Potential Biosynthetic Gene Clusters Research Articles

Decoding the diagnostic and therapeutic potential of microbiota using pan-body pan-disease microbiomics

The human microbiome emerges as a promising reservoir for diagnostic markers and therapeutics. Since host-associated microbiomes at various body sites differ and diseases do not occur in isolation, a comprehensive analysis strategy highlighting the full potential of microbiomes should include diverse specimen types and various diseases. To ensure robust data quality and comparability across specimen types and diseases, we employ standardized protocols to generate sequencing data from 1931 prospectively collected specimens, including from saliva, plaque, skin, throat, eye, and stool, with an average sequencing depth of 5.3 gigabases. Collected from 515 patients, these samples yield an average of 3.7 metagenomes per patient. Our results suggest significant microbial variations across diseases and specimen types, including unexpected anatomical sites. We identify 583 unexplored species-level genome bins (SGBs) of which 189 are significantly disease-associated. Of note, the existence of microbial resistance genes in one specimen was indicative of the same resistance genes in other specimens of the same patient. Annotated and previously undescribed SGBs collectively harbor 28,315 potential biosynthetic gene clusters (BGCs), with 1050 significant correlations to diseases. Our combinatorial approach identifies distinct SGBs and BGCs, emphasizing the value of pan-body pan-disease microbiomics as a source for diagnostic and therapeutic strategies.

Unveiling novel Neocosmospora species from Thai mangroves as potent biocontrol agents against Colletotrichum species.

Neocosmospora species are saprobes, endophytes, and pathogens belonging to the family Nectriaceae. This study aims to investigate the taxonomy, biosynthetic potential, and application of three newly isolated Neocosmospora species from mangrove habitats in the southern part of Thailand using phylogeny, bioactivity screening, genome sequencing, and bioinformatics analysis. Detailed descriptions, illustrations, and a multi-locus phylogenetic tree with large subunit ribosomal DNA (LSU), internal transcribed spacer (ITS), translation elongation factor 1-alpha (ef1-α), and RNA polymerase II second largest subunit (RPB2) regions showing the placement of three fungal strains, MFLUCC 17-0253, MFLUCC 17-0257, and MFLUCC 17-0259 clustered within the Neocosmospora clade with strong statistical support. Fungal crude extracts of the new species N. mangrovei MFLUCC 17-0253 exhibited strong antifungal activity to control Colletotrichum truncatum CG-0064, while N. ferruginea MFLUCC 17-0259 exhibited only moderate antifungal activity toward C. acutatum CC-0036. Thus, N. mangrovei MFLUCC 17-0253 was sequenced by Oxford nanopore technology. The bioinformatics analysis revealed that 49.17 Mb genome of this fungus harbors 41 potential biosynthetic gene clusters. Two fungal isolates of Neocosmospora and a new species of N. mangrovei were reported in this study. These fungal strains showed activity against pathogenic fungi causing anthracnose in chili. In addition, full genome sequencing and bioinformatics analysis of N. mangrovei MFLUCC 17-0253 were obtained.

Characterization of Bacillus amyloliquefaciens PM415 as a potential bio-preserving probiotic.

Animal feed is vulnerable to fungal infections, and the use of bio-preserving probiotics has received increasing attention. In contrast to Lactobacillus and Bifidobacteria spp., fewer Bacillus spp. have been recognized as antifungal probiotics. Therefore, our objective was to screen antifungal strains and provide more Bacillus candidates to bridge this gap. Here, we screened 56 bacterial strains for cyclic lipopeptide genes and conducted an antifungal assay with Aspergillus niger as a representative fungus. We found that a Bacillus strain Bacillus amyloliquefaciens PM415, isolated from pigeon manure, exhibited the highest fungal inhibition activity as demonstrated by the confrontation assay and morphological observation under scanning electron microscope (SEM). Preliminary safety assessment and probiotic characterization revealed its non-pathogenic feature and stress tolerance capability. Whole genome sequencing of Bacillus amyloliquefaciens PM415 revealed a genome size of 4.16 Mbp and 84 housekeeping genes thereof were used for phylogenetic analysis showing that it is most closely related to Bacillus amyloliquefaciens LFB112. The in silico analysis further supported its non-pathogenic feature at the genomic level and revealed potential biosynthetic gene clusters responsible for its antifungal property. RNA-seq analysis revealed genome-wide changes in transportation, amino acid metabolism, non-ribosomal peptides (NRPs) biosynthesis and glycan degradation during fungal antagonism. Our results suggest that Bacillus amyloliquefaciens PM415 is a safe and effective probiotic strain that can prevent fungal growth in animal feeds.

