Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast
AbstractThe development of medical applications exploiting the broad bioactivities of the diterpene therapeutic triptolide from Tripterygium wilfordii is limited by low extraction yields from the native plant. Furthermore, the extraordinarily high structural complexity prevents an economically attractive enantioselective total synthesis. An alternative production route of triptolide through engineered Saccharomyces cerevisiae (yeast) could provide a sustainable source of triptolide. A potential intermediate in the unknown biosynthetic route to triptolide is the diterpene dehydroabietic acid. Here, we report a biosynthetic route to dehydroabietic acid by transient expression of enzymes from T. wilfordii and Sitka spruce (Picea sitchensis) in Nicotiana benthamiana. The combination of diterpene synthases TwTPS9, TwTPS27, and cytochromes P450 PsCYP720B4 yielded dehydroabietic acid and a novel analog, tentatively identified as ‘miltiradienic acid’. This biosynthetic pathway was reassembled in a yeast strain engineered for increased yields of the pathway intermediates, the diterpene olefins miltiradiene and dehydroabietadiene. Introduction in that strain of PsCYP720B4 in combination with two alternative NADPH-dependent cytochrome P450 reductases resulted in scalable in vivo production of dehydroabietic acid and its analog from glucose. Approaching future elucidation of the remaining biosynthetic steps to triptolide, our findings may provide an independent platform for testing of additional recombinant candidate genes, and ultimately pave the way to biotechnological production of the high value diterpenoid therapeutic. View Full-Text
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Description: Supplementary Materials: The following are available online at www.mdpi.com/link, Table S1: Tobacco plant combinations, Table S2: Yeast expression vectors, Figure S1: MS Spectra of yeast produced products. Figure S2: Diterpene production levels, Table S3: Yeast codon optimized sequences.
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Forman, V.; Callari, R.; Folly, C.; Heider, H.; Hamberger, B. Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast. Molecules 2017, 22, 981.
Forman V, Callari R, Folly C, Heider H, Hamberger B. Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast. Molecules. 2017; 22(6):981.Chicago/Turabian Style
Forman, Victor; Callari, Roberta; Folly, Christophe; Heider, Harald; Hamberger, Björn. 2017. "Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast." Molecules 22, no. 6: 981.