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 ω3-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 ω3-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.Two decades ago, a filamentous zygomycete fungus, Mortierella alpina 1S-4, was isolated from soil as a potent producer of polyunsaturated fatty acids (PUFAs) in our laboratory and was utilized for commercial production of arachidonic acid (AA) (20:4n-6) (21). Breeding of mutants derived from the wild strain led to the production of dihomo-γ-linolenic acid (20:3n-6) and Mead acid (20:3n-9) (10-12) (Fig. ​(Fig.1).1). Furthermore, we attempted to produce other PUFAs synthesized in M. alpina 1S-4, since some fatty acids (e.g., 18:2n-9, 18:4n-3, and 20:4n-3) have limited natural sources and could have promising beneficial physiological effects (9). In particular, for microbial production of n-3 PUFAs, currently prepared from fish oil, it is necessary to achieve stable productivity and quality; however, mutation treatment caused low activity of the specific enzymes involved in PUFA biosynthesis, which is unsuitable for industrial application. In addition, gene manipulation tools have not been sufficiently developed for metabolic control of the PUFA synthetic pathway. Genetic manipulation is a new means of molecularly breeding industrial strains, analyzing their physiological properties, and clarifying the biosynthetic pathway to PUFAs. A comprehensive transformation system for this fungus has been fundamentally established. It involves a uracil-auxotrophic mutant (ura5⁻ strain) as a host strain, a homologous ura5 gene as a selectable marker gene, and transformation through the biolistic method, which is the only effective method (24).Open in a separate windowFIG. 1.Putative biosynthetic pathway of PUFAs in Mortierella alpina 1S-4. OA, oleic acid; LA, linoleic acid; ALA, α-linolenic acid; GLA, γ-linolenic acid; SDA, stearidonic acid; EDA, n-9 eicosadienoic acid; DGLA, dihomo-γ-linolenic acid; ETA, n-3 eicosatetraenoic acid; MA, Mead acid. Open and black arrows indicate elongase and desaturase reactions, respectively.Agrobacterium tumefaciens-mediated transformation (ATMT) has been employed for a wide range of plants (7, 27). Recently, it was reported that A. tumefaciens is also able to transfer its DNA to various fungi, including ascomycetes, basidiomycetes, zygomycetes, and oomycetes, as well as to plants (2, 5, 16). Additionally, this bacterium can transform intact cells and spores as well as protoplasts. Under mild conditions, the ATMT system generates a large number of stable transformants, which show vigorous growth, indicating that the ATMT system can be an efficient tool for molecular manipulation of M. alpina 1S-4. Moreover, the frequency of homologous recombination was higher than that with conventional transformation methods (8). In this study, we evaluated the external gene transfer system using the ATMT system and determined the optimal conditions for M. alpina 1S-4. Furthermore, we overexpressed the ω3-desaturase gene to improve n-3 PUFA productivity in an industrial n-6-PUFA-producing strain, M. alpina 1S-4 (18, 20), using ATMT.