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Microbiology and Biotechnology Letters

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Molecular and Cellular Microbiology / Biomedical Sciences  |  Molecular Genetics, Omics, and Systems Biology

Microbiol. Biotechnol. Lett. 2019; 47(4): 667-672

https://doi.org/10.4014/mbl.1907.07004

Received: July 9, 2019; Accepted: September 24, 2019

출아효모에서 다양한 이종 유전자의 안정적 동시발현을 위한 방법의 비교

Comparison of Methods for Stable Simultaneous Expression of Various Heterologous Genes in Saccharomyces cerevisiae

Jung Heo-Myung and Kim Yeon-Hee *

Biomedical Engineering and Biotechnology Major, Divison of Applied Bioengineering, Dong-Eui University

We compared two integration systems for stable expression of heterologous genes in Saccharomyces cerevisiae. A Candida glabrata-derived gene was used as the selective marker for the Cre/loxP system, and XYLP, XYLB, GRE3, and XYL2 genes were used as model heterologous genes and ligated into the universal pRSCMT vector. The resulting pRS-XylP, pRS-XylB, pRS-Gre3, and pRS-Xyl2 plasmids were sequentially integrated into yeast chromosome VII by four integration processes (marker rescue and gene integration). The four introduced genes were successfully expressed. Further, the pRS-PBG2 plasmid harboring expression cassettes for the four genes was constructed for one-step integration. The four genes that were introduced were stably maintained as a gene cluster and were simultaneously expressed. The one-step integration was more effective for the simultaneous integration and expression of the four genes related to xylan/xylose metabolism. This method will enable the generation of a useful biosystem through appropriate use of gene integration methods.

Keywords: Candida glabrata marker, one-step integration, Saccharomyces cerevisiae, sequential integration, xylan/xylose metabolism related genes

  1. Murray AW, Szostak JW. 1983. Pedigree analysis of plasmid segregation in yeast. Cell 34: 961-970.
    CrossRef
  2. Romanos MA, Scorer CA, Clare JJ. 1992. Foreign gene expression in yeast: a review. Yeast 8: 423-488.
    Pubmed CrossRef
  3. Lee FW, Da Silva NA. 1997. Sequential delta-integration for the regulated insertion of cloned genes in Saccharomyces cerevisiae. Biotechnol. Prog. 13: 368-373.
    Pubmed CrossRef
  4. Güldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH. 1996. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 24: 2519-2524.
    Pubmed KoreaMed CrossRef
  5. Kim YH, Sugiyama M, Yamagishi K, Kaneko Y, Fukui K, Kobayashi A, et al. 2005. A versatile and general splitting technology for generating targeted YAC subclones. Appl. Microbiol. Biotechnol. 69: 65-70.
    Pubmed CrossRef
  6. Jung HM, Kim YH. 2018. Simultaneous overexpression of integrated genes by copy number amplification of a mini-yeast artificial chromosome. J. Microbiol. Biotechnol. 28: 821-825.
    Pubmed CrossRef
  7. Choi HJ, Kim YH. 2018. Simultaneous and sequential integration by Cre/loxP site-specific recombination in Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 28: 826-830.
    Pubmed CrossRef
  8. Sugiyama M, Ikushima S, Nakazawa T, Kaneko Y, Harashima S. 2005. PCR-mediated repeated chromosome splitting in Saccharomyces cerevisiae. BioTechniques 38: 909-914.
    Pubmed CrossRef
  9. 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.
    Pubmed CrossRef
  10. Kim YH, Nam SW. 2010. Development of simultaneous YAC manipulation-amplification (SYMA) system by chromosome splitting technique harboring copy number amplification system. J. Life Sci. 20: 789-793.
    CrossRef
  11. Chun YC, Jung KH, Lee JC, Park SH, Chung HK, Yoon KH. 1998. Molecular cloning and the nucleotide sequence of a Bacillus sp. KK-1 β-xylosidase gene. J. Microbiol. Biotechnol. 8: 28-33.
  12. Lee LH, Kim DY, Han MK, Oh HW, Ham SJ, Park DS, et al. 2009. Characterization of an extracellular xylanase from Bacillus sp. HY-20, a bacterium in the gut of Apis mellifera. Korean J. Microbiol. 45: 332-338.
  13. Kim SR, Kwee NR, Kim B, Jin YS. 2013. Feasibility of xylose fermentation by engineered Saccharomyces cerevisiae overexpressing endogenous aldose reductase (GRE3), xylitol dehydrogenase (XYL2), and xylulose kinase (XYL3) from Scheffersomyces stipitis. FEMS Yeast Res. 13: 312-321.
    Pubmed CrossRef
  14. Kim MJ, Kim BH, Nam SW, Choi ES, Shin DH, Cho HY, et al. 2013. Efficient secretory expression of recombinant endoxylanase from Bacillus sp. HY-20 in Saccharomyces cerevisiae, J. Life Sci. 23: 863-868.
    CrossRef

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