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

Streptomyces clavuligerus 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). This yielded a chromosome assembly (6.75 Mb) plus assemblies for pSCL4 (1795 kb) and pSCL2 (149 kb). The strain also carries pSCL1 (12 kb), but its small size resulted in only partial sequence coverage. The previously described pSCL3 (444 kb) is not present in this isolate. Using our Cas9 vectors, we cured pSCL4 with high efficiency by targeting the plasmid's parB gene. Five of the resulting pSCL4-cured isolates were characterized and all showed impaired sporulation. Shotgun genome sequencing of each of these derivatives revealed large deletions at the ends of the chromosomes in all of them, and for two clones sufficient sequence data was obtained to show that the chromosome had circularized. Taken together, these data indicate that pSCL4 is essential for the structural stability of the linear chromosome.

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 8.2 Mb chromosome and a 158 kb plasmid, pSVJI1, which had not been reported previously. Surprisingly, while NRRL B-65442 yields green spores on MYM agar, the American Type Culture Collection (ATCC) type strain 10712 (from which NRRL B-65442 was derived) produces grey spores. While comparison of the genome sequences of the two isolates revealed almost total identity, it did reveal a single nucleotide substitution in a gene, vnz_33525, involved in spore pigment biosynthesis. Replacement of the vnz_33525 allele of ATCC 10712 with that of NRRL B-65442 resulted in green spores, explaining the discrepancy in spore pigmentation. We also applied CRISPR-Cas9 to delete the essential parB of pSVJI1 to cure the plasmid from the strain without obvious phenotypic consequences.

In Situ Activation and Heterologous Production of a Cryptic Lantibiotic from an African Plant Ant-Derived Saccharopolyspora Species.

Most clinical antibiotics are derived from actinomycete natural products discovered at least 60 years ago. However, the repeated rediscovery of known compounds led the pharmaceutical industry to largely discard microbial natural products (NPs) as a source of new chemical diversity. Recent advances in genome sequencing have revealed that these organisms have the potential to make many more NPs than previously thought. Approaches to unlock NP biosynthesis by genetic manipulation of strains, by the application of chemical genetics, or by microbial cocultivation have resulted in the identification of new antibacterial compounds. Concomitantly, intensive exploration of coevolved ecological niches, such as insect-microbe defensive symbioses, has revealed these to be a rich source of chemical novelty. Here, we report the new lanthipeptide antibiotic kyamicin, which was generated through the activation of a cryptic biosynthetic gene cluster identified by genome mining Saccharopolyspora species found in the obligate domatium-dwelling ant Tetraponera penzigi of the ant plant Vachellia drepanolobium Transcriptional activation of this silent gene cluster was achieved by ectopic expression of a pathway-specific activator under the control of a constitutive promoter. Subsequently, a heterologous production platform was developed which enabled the purification of kyamicin for structural characterization and bioactivity determination. This strategy was also successful for the production of lantibiotics from other genera, paving the way for a synthetic heterologous expression platform for the discovery of lanthipeptides that are not detected under laboratory conditions or that are new to nature.IMPORTANCE The discovery of novel antibiotics to tackle the growing threat of antimicrobial resistance is impeded by difficulties in accessing the full biosynthetic potential of microorganisms. The development of new tools to unlock the biosynthesis of cryptic bacterial natural products will greatly increase the repertoire of natural product scaffolds. Here, we report a strategy for the ectopic expression of pathway-specific positive regulators that can be rapidly applied to activate the biosynthesis of cryptic lanthipeptide biosynthetic gene clusters. This allowed the discovery of a new lanthipeptide antibiotic directly from the native host and via heterologous expression.

Heterologous Expression of a Cryptic Gene Cluster from Streptomyces leeuwenhoekii C34T Yields a Novel Lasso Peptide, Leepeptin.

