Insights into heterologous biosynthesis of Arteannuin B and artemisinin in physcomitrella patens

Nur Kusaira Khairul Ikram, Arman Beyraghdar Kashkooli, Anantha Peramuna, Alexander R. Van Der Krol, Harro Bouwmeester, Henrik Toft Simonsen*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Metabolic engineering is an integrated bioengineering approach, which has made considerable progress in producing terpenoids in plants and fermentable hosts. Here, the full biosynthetic pathway of artemisinin, originating from Artemisia annua, was integrated into the moss Physcomitrella patens. Different combinations of the five artemisinin biosynthesis genes were ectopically expressed in P. patens to study biosynthesis pathway activity, but also to ensure survival of successful transformants. Transformation of the first pathway gene, ADS, into P. patens resulted in the accumulation of the expected metabolite, amorpha-4,11-diene, and also accumulation of a second product, arteannuin B. This demonstrates the presence of endogenous promiscuous enzyme activity, possibly cytochrome P450s, in P. patens. Introduction of three pathway genes, ADSCYP71AV1- ADH1 or ADS-DBR2-ALDH1 both led to the accumulation of artemisinin, hinting at the presence of one or more endogenous enzymes in P. patens that can complement the partial pathways to full pathway activity. Transgenic P. patens lines containing the different gene combinations produce artemisinin in varying amounts. The pathway gene expression in the transgenic moss lines correlates well with the chemical profile of pathway products. Moreover, expression of the pathway genes resulted in lipid body formation in all transgenic moss lines, suggesting that these may have a function in sequestration of heterologous metabolites. This work thus provides novel insights into the metabolic response of P. patens and its complementation potential for A. annua artemisinin pathway genes. Identification of the related endogenous P. patens genes could contribute to a further successful metabolic engineering of artemisinin biosynthesis, as well as bioengineering of other high-value terpenoids in P. patens.

Original languageEnglish
Article number3822
JournalMolecules
Volume24
Issue number21
DOIs
Publication statusPublished - 23 Oct 2019

Fingerprint

Bryopsida
biosynthesis
Biosynthesis
genes
Genes
Bryophyta
Bryophytes
Artemisia annua
Metabolic Engineering
Bioengineering
bioengineering
Metabolic engineering
Terpenes
metabolites
Metabolites
Gene Expression
engineering
Biosynthetic Pathways
Enzymes
enzyme activity

Keywords

  • Artemisinin
  • Biotechnology
  • Malaria
  • Physcomitrella patens
  • Sesquiterpenoids

Cite this

Ikram, N. K. K., Kashkooli, A. B., Peramuna, A., Van Der Krol, A. R., Bouwmeester, H., & Simonsen, H. T. (2019). Insights into heterologous biosynthesis of Arteannuin B and artemisinin in physcomitrella patens. Molecules, 24(21), [3822]. https://doi.org/10.3390/molecules24213822
Ikram, Nur Kusaira Khairul ; Kashkooli, Arman Beyraghdar ; Peramuna, Anantha ; Van Der Krol, Alexander R. ; Bouwmeester, Harro ; Simonsen, Henrik Toft. / Insights into heterologous biosynthesis of Arteannuin B and artemisinin in physcomitrella patens. In: Molecules. 2019 ; Vol. 24, No. 21.
@article{8220e363ab864dcaacb050dde249fc2f,
title = "Insights into heterologous biosynthesis of Arteannuin B and artemisinin in physcomitrella patens",
abstract = "Metabolic engineering is an integrated bioengineering approach, which has made considerable progress in producing terpenoids in plants and fermentable hosts. Here, the full biosynthetic pathway of artemisinin, originating from Artemisia annua, was integrated into the moss Physcomitrella patens. Different combinations of the five artemisinin biosynthesis genes were ectopically expressed in P. patens to study biosynthesis pathway activity, but also to ensure survival of successful transformants. Transformation of the first pathway gene, ADS, into P. patens resulted in the accumulation of the expected metabolite, amorpha-4,11-diene, and also accumulation of a second product, arteannuin B. This demonstrates the presence of endogenous promiscuous enzyme activity, possibly cytochrome P450s, in P. patens. Introduction of three pathway genes, ADSCYP71AV1- ADH1 or ADS-DBR2-ALDH1 both led to the accumulation of artemisinin, hinting at the presence of one or more endogenous enzymes in P. patens that can complement the partial pathways to full pathway activity. Transgenic P. patens lines containing the different gene combinations produce artemisinin in varying amounts. The pathway gene expression in the transgenic moss lines correlates well with the chemical profile of pathway products. Moreover, expression of the pathway genes resulted in lipid body formation in all transgenic moss lines, suggesting that these may have a function in sequestration of heterologous metabolites. This work thus provides novel insights into the metabolic response of P. patens and its complementation potential for A. annua artemisinin pathway genes. Identification of the related endogenous P. patens genes could contribute to a further successful metabolic engineering of artemisinin biosynthesis, as well as bioengineering of other high-value terpenoids in P. patens.",
keywords = "Artemisinin, Biotechnology, Malaria, Physcomitrella patens, Sesquiterpenoids",
author = "Ikram, {Nur Kusaira Khairul} and Kashkooli, {Arman Beyraghdar} and Anantha Peramuna and {Van Der Krol}, {Alexander R.} and Harro Bouwmeester and Simonsen, {Henrik Toft}",
year = "2019",
month = "10",
day = "23",
doi = "10.3390/molecules24213822",
language = "English",
volume = "24",
journal = "Molecules",
issn = "1420-3049",
publisher = "MDPI",
number = "21",

