Artemisia annua mutant impaired in artemisinin synthesis demonstrates importance of nonenzymatic conversion in terpenoid metabolism

TY - JOUR

T1 - Artemisia annua mutant impaired in artemisinin synthesis demonstrates importance of nonenzymatic conversion in terpenoid metabolism

AU - Czechowski, Tomasz

AU - Larson, TR

AU - Catania, Theresa May

AU - Harvey, David

AU - Brown, Geoffrey D

AU - Graham, Ian Alexander

N1 - This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details

PY - 2016/12/27

Y1 - 2016/12/27

N2 - Artemisinin, a sesquiterpene lactone produced by Artemisia annua glandular secretory trichomes, is the active ingredient in the most effective treatment for malaria currently available. We identified a mutation that disrupts the amorpha-4,11-diene C-12 oxidase (CYP71AV1) enzyme, responsible for a series of oxidation reactions in the artemisinin biosynthetic pathway. Detailed metabolic studies of cyp71av1-1 revealed that the consequence of blocking the artemisinin biosynthetic pathway is the redirection of sesquiterpene metabolism to a sesquiterpene epoxide, which we designate arteannuin X. This sesquiterpene approaches half the concentration observed for artemisinin in wild-type plants, demonstrating high-flux plasticity in A. annua glandular trichomes and their potential as factories for the production of novel alternate sesquiterpenes at commercially viable levels. Detailed metabolite profiling of leaf maturation time-series and precursor-feeding experiments revealed that nonenzymatic conversion steps are central to both artemisinin and arteannuin X biosynthesis. In particular, feeding studies using 13C-labeled dihydroartemisinic acid (DHAA) provided strong evidence that the final steps in the synthesis of artemisinin are nonenzymatic in vivo. Our findings also suggest that the specialized subapical cavity of glandular secretory trichomes functions as a location for both the chemical conversion and the storage of phytotoxic compounds, including artemisinin. We conclude that metabolic engineering to produce high yields of novel secondary compounds such as sesquiterpenes is feasible in complex glandular trichomes. Such systems offer advantages over single-cell microbial hosts for production of toxic natural products.

AB - Artemisinin, a sesquiterpene lactone produced by Artemisia annua glandular secretory trichomes, is the active ingredient in the most effective treatment for malaria currently available. We identified a mutation that disrupts the amorpha-4,11-diene C-12 oxidase (CYP71AV1) enzyme, responsible for a series of oxidation reactions in the artemisinin biosynthetic pathway. Detailed metabolic studies of cyp71av1-1 revealed that the consequence of blocking the artemisinin biosynthetic pathway is the redirection of sesquiterpene metabolism to a sesquiterpene epoxide, which we designate arteannuin X. This sesquiterpene approaches half the concentration observed for artemisinin in wild-type plants, demonstrating high-flux plasticity in A. annua glandular trichomes and their potential as factories for the production of novel alternate sesquiterpenes at commercially viable levels. Detailed metabolite profiling of leaf maturation time-series and precursor-feeding experiments revealed that nonenzymatic conversion steps are central to both artemisinin and arteannuin X biosynthesis. In particular, feeding studies using 13C-labeled dihydroartemisinic acid (DHAA) provided strong evidence that the final steps in the synthesis of artemisinin are nonenzymatic in vivo. Our findings also suggest that the specialized subapical cavity of glandular secretory trichomes functions as a location for both the chemical conversion and the storage of phytotoxic compounds, including artemisinin. We conclude that metabolic engineering to produce high yields of novel secondary compounds such as sesquiterpenes is feasible in complex glandular trichomes. Such systems offer advantages over single-cell microbial hosts for production of toxic natural products.

U2 - 10.1073/pnas.1611567113

DO - 10.1073/pnas.1611567113

M3 - Article

SN - 0027-8424

VL - 113

SP - 15150

EP - 15155

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 52

ER -