Functional expression in-vitro and biochemical characterization of terpene synthases of Salvia fruticosa
Anssour, S.
This master thesis is part of an EU funded project (INTESY), the objective of which is to exploit the natural diversity of expression of the key biosynthetic enzymes (terpene synthases) and to obtain a detailed understanding of the molecular and structural basis of these molecules, so enabling the informed development of strategies for producing important value-added terpenoids at high levels both in vivo and in vitro. The current work focuses on the functional expression in-vitro (using E.coli) and biochemical characterization of terpene synthases of Salvia fruticosa (producer of a broad range of terpenoids). The Salvia fruticosa cDNA library made by our partner in Germany was screened in our laboratory and 1000 cDNA were then inserts end-sequenced. In the terpenoid group, one sesquiterpene synthase homologue (full length) and three monoterpene synthase homologues (two full length, one partial) were isolated and cloned in vectors for bacterial expression. Initially the terpene synthases were expressed in N-his-tagged form and purified and they were assessed for activity. From the three expressed terpene synthases only one monoterpene synthase (cineole synthase) was active. In order to study of the effect of some N-terminal residues on cineole synthase activity and substrate affinity, the enzyme was expressed three forms: his-tagged cineole synthase, CS+RR and CS-RR. The experiment revealed that the highly conserved RR on the N-terminal side are not essential for the enzymatic activity as had been proposed in the literature but that the absence of the RR motif lowers the substrate affinity, with or without the his-tag being present.
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