Metabolic flux phenotype of tobacco hairy roots engineered for increased geraniol production.
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.
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK.
Shyam K
SK
Masakapalli
VTT Technical Research Centre of Finland, P.O. Box 1000, 02044 VTT (Espoo), Finland.
Anneli
A
Ritala
Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
Lemeng
L
Dong
Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
Alexander R
AR
van der Krol
VTT Technical Research Centre of Finland, P.O. Box 1000, 02044 VTT (Espoo), Finland.
Kirsi-Marja
KM
Oksman-Caldentey
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK.
R George
RG
Ratcliffe
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK. Electronic address: lee.sweetlove@plants.ox.ac.uk.
Lee J
LJ
Sweetlove
D000080462:Acyclic Monoterpenes; D060847:Metabolic Engineering; D008968:Molecular Conformation; D010641:Phenotype; D018517:Plant Roots / Q000737:chemistry / Q000378:metabolism*; D013729:Terpenes / Q000737:chemistry / Q000378:metabolism*; D014026:Nicotiana / Q000378:metabolism*
D000080462:Acyclic Monoterpenes; D013729:Terpenes; C007836:geraniol
Flux analysis; Geraniol; Metabolic engineering; Terpenoid indole alkaloids