An Effective Strategy for Exploring Unknown Metabolic Pathways by Genome Mining

Abstract

Plants allocate an estimated 15-25% of their proteome to specialized metabolic pathways that remain largely uncharacterized. Here, we describe a genome mining strategy for exploring such unknown pathways and demonstrate this approach for triterpenoids by functionally characterizing three cytochrome P450s from Arabidopsis thaliana . Building on proven methods for characterizing oxidosqualene cyclases, we heterologously expressed in yeast known cyclases with candidate P450s chosen from gene clustering and microarray coexpression patterns. The yeast cultures produced mg/L amounts of plant metabolites in vivo without the complex phytochemical background of plant extracts. Despite this simplification, the product multiplicity and novelty overwhelmed analytical efforts by MS methods. HSQC analysis overcame this problem. Side-by-side HSQC comparisons of crude P450 extracts against a control resolved even minor P450 products among ~100 other yeast metabolites spanning a dynamic range of >10,000:1. HSQC and GC-MS then jointly guided purification and structure determination by classical NMR methods. Including our present results for P450 oxidation of thalianol, arabidiol, and marneral, the metabolic fate for most of the major triterpene synthase products in Arabidopsis is now at least partially known.

In Planta Variation of Volatile Biosynthesis: An Alternative Biosynthetic Route to the Formation of the Pathogen-Induced Volatile Homoterpene DMNT via Triterpene Degradation in Arabidopsis Roots

“…In line with these findings, a recent study by Castillo et al (2013) on triterpene-modifying enzymes in Arabidopsis showed that the C 30 -triterpene diol, arabidiol, can be degraded by CYP705A1 to a C 19 ketone product (14-apo-arabidiol). Arabidiol is produced by Pen1, also called ABDS (Xiang et al, 2006), which is highly coexpressed with CYP705A1 (Supplemental Table 2).…”

“…The reaction is induced by the root rot oomycete pathogen Pythium irregulare and treatment with the plant defense hormone jasmonic acid (JA) and contributes to Arabidopsis root defense. The CYP705A1 gene clusters with a gene encoding a pentacyclic triterpene synthase 1 (Pen1) named arabidiol synthase (here referred to as ABDS) (Xiang et al, 2006) as part of a larger triterpene biosynthetic gene cluster on chromosome 4 (Field et al, 2011;Castillo et al, 2013). Our results provide evidence for independent evolution and biosynthetic plasticity in the formation of functionally active volatiles.…”

Investigation of terpene diversification across multiple sequenced plant genomes

“…These include two diterpene clusters from Oryza sativa (rice) [the momilactone and phytocassane clusters (18,19)], three triterpene clusters [the thalianol and marneral clusters from A. thaliana (20,21), the avenacin cluster from Avena strigosa (oat) (22,23)], and clusters for steroidal glycoalkaloids and other terpenes in the Solanaceae (24,25). Potential new clusters implicated in terpene synthesis have also been reported in A. thaliana (20,(26)(27)(28) and cucumber (29). The available evidence indicates that the characterized clusters have arisen within recent evolutionary history by gene duplication, acquisition of new function and genome reorganization, and that they are not products of horizontal gene transfer from microbes (reviewed in refs.…”

Novel triterpene oxidizing activity of Arabidopsis thaliana CYP716A subfamily enzymes

“…Although all 13 OSCs have been identified in the model plant Arabidopsis thaliana (Brassicaceae) [25], only a few triterpene skeleton oxidizing P450s have been functionally characterized [3,4]. Boutanaev et al found that CYP716A1 (At5g36110) can oxidize an unknown position of tirucalla-7,24-dien-3b-ol, the main product of PEN3 (At5g36150), which is one of the 13 OSCs in A. thaliana [2,11].…”