Abstract
For development of a homologous transformation system for the zygomycete fungus, Rhizomucor pusillus, the isopropylmalate isomerase (leuA) gene was cloned from R. pusillus IFO 4578 by the DNA-probing method with the leuA sequence of Mucor circinelloides as probe. The nucleotide sequence revealed that leuA of R. pusillus encoded a 755-amino-acid protein of 82.5 kDa with no intron. The leuA gene on pUC19 (plasmid pRPLeu10) was introduced by polyethyleneglycol-assisted transformation into protoplasts of a leuA - mutant of R. pusillus that was obtained by UV mutagenesis. Transformation under optimal conditions yielded 20 Leu+ transformants (μg pRPLeu10 DNA)-1 (1×106 viable protoplasts)-1. Blot analysis of DNA from the transformants showed that the pRPLeu10 sequence was integrated into the genome by homologous recombination at the leuA locus.
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Received: 2 October 1995/Received last revision: 5 December 1995/Accepted: 11 December 1995
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Wada, M., Beppu, T. & Horinouchi, S. Integrative transformation of the zygomycete Rhizomucor pusillus by homologous recombination. Appl Microbiol Biotechnol 45, 652–657 (1996). https://doi.org/10.1007/s002530050743
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DOI: https://doi.org/10.1007/s002530050743