Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG.

Cystargolides are natural products originally isolated from Kitasatospora cystarginea NRRL B16505 as inhibitors of the proteasome. They are composed of a dipeptide backbone linked to a ß-lactone warhead. Recently, we identified the cystargolide biosynthetic gene cluster, but systematic genetic analyses had not been carried out because of the lack of a heterologous expression system. Here, we report the discovery of a homologous cystargolide biosynthetic pathway in Streptomyces durhamensis NRRL-B3309 by genome mining. The gene cluster was cloned via transformation-associated recombination and heterologously expressed in Streptomyces coelicolor M512. We demonstrate that it contains all genes necessary for the production of cystargolide A and B. Single gene deletion experiments reveal that only five of the eight genes from the initially proposed gene cluster are essential for cystargolide synthesis. Additional insights into the cystargolide pathway could be obtained from in vitro assays with CysG and chemical complementation of the respective gene knockout. This could be further supported by the in vitro investigation of the CysG homolog BelI from the belactosin biosynthetic gene cluster. Thereby, we confirm that CysG and BelI catalyze a cryptic SAM-dependent transfer of a methyl group that is critical for the construction of the cystargolide and belactosin ß-lactone warheads.

Discovery of actinomycin L, a new member of the actinomycin family of antibiotics.

Streptomycetes are major producers of bioactive natural products, including the majority of the naturally produced antibiotics. While much of the low-hanging fruit has been discovered, it is predicted that less than 5% of the chemical space of natural products has been mined. Here, we describe the discovery of the novel actinomycins L1 and L2 produced by Streptomyces sp. MBT27, via application of metabolic analysis and molecular networking. Actinomycins L1 and L2 are diastereomers, and the structure of actinomycin L2 was resolved using NMR and single crystal X-ray crystallography. Actinomycin L is formed via spirolinkage of anthranilamide to the 4-oxoproline moiety of actinomycin X2, prior to the condensation of the actinomycin halves. Such a structural feature has not previously been identified in naturally occurring actinomycins. Adding anthranilamide to cultures of the actinomycin X2 producer Streptomyces antibioticus, which has the same biosynthetic gene cluster as Streptomyces sp. MBT27, resulted in the production of actinomycin L. This supports a biosynthetic pathway whereby actinomycin L is produced from two distinct metabolic routes, namely those for actinomycin X2 and for anthranilamide. Actinomycins L1 and L2 showed significant antimicrobial activity against Gram-positive bacteria. Our work shows how new molecules can still be identified even in the oldest of natural product families.

Structural basis for non-radical catalysis by TsrM, a radical SAM methylase.

Tryptophan 2C methyltransferase (TsrM) methylates C2 of the indole ring of L-tryptophan during biosynthesis of the quinaldic acid moiety of thiostrepton. TsrM is annotated as a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase; however, TsrM does not reductively cleave SAM to the universal 5'-deoxyadenosyl 5'-radical intermediate, a hallmark of radical SAM (RS) enzymes. Herein, we report structures of TsrM from Kitasatospora setae, which are the first structures of a cobalamin-dependent radical SAM methylase. Unexpectedly, the structures show an essential arginine residue that resides in the proximal coordination sphere of the cobalamin cofactor, and a [4Fe-4S] cluster that is ligated by a glutamyl residue and three cysteines in a canonical CXXXCXXC RS motif. Structures in the presence of substrates suggest a substrate-assisted mechanism of catalysis, wherein the carboxylate group of SAM serves as a general base to deprotonate N1 of the tryptophan substrate, facilitating the formation of a C2 carbanion.

Formation of wall-less cells in Kitasatospora viridifaciens requires cytoskeletal protein FilP in oxygen-limiting conditions.

The cell wall is considered an essential component for bacterial survival, providing structural support, and protection from environmental insults. Under normal growth conditions, filamentous actinobacteria insert new cell wall material at the hyphal tips regulated by the coordinated activity of cytoskeletal proteins and cell wall biosynthetic enzymes. Despite the importance of the cell wall, some filamentous actinobacteria can produce wall-deficient S-cells upon prolonged exposure to hyperosmotic stress. Here, we performed cryo-electron tomography and live cell imaging to further characterize S-cell extrusion in Kitasatospora viridifaciens. We show that exposure to hyperosmotic stress leads to DNA compaction, membrane and S-cell extrusion, and thinning of the cell wall at hyphal tips. Additionally, we find that the extrusion of S-cells is abolished in a cytoskeletal mutant strain that lacks the intermediate filament-like protein FilP. Furthermore, micro-aerobic culturing promotes the formation of S-cells in the wild type, but the limited oxygen still impedes S-cell formation in the ΔfilP mutant. These results demonstrate that S-cell formation is stimulated by oxygen-limiting conditions and dependent on functional cytoskeleton remodeling.

