A genome-wide survey of the secondary metabolite biosynthesis genes in the wheat pathogen <i>Parastagonospora nodorum</i>

<div><p>The model pathogen <i>Parastagonospora nodorum</i> is a necrotroph and the causal agent of the wheat disease Septoria nodorum blotch (SNB). The sequenced <i>P. nodorum</i> genome has revealed that the fungus harbours a large number of secondary metabolite genes. Secondary metabolites are known to play important roles in the virulence of plant pathogens, but limited knowledge is available about the SM repertoire of this wheat pathogen. Here, we review the secondary metabolites that have been isolated from <i>P. nodorum</i> and related species of the same genus and provide an in-depth genome-wide overview of the secondary metabolite gene clusters encoded in the <i>P. nodorum</i> genome. The secondary metabolite gene survey reveals that <i>P. nodorum</i> is capable of producing a diverse range of small molecules and exciting prospects exist for discovery of novel virulence factors and bioactive molecules.</p></div

Complexity Generation in Fungal Polyketide Biosynthesis: A Spirocycle-Forming P450 in the Concise Pathway to the Antifungal Drug Griseofulvin

Griseofulvin (<b>1</b>) is a spirocyclic fungal natural product used in treatment of fungal dermatophytes. Formation of the spirocycle, or the grisan scaffold, from a benzophenone precursor is critical for the activity of <b>1</b>. In this study, we have systematically characterized each of the biosynthetic enzymes related to the biogenesis of <b>1</b>, including the characterization of a new polyketide synthase GsfA that synthesizes the benzophenone precursor and a cytochrome P450 GsfF that performs oxidative coupling between the orcinol and the phloroglucinol rings to yield the grisan structure. Notably, the finding of GsfF is in sharp contrast to the copper-dependent dihydrogeodin oxidase that performs a similar reaction in the geodin biosynthetic pathway. The biosynthetic knowledge enabled the <i>in vitro</i> total biosynthesis of <b>1</b> from malonyl-CoA using all purified enzyme components. This work therefore completely maps out the previously unresolved enzymology of the biosynthesis of a therapeutically relevant natural produc

Fungal Polyketide Synthase Product Chain-Length Control by Partnering Thiohydrolase

Fungal highly reducing polyketide synthases (HRPKSs) are an enigmatic group of multidomain enzymes that catalyze the biosynthesis of structurally diverse compounds. This variety stems from their intrinsic programming rules, which permutate the use of tailoring domains and determine the overall number of iterative cycles. From genome sequencing and mining of the producing strain <i>Eupenicillium brefeldianum</i> ATCC 58665, we identified an HRPKS involved in the biosynthesis of an important protein transport-inhibitor Brefeldin A (BFA), followed by reconstitution of its activity in <i>Saccharomyces cerevisiae</i> and in vitro. Bref-PKS demonstrated an NADPH-dependent reductive tailoring specificity that led to the synthesis of four different octaketide products with varying degrees of reduction. Furthermore, contrary to what is expected from the structure of BFA, Bref-PKS is found to be a nonaketide synthase in the absence of an associated thiohydrolase Bref-TH. Such chain-length control by the partner thiohydrolase was found to be present in other HRPKS systems and highlights the importance of including tailoring enzyme activities in predicting fungal HRPKS functions and their products

A Cytochrome P450 Serves as an Unexpected Terpene Cyclase during Fungal Meroterpenoid Biosynthesis

Viridicatumtoxin (<b>1</b>) is a tetracycline-like fungal meroterpenoid with a unique, fused spiro­bicyclic ring system. Puzzlingly, no dedicated terpene cyclase is found in the gene cluster identified in <i>Penicillium aethio­picum</i>. Cytochrome P450 enzymes VrtE and VrtK in the <i>vrt</i> gene cluster were shown to catalyze C5-hydroxylation and spiro­bicyclic ring formation, respectively. Feeding acyclic pre­viridicatum­toxin to <i>Saccharo­myces cere­visiae</i> expressing VrtK confirmed that VrtK is the sole enzyme required for cyclizing the geranyl moiety. Thus, VrtK is the first example of a P450 that can catalyze terpene cyclization, most likely via initial oxidation of C17 to an allylic carbo­cation. Quantum chemical modeling revealed a possible new tertiary carbo­cation intermediate E that forms after allylic carbo­cation formation. Intermediate E can readily undergo concerted 1,2-alkyl shift/1,3-hydride shift, either spontaneously or further aided by VrtK, followed by C7 Friedel–Crafts alkylation to afford <b>1</b>. The most likely stereochemical course of the reaction was proposed on the basis of the results of our computations

Identification and Characterization of the Echinocandin B Biosynthetic Gene Cluster from Emericella rugulosa NRRL 11440

