4 research outputs found
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<sup>-1</sup>, while integration of only the codon-optimized FgTRI5 into the chromosome of yeast improved the production to 6,535 μg L<sup>-1</sup>. 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<sup>-1</sup> 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_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<sup>-1</sup>, while integration of only the codon-optimized FgTRI5 into the chromosome of yeast improved the production to 6,535 μg L<sup>-1</sup>. 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<sup>-1</sup> 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<sup>-1</sup>, while integration of only the codon-optimized FgTRI5 into the chromosome of yeast improved the production to 6,535 μg L<sup>-1</sup>. 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<sup>-1</sup> 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
Dynamic Nature of CTCF Tandem 11 Zinc Fingers in Multivalent Recognition of DNA As Revealed by NMR Spectroscopy
The 11 zinc fingers
(ZFs) of the transcription factor CTCF play
a versatile role in the regulation of gene expression. CTCF binds
to numerous genomic sites to form chromatin loops and topologically
associated domains and thus mediates the 3D architecture of chromatin.
Although CTCF inter-ZF plasticity is essential for the recognition
of multiple genomic sites, the dynamic nature of its 11 ZFs remains
unknown. We assigned the chemical shifts of the CTCF ZFs 1–11
and solved the solution structures of each ZF. NMR backbone dynamics,
residual dipolar couplings, and small-angle X-ray scattering experiments
suggest a high inter-ZF plasticity of the free-form ZFs 1–11.
As exemplified by two different protocadherin DNA sequences, the titration
of DNAs to <sup>15</sup>N-labeled CTCF ZFs 1–11 enabled systematic
mapping of binding of CTCF ZFs to various chromatin sites. Our work
paves the way for illustrating the molecular basis of the versatile
DNA recognized by CTCF and has interesting implications for its conformational
transition during DNA binding