Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Lei Zhang, Ruxian Ding, Yourong Chai, Mercedes Bonfill, Elisabet Moyano, Kirsi-Marja Oksman-Caldentey, Tiefeng Xu, Yan Pi, Zinan Wang, Hanming Zhang, Guoyin Kai, Zhihua Liao, Xiaofen Sun, Kexuan Tang (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

204 Citations (Scopus)

Abstract

Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent. Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 β-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures. Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.05) levels of scopolamine compared with the wild-type and transgenic lines harboring a single gene (pmt or h6h). The best line (T3) produced 411 mg/liter scopolamine, which was over nine times more than that in the wild type (43 mg/liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H11 (184 mg/liter). To our knowledge, this is the highest scopolamine content achieved through genetic engineering of a plant. We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield, which was more efficient than plants harboring only one of the two genes. It seems that the pulling force of the downstream enzyme (the faucet enzyme) H6H plays a more important role in stimulating scopolamine accumulation in H. niger whereas the functioning of the upstream enzyme PMT is increased proportionally. This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.
Original languageEnglish
Pages (from-to)6786 - 6791
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number17
DOIs
Publication statusPublished - 2004
MoE publication typeA1 Journal article-refereed

Fingerprint

Hyoscyamus niger
scopolamine
biochemical pathways
engineering
putrescine N-methyltransferase
genetically modified organisms
tropane alkaloids
enzymes
atropine
gene overexpression
genes
bioreactors
genetic engineering
transgenic plants
biosynthesis

Keywords

  • Agrobacterium
  • hyoscyamine 6-beta-hydroxylase
  • putrescine
  • N-methyltransferse
  • scopolamine
  • transformation

Cite this

Zhang, Lei ; Ding, Ruxian ; Chai, Yourong ; Bonfill, Mercedes ; Moyano, Elisabet ; Oksman-Caldentey, Kirsi-Marja ; Xu, Tiefeng ; Pi, Yan ; Wang, Zinan ; Zhang, Hanming ; Kai, Guoyin ; Liao, Zhihua ; Sun, Xiaofen ; Tang, Kexuan. / Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures. In: Proceedings of the National Academy of Sciences of the United States of America. 2004 ; Vol. 101, No. 17. pp. 6786 - 6791.
@article{77f9fc6537264d3ab348825417e357a0,
title = "Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures",
abstract = "Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent. Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 β-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures. Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.05) levels of scopolamine compared with the wild-type and transgenic lines harboring a single gene (pmt or h6h). The best line (T3) produced 411 mg/liter scopolamine, which was over nine times more than that in the wild type (43 mg/liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H11 (184 mg/liter). To our knowledge, this is the highest scopolamine content achieved through genetic engineering of a plant. We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield, which was more efficient than plants harboring only one of the two genes. It seems that the pulling force of the downstream enzyme (the faucet enzyme) H6H plays a more important role in stimulating scopolamine accumulation in H. niger whereas the functioning of the upstream enzyme PMT is increased proportionally. This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.",
keywords = "Agrobacterium, hyoscyamine 6-beta-hydroxylase, putrescine, N-methyltransferse, scopolamine, transformation",
author = "Lei Zhang and Ruxian Ding and Yourong Chai and Mercedes Bonfill and Elisabet Moyano and Kirsi-Marja Oksman-Caldentey and Tiefeng Xu and Yan Pi and Zinan Wang and Hanming Zhang and Guoyin Kai and Zhihua Liao and Xiaofen Sun and Kexuan Tang",
year = "2004",
doi = "10.1073/pnas.0401391101",
language = "English",
volume = "101",
pages = "6786 -- 6791",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
publisher = "National Academy of Sciences",
number = "17",

}

Zhang, L, Ding, R, Chai, Y, Bonfill, M, Moyano, E, Oksman-Caldentey, K-M, Xu, T, Pi, Y, Wang, Z, Zhang, H, Kai, G, Liao, Z, Sun, X & Tang, K 2004, 'Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures', Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 17, pp. 6786 - 6791. https://doi.org/10.1073/pnas.0401391101

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures. / Zhang, Lei; Ding, Ruxian; Chai, Yourong; Bonfill, Mercedes; Moyano, Elisabet; Oksman-Caldentey, Kirsi-Marja; Xu, Tiefeng; Pi, Yan; Wang, Zinan; Zhang, Hanming; Kai, Guoyin; Liao, Zhihua; Sun, Xiaofen; Tang, Kexuan (Corresponding Author).

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 101, No. 17, 2004, p. 6786 - 6791.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

AU - Zhang, Lei

AU - Ding, Ruxian

AU - Chai, Yourong

AU - Bonfill, Mercedes

AU - Moyano, Elisabet

AU - Oksman-Caldentey, Kirsi-Marja

AU - Xu, Tiefeng

AU - Pi, Yan

AU - Wang, Zinan

AU - Zhang, Hanming

AU - Kai, Guoyin

AU - Liao, Zhihua

AU - Sun, Xiaofen

AU - Tang, Kexuan

PY - 2004

Y1 - 2004

N2 - Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent. Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 β-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures. Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.05) levels of scopolamine compared with the wild-type and transgenic lines harboring a single gene (pmt or h6h). The best line (T3) produced 411 mg/liter scopolamine, which was over nine times more than that in the wild type (43 mg/liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H11 (184 mg/liter). To our knowledge, this is the highest scopolamine content achieved through genetic engineering of a plant. We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield, which was more efficient than plants harboring only one of the two genes. It seems that the pulling force of the downstream enzyme (the faucet enzyme) H6H plays a more important role in stimulating scopolamine accumulation in H. niger whereas the functioning of the upstream enzyme PMT is increased proportionally. This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.

AB - Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent. Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 β-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures. Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.05) levels of scopolamine compared with the wild-type and transgenic lines harboring a single gene (pmt or h6h). The best line (T3) produced 411 mg/liter scopolamine, which was over nine times more than that in the wild type (43 mg/liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H11 (184 mg/liter). To our knowledge, this is the highest scopolamine content achieved through genetic engineering of a plant. We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield, which was more efficient than plants harboring only one of the two genes. It seems that the pulling force of the downstream enzyme (the faucet enzyme) H6H plays a more important role in stimulating scopolamine accumulation in H. niger whereas the functioning of the upstream enzyme PMT is increased proportionally. This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.

KW - Agrobacterium

KW - hyoscyamine 6-beta-hydroxylase

KW - putrescine

KW - N-methyltransferse

KW - scopolamine

KW - transformation

U2 - 10.1073/pnas.0401391101

DO - 10.1073/pnas.0401391101

M3 - Article

VL - 101

SP - 6786

EP - 6791

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

SN - 0027-8424

IS - 17

ER -