Genomic Insights and Synthetic Biology Applications of Marine Actinomycete Streptomyces griseoincarnatus HNS054.

The marine bacterium Streptomyces sp. HNS054 shows promise as a platform for producing natural products. Isolated from a marine sponge, HNS054 possesses several desirable traits for bioengineering: rapid growth, salt tolerance, and compatibility with genetic tools. Its genome contains 21 potential biosynthetic gene clusters, offering a rich source of natural products. We successfully engineered HNS054 to increase the production of aborycin and actinorhodin by 4.5-fold and 1.2-fold, respectively, compared to S. coelicolor M1346 counterparts. With its unique features and amenability to genetic manipulation, HNS054 emerges as a promising candidate for developing novel marine-derived drugs and other valuable compounds.

Effective binning of metagenomic contigs using contrastive multi-view representation learning

Contig binning plays a crucial role in metagenomic data analysis by grouping contigs from the same or closely related genomes. However, existing binning methods face challenges in practical applications due to the diversity of data types and the difficulties in efficiently integrating heterogeneous information. Here, we introduce COMEBin, a binning method based on contrastive multi-view representation learning. COMEBin utilizes data augmentation to generate multiple fragments (views) of each contig and obtains high-quality embeddings of heterogeneous features (sequence coverage and k-mer distribution) through contrastive learning. Experimental results on multiple simulated and real datasets demonstrate that COMEBin outperforms state-of-the-art binning methods, particularly in recovering near-complete genomes from real environmental samples. COMEBin outperforms other binning methods remarkably when integrated into metagenomic analysis pipelines, including the recovery of potentially pathogenic antibiotic-resistant bacteria (PARB) and moderate or higher quality bins containing potential biosynthetic gene clusters (BGCs).

The Isolation and Structure Elucidation of Spirotetronate Lobophorins A, B, and H8 from Streptomyces sp. CB09030 and Their Biosynthetic Gene Cluster.

Lobophorins (LOBs) are a growing family of spirotetronate natural products with significant cytotoxicity, anti-inflammatory, and antibacterial activities. Herein, we report the transwell-based discovery of Streptomyces sp. CB09030 from a panel of 16 in-house Streptomyces strains, which has significant anti-mycobacterial activity and produces LOB A (1), LOB B (2), and LOB H8 (3). Genome sequencing and bioinformatic analyses revealed the potential biosynthetic gene cluster (BGC) for 1-3, which is highly homologous with the reported BGCs for LOBs. However, the glycosyltransferase LobG1 in S. sp. CB09030 has certain point mutations compared to the reported LobG1. Finally, LOB analogue 4 (O-β-D-kijanosyl-(1→17)-kijanolide) was obtained through an acid-catalyzed hydrolysis of 2. Compounds 1-4 showed different antibacterial activities against Mycobacterium smegmatis and Bacillus subtilis, which revealed the varying roles of different sugars in their antibacterial activities.

Complete Genome Sequence Resource of Pseudomonas koreensis FP1691, a Plant Growth-Promoting Rhizobacterium Isolated from Cowpea

Pseudomonas koreensis is a notable colonizer that acts as a plant growth-promoting rhizobacteria and has extensive significance in soil health improvement, sustainable agricultural practices, and maintenance of rhizospheric microbial diversity. Here, we report high-quality genome information of P. koreensis FP1691, isolated from the cowpea rhizosphere. The genome was assembled into a single circular chromosome of 6.30 Mb with a G + C content of 60.23%. FP1691 was predicted to contain 5,548 genes, including 642 genes related to general function prediction only, 535 genes associated with amino acid transport and metabolism, and 119 genes linked to secondary metabolite biosynthesis, transport, and catabolism. Furthermore, there are 12 secondary metabolite gene clusters in the FP1691 genome, including a potential biosynthetic gene cluster responsible for lokisin synthesis that exhibited more than 95% identity with the known clusters. Therefore, we inferred that the synthesis of antimicrobial peptides is an important mechanism of FP1691 as an invaluable resource for biocontrol agents. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Complete genome sequence of Streptomyces malaysiensis HNM0561, a marine sponge-associated actinomycete producing malaymycin and mccrearamycin E

