Challenging old microbiological treasures for natural compound biosynthesis capacity.

Strain collections are a treasure chest of numerous valuable and taxonomically validated bioresources. The Leibniz Institute DSMZ is one of the largest and most diverse microbial strain collections worldwide, with a long tradition of actinomycetes research. Actinomycetes, especially the genus Streptomyces, are renowned as prolific producers of antibiotics and many other bioactive natural products. In light of this, five Streptomyces strains, DSM 40971T, DSM 40484T, DSM 40713T, DSM 40976T, and DSM 40907T, which had been deposited a long time ago without comprehensive characterization, were the subject of polyphasic taxonomic studies and genome mining for natural compounds based on in vitro and in silico analyses. Phenotypic, genetic, and phylogenomic studies distinguished the strains from their closely related neighbors. The digital DNA-DNA hybridization and average nucleotide identity values between the five strains and their close, validly named species were below the threshold of 70% and 95%-96%, respectively, determined for prokaryotic species demarcation. Therefore, the five strains merit being considered as novel Streptomyces species, for which the names Streptomyces kutzneri sp. nov., Streptomyces stackebrandtii sp. nov., Streptomyces zähneri sp. nov., Streptomyces winkii sp. nov., and Streptomyces kroppenstedtii sp. nov. are proposed. Bioinformatics analysis of the genome sequences of the five strains revealed their genetic potential for the production of secondary metabolites, which helped identify the natural compounds cinerubin B from strain DSM 40484T and the phosphonate antibiotic phosphonoalamide from strain DSM 40907T and highlighted strain DSM 40976T as a candidate for regulator-guided gene cluster activation due to the abundance of numerous "Streptomyces antibiotic regulatory protein" (SARP) genes.

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.

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.

In response to the FDA warning about the use of photomedicine in gynecology.

To alert patients and health care providers about the use of energy-based devices to perform a vaginal "rejuvenation," cosmetic vaginal procedures, or nonsurgical vaginal procedures to treat symptoms related to menopause, urinary incontinence, or sexual function, the US Food and Drug Administration (FDA) has issued a warning about the effectiveness and safety of such devices. We agree with the FDA that certain devices (laser, radiofrequency, etc.) have been marketed inappropriately for uses that are outside of their cleared or approved intended uses. We want to position ourselves in the strict training of professionals so that the indications and techniques are used in the best possible way, knowing that, similar to any medical or surgical technique, the side effects can appear in the short and long term, and should be recognized and remedied.

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.

The 'gifted' actinomycete Streptomyces leeuwenhoekii.

Streptomyces leeuwenhoekii strains C34T, 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. This review focusses on this new clade of Streptomyces strains, summarises the literature and presents new information on strain C34T.

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.

A Streptomyces coelicolor host for the heterologous expression of Type III polyketide synthase genes.

BACKGROUND: Recent advances in genome sequencing, combined with bioinformatic analysis, has led to the identification of numerous novel natural product gene clusters, particularly in actinomycetes of terrestrial and marine origin. Many of these gene clusters encode uncharacterised Type III polyketide synthases. To facilitate the study of these genes and their potentially novel products, we set out to construct an actinomycete expression host specifically designed for the heterologous expression of Type III PKS genes and their gene clusters. RESULTS: A derivative of Streptomyces coelicolor A3(2) designed for the expression of Type III polyketide synthase (PKS) genes was constructed from the previously engineered expression strain S. coelicolor M1152 [Δact Δred Δcpk Δcda rpoB(C1298T)] by removal of all three of the endogenous Type III PKS genes (gcs, srsA, rppA) by PCR targeting. The resulting septuple deletion mutant, M1317, proved to be an effective surrogate host for the expression of actinobacterial Type III PKS genes: expression of the reintroduced gcs gene from S. coelicolor and of the heterologous rppA gene from Streptomyces venezuelae under the control of the constitutive ermE* promoter resulted in copious production of germicidin and flaviolin, respectively. CONCLUSIONS: The newly constructed expression host S. coelicolor M1317 should be particularly useful for the discovery and analysis of new Type III polyketide metabolites.

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.

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.

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. Heterologous expression of these cryptic gene clusters combined with comparative metabolic profiling provides an important means to identify potentially novel compounds. In this chapter, the methods and strategies used to heterologously express actinomycete gene clusters, including the techniques used for cloning secondary metabolite gene clusters, the Streptomyces hosts used for their expression, and the techniques employed to analyze their products by metabolic profiling, are described.