A multicopy vector system for genetic studies in Mucor circinelloides and other zygomycetes

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.

Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation

“…circinelloides shuttle vector pEUKA11, containing the E. coli kan R gene under the transcriptional control of the Mc. circinelloides glyceraldehyde-3-phosphate dehydrogenase gene (gpd1) promoter and terminator, was a kind gift from Dr Jose Arnau at the Bioteknologisk Institut, Hoersholm, Denmark (see Appel et al, 2004;Wolff et al, 2002).…”

Use of In Vivo and In Vitro Systems to SelectLeishmania amazonensisExpressing Green Fluorescent Protein

“…No report of the use of in vivo systems to test G418 selection in Leishmania is available for comparison with our results; however, Murphy and coworkers [22] selected high numbers of transfected Trypanosoma brucei inoculating G418 in infected mice and concluded that the extracellular trypanosome blood form was highly sensitive to G418. The reasons for the high level of sensitivity of T. brucei trypanosome compared to L. amazonensis amastigote are unclear, but may be due to the problem of intracellular antibiotic delivery to the parasite and/or the acidic pH of the parasitophorous vacuoles housing amastigotes, since G418 selection is pH-dependent [23]. …”

Inhibition of Non-Homologous End Joining and integration of DNA upon transformation of Rhizopus oryzae

“…Unlike the case in most other fungi, DNA used for transformation rarely integrates into the chromosomes of Mucorales fungi, but is replicated autonomously in high-molecular-weight (HMW) concatenated structures (i.e., chains of linked monomers). It appears that autonomous replication in Mucorales does not require a defined origin of replication (Revuelta and Jayaram 1986;van Heeswijck 1986;Wostemeyer et al 1987;Yanai et al 1990;Benito et al 1995;Skory 2002Skory , 2004Appel et al 2004), and that non-specific regions of DNA can serve to initiate replication, as is common in higher eukaryotes (Gilbert 2001). While such autonomous replication can occasionally be advantageous, sitespecific integration is typically preferred and necessary for gene disruption.…”