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Agrobacterial Transformation of Tobacco with a Genetic Module of the Biosynthesis of the Antimalarial Agent Artemisinin

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Abstract

Artemisinin-based medicines are the most effective treatment for malaria. To date, wormwood plants (Artemisia annua L.) are the main source of artemisinin. Due to the limited nature of this source, considerable efforts are directed towards the development of methods of artemisinin production via heterologous expression systems. The goal of the study was to obtain tobacco transformed with genes of the artemisinin biosynthesis pathway and to analyze their transcription in a heterologous host. Tobacco plants were transformed with the artemisinin biosynthesis genes encoding amorpha-4,11-diene synthase, artemisinic aldehyde Δ11(13) reductase, amorpha-4,11-diene monooxygenase, and cytochrome P450 reductase from A. annua and yeast 3-hydroxy-3-methylglutaryl-coenzyme A reductase cloned in the pArtemC vector; farnesyl diphosphate synthase and aldehyde dehydrogenase were used to transform the plants as parts of vector p2356. Two transgenic lines with all target genes were obtained as a result of transformation with the pArtemC and p2356 vectors. After double transformation with these vectors, six transgenic lines were obtained. Five genes of artemisinin biosynthesis and two genes of biosynthesis of its precursors were successfully transferred into the genome of transgenic tobacco lines as a result of the cotransformation with the abovementioned vectors. Thus, the entire artemisinin biosynthesis pathway was first reconstructed in heterologous plants: the transcription of the artemisinin biosynthesis genes in the tobacco plants was shown via RT-PCR. The results will be used in further research on expression systems for the production of artemisinin and other nonprotein substances in heterologous host plants.

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ACKNOWLEDGMENTS

The work was carried out using at unique scientific facility Fitotron, reg. no. 2-2.9.

Funding

The work was supported by the Russian Science Foundation, grant no. 19-14-00190.

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Authors and Affiliations

  1. Shemyakin—Ovchinnikov Institute of Bioorganic Chemistry, Pushchino Branch, Russian Academy of Sciences, 142290, Pushchino, Moscow oblast, Russia

    A. P. Firsov, T. Yu. Mitiushkina, A. S. Pushin & S. V. Dolgov

  2. Faculty of Biotechnology, Lomonosov Moscow State University, 119991, Moscow, Russia

    A. Tzareva

  3. Hebrew University of Jerusalem, 76100, Rehovot, Israel

    A. M. Vainstein

Authors
  1. A. P. Firsov
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  2. T. Yu. Mitiushkina
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  3. A. S. Pushin
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  4. A. Tzareva
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  5. A. M. Vainstein
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  6. S. V. Dolgov
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Correspondence to A. P. Firsov.

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This article does not contain any studies involving animals performed by any of the authors.

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Translated by I. Gordon

Abbreviations: AD—amorpha-4,11-diene; ADS—amorpha-4,11-diene synthase; ALDH1—aldehyde dehydrogenase; BAP—6‑benzylaminopurine; CaMV—cauliflower mosaic virus; CPR—cytochrome P450 reductase; CYP71AV1—amorpha-4,11-diene monooxygenase; DBR—2Δ11(13) reductase; FPP—farnesyl diphosphate; FDPS—farnesyl diphosphate synthase; IAA—Δ-indolylacetic acid; IPP—isopentyl diphosphate; MS medium—Murashige–Skoog medium; MVA—mevalonate; OD600—optical density at a wavelength of 600 nm; Rubisco-ribulose-1,5-bisphosphate carboxylase/oxygenase; RT-PCR—polymerase chain reaction with reverse transcription; tHMGR—3-hydroxy-3‑methylglutaryl coenzyme A reductase.

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Firsov, A.P., Mitiushkina, T.Y., Pushin, A.S. et al. Agrobacterial Transformation of Tobacco with a Genetic Module of the Biosynthesis of the Antimalarial Agent Artemisinin. Appl Biochem Microbiol 57, 808–817 (2021). https://doi.org/10.1134/S0003683821070024

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