An approach for obtaining perfect hybridization probes for unknown polyketide synthase genes: a search for the epothilone gene cluster

An approach is described for obtaining 'perfect probes' for type I modular polyketide synthase (PKS) gene clusters that in turn enables the identification of all such gene clusters in a genome. The approach involves sequencing small fragments of a random genomic DNA library containing one or more modular PKS gene clusters, and identifying which fragments emanate from PKS genes. Knowing the approximate sizes of the genome and the target gene cluster, one can predict the the frequency that a PKS gene fragment will be present in the library sequenced. Computer simulations of the approach were applied to the known PKS and non-ribosomal peptide synthetase (NRPS) gene clusters in the Bacillus subtilus genome. The approach was then used to identify PKS gene fragments in a strain of Sorangium cellulosum that produces epothilone. In addition to identifying fragments of the epothilone gene cluster, we obtained 11 unique fragments from other PKS gene clusters; the results suggest that there may be six to eight PKS gene clusters in this organism. In addition, we identified four unique fragments of NRPS genes, demonstrating that the approach is also applicable for identification of these modular gene clusters.     

Application of molecular biology for the discovery of biosynthetic genes of polyketide and peptide antibiotics produced by actinomycetes

Actinomycetes are currently the main source of antibiotics. Genome sequencing reveals the presence in these organisms of multiple gene clusters for the synthesis of yet unidentified secondary metabolites. Technological advances in DNA isolation, cloning and sequencing, as well as development of bioinformatics, facilitate large scale search for new gene clusters in organisms with unknown genome sequence and in environmental DNA. Methods used for detection of polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes are described in this article. New PKS and NRPS genes give access to new biologically active natural products which can become drugs or substrates for chemical modifications. Even more inspiring is their use in combinatorial biosynthesis to produce a variety of compounds with rationally designed structures.     

新颖的黏细菌模块化天然产物装配线

黏细菌的显著特征之一是能够合成结构多样、功能丰富的天然产物．模块化聚酮合酶(PKS)和非核糖体肽合成酶(NRPS)途径是黏细菌合成天然产物的主要方式．与经典模块PKS/NRPS相比,黏细菌来源的模块化PKS/NRPS常表现出新颖的装配特征,显示出多样化的遗传加工潜能和装配产物结构多样性．本文综合归类分析了黏细菌来源的模块化PKS/NRPS遗传装配线类型及其对应化合物的生化结构特征,图文并茂地呈现了黏细菌在遗传、生化、组合生物合成、进化和药物开发领域的生机和潜能,并展望了基因组学时代带来的契机．     

Towards Prediction of Metabolic Products of Polyketide Synthases: An In Silico Analysis

Sequence data arising from an increasing number of partial and complete genome projects is revealing the presence of the polyketide synthase (PKS) family of genes not only in microbes and fungi but also in plants and other eukaryotes. PKSs are huge multifunctional megasynthases that use a variety of biosynthetic paradigms to generate enormously diverse arrays of polyketide products that posses several pharmaceutically important properties. The remarkable conservation of these gene clusters across organisms offers abundant scope for obtaining novel insights into PKS biosynthetic code by computational analysis. We have carried out a comprehensive in silico analysis of modular and iterative gene clusters to test whether chemical structures of the secondary metabolites can be predicted from PKS protein sequences. Here, we report the success of our method and demonstrate the feasibility of deciphering the putative metabolic products of uncharacterized PKS clusters found in newly sequenced genomes. Profile Hidden Markov Model analysis has revealed distinct sequence features that can distinguish modular PKS proteins from their iterative counterparts. For iterative PKS proteins, structural models of iterative ketosynthase (KS) domains have revealed novel correlations between the size of the polyketide products and volume of the active site pocket. Furthermore, we have identified key residues in the substrate binding pocket that control the number of chain extensions in iterative PKSs. For modular PKS proteins, we describe for the first time an automated method based on crucial intermolecular contacts that can distinguish the correct biosynthetic order of substrate channeling from a large number of non-cognate combinatorial possibilities. Taken together, our in silico analysis provides valuable clues for formulating rules for predicting polyketide products of iterative as well as modular PKS clusters. These results have promising potential for discovery of novel natural products by genome mining and rational design of novel natural products.     

