An extremely promiscuous terpenoid synthase from the Lamiaceae plant Colquhounia coccinea var. mollis catalyzes the formation of sester-/di-/sesqui-/mono-terpenoids

Abstract

Terpenoids are the largest class of natural products with complex structures and extensive bioactivities; their scaffolds are generated by diverse terpenoid synthases (TPSs) from a limited number of isoprenoid diphosphate precursors. Promiscuous TPSs play important roles in the evolution of terpenoid chemodiversity, but they remain largely unappreciated. Here, an extremely promiscuous terpenoid synthase (CcTPS1) of the TPS-b subfamily was cloned and functionally characterized from a leaf-specific transcriptome of the Lamiaceae plant Colquhounia coccinea var. mollis. CcTPS1 is the first sester-/di-/sesqui-/mono-TPS identified from the plant kingdom, accepting C₂₅/C₂₀/C₁₅/C₁₀ diphosphate substrates to generate a panel of sester-/di-/sesqui-/mono-terpenoids. Engineered Escherichia coli expressing CcTPS1 produced three previously unreported terpenoids (two sesterterpenoids and a diterpenoid) with rare cyclohexane-containing skeletons, along with four sesquiterpenoids and one monoterpenoid. Their structures were elucidated by extensive nuclear magnetic resonance spectroscopy. Nicotiana benthamiana transiently expressing CcTPS1 also produced the diterpenoid and sesquiterpenoids, demonstrating the enzyme's promiscuity in planta. Its highly leaf-specific expression pattern combined with detectable terpenoid products in leaves of C. coccinea var. mollis and N. benthamiana expressing CcTPS1 suggested that CcTPS1 was mainly responsible for diterpenoid and sesquiterpenoid biosynthesis in plants. CcTPS1 expression and the terpenoid products could be induced by methyl jasmonate, suggesting their possible role in plant-environment interaction. CcTPS1 was localized to the cytosol and may differ from mono-TPSs in subcellular compartmentalization and substrate tolerance. These findings will greatly aid our understanding of plant TPS evolution and terpenoid chemodiversity; they also highlight the enormous potential of transcriptome mining and heterologous expression for the exploration of unique enzymes and natural products hidden in plants.

Keywords: Colquhounia coccinea var. mollis; promiscuous enzyme; sester-/di-/sesqui-/mono-terpenoids; terpenoid diversity; terpenoid synthase.

Figure 1

GC–MS analysis of the terpenoid profiles of Colquhounia coccinea var. mollis. (A–C) Total ion chromatogram of the n-hexane extracts of fresh leaves (A), flowers (B), and epidermis-free tender stems (C). Peaks a–t and u–x are sesquiterpenoids and diterpenoids, respectively, tentatively identified based on their mass spectra. Peaks h and x correspond to sesquiterpenoid 7 and diterpenoid 3, which were characterized as the enzymatic products of CcTPS1.

Figure 2

Phylogenetic analysis of CcTPS1 and other plant TPSs. The phylogenetic tree was constructed using the maximum-likelihood method, and information on the TPSs used is listed in Supplemental Table 6. Numbers above branches are bootstrap values.

Figure 3

GC–MS analysis of the metabolites of engineered E. coli expressing CcTPS1. (A) Extracted ion chromatogram (m/z 93) of the extracts from E. coli harboring pBbA5C-MevT-MBIS and pET-28a/MmGFDPS along with pCold-TF/CcTPS1 (a), empty pCold-TF vector (b), and purified compounds 1–8 (c–j). (B) Mass spectra of compounds 1–8.

Figure 4

Chemical structure of terpenoids 1–8 produced by CcTPS1. (A) Structures of compounds 1–8. (B) Selected key ¹H–¹H COSY and HMBC correlations for compounds 1 and 3.

Figure 5

Subcellular localization and product profile analysis of CcTPS1 using N. benthamiana. (A) GFP fusion proteins in N. benthamiana leaf epidermal cells visualized by laser confocal microscopy. (B) GC–MS analysis of the extracts of N. benthamiana leaves harboring pEAQ-HT/CcTPS1 and pEAQ-HT/AtGFDPS2 (a), pEAQ-HT/AtGFDPS2 (b), and empty vector pEAQ-HT (c).

Figure 6

The proposed cyclization mechanism of sesterterpenoids 1 and 2 biosynthesized by CcTPS1 is distinct from that of recently reported plant sester-TPSs. (A) Proposed cyclization scheme of compounds 1–8 produced by CcTPS1. (B) Illustration of sesterterpenoid cyclization mechanisms catalyzed by previously reported plant sester-TPSs.