Establishment of Agrobacterium tumefaciens-mediated transformation of an oleaginous fungus, Mortierella alpina 1S-4, and its application for eicosapentaenoic acid producer breeding.
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 omega3-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/10(8) 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 omega3-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.
Ando
Akinori
A
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
Sumida
Yosuke
Y
Negoro
Hiroaki
H
Suroto
Dian Anggraini
DA
Ogawa
Jun
J
Sakuradani
Eiji
E
Shimizu
Sakayu
S
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D015825:Chromosomes, Fungal; D015118:Eicosapentaenoic Acid / Q000096:biosynthesis*; D044943:Fatty Acid Desaturases / Q000235:genetics; D005227:Fatty Acids / Q000378:metabolism; D018628:Gene Dosage; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D042822:Genomic Instability; D020094:Mortierella / Q000235:genetics*; D011995:Recombination, Genetic; D014170:Transformation, Genetic
D005227:Fatty Acids; D015118:Eicosapentaenoic Acid; D044943:Fatty Acid Desaturases; C078565:omega-3 fatty acid desaturase