Shaping the Polypropionate Biosynthesis in the Solar‐Powered Mollusc Elysia viridis

Polypropionates that incorporate pyrones are a family of polyketides featuring the chemistry of a few marine molluscs capable of phototrophic CO₂ fixation as a result of storing viable symbiotic chloroplasts in their bodies. The role and origin of these molecules is poorly investigated, although the unusual biological activities and chemistry of these natural products have recently received renewed interest. Here, we report the results of in vivo studies on production of γ‐pyrone‐containing polypropionates in the Mediterranean mollusc Elysia viridis. Biosynthesis of the metabolites in the sacoglossan is shown to proceed through condensation of eight intact C₃ units by polyketide synthase assembly. LC–MS and NMR spectroscopic studies demonstrate that the process involves a pyrone tetraene ( 10 ) as key intermediate, whereas the levels of the final polypropionates ( 6 , 7 and 9 ) are related to each other and show a significant dependence upon light conditions.     

Unprecedented diversity of catalytic domains in the first four modules of the putative pederin polyketide synthase

Polyketides of the pederin group are highly potent antitumor compounds found in terrestrial beetles and marine sponges. Pederin is used by beetles of the genera Paederus and Paederidus as a chemical defense. We have recently identified a group of putative pederin biosynthesis genes and localized them to the genome of an as yet unculturable Pseudomonas sp. symbiont, the likely true pederin producer. However, this polyketide synthase cluster lacks several genes expected for pederin production. Here we report an additional polyketide synthase encoded on a separate region of the symbiont genome. It contains at least three novel catalytic domains that are predicted to be involved in pederin chain initiation and the formation of an unusual exomethylene bond. The region is bordered by mobility pseudogenes; this suggests that gene transposition led to the disjointed cluster organization. With this work, all putative pederin genes have been identified. Their heterologous expression in a culturable bacterium will provide important insights into how sustainable sources of invertebrate-derived drug candidates can be created.     

Examination of Potential Exceptions to the F and S Biosynthetic Classification of Fused‐Ring Aromatic Polyketides

The reported acetate‐derived labelling of the fungal naphthalene γ‐pyrone fonsecin, two streptomycete dodecaketide αpyrones TW93f and TW93g, and the streptomycete phenanthraquinones piloquinone, murayaquinone and haloquinone appear to be exceptions to the generalisation that fungi and streptomycetes produce fused‐ring aromatic polyketides by different modes of cyclisation. A review of their 1) originally assigned formulae, 2) [¹³C₂]acetate‐derived labelling patterns, and 3) modes of cyclisation leads to the recognition of feasible alternative chemical structures or biosynthetic pathways, which are in accord with the originally proposed classification system.     

Dual-PKS Cluster for Biosynthesis of a Light-Induced Secondary Metabolite Found from Genome Sequencing of Hyphodiscus hymeniophilus Fungus

Filamentous fungi are known producers of important secondary metabolites. In spite of this, the majority of these organisms have not been studied at the genome level, leaving many of the bioactive molecules they produce undiscovered. In this study, we explore the secondary metabolite potential of an understudied fungus, Hyphodiscus hymeniophilus. By sequencing and assembling the first genome from this genus, we show that this fungus has genes for at least 20 natural products and that many of these products are likely novel. One of these metabolites is identified: a new, red-pigmented member of the azaphilone class, hyphodiscorubrin. We show that this metabolite is only produced when the fungus is grown in the light. Furthermore, the biosynthetic gene cluster of hyphodiscorubrin is identified though homology to other known azaphilone producing clusters.     

Deciphering Pactamycin Biosynthesis and Engineered Production of New Pactamycin Analogues

Pactamycin is an aminocyclopentitol‐derived natural product that has potent antibacterial and antitumor activities. Sequence analysis of an 86 kb continuous region of the chromosome from Streptomyces pactum ATCC 27456 revealed a gene cluster involved in the biosynthesis of pactamycin. Gene inactivation of the Fe‐S radical SAM oxidoreductase (ptmC) and the glycosyltransferase (ptmJ), individually abrogated pactamycin biosynthesis; this confirmed the involvement of the ptm gene cluster in pactamycin biosynthesis. The polyketide synthase gene (ptmQ) was found to support 6‐methylsalicylic acid (6‐MSA) synthesis in a heterologous host, S. lividans T7. In vivo inactivation of ptmQ in S. pactum impaired pactamycin and pactamycate production but led to production of two new pactamycin analogues, de‐6‐MSA‐pactamycin and de‐6‐MSA‐pactamycate. The new compounds showed equivalent cytotoxic and antibacterial activities with the corresponding parent molecules and shed more light on the structure–activity relationship of pactamycin.     

