Iterative Assembly of Two Separate Polyketide Chains by the Same Single‐Module Bacterial Polyketide Synthase in the Biosynthesis of HSAF

Antifungal HSAF (heat‐stable antifungal factor, dihydromaltophilin) is a polycyclic tetramate macrolactam from the biocontrol agent Lysobacter enzymogenes. Its biosynthetic gene cluster contains only a single‐module polyketide synthase–nonribosomal peptide synthetase (PKS‐NRPS), although two separate hexaketide chains are required to assemble the skeleton. To address the unusual biosynthetic mechanism, we expressed the biosynthetic genes in two “clean” strains of Streptomyces and showed the production of HSAF analogues and a polyene tetramate intermediate. We then expressed the PKS module in Escherichia coli and purified the enzyme. Upon incubation of the enzyme with acyl‐coenzyme A and reduced nicotinamide adenine dinucleotide phosphate (NADPH), a polyene was detected in the tryptic acyl carrier protein (ACP). Finally, we incubated the polyene–PKS with the NRPS module in the presence of ornithine and adenosine triphosphate (ATP), and we detected the same polyene tetramate as that in Streptomyces transformed with the PKS‐NRPS alone. Together, our results provide evidence for an unusual iterative biosynthetic mechanism for bacterial polyketide–peptide natural products.     

Cytotoxic tetramic acid derivative produced by a plant type-III polyketide synthase

The tetramic acid (2,4-pyrrolidinedione) scaffold has been recognized as an important structural feature because of its mycotoxic, antibacterial, antiviral, and antioxidant activities. This important class of natural products is reportedly produced by the type-I polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) hybrid megaenzyme systems. In contrast, the benzalacetone synthase (BAS) from Rheum palmatum is a structurally simple, plant-specific type-III PKS that catalyzes the one-step decarboxylative condensation of malonyl-CoA with 4-coumaroyl-CoA. The type-III PKS exhibits unusually broad substrate specificity and notable catalytic versatility. Here we report that R. palmatum BAS efficiently produces a series of unnatural, novel tetramic acid derivatives by the condensation of malonyl-CoA with aminoacyl-CoA thioesters chemically synthesized from L- and D-amino acids. Remarkably, the novel tetramic acid dimer D-5 formed from D-phenylalanoyl-CoA showed moderate antiproliferative activity against murine leukemia P388 cells.     

The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus ATCC15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase

BACKGROUND: The structural and catalytic similarities between modular nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) inspired us to search for a hybrid NRPS-PKS system. The antitumor drug bleomycin (BLM) is a natural hybrid peptide-polyketide metabolite, the biosynthesis of which provides an excellent opportunity to investigate intermodular communication between NRPS and PKS modules. Here, we report the cloning, sequencing, and characterization of the BLM biosynthetic gene cluster from Streptomyces verticillus ATCC15003. RESULTS: A set of 30 genes clustered with the previously characterized blmAB resistance genes were defined by sequencing a 85-kb contiguous region of DNA from S. verticillus ATCC15003. The sequenced gene cluster consists of 10 NRPS genes encoding nine NRPS modules, a PKS gene encoding one PKS module, five sugar biosynthesis genes, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The substrate specificities of individual NRPS and PKS modules were predicted based on sequence analysis, and the amino acid specificities of two NRPS modules were confirmed biochemically in vitro. The involvement of the cloned genes in BLM biosynthesis was demonstrated by bioconversion of the BLM aglycones into BLMs in Streptomyces lividans expressing a part of the gene cluster. CONCLUSION: The blm gene cluster is characterized by a hybrid NRPS-PKS system, supporting the wisdom of combining individual NRPS and PKS modules for combinatorial biosynthesis. The availability of the blm gene cluster has set the stage for engineering novel BLM analogs by genetic manipulation of genes governing BLM biosynthesis and for investigating the molecular basis for intermodular communication between NRPS and PKS in the biosynthesis of hybrid peptide-polyketide metabolites.     

