MIBiG 4.0: advancing biosynthetic gene cluster curation through global collaboration.

Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015.

Biotechnological and pharmaceutical potential of twenty-eight novel type strains of from different environments worldwide.

Actinomycetes are a prolific source of bioactive natural compounds many of which are used as antibiotics or other drugs. In this study we investigated the genomic and biochemical diversity of 32 actinobacterial strains that had been deposited at the DSMZ-German Collection of Microorganisms and Cell Cultures decades ago. Genome-based phylogeny and DNA-DNA hybridization supported the assignment of these strains to 26 novel species and two novel subspecies and a reclassification of a species.

Genome editing reveals that pSCL4 is required for chromosome linearity in .

is an industrially important actinomycete whose genetic manipulation is limited by low transformation and conjugation efficiencies, low levels of recombination of introduced DNA, and difficulty in obtaining consistent sporulation. We describe the construction and application of versatile vectors for Cas9-mediated genome editing of this strain. To design spacer sequences with confidence, we derived a highly accurate genome assembly for an isolate of the type strain (ATCC 27064).

Streptomyces venezuelae NRRL B-65442: genome sequence of a model strain used to study morphological differentiation in filamentous actinobacteria.

For over a decade, Streptomyces venezuelae has been used to study the molecular mechanisms that control morphological development in streptomycetes and is now a well-established model strain. Its rapid growth and ability to sporulate in a near-synchronised manner in liquid culture, unusual among streptomycetes, greatly facilitates the application of modern molecular techniques such as ChIP-seq and RNA-seq, as well as time-lapse fluorescence imaging of the complete Streptomyces life cycle. Here we describe a high-quality genome sequence of our isolate of the strain (Northern Regional Research Laboratory [NRRL] B-65442) consisting of an 8.

Heterologous Expression of a Cryptic Gene Cluster from Streptomyces leeuwenhoekii C34 Yields a Novel Lasso Peptide, Leepeptin.

Analysis of the genome sequence of C34 identified biosynthetic gene clusters (BGCs) for three different lasso peptides (Lp1, Lp2, and Lp3) which were not known to be made by the strain. Lasso peptides represent relatively new members of the RiPP (ribosomally synthesized and posttranslationally modified peptides) family of natural products and have not been extensively studied. Lp3, whose production could be detected in culture supernatants from C34 and after heterologous expression of its BGC in , is identical to the previously characterized chaxapeptin.

Analysis of the Tunicamycin Biosynthetic Gene Cluster of Streptomyces chartreusis Reveals New Insights into Tunicamycin Production and Immunity.

The tunicamycin biosynthetic gene cluster of consists of 14 genes ( to ) with a high degree of apparent translational coupling. Transcriptional analysis revealed that all of these genes are likely to be transcribed as a single operon from two promoters, p1 and p2. In-frame deletion analysis revealed that just six of these genes () are essential for tunicamycin production in the heterologous host , while five () with likely counterparts in primary metabolism are not necessary, but presumably ensure efficient production of the antibiotic at the onset of tunicamycin biosynthesis.

The 'gifted' actinomycete Streptomyces leeuwenhoekii.

Streptomyces leeuwenhoekii strains C34, C38, C58 and C79 were isolated from a soil sample collected from the Chaxa Lagoon, located in the Salar de Atacama in northern Chile. These streptomycetes produce a variety of new specialised metabolites with antibiotic, anti-cancer and anti-inflammatory activities. Moreover, genome mining performed on two of these strains has revealed the presence of biosynthetic gene clusters with the potential to produce new specialised metabolites.

Discovery of Unusual Biaryl Polyketides by Activation of a Silent Streptomyces venezuelae Biosynthetic Gene Cluster.

Comparative transcriptional profiling of a ΔbldM mutant of Streptomyces venezuelae with its unmodified progenitor revealed that the expression of a cryptic biosynthetic gene cluster containing both type I and type III polyketide synthase genes is activated in the mutant. The 29.5 kb gene cluster, which was predicted to encode an unusual biaryl metabolite, which we named venemycin, and potentially halogenated derivatives, contains 16 genes including one-vemR-that encodes a transcriptional activator of the large ATP-binding LuxR-like (LAL) family.