Streptomyces malaysiensis HNM0561 is a marine sponge-associated actinomycete with the potential to produce potential anti-androgens against prostate cancer cells, including malaymycin and mccrearamycin E. Here, we present the complete genome sequence of S. malaysiensis HNM0561, which consists of a linear chromosome of 11,656,895bp and a circular plasmid of 32,797bp, 9849 protein coding genes, 18 rRNA genes, 66 tRNA genes, and 191 sRNA genes. Genomic annotations revealed that 72.03% of the protein-coding genes were assigned to the COG database, among which the abundant genes were predicted to be involved in transcription, replication, carbohydrate transport and metabolism, and amino acid transport and metabolism. Forty-nine putative secondary metabolite biosynthetic gene clusters were found in the genome. Among them, the potential biosynthetic gene clusters of malaymycin and mccrearamycin E have been described respectively. The complete genome information presented here will enable us to investigate the biosynthetic mechanism of two novel structures of malaymycin and mccrearamycin E and to discover novel secondary metabolites with potential against prostate cancer cell activities.

Micromonospora humida sp. nov., exhibiting antimicrobial potential, isolated from riverside soil.

An actinobacterial strain designated MMS20-R1-14T was isolated from a riverside soil sample. Colonies on agar plates were orange to strong orange brown in colour, which later became black. The cells grew at 10-40 °C (optimum, 37 °C), pH 5.0-11.0 (pH 8.0) and in the presence of 0-4 % NaCl (1 %). The 16S rRNA gene sequence of strain MMS20-R1-14T showed highest similarities to Micromonospora wenchangensis CCTCC AA 2012002T (99.51 %) and Micromonospora rifamycinica AM105T (99.37 %). The orthoANI values between strain MMS20-R1-14T and the two type strains were 95.72 and 90.99 %, and the digital DNA-DNA hybridization values were 63.6 and 40.8 %, respectively, thus confirming the distinction of strain MMS20-R1-14T from its mostly related species. The DNA G+C content of strain MMS20-R1-14T was 72.9 mol%. The strain contained meso-diaminopimelic acid as the major cell-wall amino acid, and the characteristic whole-cell sugars were arabinose, xylose, glucose, ribose and rhamnose. The main cellular fatty acids were C18 : 1 ω9c, iso-C15 : 0 and iso-C16 : 0, the diagnostic polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine, and the predominant menaquinones were MK-10(H4) and MK-10(H6), all of which were consistent with those of Micromonospora. Strain MMS20-R1-14T showed antimicrobial activity against a range of bacterial and yeast species. The genome of the strain was found to contain 33 potential biosynthetic gene clusters for secondary metabolites, thus showing a high potential as a producer of bioactive compounds. On the basis of these phenotypic, genotypic and chemotaxonomic data, strain MMS20-R1-14T merits recognition as representing a novel species of the genus Micromonospora, for which the name Micromonospora humida sp. nov. (type strain=MMS20 R1-14T=KCTC 49541T=JCM 34494T) is proposed.

In silico genome mining of potential novel biosynthetic gene clusters for drug discovery from Burkholderia bacteria

As an emerging resource, Gram-negative Burkholderia bacteria were able to produce a wide range of bioactive secondary metabolites with potential therapeutic and biotechnological applications. Genome mining has emerged as an influential platform for screening and pinpointing natural product diversity with the increasing number of Burkholderia genome sequences. Here, for genome mining of potential biosynthetic gene clusters (BGCs) and prioritizing prolific producing Burkholderia strains, we investigated the relationship between species evolution and distribution of main BGC groups using computational analysis of complete genome sequences of 248 Burkholderia species publicly available. We uncovered significantly differential distribution patterns of BGCs in the Burkholderia phyla, even among strains that are genetically very similar. We found various types of BGCs in Burkholderia, including some representative and most common BGCs for biosynthesis of encrypted or known terpenes, non-ribosomal peptides (NRPs) and some hybrid BGCs for cryptic products. We also observed that Burkholderia contain a lot of unspecified BGCs, representing high potentials to produce novel compounds. Analysis of BGCs for RiPPs (Ribosomally synthesized and posttranslationally modified peptides) and a texobactin-like BGC as examples showed wide classification and diversity of RiPP BGCs in Burkholderia at species level and metabolite predication. In conclusion, as the biggest investigation in silico by far on BGCs of the particular genus Burkholderia, our data implied a great diversity of natural products in Burkholderia and BGC distributions closely related to phylogenetic variation, and suggested different or concurrent strategies used to identify new drug molecules from these microorganisms will be important for the selection of potential BGCs and prolific producing strains for drug discovery.

