Metabolic engineering of caffeine production

Abstract

Among 12,000 alkaloids which are produced in plants, caffeine (1,3,7-trimethylxanthine) is one of the best known due to it uses as an ingredient of pharmaceuticals and beverages. In coffee plants, it is synthesized from xanthosine through three successive methylation and ribose removal steps. We have isolated all genes encoding the corresponding N-methyltransferases; xanthosine methyltransferase (XMT), 7-methylxanthine methyltransferase (MXMT) and 3,7-dimethylxanthine methyltransferase (DXMT), as well as for the 7-methylxanthosine nucleosidase. Using these genes, we have engineered caffeine production in two ways. The first is to decrease the caffeine content in coffee plants to cope with occasional health problems caused by caffeine uptake, and the other is to produce caffeine as an insect repellant in crop plants, originally not synthesizing caffeine. The first approach was performed using an RNAi for MXMT, yielding a 70% suppression of the caffeine level in leaves of transgenic coffee plants. The other approach was carried out by simultaneous introduction of three genes, XMT, MXMT and DXMT, into tobacco plants, which produced up to 5 μg caffeine per g fresh weight of leaves. This amount of caffeine was enough to repel tobacco cutworms (Spodoptera litura), suggesting the method to be practically efficient for construction of herbivore tolerant crops. The significance of the present study is discussed with reference to four topics: practical metabolic engineering; development of a genetic transformation system for tropical trees; generation of genetically modified (GM) plants with a minimal load on the environment; and providing GM foods that bring direct merits to consumers.