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Applied and Environmental Microbiology, September 2009, p. 5529-5535, Vol. 75, No. 17
0099-2240/09/$08.00+0     doi:10.1128/AEM.00648-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Establishment of Agrobacterium tumefaciens-Mediated Transformation of an Oleaginous Fungus, Mortierella alpina 1S-4, and Its Application for Eicosapentaenoic Acid Producer Breeding{triangledown}

Akinori Ando,1,2 Yosuke Sumida,1 Hiroaki Negoro,1 Dian Anggraini Suroto,1 Jun Ogawa,1,2 Eiji Sakuradani,1 and Sakayu Shimizu1*

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan,1 Research Division of Microbial Sciences, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan2

Received 19 March 2009/ Accepted 25 June 2009

Gene manipulation tools for an arachidonic-producing filamentous fungus, Mortierella alpina 1S-4, have not been sufficiently developed. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for M. alpina 1S-4 transformation, using the uracil-auxotrophic mutant (ura5– strain) of M. alpina 1S-4 as a host strain and the homologous ura5 gene as a selectable marker gene. Furthermore, the gene for {omega}3-desaturase, catalyzing the conversion of n-6 fatty acid to n-3 fatty acid, was overexpressed in M. alpina 1S-4 by employing the ATMT system. As a result, we revealed that the frequency of transformation surpassed 400 transformants/108 spores, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and most of the transformants (60 to 80%) showed mitotic stability. Moreover, the accumulation of n-3 fatty acid in transformants was observed under the conditions of optimal {omega}3-desaturase gene expression. In particular, eicosapentaenoic acid (20:5n-3), an end product of n-3 fatty acids synthesized in M. alpina 1S-4, reached a maximum of 40% of total fatty acids. In conclusion, the ATMT system was found to be effective and suitable for the industrial strain Mortierella alpina 1S-4 and will be a useful tool for basic mutagenesis research and for industrial breeding of this strain.

* Corresponding author. Mailing address: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan. Phone: 81 75 753 6122. Fax: 81 75 753 6128. E-mail: sim{at}kais.kyoto-u.ac.jp

{triangledown} Published ahead of print on 6 July 2009.

Applied and Environmental Microbiology, September 2009, p. 5529-5535, Vol. 75, No. 17
0099-2240/09/$08.00+0     doi:10.1128/AEM.00648-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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