Multiple hybrid polyketide synthase/non-ribosomal peptide synthetase gene clusters in the myxobacterium Stigmatella aurantiaca

Many bacterial and fungal secondary metabolites are produced by polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS). Recently, it has been discovered that these modular enzymatic systems can also closely cooperate to form natural products. The analysis of the corresponding biosynthetic machineries, in the form of hybrid systems, is of special interest for combinatorial biosynthesis, because the combination of PKS and NRPS can lead to an immense variety of structures that might be produced. During our screening for hybrid PKS/NRPS systems from myxobacteria, we scanned the genome of Stigmatella aurantiaca DW4/3-1 for the presence of gene loci that encode both the PKS and NRPS genes. In addition to the previously characterized myxothiazol system, we identified three further hybrid loci, three additional PKS and one further NRPS gene locus. These were analyzed by hybridization, physical mapping, PCR with degenerate oligonucleotides and sequencing of fragments of the gene clusters. The function of these genes was not known but it had already been speculated that one compound produced by the strain and detected via HPLC was a secondary metabolite. This was based on the observation that its production is dependent on an active copy of the phosphopantetheinyl transferase gene mtaA. We show here that one of the identified hybrid gene loci is responsible for the formation of this secondary metabolite. In agreement with the genetic data, the chemical structure resembles a cyclic polypeptide with a PKS sidechain. Our data show that S. aurantiaca has a broader genetic capacity to produce natural products than the number of compounds isolated from the strain so far suggests.     

Identification of trans-AT polyketide clusters in two marine bacteria reveals cryptic similarities between distinct symbiosis factors

Glutarimide-containing polyketides are known as potent antitumoral and antimetastatic agents. The associated gene clusters have only been identified in a few Streptomyces producers and Burkholderia gladioli symbiont. The new glutarimide-family polyketides, denominated sesbanimides D, E and F along with the previously known sesbanimide A and C, were isolated from two marine alphaproteobacteria Stappia indica PHM037 and Labrenzia aggregata PHM038. Structures of the isolated compounds were elucidated based on 1D and 2D homo and heteronuclear NMR analyses and ESI-MS spectrometry. All compounds exhibited strong antitumor activity in lung, breast and colorectal cancer cell lines. Subsequent whole genome sequencing and genome mining revealed the presence of the trans-AT PKS gene cluster responsible for the sesbanimide biosynthesis, described as sbn cluster. Strikingly, the modular architecture of downstream mixed type PKS/NRPS, SbnQ, revealed high similarity to PedH in pederin and Lab13 in labrenzin gene clusters, although those clusters are responsible for the production of structurally completely different molecules. The unexpected presence of SbnQ homologues in unrelated polyketide gene clusters across phylogenetically distant bacteria, raises intriguing questions about the evolutionary relationship between glutarimide-like and pederin-like pathways, as well as the functionality of their synthetic products.     

Strain-specific proteogenomics accelerates the discovery of natural products via their biosynthetic pathways

The use of proteomics for direct detection of expressed pathways producing natural products has yielded many new compounds, even when used in a screening mode without a bacterial genome sequence available. Here we quantify the advantages of having draft DNA-sequence available for strain-specific proteomics using the latest in ultrahigh-resolution mass spectrometry for both proteins and the small molecules they generate. Using the draft sequence of Streptomyces lilacinus NRRL B-1968, we show a >tenfold increase in the number of peptide identifications vs. using publicly available databases. Detected in this strain were six expressed gene clusters with varying homology to those known. To date, we have identified three of these clusters as encoding for the production of griseobactin (known), rakicidin D (an orphan NRPS/PKS hybrid cluster), and a putative thr and DHB-containing siderophore produced by a new non-ribosomal peptide sythetase gene cluster. The remaining three clusters show lower homology to those known, and likely encode enzymes for production of novel compounds. Using an interpreted strain-specific DNA sequence enables deep proteomics for the detection of multiple pathways and their encoded natural products in a single cultured bacterium.     

ClustScan: an integrated program package for the semi-automatic annotation of modular biosynthetic gene clusters and in silico prediction of novel chemical structures

The program package ‘ClustScan’ (Cluster Scanner) is designed for rapid, semi-automatic, annotation of DNA sequences encoding modular biosynthetic enzymes including polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS) and hybrid (PKS/NRPS) enzymes. The program displays the predicted chemical structures of products as well as allowing export of the structures in a standard format for analyses with other programs. Recent advances in understanding of enzyme function are incorporated to make knowledge-based predictions about the stereochemistry of products. The program structure allows easy incorporation of additional knowledge about domain specificities and function. The results of analyses are presented to the user in a graphical interface, which also allows easy editing of the predictions to incorporate user experience. The versatility of this program package has been demonstrated by annotating biochemical pathways in microbial, invertebrate animal and metagenomic datasets. The speed and convenience of the package allows the annotation of all PKS and NRPS clusters in a complete Actinobacteria genome in 2–3 man hours. The open architecture of ClustScan allows easy integration with other programs, facilitating further analyses of results, which is useful for a broad range of researchers in the chemical and biological sciences.     

