Abstract
Transformation of Mucor circinelloides is routinely achieved by using a plasmid containing the wild-type leuA gene to complement the leucine requirement of an auxotrophic host strain. As is the case for other zygomycetes, the transforming DNA is usually not integrated into the genome of M. circinelloides, but is maintained as an autonomously replicating plasmid. However, even under selective conditions, the plasmid is segregationally unstable, resulting in a rather low number of cells carrying the plasmid. We report here on a new transformation vector based on a dominant selection marker conferring resistance to geneticin, which allows for plasmid maintenance in high copy numbers. The vector was also used to transform Mucor rouxii and Rhizomucor pusillus, and should therefore be a valuable tool for gene expression studies in zygomycetes. The functionality and regulatory properties of the promoter of the M. circinelloides gpd1 gene (which codes for glyceraldehyde-3P-dehydrogenase) were demonstrated in R. pusillus using geneticin selection. In this work, we have also determined the molecular basis of the Leu- phenotype of the M. circinelloides host strain R7B. The leucine requirement is due to a single point mutation in the leuA gene that results in the replacement of a glutamic acid by a lysine residue.
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Acknowledgements
The work described here was partially funded by the Danish Agency for Trade and Industry through the EUKA-Centre, a collaborative industrial project. Ulla Poulsen is thanked for excellent technical assistance. The M. rouxii strain IM-80 was kindly provided by Prof. Felix Gutierrez-Corona (University of Guanajuato, Mexico). R. pusillus strain B49-7 was kindly provided by Dr. Sueharu Horinouchi (University of Tokyo, Japan)
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Communicated by E. Cerdà-Olmedo
On 1 January, 2004, the Biotechnological Institute became Bioneer A/S (http://www.bioneer.dk)
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Appel, K.F., Wolff, A.M. & Arnau, J. A multicopy vector system for genetic studies in Mucor circinelloides and other zygomycetes. Mol Genet Genomics 271, 595–602 (2004). https://doi.org/10.1007/s00438-004-1008-6
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DOI: https://doi.org/10.1007/s00438-004-1008-6