Establishment of an overall transformation system for an oil-producing filamentous fungus, Mortierella alpina 1S-4

Abstract

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.

Transformation of the Mycorrhizal Basidiomycetes,Suillus grevilleiandS. bovinus, by Particle Bombardment

“…Putative transformants of S. grevillei and S. bovinus were most frequently obtained at 1,100 psi, which was consistent with the results of Takeno et al 25) and Vainstein et al…”

Isolation of Uracil Auxotrophs of the FungusAcremonium cellulolyticusand the Development of a Transformation System with thepyrFGene

“…Although a screening involving 5-FOA has been reported to yield both pyrG and pyrF mutants of other fungi, [17][18][19] in the present study the three isolated auxotrophs were pyrF mutants. This has been reported in studies of other organisms, 13,20,21) but, a clear explanation of this phenomenon has not been achieved. The pyrF gene of CFP3 had one nucleotide substitution relative to the wild-type putative pyrF gene.…”

“…Nonhomologous integration into the genome is a common event reported in studies of other filamentous fungi. [20][21][22][23][24] To overcome this problem, we turned to a report that gene-targeting frequency was markedly enhanced in strains of Aspergillus oryzae, 23,24) Neurospora crassa, 25) and other filamentous fungi 26,27) exhibiting disrupted ku70 or/and ku80 genes. We have discovered the ortholog of these genes in A. cellulolyticus, and the construction of strains with disruptions in these genes is now under way.…”

Establishment of Agrobacterium tumefaciens -Mediated Transformation of an Oleaginous Fungus, Mortierella alpina 1S-4, and Its Application for Eicosapentaenoic Acid Producer Breeding

“…A comprehensive transformation system for this fungus has been fundamentally established. It involves a uracil-auxotrophic mutant (ura5 Ϫ 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).…”