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Metabolic engineering of Nicotiana benthamiana for the increased production of taxadiene

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Abstract

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We report the production of taxadiene by transformation of N. benthamiana with a taxadiene synthase gene. The production was significantly increased by an elicitor treatment or metabolic pathway shunting.

Abstract

Paclitaxel (Taxol®) was first isolated from the bark of the pacific yew tree as an anticancer agent and has been used extensively to treat various types of cancer. Taxadiene, the first committed product of paclitaxel synthesis is cyclized from geranylgeranyl diphosphate (GGPP), and further complex hydroxylation and acylation processes of the unique taxane core skeleton produce paclitaxel. To accomplish de novo production of taxadiene, we transformed Nicotiana benthamiana with a taxadiene synthase (TS) gene. The introduced TS gene under the transcriptional control of the CaMV 35S promoter was constitutively expressed in N. benthamiana, and the de novo production of taxadiene was confirmed by mass spectroscopy profiling. Transformed N. benthamiana homozygous lines produced 11–27 μg taxadiene/g of dry weight. The highest taxadiene production line TSS-8 was further treated with an elicitor, methyl jasmonate, and metabolic pathway shunting by suppression of the phytoene synthase gene expression which resulted in accumulation of increased taxadiene accumulation by 1.4- or 1.9-fold, respectively. In summary, we report that the production of taxadiene in N. benthamiana was possible by the ectopic expression of the TS gene, and higher accumulation of taxadiene could be achieved by elicitor treatment or metabolic pathway shunting of the terpenoid pathway.

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Abbreviations

TS :

Taxadiene synthase

GGPP:

Geranylgeranyl diphosphate

MJ:

Methyl jasmonate

PSY :

Phytoene synthase

PDS :

Phytoene desaturase

GFP :

Green fluorescent protein

NAA:

1-Naphthaleneacetic acid

BAP:

6-Benzylaminopurine

YEP:

Yeast extract peptone

MS:

Murashige and Skoog

RT-PCR:

Reverse transcription polymerase chain reaction

qPCR:

Quantitative polymerase chain reaction

VIGS:

Virus-induced gene silencing

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Acknowledgments

This work was supported by a grant from the Systems and Synthetic Agro-biotech Center through the Next-Generation BioGreen 21 Program (PJ00950603), Rural Development Administration, Korea. We appreciate Dr. Rodney Croteau for kindly providing the cloned taxadiene synthase gene.

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Authors and Affiliations

  1. School of Biotechnology, Yeungnam University, Gyeongbuk, Gyeongsan, 712-749, Korea

    Md. Mohidul Hasan, Sang Hee Shim & Kwang-Hyun Baek

  2. Plant System Engineering Research Center, KRIBB, 125 Gwahangno, Daejeon, Yuseong-gu, 305-806, Korea

    Hyun-Soon Kim & Jae-Heung Jeon

  3. Analysis Research Division, Daegu Center, Korea Basic Science Institute, Daegu, 702-701, Korea

    Sung Hong Kim

  4. Department of Food and Nutrition, Chung-Ang University, Gyeonggi-do, Anseongsi, 456-756, Korea

    BoKyung Moon

  5. Samyang Genexbio Corporation, 730 Daedeokdaero, Daejeon, Yuseong-gu, 305-717, Korea

    Jai-Young Song

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  1. Md. Mohidul Hasan
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  2. Hyun-Soon Kim
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Correspondence to Kwang-Hyun Baek.

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Communicated by M. Jordan.

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Hasan, M.M., Kim, HS., Jeon, JH. et al. Metabolic engineering of Nicotiana benthamiana for the increased production of taxadiene. Plant Cell Rep 33, 895–904 (2014). https://doi.org/10.1007/s00299-014-1568-9

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  • DOI: https://doi.org/10.1007/s00299-014-1568-9

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