Natural Product Gene Clusters Research Articles

Discovery of fungal onoceroid triterpenoids through domainless enzyme-targeted global genome mining

Genomics-guided methodologies have revolutionized the discovery of natural products. However, a major challenge in the field of genome mining is determining how to selectively extract biosynthetic gene clusters (BGCs) for untapped natural products from numerous available genome sequences. In this study, we developed a fungal genome mining tool that extracts BGCs encoding enzymes that lack a detectable protein domain (i.e., domainless enzymes) and are not recognized as biosynthetic proteins by existing bioinformatic tools. We searched for BGCs encoding a homologue of Pyr4-family terpene cyclases, which are representative examples of apparently domainless enzymes, in approximately 2000 fungal genomes and discovered several BGCs with unique features. The subsequent characterization of selected BGCs led to the discovery of fungal onoceroid triterpenoids and unprecedented onoceroid synthases. Furthermore, in addition to the onoceroids, a previously unreported sesquiterpene hydroquinone, of which the biosynthesis involves a Pyr4-family terpene cyclase, was obtained. Our genome mining tool has broad applicability in fungal genome mining and can serve as a beneficial platform for accessing diverse, unexploited natural products.

An efficient method for targeted cloning of large DNA fragments from Streptomyces.

Cloning of large DNA fragments from microorganisms becomes increasingly important but remains seriously challenging due to the complexity and diversity of genetic background. In particular, the methods with high precision and efficiency are in great need for obtaining intact biosynthetic gene clusters (BGCs) of microbial natural products. Here, we report a new strategy for targeted cloning of large DNA fragments (TCLD) from different bacteria. Using this method, precise cloning of desired E. coli chromosomal fragments up to 201kb was achieved with 53% positive rate. Moreover, its application in cloning of large BGCs with high G + C content and multiple repetitive sequences was also demonstrated, including the 98kb tylosin BGC (tyl), 128kb daptomycin BGC (dpt), and 127kb salinomycin BGC (sal). Subsequently, heterologous expression of the cloned tyl BGC in Streptomyces coelicolor M1146 led to the production of tylosins in the resulting recombinant strains. And also, its introduction into Streptomyces fradiae ATCC 19609, a native producer of tylosin, effectively increased tylosin yield to 230%. Hence, TCLD is a powerful tool for cloning large BGCs and would facilitate the discovery of bioactive substances from microbial resources. KEY POINTS: • TCLD is an efficient method for cloning large DNA fragments. • Repeat sequence-mediated intra-molecular cyclization improves the cloning efficiency. • TCLD combined with scarless editing allows unlimited modifications on BGCs.

Navigating and expanding the roadmap of natural product genome mining tools.

Natural products are structurally highly diverse and exhibit a wide array of biological activities. As a result, they serve as an important source of new drug leads. Traditionally, natural products have been discovered by bioactivity-guided fractionation. The advent of genome sequencing technology has resulted in the introduction of an alternative approach towards novel natural product scaffolds: Genome mining. Genome mining is an in-silico natural product discovery strategy in which sequenced genomes are analyzed for the potential of the associated organism to produce natural products. Seemingly universal biosynthetic principles have been deciphered for most natural product classes that are used to detect natural product biosynthetic gene clusters using pathway-encoded conserved key enzymes, domains, or motifs as bait. Several generations of highly sophisticated tools have been developed for the biosynthetic rule-based identification of natural product gene clusters. Apart from these hard-coded algorithms, multiple tools that use machine learning-based approaches have been designed to complement the existing genome mining tool set and focus on natural product gene clusters that lack genes with conserved signature sequences. In this perspective, we take a closer look at state-of-the-art genome mining tools that are based on either hard-coded rules or machine learning algorithms, with an emphasis on the confidence of their predictions and potential to identify non-canonical natural product biosynthetic gene clusters. We highlight the genome mining pipelines' current strengths and limitations by contrasting their advantages and disadvantages. Moreover, we introduce two indirect biosynthetic gene cluster identification strategies that complement current workflows. The combination of all genome mining approaches will pave the way towards a more comprehensive understanding of the full biosynthetic repertoire encoded in microbial genome sequences.

Genome sequences of Rhizopogon roseolus, Mariannaea elegans, Myrothecium verrucaria, and Sphaerostilbella broomeana and the identification of biosynthetic gene clusters for fungal peptide natural products.

