Polyketide Biosynthetic Gene Clusters Research Articles

Biosynthesis of a Tricyclo[6.2.2.02,7 ]dodecane System by a Berberine Bridge Enzyme-Like Aldolase.

The aldol reaction is one of the most fundamental stereocontrolled carbon-carbon bond-forming reactions and is mainly catalyzed by aldolases in nature. Despite the fact that the aldol reaction has been widely proposed to be involved in fungal secondary metabolite biosynthesis, a dedicated aldolase that catalyzes stereoselective aldol reactions has only rarely been reported in fungi. Herein, we activated a cryptic polyketide biosynthetic gene cluster that was upregulated in the fungal wheat pathogen Parastagonospora nodorum during plant infection; this resulted in the production of the phytotoxic stemphyloxin II (1). Through heterologous reconstruction of the biosynthetic pathway and in vitro assay by using cell-free lysate from Aspergillus nidulans, we demonstrated that a berberine bridge enzyme (BBE)-like protein SthB catalyzes an intramolecular aldol reaction to establish the bridged tricyclo[6.2.2.02,7 ]dodecane skeleton in the post-assembly tailoring step. The characterization of SthB as an aldolase enriches the catalytic toolbox of classic reactions and the functional diversities of the BBE superfamily of enzymes.

Nystatin-like Pseudonocardia polyene B1, a novel disaccharide-containing antifungal heptaene antibiotic

Polyene macrolides such as nystatin A1 and amphotericin B belong to a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Recently, nystatin-like Pseudonocardia polyene (NPP) A1 has been identified as a unique disaccharide-containing tetraene antifungal macrolide produced by Pseudonocardia autotrophica. Despite its significantly increased water solubility and decreased hemolytic activity, its antifungal activity remains limited compared with that of nystatin A1. In this study, we developed NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase NppC. The low level NPP B1 production yield was successfully improved by eliminating the native plasmid encoding a polyketide biosynthetic gene cluster present in P. autotrophica. In vitro and in vivo antifungal activity and toxicity studies indicated that NPP B1 exhibited comparable antifungal activity against Candida albicans and was less toxic than the most potent heptaene antifungal, amphotericin B. Moreover, NPP B1 showed improved pharmacokinetic parameters compared to those of amphotericin B, suggesting that NPP B1 could be a promising candidate for development into a pharmacokinetically improved and less-toxic polyene antifungal antibiotic.

Malleilactone Is a Burkholderia pseudomallei Virulence Factor Regulated by Antibiotics and Quorum Sensing.

Burkholderia pseudomallei, the causative agent of melioidosis, encodes almost a dozen predicted polyketide (PK) biosynthetic gene clusters. Many of these are regulated by LuxR-I-type acyl-homoserine (AHL) quorum-sensing systems. One of the PK gene clusters, the mal gene cluster, is conserved in the close relative Burkholderia thailandensis The B. thailandensis mal genes code for the cytotoxin malleilactone and are regulated by a genetically linked LuxR-type transcription factor, MalR. Although AHLs typically interact with LuxR-type proteins to modulate gene transcription, the B. thailandensis MalR does not appear to be an AHL receptor. Here, we characterize the mal genes and MalR in B. pseudomallei We use chemical analyses to demonstrate that the B. pseudomallei mal genes code for malleilactone. Our results show that MalR and the mal genes contribute to the ability of B. pseudomallei to kill Caenorhabditis elegans In B. thailandensis, antibiotics like trimethoprim can activate MalR by driving transcription of the mal genes, and we demonstrate that some of the same antibiotics induce expression of B. pseudomallei malR We also demonstrate that B. pseudomallei MalR does not respond directly to AHLs. Our results suggest that MalR is indirectly repressed by AHLs, possibly through a repressor, ScmR. We further show that malleilactone is a B. pseudomallei virulence factor and provide the foundation for understanding how malleilactone contributes to the pathology of melioidosis infections.IMPORTANCE Many bacterially produced polyketides are cytotoxic to mammalian cells and are potentially important contributors to pathogenesis during infection. We are interested in the polyketide gene clusters present in Burkholderia pseudomallei, which causes the often-fatal human disease melioidosis. Using knowledge gained by studies in the close relative Burkholderia thailandensis, we show that one of the B. pseudomallei polyketide biosynthetic clusters produces a cytotoxic polyketide, malleilactone. Malleilactone contributes to B. pseudomallei virulence in a Caenorhabditis elegans infection model and is regulated by an orphan LuxR family quorum-sensing transcription factor, MalR. Our studies demonstrate that malleilactone biosynthesis or MalR could be new targets for developing therapeutics to treat melioidosis.

