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In this study, we report the rapid characterisation of a novel microbial natural product resulting from the rational derepression of a silent gene cluster. A conserved set of five regulatory genes was used as a query to search genomic databases and identify atypical biosynthetic gene clusters (BGCs). A 20-kb BGC from the genetically intractable Streptomyces sclerotialus bacterial strain was captured using yeast-based homologous recombination and introduced into validated heterologous hosts. CRISPR/Cas9-mediated genome editing was then employed to rationally inactivate the key transcriptional repressor and trigger production of an unprecedented class of hybrid natural products exemplified by (2-(benzoyloxy)acetyl)-l-proline, named scleric acid. Subsequent rounds of CRISPR/Cas9-mediated gene deletions afforded a selection of biosynthetic gene mutant strains which led to a plausible biosynthetic pathway for scleric acid assembly. Synthetic standards of scleric acid and a key biosynthetic intermediate were also prepared to confirm the chemical structures we proposed. The assembly of scleric acid involves two unique condensation reactions catalysed by a single NRPS module and an ATP-grasp enzyme that link a proline and a benzoyl residue to each end of a rare hydroxyethyl-ACP intermediate, respectively. Scleric acid was shown to exhibit moderate inhibition activity against Mycobacterium tuberculosis , as well as inhibition of the cancer-associated metabolic enzyme nicotinamide N -methyltransferase (NNMT).

The homologous transcriptional regulators ScbR and ScbR2 have previously been identified as γ-butyrolactone (GBL) and antibiotic receptors, respectively. They regulate diverse physiological processes in Streptomyces coelicolor in response to GBL and antibiotic signals. In this study, ScbR and ScbR2 proteins were shown to interact using a bacterial two-hybrid system where adenylate cyclase activity was reconstituted in Escherichia coli BTH101. These ScbR/ScbR2 interactions in S. coelicolor were then demonstrated by co-immunoprecipitation. The ScbR/ScbR2 heterodimer was shown to co-exist with their ScbR and ScbR2 respective homodimers. When potential operator targets in S. coelicolor were investigated, the heterodimer was found to bind in the promoter region of sco5158, which however was not a target for ScbR or ScbR2 homodimers. These results revealed a new mechanism of regulation by ScbR and ScbR2 in S. coelicolor.

Precise control of gene expression using exogenous factors is of great significance. To develop ideal inducible expression systems for streptomycetes, new genetic parts, oxytetracycline responsive repressor OtrR, operator otrO, and promoter otrBp from Streptomyces rimosus, were selected de novo and characterized in vivo and in vitro. OtrR showed strong affinity to otrO (KD = 1.7 × 10(-10) M) and oxytetracycline induced dissociation of the OtrR/DNA complex in a concentration-dependent manner. On the basis of these genetic parts, a synthetic inducible expression system Potr* was optimized. Induction of Potr* with 0.01-4 μM of oxytetracycline triggered a wide-range expression level of gfp reporter gene in different Streptomyces species. Benchmarking Potr* against the widely used constitutive promoters ermE* and kasOp* revealed greatly enhanced levels of expression when Potr* was fully induced. Finally, Potr* was used as a tool to activate and optimize the expression of the silent jadomycin biosynthetic gene cluster in Streptomyces venezuelae. Altogether, the synthetic Potr* presents a new versatile tool for fine-tuning gene expression in streptomycetes.

The TetR family of microbial transcription factors directly control the expression of a diverse range of genes in bacteria by sensing specific ligands. In this issue of Chemistry & and Biology, Cuthbertson and colleagues used phylogenomics to guide the identification of TetR-like protein cognate ligands and revealed a novel inducible antibiotic resistance mechanism., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Streptomyces bacteria produce different classes of diffusible signaling molecules that trigger secondary metabolite production and/or morphological development within the cell population. The biosynthesis of gamma-butyrolactones (GBLs) and 2-alkyl-4-hydroxymethylfuran-3-carboxylic acids (AHFCAs) signaling molecules is related and involves an essential AfsA-like butenolide synthase. This chapter first describes the catalytic role of AfsA-like enzyme then provides details about methods for the discovery and characterization of potentially novel signaling molecules. In section 4, one approach for establishing the biological role of these signaling molecules is presented., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Streptomycete bacteria are renowned as a prolific source of natural products with diverse biological activities. Production of these metabolites is often subject to transcriptional regulation: the biosynthetic genes remain silent until the required environmental and/or physiological signals occur. Consequently, in the laboratory environment, many gene clusters that direct the biosynthesis of natural products with clinical potential are not expressed or at very low level preventing the production/detection of the associated metabolite. Genetic engineering of streptomycetes can unleash the production of many new natural products. This chapter describes the overexpression of pathway-specific activators, the genetic disruption of pathway-specific repressors, and the main strategy used to identify and characterize new natural products from these engineered Streptomyces strains., (Copyright © 2012 Elsevier Inc. All rights reserved.)

