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As a natural sesquiterpene compound with numerous biological activities, patchoulol has extensive applications in the cosmetic industry and potential usage in pharmaceuticals. Although several patchoulol-producing microbial strains have been constructed, the low productivity still hampers large-scale fermentation. Escherichia coli possesses the ease of genetic manipulation and simple nutritional requirements and does not comprise competing pathways for the farnesyl diphosphate (FPP) precursor, showing its potential for patchoulol biosynthesis. Here, combinatorial strategies were applied to produce patchoulol in E. coli. The initial strain was constructed, and it produced 14 mg/L patchoulol after fermentation optimization. Patchoulol synthase (PTS) was engineered by semirational design, resulting in improved substrate binding affinity and a patchoulol titer of 40.3 mg/L; the patchoulol titer reached 66.2 mg/L after fusing of PTS with FPP synthase. To further improve the patchoulol production, the genome of an efficient chassis strain was engineered by deleting the competitive routes for acetate, lactate, ethanol, and succinate synthesis and cumulatively enhancing the expression of efflux transporters, which improved patchoulol production to 338.6 mg/L. When tested in a bioreactor, the patchoulol titer and productivity were further improved to 970.1 mg/L and 199 mg/L/d, respectively, and were among the highest levels reported using mineral salt medium.

Patchoulol is a tricyclic sesquiterpene widely used in perfumes and cosmetics. Herein, comprehensive engineering strategies were employed to construct an efficient yeast strain for patchoulol production. First, a platform strain was constructed via pathway modification. Second, three off-pathway genes were deleted, which led to significant physiological changes in yeast. Further, strengthening of the ergosterol pathway, enhancement of the energy supply, and a decrease in intracellular reactive oxygen species were implemented to improve the physiological status of yeast, demonstrating a new promotive relationship between ergosterol biosynthesis and synthesis of patchoulol. Moreover, patchoulol synthase was improved through protein modification and Mg2+ addition, reaching a final titer of 141.5 mg/L in a shake flask. Finally, a two-stage fermentation with dodecane addition was employed to achieve the highest production (1632.0 mg/L, 87.0 mg/g dry cell weight, 233.1 mg/L/d) ever reported for patchoulol in a 5 L bioreactor. This work lays a foundation for green and efficient patchoulol production.

Patchoulol is a sesquiterpene alcohol found in the leaves of the patchouli plant that can be extracted by steam distillation. Notably, patchoulol is an essential natural product frequently used in the chemical industry. However, patchouli produces an insignificant amount of patchoulol, not to mention steam distillation, and requires a lot of energy and time. Recombinant microorganisms that can be cultured in mild conditions and can produce patchoulol from renewable biomass resources may be a promising alternative. We previously developed the global metabolic engineering strategy (GMES), which produces a comprehensive metabolic modification in yeast, using the cocktail δ-integration method. In this study, we aimed to produce patchoulol by modifying engineered yeast. The expression of nine genes involved in patchoulol synthesis was modulated using GMES. Regarding patchoulol production, the resultant strain, YPH499/PAT167/MVA442, showed a concentration of 42.1 mg/L, a production rate of 8.42 mg/L/d, and a yield of 2.05 mg/g-glucose, respectably. These concentration values, production rate, and yield obtained through batch-fermentation in this study were high level when compared to previously reported recombinant microorganism studies. GMES could be used as a potential strategy for producing secondary metabolites from plants in recombinant Saccharomyces cerevisiae.

Patchoulol, a natural sesquiterpene compound, is widely used in perfumes and cosmetics. Several strategies were adopted to enhance patchoulol production in Saccharomyces cerevisiae: (i) farnesyl pyrophosphate (FPP) synthase and patchoulol synthase were fused to increase the utilization of FPP precursor; (ii) expression of the limiting genes of the mevalonate pathway was enhanced; (iii) squalene synthase was weakened by a glucose-inducible promoter of HXT1 (promoter for hexose transporter) to reduce metabolic flux from FPP to ergosterol; and (iv) farnesol biosynthesis was inhibited to decrease the consumption of FPP. Glucose was used to balance the trade-off between the competitive squalene and patchoulol pathways. The patchoulol production was 59.2 ± 0.7 mg/L in a shaken flask with a final production of 466.8 ± 12.3 mg/L (20.5 ± 0.5 mg/g dry cell weight) combined with fermentation optimization, which was 7.8-fold higher than the reported maximum production. The work significantly promoted the industrialization process of patchoulol production using biobased microbial platforms.