Next Generation Sequencing of Actinobacteria for the Discovery of Novel Natural Products.

Like many fields of the biosciences, actinomycete natural products research has been revolutionised by next-generation DNA sequencing (NGS). Hundreds of new genome sequences from actinobacteria are made public every year, many of them as a result of projects aimed at identifying new natural products and their biosynthetic pathways through genome mining. Advances in these technologies in the last five years have meant not only a reduction in the cost of whole genome sequencing, but also a substantial increase in the quality of the data, having moved from obtaining a draft genome sequence comprised of several hundred short contigs, sometimes of doubtful reliability, to the possibility of obtaining an almost complete and accurate chromosome sequence in a single contig, allowing a detailed study of gene clusters and the design of strategies for refactoring and full gene cluster synthesis.

The Streptomyces leeuwenhoekii genome: de novo sequencing and assembly in single contigs of the chromosome, circular plasmid pSLE1 and linear plasmid pSLE2.

Background: Next Generation DNA Sequencing (NGS) and genome mining of actinomycetes and other microorganisms is currently one of the most promising strategies for the discovery of novel bioactive natural products, potentially revealing novel chemistry and enzymology involved in their biosynthesis. This approach also allows rapid insights into the biosynthetic potential of microorganisms isolated from unexploited habitats and ecosystems, which in many cases may prove difficult to culture and manipulate in the laboratory. Streptomyces leeuwenhoekii (formerly Streptomyces sp.

Identification and Heterologous Expression of the Chaxamycin Biosynthesis Gene Cluster from Streptomyces leeuwenhoekii.

Streptomyces leeuwenhoekii, isolated from the hyperarid Atacama Desert, produces the new ansamycin-like compounds chaxamycins A to D, which possess potent antibacterial activity and moderate antiproliferative activity. We report the development of genetic tools to manipulate S. leeuwenhoekii and the identification and partial characterization of the 80.

A relA-dependent regulatory cascade for auto-induction of microbisporicin production in Microbispora corallina.

Microbisporicin is a potent type I lantibiotic produced by the rare actinomycete Microbispora corallina that is in preclinical trials for the treatment of infections caused by methicillin-resistant isolates of Staphylococcus aureus (MRSA). Analysis of the gene cluster for the biosynthesis of microbisporicin, which contains two unique post-translationally modified residues (5-chlorotryptophan and 3, 4-dihydroxyproline), has revealed an unusual regulatory mechanism that involves a pathway-specific extracytoplasmic function sigma factor (MibX)/anti-sigma factor (MibW) complex and an additional transcriptional regulator MibR. A model for the regulation of microbisporicin biosynthesis derived from transcriptional, mutational and quantitative reverse transcription polymerase chain reaction analyses suggests that MibR, which contains a C-terminal DNA-binding domain found in the LuxR family of transcriptional activators, functions as an essential master regulator to trigger microbisporicin production while MibX and MibW induce feed-forward biosynthesis and producer immunity.

Heterologous expression of natural product biosynthetic gene clusters in Streptomyces coelicolor: from genome mining to manipulation of biosynthetic pathways.

Heterologous gene expression is one of the main strategies used to access the full biosynthetic potential of actinomycetes, as well as to study the metabolic pathways of natural product biosynthesis and to create unnatural pathways. Streptomyces coelicolor A3(2) is the most studied member of the actinomycetes, bacteria renowned for their prolific capacity to synthesize a wide range of biologically active specialized metabolites. We review here the use of strains of this species for the heterologous production of structurally diverse actinomycete natural products.

Streptomyces coelicolor as an expression host for heterologous gene clusters.

The expression of a gene or a set of genes from one organism in a different species is known as "heterologous expression." In actinomycetes, prolific producers of natural products, heterologous gene expression has been used to confirm the clustering of secondary metabolite biosynthetic genes, to analyze natural product biosynthesis, to produce variants of natural products by genetic engineering, and to discover new compounds by screening genomic libraries. Recent advances in DNA sequencing have enabled the rapid and affordable sequencing of actinomycete genomes and revealed a large number of secondary metabolite gene clusters with no known products.

Biosynthesis of the tunicamycin antibiotics proceeds via unique exo-glycal intermediates.