Bacteria of the Roseobacter Clade Show Potential for Secondary Metabolite Production

Members of the Roseobacter clade are abundant and widespread in marine habitats and have very diverse metabolisms. Production of acylated homoserine lactones (AHL) and secondary metabolites, e.g., antibiotics has been described sporadically. This prompted us to screen 22 strains of this group for production of signaling molecules, antagonistic activity against bacteria of different phylogenetic groups, and the presence of genes encoding for nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS), representing enzymes involved in the synthesis of various pharmaceutically important natural products. The screening approach for NRPS and PKS genes was based on polymerase chain reaction (PCR) with degenerate primers specific for conserved sequence motifs. Additionally, sequences from whole genome sequencing projects of organisms of the Roseobacter clade were considered. Obtained PCR products were cloned, sequenced, and compared with genes of known function. With the PCR approach genes showing similarity to known NRPS and PKS genes were found in seven and five strains, respectively, and three PKS and NRPS sequences from genome sequencing projects were obtained. Three strains exhibited antagonistic activity and also showed production of AHL. Overall production of AHL was found in 10 isolates. Phylogenetic analysis of the 16S rRNA gene sequences of the tested organisms showed that several of the AHL-positive strains clustered together. Three strains were positive for three or four categories tested, and were found to be closely related within the genus Phaeobacter. The presence of a highly similar hybrid PKS/NRPS gene locus of unknown function in sequenced genomes of the Roseobacter clade plus the significant similarity of gene fragments from the strains studied to these genes argues for the functional requirement of the encoded hybrid PKS/NRPS complex. Our screening results therefore suggest that the Roseobacter clade is indeed employing PKS/NRPS biochemistry and should thus be further studied as a potential and largely untapped source of secondary metabolites.     

Single cell genome amplification accelerates identification of the apratoxin biosynthetic pathway from a complex microbial assemblage

Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA) and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites.     

Rapid Genome Mapping in Nanochannel Arrays for Highly Complete and Accurate De Novo Sequence Assembly of the Complex Aegilops tauschii Genome

Next-generation sequencing (NGS) technologies have enabled high-throughput and low-cost generation of sequence data; however, de novo genome assembly remains a great challenge, particularly for large genomes. NGS short reads are often insufficient to create large contigs that span repeat sequences and to facilitate unambiguous assembly. Plant genomes are notorious for containing high quantities of repetitive elements, which combined with huge genome sizes, makes accurate assembly of these large and complex genomes intractable thus far. Using two-color genome mapping of tiling bacterial artificial chromosomes (BAC) clones on nanochannel arrays, we completed high-confidence assembly of a 2.1-Mb, highly repetitive region in the large and complex genome of Aegilops tauschii, the D-genome donor of hexaploid wheat (Triticum aestivum). Genome mapping is based on direct visualization of sequence motifs on single DNA molecules hundreds of kilobases in length. With the genome map as a scaffold, we anchored unplaced sequence contigs, validated the initial draft assembly, and resolved instances of misassembly, some involving contigs <2 kb long, to dramatically improve the assembly from 75% to 95% complete.     

Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42

The environmental strain Bacillus amyloliquefaciens FZB42 promotes plant growth and suppresses plant pathogenic organisms present in the rhizosphere. We sampled sequenced the genome of FZB42 and identified 2,947 genes with >50% identity on the amino acid level to the corresponding genes of Bacillus subtilis 168. Six large gene clusters encoding nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) occupied 7.5% of the whole genome. Two of the PKS and one of the NRPS encoding gene clusters were unique insertions in the FZB42 genome and are not present in B. subtilis 168. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis revealed expression of the antibiotic lipopeptide products surfactin, fengycin, and bacillomycin D. The fengycin (fen) and the surfactin (srf) operons were organized and located as in B. subtilis 168. A large 37.2-kb antibiotic DNA island containing the bmy gene cluster was attributed to the biosynthesis of bacillomycin D. The bmy island was found inserted close to the fen operon. The responsibility of the bmy, fen, and srf gene clusters for the production of the corresponding secondary metabolites was demonstrated by cassette mutagenesis, which led to the loss of the ability to produce these peptides. Although these single mutants still largely retained their ability to control fungal spread, a double mutant lacking both bacillomycin D and fengycin was heavily impaired in its ability to inhibit growth of phytopathogenic fungi, suggesting that both lipopeptides act in a synergistic manner.     

