Metabolic Engineering of the Native Monoterpene Pathway in Spearmint for Production of Heterologous Monoterpenes Reveals Complex Metabolism and Pathway Interactions.

Spearmint produces and stores large amounts of monoterpenes, mainly limonene and carvone, in glandular trichomes and is the major natural source of these compounds. Towards producing heterologous monoterpenes in spearmint, we first reduced the flux into the native limonene pathway by knocking down the expression of limonene synthase (MsLS) by RNAi method. TheMsLSRNAi lines exhibited a huge reduction in the synthesis of limonene and carvone. Detailed GC-MS and LC-MS analysis revealed thatMsLSRNAi plants also showed an increase in sesquiterpene, phytosterols, fatty acids, flavonoids, and phenolic metabolites, suggesting an interaction between the MEP, MVA shikimate and fatty acid pathways in spearmint. Three different heterologous monoterpene synthases namely, linalool synthase and myrcene synthase fromPicea abiesand geraniol synthase fromCananga odoratawere cloned and introduced independently into theMsLSRNAi mutant background. The expression of these heterologous terpene synthases resulted mainly in production of monoterpene derivatives. Of all the introduced monoterpenes geraniol showed the maximum number of derivatives. Our results provide new insights into MEP pathway interactions and regulation and reveals the existence of mechanisms for complex metabolism of monoterpenes in spearmint.