The exquisite chemodiversity of terpenoids is the product of the large diverse terpene synthase (TPS) superfamily. Here, by using structural and phylogenetic analyses and site-directed mutagenesis, we identified a residue (Cys440 in Nicotiana tabacum 5-epi-aristolochene synthase) proximal to an ion-binding motif common to all TPSs and named the preNSE/DTE residue, which determines the product specificity of sesquiterpene synthases from different plant species. In sesquiterpene synthases catalyzing 1,10-cyclization (1,10-cyclases) of farnesyl diphosphate, mutation of the residue in both specific and promiscuous 1,10-cyclases from different lineages leads to the accumulation of monocyclic germacrene A-11-ol, which is "short-circuited" from complex cyclization cascades, suggesting a key role of this residue in generating the first common intermediate of 1,10-cyclization. Altering this residue in a specific 1,11-cyclase results in alternative 1,10-cyclization products. Moreover, the preNSE/DTE residue can be harnessed to engineer highly specific sesquiterpene synthases for an improved proportion of high-value terpenoids, such as patchoulol, a main constituent of several traditional Chinese medicines that could treat SARS-CoV-2.

Root-knot nematode, Meloidogyne incognita is one of the most destructive nematodes worldwide. Essential oils (EOs) are being extensively utilized as eco-benign bionematicides, although the precise mechanism of action remains unclear. Pogostemon cablin Benth. is well-known as "Patchouli". It is native to South East Asia and known for ethno-pharmacological properties. In this study, chemical composition and potential nematicidal effect of EOs hydrodistilled from the leaves of P. cablin grown at three different locations in India were comprehensively investigated to correlate their mechanism of action for target specific binding affinities toward nematode proteins. Aromatic volatile Pogostemon essential oils (PEO) from Northern India (PEO-NI), Southern India (PEO-SI) and North Eastern India (PEO-NEI) were analyzed by Gas Chromatography-Mass Spectrometry (GC/MS) to characterize forty volatile compounds. Maximum thirty-three components were identified in PEO-NEI. Sesquiterpenes were predominant with higher content of α-guaiene (2.3-24.4 %), patchoulol (6.1-32.7 %) and α-bulnesene (5.9-27.1 %). Patchoulol was the major component in PEO-SI (32.7±1.2 %) and PEO-NEI (29.2±1.1 %), while α-guaiene in PEO-NI (24.4±1.2 %). In vitro nematicidal assay revealed significant nematicidal action (LC50 44.6-87.0 μg mL-1 ) against juveniles of M. incognita within 24 h exposure. Mortality increases with increasing time to 48 h (LC50 33.6-71.6 μg mL-1 ) and 72 h (LC50 27.7-61.2 μg mL-1 ). Molecular modelling and in silico studies revealed multi-modal inhibitive action of α-bulnesene (-22 to -13 kJ mol-1 ) and α-guaiene (-22 to -12 kJ mol-1 ) against three target proteins namely, acetyl cholinesterase (AChE), odorant response gene-1 (ODR1), odorant response gene-3 (ODR3). Most preferable binding mechanism was observed against AChE due to pi-alkyl, pi-sigma, and hydrophobic interactions. Structure nematicidal activity relationship suggested the presence of hydroxy group for nematicidal activity is nonessential, rather highly depends on synergistic composition of sesquiterpene hydrocarbons.

