Linalool Dehydratase-Isomerase,a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

Castellaniella (ex Alcaligenes) defragrans strain 65Phen mineralizes monoterpenes in the absence of oxygen. Soluble cell extracts anaerobically catalyzed the isomerization of geraniol to linalool and the dehydration of linalool to myrcene. The linalool dehydratase was present in cells grown on monoterpenes, but not if grown on acetate. We purified the novel enzyme ∼1800-fold to complete homogeneity. The native enzyme had a molecular mass of 160 kDa. Denaturing gel electrophoresis revealed one single protein band with a molecular mass of 40 kDa, which indicated a homotetramer as native conformation. The aerobically purified enzyme was anaerobically activated in the presence of 2 mm DTT. The linalool dehydratase catalyzed in vitro two reactions in both directions depending on the thermodynamic driving forces: a water secession from the tertiary alcohol linalool to the corresponding acyclic monoterpene myrcene and an isomerization of the primary allylalcohol geraniol in its stereoisomer linalool. The specific activities (V_max) were 140 nanokatals mg⁻¹ for the linalool dehydratase and 410 nanokatals mg⁻¹ for the geraniol isomerase, with apparent K_m values of 750 μm and 500 μm, respectively. The corresponding open reading frame was identified and revealed a precursor protein with a signal peptide for a periplasmatic location. The amino acid sequence did not affiliate with any described enzymes. We suggest naming the enzyme linalool dehydratase-isomerase according to its bifunctionality and placing it as a member of a new protein family within the hydrolyases (EC 4.2.1.X).     

Biotransformation of geraniol, nerol and citral by sporulated surface cultures of Aspergillus niger and Penicillium sp

The biotransformation of geraniol, nerol and citral by Aspergillus niger was studied. A comparison was made between submerged liquid, sporulated surface cultures and spore suspensions. This bioconversion was also carried out with surface cultures of Penicillium sp. The main bioconversion products obtained from geraniol and nerol by liquid cultures of A. niger were linalool and alpha-terpineol. Linalool, alpha-terpineol and limonene were the main products obtained from nerol and citral by sporulated surface cultures, whereas geraniol was converted predominantly to linalool, also resulting in higher yields. Bioconversion of nerol with Penicillium chrysogenum yielded mainly alpha-terpineol and some unidentified compounds. With P. rugulosum the major bioconversion product from nerol and citral was linalool. The bioconversion of nerol to alpha-terpineol and linalool by spore suspensions of A. niger was also investigated. Finally the biotransformation with sporulated surface cultures was also monitored by solid phase microextraction (SPME). It was found that SPME is a very fast and efficient screening technique for biotransformation experiments.     

Transformations of acetates of citronellol,geraniol, and linalool by Aspergillus niger: regiospecific hydroxylation of citronellol by a cell-free system

Summary Incubation of acetates of geraniol, citronellol and linalool with Aspergillus niger resulted in their hydrolysis to corresponding alcohols which were further hydroxylated to their respective 8-hydroxy derivatives. In the case of linalyl acetate, besides linalool and 8-hydroxylinalool, small amounts of geraniol and -terpineol were also formed. Microsomes (105 000xg sediment) prepared from induced cells of A. niger were found to convert (1-³H)citronellol to 8-hydroxy citronellol in the presence of NADPH and O₂. The pH optimum for the hydroxylase was found to be 7.6.     

Biotransformation of menthol and geraniol by hairy root cultures of <Emphasis Type="Italic">Anethum graveolens</Emphasis>: effect on growth and volatile components

Two oxygen-containing monoterpene substrates, menthol or geraniol (25 mg l⁻¹), were added to Anethum graveolens hairy root cultures to evaluate the influence of the biotransformation capacity on growth and production of volatile compounds. Growth was assessed by the dissimilation method and by fresh and dry weight measurement. The volatiles were analyzed by GC and GC–MS. The total constitutive volatile component was composed, in more than 50%, by falcarinol (17–52%), apiole (11–24%), palmitic acid (7–16%), linoleic acid (4–9%), myristicin (4-8%) and n-octanal (2-5%). Substrate addition had no negative influence on growth. The relative amount of menthol quickly decreased 48 h after addition, and the biotransformation product menthyl acetate was concomitantly formed. Likewise, the added geraniol quickly decreased over 48 h alongside with the production of the biotransformation products. The added geraniol was biotransformed in 10 new products, the alcohols linalool, α-terpineol and citronellol, the aldehydes neral and geranial, the esters citronellyl, neryl and geranyl acetates and linalool and nerol oxides. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enhanced biotransformation of terpenes in plant cell suspensions using controlled release polymer

