TY - JOUR

T1 - Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis

T2 - Similar activity but difference in subcellular localization

AU - Dong, Lemeng

AU - Miettinen, Karel

AU - Goedbloed, Miriam

AU - Verstappen, Francel W.A.

AU - Voster, Alessandra

AU - Jongsma, Maarten A.

AU - Memelink, Johan

AU - Krol, Sander van der

AU - Bouwmeester, Harro J.

N1 - Funding Information: This research was funded by the European Community's Framework VII Program FP7/2007-2013 to the SMARTCELL project KBBE-2007-3-1-01. Lemeng Dong was supported by a PhD fellowship from China Scholarship Council . We thank Bert Schipper and Ric de Vos for their help with LC–MS analysis.

PY - 2013

Y1 - 2013

N2 - 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 32μM and 51μM 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.

AB - 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 32μM and 51μM 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.

KW - Engineering

KW - Enzyme assay

KW - Geraniol synthase

KW - Metabolic profiling

KW - Subcellular localization

U2 - 10.1016/j.ymben.2013.09.002

DO - 10.1016/j.ymben.2013.09.002

M3 - Journal article

C2 - 24060453

AN - SCOPUS:84887012273

VL - 20

SP - 198

EP - 211

JO - Metabolic Engineering

JF - Metabolic Engineering

SN - 1096-7176

ER -