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Establishment of an overall transformation system for an oil-producing filamentous fungus, Mortierella alpina 1S-4

  • Applied Genetics and Molecular Biotechnology
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Abstract

Oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. To determine its physiological properties and to clarify the biosynthetic pathways for polyunsaturated fatty acids, a transformation system for this fungus was established using a derivative of it, i.e., a ura5 − mutant lacking orotate phosphoribosyl transferase (OPRTase, EC.2.4.2.10) activity. Transformation with a vector containing the homologous ura5 gene as a marker was successfully performed using microprojectile bombardment, other methods frequently used for transformation, such as the protoplasting, lithium acetate, or electroporation methods, not giving satisfactory results. As a result, two types of transformants were obtained: a few stable transformants overexpressing the ura5 gene, and many unstable transformants showing OPRTase activity comparable to that of the wild-type strain. The results of quantitative PCR indicated that the stable transformants could retain the ura5 genes originating from the transformation vector regardless of the culture conditions. On the other hand, unstable transformants easily lost the marker gene under uracil-containing conditions, as expected. In this paper, we report that an overall transformation system for this fungus was successfully established, and propose how to select useful transformants as experimental and industrial strains.

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Acknowledgements

We wish to thank Dr. David B. Archer (School of Life and Environmental Sciences, University of Nottingham, United Kingdom) for providing the Mortierella transformation vector. This work was supported in part by the New Energy and Industrial Technology Development Organization (NEDO), and a Grant-in-Aid for Scientific Research (No. 15658024 to S.S.) from the Ministry of Education, Science, Sports, and Culture, Japan.

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Authors and Affiliations

  1. Laboratory of Fermentation Physiology and Applied Microbiology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan

    Seiki Takeno, Eiji Sakuradani, Shoichi Murata & Sakayu Shimizu

  2. Institute for Advanced Technology, Suntory Ltd, Osaka, 618-0001, Japan

    Misa Inohara-Ochiai & Toshihiko Ashikari

  3. Institute for Food and Beverage Health Care Science Laboratory, Suntory Ltd, Osaka, 618-0001, Japan

    Hiroshi Kawashima

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  1. Seiki Takeno
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  2. Eiji Sakuradani
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  3. Shoichi Murata
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  4. Misa Inohara-Ochiai
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  5. Hiroshi Kawashima
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  6. Toshihiko Ashikari
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  7. Sakayu Shimizu
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Correspondence to Sakayu Shimizu.

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Takeno, S., Sakuradani, E., Murata, S. et al. Establishment of an overall transformation system for an oil-producing filamentous fungus, Mortierella alpina 1S-4. Appl Microbiol Biotechnol 65, 419–425 (2004). https://doi.org/10.1007/s00253-004-1622-6

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  • DOI: https://doi.org/10.1007/s00253-004-1622-6

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