Terpenes constitute the largest class of secondary metabolites in plants. Some terpenes are essential for plant growth and development, membrane components, and photosynthesis. Terpenes are also economically useful for industry, agriculture, and pharmaceuticals. However, there is very low content of most terpenes in microbes and plants. Chemical or microbial synthesis of terpenes are often costly. Plants have the elaborate and economic biosynthetic way of producing high-value terpenes through photosynthesis. Here we engineered the heterogenous sesquiterpenoid patchoulol production in A. annua. When using a strong promoter such as 35S to over express the avian farnesyl diphosphate synthase gene and patchoulol synthase gene, the highest content of patchoulol was 52.58 μg/g DW in transgenic plants. When altering the subcellular location of the introduced sesquiterpene synthetase via a signal peptide, the accumulation of patchoulol was observably increased to 273 μg/g DW. This case demonstrates that A. annua plant with glandular trichomes is a useful platform for synthetic biology studies.

The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes by conversion of the natural substrate farnesyl pyrophosphate (FPP). PTS has the potential to be used as a biocatalyst for the production of valuable sesquiterpenes such as (-)-patchoulol. The objective of the present study is to develop an efficient biotransformation and to characterize the biocatalytic mechanism of the PTS in detail. For this purpose, soluble PTS was prepared using an optimized cultivation protocol and continuous downstream process with a purity of 98%. The PTS biotransformation was then optimized regarding buffer composition, pH-value, and temperature for biotransformation as well as functional and kinetic properties to improve productivity. For the bioconversion of FPP, the highest enzyme activity was reached with the 2-(N-morphlino)ethanesulfonic acid (MES) buffer containing 10% (v/v) glycerol and 10 mM MgCl2 at pH 6.4 and 34°C. The PTS showed an unusual substrate inhibition for sesquiterpene synthases indicating an intermediate sesquiterpene formed in the active center. Deuteration experiments were used to gain further insights into the biocatalytic mechanism described in literature. Thus it could be shown that a second substrate binding site must be responsible for substrate inhibition and that further protonation and deprotonation steps are involved in the reaction mechanism.

Plant-derived terpenoids are extensively used in perfume, food, cosmetic and pharmaceutical industries, and several attempts are being made to produce terpenes in heterologous hosts. Native hosts have evolved to accumulate large quantities of terpenes in specialized cells. However, heterologous cells lack the capacity needed to produce and store high amounts of non-native terpenes, leading to reduced growth and loss of volatile terpenes by evaporation. Here, we describe how to direct the sesquiterpene patchoulol production into cytoplasmic lipid droplets (LDs) in Physcomitrium patens (syn. Physcomitrella patens), by attaching patchoulol synthase (PTS) to proteins linked to plant LD biogenesis. Three different LD-proteins: Oleosin (PpOLE1), Lipid Droplet Associated Protein (AtLDAP1) and Seipin (PpSeipin325) were tested as anchors. Ectopic expression of PTS increased the number and size of LDs, implying an unknown mechanism between heterologous terpene production and LD biogenesis. The expression of PTS physically linked to Seipin increased the LD size and the retention of patchoulol in the cell. Overall, the expression of PTS was lower in the anchored mutants than in the control, but when normalized to the expression the production of patchoulol was higher in the seipin-linked mutants.

The constituent of Cananga oil is caryophyllene, linalool, and germacrene D, and the main constituent of patchouli oil is the sesquiterpene patchoulol, all have a repellent effect. Moreover, the patchouli oil has a fixative property. The combination of cananga and patchouli oil expected to give a more durable repellent effect. This study conducted to investigate the characteristics and the effectiveness of repellent lotion that contained cananga and patchouli oil. Made lotions that contained cananga oil 7.5% combined with patchouli oil 1%, 2%, and 3%. The characteristic test consists of organoleptic, homogeneity, emulsion type, viscosity, pH, and stability with the freeze-thaw cycle method. The activity test as a repellent using Aedes aegypti mosquitoes for 150 minutes, each period for 30minutes with 3minute exposure. The repellent lotion all the formula has the same characteristic, they are homogeneous, soft, aromatic textured liquid, an oil-in-water type emulsion, and easily dispersed, they have a good pH and viscosity, remain stable after the freeze-thaw cycle testing. While the result repellent test showed the protective power all formula is not different (Two-way ANOVA; a=0.05), they are effective as a repellent for 60 minutes.