Novel soil bacteria possess diverse genes for secondary metabolite biosynthesis.

In soil ecosystems, microorganisms produce diverse secondary metabolites such as antibiotics, antifungals and siderophores that mediate communication, competition and interactions with other organisms and the environment1,2. Most known antibiotics are derived from a few culturable microbial taxa 3 , and the biosynthetic potential of the vast majority of bacteria in soil has rarely been investigated 4 . Here we reconstruct hundreds of near-complete genomes from grassland soil metagenomes and identify microorganisms from previously understudied phyla that encode diverse polyketide and nonribosomal peptide biosynthetic gene clusters that are divergent from well-studied clusters. These biosynthetic loci are encoded by newly identified members of the Acidobacteria, Verrucomicobia and Gemmatimonadetes, and the candidate phylum Rokubacteria. Bacteria from these groups are highly abundant in soils5-7, but have not previously been genomically linked to secondary metabolite production with confidence. In particular, large numbers of biosynthetic genes were characterized in newly identified members of the Acidobacteria, which is the most abundant bacterial phylum across soil biomes 5 . We identify two acidobacterial genomes from divergent lineages, each of which encodes an unusually large repertoire of biosynthetic genes with up to fifteen large polyketide and nonribosomal peptide biosynthetic loci per genome. To track gene expression of genes encoding polyketide synthases and nonribosomal peptide synthetases in the soil ecosystem that we studied, we sampled 120 time points in a microcosm manipulation experiment and, using metatranscriptomics, found that gene clusters were differentially co-expressed in response to environmental perturbations. Transcriptional co-expression networks for specific organisms associated biosynthetic genes with two-component systems, transcriptional activation, putative antimicrobial resistance and iron regulation, linking metabolite biosynthesis to processes of environmental sensing and ecological competition. We conclude that the biosynthetic potential of abundant and phylogenetically diverse soil microorganisms has previously been underestimated. These organisms may represent a source of natural products that can address needs for new antibiotics and other pharmaceutical compounds.

Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system

BackgroundHeterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes.ResultsTo validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem clusters of pikromycin biosynthetic gene clusters.ConclusionsThe 60 kb pikromycin biosynthetic gene cluster was isolated in a single integration pSBAC vector. Introduction of the pikromycin biosynthetic gene cluster into the pikromycin non-producing strains resulted in higher pikromycin production. The utility of the pSBAC system as a precise cloning tool for large-sized biosynthetic gene clusters was verified through heterologous expression of the pikromycin biosynthetic gene cluster. Moreover, this pSBAC-driven heterologous expression strategy was confirmed to be an ideal approach for production of low and inconsistent natural products such as pikromycin in S. venezuelae, implying that this strategy could be employed for development of a custom overexpression scheme of natural product biosynthetic gene clusters in actinomycetes.

Biosynthesis, regulation, and engineering of a linear polyketide tautomycetin: a novel immunosuppressant in Streptomyces sp. CK4412.

Tautomycetin (TMC) is a natural product with a linear structure that includes an ester bond connecting a dialkylmaleic moiety to a type I polyketide chain. Although TMC was originally identified as an antifungal antibiotic in the late 1980s, follow-up studies revealed its novel immunosuppressant activity. Specifically, TMC exhibited a mechanistically unique immunosuppressant activity about 100 times higher than that of cyclosporine A, a widely used immunosuppressant drug. Interestingly, a structurally close relative, tautomycin (TTM), was reported to not possess TMC-like immunosuppressant activity, suggesting that a distinctive polyketide moiety of TMC plays a critical role in immunosuppressant activity. Cloning and engineering of a TMC polyketide biosynthetic gene cluster generated several derivatives showing different biological activities. TMC was also found to be biosynthesized as a linear structure without forming a lactone ring, unlike the most polyketide-based compounds, implying the presence of a unique polyketide thioesterase in the cluster. Although TMC biosynthesis was limited due to its tight regulation by two pathway-specific regulatory genes located in the cluster, its production was significantly stimulated through homologous and heterologous expression of its entire biosynthetic gene cluster using a Streptomyces artificial chromosome vector system. In this mini-review, we summarize recent advances in the biosynthesis, regulation, and pathway engineering of a linear polyketide, TMC, in Streptomyces sp. CK4412.

