dRNA-seq transcriptional profiling of the FK506 biosynthetic gene cluster in Streptomyces tsukubaensis NRRL18488 and general analysis of the transcriptome.

FK506 (tacrolimus) is a valuable immunosuppressant produced by several Streptomyces strains. In the genome of the wild type producer Streptomyces tsukubaensis NRRL18488, FK506 biosynthesis is encoded by a gene cluster that spans 83.5 (kb). A whole transcriptome differential shotgun sequencing (dRNA-seq) of S. tsukubaensis was performed to analyze transcription at 2 different time points; before and during active FK506 production. In total, 8,914 transcription start sites were identified in either condition, which enabled precise determination of the 5'-UTR length of the corresponding transcripts as well as the identification of 2 consensus sequence motifs in the promoter regions. The transcription start sites of all gene operons within the FK506 cluster were identified, including 3 examples of leaderless RNA transcripts. These data provide detailed insight into the transcription of the FK506 biosynthetic gene cluster to support future regulatory studies, genetic manipulation, and industrial production.

Challenges in the Heterologous Production of Antibiotics in Streptomyces.

The fast growing genome databases provide us with a large number of so far unknown secondary metabolite biosynthetic gene clusters. A key method to study these gene clusters is their heterologous expression in an engineered host strain. Gene clusters derived from actinomycetes are usually expressed in a Streptomyces host strain to identify and investigate the corresponding compounds. However, heterologous expression is often accompanied with some challenges affecting the production rates of secondary metabolites. The first step is therefore the selection of a suitable expression vector and host strain. Once production has been established, there are several possibilities to improve compound yields either by media screens, by overexpression of regulatory or transport genes or by introduction of constitutive or inducible promoters. A surely important, but hitherto little studied factor is also the regulation of a heterologously expressed gene cluster by its host strain. This review gives a short overview on the chances and challenges provided by heterologous production of secondary metabolites in Streptomyces.

Two transcription factors, CabA and CabR, are independently involved in multilevel regulation of the biosynthetic gene cluster encoding the novel aminocoumarin, cacibiocin.

Aminocoumarins are potent antibiotics belonging to a relatively small group of secondary metabolites produced by actinomycetes. Genome mining of Catenulispora acidiphila has recently led to the discovery of a gene cluster responsible for biosynthesis of novel aminocoumarins, cacibiocins. However, regulation of the expression of this novel gene cluster has not yet been analyzed. In this study, we identify transcriptional regulators of the cacibiocin gene cluster. Using a heterologous expression system, we show that the CabA and CabR proteins encoded by cabA and cabR genes in the cacibiocin gene cluster control the expression of genes involved in the biosynthesis, modification, regulation, and potentially, efflux/resistance of cacibiocins. CabA positively regulates the expression of cabH (the first gene in the cabHIYJKL operon) and cabhal genes encoding key enzymes responsible for the biosynthesis and halogenation of the aminocoumarin moiety, respectively. We provide evidence that CabA is a direct inducer of cacibiocin production, whereas the second transcriptional factor, CabR, is involved in the negative regulation of its own gene and cabT-the latter of which encodes a putative cacibiocin transporter. We also demonstrate that CabR activity is negatively regulated in vitro by aminocoumarin compounds, suggesting the existence of analogous regulation in vivo. Finally, we propose a model of multilevel regulation of gene transcription in the cacibiocin gene cluster by CabA and CabR.

Heterologous expression of the thiopeptide antibiotic GE2270 from Planobispora rosea ATCC 53733 in Streptomyces coelicolor requires deletion of ribosomal genes from the expression construct.

GE2270 is a thiopeptide antibiotic generated by extensive posttranslational modifications of a ribosomally generated precursor peptide. Thiopeptides are especially active against Gram-positive bacteria, including methicillin resistant Staphylococcus aureus (MRSA). In this study the GE2270 biosynthetic gene cluster (pbt) from Planobispora rosea ATCC 53733 was successfully expressed in the heterologous host strain Streptomyces coelicolor M1146. Notably, exconjugants containing the pbt gene cluster could only be obtained after deletion of the major part of the ribosomal genes flanking the gene cluster. This is a striking example that genes belonging to primary metabolism can prevent the successful conjugative transfer of DNA from phylogenetic distant species and thus complicate heterologous expression of secondary metabolite gene clusters. GE2270 production in the heterologous producer strain increased after introduction of the constitutive ermE* promoter upstream of the GE2270 resistance gene tuf from P. rosea. Insertion of the inducible tcp830 promoter resulted in inducible GE2270 production. When the regulatory gene pbtR was deleted, the resulting strain ceased to produce GE2270, suggesting an essential role of PbtR as a putative transcriptional activator of GE2270 expression.