}

Ikram, NKK, Kashkooli, AB, Peramuna, A, Van Der Krol, AR, Bouwmeester, H & Simonsen, HT 2019, 'Insights into heterologous biosynthesis of Arteannuin B and artemisinin in physcomitrella patens', Molecules, vol. 24, no. 21, 3822. https://doi.org/10.3390/molecules24213822

Insights into heterologous biosynthesis of Arteannuin B and artemisinin in physcomitrella patens. / Ikram, Nur Kusaira Khairul; Kashkooli, Arman Beyraghdar; Peramuna, Anantha; Van Der Krol, Alexander R.; Bouwmeester, Harro; Simonsen, Henrik Toft.

In: Molecules, Vol. 24, No. 21, 3822, 23.10.2019.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Insights into heterologous biosynthesis of Arteannuin B and artemisinin in physcomitrella patens

AU - Ikram, Nur Kusaira Khairul

AU - Kashkooli, Arman Beyraghdar

AU - Peramuna, Anantha

AU - Van Der Krol, Alexander R.

AU - Bouwmeester, Harro

AU - Simonsen, Henrik Toft

PY - 2019/10/23

Y1 - 2019/10/23

N2 - Metabolic engineering is an integrated bioengineering approach, which has made considerable progress in producing terpenoids in plants and fermentable hosts. Here, the full biosynthetic pathway of artemisinin, originating from Artemisia annua, was integrated into the moss Physcomitrella patens. Different combinations of the five artemisinin biosynthesis genes were ectopically expressed in P. patens to study biosynthesis pathway activity, but also to ensure survival of successful transformants. Transformation of the first pathway gene, ADS, into P. patens resulted in the accumulation of the expected metabolite, amorpha-4,11-diene, and also accumulation of a second product, arteannuin B. This demonstrates the presence of endogenous promiscuous enzyme activity, possibly cytochrome P450s, in P. patens. Introduction of three pathway genes, ADSCYP71AV1- ADH1 or ADS-DBR2-ALDH1 both led to the accumulation of artemisinin, hinting at the presence of one or more endogenous enzymes in P. patens that can complement the partial pathways to full pathway activity. Transgenic P. patens lines containing the different gene combinations produce artemisinin in varying amounts. The pathway gene expression in the transgenic moss lines correlates well with the chemical profile of pathway products. Moreover, expression of the pathway genes resulted in lipid body formation in all transgenic moss lines, suggesting that these may have a function in sequestration of heterologous metabolites. This work thus provides novel insights into the metabolic response of P. patens and its complementation potential for A. annua artemisinin pathway genes. Identification of the related endogenous P. patens genes could contribute to a further successful metabolic engineering of artemisinin biosynthesis, as well as bioengineering of other high-value terpenoids in P. patens.

AB - Metabolic engineering is an integrated bioengineering approach, which has made considerable progress in producing terpenoids in plants and fermentable hosts. Here, the full biosynthetic pathway of artemisinin, originating from Artemisia annua, was integrated into the moss Physcomitrella patens. Different combinations of the five artemisinin biosynthesis genes were ectopically expressed in P. patens to study biosynthesis pathway activity, but also to ensure survival of successful transformants. Transformation of the first pathway gene, ADS, into P. patens resulted in the accumulation of the expected metabolite, amorpha-4,11-diene, and also accumulation of a second product, arteannuin B. This demonstrates the presence of endogenous promiscuous enzyme activity, possibly cytochrome P450s, in P. patens. Introduction of three pathway genes, ADSCYP71AV1- ADH1 or ADS-DBR2-ALDH1 both led to the accumulation of artemisinin, hinting at the presence of one or more endogenous enzymes in P. patens that can complement the partial pathways to full pathway activity. Transgenic P. patens lines containing the different gene combinations produce artemisinin in varying amounts. The pathway gene expression in the transgenic moss lines correlates well with the chemical profile of pathway products. Moreover, expression of the pathway genes resulted in lipid body formation in all transgenic moss lines, suggesting that these may have a function in sequestration of heterologous metabolites. This work thus provides novel insights into the metabolic response of P. patens and its complementation potential for A. annua artemisinin pathway genes. Identification of the related endogenous P. patens genes could contribute to a further successful metabolic engineering of artemisinin biosynthesis, as well as bioengineering of other high-value terpenoids in P. patens.

KW - Artemisinin

KW - Biotechnology

KW - Malaria

KW - Physcomitrella patens

KW - Sesquiterpenoids

U2 - 10.3390/molecules24213822

DO - 10.3390/molecules24213822

M3 - Article

VL - 24

JO - Molecules

JF - Molecules

SN - 1420-3049

IS - 21

M1 - 3822

ER -

Ikram NKK, Kashkooli AB, Peramuna A, Van Der Krol AR, Bouwmeester H, Simonsen HT. Insights into heterologous biosynthesis of Arteannuin B and artemisinin in physcomitrella patens. Molecules. 2019 Oct 23;24(21). 3822. https://doi.org/10.3390/molecules24213822