Metagenomic Analysis Reveals Diverse Polyketide Synthase Gene Clusters in Microorganisms Associated with the Marine Sponge Discodermia dissoluta

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up <1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.     

A novel docking domain interface model predicting recombination between homoeologous modular biosynthetic gene clusters

An in silico model for homoeologous recombination between gene clusters encoding modular polyketide synthases (PKS) or non-ribosomal peptide synthetases (NRPS) was developed. This model was used to analyze recombination between 12 PKS clusters from Streptomyces species and related genera to predict if new clusters might give rise to new products. In many cases, there were only a limited number of recombination sites (about 13 per cluster pair), suggesting that recombination may pose constraints on the evolution of PKS clusters. Most recombination events occurred between pairs of ketosynthase (KS) domains, allowing the biosynthetic outcome of the recombinant modules to be predicted. About 30% of recombinants were predicted to produce polyketides. Four NRPS clusters from Streptomyces strains were also used for in silico recombination. They yielded a comparable number of recombinants to PKS clusters, but the adenylation (A) domains contained the largest proportion of recombination events; this might be a mechanism for producing new substrate specificities. The extreme G + C-content, the presence of linear chromosomes and plasmids, as well as the lack of a mutSL-mismatch repair system should favor production of recombinants in Streptomyces species.     

Phylum-wide comparative genomics unravel the diversity of secondary metabolism in Cyanobacteria

Background

Cyanobacteria are an ancient lineage of photosynthetic bacteria from which hundreds of natural products have been described, including many notorious toxins but also potent natural products of interest to the pharmaceutical and biotechnological industries. Many of these compounds are the products of non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways. However, current understanding of the diversification of these pathways is largely based on the chemical structure of the bioactive compounds, while the evolutionary forces driving their remarkable chemical diversity are poorly understood.

Results

We carried out a phylum-wide investigation of genetic diversification of the cyanobacterial NRPS and PKS pathways for the production of bioactive compounds. 452 NRPS and PKS gene clusters were identified from 89 cyanobacterial genomes, revealing a clear burst in late-branching lineages. Our genomic analysis further grouped the clusters into 286 highly diversified cluster families (CF) of pathways. Some CFs appeared vertically inherited, while others presented a more complex evolutionary history. Only a few horizontal gene transfers were evidenced amongst strongly conserved CFs in the phylum, while several others have undergone drastic gene shuffling events, which could result in the observed diversification of the pathways.

Conclusions

Therefore, in addition to toxin production, several NRPS and PKS gene clusters are devoted to important cellular processes of these bacteria such as nitrogen fixation and iron uptake. The majority of the biosynthetic clusters identified here have unknown end products, highlighting the power of genome mining for the discovery of new natural products.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-977) contains supplementary material, which is available to authorized users.     

Distribution and diversity of natural product genes in marine and freshwater cyanobacterial cultures and genomes

Natural products are a functionally diverse class of biochemically synthesized compounds, which include antibiotics, toxins, and siderophores. In this paper, we describe both the detection of natural product activities and the sequence identification of gene fragments from two molecular systems that have previously been implicated in natural product production, i.e., nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), in diverse marine and freshwater cyanobacterial cultures. Using degenerate PCR and the sequencing of cloned products, we show that NRPSs and PKSs are common among the cyanobacteria tested. Our molecular data, when combined with genomic searches of finished and progressing cyanobacterial genomes, demonstrate that not all cyanobacteria contain NRPS and PKS genes and that the filamentous and heterocystous cyanobacteria are the richest sources of these genes and the most likely sources of novel natural products within the phylum. In addition to validating the use of degenerate primers for the identification of PKS and NRPS genes in cyanobacteria, this study also defines numerous gene fragments that will be useful as probes for future studies of the synthesis of natural products in cyanobacteria. Phylogenetic analyses of the cyanobacterial NRPS and PKS fragments sequenced in this study, as well as those from the cyanobacterial genome projects, demonstrate that there is remarkable diversity and likely novelty of these genes within the cyanobacteria. These results underscore the potential variety of novel products being produced by these ubiquitous organisms.     

Genome sequencing of a yeast-like fungal strain P6, a novel species of Aureobasidium spp.: insights into its taxonomy,evolution, and biotechnological potentials

Purpose

This study aimed to look insights into taxonomy, evolution, and biotechnological potentials of a yeast-like fungal strain P6 isolated from a mangrove ecosystem.