Evidence for a monomeric structure of nonribosomal Peptide synthetases

Nonribosomal peptide synthetases (NRPS) are multimodular biocatalysts that bacteria and fungi use to assemble many complex peptides with broad biological activities. The same modular enzymatic assembly line principles are found in fatty acid synthases (FAS), polyketide synthases (PKS), and most recently in hybrid NRPS/PKS multienzymes. FAS as well as PKS are known to function as homodimeric enzyme complexes, raising the question of whether NRPS may also act as homodimers. To test this hypothesis, biophysical methods (size exclusion chromatography, analytical equilibrium ultracentrifugation, and chemical crosslinking) and biochemical methods (two-affinity-tag-system and complementation studies with enzymes being inactivated in different catalytic domains) were applied to NRPS subunits from the gramicidin S (GrsA-ATE), tyrocidine (TycB(1)-CAT and TycB(2-3)-AT.CATE), and enterobactin (EntF-CATTe) biosynthetic systems. These methods had revealed the dimeric structure of FAS and PKS previously, but all three NRPS systems investigated are functionally active as monomers.     

Molecular analysis of the kirromycin biosynthetic gene cluster revealed beta-alanine as precursor of the pyridone moiety

Kirromycin is a complex linear polyketide that acts as a protein biosynthesis inhibitor by binding to the bacterial elongation factor Tu. The kirromycin biosynthetic gene cluster was isolated from the producer, Streptomyces collinus Tü 365, and confirmed by targeted disruption of essential biosynthesis genes. Kirromycin is synthesized by a large hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) encoded by the genes kirAI-kirAVI. This complex involves some very unusual features, including the absence of internal acyltransferase (AT) domains in KirAI-KirAV, multiple split-ups of PKS modules on separate genes, and swapping in the domain organization. Interestingly, one PKS enzyme, KirAVI, contains internal AT domains. Based on in silico analysis, a route to pyridone formation involving PKS and NRPS steps was postulated. This hypothesis was experimentally proven by feeding studies with [U-13C3(15)N]beta-alanine and NMR and MS analyses of the isolated pure kirromycin.     

Novel features in a combined polyketide synthase/non-ribosomal peptide synthetase: the myxalamid biosynthetic gene cluster of the myxobacterium Stigmatella aurantiaca Sga15

BACKGROUND: Myxobacteria have been well established as a potent source for natural products with biological activity. They produce a considerable variety of compounds which represent typical polyketide structures with incorporated amino acids (e.g. the epothilons, the myxothiazols and the myxalamids). Several of these secondary metabolites are effective inhibitors of the electron transport via the respiratory chain and have been widely used. Molecular cloning and characterization of the genes governing the biosynthesis of these structures is of considerable interest, because such information adds to the limited knowledge as to how polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) interact and how they might be manipulated in order to form novel antibiotics. RESULTS: A DNA region of approximately 50000 base pairs from Stigmatella aurantiaca Sga15 was sequenced and shown by gene disruption to be involved in myxalamid biosynthesis. Sequence analysis reveals that the myxalamids are formed by a combined PKS/NRPS system. The terminal NRPS MxaA extends the assembled polyketide chain of the myxalamids with alanine. MxaA contains an N-terminal domain with homology to NAD binding proteins, which is responsible during the biogenesis for a novel type of reductive chain release giving rise to the 2-amino-propanol moiety of the myxalamids. The last module of the PKS reveals an unprecedented genetic organization; it is encoded on two genes (mxaB1 and mxaB2), subdividing the domains of one module from each other. A sequence comparison of myxobacterial acyl-transferase domains with known systems from streptomycetes and bacilli reveals that consensus sequences proposed to be specific for methylmalonyl-CoA and malonyl-CoA are not always reliable. CONCLUSIONS: The complete biosynthetic gene cluster of the myxalamid-type electron transport inhibitor from S. aurantiaca Sga15 has been cloned and analyzed. It represents one of the few examples of combined PKS/NRPS systems, the analysis and manipulation of which has the potential to generate novel hybrid structures via combinatorial biosynthesis (e.g. via module-swapping techniques). Additionally, a new type of reductive release from PKS/NRPS systems is described.     

Genome-wide high-throughput mining of natural-product biosynthetic gene clusters by phage display

We have developed a phage-display method for high-throughput mining of bacterial gene clusters encoding the natural-product biosynthetic enzymes, polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). This method uses the phosphopantetheinyl transferase activity of Sfp to specifically biotinylate NRPS and PKS carrier-protein domains expressed from a library of random genome fragments fused to a gene encoding a phage coat protein. Subsequently, the biotinylated phages are enriched through selection on streptavidin-coated plates. Using this method, we isolated phage clones from the multiple NRPS and PKS gene clusters encoded in the genomes of Bacillus subtilis and Myxococcus xanthus. Due to the rapid and unambiguous identification of carrier domains, this method will provide an efficient tool for high-throughput cloning of NRPS and PKS gene clusters from many individual bacterial genomes and multigenome environmental DNA.     

