Uncovering biosynthetic relationships between antifungal nonadrides and octadrides.

Maleidrides are a class of bioactive secondary metabolites unique to filamentous fungi, which contain one or more maleic anhydrides fused to a 7-, 8- or 9- membered carbocycle (named heptadrides, octadrides and nonadrides respectively). Herein structural and biosynthetic studies on the antifungal octadride, zopfiellin, and nonadrides scytalidin, deoxyscytalidin and castaneiolide are described. A combination of genome sequencing, bioinformatic analyses, gene disruptions, biotransformations, isotopic feeding studies, NMR and X-ray crystallography revealed that they share a common biosynthetic pathway, diverging only after the nonadride deoxyscytalidin. 5-Hydroxylation of deoxyscytalidin occurs prior to ring contraction in the zopfiellin pathway of Diffractella curvata. In Scytalidium album, 6-hydroxylation - confirmed as being catalysed by the α-ketoglutarate dependent oxidoreductase ScyL2 - converts deoxyscytalidin to scytalidin, in the final step in the scytalidin pathway. Feeding scytalidin to a zopfiellin PKS knockout strain led to the production of the nonadride castaneiolide and two novel ring-open maleidrides.

Identification of key enzymes responsible for protolimonoid biosynthesis in plants: Opening the door to azadirachtin production.

Limonoids are natural products made by plants belonging to the Meliaceae (Mahogany) and Rutaceae (Citrus) families. They are well known for their insecticidal activity, contribution to bitterness in citrus fruits, and potential pharmaceutical properties. The best known limonoid insecticide is azadirachtin, produced by the neem tree (Azadirachta indica). Despite intensive investigation of limonoids over the last half century, the route of limonoid biosynthesis remains unknown. Limonoids are classified as tetranortriterpenes because the prototypical 26-carbon limonoid scaffold is postulated to be formed from a 30-carbon triterpene scaffold by loss of 4 carbons with associated furan ring formation, by an as yet unknown mechanism. Here we have mined genome and transcriptome sequence resources for 3 diverse limonoid-producing species (A. indica, Melia azedarach, and Citrus sinensis) to elucidate the early steps in limonoid biosynthesis. We identify an oxidosqualene cyclase able to produce the potential 30-carbon triterpene scaffold precursor tirucalla-7,24-dien-3ß-ol from each of the 3 species. We further identify coexpressed cytochrome P450 enzymes from M. azedarach (MaCYP71CD2 and MaCYP71BQ5) and C. sinensis (CsCYP71CD1 and CsCYP71BQ4) that are capable of 3 oxidations of tirucalla-7,24-dien-3ß-ol, resulting in spontaneous hemiacetal ring formation and the production of the protolimonoid melianol. Our work reports the characterization of protolimonoid biosynthetic enzymes from different plant species and supports the notion of pathway conservation between both plant families. It further paves the way for engineering crop plants with enhanced insect resistance and producing high-value limonoids for pharmaceutical and other applications by expression in heterologous hosts.

Structure revision of cryptosporioptides and determination of the genetic basis for dimeric xanthone biosynthesis in fungi.

Three novel dimeric xanthones, cryptosporioptides A-C were isolated from Cryptosporiopsis sp. 8999 and their structures elucidated. Methylation of cryptosporioptide A gave a methyl ester with identical NMR data to cryptosporioptide, a compound previously reported to have been isolated from the same fungus. However, HRMS analysis revealed that cryptosporioptide is a symmetrical dimer, not a monomer as previously proposed, and the revised structure was elucidated by extensive NMR analysis. The genome of Cryptosporiopsis sp. 8999 was sequenced and the dimeric xanthone (dmx) biosynthetic gene cluster responsible for the production of the cryptosporioptides was identified. Gene disruption experiments identified a gene (dmxR5) encoding a cytochrome P450 oxygenase as being responsible for the dimerisation step late in the biosynthetic pathway. Disruption of dmxR5 led to the isolation of novel monomeric xanthones. Cryptosporioptide B and C feature an unusual ethylmalonate subunit: a hrPKS and acyl CoA carboxylase are responsible for its formation. Bioinformatic analysis of the genomes of several fungi producing related xanthones, e.g. the widely occurring ergochromes, and related metabolites allows detailed annotation of the biosynthetic genes, and a rational overall biosynthetic scheme for the production of fungal dimeric xanthones to be proposed.

