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Title: Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast

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 pavemore » the way to biotechnological production of the high value diterpenoid therapeutic.« less
Authors:
ORCiD logo [1] ;  [2] ;  [2] ;  [2] ;  [3]
  1. Evolva A/S, Copenhagen (Denmark); Evolva SA, Reinach (Switzerland)
  2. Evolva SA, Reinach (Switzerland)
  3. Michigan State Univ., East Lansing, MI (United States). Department of Biochemistry and Molecular Biology
Publication Date:
Grant/Contract Number:
FC02-07ER64494
Type:
Accepted Manuscript
Journal Name:
Molecules
Additional Journal Information:
Journal Volume: 22; Journal Issue: 6; Journal ID: ISSN 1420-3049
Publisher:
MDPI
Research Org:
Univ. of Wisconsin, Madison, WI (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; triptolide; dehydroabietic acid; miltiradienic acid; miltiradiene; dehydroabietadiene; medicinal diterpenes; Saccharomyces cerevisiae; Nicotiana benthamiana; Tripterygium wilfordii
OSTI Identifier:
1427579

Forman, Victor, Callari, Roberta, Folly, Christophe, Heider, Harald, and Hamberger, Bjorn. Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast. United States: N. p., Web. doi:10.3390/molecules22060981.
Forman, Victor, Callari, Roberta, Folly, Christophe, Heider, Harald, & Hamberger, Bjorn. Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast. United States. doi:10.3390/molecules22060981.
Forman, Victor, Callari, Roberta, Folly, Christophe, Heider, Harald, and Hamberger, Bjorn. 2017. "Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast". United States. doi:10.3390/molecules22060981. https://www.osti.gov/servlets/purl/1427579.
@article{osti_1427579,
title = {Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast},
author = {Forman, Victor and Callari, Roberta and Folly, Christophe and Heider, Harald and Hamberger, Bjorn},
abstractNote = {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.},
doi = {10.3390/molecules22060981},
journal = {Molecules},
number = 6,
volume = 22,
place = {United States},
year = {2017},
month = {6}
}