Establishment of an overall transformation system for an oil-producing filamentous fungus, Mortierella alpina 1S-4.
Oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. To determine its physiological properties and to clarify the biosynthetic pathways for polyunsaturated fatty acids, a transformation system for this fungus was established using a derivative of it, i.e., a ura5- mutant lacking orotate phosphoribosyl transferase (OPRTase, EC.2.4.2.10) activity. Transformation with a vector containing the homologous ura5 gene as a marker was successfully performed using microprojectile bombardment, other methods frequently used for transformation, such as the protoplasting, lithium acetate, or electroporation methods, not giving satisfactory results. As a result, two types of transformants were obtained: a few stable transformants overexpressing the ura5 gene, and many unstable transformants showing OPRTase activity comparable to that of the wild-type strain. The results of quantitative PCR indicated that the stable transformants could retain the ura5 genes originating from the transformation vector regardless of the culture conditions. On the other hand, unstable transformants easily lost the marker gene under uracil-containing conditions, as expected. In this paper, we report that an overall transformation system for this fungus was successfully established, and propose how to select useful transformants as experimental and industrial strains.
Takeno
Seiki
S
Laboratory of Fermentation Physiology and Applied Microbiology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, 606-8502, Kyoto, Japan.
Sakuradani
Eiji
E
Murata
Shoichi
S
Inohara-Ochiai
Misa
M
Kawashima
Hiroshi
H
Ashikari
Toshihiko
T
Shimizu
Sakayu
S
D000085:Acetates / Q000494:pharmacology; D003470:Culture Media / Q000737:chemistry; D018274:Electroporation; D005800:Genes, Fungal; D005822:Genetic Vectors; D020094:Mortierella / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D009821:Oils / Q000378:metabolism; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D011523:Protoplasts; D012641:Selection, Genetic; D014170:Transformation, Genetic
D000085:Acetates; D003470:Culture Media; D009821:Oils; D009962:Orotate Phosphoribosyltransferase; C488804:lithium acetate