Abstract
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.
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References
Barth G, Gaillardin C (1996) Yarrowia lipolytica. In: Wolf K (ed) Non conventional yeasts in biotechnology, vol 1. Springer, New York, pp 313–388
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 48:375–387
Beopoulos A, Nicaud JM, Gaillardin C (2011) An overview of lipid metabolism in yeasts and its impact on biotechnological processes. Appl Microbiol Biotechnol 90:1193–1206
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 70:493–502
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 17:1819–1828
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:e39976
Dear S, Staden R (1991) A sequence assembly and editing program for efficient management of large projects. Nucleic Acids Res 19:3907–3911
Doherty AJ, Jackson SP, Weller GR (2001) Identification of bacterial homologues of the Ku DNA repair proteins. FEBS Lett 500:186–188
Dujon B, Sherman D et al (2004) Genome evolution in yeasts. Nature 430:35–44
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 55:727–737
Gaillardin C, Ribet AM, Heslot H (1985) Integrative transformation of the yeast Yarrowia lipolytica. Curr Genet 10:49–58
Kooistra R, Hooykaas PJ, Steensma HY (2004) Efficient gene targeting in Kluyveromyces lactis. Yeast 21:781–792
Lustig AJ (1999) The kudos of non-homologous end-joining. Nat Genet 23:130–131
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 70:3918–3924
Nicaud JM (2012) Yarrowia lipolytica. Yeast 29:409–418
Querol A, Barrio E, Huerta T, Ramon D (1992) Molecular monitoring of wine fermentations conducted by active dry yeast strains. Appl Environ Microbiol 58:2948–2953
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 22:1011–1023
Sambrook J, Maniatis T, Fritsch EF (1989) Molecular cloning : a laboratory manual. Cold Spring Harbor, New York
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 275:460–470
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 13:763–768
Van Dyck E, Stasiak AZ, Stasiak A, West SC (1999) Binding of double-strand breaks in DNA by human Rad52 protein. Nature 398:728–731
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 46:1436–1441
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 273:167–176
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 39:917–925
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Verbeke, J., Beopoulos, A. & Nicaud, JM. Efficient homologous recombination with short length flanking fragments in Ku70 deficient Yarrowia lipolytica strains. Biotechnol Lett 35, 571–576 (2013). https://doi.org/10.1007/s10529-012-1107-0
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DOI: https://doi.org/10.1007/s10529-012-1107-0