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Molecules 2017, 22(6), 981; doi:10.3390/molecules22060981

Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast

1
Evolva A/S, Lersø Park Allé 42-44, Copenhagen Ø DK-2100, Denmark
2
Evolva SA, Duggingerstrasse 23, Reinach CH-4153, Switzerland
3
Department of Biochemistry and Molecular Biology, Michigan State University, 603 Wilson Road, East Lansing, MI 48824, USA
*
Author to whom correspondence should be addressed.
Academic Editor: Derek J. McPhee
Received: 7 April 2017 / Accepted: 7 June 2017 / Published: 13 June 2017
(This article belongs to the Special Issue Isoprenoid Biosynthesis)
View Full-Text   |   Download PDF [1041 KB, uploaded 13 June 2017]   |  

Abstract

The 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
Keywords: triptolide; dehydroabietic acid; miltiradienic acid; miltiradiene; dehydroabietadiene; medicinal diterpenes; Saccharomyces cerevisiae; Nicotiana benthamiana; Tripterygium wilfordii triptolide; dehydroabietic acid; miltiradienic acid; miltiradiene; dehydroabietadiene; medicinal diterpenes; Saccharomyces cerevisiae; Nicotiana benthamiana; Tripterygium wilfordii
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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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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MDPI and ACS Style

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.

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