Precise cloning and tandem integration of large polyketide biosynthetic gene cluster using Streptomyces artificial chromosome system

BackgroundDirect cloning combined with heterologous expression of a secondary metabolite biosynthetic gene cluster has become a useful strategy for production improvement and pathway modification of potentially valuable natural products present at minute quantities in original isolates of actinomycetes. However, precise cloning and efficient overexpression of an entire biosynthetic gene cluster remains challenging due to the ineffectiveness of current genetic systems in manipulating large-sized gene clusters for heterologous as well as homologous expression.ResultsA versatile Escherichia coli-Streptomyces shuttle bacterial artificial chromosomal (BAC) conjugation vector, pSBAC, was used along with a cluster tandem integration approach to carry out homologous and heterologous overexpression of a large 80-kb polyketide biosynthetic pathway gene cluster of tautomycetin (TMC), which is a protein phosphatase PP1/PP2A inhibitor and T cell-specific immunosuppressant. Unique XbaI restriction sites were precisely inserted at both border regions of the TMC biosynthetic gene cluster within the chromosome of TMC-producing Streptomyces sp. CK4412, followed by site-specific recombination of pSBAC into the flanking region of the TMC gene cluster. The entire TMC gene cluster was then rescued as a single giant recombinant pSBAC by XbaI digestion of the chromosomal DNA as well as subsequent self-ligation. Next, the recombinant pSBAC construct containing the entire TMC cluster in E. coli was directly conjugated into model Streptomyces strains, resulting in rapid and enhanced TMC production. Moreover, introduction of the TMC cluster-containing pSBAC into wild-type Streptomyces sp. CK4412 as well as a recombinant S. coelicolor strain resulted in a chromosomal tandem repeat of the entire TMC cluster with 14-fold and 5.4-fold enhanced TMC productivities, respectively.ConclusionsThe 80-kb TMC biosynthetic gene cluster was isolated in a single integration vector, pSBAC. Introduction of TMC biosynthetic gene cluster in TMC non-producing strains has resulted in similar amount of TMC production yield. Moreover, over-expression of TMC biosynthetic gene cluster in original producing strain and recombinant S. coelicolor dramatically increased TMC production. Thus, this strategy can be employed to develop a custom overexpression scheme of entire metabolite pathway clusters present in actinomycetes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0325-2) contains supplementary material, which is available to authorized users.

Expanding our understanding of sequence-function relationships of type II polyketide biosynthetic gene clusters: bioinformatics-guided identification of Frankiamicin A from Frankia sp. EAN1pec.

A large and rapidly increasing number of unstudied “orphan” natural product biosynthetic gene clusters are being uncovered in sequenced microbial genomes. An important goal of modern natural products research is to be able to accurately predict natural product structures and biosynthetic pathways from these gene cluster sequences. This requires both development of bioinformatic methods for global analysis of these gene clusters and experimental characterization of select products produced by gene clusters with divergent sequence characteristics. Here, we conduct global bioinformatic analysis of all available type II polyketide gene cluster sequences and identify a conserved set of gene clusters with unique ketosynthase α/β sequence characteristics in the genomes of Frankia species, a group of Actinobacteria with underexploited natural product biosynthetic potential. Through LC-MS profiling of extracts from several Frankia species grown under various conditions, we identified Frankia sp. EAN1pec as producing a compound with spectral characteristics consistent with the type II polyketide produced by this gene cluster. We isolated the compound, a pentangular polyketide which we named frankiamicin A, and elucidated its structure by NMR and labeled precursor feeding. We also propose biosynthetic and regulatory pathways for frankiamicin A based on comparative genomic analysis and literature precedent, and conduct bioactivity assays of the compound. Our findings provide new information linking this set of Frankia gene clusters with the compound they produce, and our approach has implications for accurate functional prediction of the many other type II polyketide clusters present in bacterial genomes.