Streptomonospora litoralis sp. nov., a halophilic thiopeptides producer isolated from sand collected at Cuxhaven beach

Strain M2T was isolated from the beach of Cuxhaven, Wadden Sea, Germany, in course of a program to attain new producers of bioactive natural products. Strain M2T produces litoralimycin and sulfomycin-type thiopeptides. Bioinformatic analysis revealed a potential biosynthetic gene cluster encoding for the M2T thiopeptides. The strain is Gram-stain-positive, rod shaped, non-motile, spore forming, showing a yellow colony color and forms extensively branched substrate mycelium and aerial hyphae. Inferred from the 16S rRNA gene phylogeny strain M2T affiliates with the genus Streptomonospora. It shows 96.6% 16S rRNA gene sequence similarity to the type species Streptomonospora salina DSM 44593 T and forms a distinct branch with Streptomonospora sediminis DSM 45723 T with 97.0% 16S rRNA gene sequence similarity. Genome-based phylogenetic analysis revealed that M2T is closely related to Streptomonospora alba YIM 90003 T with a digital DNA-DNA hybridisation (dDDH) value of 26.6%. The predominant menaquinones of M2T are MK-10(H6), MK-10(H8), and MK-11(H6) (> 10%). Major cellular fatty acids are iso-C16:0, anteiso C17:0 and C18:0 10-methyl. The polar lipid profile consisted of diphosphatidylglycerol phosphatidyl glycerol, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, three glycolipids, two unknown phospholipids, and two unknown lipids. The genome size of type strain M2T is 5,878,427 bp with 72.1 mol % G + C content. Based on the results obtained from phylogenetic and chemotaxonomic studies, strain M2T (= DSM 106425 T = NCCB 100650 T) is considered to represent a novel species within the genus Streptomonospora for which the name Streptomonospora litoralis sp. nov. is proposed.

A Community Effort: Combining Functional Amplicon Sequencing and Metagenomics Reveals Potential Biosynthetic Gene Clusters Associated with Protective Phenotypes in Rhizosphere Microbiomes.

ABSTRACTSoil-dwelling microorganisms associated with plant roots carry out essential processes that promote plant growth and productivity. In addition to these beneficial functions, the rhizosphere microbiome also serves as the first line of defense against many plant pathogens. While many rhizobacteria are capable of producing antifungal natural products, fungal pathogens, such as those belonging to the genus Fusarium, continue to be a major threat to agricultural crops worldwide. In this issue, Tracanna and coworkers (V. Tracanna, A. Ossowicki, M. L. C. Petrus, S. Overduin, et al., mSystems 6:e01116-20, 2021, https://doi.org/10.1128/mSystems.01116-20) implement a targeted amplicon sequencing approach to identify conserved domains and specific metabolic pathways shared among soil samples with antagonistic activities against Fusarium culmorum. They also introduce dom2BGC, an open-source annotation platform that builds co-occurrence networks of natural product-associated domains across samples and aids in putative gene cluster reconstruction. When coupled with metagenomics, functional amplicon sequencing and the dom2BGC pipeline can aid in identifying mechanisms and potential metabolites associated with particular microbiome-associated phenotypes.

Peeling the Layers Away: The Genomic Characterization of Bacillus pumilus 64-1, an Isolate With Antimicrobial Activity From the Marine Sponge Plakina cyanorosea (Porifera, Homoscleromorpha)

Bacillus pumilus 64-1, a bacterial strain isolated from the marine sponge Plakina cyanorosea, which exhibits antimicrobial activity against both pathogenic and drug-resistant Gram-positive and Gram-negative bacteria. This study aimed to conduct an in-depth genomic analysis of this bioactive sponge-derived strain. The nearly complete genome of strain 64-1 consists of 3.6 Mbp (41.5% GC), which includes 3,705 coding sequences (CDS). An open pangenome was observed when limiting to the type strains of the B. pumilus group and aquatic-derived B. pumilus representatives. The genome appears to encode for at least 12 potential biosynthetic gene clusters (BGCs), including both types I and III polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS), and one NRPS-T1PKS hybrid, among others. In particular, bacilysin and other bacteriocin-coding genes were found and may be associated with the detected antimicrobial activity. Strain 64-1 also appears to possess a broad repertoire of genes encoding for plant cell wall-degrading carbohydrate-active enzymes (CAZymes). A myriad of genes which may be involved in various process required by the strain in its marine habitat, such as those encoding for osmoprotectory transport systems and the biosynthesis of compatible solutes were also present. Several heavy metal tolerance genes are also present, together with various mobile elements including a region encoding for a type III-B Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) region, four prophage segments and transposase elements. This is the first report on the genomic characterization of a cultivable bacterial member of the Plakina cyanorosea holobiont.

