Metabolic flux phenotype of tobacco hairy roots engineered for increased geraniol production

S K Masakapalli, Anneli Ritala, L Dong, A R van der Krol, Kirsi-Marja Oksman-Caldentey, R G Ratcliffe, L J Sweetlove (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

20 Citations (Scopus)

Abstract

The goal of this study was to characterise the metabolic flux phenotype of transgenic tobacco (Nicotiana tabacum) hairy roots engineered for increased biosynthesis of geraniol, an intermediate of the terpenoid indole alkaloid pathway. Steady state, stable isotope labelling was used to determine flux maps of central carbon metabolism for transgenic lines over-expressing (i) plastid-targeted geraniol synthase (pGES) from Valeriana officinalis, and (ii) pGES in combination with plastid-targeted geranyl pyrophosphate synthase from Arabidopsis thaliana (pGES + pGPPS), as well as for wild type and control-vector-transformed roots. Fluxes were constrained by the redistribution of label from [1-13C]-, [2-13C]- or [13C6]glucose into amino acids, sugars and organic acids at isotopic steady state, and by biomass output fluxes determined from the fractionation of [U-14C]glucose into insoluble polymers. No significant differences in growth and biomass composition were observed between the lines. The pGES line accumulated significant amounts of geraniol/geraniol glycosides (151 ± 24 ng/mg dry weight) and the de novo synthesis of geraniol in pGES was confirmed by 13C labelling analysis. The pGES + pGPPS also accumulated geraniol and geraniol glycosides, but to lower levels than the pGES line. Although there was a distinct impact of the transgenes at the level of geraniol synthesis, other network fluxes were unaffected, reflecting the capacity of central metabolism to meet the relatively modest demand for increased precursors in the transgenic lines. It is concluded that re-engineering of the terpenoid indole alkaloid pathway will only require simultaneous manipulation of the steps producing the pathway precursors that originate in central metabolism in tissues engineered to produce at least an order of magnitude more geraniol than has been achieved so far
Original languageEnglish
Pages (from-to)73-85
Number of pages13
JournalPhytochemistry
Volume99
DOIs
Publication statusPublished - 2014
MoE publication typeA1 Journal article-refereed

Fingerprint

geraniol
Tobacco
tobacco
Fluxes
Phenotype
phenotype
Plastids
plastids
Secologanin Tryptamine Alkaloids
Metabolism
indole alkaloids
genetically modified organisms
Glycosides
terpenoids
Biomass
Labeling
metabolism
glycosides
Valeriana officinalis
Valerian

Keywords

  • Flux analysis
  • geraniol
  • metabolic engineering
  • terpenoid indole alkoloids

Cite this

Masakapalli, S K ; Ritala, Anneli ; Dong, L ; van der Krol, A R ; Oksman-Caldentey, Kirsi-Marja ; Ratcliffe, R G ; Sweetlove, L J. / Metabolic flux phenotype of tobacco hairy roots engineered for increased geraniol production. In: Phytochemistry. 2014 ; Vol. 99. pp. 73-85.
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abstract = "The goal of this study was to characterise the metabolic flux phenotype of transgenic tobacco (Nicotiana tabacum) hairy roots engineered for increased biosynthesis of geraniol, an intermediate of the terpenoid indole alkaloid pathway. Steady state, stable isotope labelling was used to determine flux maps of central carbon metabolism for transgenic lines over-expressing (i) plastid-targeted geraniol synthase (pGES) from Valeriana officinalis, and (ii) pGES in combination with plastid-targeted geranyl pyrophosphate synthase from Arabidopsis thaliana (pGES + pGPPS), as well as for wild type and control-vector-transformed roots. Fluxes were constrained by the redistribution of label from [1-13C]-, [2-13C]- or [13C6]glucose into amino acids, sugars and organic acids at isotopic steady state, and by biomass output fluxes determined from the fractionation of [U-14C]glucose into insoluble polymers. No significant differences in growth and biomass composition were observed between the lines. The pGES line accumulated significant amounts of geraniol/geraniol glycosides (151 ± 24 ng/mg dry weight) and the de novo synthesis of geraniol in pGES was confirmed by 13C labelling analysis. The pGES + pGPPS also accumulated geraniol and geraniol glycosides, but to lower levels than the pGES line. Although there was a distinct impact of the transgenes at the level of geraniol synthesis, other network fluxes were unaffected, reflecting the capacity of central metabolism to meet the relatively modest demand for increased precursors in the transgenic lines. It is concluded that re-engineering of the terpenoid indole alkaloid pathway will only require simultaneous manipulation of the steps producing the pathway precursors that originate in central metabolism in tissues engineered to produce at least an order of magnitude more geraniol than has been achieved so far",
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Metabolic flux phenotype of tobacco hairy roots engineered for increased geraniol production. / Masakapalli, S K; Ritala, Anneli; Dong, L; van der Krol, A R; Oksman-Caldentey, Kirsi-Marja; Ratcliffe, R G; Sweetlove, L J (Corresponding Author).