Seamless stitching of biosynthetic gene cluster containing type I polyketide synthases using Red/ET mediated recombination for construction of stably co-existing plasmids

Type I polyketides are natural products with diverse functions that are important for medical and agricultural applications. Manipulation of large biosynthetic gene clusters containing type I polyketide synthases (PKS) for heterologous expression is difficult due to the existence of conservative sequences of PKS in multiple modules. Red/ET mediated recombination has permitted rapid manipulation of large fragments; however, it requires insertion of antibiotic selection marker in the cassette, raising the problem of interference of expression by leaving “scar” sequence. Here, we report a method for precise seamless stitching of large polyketide biosynthetic gene cluster using a 48.4kb fragment containing type I PKS involved in fostriecin biosynthesis as an example. rpsL counter-selection was used to assist seamless stitching of large fragments, where we have overcome both the size limitations and the restriction on endonuclease sites during the Red/ET recombination. The compatibility and stability of the co-existing vectors (p184 and pMT) which respectively accommodate 16kb and 32.4kb inserted fragments were demonstrated. The procedure described here is efficient for manipulation of large DNA fragments for heterologous expression.

Draft Genome Sequence of the Rifamycin Producer Amycolatopsis rifamycinica DSM 46095.

Amycolatopsis rifamycinica DSM 46095 is an actinobacterium that produces rifamycin SV, an antibiotic used against Mycobacterium tuberculosis. Here, we present the draft genome of DSM 46095, which harbors a novel rifamycin polyketide biosynthetic gene cluster (rif PKS) that differed by 10% in nucleotide sequence from the already reported rif PKS cluster of Amycolatopsis mediterranei S699.

Multiple Effects of a Novel Epothilone Analog on Cellular Processes and Signaling Pathways Regulated by Rac1 GTPase in the Human Breast Cancer Cells.

The epothilones are a class of microtubule inhibitors that exhibit a strong antitumor activity. UTD2 is a novel epothilone analog generated by genetic manipulation of the polyketide biosynthetic gene cluster. This study investigated the effects of UTD2 on the actin cytoskeleton and its critical regulators, and the signaling pathways which are essential for cell motility, growth and survival in MCF-7 breast cancer cells. Results showed that UTD2 inhibited the cellular functions of actin cytoskeleton, such as wound-closure, migration and invasion, as well as adhesion. Our study further demonstrated that UTD2 suppressed Rac1 GTPase activation and reduced the activity of PAK1, which is a downstream effector of Rac1, while the activity of Cdc42 was not affected. Additionally, the phosphorylation of p38 and ERK were significantly inhibited, but the phosphorylation of JNK remained the same after UTD2 treatment. Moreover, UTD2 inhibited the activity and mRNA expression of MMP-2, which plays a key role in cell motility. UTD2 also reduced the phosphorylation of Akt, which is an important signaling kinase regulating the cell survival through Rac1. Furthermore, UTD2 interrupted the synergy between Rac1 and Raf in focus formation assays. Taken together, these results indicated that UTD2 exerted multiple effects on the actin cytoskeleton and signaling pathways associated with Rac1. This study provided novel insights into the molecular mechanism of the antineoplastic and antimetastatic activities of epothilones. Our findings also suggest that the signaling pathways regulated by Rac1 may be evaluated as biomarkers for the response to therapy in clinical trials of epothilones.

Cytochrome P450-mediated hydroxylation is required for polyketide macrolactonization in stambomycin biosynthesis

Many polyketide antibiotics contain macrolactones that arise from polyketide synthase chain release via thioesterase (TE) domain-catalyzed macrolactonization. The hydroxyl groups utilized in such macrolactonization reactions typically derive from reduction of β-ketothioester intermediates in polyketide chain assembly. The stambomycins are a group of novel macrolide antibiotics with promising anticancer activity that we recently discovered via rational activation of a silent polyketide biosynthetic gene cluster in Streptomyces ambofaciens. Here we report that the hydroxyl group utilized for formation of the macrolactone in the stambomycins is derived from cytochrome P450-catalyzed hydroxylation of the polyketide chain rather than keto reduction during chain assembly. This is a novel mechanism for macrolactone formation in polyketide antibiotic biosynthesis.