There is a constant need for new and improved drugs to combat infectious diseases, cancer, and other major life-threatening conditions. The recent development of genomics-guided approaches for novel natural product discovery has stimulated renewed interest in the search for natural product-based drugs. Genome sequence analysis of Streptomyces ambofaciens ATCC23877 has revealed numerous secondary metabolite biosynthetic gene clusters, including a giant type I modular polyketide synthase (PKS) gene cluster, which is composed of 25 genes (nine of which encode PKSs) and spans almost 150 kb, making it one of the largest polyketide biosynthetic gene clusters described to date. The metabolic product(s) of this gene cluster are unknown, and transcriptional analyses showed that it is not expressed under laboratory growth conditions. The constitutive expression of a regulatory gene within the cluster, encoding a protein that is similar to Large ATP binding of the LuxR (LAL) family proteins, triggered the expression of the biosynthetic genes. This led to the identification of four 51-membered glycosylated macrolides, named stambomycins A-D as metabolic products of the gene cluster. The structures of these compounds imply several interesting biosynthetic features, including incorporation of unusual extender units into the polyketide chain and in trans hydroxylation of the growing polyketide chain to provide the hydroxyl group for macrolide formation. Interestingly, the stambomycins possess promising antiproliferative activity against human cancer cell lines. Database searches identify genes encoding LAL regulators within numerous cryptic biosynthetic gene clusters in actinomycete genomes, suggesting that constitutive expression of such pathway-specific activators represents a powerful approach for novel bioactive natural product discovery.

Reports of anticancer and immunosuppressive properties have spurred recent interest in the bacterially produced prodiginines. We use electrospray tandem mass spectrometry (ES-MS/MS) to investigate prodigiosin, undecylprodiginine, and streptorubin B (butyl-meta-cycloheptylprodiginine) and to explore their fragmentation pathways to explain the unusual methyl radical loss and consecutive fragment ions that dominate low-energy collision-induced dissociation (CID) mass spectra. The competition between the formation of even-electron ions and radical ions is examined in detail. Theoretical calculations are used to optimize the structures and calculate the energies of both reactants and products using the Gaussian 03 program. Results indicate that protonation occurs on the nitrogen atom that initially held no hydrogen, thus allowing formation of a pseudo-seven-membered ring that constitutes the most stable ground state [M + H](+) structure. From this precursor, experimental data show that methyl radical loss has the lowest apparent threshold but, alternatively, even-electron fragment ions can be formed by loss of a methanol molecule. Computational modeling indicates that methyl radical loss is the more endothermic process in this competition, but the lower apparent threshold associated with methyl radical loss points to a lower kinetic barrier. Additionally, this characteristic and unusual loss of methyl radical (in combination with weaker methanol loss) from each prodiginine is useful for performing constant neutral loss scans to quickly and efficiently identify all prodiginines in a complex biological mixture without any clean-up or purification. The feasibility of this approach has been proven through the identification of a new, low-abundance prodigiosin analog arising from Hahella chejuensis.

All of the genetic elements necessary for the production of the antibiotic methylenomycin (Mm) and its regulation are contained within the 22-kb mmy-mmf gene cluster, which is located on the 356-kb linear plasmid SCP1 of Streptomyces coelicolor A3(2). A putative operon of 3 genes within this gene cluster, mmfLHP, was proposed to direct the biosynthesis of an A-factor-like signaling molecule, which could play a role in the regulation of Mm biosynthesis. The mmfLHP operon was expressed under the control of its native promoter in S. coelicolor M512, a host lacking the SCP1 plasmid, and the ability to produce prodiginine and actinorhodin antibiotics. Comparative metabolic profiling led to the identification and structure elucidation of a family of 5 new 2-alkyl-4-hydroxymethylfuran-3-carboxylic acids (AHFCAs), collectively termed Mm furans (MMFs), as the products of the mmfLHP genes. MMFs specifically induce the production of the Mm antibiotics in S. coelicolor. Comparative genomics analyses and searches of the natural product chemistry literature indicated that other streptomycetes may produce AHFCAs, suggesting that they could form a general class of antibiotic biosynthesis inducers in Streptomyces species, with analogous functions to the better known gamma-butyrolactone regulatory molecules.