Patchoulol is a sesquiterpene alcohol and an important natural product for the perfume industry. Corynebacterium glutamicum is the prominent host for the fermentative production of amino acids with an average annual production volume of ~6 million tons. Due to its robustness and well established large-scale fermentation, C. glutamicum has been engineered for the production of a number of value-added compounds including terpenoids. Both C40 and C50 carotenoids, including the industrially relevant astaxanthin, and short-chain terpenes such as the sesquiterpene valencene can be produced with this organism. In this study, systematic metabolic engineering enabled construction of a patchoulol producing C. glutamicum strain by applying the following strategies: (i) construction of a farnesyl pyrophosphate-producing platform strain by combining genomic deletions with heterologous expression of ispA from Escherichia coli; (ii) prevention of carotenoid-like byproduct formation; (iii) overproduction of limiting enzymes from the 2-c-methyl-d-erythritol 4-phosphate (MEP)-pathway to increase precursor supply; and (iv) heterologous expression of the plant patchoulol synthase gene PcPS from Pogostemon cablin. Additionally, a proof of principle liter-scale fermentation with a two-phase organic overlay-culture medium system for terpenoid capture was performed. To the best of our knowledge, the patchoulol titers demonstrated here are the highest reported to date with up to 60 mg L−1 and volumetric productivities of up to 18 mg L−1 d−1.

Patchoulol is a sesquiterpene alcohol and an important natural product for the perfume industry. Corynebacterium glutamicum is the prominent host for the fermentative production of amino acids with an average annual production volume of ~6 million tons. Due to its robustness and well established large-scale fermentation, C. glutamicum has been engineered for the production of a number of value-added compounds including terpenoids. Both C40 and C50 carotenoids, including the industrially relevant astaxanthin, and short-chain terpenes such as the sesquiterpene valencene can be produced with this organism. In this study, systematic metabolic engineering enabled construction of a patchoulol producing C. glutamicum strain by applying the following strategies: (i) construction of a farnesyl pyrophosphate-producing platform strain by combining genomic deletions with heterologous expression of ispA from Escherichia coli; (ii) prevention of carotenoid-like byproduct formation; (iii) overproduction of limiting enzymes from the 2-c-methyl-d-erythritol 4-phosphate (MEP)-pathway to increase precursor supply; and (iv) heterologous expression of the plant patchoulol synthase gene PcPS from Pogostemon cablin. Additionally, a proof of principle liter-scale fermentation with a two-phase organic overlay-culture medium system for terpenoid capture was performed. To the best of our knowledge, the patchoulol titers demonstrated here are the highest reported to date with up to 60 mg L−1 and volumetric productivities of up to 18 mg L−1 d−1.

The patchoulol synthase (PTS) is a multi-product sesquiterpene synthases which is the central enzyme for biosynthesis of patchouli essential oil in the patchouli plant. Sesquiterpene synthases catalyse the formation of various complex carbon backbones difficult to approach by organic synthesis. Here, we report the characterisation of a recombinant patchoulol synthase complementary DNA (cDNA) variant (PTS var. 1), exhibiting significant amino acid exchanges compared to the native PTS. The product spectrum using the natural substrate E,E-farnesyl diphosphate (FDP) as well as terpenoid products resulting from conversions employing alternative substrates was analysed by GC-MS. In respect to a potential use as a biocatalyst, important enzymatic parameters such as the optimal reaction conditions, kinetic behaviour and the product selectivity were studied as well. Adjusting the reaction conditions, an increased patchoulol ratio in the recombinant essential oil was achieved. Nevertheless, the ratio remained lower than in plant-derived patchouli oil. As alternative substrates, several prenyl diposphates were accepted and converted in numerous compounds by the PTS var. 1, revealing its great biocatalytic potential. The final publication is available at Springer via http://dx.doi.org/10.1007/s12010-015-1707-y EFRE/ZW-8-80130940

