Evaluating Different Methodologies for Bioprospecting Actinomycetes in Canary Islands Soils.

Actinomycetes are a wide group of Gram positive prokaryotes, the soil being their most characteristic habitat, where they play important ecological functions. Their immense biotechnological potential as producers of bioactive molecules of great commercial and industrial interest is exemplified by most antibiotics for clinical use being derived from this group of bacteria. In this work several methodologies and culture media were tested for the isolation of actinomycetes in soils from three different edafoclimatic areas of Tenerife (Canary Islands, Spain): an arid zone of the southeast coast of the island, a humid area in the laurel forest and the Canary high mountain. The results of this work evidenced that just an air drying of the sample during 7 days (pretreatment) produces high counts of actinomycetes versus non-actinomycetes in the three soils and in any of the culture media studied, making unnecessary post physical and chemical treatments. However, the only method that produced an exclusive isolation of actinomycetes was the use of 0.22-µm filters as a physical barrier, and hence being a tool of unquestionable utility to estimate actinomycetes populations in soil.The analysis by BOX rep-PCR determined that the richness and genetic diversity of the isolates of these localities are very high. The greatest similarities were, with one exception, found between strains from the same locations. Izaña was one that obtained the highest diversity. Regarding the biotecnology potential as antibiotic produces isolates from Güimar soils are the most promising as 7 out of 10 isolates showed some antibiotic activity.

New tools for high-throughput expression of fungal secretory proteins in Saccharomyces cerevisiae and Pichia pastoris.

Heterologous protein expression in yeast, mostly in Saccharomyces cerevisiae and Pichia pastoris, is a well-established and widely used technique. It typically requires the construction of an expression vector in Escherichia coli containing the foreign gene and its subsequent transformation into yeast. Although simple, this procedure has important limitations for the expression of large numbers of proteins, that is, for the generation of protein libraries. We describe here the development of a novel system for the easy and fast expression of heterologous proteins both in S. cerevisiae and in P. pastoris, under the control of the GAL1 and AOX1 promoters respectively. Expression in S. cerevisiae requires only the transformation of yeast cells with an unpurified PCR product carrying the gene to be expressed, and the expression of the same gene in P. pastoris requires only the isolation of the plasmid generated in S. cerevisiae and its transformation into this second yeast, thus making this system suitable for high-throughput projects. The system has been tested by the extracellular expression of 30 secretory fungal proteins.