Current Genetics

, Volume 60, Issue 3, pp 223–230 | Cite as

An optimized transformation protocol for Lipomyces starkeyi

  • Christopher H. Calvey
  • Laura B. Willis
  • Thomas W. Jeffries
Research Article


We report the development of an efficient genetic transformation system for Lipomyces starkeyi based on a modified lithium acetate transformation protocol. L. starkeyi is a highly lipogenic yeast that grows on a wide range of substrates. The initial transformation rate for this species was extremely low, and required very high concentrations of DNA. A systematic approach for optimizing the protocol resulted in an increase in the transformation efficiency by four orders of magnitude. Important parameters included cell density, the duration of incubation and recovery periods, the heat shock temperature, and the concentration of lithium acetate and carrier DNA within the transformation mixture. We have achieved efficiencies in excess of 8,000 transformants/µg DNA, which now make it possible to screen libraries in the metabolic engineering of this yeast. Metabolic engineering based on this transformation system could improve lipogenesis and enable formation of higher value products.


Lipomyces starkeyi Lithium acetate Transformation Oleaginous yeast 



This work was funded in part by the Department of Energy Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494). CHC was supported in part through a Grant entitled “Investigation of Lipid Accumulation in Lipomyces starkeyi” awarded by the Graduate School of University of Wisconsin-Madison to TWJ. CHC gratefully acknowledges Kenneth Hammel for sponsorship at the USDA Forest Products Laboratory.

Conflict of interest

TWJ is the president and owner of Xylome, a private entity. However, the research carried out in this program was conducted independently of Xylome, and the authors declare that they have no conflict of interest.


  1. Angerbauer C, Siebenhofer M, Mittelbach M, Guebitz GM (2008) Conversion of sewage sludge into lipids by Lipomyces starkeyi for biodiesel production. Bioresour Technol 99:3051–3056PubMedCrossRefGoogle Scholar
  2. Berardi E, Thomas DY (1990) An effective transformation method for Hansenula polymorpha. Curr Genet 18:169–170. doi: 10.1007/BF00312606 CrossRefGoogle Scholar
  3. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345. doi: 10.1038/nmeth.1318 PubMedCrossRefGoogle Scholar
  4. Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/ss carrier DNA/PEG method. Nat Protoc 2:31–34. doi: 10.1038/nprot.2007.13 PubMedCrossRefGoogle Scholar
  5. Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96PubMedCrossRefGoogle Scholar
  6. Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/ss-DNA/PEG procedure. Yeast 11:355–360. doi: 10.1002/yea.320110408 PubMedCrossRefGoogle Scholar
  7. Jeffries TW, Nelson SS, Mahan SD (2011) Metabolically engineered yeasts for the production of ethanol and other products from xylose and cellobiose. US Patent Application 20110262983Google Scholar
  8. Laplaza JM, Torres BR, Jin YS, Jeffries TW (2006) Sh Ble and Cre adapted for functional genomics and metabolic engineering of Pichia stipitis. Enzym Microb Technol 38:741–747. doi: 10.1016/j.enzmictec.2005.07.024 CrossRefGoogle Scholar
  9. Lin J, Shen H, Tan H, Zhao X, Wu S, Hu C, Zhao SK (2011) Lipid production by Lipomyces starkeyi cells in glucose solution without auxiliary nutrients. J Biotechnol 152:184–188. doi: 10.1016/j.jbiotec.2011.02.010 PubMedCrossRefGoogle Scholar
  10. Pham TA, Kawai S, Kono E, Murata K (2011) Visualization of the synergistic effect of lithium acetate and single-stranded carrier DNA on Saccharomyces cerevisiae transformation. Curr Genet 57:233–239. doi: 10.1007/s00294-011-0341-7 PubMedCrossRefGoogle Scholar
  11. Schiestl RH, Gietz RD (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet 16:339–346. doi: 10.1007/BF00340712 PubMedCrossRefGoogle Scholar
  12. Schiestl RH, Manivasakam P, Woods RA, Gietz RD (1993) Introducing DNA into yeast by transformation. Methods 5:79–85. doi: 10.1006/meth.1993.1011 CrossRefGoogle Scholar
  13. Tai M, Stephanopoulos G (2013) Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. Metab Eng 15:1–9. doi: 10.1016/j.ymben.2012.08.007 PubMedCrossRefGoogle Scholar
  14. Tripp JD, Lilley JL, Wood WN, Lewis LK (2013) Enhancement of plasmid DNA transformation efficiencies in early stationary-phase yeast cell cultures. Yeast 30:191–200. doi: 10.1002/yea.2951 PubMedCentralPubMedCrossRefGoogle Scholar
  15. Walther A, Wendland J (2003) An improved transformation protocol for the human fungal pathogen Candida albicans. Curr Genet 42:339–343. doi: 10.1007/s00294-002-0349-0 PubMedCrossRefGoogle Scholar
  16. Wang TT, Choi YJ, Lee BH (2001) Transformation systems of non-Saccharomyces yeasts. Crit Rev Biotechnol 21:177–218PubMedCrossRefGoogle Scholar
  17. Wang P, Wan X, Zhang Y, Jiang M (2011) Production of γ-linolenic acid using a novel heterologous expression system in the oleaginous yeast Lipomyces kononenkoae. Biotechnol Lett 33:1993–1998PubMedCrossRefGoogle Scholar
  18. Wild R, Patil S, Popovi M, Zappi M, Dufreche S, Bajpai R (2010) Lipids from Lipomyces starkeyi. Food Technol Biotechnol 48:329–335Google Scholar
  19. Yehuda H, Droby S, Wisniewski M, Goldway M (2001) A transformation system for the biocontrol yeast, Candida oleophila, based on hygromycin B resistance. Curr Genet 40:282–287PubMedCrossRefGoogle Scholar
  20. Zhao X, Kong X, Hua Y, Feng B, Zhao ZK (2008) Medium optimization for lipid production through co-fermentation of glucose and xylose by the oleaginous yeast Lipomyces starkeyi. Eur J Lipid Sci Tech 110:405–412. doi: 10.1002/ejlt.200700224 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Christopher H. Calvey
    • 1
    • 2
  • Laura B. Willis
    • 1
    • 2
    • 3
  • Thomas W. Jeffries
    • 1
    • 2
    • 3
  1. 1.Department of BacteriologyUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.Great Lakes Bioenergy Research CenterUniversity of Wisconsin-MadisonMadisonUSA
  3. 3.Institute for Microbial and Biochemical Technology, Forest Products LaboratoryUSDA Forest ServiceMadisonUSA

Personalised recommendations