Dissecting non-ribosomal and polyketide biosynthetic machineries using electrospray ionization Fourier-Transform mass spectrometry

Many virulence factors and bioactive compounds with antifungal, antimicrobial, and antitumor properties are produced via the non-ribosomal peptide synthetase (NRPS) or polyketide synthase(PKS) paradigm. During the biosynthesis of these natural products, substrates, intermediates and side products are covalently tethered to the NRPS or PKS catalyst, introducing mass changes, making these biosynthetic systems ideal candidates for interrogation by large molecule mass spectrometry. This review serves as an introduction into the application of electrospray ionization Fourier-Transform massspectrometry (ESI-FTMS) to investigate NRPS and PKS systems. ESI-FTMS can be used to understand substrate tolerance, timing of covalent linkages, timing of tailoring reactions and the transfer of substrates and biosynthetic intermediates from domain to domain. Therefore we not only highlight key mechanistic insights for thiotemplate systems as found on the enterobactin,yersiniabactin, epothilone, clorobiocin, coumermycin, pyoluteorin, gramicidin, mycosubtilin, C-1027,6-deoxyerythronolide B and FK520 biosynthetic pathways, but we also explain the approaches taken to identify active sites from complex digests and compare the FTMS based assay to traditional assays and other mass spectrometric techniques. Although mass spectrometry was introduced over two decades ago to investigate NRPS and PKS biosynthetic systems, this is the first review devoted to this methodology.     

Cyclization mechanism for the synthesis of macrocyclic antibiotic lankacidin in Streptomyces rochei

The lankacidin biosynthetic gene cluster in Streptomyces rochei strain 7434AN4 was found to span 31 kb of the giant linear plasmid pSLA2-L and contain a polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) hybrid gene (lkcA), type I PKS genes, and pyrroloquinoline quinone (PQQ) biosynthetic genes (lkcK-lkcO). Feeding of PQQ to a pqq mutant restored the lankacidin production, suggesting its crucial role in an oxidation process. However, formation of the 17-membered macrocyclic ring was not catalyzed by PQQ-dependent dehydrogenase (Orf23), but was by flavin-dependent amine oxidase (LkcE). Compound LC-KA05 isolated from an lkcE disruptant was an acyclic intermediate lacking the C2-C18 linkage. These results suggested a cyclization mechanism for the synthesis of the lankacidin macrocyclic skeleton.     

Leinamycin biosynthesis revealing unprecedented architectural complexity for a hybrid polyketide synthase and nonribosomal peptide synthetase

A 135,638 bp DNA region that encompasses the leinamycin (LNM) biosynthetic gene cluster was sequenced from Streptomyces atroolivaceus S-140. The boundaries of the lnm cluster were defined by systematic inactivation of open reading frames within the sequenced region. The lnm cluster spans 61.3 kb of DNA and consists of 27 genes encoding nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), hybrid NRPS-PKS, resistance, regulatory, and tailoring enzymes, as well as proteins of unknown function. A model for LNM biosynthesis is proposed, central to which is the LNM hybrid NRPS-PKS megasynthetase consisting of discrete (LnmQ and LnmP) and modular (LnmI) NRPS, acyltransferase-less PKS (LnmG, LnmI, and LnmJ), and PKS modules with unusual domain organization. These studies unveil an unprecedented architectural complexity for the LNM hybrid NRPS-PKS megasynthetase and set the stage to investigate the molecular basis for LNM biosynthesis.     

Cloning and elucidation of the FR901464 gene cluster revealing a complex acyltransferase-less polyketide synthase using glycerate as starter units

