Mervyn J. Bibb

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.

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

Significance A mechanism for regulating production of the antibiotic planosporicin by Planomonospora alba is described. A low level of planosporicin biosynthesis, probably stimulated by nutrient limitation, functions in a feed-forward mechanism to release a key regulatory protein required for high level production. Planosporicin also functions as an extracellular signaling molecule to induce its own synchronous production, presumably ensuring ecologically effective levels of the antibiotic by coordinating biosynthesis in the multicellular colony, regions of which have reached different levels of maturity. This information was used to increase the level of planosporicin production, with important implications for strain improvement. 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.

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

BackgroundNext 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.ResultsHere we present the de novo sequencing of the S. leeuwenhoekii linear chromosome (8xa0Mb) and two extrachromosomal replicons, the circular pSLE1 (86xa0kb) and the linear pSLE2 (132xa0kb), 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.ConclusionsThe 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 >99xa0% 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.

Cloning and Analysis of the Planosporicin Lantibiotic Biosynthetic Gene Cluster of Planomonospora alba

The increasing prevalence of antibiotic resistance in bacterial pathogens has renewed focus on natural products with antimicrobial properties. Lantibiotics are ribosomally synthesized peptide antibiotics that are posttranslationally modified to introduce (methyl)lanthionine bridges. Actinomycetes are renowned for their ability to produce a large variety of antibiotics, many with clinical applications, but are known to make only a few lantibiotics. One such compound is planosporicin produced by Planomonospora alba, which inhibits cell wall biosynthesis in Gram-positive pathogens. Planosporicin is a type AI lantibiotic structurally similar to those which bind lipid II, the immediate precursor for cell wall biosynthesis. The gene cluster responsible for planosporicin biosynthesis was identified by genome mining and subsequently isolated from a P. alba cosmid library. A minimal cluster of 15 genes sufficient for planosporicin production was defined by heterologous expression in Nonomuraea sp. strain ATCC 39727, while deletion of the gene encoding the precursor peptide from P. alba, which abolished planosporicin production, was also used to confirm the identity of the gene cluster. Deletion of genes encoding likely biosynthetic enzymes identified through bioinformatic analysis revealed that they, too, are essential for planosporicin production in the native host. Reverse transcription-PCR (RT-PCR) analysis indicated that the planosporicin gene cluster is transcribed in three operons. Expression of one of these, pspEF, which encodes an ABC transporter, in Streptomyces coelicolor A3(2) conferred some degree of planosporicin resistance on the heterologous host. The inability to delete these genes from P. alba suggests that they play an essential role in immunity in the natural producer.

New Insights into Chloramphenicol Biosynthesis in Streptomyces venezuelae ATCC 10712

ABSTRACT 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.

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 organisms 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.

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.

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.