The tunicamycins are archetypal nucleoside antibiotics targeting bacterial peptidoglycan biosynthesis and eukaryotic protein N-glycosylation. Understanding the biosynthesis of their unusual carbon framework may lead to variants with improved selectivity. Here, we demonstrate in vitro recapitulation of key sugar-manipulating enzymes from this pathway.

Identification and characterization of CdgB, a diguanylate cyclase involved in developmental processes in Streptomyces coelicolor.

We describe the identification and functional characterization of cdgB (SCO4281), a recently discovered target of BldD, a key regulator of morphological differentiation and antibiotic production in the mycelial bacteria of the genus Streptomyces. cdgB (cyclic dimeric GMP [c-di-GMP] B) encodes a GGDEF-containing protein that has diguanylate cyclase activity in vitro. Consistent with this enzymatic activity, heterologous expression of cdgB in Escherichia coli resulted in increased production of extracellular matrix in colonies and enhanced surface attachment of cells in standing liquid cultures.

Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters.

We have constructed derivatives of Streptomyces coelicolor M145 as hosts for the heterologous expression of secondary metabolite gene clusters. To remove potentially competitive sinks of carbon and nitrogen, and to provide a host devoid of antibiotic activity, we deleted four endogenous secondary metabolite gene clusters from S. coelicolor M145--those for actinorhodin, prodiginine, CPK and CDA biosynthesis.

Heterologous expression of the biosynthetic gene clusters of coumermycin A(1), clorobiocin and caprazamycins in genetically modified Streptomyces coelicolor strains.

The biosynthetic gene clusters of the aminocoumarin antibiotics clorobiocin and coumermycin A(1) and of the liponucleoside antibiotic caprazamycin were stably integrated into the genomes of different host strains derived from Streptomyces coelicolor A3(2). For the heterologous expression of clorobiocin derivatives in a chemically defined medium, inclusion of 0.6% of the siloxylated ethylene oxide/propylene oxide copolymer Q2-5247 into the growth medium proved to result in a 4.

Deletion of a regulatory gene within the cpk gene cluster reveals novel antibacterial activity in Streptomyces coelicolor A3(2).

Genome sequencing of Streptomyces coelicolor A3(2) revealed an uncharacterized type I polyketide synthase gene cluster (cpk). Here we describe the discovery of a novel antibacterial activity (abCPK) and a yellow-pigmented secondary metabolite (yCPK) after deleting a presumed pathway-specific regulatory gene (scbR2) that encodes a member of the gamma-butyrolactone receptor family of proteins and which lies in the cpk gene cluster. Overproduction of yCPK and abCPK in a scbR2 deletion mutant, and the absence of the newly described compounds from cpk deletion mutants, suggest that they are products of the previously orphan cpk biosynthetic pathway in which abCPK is converted into the yellow pigment.

Regulatory mechanisms controlling antibiotic production in Streptomyces clavuligerus.

Streptomyces clavuligerus produces a large array of natural compounds with antibiotic, antitumor, beta-lactamase inhibition or inmunomodulating activities. The production of cephamycin C, clavulanic acid and other compounds with a clavam structure has been studied for many years. A network of regulatory mechanisms is present in S.

Streptomyces clavuligerus relA-null mutants overproduce clavulanic acid and cephamycin C: negative regulation of secondary metabolism by (p)ppGpp.

The (p)ppGpp synthetase gene, relA, of Streptomyces clavuligerus was cloned, sequenced and shown to be located in a genomic region that is highly conserved in other Streptomyces species. relA-disrupted and relA-deleted mutants of S. clavuligerus were constructed, and both were unable to form aerial mycelium or to sporulate, but regained these abilities when complemented with wild-type relA.

An rplKDelta29-PALG-32 mutation leads to reduced expression of the regulatory genes ccaR and claR and very low transcription of the ceaS2 gene for clavulanic acid biosynthesis in Streptomyces clavuligerus.

The transcriptional and translational control of the biosynthesis of the beta-lactamase inhibitor clavulanic acid is a subject of great scientific and industrial interest. To study the role of the ribosomal protein L11 on control of clavulanic acid gene transcription, the DNA region aspC-tRNA(trp)-secE-rplK-rplA-rplJ-rplL of Streptomyces clavuligerus was cloned and characterized. An S.