Characterisation of the biosynthetic pathway to agnestins A and B reveals the reductive route to chrysophanol in fungi.

Two new dihydroxy-xanthone metabolites, agnestins A and B, were isolated from Paecilomyces variotii along with a number of related benzophenones and xanthones including monodictyphenone. The structures were elucidated by NMR analyses and X-ray crystallography. The agnestin (agn) biosynthetic gene cluster was identified and targeted gene disruptions of the PKS, Baeyer-Villiger monooxygenase, and other oxido-reductase genes revealed new details of fungal xanthone biosynthesis. In particular, identification of a reductase responsible for in vivo anthraquinone to anthrol conversion confirms a previously postulated essential step in aromatic deoxygenation of anthraquinones, e.g. emodin to chrysophanol.

The cycloaspeptides: uncovering a new model for methylated nonribosomal peptide biosynthesis.

The cycloaspeptides are bioactive pentapeptides produced by various filamentous fungi, which have garnered interest from the agricultural industry due to the reported insecticidal activity of the minor metabolite, cycloaspeptide E. Genome sequencing, bioinformatics and heterologous expression confirmed that the cycloaspeptide gene cluster contains a minimal 5-module nonribosomal peptide synthetase (NRPS) and a new type of trans-acting N-methyltransferase (N-MeT). Deletion of the N-MeT encoding gene and subsequent feeding studies determined that two modules of the NRPS preferentially accept and incorporate N-methylated amino acids. This discovery allowed the development of a system with unprecedented control over substrate supply and thus output, both increasing yields of specific metabolites and allowing the production of novel fluorinated analogues. Furthermore, the biosynthetic pathway to ditryptophenaline, another fungal nonribosomal peptide, was shown to be similar, in that methylated phenylalanine is accepted by the ditryptophenaline NRPS. Again, this allowed the directed biosynthesis of a fluorinated analogue, through the feeding of a mutant strain. These discoveries represent a new paradigm for the production of N-methylated cyclic peptides via the selective incorporation of N-methylated free amino acids.

Genetic and chemical characterisation of the cornexistin pathway provides further insight into maleidride biosynthesis.

The biosynthesis of the herbicide cornexistin in the fungus Paecilomyces variotii was investigated by full sequencing of its genome, knockout of key genes within its biosynthetic gene cluster and isolation and identification of intermediate compounds. The general biosynthetic pathway resembles that of byssochlamic acid and other nonadrides in the early stages, but differs in requiring fewer enzymes in the key nonadride dimerisation step, and in the removal of one maleic anhydride moiety.

Heterologous Production of Fungal Maleidrides Reveals the Cryptic Cyclization Involved in their Biosynthesis.

Fungal maleidrides are an important family of bioactive secondary metabolites that consist of 7, 8, or 9-membered carbocycles with one or two fused maleic anhydride moieties. The biosynthesis of byssochlamic acid (a nonadride) and agnestadride A (a heptadride) was investigated through gene disruption and heterologous expression experiments. The results reveal that the precursors for cyclization are formed by an iterative highly reducing fungal polyketide synthase supported by a hydrolase, together with two citrate-processing enzymes. The enigmatic ring formation is catalyzed by two proteins with homology to ketosteroid isomerases, and assisted by two proteins with homology to phosphatidylethanolamine-binding proteins.

Novel nonadride, heptadride and maleic acid metabolites from the byssochlamic acid producer Byssochlamys fulva IMI 40021 - an insight into the biosynthesis of maleidrides.

The filamentous fungus Byssochlamys fulva strain IMI 40021 produces (+)-byssochlamic acid 1, its novel dihydroanalogue 2 and four related secondary metabolites. Agnestadrides A, 17 and B, 18 constitute a novel class of seven-membered ring, maleic anhydride-containing (hence termed heptadride) natural products. The putative maleic anhydride precursor 5 for both nonadride and heptadride biosynthesis was isolated as a fermentation product for the first time and its structure confirmed by synthesis. Acid 5 undergoes facile decarboxylation to anhydride 6. The generic term maleidrides is proposed to encompass biosynthetically-related compounds containing maleic anhydride moieties fused to an alicyclic ring, varying in size and substituents.