Use of the Saccharomycopsis schoenii MET17 promoter for regulated heterologous gene expression.

The ability to regulate the expression of genes is a central tool for the characterization of fungal genes. This is of particular interest to study genes required for specific processes or the effect of genes expressed only under specific conditions. Saccharomycopsis species show a unique property of necrotrophic mycoparasitism that is activated upon starvation. Here we describe the use of the MET17 promoter of S. schoenii as a tool to regulate gene expression based on the availability of methionine. Conditional expression was tested using lacZ and GFP reporter genes. Gene expression could be strongly down-regulated by the addition of methionine or cysteine to the growth medium and upregulated by starvation for methionine. We used X-gal (5-bromo-4-chloro-3-indolyl-ß-d-galactopyranoside) to detect lacZ-expression in plate assays and ONPG (ortho-nitrophenyl-ß-galactopyranoside) as a substrate for ß-galactosidase in liquid-phase assays. For in vivo expression analyses we used fluorescence microscopy for the detection and localization of a MET17-driven histone H4-GFP reporter gene. With these assays we demonstrated the usefulness of the MET17 promoter to regulate expression of genes based on methionine availability. In silico analyses revealed similar promoter motifs as found in MET3 genes of Saccharomyces cerevisiae and Ashbya gossypii. This suggests a regulation of the MET17 promoter by CBF1 and MET31/MET32 in conjunction with the transcriptional activator MET4, which were also identified in the S. schoenii genome.

This article describes the characterization of the S. schoenii MET17 promoter for regulated gene expression.

Isolation of Saccharomycopsis species from plant material.

Saccharomycopsis species are natural organic sulphur auxotrophs. Their genomes do not encode genes for the uptake and assimilation of sulphate and thus these species cannot grow on media lacking e.g. methionine. Due to the similarity between sulphate and selenate, uptake and assimilation of selenate occurs through the same pathway starting from sulphate transporters encoded by the homologs of the SUL1 and SUL2 genes in S. cerevisiae. Lack of these transporters renders Saccharomycopsis species resistant to selenate levels that are toxic to other microorganisms. We used this feature to enrich environmental samples for Saccharomycopsis species. This led to the isolation of S. schoenii, S. lassenensis and a hitherto undescribed Saccharomycopsis species with limited by-catch of other yeasts, mainly belonging to Metschnikowia and Hanseniaspora. We performed growth and predation assays to characterize the potential of these new isolates as predacious yeasts. Most Saccharomycopsis species are temperature sensitive and cannot grow at 37°C; with the exception of S. lassenensis strains. Predation assays with S. schoenii and S. cerevisiae as prey indicated that predation was enhanced at 20°C compared to 30°C. We crossed an American isolate of S. schoenii with our German isolate using marker directed breeding. Viable progeny indicated that both strains are interfertile and belong to the same biological species. S. lassenensis is heterothallic, while S. schoenii and the new Saccharomycopsis isolate, for which we suggest the name S. geisenheimensis sp. nov., are homothallic.

Improved gene-targeting efficiency upon starvation in Saccharomycopsis.

Commonly used fungal transformation protocols rely on the use of either electroporation or the lithium acetate/single strand carrier DNA/Polyethylene glycol/heat shock method. We have used the latter method previously in establishing DNA-mediated transformation in Saccharomycopsis schoenii, a CTG-clade yeast that exhibits necrotrophic mycoparasitism. To elucidate the molecular mechanisms of predation by Saccharomycopsis we aim at gene-function analyses to identify virulence-related pathways and genes. However, in spite of a satisfactory transformation efficiency our efforts were crippled by high frequency of ectopic integration of disruption cassettes. Here, we show that overnight starvation of S. schoenii cells, while reducing the number of transformants, resulted in a substantial increase in gene-targeting via homologous recombination. To demonstrate this, we have deleted the S. schoenii CHS1, HIS3 and LEU2 genes and determined the required size of the flanking homology regions. Additionally, we complemented the S. schoenii leu2 mutant with heterologous LEU2 gene from Saccharomycopsis fermentans. To demonstrate the usefulness of our approach we also generated a S. fermentans leu2 strain, suggesting that this approach may have broader applicability.