FR901464, an antitumor natural product, represents a new class of potent anticancer small molecules targeting spliceosome and inhibiting both splicing and nuclear retention of pre-mRNA. Herein we describe the biosynthetic gene cluster of FR901464, identified by degenerate primer PCR amplification of a gene encoding the 3-hydroxy-3-methylglutaryl-CoA synthase (HCS) postulated to be involved in the biosynthesis of a β-branched polyketide from Pseudomonas sp. No. 2663. This cluster consists of twenty open reading frames (ORFs) and was localized to 93-kb DNA segment, and its involvement in FR901464 biosynthesis was confirmed by gene inactivation and complementation. FR901464 is biosynthesized by a hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS), HCS, and acyltransferases (AT)-less system. The PKS/NRPS modules feature unusual domain organization including multiple domain redundancy, inactivation, and tandem. Biochemical characterization of a glyceryl transferase and an acyl carrier protein (ACP) in the start module revealed that it incorporates D-1,3-bisphosphoglycerate, which is dephosphorylated and transferred to ACP as the starter unit. Furthermore, an oxidative Baeyer-Villiger reaction followed by chain release was postulated to form a pyran moiety. On the basis of in silico analysis and genetic and biochemical evidances, a biosynthetic pathway for FR901464 was proposed, which sets the stage to further investigate the complex PKS biochemically and engineer the biosynthetic machinery for the production of novel analogues.     

Yersiniabactin synthetase: a four-protein assembly line producing the nonribosomal peptide/polyketide hybrid siderophore of Yersinia pestis

Yersiniabactin synthetase comprises four proteins, YbtE, HMWP1, HMWP2, and YbtU, encompassing seventeen functional domains, twelve catalytic and five carrier, to select, activate, and incorporate salicylate, three cysteines, and one malonyl moiety into the iron chelator yersiniabactin (Ybt). In the present study, yersiniabactin has been reconstituted in vitro from the 4 protein assembly line by the use of eight biosynthetic precursors. The rate of one turnover, comprising 22 chemical operations performed by the assembly line to release the completed Ybt molecule, was determined at 1.4 min(-1). During the course of Ybt production, the elongating acyl-S-enzyme chain was shown to transfer across a nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) interprotein interface and then a PKS/NRPS intraprotein interface. This study on the Ybt synthetase assembly line represents the first complete in vitro reconstitution of a nonribosomal peptide/polyketide hybrid system.     

Paenilamicin: Structure and Biosynthesis of a Hybrid Nonribosomal Peptide/Polyketide Antibiotic from the Bee Pathogen Paenibacillus larvae

The spore‐forming bacterium Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a fatal disease of honey bees that occurs worldwide. Previously, we identified a complex hybrid nonribosomal peptide/polyketide synthesis (NRPS/PKS) gene cluster in the genome of P. larvae. Herein, we present the isolation and structure elucidation of the antibacterial and antifungal products of this gene cluster, termed paenilamicins. The unique structures of the paenilamicins give deep insight into the underlying complex hybrid NRPS/PKS biosynthetic machinery. Bee larval co‐infection assays reveal that the paenilamicins are employed by P. larvae in fighting ecological niche competitors and are not directly involved in killing the bee larvae. Their antibacterial and antifungal activities qualify the paenilamicins as attractive candidates for drug development.     

Nature's Combinatorial Biosynthesis Produces Vatiamides A–F

Hybrid type I PKS/NRPS biosynthetic pathways typically proceed in a collinear manner wherein one molecular building block is enzymatically incorporated in a sequence that corresponds to gene arrangement. In this work, genome mining combined with the use of a fluorogenic azide‐based click probe led to the discovery and characterization of vatiamides A–F, three structurally diverse alkynylated lipopeptides, and their brominated analogues, from the cyanobacterium Moorea producens ASI16Jul14‐2. These derive from a unique combinatorial non‐collinear PKS/NRPS system encoded by a 90 kb gene cluster in which an upstream PKS cassette interacts with three separate cognate NRPS partners. This is facilitated by a series of promiscuous intermodule PKS‐NRPS docking motifs possessing identical amino acid sequences. This interaction confers a new type of combinatorial capacity for creating molecular diversity in microbial systems.     

DKxanthene biosynthesis--understanding the basis for diversity-oriented synthesis in myxobacterial secondary metabolism

The DKxanthenes are a family of yellow pigments which play a critical role in myxobacterial development. Thirteen unique structures from Myxococcus xanthus DK1622 differ in the length of their characteristic polyene functionality, as well as the extent of methyl branching. We aimed to understand the mechanistic basis for this "molecular promiscuity" by analyzing the gene cluster in DK1622, and comparing it to the DKxanthene biosynthetic locus in a second myxobacterium, Stigmatella aurantiaca DW4/3-1, which produces a more limited range of compounds. While the core biosynthetic machinery is highly conserved, M. xanthus contains a putative asparagine hydroxylase function which is not present in S. aurantiaca. This observation accounts, in part, for the significantly larger metabolite family in M. xanthus. Detailed analysis of the encoded hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) assembly line provides direct evidence for the mechanism underlying the variable polyene length and the observed pattern of methyl functionalities.     

