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

Abstract

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. The MsLS RNAi lines exhibited a huge reduction in the synthesis of limonene and carvone. Detailed GC-MS and LC-MS analysis revealed that MsLS RNAi 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 from Picea abies and geraniol synthase from Cananga odorata were cloned and introduced independently into the MsLS RNAi 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.

Keywords: derivatives; limonene synthase; metabolic engineering; pathway flux analyses; secondary metabolites; terpenes.

Figure 1

Functional characterization of recombinant MsLS in vitro (A) GC-MS profile of recombinant MsLS protein activity with (in blue) and without GDP (red). (B) Mass spectrum of peak A compared to the matched limonene peak from NIST library.

Figure 2

Characterization of MsLS RNAi lines (A) Southern blot analysis of MsLS RNAi lines. DNA from transgenic plants and wild type (WT) were digested with EcoRI (E) and Xbal (X). M indicates DNA Molecular Weight Marker Vii. (B) MsLS transcripts level analysis by qRT-PCR. Gene expression is presented as relative to that of WT (%). Data represent as mean ± SD for three biological replicates. (**, p ≤ 0.01).

Figure 3

Volatile component analysis of three MsLS RNAi plant (LS2, LS4 and LS5) and wild type (WT) by GC-MS (A) Quantitative analysis of two major monoterpenes. (B) Quantitative analysis of sesquiterpenes. Amount of terpenes production are presented as relative to that of WT. Data represent as mean ± SD for three biological replicates. (*, p ≤ 0.05; **, p ≤ 0.01).

Figure 4

Percent composition pf fatty acids, sterroids, and flavonoids in wildtype and MsLS RNAi lines using GC-MS and LC-MS analysis. Data represent mean ± SD for three biological replicates. (**, p ≤ 0.01).

Figure 5

Amounts of the introduced monoterpene derivatives quantified using LC MS analysis for (A) CoGers transgenic lines (B) PaLinS transgenic lines and (C) PaMyrs transgenic lines respectively. Data represent mean ± SD for three biological replicates. (**, p ≤ 0.01).

Figure 6

Principal component analysis for LC-MS data obtained. Score plot of (A) MsLS RNAi4 with wild type (B) MsLS RNAi4 with CoGerS9 transgenic line (C) MsLS RNAi4 with PaLinS18 transgenic line and (D) MsLS RNAi4 with PaMyrS2 transgenic line. Explained variance for PC1 and PC2 are indicated as spercentages in X and Y axis respectively.