Surrogate Splicing for Functional Analysis of Sesquiterpene Synthase Genes

Abstract

A method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns is described. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product. The method was optimized by examining the expression of an intron-containing b-glucuronidase gene agroinfiltrated into petunia (Petunia hybrida) leaves, and its utility was demonstrated by defining the function of two previously uncharacterized terpene synthases. A tobacco (Nicotiana tabacum) terpene synthase-like gene containing six predicted introns was characterized as having 5-epi-aristolochene synthase activity, while an Arabidopsis (Arabidopsis thaliana) gene previously annotated as a terpene synthase was shown to possess a novel sesquiterpene synthase activity for a-barbatene, thujopsene, and b-chamigrene biosynthesis.Terpene synthases are a class of enzymes that catalyze the conversion of prenyl diphosphates to mono-, sesqui-, and diterpenoid compounds (Chappell, 1995;Davis and Croteau, 2000). The reactions catalyzed by many of these enzymes are stereospecific and complex, often generating one to upwards of 30 reaction products (Steele et al., 1998), many of which may contain one or more ring structures. Terpene synthases are of particular interest to phytochemists for their role in the synthesis of natural products, including flavors and fragrances, such as nootkatone and patchouli alcohol and antimicrobial phytoalexins like capsidiol (Chappell, 1995). Sesquiterpene synthases, a subset of terpene synthases catalyzing the biosynthesis of products derived from farnesyl diphosphate, have been characterized from many plant species including tobacco (Nicotiana tabacum;Facchini and Chappell, 1992) Many of the initial molecular studies of sesquiterpene synthases were based on the identification of cDNA clones encoding for enzymes catalyzing the biosynthesis of particular sesquiterpene compounds (Facchini and Chappell, 1992). More recently, many of the plant genetic databases contain genomic sequences bearing significant similarity to known terpene synthases. However, the actual biochemical function for many of these genes is unknown. For example, the Arabidopsis genome contains between 34 and 40 genes annotated as having similarity to terpene synthases (Aubourg et al., 2002; Supplemental Figs. 1 and 2), though less than one-third of these have been confirmed through direct demonstration of catalytic function (Bohlmann et al., 2000;Chen et al., 2003Chen et al., , 2004 Fäldt et al., 2003). Furthermore, genome sequencing projects, by virtue of their comprehensive nature, are bound to reveal genes that may not be expressed because of any number of molecular alterations, yet are nonetheless of interest for mechanistic and evolutionary studies of catalytic ...

Metabolic engineering of sesquiterpene metabolism in yeast

“…However, an alternative means towards uncovering the breadth of this diversity is also apparent from recent advances in the isolation and characterization of the genes encoding for these biosynthetic systems. Functional identification of the terpene biosynthetic genes necessarily entails expression of the corresponding gene and most routinely an in vitro characterization of the isolated enzyme (i.e., Wu et al, 2005). Mechanisms for in vivo assessment have also been described including the use of knockout mutations and RNA suppression technologies and the observed loss of a particular metabolite profile (Tholl et al, 2005;Wang and Wagner, 2003).…”

“…We describe here the utilization of yeast strains engineered to enhance carbon flux through the mevalonate pathway and accumulate high intracellular levels of farnesyl diphosphate (FPP), a key intermediate in sesquiterpene biosynthesis; the diversion of this intermediate for high-level production of diverse sesquiterpene hydrocarbons in lines engineered with terpene synthase genes; and finally the functional hydroxylation of a sesquiterpene scaffold by coexpression of a cognate cytochrome P450 hydroxylase and cytochrome P450 reductase. Such developments support long range objectives to facilitate functional characterization of putative terpene biosynthetic genes identified in genomic sequencing efforts (Fazio et al, 2004;Shibuya et al, 2006;Wu et al, 2005), to generate large quantities of end-product terpenes sufficient for detailed chemical analyses and diverse biological and industrial testing, as well as to combine tools for molecular evolution of terpene biosynthetic enzymes (Greenhagen, 2003;Greenhagen et al, 2006;Yoshikuni et al, 2006) with a production platform suitable for the molecular dissection of catalytic activities and the discovery of novel terpene compounds.…”

Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha

“…In earlier work aimed at the functional characterization of putative terpene synthase genes in Arabidopsis thaliana, Wu et al (2005) and Tholl et al (2005) described a sesquiterpene synthase catalyzing the biosynthesis of a-barbatene, thujopsene, and b-chamigrene. While the observation of terpene synthases capable of generating more than one reaction product was not unusual (Degenhardt et al, 2009), the particular mix of sesquiterpene reaction products was unexpected.…”

“…While the observation of terpene synthases capable of generating more than one reaction product was not unusual (Degenhardt et al, 2009), the particular mix of sesquiterpene reaction products was unexpected. These particular sesquiterpenes are commonly found in liverworts rather than in vascular plants (Wu et al, 2005), leaving the impression that at least some of the vascular plant terpene synthases could have arisen from an ancestral gene in common with liverworts. Hence, the aim of this work was to first identify the terpene synthase genes within the model liverwort species M. polymorpha and then to assess the phylogenetic relationships between the vascular plants and liverwort genes with hopes of uncovering peptide residues or domains that might mediate various facets of their catalytic specificity.…”

Novel intron-containing luciferase genes for quantitative analysis of mRNA levels in transient gene expression assays

“…These methods are also applicable to cDNA derived from unspliced bacterial or plant mRNA. The introduction of eukaryotic introns into reporter genes can be used to enhance reporter gene expression (Le Hir et al 2003), to shut off reporter translation within Agrobacterium cells (of mRNA leakily transcribed from plant promoters within bacterial cells) (Vancanneyt et al 1990), and to isolate spliced cDNA from transiently expressed genomic clones (Wu et al 2005). Here, we introduced introns to repress the PCR amplification from contaminating DNA and unspliced mRNA.…”