Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization.
Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed.
Laboratory of Plant Physiology, Wageningen UR, P.O. Box 658, 6700 AR Wageningen, The Netherlands.
Lemeng
L
Dong
Karel
K
Miettinen
Miriam
M
Goedbloed
Francel W A
FW
Verstappen
Alessandra
A
Voster
Maarten A
MA
Jongsma
Johan
J
Memelink
Sander
S
van der Krol
Harro J
HJ
Bouwmeester
D000080462:Acyclic Monoterpenes; D060365:Chloroplast Proteins / Q000096:biosynthesis* / Q000235:genetics; D003600:Cytosol / Q000201:enzymology*; D032411:Lippia / Q000201:enzymology* / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D018087:Plastids / Q000201:enzymology* / Q000235:genetics; D013045:Species Specificity; D013729:Terpenes / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014632:Valerian / Q000201:enzymology* / Q000235:genetics
D000080462:Acyclic Monoterpenes; D060365:Chloroplast Proteins; D013729:Terpenes; D010744:Phosphoric Monoester Hydrolases; C007836:geraniol
Engineering; Enzyme assay; Geraniol synthase; Metabolic profiling; Subcellular localization