92NISR RESEARCH GRANT
2004 Young Investigator Research Grant

Studies of Metabolic Engineering of Useful Lipid-producing
Microorganisms
Eiji Sakuradani
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University
Research aims
Polyunsaturated fatty acids (PUFAs), such as arachidonic
acid (AA), can serve not only as structural components ofmembrane phospholipids but also as precursors of theeicosanoids of signaling molecules in mammals. Therefore,studies on PUFA production are important for the medicaland pharmaceutical fields. Mortierella alpina 1S-4, belonging to Zygomycetes, was found as the best producer oflipids containing more than 40% of AA in the total fatty
acids. A host-vector system of this fungus to enhance thePUFA productivity was established. In this study, I have improved the fatty acid composition and the lipid productivity
by means of overexpression of an enzyme gene involved in
PUFA biosynthesis.
Methods
Transformation of the M. alpina 1S-4 ura5 strain withthe expression vectors was performed with a PDS-1000/He
Particle Delivery System (Bio-Rad Laboratory Inc., Hercules, CA) as described previously.1(GLA) to dihomo-g -linolenic acid (DGLA) and involved inAA biosynthesis in M. alpina 1S-4. A transformation vector, pDura5GLELO, was constructed and used for transformation of the ura5 strain to express the GLELO gene. Outof the 36 transformants obtained, 9 were determined to bestable transformants with pDura5GLELO. The transformant #3 selected from the stable transformants exhibitedthe different fatty acid composition from that of the hostcells: GLA, which serves as a substrate for GLELOp, wasdecreased and AA was increased. For further investigation,
time course experiments were performed with transformant#3 and the host cells (Fig. 1).
The accumulation of AA continued after 4 days in bothstrains, regardless of the culture conditions. In the case of5% glucose in the medium, AA production remained low incomparison with that with 2% glucose. Although this observation was in accordance with results previously reported, the AA productivity (mg/ml of culture broth) wasproportional to the glucose concentration. In the case of 2%
glucose (Fig. 1A and B), the AA content increased linearly

Results1) Construction of an improved transformation systemfor expression of a target gene
The first trial involved expression of the enhanced green
fluorescent protein (EGFP) gene in the ura5 strain (uracilauxotroph) derived from M. alpina 1S-4 by use of a homologous ura5 gene-containing vector (pDura5). The EGFP
gene was placed between the same promoter and terminator
as used in pDura5, resulting in construction of an EGFP-expression vector, pDura5GFP. Transformation was successfully performed, and six stable transformants were selectedfrom the 45 isolates. All the stable ones were determined to
be transformed through vector insertion into the rDNA
locus. Under microscopic fluorescent observation, a strong
fluorescence was detected only in spores of the transformants, not in those of the control strain (ura5 strain). On
the other hand, we could not observe the difference of fluorescent intensity of the fungal filaments between the transformant and the control strain, because of the strong selffluorescence of the filaments of this fungus.
2) Improvement of fatty acid composition and AA productivity2
GLELOp has an elongation activity of g -linolenic acid

Fig. 1. Time courses of changes in the mycelial fatty acid content in theGLELO-overexpressing M. alpina 1S-4 transformant and the host cells.
All cultivations were started by inoculation of the respective spores andperformed with reciprocal shaking (120 strokes/min) at 28°C. The culture
conditions were as follows: (A, B) GY medium containing 2% glucose.
(C, D) GY medium containing 5% glucose. All media also contained 1%
yeast extract and 0.05 mg/ml of uracil. The strains used were as follows:(A, C) transformant. (B, D) host cells. Fatty acid analysis was performed
with total biomass obtained from each cultivation. The values are themeans of triplicate experiments. GLA, g -linolenic acid; DGLA, dihomog -linolenic acid; AA, arachidonic acid.

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after 4 days in the transformant but not in the host cells.
The final content of AA in the transformant was higher thanthat in the host cells. On the other hand, in the case of 5%
glucose (Fig. 1C and D), differences clearly appeared: theAA content of transformant #3 increased more greatly thanthat of the host cells after 5 days. The GLA content of thetransformant cells remained lower than that of the hostcells, especially after 4 days. The DGLA content increased
at the beginning of cultivation and then decreased concomitantly with the increase in the AA content. The AA contentof transformant #3 increased by 8.3% at the end point. As afurther analysis, real-time quantitative PCR (RTQ-PCR)
showed that the quantity of GLELO RNA in transformant#3 was 7.4-fold higher than that of the host strain on 4 days.
Therefore, the results obtained for stable transformant #3
must be directly due to GLELO gene expression.

Conclusion
We succeeded in expression of target genes such as
EGFP or GLELO genes in M. alpina 1S-4 ura5 strain.
Overexpression of GLELO gene led to enhance AA production in the transformant. Furthermore, GLELOp wasshown to serve as the rate-limiting step in AA biosynthesis.
Reference
1.

2.

Takeno S., Sakuradani E., Murata S., Inohara-Ochiai M., Kawashima
H., Ashikari T., and Shimizu S. (2004) Establishment of an overall
transformation system for an oil-producing filamentous fungus,
Mortierella alpina 1S-4. Appl. Microbiol. Biotechnol., 65, 419–425.
Takeno S., Sakuradani E., and Shimizu S. (2004) Creation and application of host-vector system in oleaginous microorganism
Mortierella alpina 1S-4. Japan Appl. Microbiol. Soc. 2, 82–87.
http://www.kuicr.kyoto-u.ac.jp/labos/bm2/oubikyo/2004120107.pdf