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Increased homologous integration frequency in Yarrowia lipolytica strains defective in non-homologous end-joining

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Abstract

The ascomycetous yeast Yarrowia lipolytica has been established as model system for studies of several research topics as well as for biotechnological processes in the last two decades. However, frequency of heterologous recombination is high in this yeast species, and so knockouts of genes are laborious to achieve. Therefore, the aim of this study was to check whether a reduction of non-homologous end-joining (NHEJ) of double strand breaks (DSB) results in a strong increase of proportion of homologous recombinants. The Ku70–Ku80 heterodimer is known as an essential protein complex of the NHEJ. We show that deletion of YlKU70 and/or YlKU80 results in an increase of the rate of transformants with homologous recombination (HR) up to 85 % in each case. However, it never reaches near 100 % of HR in any case as described for some other yeast. Furthermore, we demonstrated that growth of Δylku strains was similar to that of the wild-type strain. In addition, no differences were detected between the Δylku strains and the parent strain in respect to sensitivity to the mutagenic agent EMS as well as to the antibiotics hygromycin, bleomycin and nourseothricin. However, Δylku70 and Δylku80 strain showed a slightly higher sensitivity against UV rays. Thus, the new constructed Δylku strains are attractive recipient strains for homologous integration of DNA fragments and a valuable tool for directed knockouts of genes. Nevertheless, our data suggest the existence of another system of non-homologous recombination what may be subject of further investigation.

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Acknowledgments

We would like to thank Maria Sperrhake and Thomas Uhlig for supplying the vectors pTRPPTU and pUC-HIS3-DK as well as Annett Buchwald for her technical help during this work.

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  1. Anne Kretzschmar

    Present address: Laboratoriumsmedizin Köln, Dres. Med. Wisplinghoff and Kollegen, Classen-Kappelmann-Straße 24, 50931, Köln, Germany

  2. Severine Werner

    Present address: CUP Laboratorien Dr. Freitag GmbH, Carl-Eschebach-Str. 7, 01454, Radeberg, Germany

Authors and Affiliations

  1. Institut für Mikrobiologie, Technische Universität Dresden, 01062, Dresden, Germany

    Christina Otto, Martina Holz, Linda Hübner & Gerold Barth

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  1. Anne Kretzschmar
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Correspondence to Christina Otto.

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Communicated by C. Gaillardin.

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Kretzschmar, A., Otto, C., Holz, M. et al. Increased homologous integration frequency in Yarrowia lipolytica strains defective in non-homologous end-joining. Curr Genet 59, 63–72 (2013). https://doi.org/10.1007/s00294-013-0389-7

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