Abstract
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.
Highlights
Terpenoids are the largest group of natural products mostly isolated from the plants (Sun et al, 2006; Fraga, 2011), but are frequently encountered as fungal secondary metabolites (Geris and Simpson, 2009)
Evaluation of in vitro enzymatic activity of the His6-tagged FgTRI5 synthase from E. coli BL21 (DE3) verified that the it catalyzed the conversion from farnesyl diphosphate (FPP) to trichodiene (Methods; Supplementary Figure 1)
A titer of 683 μg L−1 was determined for trichodiene synthesized in TD2 based on the results from GC analysis of the organic phase
Summary
Terpenoids are the largest group of natural products mostly isolated from the plants (Sun et al, 2006; Fraga, 2011), but are frequently encountered as fungal secondary metabolites (Geris and Simpson, 2009). They have been widely used in pharmaceuticals, food additives, and fragrance due to highly diverse structures, physical properties, and biological functions. Multicopy integration targeting repetitive chromosomal DNA sequences, and long terminal repeats of Ty element (δDNA sequence) to stabilize gene expression and to achieve high average copies (Lee and Da Silva, 1997; Tokuhiro et al, 2009; Tyo et al, 2009) were employed
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