Biotechnology Letters

, Volume 35, Issue 4, pp 571–576 | Cite as

Efficient homologous recombination with short length flanking fragments in Ku70 deficient Yarrowia lipolytica strains

  • Jonathan Verbeke
  • Athanasios Beopoulos
  • Jean-Marc NicaudEmail author
Original Research Paper


In Yarrowia lipolytica, targeted gene replacement occurs only with long length (1 kb) homologous flanking fragments, as this yeast preferentially uses the non-homologous end-joining mechanism (NHEJ) for DNA repair over homologous recombination (HR). To improve the frequency of HR, we identified and disrupted the KU70 and KU80 genes responsible for double strand break repair in the NHEJ pathway in Y. lipolytica. In ku70∆ HR of URA3 marker at the ADE2 locus occurred with 43 % frequency with as little as 50 bp long flanking regions. The number of Ura+ transformants was reduced to 1 % of the Po1d (ura3-302) wild type-like strain level, regardless of the flanking fragment length. On the contrary, even though HR was not improved in ku80∆, Ura+ transformants was 60 % lower compared to the wild type.


Gene targeting Homologous recombination Ku complex NHEJ Yarrowia lipolytica 

Supplementary material

10529_2012_1107_MOESM1_ESM.docx (93 kb)
Supplementary material 1 (DOCX 92 kb)


  1. Barth G, Gaillardin C (1996) Yarrowia lipolytica. In: Wolf K (ed) Non conventional yeasts in biotechnology, vol 1. Springer, New York, pp 313–388CrossRefGoogle Scholar
  2. Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, Nicaud JM (2009) Yarrowia lipolytica as a model for bio-oil production. Prog Lipid Res 48:375–387PubMedCrossRefGoogle Scholar
  3. Beopoulos A, Nicaud JM, Gaillardin C (2011) An overview of lipid metabolism in yeasts and its impact on biotechnological processes. Appl Microbiol Biotechnol 90:1193–1206PubMedCrossRefGoogle Scholar
  4. Bordes F, Fudalej F, Dossat V, Nicaud JM, Marty A (2007) A new recombinant protein expression system for high-throughput screening in the yeast Yarrowia lipolytica. J Microbiol Methods 70:493–502PubMedCrossRefGoogle Scholar
  5. Boulton SJ, Jackson SP (1998) Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing. EMBO J 17:1819–1828PubMedCrossRefGoogle Scholar
  6. De Pourcq K, Tiels P, Van Hecke A, Geysens S, Vervecken W, Callewaert N (2012) Engineering Yarrowia lipolytica to produce glycoproteins homogeneously modified with the universal man3glcnac2 n-glycan core. PLoS ONE 7:e39976PubMedCrossRefGoogle Scholar
  7. Dear S, Staden R (1991) A sequence assembly and editing program for efficient management of large projects. Nucleic Acids Res 19:3907–3911PubMedCrossRefGoogle Scholar
  8. Doherty AJ, Jackson SP, Weller GR (2001) Identification of bacterial homologues of the Ku DNA repair proteins. FEBS Lett 500:186–188PubMedCrossRefGoogle Scholar
  9. Dujon B, Sherman D et al (2004) Genome evolution in yeasts. Nature 430:35–44PubMedCrossRefGoogle Scholar
  10. Fickers P, Le Dall MT, Gaillardin C, Thonart P, Nicaud JM (2003) New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J Microbiol Methods 55:727–737PubMedCrossRefGoogle Scholar
  11. Gaillardin C, Ribet AM, Heslot H (1985) Integrative transformation of the yeast Yarrowia lipolytica. Curr Genet 10:49–58CrossRefGoogle Scholar
  12. Kooistra R, Hooykaas PJ, Steensma HY (2004) Efficient gene targeting in Kluyveromyces lactis. Yeast 21:781–792PubMedCrossRefGoogle Scholar
  13. Lustig AJ (1999) The kudos of non-homologous end-joining. Nat Genet 23:130–131PubMedCrossRefGoogle Scholar
  14. Mlickova K, Roux E, Athenstaedt K, d’Andrea S, Daum G, Chardot T, Nicaud JM (2004) Lipid accumulation, lipid body formation, and acyl coenzyme A oxidases of the yeast Yarrowia lipolytica. Appl Environ Microbiol 70:3918–3924PubMedCrossRefGoogle Scholar
  15. Nicaud JM (2012) Yarrowia lipolytica. Yeast 29:409–418PubMedCrossRefGoogle Scholar
  16. Querol A, Barrio E, Huerta T, Ramon D (1992) Molecular monitoring of wine fermentations conducted by active dry yeast strains. Appl Environ Microbiol 58:2948–2953PubMedGoogle Scholar
  17. Richard GF, Kerrest A, Lafontaine I, Dujon B (2005) Comparative genomics of hemiascomycete yeasts: genes involved in DNA replication, repair, and recombination. Mol Biol Evol 22:1011–1023PubMedCrossRefGoogle Scholar
  18. Sambrook J, Maniatis T, Fritsch EF (1989) Molecular cloning : a laboratory manual. Cold Spring Harbor, New YorkGoogle Scholar
  19. Takahashi T, Masuda T, Koyama Y (2006) Enhanced gene targeting frequency in ku70 and ku80 disruption mutants of Aspergillus sojae and Aspergillus oryzae. Mol Genet Genomics 275:460–470PubMedCrossRefGoogle Scholar
  20. Takita Y, Takahara M, Nogami S, Anraku, Ohya Y (1997) Applications of the long and accurate polymerase chain reaction method in yeast molecular biology: direct sequencing of the amplified DNA and its introduction into yeast. Yeast 13:763–768PubMedCrossRefGoogle Scholar
  21. Van Dyck E, Stasiak AZ, Stasiak A, West SC (1999) Binding of double-strand breaks in DNA by human Rad52 protein. Nature 398:728–731PubMedCrossRefGoogle Scholar
  22. Wang J, Zhang B, Chen S (2011) Oleaginous yeast Yarrowia lipolytica mutants with a disrupted fatty acyl-coa synthetase gene accumulate saturated fatty acid. Proc Biochem 46:1436–1441CrossRefGoogle Scholar
  23. Yamana Y, Maeda T, Ohba H, Usui T, Ogawa HI, Kusano K (2005) Regulation of homologous integration in yeast by the DNA repair proteins Ku70 and RecQ. Mol Genet Genomics 273:167–176PubMedCrossRefGoogle Scholar
  24. Zhang X, Chen W, Zhang Y, Jiang L, Chen Z, Wen Y, Li J (2012) Deletion of ku homologs increases gene targeting frequency in Streptomyces avermitilis. J Ind Microbiol Biotechnol 39:917–925PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Jonathan Verbeke
    • 1
  • Athanasios Beopoulos
    • 1
  • Jean-Marc Nicaud
    • 1
    • 2
    Email author
  1. 1.INRA, MicalisJouy-en-JosasFrance
  2. 2.CNRS, MicalisJouy-en-JosasFrance

Personalised recommendations