In recent years, a variety of fungal cyclic peptides with interesting bioactivities have been discovered. For many of these peptides, the biosynthetic pathways are unknown and their elucidation often holds surprises. The cyclic and backbone N-methylated omphalotins from Omphalotus olearius were recently shown to constitute a novel class (borosins) of ribosomally synthesized and posttranslationally modified peptides, members of which are produced by many fungi, including species of the genus Rhizopogon. Other recently discovered fungal peptide macrocycles include the mariannamides from Mariannaea elegans and the backbone N-methylated verrucamides and broomeanamides from Myrothecium verrucaria and Sphaerostilbella broomeana, respectively. Here, we present draft genome sequences of four fungal species Rhizopogon roseolus, Mariannaea elegans, Myrothecium verrucaria, and Sphaerostilbella broomeana. We screened these genomes for precursor proteins or gene clusters involved in the mariannamide, verrucamide, and broomeanamide biosynthesis including a general screen for borosin-producing precursor proteins. While our genomic screen for potential ribosomally synthesized and posttranslationally modified peptide precursor proteins of mariannamides, verrucamides, broomeanamides, and borosins remained unsuccessful, antiSMASH predicted nonribosomal peptide synthase gene clusters that may be responsible for the biosynthesis of mariannamides, verrucamides, and broomeanamides. In M. verrucaria, our antiSMASH search led to a putative NRPS gene cluster with a predicted peptide product of 20 amino acids, including multiple nonproteinogenic isovalines. This cluster likely encodes a member of the peptaibols, an antimicrobial class of peptides previously isolated primarily from the Genus Trichoderma. The nonribosomal peptide synthase gene clusters discovered in our screenings are promising candidates for future research.

Cross-species transcriptomics identifies core regulatory changes differentiating the asymptomatic asexual and virulent sexual life cycles of grass-symbiotic Epichloë fungi.

Fungi from the genus Epichloë form systemic endobiotic infections of cool season grasses, producing a range of host-protective natural products in return for access to nutrients. These infections are asymptomatic during vegetative host growth, with associations between asexual Epichloë spp. and their hosts considered mutualistic. However, the sexual cycle of Epichloë spp. involves virulent growth, characterized by the envelopment and sterilization of a developing host inflorescence by a dense sheath of mycelia known as a stroma. Microscopic analysis of stromata revealed a dramatic increase in hyphal propagation and host degradation compared with asymptomatic tissues. RNAseq was used to identify differentially expressed genes in asymptomatic vs stromatized tissues from 3 diverse Epichloë–host associations. Comparative analysis identified a core set of 135 differentially expressed genes that exhibited conserved transcriptional changes across all 3 associations. The core differentially expressed genes more strongly expressed during virulent growth encode proteins associated with host suppression, digestion, adaptation to the external environment, a biosynthetic gene cluster, and 5 transcription factors that may regulate Epichloë stroma formation. An additional 5 transcription factor encoding differentially expressed genes were suppressed during virulent growth, suggesting they regulate mutualistic processes. Expression of biosynthetic gene clusters for natural products that suppress herbivory was universally suppressed during virulent growth, and additional biosynthetic gene clusters that may encode production of novel host-protective natural products were identified. A comparative analysis of 26 Epichloë genomes found a general decrease in core differentially expressed gene conservation among asexual species, and a specific decrease in conservation for the biosynthetic gene cluster expressed during virulent growth and an unusual uncharacterized gene.

Bacillibactin class of siderophore antibiotics from a marine symbiotic Bacillus as promising antibacterial agents

Preliminary antibacterial metabolite production screening unveiled thatB. amyloliquefaciens MTCC 12,713 associated with the intertidal red alga Kappaphycus alverezii exhibited potential inhibitory effects against drug-resistant pathogens methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Four homologous siderophore types of bacillibactins were isolated from a heterotrophic marine bacterium through bioactivity-guided purification. All detectable natural product gene clusters in B. amyloliquefaciens MTCC 12,713 were analyzed by sequencing the complete genome of the bacterium. The studied compounds displayed broad spectrum bactericidal activity against multidrug-resistant strains with a range of minimum inhibitory concentration values from 1.56 to 6.25µg/mL, whereas standard antibiotic chloramphenicol was active at 6.25 to 12.5µg/mL. Structure-bioactivity relationship assessment showed that higher electronic values were responsible for antibacterial properties against the nosocomial pathogens. The 2, 3-dihydroxybenzoate (dhb)-assisted biosynthetic pathway of catecholate-enclosed bacillibactins was proposed through the bacillibactin synthase multienzyme complex catalysis followed by dimerization of dhbACEBF operons with 16 genes (~ 12kb bacterial genome). The present findings recognized an undescribed 4-methoxy-11'-pentanoyloxy-bacillibactin C as a source of potential antibacterial agent for use against drug-resistant pathogens for pharmaceutical applications. KEY POINTS: • Bacillus amyloliquefaciens in association with Kappaphycus alverezii was isolated • Four antibacterial bacillibactin analogs were identified from symbiotic bacterium • 4-Methoxy-11'-pentanoyloxy-bacillibactin C showed potential antibacterial activity.

