyces stipitis. FEMS Yeast Res. 13, 312-321. https://doi.org/10.1111/1567-1364.12036
  17. Latchinian-Sadek, L. and Thomas, D. Y. 1993. Expression, purification, and characterization of the yeast KEX1 gene product, a polypeptide precursor processing carboxypeptidase. J. Biol. Chem. 268, 534-540.
  18. Lim, C. K., Kim, Y. K., Kim, K. H., Kim, C. H., Rhee, S. K. and Nam, S. W. 2004. Expression and secretion of Zymononas mobilis levansucrase in Saccharomyces cerevisiae. J. Life Sci. 14, 429-434. https://doi.org/10.5352/JLS.2004.14.3.429
  19. Makinen, K. K. 2011. Sugar alcohol sweeteners as alternatives to sugar with special consideration of xylitol. Med. Princ. Pract. 20, 303-320. https://doi.org/10.1159/000324534
  20. Mattam, A. J., Kuila, A., Suralikerimath, N., Choudary, N., Rao, P. V. and Velankar, H. R. 2016. Cellulolytic enzyme expression and simultaneous conversion of lignocellulosic sugars into ethanol and xylitol by a new Candida tropicalis strain. Biotechnol. Biofuels 9, 157-169. https://doi.org/10.1186/s13068-016-0575-1
  21. Nidetzky, B., Neuhauser, W., Haltrich, D. and Kulbe, K. D. 1996. Continuous enzymatic production of xylitol with simultaneous coenzyme regeneration in a charged membrane reactor. Biotechnol. Bioeng. 52. 387-396.
  22. Park, E., Park, M. H., Na, H. S. and Chung, J. 2015. Xylitol induces cell death in lung cancer A549 cells by autophagy. Biotechnol. Lett. 37, 983-990. https://doi.org/10.1007/s10529-014-1757-1
  23. Vernet, T., Dignard, D. and Thomas, D. Y. 1987. A family of yeast expression vectors containing the phage f1 intergenic region. Gene 52, 225-233. https://doi.org/10.1016/0378-1119(87)90049-7
  24. Wisniak, J., Hershkowitz, M., Leibowitz, R. and Stein, S. 1974. Hydrogenation of xylose to xylitol. Ind. Eng. Chem. Res. 13, 75-79. https://doi.org/10.1021/i360049a015
  25. Yadav, M., Mishra, D. K. and Hwang, J. S. 2012. Catalytic hydrogenation of xylose to xylitol using ruthenium catalyst on NiO modified TiO2 support. Appl. Catal., A 425, 110-116.