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Functional Characterization of <i>ent</i>-Copalyl Diphosphate Synthase and Kaurene Synthase Genes from <i>Coffea arabica</i> L
The biochemical profile of coffee beans translates directly
into
quality traits, nutraceutical and health promoting properties of the
coffee beverage. Ent-kaurene is the ubiquitous precursor
for gibberellin biosynthesis in plants, but it also serves as an intermediate
in specialized (i.e., secondary) diterpenoid metabolism that leads
to a diversity of more than 1,000 different metabolites. Nutraceutical
effects on human health attributed to diterpenes include antioxidant,
anticarcinogenic, and anti-inflammatory properties. Cafestol (CAF)
and kahweol (KAH) are two diterpenes found exclusively in the Coffea genus. Our objective was to identify and functionally
characterize genes involved in the central step of ent-kaurene production. We identified 17 putative terpene synthase genes
in the transcriptome of Coffea arabica. Two ent-copalyl diphosphate synthase (CaCPS) and three kaurene synthase (CaKS) were selected
and manually annotated. Transcript expression profiles of CaCPS1 and CaKS3 best matched the CAF and
KAH metabolite profiles in different tissues. CaCPS1 and CaKS3 proteins
were heterologously expressed and functionally characterized. CaCPS1
catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to ent-copalyl diphosphate (ent-CPP), which
is converted to ent-kaurene by CaKS3. Knowledge about
the central steps of diterpene formation in coffee provides a foundation
for future characterization of the subsequent enzymes involved in
CAF and KAH biosynthesis