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Engineering a mevalonate pathway in Escherichia coli for production of terpenoids

Abstract

Isoprenoids are the most numerous and structurally diverse family of natural products. Terpenoids, a class of isoprenoids often isolated from plants, are used as commercial flavor and fragrance compounds and antimalarial or anticancer drugs. Because plant tissue extractions typically yield low terpenoid concentrations, we sought an alternative method to produce high-value terpenoid compounds, such as the antimalarial drug artemisinin, in a microbial host. We engineered the expression of a synthetic amorpha-4,11-diene synthase gene and the mevalonate isoprenoid pathway from Saccharomyces cerevisiae in Escherichia coli. Concentrations of amorphadiene, the sesquiterpene olefin precursor to artemisinin, reached 24 μg caryophyllene equivalent/ml. Because isopentenyl and dimethylallyl pyrophosphates are the universal precursors to all isoprenoids, the strains developed in this study can serve as platform hosts for the production of any terpenoid compound for which a terpene synthase gene is available.

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Figure 1: Production of amorphadiene via the DXP or mevalonate isoprenoid pathways and depiction of the synthetic operons used in this study.
Figure 2: Comparison of the production of amorphadiene in LB medium.
Figure 3: Growth curves of E. coli showing the inhibition effect caused by increasing concentrations of DL-mevalonate in the LB medium.
Figure 4: Prenyl pyrophosphate accumulation in resting cells harboring various mevalonate operons.
Figure 5: Effect of amorphadiene synthase (ADS) expression on the growth of E. coli harboring pMBIS.
Figure 6: Comparison of amorphadiene production between E. coli expressing the native DXP pathway and the engineered isoprenoid pathways.

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Acknowledgements

The authors would like to acknowledge Seon-Won Kim, Stephen del Cardayré, Ranjini Chaterjee and David Williams for their contributions and Ahamindra Jain for the synthesis of 2-C-methyl-D-erythritol. This work was supported by research grants from the US National Science Foundation (grant number BES-9911463), University of California BioSTAR (grant number 99-10044), Maxygen and the Office of Naval Research (grant number FDN00014-99-0182). D.J.P. is the recipient of a National Science Foundation graduate fellowship.

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Correspondence to Jay D Keasling.

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Martin, V., Pitera, D., Withers, S. et al. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 21, 796–802 (2003). https://doi.org/10.1038/nbt833

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