The leaves of Pogostemon cablin (Blanco) Benth. (Lamiaceae) are the source of patchouli essential oil, which is - with an annual production of about 1300 tonnes - an important and unique commodity in the fragrance industry. All the literature pertaining to patchouli was critically reviewed with an emphasis on the qualitative and quantitative chemical analysis of the oil but also harvesting, fermentation, drying, distillation, used analytical techniques, sensory aspects including molecules responsible for the odour, adulteration and toxicological aspects, i.e., skin sensitisation, are discussed. In total 72 constituents have been convincingly identified in the oil and another 58 tentatively. The main constituent is the sesquiterpene patchoulol. For this review over 600 papers were consulted and in the supplementary information all patchouli-related references not relevant enough to be cited in the paper itself are listed.

The sesquiterpene alcohols cis-nerolidol, α-bisabolol, cedrol, patchoulol, and santalol were screened for cytotoxicity and cytoproliferative activity on HeLa cell using Cytotoxicity Detection Kit (LDH) and the Cell Proliferation Reagent WST-1, respectively. Actinomycin D and curcumin as well as the plant acids geranic and salicylic acid were used as reference samples, all known for their cytotoxic properties. Among the sesquiterpene alcohols studied, cis-nerolidol possessed the strongest cytotoxic properties (16.5 μM ± 6.7). Low cytotoxicity and influence on HeLa cell viability was demonstrated for geranic and especially salicylic acid.

Incubations of isotopically pure [2-(2)H(1)](E,E)-farnesyl diphosphate with recombinant patchoulol synthase (PTS) from Pogostemon cablin afforded a 65:35 mixture of monodeuterated and dideuterated patchoulols as well as numerous sesquiterpene hydrocarbons. Extensive NMR analyses ((1)H and (13)C NMR, (1)H homodecoupling NMR, HMQC, and (2)H NMR) of the labeled patchoulol mixture and comparisons of the spectra with those of unlabeled alcohol led to the conclusion that the deuterium label was located at positions (patchoulol numbering system) C5 (both isotopomers, ca. 100%) and C12 (minor isotopomer, 30-35%), that is, an approximately 2:1 mixture of [5-(2)H(1)]- and [5,12-(2)H(2)]-patchoulols. Low-resolution FIMS analyses and isotope ratio calculations further corroborated the composition of the mixture as mainly one singly deuterated and one doubly deuterated patchoulol. From a mechanistic point of view, the formation of [5,12-(2)H(2)]patchoulol is rationalized through the intermediacy of an unknown exocyclic [7,10:1,5]patchoul-4(12)-ene (15-d(1)), which could incorporate a deuteron at the C-12 position on the pathway to doubly labeled patchoulol. The corresponding depletion of deuterium content observed in the hydrocarbon coproducts, beta-patchoulene and alpha-guaiene (55% d(0)), identified the source of the excess label found in patchoulol-d(2). Comparison of the PTS amino acid sequence with those of other sesquiterpene synthases, and examination of an active site model, suggested that re-orientation of leucine 410 side chain in PTS might facilitate the creation of a 2-pocket active site where the observed deuteron transfers could occur. The retention of deuterium at C5 in the labeled patchoulol and its absence at C4 rule out an alternative mechanism involving two consecutive 1,2-hydride shifts and appears to confirm the previously proposed occurrence of a 1,3-hydride shift across the 5-membered ring. A new, semisystematic nomenclature is presented for the purpose of distinguishing the three different skeletal structures of the patchoulane sesquiterpenes.