Analysis of the genome sequence of Streptomyces leeuwenhoekii C34T 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 S. leeuwenhoekii C34T and after heterologous expression of its BGC in Streptomyces coelicolor, is identical to the previously characterized chaxapeptin. Lp1, whose production could not be detected or achieved heterologously, appears to be identical to a recently identified member of the citrulassin family of lasso peptides. Since production of Lp2 by S. leeuwenhoekii C34T was not observed, its BGC was also expressed in S. coelicolor The lasso peptide was isolated and its structure confirmed by mass spectrometry and nuclear magnetic resonance analyses, revealing a novel structure that appears to represent a new family of lasso peptides.IMPORTANCE Recent developments in genome sequencing combined with bioinformatic analysis have revealed that actinomycetes contain a plethora of unexpected BGCs and thus have the potential to produce many more natural products than previously thought. This reflects the inability to detect the production of these compounds under laboratory conditions, perhaps through the use of inappropriate growth media or the absence of the environmental cues required to elicit expression of the corresponding BGCs. One approach to overcoming this problem is to circumvent the regulatory mechanisms that control expression of the BGC in its natural host by deploying heterologous expression. The generally compact nature of lasso peptide BGCs makes them particularly amenable to this approach, and, in the example given here, analysis revealed a new member of the lasso peptide family of RiPPs. This approach should be readily applicable to other cryptic lasso peptide gene clusters and would also facilitate the design and production of nonnatural variants by changing the sequence encoding the core peptide, as has been achieved with other classes of RiPPs.

Structures of DPAGT1 Explain Glycosylation Disease Mechanisms and Advance TB Antibiotic Design.

Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.

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

The tunicamycin biosynthetic gene cluster of Streptomyces chartreusis consists of 14 genes (tunA to tunN) 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, tunp1 and tunp2. In-frame deletion analysis revealed that just six of these genes (tunABCDEH) are essential for tunicamycin production in the heterologous host Streptomyces coelicolor, while five (tunFGKLN) with likely counterparts in primary metabolism are not necessary, but presumably ensure efficient production of the antibiotic at the onset of tunicamycin biosynthesis. Three genes are implicated in immunity, namely, tunI and tunJ, which encode a two-component ABC transporter presumably required for export of the antibiotic, and tunM, which encodes a putative S-adenosylmethionine (SAM)-dependent methyltransferase. Expression of tunIJ or tunM in S. coelicolor conferred resistance to exogenous tunicamycin. The results presented here provide new insights into tunicamycin biosynthesis and immunity.

Watasemycin biosynthesis in Streptomyces venezuelae: thiazoline C-methylation by a type B radical-SAM methylase homologue.

2-Hydroxyphenylthiazolines are a family of iron-chelating nonribosomal peptide natural products that function as virulence-conferring siderophores in various Gram-negative bacteria. They have also been reported as metabolites of Gram-positive Streptomyces species. Transcriptional analyses of Streptomyces venezuelae ATCC 10712 revealed that its genome contains a putative 2-hydroxyphenylthiazoline biosynthetic gene cluster. Heterologous expression of the gene cluster in Streptomyces coelicolor M1152 showed that the mono- and dimethylated derivatives, thiazostatin and watasemycin, respectively, of the 2-hydroxyphenylthiazoline enantiopyochelin are two of its metabolic products. In addition, isopyochelin, a novel isomer of pyochelin containing a C-methylated thiazolidine, was identified as a third metabolic product of the cluster. Metabolites with molecular formulae corresponding to aerugine and pulicatins A/B were also detected. The structure and stereochemistry of isopyochelin were confirmed by comparison with synthetic standards. The role of two genes in the cluster encoding homologues of PchK, which is proposed to catalyse thiazoline reduction in the biosynthesis of enantiopyochelin in Pseudomonas protegens, was investigated. One was required for the production of all the metabolic products of the cluster, whereas the other appears not to be involved in the biosynthesis of any of them. Deletion of a gene in the cluster encoding a type B radical-SAM methylase homologue abolished the production of watasemycin, but not thiazostatin or isopyochelin. Feeding of thiazostatin to the mutant lacking the functional PchK homologue resulted in complete conversion to watasemycin, demonstrating that thiazoline C-methylation by the type B radical-SAM methylase homologue is the final step in watasemycin biosynthesis.

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. Constitutive expression of vemR in the ΔbldM mutant led to the production of sufficient venemycin for structural characterisation, confirming its unusual biaryl structure. Co-expression of the venemycin biosynthetic gene cluster and vemR in the heterologous host Streptomyces coelicolor also resulted in venemycin production. Although the gene cluster encodes two halogenases and a flavin reductase, constitutive expression of all three genes led to the accumulation only of a monohalogenated venemycin derivative, both in the native producer and the heterologous host. A competition experiment in which equimolar quantities of sodium chloride and sodium bromide were fed to the venemycin-producing strains resulted in the preferential incorporation of bromine, thus suggesting that bromide is the preferred substrate for one or both halogenases.

