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Gene Targeting by RNAi-Mediated Knockdown of Potent DNA Ligase IV Homologue in the Cellulase-Producing Fungus Talaromyces cellulolyticus

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Abstract

The genome of the cellulase-producing fungus Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) was screened for a potent DNA ligase IV gene (ligD homologue). Homologous recombination efficiency in T. cellulolyticus is very low. Therefore, suppression of a non-homologous end-joining system was attempted to enable specific gene knockouts for molecular breeding. The transcript levels of ligD homologue were 0.037 of those of the parental YP-4 strain in the Li20 transformant carrying the RNAi construct targeting the ligD homologue. Transformation of the hairpin-type RNAi vector into T. cellulolyticus could be useful in fungal gene knockdown experiments. Cellulase production and protein secretion were similar in the parental YP-4 strain and the Li20 transformant. Knockout transformation of ligD homologue using the Li20 transformant led to 23.1 % double crossover gene targeting. Our results suggest that the potent DNA ligase IV gene of T. cellulolyticus is related to non-homologous end joining and that the knockdown of the ligD homologue is useful in gene targeting.

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Acknowledgments

This study was supported by the New Energy and Industrial Technology Development Organization (NEDO), Japan.

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Authors and Affiliations

  1. Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan

    Koutarou Hayata, Seiya Asada & Shigeki Sawayama

  2. Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-0046, Japan

    Tatsuya Fujii, Hiroyuki Inoue & Kazuhiko Ishikawa

Authors
  1. Koutarou Hayata
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  2. Seiya Asada
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  3. Tatsuya Fujii
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  4. Hiroyuki Inoue
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  5. Kazuhiko Ishikawa
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  6. Shigeki Sawayama
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Correspondence to Shigeki Sawayama.

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Hayata, K., Asada, S., Fujii, T. et al. Gene Targeting by RNAi-Mediated Knockdown of Potent DNA Ligase IV Homologue in the Cellulase-Producing Fungus Talaromyces cellulolyticus . Appl Biochem Biotechnol 174, 1697–1704 (2014). https://doi.org/10.1007/s12010-014-1142-5

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  • DOI: https://doi.org/10.1007/s12010-014-1142-5

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