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Plant metabolites vary at different stages of their life cycle. Although it is well documented that environmental factors stimulate biosynthesis of secondary metabolites, the regulation by endogenous developmental cues remains poorly understood. The microRNA156 (miR156)-targeted squamosa promoter binding protein-like (SPL) factors function as a major age cue in regulating developmental phase transition and flowering. We show here that the miR156-targeted SPL transcription factor plays an important role in the spatiotemporal regulation of sesquiterpene biosynthesis. In Arabidopsis thaliana, the miR156-SPL module regulates the formation of (E)-β-caryophyllene in the flowering stage through modulating expression of the sesquiterpene synthase gene TPS21. We demonstrated that SPL9 directly binds to TPS21 promoter and activates its expression. In the perennial fragrant herb Pogostemon cablin, the accumulation of patchouli oil, largely composed of sesquiterpenes dominated by (-)-patchoulol, is also age-regulated, and the SPL promotes biosynthesis of sesquiterpenes in elder plants by upregulating patchoulol synthase (PatPTS) gene expression. As miR156-SPLs are highly conserved in plants, our finding not only uncovers a molecular link between developmental timing and sesquiterpene production but also suggests a new strategy to engineer plants for accelerated growth with enhanced production of terpenoids.

The antifungal activity of natural sesquiterpenoids patchoulol, ginsenol, cedrol and globulol against the fungus Botrytis cinerea has been determined. The diminishing of the effect after 3–6 days suggests that a mechanism of detoxification is present. Biotransformation of these fungistatic compounds has been investigated as a method of studying this mechanism.

The microbiological hydroxylation of patchoulol by Mucor plumbeus gave the 5α- and 9α-hydroxy derivatives. The 5α-hydroxy compound was also obtained using Cephalosporium aphidicola and its structure was established by X-ray crystallography.

The methyl-polysiloxane phase “Chirasil-Val” containing about 6% branched aliphatic side chains with l -valine in diamide linkage, has had its response assessed to various volatile oil constituents. Changes in relative retention times with temperature increase were distinctively large for camphor and fenchone. There was a greater spread of retention indices for seven solutes than on fully methyl polysiloxane, although less difference for another three solutes. Isothermally at 130°C, Chirasil-Val gave better resolution of the sesquiterpene hydrocarbon mix in patchouli oil than methyl polysiloxane, and could then be temperature programmed to evaluate patchoulol, etc. Carbowax 20M is a poor phase for patchouli analysis.

The sesquiterpene cyclase, patchoulol synthase, from Pogostemon cablin (patchouli) leaves was purified to apparent homogeneity by chromatofocusing, anion exchange, gel permeation, and hydroxylapatite chromatography. The enzyme showed a maximum specific activity of about 20 nmol/min/mg protein, and a native molecular weight of 80,000 as determined by gel permeation chromatography. The protein was very hydrophobic, as judged by chromatographic behavior on several matrices, and possessed a pI value of about 5.0, as determined by isoelectric and chromatofocusing. SDS-PAGE showed the enzyme to be composed of two apparently identical subunits of Mr approximately 40,000. Maximum activity was observed at pH 6.7 in the presence of Mg2+ (Km approximately 1.7 mM); other divalent metal ions were ineffective in promoting catalysis. The Km value for the substrate, farnesyl pyrophosphate, was 6.8 microM. Patchoulol synthase copurified with the ability to transform farnesyl pyrophosphate to cyclic olefins (alpha- and beta-patchoulene, alpha-bulnesene, and alpha-guiaene) and this observation, plus evidence based on differential inhibition and inactivation studies, suggested that these structurally related products are synthesized by the same cyclase enzyme. In general properties, the patchoulol synthase from patchouli leaves resembles fungal sesquiterpene olefin cyclases except for the ability to synthesize multiple products, a property more typical of monoterpene cyclases of higher plant origin.

In continuation of studying structure/activity relationships of odorous compounds, the influence of the bridgehead-bonded Me group of (+)-norpatchoulenol (1) and (−)-patchoulol (2) and the olfactory properties of the corresponding unsaturated and saturated derivatives (±)−3, (±)−4 and (±)−5, (±)−6, respectively, are studied. The key odour descriptors-wood, earth, and camphor-are used for classification.