Azohydromonas aeria sp. nov., isolated from air.

A grey pink colored bacterium, strain t3-1-3T, was isolated from the air at the foot of the Xiangshan Mountain in Beijing, China. The cells are aerobic, Gram-stain-negative, non-spore-forming, motile and coccoid-rod shaped (0.9-1.2 × 1.9-2.1 μm). Strain t3-1-3T was catalase-positive and oxidase-negative and this strain grew at 4-42°C (optimum 28°C), a pH of 4.0-9.0 (optimum pH 7.0) and under 0-2% (w/v) NaCl (optimum 0-1% NaCl). A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain t3-1-3T was closely related to Azohydromonas riparia UCM-11T (97.4% similarity), followed by Azohydromonas australica G1-2T (96.8%) and Azohydromonas ureilytica UCM-80T (96.7%). The genome of strain t3-1-3T contains 6,895 predicted protein-encoding genes, 8 rRNA genes, 62 tRNA genes and one sRNA gene, as well as five potential biosynthetic gene clusters, including clusters of genes coding for non-ribosomal peptide synthetase (NRPS), bacteriocin and arylpolyene and two clusters of genes for terpene. The predominant cellular fatty acids (> 10.0% of the total) in strain t3-1-3T were summed feature 3 (C16:1ω7c and/or C16:1ω6c, 37.8%), summed feature 8 (C18:1ω7c and/or C18:1ω6c, 29.7%) and C16:0 (17.3%). Strain t3-1-3T contained ubiquinone-8 (Q-8) as the predominant respiratory quinone. The polar lipids of strain t3-1-3T comprised phosphatidyl ethanolamine (PE), phosphatidyl glycerol (PG), diphosphatidyl glycerol (DPG), an unidentified glycolipid (GL), an unidentified aminophospholipid (APL), two unidentified phospholipid (PL1-2) and five unidentified lipid (L1-5). The DNA G + C content of the type strain is 70.3%. The broader range of growth temperature, assimilation of malic acid and trisodium citrate, presence of C18:3ω6c and an unidentified glycolipid and absence of C12:0 2-OH and C16:0iso differentiate strain t3-1-3T from related species. Based on the taxonomic data presented in this study, we suggest that strain t3-1-3T represents a novel species within the genus Azohydromonas, for which the name Azohydromonas aeria sp. nov. is proposed. The type strain of Azohydromonas aeria is t3-1-3T (= CFCC 13393T = LMG 30135T).

Nature's combinatorial biosynthesis and recently engineered production of nucleoside antibiotics in Streptomyces.

Modified nucleosides produced by Streptomyces and related actinomycetes are widely used in agriculture and medicine as antibacterial, antifungal, anticancer and antiviral agents. These specialized small-molecule metabolites are biosynthesized by complex enzymatic machineries encoded within gene clusters in the genome. The past decade has witnessed a burst of reports defining the key metabolic processes involved in the biosynthesis of several distinct families of nucleoside antibiotics. Furthermore, genome sequencing of various Streptomyces species has dramatically increased over recent years. Potential biosynthetic gene clusters for novel nucleoside antibiotics are now apparent by analysis of these genomes. Here we revisit strategies for production improvement of nucleoside antibiotics that have defined mechanisms of action, and are in clinical or agricultural use. We summarize the progress for genetically manipulating biosynthetic pathways for structural diversification of nucleoside antibiotics. Microorganism-based biosynthetic examples are provided and organized under genetic principles and metabolic engineering guidelines. We show perspectives on the future of combinatorial biosynthesis, and present a working model for discovery of novel nucleoside natural products in Streptomyces.

Identification of a novel endophytic Bacillus sp. from Capsicum annuum with highly efficient and broad spectrum plant probiotic effect.