Activation of Naringenin and Kaempferol through Pathway Refactoring in the Endophyte Phomopsis Liquidambaris.

Abundant gene clusters of natural products are observed in the endophytic fungus Phomopsis liquidambaris; however, most of them are silent. Herein, a plug-and-play DNA assembly tool has been applied for flavonoid synthesis in P. liquidambaris. A shuttle plasmid was constructed based on S. cerevisiae, E. coli, and P. liquidambaris with screening markers URA, Amp, and hygR, respectively. Each fragment or cassette was successively assembled by overlap extension PCR with at least 40-50 bp homologous arms in S. cerevisiae for generating a new vector. Seven native promoters were screened by the DNA assembly based on the fluorescence intensity of the mCherry reporter gene in P. liquidambaris, and two of them were new promoters. The key enzyme chalcone synthase was the limiting step of the pathway. The naringenin and kaempferol pathways were refactored and activated with the titers of naringenin and kaempferol of 121.53 mg/L and 75.38 mg/L in P. liquidambaris using fed-batch fermentation, respectively. This study will be efficient and helpful for the biosynthesis of secondary metabolites.

The \u03c354 system directly regulates bacterial natural product genes

Bacterial-derived polyketide and non-ribosomal peptide natural products are crucial sources of therapeutics and yet little is known about the conditions that favor activation of natural product genes or the regulatory machinery controlling their transcription. Recent findings suggest that the σ54 system, which includes σ54-loaded RNA polymerase and transcriptional activators called enhancer binding proteins (EBPs), might be a common regulator of natural product genes. Here, we explored this idea by analyzing a selected group of putative σ54 promoters identified in Myxococcus xanthus natural product gene clusters. We show that mutations in putative σ54-RNA polymerase binding regions and in putative Nla28 EBP binding sites dramatically reduce in vivo promoter activities in growing and developing cells. We also show in vivo promoter activities are reduced in a nla28 mutant, that Nla28 binds to wild-type fragments of these promoters in vitro, and that in vitro binding is lost when the Nla28 binding sites are mutated. Together, our results indicate that M. xanthus uses σ54 promoters for transcription of at least some of its natural product genes. Interestingly, the vast majority of experimentally confirmed and putative σ54 promoters in M. xanthus natural product loci are located within genes and not in intergenic sequences.

Recent developments of tools for genome and metabolome studies in basidiomycete fungi and their application to natural product research.

ABSTRACTBasidiomycota are a large and diverse phylum of fungi. They can make bioactive metabolites that are used or have inspired the synthesis of antibiotics and agrochemicals. Terpenoids are the most abundant class of natural products encountered in this taxon. Other natural product classes have been described, including polyketides, peptides, and indole alkaloids. The discovery and study of natural products made by basidiomycete fungi has so far been hampered by several factors, which include their slow growth and complex genome architecture. Recent developments of tools for genome and metabolome studies are allowing researchers to more easily tackle the secondary metabolome of basidiomycete fungi. Inexpensive long-read whole-genome sequencing enables the assembly of high-quality genomes, improving the scaffold upon which natural product gene clusters can be predicted. CRISPR/Cas9-based engineering of basidiomycete fungi has been described and will have an important role in linking natural products to their genetic determinants. Platforms for the heterologous expression of basidiomycete genes and gene clusters have been developed, enabling natural product biosynthesis studies. Molecular network analyses and publicly available natural product databases facilitate data dereplication and natural product characterisation. These technological advances combined are prompting a revived interest in natural product discovery from basidiomycete fungi.This article has an associated Future Leader to Watch interview with the first author of the paper.

Heterologous production of fungal natural products: Reconstitution of biosynthetic gene clusters in model host Aspergillus oryzae.

