prodiginine and actinorhodin

In the genus Streptomyces, carbon utilization is of significant importance for the expression of genes involved in morphological differentiation and antibiotic production. However, there is little information about the mechanism involved in these effects. In the present work, it was found that glucose exerted a suppressive effect on the Streptomyces coelicolor actinorhodin (Act) and undecylprodigiosin (Red) production, as well as in its morphological differentiation. Accordingly, using a high-density microarray approach in S. coelicolor grown under glucose repression, at early growth stages, a negative effect was exerted on the transcription of genes involved in Act and Red production, when compared with non-repressive conditions. Seven genes of Act and at least ten genes of Red production were down-regulated by glucose. Stronger repression was observed on the initial steps of antibiotics formation. On the contrary, the coelimycin P1 cluster was up-regulated by glucose. Regarding differentiation, no sporulation was observed in the presence of glucose and expression of a set of genes of the bld cascade was repressed as well as chaplins and rodlins genes. Finally, a series of transcriptional regulators involved in both processes were up- or down-regulated by glucose. This is the first global transcriptomic approach performed to understand the molecular basis of the glucose effect on the synthesis of secondary metabolism and differentiation in the genus Streptomyces. The results of this study are opening new avenues for further exploration.

Topics: Anthraquinones; Anti-Bacterial Agents; Carbon; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genes, Bacterial; Glucose; Prodigiosin; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Secondary Metabolism; Streptomyces coelicolor

Streptomyces bacteria are known for producing important natural compounds by secondary metabolism, especially antibiotics with novel biological activities. Functional studies of antibiotic-biosynthesizing gene clusters are generally through homologous genomic recombination by gene-targeting vectors. Here, we present a rapid and efficient method for construction of gene-targeting vectors. This approach is based on Streptomyces phage φBT1 integrase-mediated multisite in vitro site-specific recombination. Four 'entry clones' were assembled into a circular plasmid to generate the destination gene-targeting vector by a one-step reaction. The four 'entry clones' contained two clones of the upstream and downstream flanks of the target gene, a selectable marker and an E. coli-Streptomyces shuttle vector. After targeted modification of the genome, the selectable markers were removed by φC31 integrase-mediated in vivo site-specific recombination between pre-placed attB and attP sites. Using this method, part of the calcium-dependent antibiotic (CDA) and actinorhodin (Act) biosynthetic gene clusters were deleted, and the rrdA encoding RrdA, a negative regulator of Red production, was also deleted. The final prodiginine production of the engineered strain was over five times that of the wild-type strain. This straightforward φBT1 and φC31 integrase-based strategy provides an alternative approach for rapid gene-targeting vector construction and marker removal in streptomycetes.

Topics: Anthraquinones; Gene Deletion; Gene Order; Gene Targeting; Genes, Bacterial; Genetic Vectors; Homologous Recombination; Mutagenesis; Mutation; Phenotype; Prodigiosin; Streptomyces

Bacterially produced secondary metabolites are used as antibiotics, anticancer drugs, and for many other medicinal applications. The mechanisms that limit the production of these molecules in the laboratory are not well understood, and this has impeded the discovery of many important compounds. We have identified small molecules that remodel the yields of secondary metabolites in many actinomycetes and show that one set of these molecules does so by inhibiting fatty acid biosynthesis. This demonstrates a particularly intimate relationship between this primary metabolic pathway and secondary metabolism and suggests an approach to enhance the yields of metabolites for discovery and biochemical characterization.

Topics: Actinobacteria; Anthraquinones; Anti-Bacterial Agents; Chromatography, High Pressure Liquid; Fatty Acids; Mass Spectrometry; Prodigiosin; Small Molecule Libraries; Spectrophotometry; Streptomyces coelicolor