Functional genomics for the discovery of genes affecting lemon basil aroma and their use in flavor engineering of tomato

Aromatic, medicinal and spice plants have been valued since antiquity as food supplements, preservatives and sources of flavors, aromas, medicinal drugs and essential oils. We have embarked on a long-term project to identify and isolate genes that encode key enzymes in aroma formation in plants. Lemon basil (Ocimum basilicum L. Lamiaceae) is known to accumulate essential oils rich in monoterpenes such as citral (geranial and neral) and sesquiterpenes, such as germacrene D, β-caryophyllene, α-bisabolene and α-zingiberene in glandular trichomes localized on the surface of the aerial parts of the plant. We have isolated mRNA from such glandular trichomes and produced a database of the genes that are expressed in them. Information on three basil lines with different chemical compositions was thus generated. Bioinformatic analysis of these databases yielded genes encoding key enzymes involved in mono- and sesquiterpene formation. cDNAs of these genes were functionally expressed in E. coli to validate their biochemical role. They include geraniol synthase (GES), a gene encoding the enzyme that converts geranyl diphosphate into the rose-scented compound geraniol, and α-zingiberene synthase (ZIS), a gene that encodes a multifunctional enzyme able to produce 13 monoterpenes from geranyl diphosphate and 15 sesquiterpenes from farnesyl diphosphate. To test the potential of metabolic engineering to effectively modify aroma and taste properties of tomato, we diverted the tomato terpenoid pathways by expression the lemon basil GES and ZIS genes. Expression of GES in tomato fruit caused elevated levels of geraniol and several other geraniol derivatives, resulting in dramatic changes in the aroma and overall flavor of the transgenic fruits, as judged by a panel of untrained taste panelists. Expression of the lemon basil ZIS gene in tomato fruit resulted in the enhanced accumulation of mono- and sesquiterpenes, bringing about improved aroma characteristics of the fruits. Our data clearly indicate that the utilization of genes originating in lemon basil for the genetic engineering of tomato fruit aroma and taste is feasible and that this approach has the potential for modifying the aroma and taste of other carotenoid-accumulating vegetables and fruits.