While exploring phytotoxic metabolites from phytopathogenic fungi in the 1970s, we became interested in biosynthetic enzymes that catalyze Diels–Alder reactions involving biosynthesis of several phytotoxins that we isolated. Target enzymes were successfully characterized, and this triggered the identification of various Diels–Alderases in a recent decade. Through our Diels–Alderase project in 1990s, we recognized a highly efficient expression system of various biosynthetic genes with Aspergillus oryzae as a host. With the development of tools such as genomic data and bioinformatics analysis to identify biosynthetic gene clusters for natural products, we developed a highly reliable methodology such as hot spot knock-in to elucidate the biosynthetic pathways of representative fungal metabolites including phytotoxic substances. This methodology allows total biosynthesis of natural products and genome mining using silent biosynthetic gene clusters to obtain novel bioactive metabolites. Further applications of this technology are discussed.

Total in vitro biosynthesis of the nonribosomal macrolactone peptide valinomycin

Natural products are important because of their significant pharmaceutical properties such as antiviral, antimicrobial, and anticancer activity. Recent breakthroughs in DNA sequencing reveal that a great number of cryptic natural product biosynthetic gene clusters are encoded in microbial genomes, for example, those of Streptomyces species. However, it is still challenging to access compounds from these clusters because many source organisms are uncultivable or the genes are silent during laboratory cultivation. To address this challenge, we develop an efficient cell-free platform for the rapid, in vitro total biosynthesis of the nonribosomal peptide valinomycin as a model. We achieve this goal in two ways. First, we used a cell-free protein synthesis (CFPS) system to express the entire valinomycin biosynthetic gene cluster (>19 kb) in a single-pot reaction, giving rise to approximately 37 μg/L of valinomycin after optimization. Second, we coupled CFPS with cell-free metabolic engineering system by mixing two enzyme-enriched cell lysates to perform a two-stage biosynthesis. This strategy improved valinomycin production ~5000-fold to nearly 30 mg/L. We expect that cell-free biosynthetic systems will provide a new avenue to express, discover, and characterize natural product gene clusters of interest in vitro.

Reconstitution of biosynthetic machinery of fungal natural products in heterologous hosts.

Ascomycota and basidiomycota fungi are prolific sources of biologically active natural products. Recent genomic data and bioinformatic analysis indicate that fungi possess a large number of biosynthetic gene clusters for bioactive natural products but more than 90% are silent. Heterologous expression in the filamentous fungi as hosts is one of the powerful tools to expression of the silent gene clusters. This review introduces recent studies on the total biosynthesis of representative family members via common platform intermediates, genome mining of novel di- and sesterterpenoids including detailed cyclization pathway, and development of expression host for basidiomycota genes with efficient genome editing method. In addition, this review will discuss the several strategies, for the generation of structural diversity, which are found through these studies.

Oxygenated elansolid-type of polyketide spanned macrolides from a marine heterotrophic Bacillus as prospective antimicrobial agents against multidrug-resistant pathogens

Three homologous oxygenated elansolid-type of polyketide spanned macrolides were isolated from a heterotrophic marine bacterium, Bacillus amyloliquefaciens MTCC 12716, associated with an intertidal red alga Hypnea valentiae. The complete genome of the bacterium was sequenced and all detectable natural product gene clusters were analysed. The B. amyloliquefaciens MTCC 12716 genome features polyketide synthase (pks) systems of every known formally classified family, nonribosomal peptide synthetases and hybrid clusters. Comprehensive spectroscopic studies revealed the compounds to possess isobenzofuranyl benzoate and 1H-furopyrano[2,3-c]oxacyclononadecine-6-carboxylate moieties. The identified compounds displayed broad-spectrum bactericidal activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, and drug-resistant strains of Pseudomonas aeruginosa and Klebsiella pneumoniae with minimum inhibitory concentrations (MICs) of ≤1.0 µg/mL, whereas the standard antibiotics ampicillin and chloramphenicol were active only at concentrations of ≥6.25 µg/mL. The plausible mechanism of elansolid-type macrolide biosynthesis by trans-AT polyketide synthases through the pks starter unit para-hydroxybenzoic acid was hypothesised, and the structures were correlated with the gene organisation, with the predicted gene cluster comprising 16 genes (~81 kb in size). The best binding poses for each compound with the peptide deformylase (PDF) protein of S. aureus revealed docking scores (>11.30 kcal/mol) greater than actinonin (6.96 kcal/mol), a natural PDF inhibitor. The higher electronic values along with optimum lipophilic parameters support the potential anti-infective properties of the studied macrolides. These antibacterial elansolid-type of polyketide spanned macrolides in marine symbiotic B. amyloliquefaciens could be potential leads for biotechnological and pharmaceutical applications against emerging multidrug-resistant pathogens.

