Development of molecular tools for the mulundocandin producer Aspergillus sydowii: DNA-mediated transformation and reporter gene expression.
The echinocandin-type antimycotic mulundocandin and its derivatives are produced by the filamentous fungus Aspergillus sydowii (strain FH2551). These agents have been considered as a potential drug to treat immunocompromised patients who suffer from severe opportunistic fungal infections. In order to generate strains with a modified mulundocandin biosynthesis, we developed molecular tools for genetic engineering of A. sydowii as an alternative to conventional strain improvement procedures. For our experiments, we used strain FH2551, which was discriminated from other Aspergillus strains by determining the sequence of the two internal transcribed spacers (ITS1 and ITS2) of the rDNA locus. In addition, the electrophoretic karyotype of A. sydowii was established using pulsed-field gel electrophoresis (PFGE), leading to a calculated genomic size of about 40 Mb. For gene mapping, chromosomes were subjected to PFGE either unrestricted or after incubation with rare cutting enzymes and probed with heterologous genes. Using the bacterial hygromycin B phosphotransferase gene as a selectable marker for transformation of A. sydowii, we generated transformants with single and multiple copies of plasmid DNA. Subsequently, the heterologous lacZ and gfp genes were efficiently transferred and expressed in A. sydowii. The majority of lacZ-transformants showed more than 6 pkat beta-galactosidase activity/mg protein, while the control strains had no significant background activity. Fluorescence microscopy of gfp-transformants demonstrated that the green-fluorescent protein is present in a stable and active form in the cytoplasm of vegetative hyphae and conidiospores.
Schmitt
E K
EK
Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, 44801 Bochum, Germany.
Eilinghoff
B
B
Olliger
R
R
Decker
H
H
Kück
U
U
D000935:Antifungal Agents / Q000096:biosynthesis* / Q000494:pharmacology; D001230:Aspergillus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D054714:Echinocandins; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006921:Hygromycin B / Q000494:pharmacology; D007621:Karyotyping; D008164:Luminescent Proteins / Q000032:analysis; D010456:Peptides, Cyclic / Q000096:biosynthesis* / Q000494:pharmacology; D010802:Phylogeny; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D000935:Antifungal Agents; D054714:Echinocandins; D008164:Luminescent Proteins; D010456:Peptides, Cyclic; D049452:Green Fluorescent Proteins; D006921:Hygromycin B; D001616:beta-Galactosidase; C052098:mulundocandin