Identification and Characterization of the Cuevaene A Biosynthetic Gene Cluster in Streptomyces sp. LZ35

Genome sequence analysis of Streptomyces sp. LZ35 has revealed a large number of secondary metabolite pathways, including one encoded in an orphan type I polyketide synthase gene cluster that contains a putative chorismatase/3-hydroxybenzoate synthase gene. Mutagenesis and comparative metabolic profiling led to the identification of cuevaene A as the metabolic product of the gene cluster, thus making it the first 3-HBA containing polyketide biosynthetic gene cluster described to date. Cuv10 was proven to be responsible for the conversion of chorismate into 3-HBA; Cuv18 is speculated to be responsible for the 6-hydroxylation of 3-HBA during polyketide chain elongation. Additionally, several pathway-specific regulatory factors that affect the production of cuevaene A were identified. Our results indicate that targeted inactivation of a gene followed by comparative metabolic profiling is a useful approach to identify and characterize cryptic biosynthetic gene clusters.

Alternative Sigma Factor Over-Expression Enables Heterologous Expression of a Type II Polyketide Biosynthetic Pathway in Escherichia coli

BackgroundHeterologous expression of bacterial biosynthetic gene clusters is currently an indispensable tool for characterizing biosynthetic pathways. Development of an effective, general heterologous expression system that can be applied to bioprospecting from metagenomic DNA will enable the discovery of a wealth of new natural products.MethodologyWe have developed a new Escherichia coli-based heterologous expression system for polyketide biosynthetic gene clusters. We have demonstrated the over-expression of the alternative sigma factor σ54 directly and positively regulates heterologous expression of the oxytetracycline biosynthetic gene cluster in E. coli. Bioinformatics analysis indicates that σ54 promoters are present in nearly 70% of polyketide and non-ribosomal peptide biosynthetic pathways.ConclusionsWe have demonstrated a new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where high-level pleiotropic sigma factors from the heterologous host directly and positively regulate transcription of the non-native biosynthetic gene cluster. Our bioinformatics analysis is consistent with the hypothesis that heterologous expression mediated by the alternative sigma factor σ54 may be a viable method for the production of additional polyketide products.

The role of transcription in heterologous expression of polyketides in bacterial hosts

Heterologous expression of biosynthetic pathways is an indispensable tool in the discovery, production, engineering, and characterization of bacterial polyketides and the complex enzymology involved in their biosynthesis. Ensuring transcription of polyketide biosynthetic gene clusters in heterologous hosts is a pressing problem. This review evaluates the two strategies used to ensure transcription. The first is a promoter replacement approach where promoters known to function in the heterologous host are inserted into the biosynthetic gene cluster. The second is an approach that relies on the heterologous host recognizing and utilizing promoters native to the gene cluster. Both have been successful methodologies and have different strengths and weaknesses, which are highlighted and discussed.

Tetarimycin A, an MRSA-Active Antibiotic Identified through Induced Expression of Environmental DNA Gene Clusters

The propagation of DNA extracted directly from environmental samples in laboratory-grown bacteria provides a means to study natural products encoded in the genomes of uncultured bacteria. However, gene silencing often hampers the functional characterization of gene clusters captured on environmental DNA clones. Here we show that the overexpression of transcription factors found in sequenced environmental DNA-derived biosynthetic gene clusters, in conjunction with traditional culture-broth extract screening, can be used to identify new bioactive secondary metabolites from otherwise-silent gene clusters. Tetarimycin A, a tetracyclic methicillin-resistant Staphylococcus aureus (MRSA)-active antibiotic, was isolated from the culture-broth extract of Streptomyces albus cultures cotransformed with an environmentally derived type-II polyketide biosynthetic gene cluster and its pathway-specific Streptomyces antibiotic regulatory protein (SARP) cloned under the control of the constitutive ermE* promoter.