Activation of paulomycin production by exogenous γ-butyrolactone signaling molecules in Streptomyces albidoflavus J1074.

The interspecies communication roles of γ-butyrolactones (GBLs) have been described for a long time but are still poorly understood. Herein, we analyzed more than 1000 Streptomyces strains and noticed a big quantitative gap between the strains with GBL biosynthetic genes and the strains with GBL receptor genes, which implies the wide-spread of GBLs as interspecies signals in Streptomyces and their great potential in the activation of silent natural product gene clusters. Streptomyces albidoflavus J1074, which has one GBL receptor gene but no GBL biosynthetic gene, was chosen as a target to study the possible interspecies communication roles of GBLs. At first, the GBL biosynthetic genes from Streptomyces coelicolor M145 were expressed in S. albidoflavus J1074, which enabled the S. albidoflavus strains to synthesize Streptomyces coelicolor butanolides (SCBs) and activated the production of paulomycins. Further studies showed that this activation process requires the participation of the GBL receptor gene XNR_4681. The results suggest that the expression of exogenous GBL biosynthetic genes can modulate the metabolisms of GBL non-producing strains, and this regulation role might be meaningful for silent gene cluster activation in Streptomyces. At final, we synthesized racemic-SCB2 and tried to simplify the activation process by adding SCB2 directly to S. albidoflavus J1074, which unfortunately failed to induce paulomycin production.

A computational framework to explore large-scale biosynthetic diversity.

Genome mining has become a key technology to exploit natural product diversity. While initially performed on a single-genome basis, the process is now being scaled up to mine entire genera, strain collections and microbiomes. However, no bioinformatic framework is currently available for effectively analyzing datasets of this size and complexity. Here, we provide a streamlined computational workflow consisting of two new software tools: The ‘Biosynthetic Gene Similarity Clustering And Prospecting Engine’ (BiG-SCAPE) facilitates fast and interactive sequence similarity network analysis of biosynthetic gene clusters and gene cluster families. ‘CORe Analysis of Syntenic Orthologues to prioritize Natural product gene clusters’ (CORASON) elucidates phylogenetic relationships within and across these families. We validate BiG-SCAPE by correlating its output to metabolomic data across 363 actinobacterial strains and demonstrate the discovery potential of CORASON by comprehensively mapping biosynthetic diversity across a range of detoxin/rimosamide-related gene cluster families, culminating in the characterization of seven novel analogues.

New voyages to explore the natural product galaxy.

Natural products are a large family of diverse and complex chemical molecules that have roles in both primary and secondary metabolism, and over 210,000 natural products have been described. Secondary metabolite natural products are of high commercial and societal value with therapeutic uses as antibiotics, antifungals, antitumor and antiparasitic products and in agriculture as products for crop protection and animal health. There is a resurgence of activity in exploring natural products for a wide range of applications, due to not only increasing antibiotic resistance, but the advent of next-generation genome sequencing and new technologies to interrogate and investigate natural product biosynthesis. Genome mining has revealed a previously undiscovered richness of biosynthetic potential in novel biosynthetic gene clusters for natural products. Complementing these computational processes are new experimental platforms that are being developed and deployed to access new natural products.

Genome-led discovery of novel microbial natural products

Microorganisms have been increasingly exploited for their remarkable ability to produce diverse natural products, which display bioactivities ranging from antimicrobial and anticancer to signalling and developmental. Genome sequencing has revealed that bacteria harbour many more biosynthetic gene clusters (BGCs) for natural products than are currently characterised. Whilst several genome mining tools have been developed to aid discovery, some classes of BGCs remain difficult to identify. One such class is ribosomally-synthesised and post-translationally modified peptides (RiPPs). RiPP BGCs are small with few regions of homology, and the short precursor peptides are rarely annotated in genomes. After developing a targeted genome mining approach for RiPPs, we identified a novel family of BGCs spanning over 200 actinobacterial genomes. The presence of diverse biosynthetic enzymes and sequence variation of the precursor peptides suggest that these BGCs may produce several structurally diverse molecules. We have successfully TAR cloned one such BGC from Streptomycesand expressed it heterologously, and metabolomic analysis led to identification of the pathway product. Gene deletion experiments have also confirmed the involvement of individual biosynthetic enzymes. The compound has been purified and the structure elucidated by NMR. Further work will focus on exploring RiPP BGCs from other species and investigating the biological role of these molecules. The discovery of such a diverse and highly conserved group of BGCs highlights that we are still scratching the surface of the huge biosynthetic capacity of microorganisms, and the use of more sophisticated genome mining tools could help unveil many more important molecules in the future.