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 impact that these technologies are having in the discovery and study of natural products from actinobacteria, including those from the marine environment, is only starting to be realised. In this review we provide a historical perspective of the field, analyse the strengths and limitations of the most relevant technologies, and share the insights acquired during our genome mining projects.

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. strain C34) was isolated from the hyper-arid high-altitude Atacama Desert in Chile and shown to produce novel polyketide antibiotics. RESULTS: Here we present the de novo sequencing of the S. leeuwenhoekii linear chromosome (8 Mb) and two extrachromosomal replicons, the circular pSLE1 (86 kb) and the linear pSLE2 (132 kb), all in single contigs, obtained by combining Pacific Biosciences SMRT (PacBio) and Illumina MiSeq technologies. We identified the biosynthetic gene clusters for chaxamycin, chaxalactin, hygromycin A and desferrioxamine E, metabolites all previously shown to be produced by this strain (J Nat Prod, 2011, 74:1965) and an additional 31 putative gene clusters for specialised metabolites. As well as gene clusters for polyketides and non-ribosomal peptides, we also identified three gene clusters encoding novel lasso-peptides. CONCLUSIONS: The S. leeuwenhoekii genome contains 35 gene clusters apparently encoding the biosynthesis of specialised metabolites, most of them completely novel and uncharacterised. This project has served to evaluate the current state of NGS for efficient and effective genome mining of high GC actinomycetes. The PacBio technology now permits the assembly of actinomycete replicons into single contigs with >99 % accuracy. The assembled Illumina sequence permitted not only the correction of omissions found in GC homopolymers in the PacBio assembly (exacerbated by the high GC content of actinomycete DNA) but it also allowed us to obtain the sequences of the termini of the chromosome and of a linear plasmid that were not assembled by PacBio. We propose an experimental pipeline that uses the Illumina assembled contigs, in addition to just the reads, to complement the current limitations of the PacBio sequencing technology and assembly software.

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 80.2-kb chaxamycin biosynthesis gene cluster, which was achieved by both mutational analysis in the natural producer and heterologous expression in Streptomyces coelicolor A3(2) strain M1152. Restoration of chaxamycin production in a nonproducing ΔcxmK mutant (cxmK encodes 3-amino-5-hydroxybenzoic acid [AHBA] synthase) was achieved by supplementing the growth medium with AHBA, suggesting that mutasynthesis may be a viable approach for the generation of novel chaxamycin derivatives.

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. Moreover, we demonstrate that initial expression of mibR, and thus microbisporicin production, is dependent on the ppGpp synthetase gene (relA) of M. corallina. In addition, we show that constitutive expression of either of the two positively acting regulatory genes, mibR or mibX, leads to precocious and enhanced microbisporicin production.

Use of the meganuclease I-SceI of Saccharomyces cerevisiae to select for gene deletions in actinomycetes.

The search for new natural products is leading to the isolation of novel actinomycete species, many of which will ultimately require genetic analysis. Some of these isolates will likely exhibit low intrinsic frequencies of homologous recombination and fail to sporulate under laboratory conditions, exacerbating the construction of targeted gene deletions and replacements in genetically uncharacterised strains. To facilitate the genetic manipulation of such species, we have developed an efficient method to generate gene or gene cluster deletions in actinomycetes by homologous recombination that does not introduce any other changes to the targeted organism's genome. We have synthesised a codon optimised I-SceI gene for expression in actinomycetes that results in the production of the yeast I-SceI homing endonuclease which produces double strand breaks at a unique introduced 18 base pair recognition sequence. Only those genomes that undergo homologous recombination survive, providing a powerful selection for recombinants, approximately half of which possess the desired mutant genotype. To demonstrate the efficacy and efficiency of the system, we deleted part of the gene cluster for the red-pigmented undecylprodiginine complex of compounds in Streptomyces coelicolor M1141. We believe that the system we have developed will be broadly applicable across a wide range of actinomycetes.

New insights into chloramphenicol biosynthesis in Streptomyces venezuelae ATCC 10712.