Molecular and biochemical studies of chondramide formation-highly cytotoxic natural products from Chondromyces crocatus Cm c5

The jaspamide/chondramide family of depsipeptides are mixed PKS/NRPS natural products isolated from marine sponges and a terrestrial myxobacterium that potently affect the function of the actin cytoskeleton. As a first step to improve production in heterologous host cells and permit genetic approaches to novel analogs, we have cloned and characterized the chondramide biosynthetic genes from the myxobacterium Chondromyces crocatus Cm c5. In addition to the expected PKS and NRPS genes, the cluster encodes a rare tyrosine aminomutase for beta-tyrosine formation and a previously unknown tryptophan-2-halogenase. Conditions for gene transfer into C. crocatus Cm c5 were developed, and inactivation of several genes corroborated their proposed function and served to define the boundaries of the cluster. Biochemical characterization of the final NRPS adenylation domain confirmed the direct activation of beta-tyrosine, and fluorinated chondramides were produced through precursor-directed biosynthesis.     

Hybrid nonribosomal peptide-polyketide interfaces in epothilone biosynthesis: minimal requirements at N and C termini of EpoB for elongation

Epothilone (Epo) D, an antitumor agent currently in clinical trials, is a hybrid natural product produced by the combined action of nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS). In the epothilone biosynthetic pathway, EpoB, a 165 kDa NRPS is inserted into an otherwise entirely PKS assembly line, forming two hybrid NRPS-PKS interfaces. In light of the terminal linker effect previously identified in PKS, the N- and C-terminal sequences of EpoB were examined for their roles in propagating the incipient natural product. Eight amino acid residues at EpoB C terminus, in which six are positively charged, were found to be a key component of the C-terminal linker effect. A minimal sequence of 56 residues at EpoB N terminus was required for elongating the acetyl group from the acyl carrier protein (ACP) of EpoA to form methylthiazolyl-S-EpoB.     

Polyketide synthases and nonribosomal peptide synthetases: the emerging view from bacterial genomics

A total of 223 complete bacterial genomes are analyzed, with 281 citations, for the presence of genes encoding modular polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). We report on the distribution of these systems in different bacterial taxa and, whenever known, the metabolites they synthesize. We also highlight, in the different bacterial lineages, the PKS and NRPS genes and, whenever known, the corresponding products.     

Characterization of the maduropeptin biosynthetic gene cluster from Actinomadura madurae ATCC 39144 supporting a unifying paradigm for enediyne biosynthesis

The biosynthetic gene cluster for the enediyne antitumor antibiotic maduropeptin (MDP) from Actinomadura madurae ATCC 39144 was cloned and sequenced. Cloning of the mdp gene cluster was confirmed by heterologous complementation of enediyne polyketide synthase (PKS) mutants from the C-1027 producer Streptomyces globisporus and the neocarzinostatin producer Streptomyces carzinostaticus using the MDP enediyne PKS and associated genes. Furthermore, MDP was produced, and its apoprotein was isolated and N-terminal sequenced; the encoding gene, mdpA, was found to reside within the cluster. The biosynthesis of MDP is highlighted by two iterative type I PKSs--the enediyne PKS and a 6-methylsalicylic acid PKS; generation of (S)-3-(2-chloro-3-hydroxy-4-methoxyphenyl)-3-hydroxypropionic acid derived from L-alpha-tyrosine; a unique type of enediyne apoprotein; and a convergent biosynthetic approach to the final MDP chromophore. The results demonstrate a platform for engineering new enediynes by combinatorial biosynthesis and establish a unified paradigm for the biosynthesis of enediyne polyketides.     

Identification of caerulomycin A gene cluster implicates a tailoring amidohydrolase

The biosynthetic gene cluster for caerulomycin A (1) was cloned and characterized from the marine actinomycete Actinoalloteichus cyanogriseus WH1-2216-6, which revealed an unusual hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) system. The crmL disruption mutant accumulated caerulomycin L (2) with an extended L-leucine at C-7, implicating an amidohydrolase activity for CrmL. The leucine-removing activity was confirmed for crude CrmL enzymes. Heterologous expression of the 1 gene cluster led to 1 production in Streptomyces coelicolor.