Functional Dissection of a Multimodular Polypeptide of the Pikromycin Polyketide Synthase into Monomodules by Using a Matched Pair of Heterologous Docking Domains

Working together or apart : Separating multimodular PKS enzymes into their respective monomodules by replacing the natural intraprotein linkers (illustrated in red in the figure) with a matched docking domain pair from a heterologous PKS system, leads to only small losses in overall in vivo polyketide product and increased efficiency at utilizing polyketide pathway intermediates to prime the biosynthetic process.

     

Discovery and Elucidation of the Biosynthesis of Aspernidgulenes: Novel Polyenes from Aspergillus Nidulans by Using Serial Promoter Replacement

Through serial promoter exchanges, we isolated several novel polyenes, the aspernidgulenes, from Aspergillus nidulans and uncovered their succinct biosynthetic pathway involving only four enzymes. An enoyl reductase (ER)-less highly reducing polyketide synthase (HR-PKS) putatively produces a 5,6-dihydro-α-pyrone polyene, which undergoes bisepoxidation, epoxide ring opening, cyclization, and hydrolytic cleavage by three tailoring enzymes to generate aspernidgulene A1 and A2. Our findings demonstrate the prowess of fungal-tailoring enzymes to transform a polyketide scaffold concisely and efficiently into complex structures. Moreover, comparison with citreoviridin and aurovertin biosynthesis suggests that methylation of the α-pyrone hydroxy group by methyltransferase (CtvB or AurB) is the branching point at which the biosynthesis of these two classes of compounds diverge. Therefore, scanning for the presence or absence of the gatekeeping α-pyrone methyltransferase gene in homologous clusters might be a potential way to classify the product bioinformatically as belonging to methylated α-pyrone polyenes or polyenes containing rings derived from the cyclization of the unmethylated 5,6-dihydro-α-pyrone, such as 2,3-dimethyl-γ-lactone and oxabicyclo[2.2.1]heptane.     

Functional analysis of the aureothin iterative type I polyketide synthase

The modular-type polyketide synthase (PKS) that is involved in aureothin (aur) biosynthesis represents one of the first examples in which a single PKS module (AurA) is used in an iterative fashion. Here we report on the heterologous expression of an engineered AurAB fusion protein that unequivocally proves the iterative nature of AurA. In addition, point mutations reveal that aur PKS module 4 participates in polyketide biosynthesis despite its aberrant acyltransferase domain.     

Induction of Nectriapyrone Biosynthesis in the Rice Blast Fungus Pyricularia oryzae by Disturbance of the Two-Component Signal Transduction System

Most fungal secondary metabolism genes are poorly expressed under laboratory conditions. Nectriapyrones are known as secondary metabolites produced mainly by symbiotic fungi, including endophytes and plant pathogens. Herein, we show the induction of nectriapyrone production in the rice blast fungus Pyricularia oryzae. The two-component signal transduction system was disturbed by disrupting OSM1 and PoYPD1, which encoded a HOG MAP kinase and a His-containing phosphotransfer (HPt) protein, respectively. This induced the production of two polyketide compounds: nectriapyrone and its hydroxylated analogue. The nectriapyrone biosynthetic gene cluster consists of a polyketide synthase gene (NEC1) and an O-methyltransferase gene (NEC2). Overexpression of the two genes induced overproduction of nectriapyrone and five nectriapyrone analogues, including a new derivative. Nectriapyrone production was not required for the infection of rice. The structure of nectriapyrone is similar to that of the germicidins produced by Streptomyces spp., and nectriapyrone inhibited the growth of Streptomyces griseus.     

Parallel Post‐Polyketide Synthase Modification Mechanism Involved in FD‐891 Biosynthesis in Streptomyces graminofaciens A‐8890

To isolate a key polyketide biosynthetic intermediate for the 16‐membered macrolide FD‐891 ( 1 ), we inactivated two biosynthetic genes coding for post‐polyketide synthase (PKS) modification enzymes: a methyltransferase (GfsG) and a cytochrome P450 (GfsF). Consequently, FD‐892 ( 2 ), which lacks the epoxide moiety at C8–C9, the hydroxy group at C10, and the O‐methyl group at O‐25 of FD‐891, was isolated from the gfsF/gfsG double‐knockout mutant. In addition, 25‐O‐methyl‐FD‐892 ( 3 ) and 25‐O‐demethyl‐FD‐891 ( 4 ) were isolated from the gfsF and gfsG mutants, respectively. We also confirmed that GfsG efficiently catalyzes the methylation of 2 and 4 in vitro. Further, GfsF catalyzed the epoxidation of the double bond at C8‐C9 of 2 and 3 and subsequent hydroxylation at C10, to afford 4 and 1 , respectively. These results suggest that a parallel post‐PKS modification mechanism is involved in FD‐891 biosynthesis.     

