BioMatNet Item: QLK3-2002-01930 - Investigation of biochemical and genetical diversity of terpenoid biosynthesis for the production of high value-added compounds (INTESY)

[BioMatNet Database - FP5 Quality of Life Programme]

QLK3-2002-01930
Investigation of biochemical and genetical diversity of terpenoid biosynthesis for the production of high value-added compounds (INTESY)

Contacts

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Type of Project	Shared Cost Research
Contract No	QLK3-2002-01930
Total Cost
EC Contribution	1,298,108 EUR
Start Date	01-12-2002
Duration	36 Months

Abstract Terpene synthases are responsible for a key biosynthetic step in the formation of many mono- and sesquiterpenes produced by aromatic plants that are valuable in medicine, in food and in the cosmetic industry. The overall objectives of the project are:

to exploit the natural diversity of expression of terpene synthases
to obtain a detailed understanding of the molecular and structural basis of terpene synthase function, so enabling the informed development of strategies for producing important value-added terpenoids at high levels both in vivo and in vitro.

The terpene synthase genes of three species will be cloned and expressed, the encoded enzymes characterised and the role of these catalysts in producing diverse terpene profiles investigated. The elucidation of structure-function relationships within terpene synthase families will provide the theoretical basis to develop the possibility of protein engineering.

Objectives

Within each genus of the Lamiaceae a similar spectrum of terpenes is often produced, but in different species and populations they are found in very different proportions that are further modified by environmental and developmental factors. Thus each genus presents a tool for investigating both the structural basis for differences in terpene synthase function, and the genetic and physiological control of terpene synthesis.

Activities

The mRNA of glandular trichomes from Salvia and Origanum species will be extra cted and cDNA libraries constructed. After a representative number of cDNA clones from each library has been end-sequenced, the sequences homologous to known terpene synthases will be isolated as full-length clones. Identifying selected synthases will be achieved using two strategies:

specific mRNA and terpene end product levels will be compared under a range of conditions.
functional expression of the genes will be obtained using a bacterial expression system and the product spectrum of each identified.

The bacterially expressed genes will be further purified and characterised to assess the extent to which variation in their kinetic properties contributes to the expression patterns observed in the different species, and to evaluate how changes in structure (see below) are related to changes in kinetic properties.

Structural elucidation of related terpene synthase genes will be carried out by a combination of modelling, based on a comparison of sequence information with the few known terpene synthase structures and experiment, involving crystallisation of purified terpene synthases, their use to diffract X-rays, construction of electron density maps, etc.

Further investigation will be carried out to establish how these enzymes regulate monoterpene accumulation in vivo by comparing levels of terpene synthase mRNA, protein and enzyme activity with accumulation of products in plants at different developmental stages.

Deliverables

Deliverables from the project will include:

DNA libraries from glandular trichomes of Salvia and Origanum spp.
Full-length DNA sequences of putative monoterpene synthase genes.
Bacterial strains expressing terpene synthases for large-scale delivery and purification of monoterpene synthase proteins. Comparison of the biochemical properties of related Salvia and Origanum synthases.
Mutant monoterpene synthase proteins with alterations in amino acid residues at the catalytic site.
Crystal structure elucidation and modelling of monoterpene synthase structures, including analysis of amino acids present at the active site and their roles in the reaction mechanism.
Development of a low-cost method for the industrial production of valuable terpenoid compounds using yeast cells as a "cell factory".
Elucidation of the mode of inheritance of chemical and morphological characters and assessment of variation in monoterpene composition caused by changes in development and environment, including genotype x environment interaction.
Elucidation of how terpene synthase activity regulates monoterpene accumulation and of how terpene synthase activity itself is regulated by gene expression and the level of the corresponding proteins.
IPR assessment, including possible patents, marketing strategies and market research leading to patent applications in respect of inventions made during the course of the project.

Contacts

Coordinator

Johannes NOVAK / University of Veterinary Medicine Institute for Applied Botany / AUSTRIA

Participant