Genome Mining of the Genus Streptacidiphilus for Biosynthetic and Biodegradation Potential.

The genus Streptacidiphilus represents a group of acidophilic actinobacteria within the family Streptomycetaceae, and currently encompasses 15 validly named species, which include five recent additions within the last two years. Considering the potential of the related genera within the family, namely Streptomyces and Kitasatospora, these relatively new members of the family can also be a promising source for novel secondary metabolites. At present, 15 genome data for 11 species from this genus are available, which can provide valuable information on their biology including the potential for metabolite production as well as enzymatic activities in comparison to the neighboring taxa. In this study, the genome sequences of 11 Streptacidiphilus species were subjected to the comparative analysis together with selected Streptomyces and Kitasatospora genomes. This study represents the first comprehensive comparative genomic analysis of the genus Streptacidiphilus. The results indicate that the genomes of Streptacidiphilus contained various secondary metabolite (SM) producing biosynthetic gene clusters (BGCs), some of them exclusively identified in Streptacidiphilus only. Several of these clusters may potentially code for SMs that may have a broad range of bioactivities, such as antibacterial, antifungal, antimalarial and antitumor activities. The biodegradation capabilities of Streptacidiphilus were also explored by investigating the hydrolytic enzymes for complex carbohydrates. Although all genomes were enriched with carbohydrate-active enzymes (CAZymes), their numbers in the genomes of some strains such as Streptacidiphilus carbonis NBRC 100919T were higher as compared to well-known carbohydrate degrading organisms. These distinctive features of each Streptacidiphilus species make them interesting candidates for future studies with respect to their potential for SM production and enzymatic activities.

Genome rearrangements and megaplasmid loss in the filamentous bacterium Kitasatospora viridifaciens are associated with protoplast formation and regeneration.

Filamentous Actinobacteria are multicellular bacteria with linear replicons. Kitasatospora viridifaciens DSM 40239 contains a linear 7.8 Mb chromosome and an autonomously replicating plasmid KVP1 of 1.7 Mb. Here we show that lysozyme-induced protoplast formation of the multinucleated mycelium of K. viridifaciens drives morphological diversity. Characterisation and sequencing of an individual revertant colony that had lost the ability to differentiate revealed that the strain had not only lost most of KVP1 but also carried deletions in the right arm of the chromosome. Strikingly, the deletion sites were preceded by insertion sequence elements, suggesting that the rearrangements may have been caused by replicative transposition and homologous recombination between both replicons. These data indicate that protoplast formation is a stressful process that can lead to profound genetic changes.

Secondary nucleotide messenger c-di-GMP exerts a global control on natural product biosynthesis in streptomycetes.

Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, is a ubiquitous second messenger controlling diverse cellular processes in bacteria. In streptomycetes, c-di-GMP plays a crucial role in a complex morphological differentiation by modulating an activity of the pleiotropic regulator BldD. Here we report that c-di-GMP plays a key role in regulating secondary metabolite production in streptomycetes by altering the expression levels of bldD. Deletion of cdgB encoding a diguanylate cyclase in Streptomycesghanaensis reduced c-di-GMP levels and the production of the peptidoglycan glycosyltransferase inhibitor moenomycin A. In contrast to the cdgB mutant, inactivation of rmdB, encoding a phosphodiesterase for the c-di-GMP hydrolysis, positively correlated with the c-di-GMP and moenomycin A accumulation. Deletion of bldD adversely affected the synthesis of secondary metabolites in S. ghanaensis, including the production of moenomycin A. The bldD-deficient phenotype is partly mediated by an increase in expression of the pleiotropic regulatory gene wblA. Genetic and biochemical analyses demonstrate that a complex of c-di-GMP and BldD effectively represses transcription of wblA, thus preventing sporogenesis and sustaining antibiotic synthesis. These results show that manipulation of the expression of genes controlling c-di-GMP pool has the potential to improve antibiotic production as well as activate the expression of silent gene clusters.