Overexpressing Transcriptional Regulator in Aspergillus oryzae Activates a Silent Biosynthetic Pathway to Produce a Novel Polyketide

Fungal genomes carry many gene clusters seemingly capable of natural product biosynthesis, yet most clusters remain silent. This places a major constraint on the conventional approach of cloning these genes in more amenable heterologous host for the natural product biosynthesis. One way to overcome this difficulty is to activate the silent gene clusters within the context of the target fungus. Here, we successfully activated a silent polyketide biosynthetic gene cluster in Aspergillus oryzae by overexpressing a transcriptional regulator found within the cluster from a plasmid. This strategy allowed us to isolate a new polyketide product and to efficiently decipher its biosynthetic pathway. Through this exercise, we also discovered unexpected activities of the biosynthetic enzymes found in the cluster. These results indicate that our approach would be valuable for isolating novel natural products and engineering analogues of comparable, if not more potent, bioactivity.

Environmental DNA-Encoded Antibiotics Fasamycins A and B Inhibit FabF in Type II Fatty Acid Biosynthesis

In a recent study of polyketide biosynthetic gene clusters cloned directly from soil, we isolated two antibiotics, fasamycins A and B, which showed activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. To identify the target of the fasamycins, mutants with elevated fasamycin A minimum inhibitory concentrations were selected from a wild-type culture of E. faecalis OG1RF. Next-generation sequencing of these mutants, in conjunction with in vitro biochemical assays, showed that the fasamycins inhibit FabF of type II fatty acid biosynthesis (FASII). Candidate gene overexpression studies also showed that fasamycin resistance is conferred by fabF overexpression. On the basis of comparisons with known FASII inhibitors and in silico docking studies, the chloro-gem-dimethyl-anthracenone substructure seen in the fasamycins is predicted to represent a naturally occurring FabF-specific antibiotic pharmacophore. Optimization of this pharmacophore should yield FabF-specific antibiotics with increased potencies and differing spectra of activity. This study demonstrates that culture-independent antibiotic discovery methods have the potential to provide access to novel metabolites with modes of action that differ from those of antibiotics currently in clinical use.

Structure and biosynthesis of the unusual polyketide alkaloid coelimycin P1, a metabolic product of the cpk gene cluster of Streptomyces coelicolor M145

Cryptic natural product biosynthetic pathways discovered by genome mining are a promising source of novel bioactive natural products. Here we report the identification, isolation and structure elucidation of coelimycin P1, an unusual yellow-pigmented metabolic product of the cpk cryptic polyketide biosynthetic gene cluster of Streptomyces coelicolor M145, using a genetic engineering strategy designed to increase metabolic flux through the biosynthetic pathway. This resulted in overproduction of the yellow pigment, which was identified by HPLC comparison of the metabolite profile of culture supernatants of the engineered strain and an equivalent strain from which the cpk gene cluster had been deleted. Isolation and structure elucidation of the pigment revealed that it is a novel alkaloid containing a unique functionalized 1,5-oxathiocane. Sequence analysis of the enzymes encoded by the cpk gene cluster led us to propose a pathway for coelimycin P1 biosynthesis that is fully consistent with the results of incorporation experiments utilizing isotope-labelled precursors.

Biochemical Characterization of a Type III Polyketide Biosynthetic Gene Cluster from Streptomyces toxytricini

A type III polyketide biosynthetic gene cluster has been discovered in the industrially important strain Streptomyces toxytricini NRRL 15443, including four genes stp450-1, stts, stp450-2, and stmo. The stts gene encodes a putative type III polyketide synthase that is homologous to RppA, a 1,3,6,8-tetrahydroxynaphthalene (THN) synthase from Streptomyces griseus. The deduced protein product of stmo resembles the cupin-containing monooxygenase MomA from Streptomyces antibioticus that oxidizes THN into flaviolin. Two cytochrome P450s (CYPs), StP450-1 and StP450-2, are present in the gene cluster. StTS was overexpressed in Escherichia coli BL21(DE3) and identified as a THN synthase. The synthesized THN can be easily oxidized into flaviolin by air. Both CYPs were reconstituted in E. coli BL21(DE3) and can oxidize flaviolin to form oligomers. The k(cat)/K(m) values for StP450-1 and StP450-2 were 0.28 and 0.71 min⁻¹ mM⁻¹, respectively. UV irradiation test showed that expression of StTS in E. coli BL21(DE3) significantly protects the cells from UV radiation, and coexpression of StTS and StP450-1 provides even stronger protection.