High frequency transformation of Mucor with recombinant plasmid DNA

Abstract

Genetic transformation of Mucor circinelloides has been achieved by incubating protoplasts from a leucine requiring strain with recombinant plasmid DNA in the presence of polyethylene glycol and CaCl2. The donor DNA in the initial experiments consisted of pools of recombinant plasmid DNA representing a Mucor genomic library. The library was constructed by insertion of quasi-random fragments of Mucor DNA into the yeast-E. coli shuttle vector YRp17. Transformation of Mucor gave rise to Leu+ prototrophic colonies which were found, by Southern hybridisation analysis, to contain vector DNA sequences. Transformation of E. coli with DNA isolated from the Mucor transformants resulted in ampicillin resistant colonies containing plasmid DNA. One of the plasmids, pMCL1302, consisted of YRp17 with an insert of 10.5 kb. It hybridised to Mucor genomic DNA and was capable of complementing the leu mutation. The frequency of transformation obtained with pMCL1302 reached 600 Leu+ transformants per μg DNA and 3.2×106 viable cells. This is the first report of the cloning of a gene by direct selection in a filamentous fungus, and demonstrates the successful transformation of a fungus of the Class Zygomycetes with plasmid DNA. © 1984 Carlsberg Laboratory.