Versatile Oxidase and Dehydrogenase Activities of Bacterial Pyranose 2-Oxidase Facilitate Redox Cycling with Manganese Peroxidase In Vitro.

Pyranose 2-oxidase (POx) has long been accredited a physiological role in lignin degradation, but evidence to provide insights into the biochemical mechanisms and interactions is insufficient. There are ample data in the literature on the oxidase and dehydrogenase activities of POx, yet the biological relevance of this duality could not be established conclusively. Here we present a comprehensive biochemical and phylogenetic characterization of a novel pyranose 2-oxidase from the actinomycetous bacterium Kitasatospora aureofaciens (KaPOx) as well as a possible biomolecular synergism of this enzyme with peroxidases using phenolic model substrates in vitro A phylogenetic analysis of both fungal and bacterial putative POx-encoding sequences revealed their close evolutionary relationship and supports a late horizontal gene transfer of ancestral POx sequences. We successfully expressed and characterized a novel bacterial POx gene from K. aureofaciens, one of the putative POx genes closely related to well-known fungal POx genes. Its biochemical characteristics comply with most of the classical hallmarks of known fungal pyranose 2-oxidases, i.e., reactivity with a range of different monosaccharides as electron donors as well as activity with oxygen, various quinones, and complexed metal ions as electron acceptors. Thus, KaPOx shows the pronounced duality of oxidase and dehydrogenase similar to that of fungal POx. We further performed efficient redox cycling of aromatic lignin model compounds between KaPOx and manganese peroxidase (MnP). In addition, we found a Mn(III) reduction activity in KaPOx, which, in combination with its ability to provide H2O2, implies this and potentially other POx as complementary enzymatic tools for oxidative lignin degradation by specialized peroxidases.IMPORTANCE Establishment of a mechanistic synergism between pyranose oxidase and (manganese) peroxidases represents a vital step in the course of elucidating microbial lignin degradation. Here, the comprehensive characterization of a bacterial pyranose 2-oxidase from Kitasatospora aureofaciens is of particular interest for several reasons. First, the phylogenetic analysis of putative pyranose oxidase genes reveals a widespread occurrence of highly similar enzymes in bacteria. Still, there is only a single report on a bacterial pyranose oxidase, stressing the need of closing this gap in the scientific literature. In addition, the relatively small K. aureofaciens proteome supposedly supplies a limited set of enzymatic functions to realize lignocellulosic biomass degradation. Both enzyme and organism therefore present a viable model to study the mechanisms of bacterial lignin decomposition, elucidate physiologically relevant interactions with specialized peroxidases, and potentially realize biotechnological applications.

Discovery of a Short-Chain ε-Poly-l-lysine and Its Highly Efficient Production via Synthetase Swap Strategy.

ε-Poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide, which is generally recognized as safe for use as a food preservative. To date, the production capacity of strains that produce low-molecular weight ε-PL remains very low and thus unsuitable for industrial production. Here, we report a new low-molecular weight ε-PL-producing Kitasatospora aureofaciens strain. The ε-PL synthase gene of this strain was cloned into a high ε-PL-producing Streptomyces albulus strain. The resulting recombinant strain efficiently produced ε-PL with a molecular weight of 1.3-2.3 kDa and yielded of 23.6 g/L following fed-batch fermentation in a 5 L bioreactor. In addition, circular dichroism spectra showed that this ε-PL takes on a conformation similar to an antiparallel pleated-sheet. Moreover, it demonstrated better antimicrobial activity against yeast compared to the 3.2-4.5 kDa ε-PL. This study provides a highly efficient strategy for production of the low-molecular weight ε-PL, which helps to expand its potential applications.

Exclusivity offers a sound yet practical species criterion for bacteria despite abundant gene flow.