The study mainly aimed the isolation and characterization of plant probiotic endophytic bacteria from Capsicum annuum to explore its multipotent agricultural applications. Endophytic bacteria were isolated from the surface sterilized fruit tissue. The isolates were then subjected to PCR-based screening for the presence of potential biosynthetic gene clusters. The PCR positive isolate was then analysed for its inhibitory effect towards fungal and bacterial pathogens. The compounds responsible for the antimicrobial activity was purified from large scale culture and subjected to identification by LC-MS/MS. The ability of the selected isolate in plant growth enhancement was also done using Vigna radiata seedlings. In this study, an endophytic bacterium isolated from C.annuum was found to have the phenotypic and genetic basis for broad antimicrobial property. PCR-based sequence analysis has resulted in the identification of nonribosomal peptide synthases, PKS Type I, Iturin, surfactin, DAPG and gacA genes in the selected isolate CaB 5. The bioactivity-guided fractionation using column and HPLC purification of active fraction followed by LC-MS/MS analysis has proved the presence of surfactin derivatives (M+H(+) - 1008 & 1036) and iturin (M+H(+) - 1058) as the basis of antimicrobial activity of CaB 5. The isolate was identified as a novel Bacillus sp. because of its low (76%) identity to the reported sequences. Endophytes are considered to have the genetic basis for a diverse array of bioactive metabolites which can have significant applications in both pharmaceutical industry and agriculture. The identification of CaB 5 with broad bioactivity and excellent plant growth enhancement on taxonomically distinct plant species as explained in current study and our previous reports highlights its plant probiotic applicability. This proves the potential of the isolate obtained in the study to be an excellent plant probiotic.

New natural products isolated from Metarhizium robertsii ARSEF 23 by chemical screening and identification of the gene cluster through engineered biosynthesis in Aspergillus nidulans A1145.

To rapidly identify novel natural products and their associated biosynthetic genes from underutilized and genetically difficult-to-manipulate microbes, we developed a method that uses (1) chemical screening to isolate novel microbial secondary metabolites, (2) bioinformatic analyses to identify a potential biosynthetic gene cluster and (3) heterologous expression of the genes in a convenient host to confirm the identity of the gene cluster and the proposed biosynthetic mechanism. The chemical screen was achieved by searching known natural product databases with data from liquid chromatographic and high-resolution mass spectrometric analyses collected on the extract from a target microbe culture. Using this method, we were able to isolate two new meroterpenes, subglutinols C (1) and D (2), from an entomopathogenic filamentous fungus Metarhizium robertsii ARSEF 23. Bioinformatics analysis of the genome allowed us to identify a gene cluster likely to be responsible for the formation of subglutinols. Heterologous expression of three genes from the gene cluster encoding a polyketide synthase, a prenyltransferase and a geranylgeranyl pyrophosphate synthase in Aspergillus nidulans A1145 afforded an α-pyrone-fused uncyclized diterpene, the expected intermediate of the subglutinol biosynthesis, thereby confirming the gene cluster to be responsible for the subglutinol biosynthesis. These results indicate the usefulness of our methodology in isolating new natural products and identifying their associated biosynthetic gene cluster from microbes that are not amenable to genetic manipulation. Our method should facilitate the natural product discovery efforts by expediting the identification of new secondary metabolites and their associated biosynthetic genes from a wider source of microbes.

Identification of the gene cluster for the dithiolopyrrolone antibiotic holomycin in Streptomyces clavuligerus

Streptomyces clavuligerus, an industrially important producer of clavulanate as well as cephem antibiotics, also produces the N-acylated dithiolopyrrolone antibiotic holomycin, a reported inhibitor of RNA synthesis. The genome sequence of S. clavuligerus ATCC 27064 was examined for a potential biosynthetic gene cluster, assuming that holomycin arises from some derivative of an L-Cys-L-Cys dipeptide that has undergone eight-electron oxidation, fused five-five ring formation, and decarboxylation. ORFs 3483-3492 comprise a candidate cluster, with a predicted acyltransferase, a stand-alone nonribosomal peptide synthetase (NRPS) module, and four flavin-dependent oxidoreductases. Deletions of ORF3488, the NRPS module, and ORF3489, a phosphopantothenoylcysteine decarboxylase homolog, abolished holomycin production both in wild type and in a holomycin-overproducing mutant. Heterologous expression and purification of ORF3488 allowed demonstration of L-Cys-AMP formation and subsequent covalent tethering of Cys to the phosphopantetheinyl arm of the thiolation domain of this NRPS protein. Purified ORF3483 shows acyltransferase activity, converting holothin to holomycin and longer acylated homologs as the last step in antibiotic assembly.