Methods

The genome sequencing for the yeast-like fungal strain P6 was conducted on a Hiseq sequencing platform, and the genomic characteristics and annotations were analyzed. The central metabolism and gluconate biosynthesis pathway were studied through the genome sequence data by using the GO, KOG, and KEGG databases. The secondary metabolite potentials were also evaluated.

Results

The whole genome size of the P6 strain was 25.41Mb and the G + C content of its genome was 50.69%. Totally, 6098 protein-coding genes and 264 non-coding RNA genes were predicted. The annotation results showed that the yeast-like fungal strain P6 had complete metabolic pathways of TCA cycle, EMP pathway, pentose phosphate pathway, glyoxylic acid cycle, and other central metabolic pathways. Furthermore, the inulinase activity associated with β-fructofuranosidase and high glucose oxidase activity in this strain have been demonstrated. It was found that this yeast-like fungal strain was located at root of most species of Aureobasidium spp. and at a separate cluster of all the phylogenetic trees. The P6 strain was predicted to contain three NRPS gene clusters, five type-I PKS gene clusters, and one type-I NRPS/PKS gene cluster via analysis at the antiSMASH Website. It may synthesize epichloenin A, fusaric acid, elsinochromes, and fusaridione A.

Conclusions

Based on its unique DNA sequence, taxonomic position in the phylogenetic tree and evolutional position, the yeast-like fungal strain P6 was identified as a novel species Aureobasidium hainanensis sp. nov. P6 isolate and had highly potential applications.

     

Relationship between homoeologous regulatory and structural genes in allopolyploid genome – A case study in bread wheat

Background

Whole-genome physical maps facilitate genome sequencing, sequence assembly, mapping of candidate genes, and the design of targeted genetic markers. An automated protocol was used to construct a Vitis vinifera 'Cabernet Sauvignon' physical map. The quality of the result was addressed with regard to the effect of high heterozygosity on the accuracy of contig assembly. Its usefulness for the genome-wide mapping of genes for disease resistance, which is an important trait for grapevine, was then assessed.

Results

The physical map included 29,727 BAC clones assembled into 1,770 contigs, spanning 715,684 kbp, and corresponding to 1.5-fold the genome size. Map inflation was due to high heterozygosity, which caused either the separation of allelic BACs in two different contigs, or local mis-assembly in contigs containing BACs from the two haplotypes. Genetic markers anchored 395 contigs or 255,476 kbp to chromosomes. The fully automated assembly and anchorage procedures were validated by BAC-by-BAC blast of the end sequences against the grape genome sequence, unveiling 7.3% of chimerical contigs. The distribution across the physical map of candidate genes for non-host and host resistance, and for defence signalling pathways was then studied. NBS-LRR and RLK genes for host resistance were found in 424 contigs, 133 of them (32%) were assigned to chromosomes, on which they are mostly organised in clusters. Non-host and defence signalling genes were found in 99 contigs dispersed without a discernable pattern across the genome.

Conclusion

Despite some limitations that interfere with the correct assembly of heterozygous clones into contigs, the 'Cabernet Sauvignon' physical map is a useful and reliable intermediary step between a genetic map and the genome sequence. This tool was successfully exploited for a quick mapping of complex families of genes, and it strengthened previous clues of co-localisation of major NBS-LRR clusters and disease resistance loci in grapevine.     

A PKS/NRPS/FAS Hybrid Gene Cluster from Serratia plymuthica RVH1 Encoding the Biosynthesis of Three Broad Spectrum,Zeamine-Related Antibiotics

Serratia plymuthica strain RVH1, initially isolated from an industrial food processing environment, displays potent antimicrobial activity towards a broad spectrum of Gram-positive and Gram-negative bacterial pathogens. Isolation and subsequent structure determination of bioactive molecules led to the identification of two polyamino antibiotics with the same molecular structure as zeamine and zeamine II as well as a third, closely related analogue, designated zeamine I. The gene cluster encoding the biosynthesis of the zeamine antibiotics was cloned and sequenced and shown to encode FAS, PKS as well as NRPS related enzymes in addition to putative tailoring and export enzymes. Interestingly, several genes show strong homology to the pfa cluster of genes involved in the biosynthesis of long chain polyunsaturated fatty acids in marine bacteria. We postulate that a mixed FAS/PKS and a hybrid NRPS/PKS assembly line each synthesize parts of the backbone that are linked together post-assembly in the case of zeamine and zeamine I. This interaction reflects a unique interplay between secondary lipid and secondary metabolite biosynthesis. Most likely, the zeamine antibiotics are produced as prodrugs that undergo activation in which a nonribosomal peptide sequence is cleaved off.