Echinocandins are a family of fungal lipidated cyclic hexapeptide natural products. Due to their effectiveness as antifungal agents, three semisynthetic derivatives have been developed and approved for treatment of human invasive candidiasis. All six of the amino acid residues are hydroxylated, including 4<i>R</i>,5<i>R</i>-dihydroxy-l-ornithine, 4<i>R</i>-hydroxyl-l-proline, 3<i>S</i>,4<i>S</i>-dihydroxy-l-homotyrosine, and 3<i>S</i>-hydroxyl-4<i>S</i>-methyl-l-proline. We report here the biosynthetic gene cluster of echinocandin B <b>1</b> from Emericella rugulosa NRRL 11440 containing genes encoding for a six-module nonribosomal peptide synthetase EcdA, an acyl-AMP ligase EcdI, and oxygenases EcdG, EcdH, and EcdK. We showed EcdI activates linoleate as linoleyl-AMP and installs it on the first thiolation domain of EcdA. We have also established through ATP–PP_i exchange assay that EcdA loads l-ornithine in the first module. A separate <i>hty</i> gene cluster encodes four enzymes for de novo generation of l-homotyrosine from acetyl-CoA and 4-hydroxyphenyl-pyruvate is found from the sequenced genome. Deletions in the <i>ecdA</i>, and <i>htyA</i> genes validate their essential roles in echinocandin B production. Five predicted iron-centered oxygenase genes, <i>ecdG</i>, <i>ecdH</i>, <i>ecdK</i>, <i>htyE</i>, and <i>htyF</i>, in the two separate <i>ecd</i> and <i>hty</i> clusters are likely to be the tailoring oxygenases for maturation of the nascent NRPS lipohexapeptidolactam product

Genome Mining of a Prenylated and Immunosuppressive Polyketide from Pathogenic Fungi

Activation of the polycyclic polyketide prenyltransferase (pcPTase)-containing silent clusters in <i>Aspergillus fumigatus</i> and <i>Neosartorya fischeri</i> led to isolation of a new metabolite neosartoricin (<b>3</b>). The structure of <b>3</b> was solved by X-ray crystallography and NMR to be a prenylated anthracenone. <b>3</b> exhibits T-cell antiproliferative activity with an IC₅₀ of 3 μM, suggestive of a physiological role as an immunosuppressive agent

Nerita virginea

Activation of the polycyclic polyketide prenyltransferase (pcPTase)-containing silent clusters in <i>Aspergillus fumigatus</i> and <i>Neosartorya fischeri</i> led to isolation of a new metabolite neosartoricin (<b>3</b>). The structure of <b>3</b> was solved by X-ray crystallography and NMR to be a prenylated anthracenone. <b>3</b> exhibits T-cell antiproliferative activity with an IC₅₀ of 3 μM, suggestive of a physiological role as an immunosuppressive agent

Discovery and Characterization of a Group of Fungal Polycyclic Polyketide Prenyltransferases

The prenyltransferase (PTase) gene <i>vrtC</i> was proposed to be involved in viridicatumtoxin (<b>1</b>) biosynthesis in <i>Penicillium aethiopicum</i>. Targeted gene deletion and reconstitution of recombinant VrtC activity in vitro established that VrtC is a geranyl transferase that catalyzes a regiospecific Friedel–Crafts alkylation of the naphthacenedione carboxamide intermediate <b>2</b> at carbon 6 with geranyl diphosphate. VrtC can function in the absence of divalent ions and can utilize similar naphthacenedione substrates, such as the acetyl-primed TAN-1612 (<b>4</b>). Genome mining using the VrtC protein sequence leads to the identification of a homologous group of PTase genes in the genomes of human and animal-associated fungi. Three enzymes encoded by this new subgroup of PTase genes from <i>Neosartorya fischeri</i>, <i>Microsporum canis</i>, and <i>Trichophyton tonsurans</i> were shown to be able to catalyze transfer of dimethylallyl to several tetracyclic naphthacenedione substrates in vitro. In total, seven C₅- or C₁₀-prenylated naphthacenedione compounds were generated. The regioselectivity of these new polycyclic PTases (pcPTases) was confirmed by characterization of product <b>9</b> obtained from biotransformation of <b>4</b> in <i>Escherichia coli</i> expressing the <i>N. fischeri</i> pcPTase gene. The discovery of this new subgroup of PTases extends our enzymatic tools for modifying polycyclic compounds and enables genome mining of new prenylated polyketides

The Fumagillin Biosynthetic Gene Cluster in <i>Aspergillus fumigatus</i> Encodes a Cryptic Terpene Cyclase Involved in the Formation of β-<i>trans</i>-Bergamotene

Fumagillin <b>1</b> is a meroterpenoid from <i>Aspergillus fumigatus</i> that is known for its anti-angiogenic activity by binding to human methionine aminopeptidase 2. The genetic and molecular basis for biosynthesis of <b>1</b> had been an enigma despite the availability of the <i>A. fumigatus</i> genome sequence. Here, we report the identification and verification of the <i>fma</i> gene cluster, followed by characterization of the polyketide synthase and acyltransferase involved in biosynthesis of the dioic acid portion of <b>1</b>. More significantly, we uncovered the elusive β-<i>trans</i>-bergamotene synthase in <i>A. fumigatus</i> as a membrane-bound terpene cyclase

Identification and Heterologous Production of a Benzoyl-Primed Tricarboxylic Acid Polyketide Intermediate from the Zaragozic Acid A Biosynthetic Pathway

Zaragozic acid A (<b>1</b>) is a potent cholesterol lowering, polyketide natural product made by various filamentous fungi. The reconstitution of enzymes responsible for the initial steps of the biosynthetic pathway of <b>1</b> is accomplished using an engineered fungal heterologous host. These initial steps feature the priming of a benzoic acid starter unit onto a highly reducing polyketide synthase (HRPKS), followed by oxaloacetate extension and product release to generate a tricarboxylic acid containing product <b>2</b>. The reconstitution studies demonstrated that only three enzymes, HRPKS, citrate synthase, and hydrolase, are needed in <i>A. nidulans</i> to produce the structurally complex product