BACKGROUND: The question of whether bacterial species objectively exist has long divided microbiologists. A major source of contention stems from the fact that bacteria regularly engage in horizontal gene transfer (HGT), making it difficult to ascertain relatedness and draw boundaries between taxa. A natural way to define taxa is based on exclusivity of relatedness, which applies when members of a taxon are more closely related to each other than they are to any outsider. It is largely unknown whether exclusive bacterial taxa exist when averaging over the genome or are rare due to rampant hybridization. RESULTS: Here, we analyze a collection of 701 genomes representing a wide variety of environmental isolates from the family Streptomycetaceae, whose members are competent at HGT. We find that the presence/absence of auxiliary genes in the pan-genome displays a hierarchical (tree-like) structure that correlates significantly with the genealogy of the core-genome. Moreover, we identified the existence of many exclusive taxa, although individual genes often contradict these taxa. These conclusions were supported by repeating the analysis on 1,586 genomes belonging to the genus Bacillus. However, despite confirming the existence of exclusive groups (taxa), we were unable to identify an objective threshold at which to assign the rank of species. CONCLUSIONS: The existence of bacterial taxa is justified by considering average relatedness across the entire genome, as captured by exclusivity, but is rejected if one requires unanimous agreement of all parts of the genome. We propose using exclusivity to delimit taxa and conventional genome similarity thresholds to assign bacterial taxa to the species rank. This approach recognizes species that are phylogenetically meaningful, while also establishing some degree of comparability across species-ranked taxa in different bacterial clades.

Streptacidiphilus pinicola sp. nov., isolated from pine grove soil.

A moderately acidophilic actinobacterial strain, designated MMS16-CNU450T, was isolated from pine grove soil, and its taxonomic position was analysed using a polyphasic approach. The isolate showed best growth at 30 °C, pH 6 and 0.5 % (w/v) NaCl. On the basis of 16S rRNA gene sequence similarity, the isolate was assigned to the genus Streptacidiphilus, and the closest species were Streptacidiphilus rugosus AM-16T (sequence similarity, 98.61 %), Streptacidiphilus melanogenes NBRC 103184T (98.53 %), Streptacidiphilus jiangxiensis NBRC 100920T (98.19 %) and Streptacidiphilus anmyonensis NBRC 103185T (98.05 %). The isolate formed a distinct cluster of its own within the Streptacidiphilusclade in the phylogenetic tree. Based on whole-genome comparison between the strain MMS16-CNU450T and the type strains of related species, the orthologous average nucleotide identity and in silico DNA-DNA hybridization values were in the range of 77.9-87.0 and 22.3-32.7 %, respectively. The DNA G+C content of the isolate was 68.6 mol%. The phylogenetic, phenotypic, chemotaxonomic and genomic data supported the affiliation of the strain to Streptacidiphilus, and the name Streptacidiphilus pinicola sp. nov. (type strain, MMS16-CNU450T=KCTC 49008T=JCM 32300T) is proposed accordingly.

Biosynthesis of 2-amino-3-hydroxycyclopent-2-enone moiety of bafilomycin in Kitasatospora cheerisanensis KCTC2395.

Bafilomycins produced by Kitasatospora cheerisanensis KCTC- 2395 belong to the 16-membered macrolactone family plecomacrolide antibiotics. Bafilomycin B1 contains 2-amino- 3-hydroxycyclopent-2-enone (C5N), a five membered ring, which gets condensed via an amide linkage to bafilomycin polyketide. To study the biosynthetic pathway of C5N during bafilomycin biosynthesis in K. cheerisanensis KCTC2395, we attempted the functional analysis of two putative genes, encoding 5-aminolevulinic acid synthase (ALAS) and acyl- CoA ligase (ACL). The amplified putative genes for ALAS and ACL were cloned into the E. coli expression vector pET- 32a(+) plasmid, following which the soluble recombinant ALAS and ACL proteins were purified through nickel-affinity column chromatography. Through HPLC analysis of the enzyme reaction mixture, we confirmed the products of putative ALAS and ACL reaction as 5-aminolevulinic acid (5-ALA) and 5-ALA-CoA, respectively. The optimal pH for the putative ALAS reaction was 7.5, and for putative ACL reaction was 7.0, as confirmed by the colorimetric assay. Furthermore, pyridoxal 5'-phosphate (PLP) was found to be an essential cofactor in the putative ALAS reaction, and ATP was a cofactor for the putative ACL catalysis. Finally, we also confirmed that the simultaneous treatment of putative ACL and putative ALAS enzymes resulted in the production of C5N compound from 5-ALA.

Engineered production of kitasetalic acid, a new tetrahydro-ß-carboline with the ability to suppress glucose-regulated protein synthesis.

ß-Carboline alkaloids and related compounds show a broad spectrum of biological activities. We previously identified new members of the ß-carboline alkaloid family by using an engineered Kitasatospora setae strain and a heterologous Streptomyces host expressing the plausible biosynthetic genes, including the hypothetical gene kse_70640 (kslB). Here, we elucidated the chemical structure of a new tetrahydro-ß-carboline compound (named kitasetalic acid) that appeared in a heterologous Streptomyces host expressing the kslB gene alone. Kitasetalic acid suppressed the expression of glucose-regulated protein 78 (GRP78) without inducing cell death. This is the first report to show that a tetrahydro-ß-carboline compound regulates the expression of the GRP78 protein in cancer cell lines.