Comparative genome analysis revealed seven uncharacterized genes, sven0909 to sven0915, adjacent to the previously identified chloramphenicol biosynthetic gene cluster (sven0916-sven0928) of Streptomyces venezuelae strain ATCC 10712 that was absent in a closely related Streptomyces strain that does not produce chloramphenicol. Transcriptional analysis suggested that three of these genes might be involved in chloramphenicol production, a prediction confirmed by the construction of deletion mutants. These three genes encode a cluster-associated transcriptional activator (Sven0913), a phosphopantetheinyl transferase (Sven0914), and a Na(+)/H(+) antiporter (Sven0915). Bioinformatic analysis also revealed the presence of a previously undetected gene, sven0925, embedded within the chloramphenicol biosynthetic gene cluster that appears to encode an acyl carrier protein, bringing the number of new genes likely to be involved in chloramphenicol production to four. Microarray experiments and synteny comparisons also suggest that sven0929 is part of the biosynthetic gene cluster. This has allowed us to propose an updated and revised version of the chloramphenicol biosynthetic pathway.

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.

Understanding and manipulating antibiotic production in actinomycetes.

Actinomycetes are prolific producers of natural products with a wide range of biological activities. Many of the compounds that they make (and derivatives thereof) are used extensively in medicine, most notably as clinically important antibiotics, and in agriculture. Moreover, these organisms remain a source of novel and potentially useful molecules, but maximizing their biosynthetic potential requires a better understanding of natural product biosynthesis. Recent developments in genome sequencing have greatly facilitated the identification of natural product biosynthetic gene clusters. In the present article, I summarize the recent contributions of our laboratory in applying genomic technologies to better understand and manipulate natural product biosynthesis in a range of different actinomycetes.

Synthetic RNA Silencing of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2).

We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.

Phage p1-derived artificial chromosomes facilitate heterologous expression of the FK506 gene cluster.

We describe a procedure for the conjugative transfer of phage P1-derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (≥70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus), a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L(-1) to 5.5 mg L(-1)) in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.

The antibiotic planosporicin coordinates its own production in the actinomycete Planomonospora alba.

Planosporicin is a ribosomally synthesized, posttranslationally modified peptide lantibiotic produced by the actinomycete Planomonospora alba. It contains one methyl-lanthionine and four lanthionine bridges and inhibits cell wall biosynthesis in other Gram-positive bacteria probably by binding to lipid II, the immediate precursor for cell wall biosynthesis. Planosporicin production, which is encoded by a cluster of 15 genes, is confined to stationary phase in liquid culture and to the onset of morphological differentiation when P. alba is grown on agar. This growth phase-dependent gene expression is controlled transcriptionally by three pathway-specific regulatory proteins: an extracytoplasmic function σ factor (PspX), its cognate anti-σ factor (PspW), and a transcriptional activator (PspR) with a C-terminal helix-turn-helix DNA-binding domain. Using mutational analysis, S1 nuclease mapping, quantitative RT-PCR, and transcriptional fusions, we have determined the direct regulatory dependencies within the planosporicin gene cluster and present a model in which subinhibitory concentrations of the lantibiotic function in a feed-forward mechanism to elicit high levels of planosporicin production. We show that in addition to acting as an antibiotic, planosporicin can function as an extracellular signaling molecule to elicit precocious production of the lantibiotic, presumably ensuring synchronous and concerted lantibiotic biosynthesis in the wider population and, thus, the production of ecologically effective concentrations of the antibiotic.

Investigation of DNA sequence recognition by a streptomycete MarR family transcriptional regulator through surface plasmon resonance and X-ray crystallography.

Consistent with their complex lifestyles and rich secondary metabolite profiles, the genomes of streptomycetes encode a plethora of transcription factors, the vast majority of which are uncharacterized. Herein, we use Surface Plasmon Resonance (SPR) to identify and delineate putative operator sites for SCO3205, a MarR family transcriptional regulator from Streptomyces coelicolor that is well represented in sequenced actinomycete genomes. In particular, we use a novel SPR footprinting approach that exploits indirect ligand capture to vastly extend the lifetime of a standard streptavidin SPR chip. We define two operator sites upstream of sco3205 and a pseudopalindromic consensus sequence derived from these enables further potential operator sites to be identified in the S. coelicolor genome. We evaluate each of these through SPR and test the importance of the conserved bases within the consensus sequence. Informed by these results, we determine the crystal structure of a SCO3205-DNA complex at 2.8 Å resolution, enabling molecular level rationalization of the SPR data. Taken together, our observations support a DNA recognition mechanism involving both direct and indirect sequence readout.