Genetic Manipulation of the Pneumocandin Biosynthetic Pathway for Generation of Analogues and Evaluation of Their Antifungal Activity

Pneumocandins are lipohexapeptides of the echinocandin family that potently interrupt fungal cell wall biogenesis by noncompetitive inhibition of 1,3-β-glucan synthase. The pneumocandin biosynthetic gene cluster was previously elucidated by whole genome sequencing. In addition to the core nonribosomal peptide synthetase and polyketide synthase (<i>GLNRPS4</i> and <i>GLPKS4</i>), the pneumocandin biosynthetic cluster includes two P450-type hemeprotein monooxygenase genes (<i>GLP450-1</i> and <i>GLP450-2</i>) and four nonheme mononuclear iron oxygenase genes (<i>GLOXY1</i>, <i>GLOXY2</i>, <i>GLOXY3</i>, and <i>GLOXY4</i>), which function to biosynthesize and create the unusual sequence of hydroxylated amino acids of the mature pneumocandin peptide. Insertional inactivation of three of these genes (<i>GLP450-1</i>, <i>GLP450-2</i>, and <i>GLOXY1</i>) generated 13 different pneumocandin analogues that lack one, two, three, or four hydroxyl groups on 4<i>R</i>,5<i>R</i>-dihydroxy-ornithine and 3<i>S</i>,4<i>S</i>-dihydroxy-homotyrosine of the parent hexapeptide. Among them, seven analogues are previously unreported genetically engineered pneumocandins whose structures were established by NMR experiments. These new pneumocandins afforded a unique opportunity for side-by-side exploration of the effects of hydroxylation on pneumocandin antifungal activity. All of these cyclic lipopeptides showed potent antifungal activities, and two new metabolites pneumocandins F (<b>3</b>) and G (<b>4</b>) were more potent <i>in vitro</i> against <i>Candida</i> species and <i>Aspergillus fumigatus</i> than the principal fermentation products, pneumocandins A₀ and B<sub>0.</sub

Emestrins: Anti-<i>Cryptococcus</i> Epipolythiodioxopiperazines from <i>Podospora australis</i>

Eleven emestrin-type epipolythiodioxo­piperazines, including four new compounds, emestrins H–K (<b>1</b>–<b>4</b>), were isolated from the crude extracts of two strains of the coprophilous fungus <i>Podospora australis.</i> The structures of <b>1</b>–<b>4</b> were established primarily by analysis of NMR data, and the absolute configuration of C-6 in <b>1</b> was independently assigned using the modified Mosher method. Four of the known emestrins obtained (emestrins C–E and MPC1001C) were found to selectively inhibit the growth of <i>Cryptococcus neoformans</i>. These results also represent the first report of chemistry from any strain of <i>P. australis</i>

Insect_Bayes_OutputTree.con

BI tree of ghost moths based on 2 loci (COI and wg

Fungus_Alignment_7loci_125individual_4326nt

Alignmnet file of 125 Ophiocordyceps sinensis individuals on seven nuclear loci: nrDNA ITS, MAT1-2-1, csp1, OSRC14, OSRC17, OSRC27, and OSRC32. See ReadMe file for dataset partition

Fungus_Garli_OutputTree

ML tree of Ophiocordyceps sinensis based on 6 loci (nrDNA ITS, MAT1-2-1, csp1, OSRC14, OSRC17, and OSRC27

raw data of infection trial

raw data of infection tria

Insect_Garli_OutputTree

ML tree of ghost moths based on 2 loci (COI and wg

Anti-<i>Cryptococcus</i> Phenalenones and Cyclic Tetrapeptides from <i>Auxarthron pseudauxarthron</i>

Auxarthrones A–E (<b>1</b>–<b>5</b>), five new phenalenones, and two new naturally occurring cyclic tetrapeptides, auxarthrides A (<b>7</b>) and B (<b>8</b>), were obtained from three different solvent extracts of cultures of the coprophilous fungus <i>Auxarthron pseudauxarthron</i>. Auxarthrones C (<b>3</b>) and E (<b>5</b>) possess an unusual 7a,8-dihydrocyclopenta­[<i>a</i>]­phenalene-7,9-dione ring system that has not been previously observed in natural products. Formation of <b>1</b>–<b>5</b> was found to be dependent on the solvent used for culture extraction. The structures of these new compounds were elucidated primarily by analysis of NMR and MS data. Auxarthrone A (<b>1</b>) was obtained as a mixture of chromatographically inseparable racemic diastereomers (<b>1a</b> and <b>1b</b>) that cocrystallized, enabling confirmation of their structures by X-ray crystallography. The absolute configurations of <b>7</b> and <b>8</b> were assigned by analysis of their acid hydrolysates using Marfey’s method. Compound <b>1</b> displayed moderate antifungal activity against <i>Cryptococcus neoformans</i> and <i>Candida albicans</i>, but did not affect human cancer cell lines

Anti-<i>Cryptococcus</i> Phenalenones and Cyclic Tetrapeptides from <i>Auxarthron pseudauxarthron</i>

Auxarthrones A–E (<b>1</b>–<b>5</b>), five new phenalenones, and two new naturally occurring cyclic tetrapeptides, auxarthrides A (<b>7</b>) and B (<b>8</b>), were obtained from three different solvent extracts of cultures of the coprophilous fungus <i>Auxarthron pseudauxarthron</i>. Auxarthrones C (<b>3</b>) and E (<b>5</b>) possess an unusual 7a,8-dihydrocyclopenta­[<i>a</i>]­phenalene-7,9-dione ring system that has not been previously observed in natural products. Formation of <b>1</b>–<b>5</b> was found to be dependent on the solvent used for culture extraction. The structures of these new compounds were elucidated primarily by analysis of NMR and MS data. Auxarthrone A (<b>1</b>) was obtained as a mixture of chromatographically inseparable racemic diastereomers (<b>1a</b> and <b>1b</b>) that cocrystallized, enabling confirmation of their structures by X-ray crystallography. The absolute configurations of <b>7</b> and <b>8</b> were assigned by analysis of their acid hydrolysates using Marfey’s method. Compound <b>1</b> displayed moderate antifungal activity against <i>Cryptococcus neoformans</i> and <i>Candida albicans</i>, but did not affect human cancer cell lines

Insect_Alignment_3loci_125individual_1404nt

Alignment of 125 ghost moth individuals on three loci:COI, cytb, and wg. See ReadMe file for dataset partition