Agronomic and chemical performance of field-grown tobacco engineered for triterpene and methylated triterpene metabolism.

Squalene is a linear intermediate to nearly all classes of triterpenes and sterols and is itself highly valued for its use in wide range of industrial applications. Another unique linear triterpene is botryococcene and its methylated derivatives generated by the alga Botryococcus braunii race B, which are progenitors to fossil fuel deposits. Production of these linear triterpenes was previously engineered into transgenic tobacco by introducing the key steps of triterpene metabolism into the particular subcellular compartments. In this study, the agronomic characteristics (height, biomass accumulation, leaf area), the photosynthetic capacity (photosynthesis rate, conductance, internal CO2 levels) and triterpene content of select lines grown under field conditions were evaluated for three consecutive growing seasons. We observed that transgenic lines targeting enzymes to the chloroplasts accumulated 50-150 times more squalene than the lines targeting the enzymes to the cytoplasm, without compromising growth or photosynthesis. We also found that the transgenic lines directing botryococcene metabolism to the chloroplast accumulated 10- to 33-fold greater levels than the lines where the same enzymes were targeted to in the cytoplasm. However, growth of these high botryococcene accumulators was highly compromised, yet their photosynthesis rates remained unaffected. In addition, in the transgenic lines targeting a triterpene methyltransferase (TMT) to the chloroplasts of high squalene accumulators, 55%-65% of total squalene was methylated, whereas in the lines expressing a TMT in the cytoplasm, only 6%-13% of squalene was methylated. The growth of these methylated triterpene-accumulating lines was more compromised than that of nonmethylated squalene lines.

Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha.

Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono- and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases.

Regulation of sesquiterpenoid metabolism in recombinant and elicited Valeriana officinalis hairy roots.

The medicinal properties of Valerian (Valeriana officinalis) root preparations are attributed to the anxiolytic sesquiterpenoid valerenic acid and its biosynthetic precursors valerenal and valerenadiene, as well as the anti-inflammatory sesquiterpenoid ß-caryophyllene. In order to study and engineer the biosynthesis of these pharmacologically active metabolites, a binary vector co-transformation system was developed for V. officinalis hairy roots. The relative expression levels and jasmonate-inducibility of a number of genes associated with sesquiterpenoid metabolism were profiled in roots: farnesyl pyrophosphate synthase (VoFPS), valerendiene synthase (VoVDS), germacrene C synthase (VoGCS), and a cytochrome P450 (CYP71D442) putatively associated with terpene metabolism based on sequence homology. Recombinant hairy root lines overexpressing VoFPS or VoVDS were generated and compared to control cultures. Overexpression of the VoFPS cDNA increased levels of the corresponding transcript 4- to 8-fold and sesquiterpene hydrocarbon accumulation by 1.5- to 4-fold. Overexpression of the VoVDS cDNA increased the corresponding transcript levels 5- to 9-fold and markedly increased yields of the oxygenated sesquiterpenoids valerenic acid and valerenal. Our findings suggest that the availability of cytoplasmic farnesyl diphosphate and valerenadiene are potential bottlenecks in Valeriana-specific sesquiterpenoid biosynthesis, which is also subject to regulation by methyl jasmonate elicitation.

Functional identification of triterpene methyltransferases from Botryococcus braunii race B.

Botryococcus braunii race B is a colony-forming, green algae that accumulates triterpene oils in excess of 30% of its dry weight. The composition of the triterpene oils is dominated by dimethylated to tetramethylated forms of botryococcene and squalene. Although unusual mechanisms for the biosynthesis of botryococcene and squalene were recently described, the enzyme(s) responsible for decorating these triterpene scaffolds with methyl substituents were unknown. A transcriptome of B. braunii was screened computationally assuming that the triterpene methyltransferases (TMTs) might resemble the S-adenosyl methionine-dependent enzymes described for methylating the side chain of sterols. Six sterol methyltransferase-like genes were isolated and functionally characterized. Three of these genes when co-expressed in yeast with complementary squalene synthase or botryococcene synthase expression cassettes resulted in the accumulation of mono- and dimethylated forms of both triterpene scaffolds. Surprisingly, TMT-1 and TMT-2 exhibited preference for squalene as the methyl acceptor substrate, whereas TMT-3 showed a striking preference for botryococcene as its methyl acceptor substrate. These in vivo preferences were confirmed with in vitro assays utilizing microsomal preparations from yeast overexpressing the respective genes, which encode for membrane-associated enzymes. Structural examination of the in vivo yeast generated mono- and dimethylated products by NMR identified terminal carbons, C-3 and C-22/C-20, as the atomic acceptor sites for the methyl additions to squalene and botryococcene, respectively. These sites are identical to those previously reported for the triterpenes extracted from the algae. The availability of closely related triterpene methyltransferases exhibiting distinct substrate selectivity and successive catalytic activities provides important tools for investigating the molecular mechanisms responsible for the specificities exhibited by these unique enzymes.