α'-Methylation of (−)-carvone followed by bicycloannulation with vinyltriphenylphosphonium bromide gives a 59% yield of a tricycio[3.2.1.0 2,7 ]octan-6-one which can be converted to precursors of (−)-patchouli alcohol, (−)-seychellene and (−)-cycloseycbellene.

The yeast Saccharomyces cerevisiae was chosen as a microbial host for heterologous biosynthesis of three different plant sesquiterpenes, namely valencene, cubebol, and patchoulol. The volatility and low solubility of the sesquiterpenes were major practical problems for quantification of the excreted sesquiterpenes. In situ separation of sesquiterpenes in a two-phase fermentation using dodecane as the secondary phase was therefore performed in order to enable quantitative evaluation of different strains. In order to enhance the availability of the precursor for synthesis of sesquiterpenes, farnesyl diphosphate (FPP), the ERG9 gene which is responsible for conversion of FPP to squalene was downregulated by replacing the native ERG9 promoter with the regulatable MET3 promoter combined with addition of 2 mM methionine to the medium. This strategy led to a reduced ergosterol content of the cells and accumulation of FPP derived compounds like target sesquiterpenes and farnesol. Adjustment of the methionine level during fermentations prevented relieving MET3 promoter repression and resulted in further improved sesquiterpene production. Thus, the final titer of patchoulol and farnesol in the ERG9 downregulated strain reached 16.9 and 20.2 mg/L, respectively. The results obtained in this study revealed the great potential of yeast as a cell factory for production of sesquiterpenes.

The solubility of the sesquiterpene alcohol patchoulol in supercritical carbon dioxide was measured at pressures (P) ranging from 10.0 MPa to 25.0 MPa and temperatures (T) of 40.0 and 50.0 °C using a simple microsampling-type apparatus with a 100.5 μL sample loop to remove aliquots for off-line analysis. The system was first validated using vanillin with off-line spectrophotometric analysis, then utilized for patchoulol measurements with off-line GC−MS analysis. The measured solubility of patchoulol in supercritical CO2 ranged from mole fractions of 0.43 × 10-3 at 10.0 MPa and 50.0 °C to mole fractions of 9.4 × 10-3 at 25.0 MPa and 40.0 °C.

Pogostemon cablin (patchouli), like many plants within the Lamiaceae, accumulates large amounts of essential oil. Patchouli oil is unique because it consists of over 24 different sesquiterpenes, rather than a blend of different mono-, sesqui- and di-terpene compounds. To determine if this complex mixture of sesquiterpenes arises from an equal number of unique sesquiterpene synthases, we developed a RT-PCR strategy to isolate and functionally characterize the respective patchouli oil synthase genes. Unexpectedly, only five terpene synthase cDNA genes were isolated. Four of the cDNAs encode for synthases catalyzing the biosynthesis of one major sesquiterpene, including a gamma-curcumene synthase, two germacrene D synthases, and a germacrene A synthase. The fifth cDNA encodes for a patchoulol synthase, which catalyzes the conversion of FPP to patchoulol plus at least 13 additional sesquiterpene products. Equally intriguing, the yield of the different in vitro reaction products resembles quantitatively and qualitatively the profile of sesquiterpenes found in patchouli oil extracted from plants, suggesting that a single terpene synthase is responsible for the bulk and diversity of terpene products produced in planta.

Biotransformation of the fungistatic sesquiterpenoid patchoulol (1) by the fungus Botrytis cinerea affords the 5-, 7- and (8R)-hydroxy (2, 3, and 5) derivatives as the major metabolites, together with a number of minor metabolites (4, 6-9) arising from hydroxylation at C-2, C-3, C-5, C-9, C-13, and C-14.

