Structurally Guided Reprogramming of Valerenadiene Synthase.

Valerena-1,10-diene synthase (VDS) catalyzes the conversion of the universal precursor farnesyl diphosphate into the unusual sesquiterpene valerena-1,10-diene (VLD), which possesses a unique isobutenyl substituent group. In planta, one of VLD's isobutenyl terminal methyl groups becomes oxidized to a carboxylic acid forming valerenic acid (VA), an allosteric modulator of the GABAA receptor. Because a structure-activity relationship study of VA for its modulatory activity is desired, we sought to manipulate the VDS enzyme for the biosynthesis of structurally diverse scaffolds that could ultimately lead to the generation of VA analogues. Using three-dimensional structural homology models, phylogenetic sequence comparisons to well-characterized sesquiterpene synthases, and a substrate-active site contact mapping approach, the contributions of specific amino acid residues within or near the VDS active site to possible catalytic cascades for VLD and other sesquiterpene products were assessed. An essential role of Tyr535 in a germacrenyl route to VLD was demonstrated, while its contribution to a family of other sesquiterpenes derived from a humulyl route was not. No role for Cys415 or Cys452 serving as a proton donor to reaction intermediates in VLD biosynthesis was observed. However, a gatekeeper role for Asn455 in directing farnesyl carbocations down all-trans catalytic cascades (humulyl and germacrenyl routes) versus a cisoid cascade (nerolidyl route) was demonstrated. Altogether, these results have mapped residues that establish a context for the catalytic cascades operating in VDS and future manipulations for generating more structurally constrained scaffolds.

De novo synthesis of the sedative valerenic acid in Saccharomyces cerevisiae.

Valeriana officinalis (Valerian) root extracts have been used by European and Asian cultures for millennia for their anxiolytic and sedative properties. However, the efficacy of these extracts suffers from variable yields and composition, making these extracts a prime candidate for microbial production. Recently, valerenic acid, a C15 sesquiterpenoid, was identified as the active compound that modulates the GABAA channel. Although the first committed step, valerena-4,7(11)-diene synthase, has been identified and described, the complete valerenic acid biosynthetic pathway remains to be elucidated. Sequence homology and tissue-specific expression profiles of V. officinalis putative P450s led to the discovery of a V. officinalis valerena-4,7(11)-diene oxidase, VoCYP71DJ1, which required coexpression with a V. officinalis alcohol dehydrogenase and aldehyde dehydrogenase to complete valerenic acid biosynthesis in yeast. Further, we demonstrated the stable integration of all pathway enzymes in yeast, resulting in the production of 140 mg/L of valerena-4,7(11)-diene and 4 mg/L of valerenic acid in milliliter plates. These findings showcase Saccharomyces cerevisiae's potential as an expression platform for facilitating multiply-oxidized medicinal terpenoid pathway discovery, possibly paving the way for scale up and FDA approval of valerenic acid and other active compounds from plant-derived herbal medicines.

Monoterpene biosynthesis potential of plant subcellular compartments.

Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana benthamiana indicated local GDP availability for each compartment but resulted in different product levels. A GDP synthase from Picea abies (PaGDPS1) was shown to boost GDP production. PaGDPS1 was also targeted to plastids, cytosol or mitochondria and PaGDPS1 and GES were coexpressed in all possible combinations. Geraniol and geraniol-derived products were analyzed by GC-MS and LC-MS, respectively. GES product levels were highest for plastid-targeted GES, followed by mitochondrial- and then cytosolic-targeted GES. For each compartment local boosting of GDP biosynthesis increased GES product levels. GDP exchange between compartments is not equal: while no GDP is exchanged from the cytosol to the plastids, 100% of GDP in mitochondria can be exchanged to plastids, while only 7% of GDP from plastids is available for mitochondria. This suggests a direct exchange mechanism for GDP between plastids and mitochondria. Cytosolic PaGDPS1 competes with plastidial GES activity, suggesting an effective drain of isopentenyl diphosphate from the plastids to the cytosol.

Molecular cloning and characterization of drimenol synthase from valerian plant (Valeriana officinalis).

Drimenol, a sesquiterpene alcohol, and its derivatives display diverse bio-activities in nature. However, a drimenol synthase gene has yet to be identified. We identified a new sesquiterpene synthase cDNA (VoTPS3) in valerian plant (Valeriana officinalis). Purification and NMR analyses of the VoTPS3-produced terpene, and characterization of the VoTPS3 enzyme confirmed that VoTPS3 synthesizes (-)-drimenol. In feeding assays, possible reaction intermediates, farnesol and drimenyl diphosphate, could not be converted to drimenol, suggesting that the intermediate remains tightly bound to VoTPS3 during catalysis. A mechanistic consideration of (-)-drimenol synthesis suggests that drimenol synthase is likely to use a protonation-initiated cyclization, which is rare for sesquiterpene synthases. VoTPS3 can be used to produce (-)-drimenol, from which useful drimane-type terpenes can be synthesized.

Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization.

Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed.

Functional identification of valerena-1,10-diene synthase, a terpene synthase catalyzing a unique chemical cascade in the biosynthesis of biologically active sesquiterpenes in Valeriana officinalis.

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [(13)C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes.

Enzymatic synthesis of valerena-4,7(11)-diene by a unique sesquiterpene synthase from the valerian plant (Valeriana officinalis).

Valerian (Valeriana officinalis) is a popular medicinal plant in North America and Europe. Its root extract is commonly used as a mild sedative and anxiolytic. Among dozens of chemical constituents (e.g. alkaloids, iridoids, flavonoids, and terpenoids) found in valerian root, valerena-4,7(11)-diene and valerenic acid (C15 sesquiterpenoid) have been suggested as the active ingredients responsible for the sedative effect. However, the biosynthesis of the valerena-4,7(11)-diene hydrocarbon skeleton in valerian remains unknown to date. To identify the responsible terpene synthase, next-generation sequencing (Roche 454 pyrosequencing) was used to generate ∼ 1 million transcript reads from valerian root. From the assembled transcripts, two sesquiterpene synthases were identified (VoTPS1 and VoTPS2), both of which showed predominant expression patterns in root. Transgenic yeast expressing VoTPS1 and VoTPS2 produced germacrene C/germacrene D and valerena-4,7(11)-diene, respectively, as major terpene products. Purified VoTPS1 and VoTPS2 recombinant enzymes confirmed these activities in vitro, with competent kinetic properties (K(m) of ∼ 10 µm and k(cat) of 0.01 s(-1) for both enzymes). The structure of the valerena-4,7(11)-diene produced from the yeast expressing VoTPS2 was further substantiated by (13) C-NMR and GC-MS in comparison with the synthetic standard. This study demonstrates an integrative approach involving next-generation sequencing and metabolically engineered microbes to expand our knowledge of terpenoid diversity in medicinal plants.