Transformation of oil-producing fungus, Mortierella alpina 1S-4, using Zeocin, and application to arachidonic acid production.
The arachidonic acid-producing fungus Mortierella alpina 1S-4, an industrial strain, was endowed with Zeocin resistance by integration of the Zeocin-resistance gene at the rDNA locus of genomic DNA. Plasmid DNA was introduced into spores by microprojectile bombardment. Twenty mg/ml Zeocin completely inhibited the germination of M. alpina 1S-4 spores, and decreased the growth rate of fungal filaments to some extent. It was suggested that preincubation period and temperature had a great influence on transformation efficiency. Four out of 26 isolated transformants were selected. Molecular analysis of these stable transformants showed that the plasmid DNA was integrated into the rDNA locus of the genomic DNA. We expect that this system will be applied for useful oil production by gene manipulation of M. alpina 1S-4 and its derivative mutants. On the basis of the fundamental transformation system, we also tried to overexpress a homologous polyunsaturated fatty acid elongase gene, which has been reported to be included in the rate-limiting step for arachidonic acid production, thereby leading to increased arachidonic acid production.
Laboratory of Fermentation Physiology and Applied Microbiology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Japan.
Seiki
S
Takeno
Eiji
E
Sakuradani
Akiko
A
Tomi
Misa
M
Inohara-Ochiai
Hiroshi
H
Kawashima
Sakayu
S
Shimizu
D016718:Arachidonic Acid / Q000096:biosynthesis*; D001761:Bleomycin / Q000494:pharmacology*; D018929:Cell Culture Techniques / Q000379:methods; D025141:Drug Resistance, Fungal / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods*; D020094:Mortierella / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D001761:Bleomycin; C105427:Zeocin; D016718:Arachidonic Acid