Genome mining reveals the origin of a bald phenotype and a cryptic nucleocidin gene cluster in Streptomyces asterosporus DSM 41452

Streptomyces asterosporus DSM 41452 is a producer of the polyketide annimycin and the non-ribosomal depsipeptide WS9326A. This strain is also notable for exhibiting a bald phenotype that is devoid of spores and aerial mycelium when grown on solid media. Based on the similarity of the 16S rRNA sequence to Streptomyces calvus, the only known producer of the fluorometabolite nucleocidin, the genome of S. asterosporus DSM 41452 was sequenced and analyzed. Twenty-nine natural product gene clusters were detected in the genome, including a gene cluster predicted to encode the fluorometabolite nucleocidin. Through genome analysis and gene complementation experiments, we demonstrate that the bald phenotype arises from a transposon gene inserted within the promoter sequence for the pleiotropic regulator adpA. Complementation of S. asterosporus DSM 41452 with a functional adpA sequence restored morphological differentiation and promoted the production of nucleocidin.

Leveraging synthetic biology for producing bioactive polyketides and non-ribosomal peptides in bacterial heterologous hosts.

Bacteria have historically been a rich source of natural products (e.g. polyketides and non-ribosomal peptides) that possess medically-relevant activities. Despite extensive discovery programs in both industry and academia, a plethora of biosynthetic pathways remain uncharacterized and the corresponding molecular products untested for potential bioactivities. This knowledge gap comes in part from the fact that many putative natural product producers have not been cultured in conventional laboratory settings in which the corresponding products are produced at detectable levels. Next-generation sequencing technologies are further increasing the knowledge gap by obtaining metagenomic sequence information from complex communities where production of the desired compound cannot be isolated in the laboratory. For these reasons, many groups are turning to synthetic biology to produce putative natural products in heterologous hosts. This strategy depends on the ability to heterologously express putative biosynthetic gene clusters and produce relevant quantities of the corresponding products. Actinobacteria remain the most abundant source of natural products and the most promising heterologous hosts for natural product discovery and production. However, researchers are discovering more natural products from other groups of bacteria, such as myxobacteria and cyanobacteria. Therefore, phylogenetically similar heterologous hosts have become promising candidates for synthesizing these novel molecules. The downside of working with these microbes is the lack of well-characterized genetic tools for optimizing expression of gene clusters and product titers. This review examines heterologous expression of natural product gene clusters in terms of the motivations for this research, the traits desired in an ideal host, tools available to the field, and a survey of recent progress.

Discovery of the actinoplanic acid pathway in Streptomyces rapamycinicus reveals a genetically conserved synergism with rapamycin

Actinobacteria possess a great wealth of pathways for production of bioactive compounds. Following advances in genome mining, dozens of natural product (NP) gene clusters are routinely found in each actinobacterial genome; however, the modus operandi of this large arsenal is poorly understood. During investigations of the secondary metabolome of Streptomyces rapamycinicus, the producer of rapamycin, we observed accumulation of two compounds never before reported from this organism. Structural elucidation revealed actinoplanic acid A and its demethyl analogue. Actinoplanic acids (APLs) are potent inhibitors of Ras farnesyltransferase and therefore represent bioactive compounds of medicinal interest. Supported with the unique structure of these polyketides and using genome mining, we identified a gene cluster responsible for their biosynthesis in S. rapamycinicus Based on experimental evidence and genetic organization of the cluster, we propose a stepwise biosynthesis of APL, the first bacterial example of a pathway incorporating the rare tricarballylic moiety into an NP. Although phylogenetically distant, the pathway shares some of the biosynthetic principles with the mycotoxins fumonisins. Namely, the core polyketide is acylated with the tricarballylate by an atypical nonribosomal peptide synthetase-catalyzed ester formation. Finally, motivated by the conserved colocalization of the rapamycin and APL pathway clusters in S. rapamycinicus and all other rapamycin-producing actinobacteria, we confirmed a strong synergism of these compounds in antifungal assays. Mining for such evolutionarily conserved coharboring of pathways would likely reveal further examples of NP sets, attacking multiple targets on the same foe. These could then serve as a guide for development of new combination therapies.