Phenylnannolones A–C: Biosynthesis of New Secondary Metabolites from the Myxobacterium Nannocystis exedens

Myxobacteria are gliding bacteria that belong to the δ‐Proteobacteria and are known for their unique biosynthetic capabilities. Among myxobacteria, Nannocystis spp. are most closely related to marine myxobacteria and their secondary metabolism has hardly been investigated. Phenylnannolones A ( 1 ), B ( 2 ) and C ( 3 ) were obtained from a culture of Nannocystis exedens that was isolated from the intertidal region of Crete. Compound 1 had inhibitory activity toward the ABCB1 gene product P‐glycoprotein and reversed daunorubicin resistance in cultured cancer cells. Phenylnannolone A has an unusual structural architecture; it is composed of an ethyl‐substituted polyene chain linked to a pyrone moiety on one side and to a phenyl ring on the other. The investigation of the biosynthesis with labelled precursors revealed acetate, butyrate and phenylalanine as building blocks for 1 . The labelling pattern suggested novel biochemical reactions for the biosynthesis of the starter unit.     

Biosynthesis of xyrrolin, a new cytotoxic hybrid polyketide/non-ribosomal peptide pyrroline with anticancer potential, in Xylaria sp. BCC 1067

A gene from Xylaria sp. BCC 1067, pks3, that encodes a putative 3660-residue hybrid polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) was characterised by targeted gene disruption in combination with comprehensive product identification. Studies of the features of a corresponding mutant, YA3, allowed us to demonstrate that pks3 is responsible for the synthesis of a new pyrroline compound, named xyrrolin, in the wild-type Xylaria sp. BCC 1067. The structure of xyrrolin was established by extensive spectroscopic and spectrometric analyses, including low- and high-resolution MS, IR, (1)H NMR, (13)C NMR, (13)C NMR with Dept135, HMQC 2D NMR, HMBC 2D NMR and COSY 2D NMR. On the basis of the Pks3 domain organisation and the chemical structure of xyrrolin, we proposed that biosynthesis of this compound requires the condensation of a tetraketide and an L-serine unit, followed by Dieckmann or reductive cyclisation and enzymatic removal of ketone residue(s). Bioassays of the pure xyrrolin further displayed cytotoxicity against an oral cavity (KB) cancer cell line.     

Bacterial Tropone Natural Products and Derivatives: Overview of their Biosynthesis,Bioactivities, Ecological Role and Biotechnological Potential

Tropone natural products are non-benzene aromatic compounds of significant ecological and pharmaceutical interest. Herein, we highlight current knowledge on bacterial tropones and their derivatives such as tropolones, tropodithietic acid, and roseobacticides. Their unusual biosynthesis depends on a universal CoA-bound precursor featuring a seven-membered carbon ring as backbone, which is generated by a side reaction of the phenylacetic acid catabolic pathway. Enzymes encoded by separate gene clusters then further modify this key intermediate by oxidation, CoA-release, or incorporation of sulfur among other reactions. Tropones play important roles in the terrestrial and marine environment where they act as antibiotics, algaecides, or quorum sensing signals, while their bacterial producers are often involved in symbiotic interactions with plants and marine invertebrates (e. g., algae, corals, sponges, or mollusks). Because of their potent bioactivities and of slowly developing bacterial resistance, tropones and their derivatives hold great promise for biomedical or biotechnological applications, for instance as antibiotics in (shell)fish aquaculture.     

Cellular origin of dysiherbaine, an excitatory amino acid derived from a marine sponge

The cellular origin of dysiherbaine, a marine-sponge toxin, was investigated immunohistochemically by using an anti-dysiherbaine antibody. Dysiherbaine-like immunoreactivity was found to be localized in spherical cells harbored in the sponge mesohyl. A combination of ribosomal RNA gene (rDNA) analysis and cell-morphology analysis revealed that the spherical cells were Synechocystis cyanobacteria. However, the sponge, identified as Lendenfeldia chondrodes on the basis of its rDNA sequence, appeared to contain two different chemotypes--dysiherbaine-producing (DH+) and nondysiherbaine-producing (DH-)--both of which inhabited the same region. Synechocystis cells in the DH- sponge were not labeled with antibody, although the 16S rDNA gene profile of the cyanobacteria in the DH- sponge was indistinguishable from that of the cyanobacteria in the DH+ sponge. On the basis of these results, we hypothesize that dysiherbaine is a metabolite of certain varieties of endosymbiotic Synechocystis sp.