Metabolic engineering of monoterpene biosynthesis in plants

Monoterpenes are a large group of compounds that belong to the terpenoid family of natural compounds in plants. They are small, volatile, lipophilic substances of which around one thousand different structures have been identified. Monoterpenes are involved in plant-insect, plant-microorganism and plant-plant interactions. Many monoterpenes, such as menthol, carvone, limonene and linalool, are of commercial interest as they are commonly used in foods, beverages, perfumes and cosmetics and in many cleaning products. In flowers they also contribute to the characteristic scent. Monoterpene synthases and subsequent modifying enzymes such as cytochrome P450 hydroxylases, dehydrogenases, reductases and isomerases are responsible for the production of the variety of different carbon skeletons of monoterpenes that are found in nature. In this thesis the use of genetic engineering to introduce or alter the production of monoterpenes by plants was explored.

Initially, as described in Chapter 2, S -linalool synthase from Clarkia breweri was introduced in Petunia plants regulated by a constitutive promoter. Expression was obtained in all tissues analysed, but formation of linalool was restricted to leaves, sepals, corollas, stems and ovaries, and could not be detected in nectaries, roots, pollen and style. Although it was expected that the formation of linalool would result in an alteration of the scent of the plants, no linalool was detected in the headspace. Instead, all the S -linalool produced was efficiently converted by an endogenous glucosyltransferase present in the petunia tissues to the non-volatile S -linalyl-b-D-glucopyranoside. These results showed that genetic engineering of plants for monoterpene biosynthesis is possible, but that it can lead to unexpected conversions of the produced metabolites by endogenous enzyme activities.

In order to obtain new monoterpene synthases for the genetic engineering of plants, a cDNA library was made of the fruit peel of lemon, a plant species producing many different monoterpenes. From this library four different monoterpene synthases were obtained as described in Chapter 3, which together showed to be responsible for more than 90% of the total number of components present in lemon oil. The product specificity of the enzymes could be analysed after heterologous expression in Escherichia coli . Two of the four cDNA-encoded enzymes were producing (+)-limonene, the main component present in lemon. One cDNA-encoded enzyme was mainly producing (-)-b-pinene and the fourth cDNA-encoded enzyme was mainly producingg-terpinene. The latter two enzymes were both producing traces of multiple side products as well. Also other properties of the heterologously expressed enzymes were determined, which are described in Chapter 3.

Three monoterpene synthases responsible for the production of different main products were chosen for the genetic engineering of Nicotiana tabacum 'Petit Havana' SR1, described in Chapter 4. The wild type of this tobacco variety produces one monoterpene, linalool that is only emitted from the flowers. After the transformation with the three monoterpene synthases and subsequent crossings, a plant was obtained that emitted all the three main products of the three introduced monoterpene synthases in addition to the endogenous linalool in the flowers. The levels of limonene,b-pinene andg-terpinene emitted from the leaves and flowers of the plant were higher than the level of the endogenous monoterpene. Also the side products of the monoterpene synthases were detected. The extensive modification of the volatile profile of the tobacco plants that we obtained indicates that there is a sufficient amount of substrate available to the introduced enzymes.

In Chapter 5 the transgenic tobacco plant emitting the products of three monoterpene synthases, was used in a subsequent transformation experiment in order to modify the already introduced pathway. A second step in the pathway was introduced by transformation of the plant material with a limonene-3-hydroxylase isolated from spearmint, which is supposed to be localised in the endoplasmatic reticulum (ER) in the cytosol of the plant cells, while the primarily introduced monoterpene synthases were most likely localised in the plastids in the transgenic plants. The introduction of the cytochrome P450 monoterpene hydroxylase and the resulting formation of the hydroxylated product of (+)-limonene, (+)- trans -isopiperitenol demonstrates that there is intracellular trafficking of limonene from the plastids to the ER in the cytosol. That this trafficking mechanism would be present in plants normally producing these hydroxylated monoterpenes could be expected, but that it is apparently also present in plants not specialised for the production of these compounds is an exciting discovery. Apart from the production and subsequent emission of high further oxidised conversion product isopiperitenone was detected. In addition, an increase in the p -cymene level and the formation of the new products 1,3,8- p -menthatriene and 1,5,8- p -menthatriene were detected. The occurrence of these latter two products and the increase of the p -cymene level could be a consequence of the metabolic engineering of the biosynthetic route into a cell compartment not adapted to the production of these compounds. Endogenous enzymes and pH differences were suggested to be the main cause the formation of these products.

Chapter 6 discusses the various strategies followed for the metabolic engineering of monoterpene biosynthesis in this thesis and by other groups. Functional implications are discussed such as ecological and physiological consequences of the new metabolites for the transgenic plants. The commercial aspects and interesting opportunities for further research are also discussed.