Toward Systems Metabolic Engineering of Streptomycetes for Secondary Metabolites Production.

Streptomycetes are known for their inherent ability to produce pharmaceutically relevant secondary metabolites. Discovery of medically useful, yet novel compounds has become a great challenge due to frequent rediscovery of known compounds and a consequent decline in the number of relevant clinical trials in the last decades. A paradigm shift took place when the first whole genome sequences of streptomycetes became available, from which silent or "cryptic" biosynthetic gene clusters (BGCs) were discovered. Cryptic BGCs reveal a so far untapped potential of the microorganisms for the production of novel compounds, which has spurred new efforts in understanding the complex regulation between primary and secondary metabolism. This new trend has been accompanied with development of new computational resources (genome and compound mining tools), generation of various high-quality omics data, establishment of molecular tools, and other strain engineering strategies. They all come together to enable systems metabolic engineering of streptomycetes, allowing more systematic and efficient strain development. In this review, the authors present recent progresses within systems metabolic engineering of streptomycetes for uncovering their hidden potential to produce novel compounds and for the improved production of secondary metabolites.

Allostreptomyces indica sp. nov., isolated from India.

A novel actinobacterium, designated strain YIM 75704T, was isolated from a limestone quarry located at Gulbarga, Karnataka, India. The novel strain has showed typical morphological and chemotaxonomic characteristics of the family Streptomycetaceae. Comparison of 16S rRNA gene sequences indicated that this strain represents a novel member of the family Streptomycetaceae and exhibited 99.0% 16S rRNA gene sequence similarities with the type species of the recently described novel genus Allostreptomyces, that is, Allostreptomyces psammosilenae, whereas other species of Streptomyces were below 95% sequence similarity. The cell hydrolysates contained the LL-isomer of diaminopimelic acid and the predominant quinones were MK-9 (H6, H8 and H4). The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylinositolmannosides and three unknown phospholipids. The DNA G+C content was 75.0 mol%. A polyphasic study of the strain with morphological, phenotypic, phylogenetic and with DNA-DNA hybridization evidence with related members showed that this strain represents novel species of Allostreptomyces for which the name Allostreptomyces indica sp. nov., is proposed. The type strain is YIM 75704T (= DSM 41985T=CCTCC AA 209051T= NCIM 5485T).

Streptomyces virginiae PPDC Is a New Type of Phenylpyruvate Decarboxylase Composed of Two Subunits.

Streptomyces virginiae phenylpyruvate decarboxylase (PPDC) has not been identified before. Two putative branched-chain α-keto acid dehydrogenase subunit genes bkdC and bkdD from S. virginiae are similar to halves of other PPDC coding sequences. We cloned and characterized them biochemically in this work. The two proteins formed a stable complex attested by pull-down assay, consistent with the finding that their soluble expression was obtained only when they were coexpressed in Escherichia coli. The subunits were redesignated as SvPPDCα and SvPPDCß, because the SvPPDCα/ß complex catalyzed the conversion of phenylpyruvate to phenylacetaldehyde, reflecting the nature of the enzyme. Moreover, mutations of conserved residues in either of the two subunits led to inactivation or decreased specific activity of the enzymatic reaction. All previously identified PPDCs are encoded by a single gene. Here, we identified a new type of PPDC that contains two subunits, which gives new insights into the PPDC family.

Biosynthesis of the ß-Lactone Proteasome Inhibitors Belactosin and Cystargolide.

Belactosins and cystargolides are natural product proteasome inhibitors from Actinobacteria. Both feature dipeptidic backbones and a unique ß-lactone building block. Herein, we present a detailed investigation of their biosynthesis. Identification and analysis of the corresponding gene clusters indicated that both compounds are assembled by rare single-enzyme amino acid ligases. Feeding experiments with isotope-labeled precursors and in vitro biochemistry showed that the formation of the ß-lactone warhead is unprecedented and reminiscent of leucine biosynthesis, and that it involves the action of isopropylmalate synthase homologues.

Genus Kitasatospora, taxonomic features and diversity of secondary metabolites.

