Biosynthesis of Shearinine: Diversification of a Tandem Prenyl Moiety of Fungal Indole Diterpenes

The late-stage biosynthetic pathway of the indole diterpene shearinine involving four enzymatic reactions (JanQDOJ) was elucidated by an efficient heterologous expression system using <i>Aspergillus oryzae</i>. Key oxidative cyclization, forming a characteristic A/B bicyclic shearinine core by flavoprotein oxidase, was studied using a substrate analogue and a buffer containing H₂¹⁸O. These experimental data provided evidence that JanO catalyzes two-step oxidation via a hydroxylated product and that the JanO reaction involves the hydride-transfer mechanism

The B10 SSR from <i>Epichloë uncinata</i> strains.

<p>(a) Sequences of the small B10 repeats contained within each <i>E</i>. <i>uncinata</i> strain. Strains are grouped and aligned for the different ecotypes. (b) Sequences of the large B10 repeats within <i>E</i>. <i>uncinata</i> strains. (c) B10 repeats of the proposed ancestors of <i>E</i>. <i>uncinata</i>, <i>Epichloë typhina</i> and <i>Epichloë bromicola</i>. (d) Schematic representation of the B10 repeat structures from <i>E</i>. <i>uncinata</i> and its proposed ancestors. A single B10 allele is found within <i>E</i>. <i>typhina</i> and <i>E</i>. <i>bromicola</i>, while two alleles are contained within <i>E</i>. <i>uncinata</i>. Repeat structure of <i>E</i>. <i>typhina</i> and <i>E</i>. <i>bromicola</i> are similar to that of <i>E</i>. <i>uncinata</i>. Repeat structure is colored to represent repeated units CAG (yellow), CAT (violet) and CAA (pink).</p

SSR alleles of <i>Epichloë uncinata and proposed ancestors</i>.

<p>SSR alleles of <i>Epichloë uncinata and proposed ancestors</i>.</p

Maximum-likelihood phylogenetic tree of <i>Epichloë</i> spp. B10 alleles.

<p>The appropriate likelihood ratio test support values are indicated over the branches.</p

Amino acid sequence alignment of putative bZIP transcription factors.

<p>CLUSTALW alignment of amino acid sequences showing the B10 SSR region (green box). The SSR is seen as repeated glutamine (Q) and histidine (H) residues. Gene IDs with associated GenBank protein accession numbers or gene models of protein homologues are: <i>Epichloë bromicola</i> EfP3.072790; <i>Epichloë typhina</i> EfP3.072790; <i>Epichloë festucae</i> EfM3.072790; <i>Epichloë brachyelytri</i> EfP3.072790; <i>Epichloë amarillans</i> EfP3.072790; <i>Epichloë glyceriae</i> EfP3.072790; <i>Claviceps paspali</i> EfP3.072790; <i>Claviceps fusiformis</i> EfP3.072790; <i>Periglandula ipomoeae</i> EfP3.072790; <i>Magnaporthe oryzae</i> MGG_08118.6 (XP_003715075); <i>Neurospora crassa</i> NCU03847.7 (XP_955781); <i>Fusarium oxysporum</i> FOXB_15670.1 (EGU73820).</p

Amino acid sequence alignment of putative G-protein coupled receptors.

<p>CLUSTALW alignment of amino acid sequences showing the B13 SSR region (green box). The SSR is seen as repeated glutamine (Q) and histidine (H) residues. Gene IDs with associated GenBank protein accession numbers or gene models of protein homologues are: <i>Epichloë festucae</i> EfM3.080640; <i>Epichloë typhina</i> EfP3.080640; <i>Epichloë amarillans</i> EfP3.080640; <i>Epichloë brachyelytri</i> EfP3.080640; <i>Epichloë glyceriae</i> EfP3.080640; <i>Periglandula ipomoeae</i> EfP3.080640; <i>Aciculosporium take</i> EfP3.080640; <i>Claviceps fusiformis</i> EfP3.080640; <i>Claviceps paspali</i> EfP3.080640; <i>Fusarium graminearum</i> FGSG_05239.3 (XP_011323750).</p

Consequences of B10 repeat changes on peptide sequence.

<p>Comparison of the B10 repeats of <i>Epichloë glyceriae</i> and <i>Epichloë amarillans</i> and the amino acid each tri-nucleotide encodes. Repeats are colored CAG (yellow), CAT (violet) and CAA (pink). Amino acids encoded are colored for the repeat sequence from which they are derived.</p

B10-like repeats in Sordariomycetes fungi.

<p>B10-like repeats in Sordariomycetes fungi.</p

Overview of B10 SSR.

<p>(a) Genomic region containing the B10 SSR in <i>Epichloë festucae</i> Fl1. The position of primers B10.1 and B10.2 used to amplify the B10 SSR (green) are shown in grey. The SSR is within an exon of a putative bZIP transcription factor (EfM3.072790) (red). (b) Repeat sequence of (CAG)_n(CAT)_n(CAA)_n between <i>Epichloë</i> strains is colored by tri-nucleotide repeats, CAG (yellow), CAT (violet), CAA (pink). Single nucleotide polymorphisms resulting in changes in tri-nucleotide repeats (CCG and CCA) are not colored. (c) Consensus sequences for each <i>Epichloë</i> strain identifying variety in repeat units.</p

Heterologous Biosynthesis of Nodulisporic Acid F

Nodulisporic acids comprise a group of valuable indole diterpenes that exhibit potent insecticidal activities. We report the identification of a gene cluster in the genome of the filamentous fungus <i>Hypoxylon pulicicidum</i> (<i>Nodulisporium</i> sp.) that contains genes responsible for the biosynthesis of nodulisporic acids. Using <i>Penicillium paxilli</i> as a heterologous host, and through pathway reconstitution experiments, we identified the function of four genes involved in the biosynthesis of the nodulisporic acid core compound, nodulisporic acid F (NAF). Two of these genes (<i>nodM</i> and <i>nodW</i>) are especially significant as they encode enzymes with previously unreported functionality: <i>nodM</i> encodes a 3-geranylgeranylindole epoxidase capable of catalyzing only a single epoxidation step to prime formation of the distinctive ring structure of nodulisporic acids, and <i>nodW</i> encodes the first reported gene product capable of introducing a carboxylic acid moiety to an indole diterpene core structure that acts as a reactive handle for further modification. Here, we present the enzymatic basis for the biosynthetic branch point that gives rise to nodulisporic acids