Current Microbiology

, Volume 64, Issue 4, pp 357–364 | Cite as

The Construction of a New Integrative Vector with a New Selective Marker of Copper Resistance for Glycerol Producer Candida glycerinogenes

  • Xiaona Gao
  • Bin Zhuge
  • Huiying Fang
  • Jian Zhuge


Candida glycerinogenes WL2002-5 has been used for industrial-scale fermentation of glycerol and may be a promising genetic host due to its tolerance to high osmotic pressure and fast growth. It resists many kinds of drugs, such as G418/hygromycin/cycloheximide. In previous studies, only Zeocin was used as a drug-resistant marker. But Zeocin is so expensive that it largely limits the genetic and molecular study. Here, we constructed a eukaryotic integrative vector pGAPZU, based on pGAPZB, to gain a new selectable marker of copper resistance for this strain. The results showed that the CUP1 gene of Saccharomyces cerevisiae elevated copper resistance of C. glycerinogenes. The C. glycerinogenes transformed with recombinant vector pGUC, obtained from introducing CUP1 gene into plasmid pGAPZU, could resist 21 mM copper, while the minimum inhibitory concentration (MIC) of wild type was 18 mM in solid YEPD medium. With copper resistance as a selective marker, research cost was largely reduced from 114.0 $/L with Zeocin as selective marker to 0.1 $/L. The new expression vector pGUC and selective marker of copper resistance gene establish a good foundation for further study on this industrial strain.


Polymerase Chain Reaction Amplification URA3 Gene Copper Resistance Autonomous Replication Sequence Polymerase Chain Reaction Experiment 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This study was supported by the National High Technology Research and Development Program of China (863 Program, No. 2009AA02Z210) and the 111 Project (No. 111-2-06).


  1. 1.
    Bayle D, Wangler S, Weitzenegger T, Steinhilber W, Volz J, Przybylski M et al (1998) Properties of the P-type ATPases encoded by the copAP operons of Helicobacter pylori and Helicobacter felis. J Bacteriol 180:317–329PubMedGoogle Scholar
  2. 2.
    Calmels T, Parriche M, Burand H, Tiraby G (1991) High efficiency transformation of Tolypocladium geodes conidiospores to phleomycin resistance. Curr Genet 20:309–314PubMedCrossRefGoogle Scholar
  3. 3.
    Chand-Goyal T, Eckert JW, Droby S, Glickmann E, Atkinson K (1999) Transformation of Candida oleophila and survival of a transformant on orange fruit under field conditions. Curr Genet 35:51–57PubMedCrossRefGoogle Scholar
  4. 4.
    Chen XZ, Fang HY, Rao ZM, Shen W, Zhuge B, Wang ZX, Zhuge J (2008) An efficient genetic transformation method for glycerol producer Candida glycerinogenes. Microbiol Res 163:531–553PubMedCrossRefGoogle Scholar
  5. 5.
    Drocourt D, Calmels T, Reynes JP, Baron M, Tiraby G (1990) Cassettes of the Streptoalloteichus hindustanus ble gene for transformation of lower and higher eukaryotes to phleomycin resistance. Nucleic Acids Res 18:4009PubMedCrossRefGoogle Scholar
  6. 6.
    Ge Z, Taylor DE (1996) Helicobacter pylori genes hpcopA and hpcopP constitute a cop operon involved in copper export. FEMS Microbiol Lett 145:181–188PubMedCrossRefGoogle Scholar
  7. 7.
    Halliwell B, Gutteridge JM (1990) Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol 186:1–85PubMedCrossRefGoogle Scholar
  8. 8.
    Hamer DH, Thiele DJ, Lemontt JE (1985) Function and autoregulation of yeast copperthionein. Science 228:685–690PubMedCrossRefGoogle Scholar
  9. 9.
    Hicks JB, Hinnen A, Fink GR (1979) Properties of yeast transformation. Cold Spring Harb Symp Quant Biol 43:1305–1313PubMedCrossRefGoogle Scholar
  10. 10.
    Ito H, Inouhe M, Tohoyama H, Joho M (2007) Characteristics of copper tolerance in Yarrowia lipolytica. Biometals 20:773–780PubMedCrossRefGoogle Scholar
  11. 11.
    Jayaram M, Li YY, Broach JR (1983) The yeast plasmid 2 [mu] circle encodes components required for its high copy propagation. Cell 34:95–104PubMedCrossRefGoogle Scholar
  12. 12.
    Jin HR, Fang HY, Zhuge J (2003) By-product formation by a novel glycerol-producing yeast, Candida glycerinogenes, with different O2 supplies. Biotechnol Lett 25:311–314PubMedCrossRefGoogle Scholar
  13. 13.
    Kim DY, Song WY, Yang YY (2001) The role of PDR13 in tolerance to high copper stress in budding yeast. FEBS Lett 508:99–102PubMedCrossRefGoogle Scholar
  14. 14.
    Liu XD, Thiele DJ (1997) Yeast metallothionein gene expression in response to metals and oxidative stress. Methods 11:289–299PubMedCrossRefGoogle Scholar
  15. 15.
    Lundblad V (2001) Yeast cloning vectors and genes. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current protocols in molecular biology. John Wiley & Sons, Inc., New York, pp 13.4.1–13.4.10Google Scholar
  16. 16.
    Odermatt A, Suter H, Krapf R, Solioz M (1992) An ATPase operon involved in copper resistance by Enterococcus hirae. Ann N Y Acad Sci 671:484–486PubMedCrossRefGoogle Scholar
  17. 17.
    Shen W, Wang ZX, Rao ZM, Zhuge J, Zhuge B (2010) A genetic transformation system based on trp1 complementation in C. glycerinogenes. World J Microbiol Biotechnol 27:1005–1008CrossRefGoogle Scholar
  18. 18.
    Wang ZX, Zhuge J, Fang HY (1999) A new osmotolerant and glycerol-highly-producing species–Candida glycerolgenesis Zhuge sp. nov. Wei Sheng Wu Xue Bao 39:68–74 [Article in Chinese]Google Scholar
  19. 19.
    Xie T, Fang HY, Zhuge B, Zhuge J (2009) Promotional mechanism of high glycerol productivity in the aerobic batch fermentation of Candida glycerinogenes after feeding several amino acids. Appl Biochem Micro 45:303–308CrossRefGoogle Scholar
  20. 20.
    Xu J, Tian YS, Peng RH (2009) Yeast copper-dependent transcription factor ACE1 enhanced copper stress tolerance in Arabidopsis. BMB Rep 42:752–757PubMedCrossRefGoogle Scholar
  21. 21.
    Zhuge J, Fang HY, Wang ZX, Chen DZ, Jin HR, Gu HL (2001) Glycerol production by a novel osmotolerant yeast C. glycerinogenes. Appl Microbiol Biotechnol 55:686–692PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Xiaona Gao
    • 1
  • Bin Zhuge
    • 1
  • Huiying Fang
    • 1
  • Jian Zhuge
    • 1
  1. 1.The Key Laboratory of Industrial Biotechnology, Ministry of Education, Research Centre of Industrial Microorganisms, School of BiotechnologyJiangnan UniversityWuxiChina

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