Data_Sheet_1_Heterologous Biosynthesis of the Fungal Sesquiterpene Trichodermol in Saccharomyces cerevisiae.XLS

<p>Trichodermol, a fungal sesquiterpene derived from the farnesyl diphosphate pathway, is the biosynthetic precursor for trichodermin, a member of the trichothecene class of fungal toxins produced mainly by the genera of Trichoderma and Fusarium. Trichodermin is a promising candidate for the development of fungicides and antitumor agents due to its significant antifungal and cytotoxic effects. It can also serve as a scaffold to generate new congeners for structure-activity relationship (SAR) study. We reconstructed the biosynthetic pathway of trichodermol in Saccharomyces cerevisiae BY4741, and investigated the effect of produced trichodermol on the host by de novo RNA sequencing (RNA-Seq) and quantitative Real-time PCR analyses. Co-expression of pESC::FgTRI5 using plasmid pLLeu-tHMGR-UPC2.1 led to trichodiene production of 683 μg L^-1, while integration of only the codon-optimized FgTRI5 into the chromosome of yeast improved the production to 6,535 μg L^-1. Subsequent expression of the codon-optimized cytochrome P450 monooxygenase encoding genes, TaTRI4 and TaTRI11, resulted in trichodermol, with an estimated titer of 252 μg L^-1 at shake flask level. RNA-Seq and qPCR analyses revealed that the produced trichodermol downregulated the expression of the genes involved in ergosterol biosynthesis, but significantly upregulated the expression of PDR5 related to membrane transport pathway in S. cerevisiae. Collectively, we achieved the first heterologous biosynthesis of trichodermol by reconstructing its biosynthetic pathway in yeast, and the reconstructed pathway will serve as a platform to generate trichodermin analogs as potential candidates for agrochemicals and anticancer agents through further optimizations.</p

Data_Sheet_2_Heterologous Biosynthesis of the Fungal Sesquiterpene Trichodermol in Saccharomyces cerevisiae.XLS

<p>Trichodermol, a fungal sesquiterpene derived from the farnesyl diphosphate pathway, is the biosynthetic precursor for trichodermin, a member of the trichothecene class of fungal toxins produced mainly by the genera of Trichoderma and Fusarium. Trichodermin is a promising candidate for the development of fungicides and antitumor agents due to its significant antifungal and cytotoxic effects. It can also serve as a scaffold to generate new congeners for structure-activity relationship (SAR) study. We reconstructed the biosynthetic pathway of trichodermol in Saccharomyces cerevisiae BY4741, and investigated the effect of produced trichodermol on the host by de novo RNA sequencing (RNA-Seq) and quantitative Real-time PCR analyses. Co-expression of pESC::FgTRI5 using plasmid pLLeu-tHMGR-UPC2.1 led to trichodiene production of 683 μg L^-1, while integration of only the codon-optimized FgTRI5 into the chromosome of yeast improved the production to 6,535 μg L^-1. Subsequent expression of the codon-optimized cytochrome P450 monooxygenase encoding genes, TaTRI4 and TaTRI11, resulted in trichodermol, with an estimated titer of 252 μg L^-1 at shake flask level. RNA-Seq and qPCR analyses revealed that the produced trichodermol downregulated the expression of the genes involved in ergosterol biosynthesis, but significantly upregulated the expression of PDR5 related to membrane transport pathway in S. cerevisiae. Collectively, we achieved the first heterologous biosynthesis of trichodermol by reconstructing its biosynthetic pathway in yeast, and the reconstructed pathway will serve as a platform to generate trichodermin analogs as potential candidates for agrochemicals and anticancer agents through further optimizations.</p

Presentation_1_Heterologous Biosynthesis of the Fungal Sesquiterpene Trichodermol in Saccharomyces cerevisiae.pdf

<p>Trichodermol, a fungal sesquiterpene derived from the farnesyl diphosphate pathway, is the biosynthetic precursor for trichodermin, a member of the trichothecene class of fungal toxins produced mainly by the genera of Trichoderma and Fusarium. Trichodermin is a promising candidate for the development of fungicides and antitumor agents due to its significant antifungal and cytotoxic effects. It can also serve as a scaffold to generate new congeners for structure-activity relationship (SAR) study. We reconstructed the biosynthetic pathway of trichodermol in Saccharomyces cerevisiae BY4741, and investigated the effect of produced trichodermol on the host by de novo RNA sequencing (RNA-Seq) and quantitative Real-time PCR analyses. Co-expression of pESC::FgTRI5 using plasmid pLLeu-tHMGR-UPC2.1 led to trichodiene production of 683 μg L^-1, while integration of only the codon-optimized FgTRI5 into the chromosome of yeast improved the production to 6,535 μg L^-1. Subsequent expression of the codon-optimized cytochrome P450 monooxygenase encoding genes, TaTRI4 and TaTRI11, resulted in trichodermol, with an estimated titer of 252 μg L^-1 at shake flask level. RNA-Seq and qPCR analyses revealed that the produced trichodermol downregulated the expression of the genes involved in ergosterol biosynthesis, but significantly upregulated the expression of PDR5 related to membrane transport pathway in S. cerevisiae. Collectively, we achieved the first heterologous biosynthesis of trichodermol by reconstructing its biosynthetic pathway in yeast, and the reconstructed pathway will serve as a platform to generate trichodermin analogs as potential candidates for agrochemicals and anticancer agents through further optimizations.</p

Hypocriols A–F, Heterodimeric Botryane Ethers from <i>Hypocrea</i> sp., an Insect-Associated Fungus

The new heterodimeric botryane ethers hypocriols A–F (<b>1</b>–<b>6</b>) and the known compounds 4β-acetoxy-9β,10β,15α-trihydroxy­probotrydial (<b>7</b>), dihydrobotrydial (<b>8</b>), 10-oxodehydro­dihydrobotrydial (<b>9</b>), and dehydro­botrydienol (<b>10</b>) were isolated from the solid cultures of an insect-associated fungus <i>Hypocrea</i> sp. The structures of <b>1</b>–<b>6</b> were elucidated primarily by NMR experiments. The absolute configuration of <b>1</b> was assigned using the modified Mosher method and electronic circular dichroism (ECD) calculations, whereas those for <b>3</b>–<b>5</b>, and <b>2</b> and <b>6</b> were deduced via ECD calculations and circular dichroism data, respectively. Compounds <b>1</b>–<b>6</b> appear to be the first heterodimeric botryane ethers and showed antiproliferative effects against a small panel of four human tumor cell lines

Hawaiienols A–D, Highly Oxygenated <i>p</i>‑Terphenyls from an Insect-Associated Fungus, <i>Paraconiothyrium hawaiiense</i>

Four new highly oxygenated <i>p</i>-terphenyls, hawaiienols A–D (<b>1</b>–<b>4</b>), have been isolated from cultures of <i>Paraconiothyrium hawaiiense</i>, a fungus associated with the <i>Septobasidium</i>-infected insect <i>Diaspidiotus</i> sp.; their structures were elucidated primarily by NMR experiments. The absolute configurations of <b>1</b> and <b>2</b>–<b>4</b> were assigned by single-crystal X-ray diffraction analysis using Cu Kα radiation and via electronic circular dichroism calculations, respectively. Compound <b>1</b> incorporated the first naturally occurring 4,7-dioxatricyclo­[3.2.1.0^3,6]­octane unit in its <i>p</i>-terphenyl skeleton and showed cytotoxicity toward six human tumor cell lines