In: Phytochemistry, Vol. 99, 2014, p. 73-85.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Metabolic flux phenotype of tobacco hairy roots engineered for increased geraniol production

AU - Masakapalli, S K

AU - Ritala, Anneli

AU - Dong, L

AU - van der Krol, A R

AU - Oksman-Caldentey, Kirsi-Marja

AU - Ratcliffe, R G

AU - Sweetlove, L J

PY - 2014

Y1 - 2014

N2 - The goal of this study was to characterise the metabolic flux phenotype of transgenic tobacco (Nicotiana tabacum) hairy roots engineered for increased biosynthesis of geraniol, an intermediate of the terpenoid indole alkaloid pathway. Steady state, stable isotope labelling was used to determine flux maps of central carbon metabolism for transgenic lines over-expressing (i) plastid-targeted geraniol synthase (pGES) from Valeriana officinalis, and (ii) pGES in combination with plastid-targeted geranyl pyrophosphate synthase from Arabidopsis thaliana (pGES + pGPPS), as well as for wild type and control-vector-transformed roots. Fluxes were constrained by the redistribution of label from [1-13C]-, [2-13C]- or [13C6]glucose into amino acids, sugars and organic acids at isotopic steady state, and by biomass output fluxes determined from the fractionation of [U-14C]glucose into insoluble polymers. No significant differences in growth and biomass composition were observed between the lines. The pGES line accumulated significant amounts of geraniol/geraniol glycosides (151 ± 24 ng/mg dry weight) and the de novo synthesis of geraniol in pGES was confirmed by 13C labelling analysis. The pGES + pGPPS also accumulated geraniol and geraniol glycosides, but to lower levels than the pGES line. Although there was a distinct impact of the transgenes at the level of geraniol synthesis, other network fluxes were unaffected, reflecting the capacity of central metabolism to meet the relatively modest demand for increased precursors in the transgenic lines. It is concluded that re-engineering of the terpenoid indole alkaloid pathway will only require simultaneous manipulation of the steps producing the pathway precursors that originate in central metabolism in tissues engineered to produce at least an order of magnitude more geraniol than has been achieved so far

AB - The goal of this study was to characterise the metabolic flux phenotype of transgenic tobacco (Nicotiana tabacum) hairy roots engineered for increased biosynthesis of geraniol, an intermediate of the terpenoid indole alkaloid pathway. Steady state, stable isotope labelling was used to determine flux maps of central carbon metabolism for transgenic lines over-expressing (i) plastid-targeted geraniol synthase (pGES) from Valeriana officinalis, and (ii) pGES in combination with plastid-targeted geranyl pyrophosphate synthase from Arabidopsis thaliana (pGES + pGPPS), as well as for wild type and control-vector-transformed roots. Fluxes were constrained by the redistribution of label from [1-13C]-, [2-13C]- or [13C6]glucose into amino acids, sugars and organic acids at isotopic steady state, and by biomass output fluxes determined from the fractionation of [U-14C]glucose into insoluble polymers. No significant differences in growth and biomass composition were observed between the lines. The pGES line accumulated significant amounts of geraniol/geraniol glycosides (151 ± 24 ng/mg dry weight) and the de novo synthesis of geraniol in pGES was confirmed by 13C labelling analysis. The pGES + pGPPS also accumulated geraniol and geraniol glycosides, but to lower levels than the pGES line. Although there was a distinct impact of the transgenes at the level of geraniol synthesis, other network fluxes were unaffected, reflecting the capacity of central metabolism to meet the relatively modest demand for increased precursors in the transgenic lines. It is concluded that re-engineering of the terpenoid indole alkaloid pathway will only require simultaneous manipulation of the steps producing the pathway precursors that originate in central metabolism in tissues engineered to produce at least an order of magnitude more geraniol than has been achieved so far

KW - Flux analysis

KW - geraniol

KW - metabolic engineering

KW - terpenoid indole alkoloids

U2 - 10.1016/j.phytochem.2013.12.007

DO - 10.1016/j.phytochem.2013.12.007

M3 - Article

VL - 99

SP - 73

EP - 85

JO - Phytochemistry

JF - Phytochemistry

SN - 0031-9422

ER -