Several mechanistic alternatives have been proposed for the enzyme-catalyzed, electrophilic cyclization of farnesyl pyrophosphate to the tricyclic sesquiterpene alcohol patchoulol, which is the characteristic component of the essential oil of Pogostemam cablin (patchouli). These alternatives include schemes involving deprotonation-reprotonation steps and the intermediacy of the monocyclic and bicyclic olefins germacrene and bulnesene, respectively, and involving a 1,3-hydride shift with only tertiary cationic intermediates and without any deprotonation-reprotonation steps. Analytical studies, based on analyses of P. cablin leaf oil at different stages of plant development, and in vivo time-course investigations, using14CO2 and [14C]sucrose, gave no indication that germacrene and bulnesene were intermediates in patchoulol biosynthesis. A soluble enzyme system from P. cablin leaves was prepared, which was capable of converting farnesyl pyrophosphate to patchoulol, and isotopic dilution experiments with both labeled and unlabeled olefins were carried out with this system to confirm that sesquiterpene olefins did not participate as free intermediates in the transformation of the acyclic precursor to patchoulol. Patchoulol derived biosynthetically from [12,13-14C;1-3H]farnesyl pyrophosphate was chemically degraded to establish the overall construction pattern of the product. Similar studies with 12,13-14C;6-3H farnesyl pyrophosphate as a precursor eliminated deprotonation steps to form bound olefinic intermediates in the biosynthesis of patchoulol, while providing supporting evidence for the hydride shift mechanism.

Of some 350 microorganisms screened, four strains of Pithomyces species were found to carry out regio-selective hydroxylation of patchoulol, a sesquiterpene, to 10-hydroxypatchoulol: Pithomyces sp. NRJ201, P. chartarum NRJ210, and, to a lesser extent, P. cynodontis ATCC 26150 and P. atro-olivaceus IFO 6651 were found to catalyze this reaction. A method has been developed by which 10-hydroxypatchoulol was obtained in 25 to 45% yields in 1- to 5-liter fermentation jars at 2 to 4 g of patchoulol per liter and isolated as pure material in 30% yields.

Sesquiterpenoids are a large class of natural compounds offering manifold properties valuable for food, cosmetics, agriculture and pharma industry. Production in microorganisms is a sustainable approach to provide sesquiterpenoids for research and industrial use independent of their natural sources. This requires the functional transfer of the respective biocatalytic pathways in an adequate host microorganism offering a sufficient supply of precursors that is ideally adjusted to the individual demand of the recombinant biosynthesis route. The phototrophic purple bacterium Rhodobacter capsulatus offers unique physiological properties that are favorable for biosynthesis of hydrophobic terpenes. Under phototrophic conditions, it develops a large intracytoplasmic membrane suitable for hosting membrane-bound enzymes and metabolites of respective biosynthetic pathways. In addition, Rhodobacter harbors an intrinsic carotenoid biosynthesis that can be engineered towards the production of foreign terpenes. Here, we evaluate R. capsulatus as host for the production of plant sesquiterpenoids under phototrophic conditions using patchoulol and valencene as a proof of concept. The heterologous expression of patchoulol synthase PcPS from Pogostemon cablin as well as the valencene synthases CsVS from Citrus sinensis and CnVS from Callitropsis nootkatensis led to the production of the respective sesquiterpenoids in R. capsulatus. To analyze, if gradually adjustable formation of the key precursor farnesylpyrophosphate (FPP) is beneficial for sesquiterpene synthesis under phototrophic conditions, the intrinsic 1-deoxy-D-xylulose 5-phosphate (DXP) pathway genes as well as the heterologous mevalonate pathway genes were modularly expressed in various combinations. To this end, different plasmids and chromosomally integrated expression tools were developed harboring the strong and tightly controlled Pnif promoter for heterologous gene expression. Notably, comparative studies identified a distinct combination of precursor biosynthetic genes as best-performing setup for each of the tested sesquiterpene synthases. In summary, we could demonstrate that R. capsulatus is a promising alternative platform organism that is suited for sustainable sesquiterpenoid formation under phototrophic cultivation conditions. A modular engineering of R. capsulatus strains via tailored co-expression of FPP biosynthetic genes further allowed adaptation of sesquiterpene precursor formation to its catalytic conversion by different plant terpene synthases.