Knowledge of the flowering process - an important trait in ornamental plants such as roses - is necessary for efficient control of flowering. This study was carried out to develop and characterize new resources to gain further insight into floral control in rose. We studied floral initiation in a nonrecurrent blooming rose (hybrid of Rosa wichurana) and a recurrent blooming rose (Rosa hybrida Black Baccara. In Black Baccara, floral initiation took place rapidly after bud burst, whereas in the greenhouse R. wichurana remained vegetative. During floral initiation, the apex enlarged and domed quickly and concomitantly. This is the first description of this transition between the vegetative and floral bud stages in rose. From these vegetative and pre-floral tissues, two cDNA libraries were constructed and 5,000 ESTs sequenced. By collecting our ESTs and those available in public databases, we developed a comprehensive database representing approximately 5,000 unique sequences after clustering. By screening this database for candidate genes involved in the flowering process, we identified 13 genes potentially involved in gibberellic acid signalling, photoperiod pathways, and floral development. Based on expression data, we put forward different hypotheses on the control of flowering in rose (photoperiod control and involvement of gibberellins) relative to what is already known in Arabidopsis.

The red gene cluster of Streptomyces coelicolor directs production of undecylprodiginine. Here we report that this gene cluster also directs production of streptorubin B and show that 2-undecylpyrrole (UP) is an intermediate in the biosynthesis of undecylprodiginine and streptorubin B. The redPQRKL genes are involved in UP biosynthesis. RedL and RedK are proposed to generate UP from dodecanoic acid or a derivative. A redK(-) mutant produces a hydroxylated undecylprodiginine derivative, whereas redL(-) and redK(-) mutants require addition of chemically synthesized UP for production of undecylprodiginine and streptorubin B. Fatty acid biosynthetic enzymes can provide dodecanoic acid, but efficient and selective prodiginine biosynthesis requires RedPQR. Deletion of redP, redQ, or redR leads to an 80%-95% decrease in production of undecylprodiginine and an array of prodiginine analogs with varying alkyl chains. In a redR(-) mutant, the ratio of these can be altered in a logical manner by feeding various fatty acids.

In this issue of Chemistry & Biology, Gross et al. report development of a novel genome mining method for isolating products of orphan biosynthetic gene clusters, and the application of this method to the isolation of orfamide A, a novel cyclic lipopeptide.

Polyketide synthases (PKSs) are involved in the biosynthesis of many important natural products. In bacteria, type III PKSs typically catalyze iterative decarboxylation and condensation reactions of malonyl-CoA building blocks in the biosynthesis of polyhydroxyaromatic products. Here it is shown that Gcs, a type III PKS encoded by the sco7221 ORF of the bacterium Streptomyces coelicolor, is required for biosynthesis of the germicidin family of 3,6-dialkyl-4-hydroxypyran-2-one natural products. Evidence consistent with Gcs-catalyzed elongation of specific beta-ketoacyl-ACP products of the fatty acid synthase FabH with ethyl- or methylmalonyl-CoA in the biosynthesis of germicidins is presented. Selectivity for beta-ketoacyl-ACP starter units and ethylmalonyl-CoA as an extender unit is unprecedented for type III PKSs, suggesting these enzymes may be capable of utilizing a far wider range of starter and extender units for natural product assembly than believed until now.

Several polyketide secondary metabolites are shown by feeding experiments to incorporate glycerol-derived 3-carbon starter units, 2-carbon extender units, or 3-carbon branches into their hydrocarbon chains. In recent years, genetic studies have begun to elucidate the mechanisms by which this occurs. In this article we review the incorporation of glycerol-derived precursors into polyketides and propose new mechanisms for the incorporation processes.