New aminocoumarins from the rare actinomycete Catenulispora acidiphila DSM 44928: identification, structure elucidation, and heterologous production.

Genome mining led to the discovery of a novel aminocoumarin gene cluster in the rare actinomycete Catenulispora acidiphila DSM 44928. Sequence analysis revealed the presence of genes putatively involved in export/resistance, regulation, and biosynthesis of the aminocoumarin moiety and its halogenation, as well as several genes with so far unknown function. Two new aminocoumarins, cacibiocin A and B, were identified in the culture broth of C. acidiphila. Heterologous expression of the putative gene cluster in Streptomyces coelicolor M1152 confirmed that this cluster is responsible for cacibiocin biosynthesis. Furthermore, total production levels of cacibiocins could be increased by heterologous expression and screening of different culture media from an initial yield of 4.9 mg L(-1) in C. acidiphila to 60 mg L(-1) in S. coelicolor M1152. By HR-MS and NMR analysis, cacibiocin A was found to contain a 3-amino-4,7-dihydroxycoumarin moiety linked by an amide bond to a pyrrole-2,5-dicarboxylic acid. The latter structural motif has not been identified previously in any natural compound. Additionally, cacibiocin B contains two chlorine atoms at positions 6' and 8' of the aminocoumarin moiety.

Phosphate and carbon source regulation of two PhoP-dependent glycerophosphodiester phosphodiesterase genes of Streptomyces coelicolor.

Glycerophosphodiesters are formed by deacylation of phospholipids. Streptomyces coelicolor and other soil-dwelling actinomycetes utilize glycerophosphodiesters as phosphate and carbon sources by the action of glycerophosphodiester phosphodiesterases (GDPDs). Seven genes encoding putative GDPDs occur in the S. coelicolor genome. Two of these genes, glpQ1 and glpQ2, encoding extracellular GDPDs, showed a PhoP-dependent upregulated profile in response to phosphate shiftdown. Expression studies using the luxAB genes as reporter confirmed the PhoP dependence of both glpQ1 and glpQ2. Footprinting analyses with pure GST-PhoP of the glpQ1 promoter revealed four protected direct repeat units (DRu). PhoP binding affinity to the glpQ2 promoter was lower and revealed a protected region containing five DRu. As expected for pho regulon genes, inorganic phosphate, and also glycerol 3-phosphate, inhibited the expression from both glpQ1 and glpQ2. The expression of glpQ1 was also repressed by serine and inositol but expression of glpQ2 was not. In contrast, glucose, fructose and glycerol increased expression of glpQ2 but not that of glpQ1. In summary, our results suggest an interaction of phosphate control mediated by PhoP and carbon source regulation of the glpQ1 and glpQ2 genes involving complex operator structures.

Phosphate control of phoA, phoC and phoD gene expression in Streptomyces coelicolor reveals significant differences in binding of PhoP to their promoter regions.

Three putative alkaline phosphatase genes, phoA, phoC and phoD, were identified in the genome of Streptomyces coelicolor by homology with the amino acid sequence obtained from the PhoA protein of Streptomyces griseus. PhoA and PhoC correspond to broad-spectrum alkaline phosphatases whereas PhoD is similar to a Ca(2+)-dependent phospholipase D of Streptomyces chromofuscus. The phoA and phoD genes were efficiently expressed in R5 medium under phosphate-limited conditions, as shown by studies using the xylE reporter gene, whereas phoC was poorly transcribed under the same conditions. Expression of phoA was clearly PhoP-dependent since it was not transcribed in the S. coelicolor DeltaphoP mutant and was strongly activated under low phosphate concentrations. Similarly, expression of phoD was PhoP-dependent and highly sensitive to phosphate availability. By contrast, expression of phoC was not PhoP-dependent. Electrophoretic mobility shift assays showed that PhoP binds to the phoA and phoD promoters, but not to that of phoC. Footprinting studies with GST-PhoP revealed the presence of a PHO box (two direct 11 nt repeats) in the phoA promoter and two PHO boxes in the promoter of phoD. The transcription start points of the three promoters were identified by primer extension. The transcription start point of phoD coincides with the G of its translation start codon, indicating that this gene is transcribed as a leaderless mRNA. The deduced -10 and -35 regions of phoD (but not those of phoA) overlapped with the PHO boxes in this promoter, suggesting that an excess of PhoP interferes with binding of the RNA polymerase to this promoter. In summary, the three promoters showed clear differences in the modulation of their expression by PhoP.