The genus Kitasatospora was proposed in 1982. Although Kitasatospora strains resemble Streptomyces strains in morphology, they are clearly different in cell-wall composition, as they contain both LL- and meso-diaminopimelic acid. Aerial and submerged spores contain LL-, while vegetative and submerged mycelia contain mainly meso- in their cell walls. Currently, 23 species have been validly proposed. Members of the genus Kitasatospora form a tight cluster and represent a legitimate genus distinct from Streptomyces on the basis of phylogenetic analysis of 16S rRNA gene sequences. A variety of biologically active compounds have been found from Kitasatospora strains and structures of these compounds are extremely diverse. Genome sequences of 15 strains published so far are about 7-9 Mb in size and contain many genes governing secondary metabolites.

Allostreptomyces psammosilenae gen. nov., sp. nov., an endophytic actinobacterium isolated from the roots of Psammosilene tunicoides and emended description of the family Streptomycetaceae [Waksman and Henrici (1943)AL] emend. Rainey et al. 1997, emend. Kim et al. 2003, emend. Zhi et al. 2009.

A Gram-stain-positive actinobacterium, designated strain YIM DR4008T, was isolated from the root sample of Psammosilene tunicoides collected from Lijiang, Yunnan, China. Strain YIM DR4008T could grow at temperatures ranging from 10 to 50 °C (optimum 28-30 °C), at pH 5.0-11.0 (optimum pH 7.0) and in the presence of up to 4 % (w/v) NaCl. Sequence analysis of the 16S ribosomal RNA gene revealed that strain YIM DR4008T shared highest similarity (95.0 %) with Streptomyces griseoplanus NBRC 12779T and <95 % similarity with other known members of the genera Streptomyces, Kitasatospora and Streptacidiphilus. The diagnostic cell-wall diamino acid of strain YIM DR4008T was found to be ll-diaminopimelic acid. The whole-cell hydrolysates contained a major amount of galactose and mannose along with a small proportion of fucose, glucose, rhamnose and ribose. The polar lipids consisted of diphosphatidylglycerol, phosphatidylinositol mannosides and three unidentified phospholipids. The respiratory menaquinones were MK-9(H6) and MK-9(H8), while the major cellular fatty acids (>10 %) were anteiso-C15 : 0, C16 : 0, iso-C16 : 0, iso-C15 : 0 and anteiso-C17 : 0. The genomic DNA G+C content was determined to be 75.3 mol%. Based on the phenotypic, chemotaxonomic and molecular characteristics, strain YIM DR4008T is proposed to be recognized as a novel species of a new genus in the family Streptomycetaceae, with the name Allostreptomyces psammosilenae gen. nov., sp. nov. The type strain of the type species is YIM DR4008T (=DSM 42178T=CGMCC 4.7247T). An emended description of the family Streptomycetaceae is also provided.

Phylogenomic Analysis of Natural Products Biosynthetic Gene Clusters Allows Discovery of Arseno-Organic Metabolites in Model Streptomycetes.

Natural products from microbes have provided humans with beneficial antibiotics for millennia. However, a decline in the pace of antibiotic discovery exerts pressure on human health as antibiotic resistance spreads, a challenge that may better faced by unveiling chemical diversity produced by microbes. Current microbial genome mining approaches have revitalized research into antibiotics, but the empirical nature of these methods limits the chemical space that is explored.Here, we address the problem of finding novel pathways by incorporating evolutionary principles into genome mining. We recapitulated the evolutionary history of twenty-three enzyme families previously uninvestigated in the context of natural product biosynthesis in Actinobacteria, the most proficient producers of natural products. Our genome evolutionary analyses where based on the assumption that expanded-repurposed enzyme families-from central metabolism, occur frequently and thus have the potential to catalyze new conversions in the context of natural products biosynthesis. Our analyses led to the discovery of biosynthetic gene clusters coding for hidden chemical diversity, as validated by comparing our predictions with those from state-of-the-art genome mining tools; as well as experimentally demonstrating the existence of a biosynthetic pathway for arseno-organic metabolites in Streptomyces coelicolor and Streptomyces lividans, Using a gene knockout and metabolite profile combined strategy.As our approach does not rely solely on sequence similarity searches of previously identified biosynthetic enzymes, these results establish the basis for the development of an evolutionary-driven genome mining tool termed EvoMining that complements current platforms. We anticipate that by doing so real 'chemical dark matter' will be unveiled.