Plant cell cultures of Peganum harmala converted geranyl acetate to geraniol. Although the reaction started immediately after feeding, there was disappearance of both product and substrate. Geranyl acetate at 100 mg l^–1 when fed to 100 ml Peganum harmala suspensions (16% packed cell volume) was completely used within 24 h without accumulation of any product. Similarly, linalyl acetate and its biotransformation products, linalool and -terpineol, disappeared. Controlled-release polymer discs made from poly-2-hydroxyethyl methacrylate and containing concentrations of geranyl acetate or linalyl acetate produced greatly extended concentrations of these substrates and their biotransformation products (from about 1 day to over 12 days). The concentrations of substrates remained at around 5 mg l^–1throughout the experiments, while the concentrations of biotransformation products increased from 10 mg l^–1to 55.5 mg l^–1 for geraniol, from 5 mg l^–1 to 14 mg l^–1 for linalool, and 5 mg l^–1 to 12 mg l^–1 for -terpineol compared to the control value. Also low concentrations (30–200 g/disc) of product were taken up by the polymer over 10 days.     

Microbial degradation of monoterpenes in the absence of molecular oxygen.

Anaerobic degradation of natural monoterpenes by microorganisms was evaluated by using Pseudomonas citronellolis DSM 50332 and enrichment cultures containing nitrate as an electron acceptor. P. citronellolis grew anaerobically on 3,7-dimethyl-1-octanol and citronellol but not on geraniol, nerol, and alicyclic monoterpenes. In contrast, several a-, mono-, and bicyclic monoterpenes supported microbial growth and denitrification in enrichment cultures. We found that consumption of linalool, menthol, menth-1-ene, alpha-phellandrene, limonene, 2-carene, alpha-pinene, and fenchone in enrichment cultures depended on the presence of living microorganisms and nitrate. In these experiments, the ratios of number of electrons derived from complete substrate oxidation to number of electrons derived from nitrate reduction ranged from 1.2:1 to 2.9:1. Microbial degradation was accompanied by the formation of small traces of monoterpenes, which were characterized by gas chromatography-mass spectroscopy. The formation of geraniol and geranial from linalool suggested that a 3,1-hydroxyl-delta 1-delta 2-mutase reaction initiates linalool degradation. Seven strains of motile, oval to rod-shaped, facultatively denitrifying bacteria were isolated on agar bottle plates by using linalool, menthol, menth-1-ene, alpha-phellandrene, 2-carene, eucalyptol, and alpha-pinene as sole carbon and energy sources.     

Geraniol and linalool synthases from wild species of perilla

Geraniol and linalool synthases were isolated from three pure strains of Perilla hirtella and Perilla setoyensis, which are wild species of perilla. Their amino acid sequences were very similar to those of Perilla citriodora and Perilla frutescens that were reported previously. However, comparison of the sequences of the same functional synthases derived from different species of Perilla demonstrated that the similarities were high among P. citriodora, P. hirtella and P. frutescens, but low between P. setoyensis and any of the others. This result corresponds well with our previous results showing that P. setoyensis is remotely related to the other perilla species. Both geraniol and linalool synthases utilize geranyl diphosphate (GDP) as their catalytic substrate and they were expressed simultaneously in perilla. The linalool synthase is considered to be the enzyme whose metabolite seems not to be oxidized nor reduced in the plant body and the geraniol and limonene synthases are the initial-step-catalyzing enzymes for a variety of oil compounds. The regulation of the substrate flow between them would be interesting for further study.     

A Primeverosidase as a Main Glycosidase Concerned with the Alcoholic Aroma Formation in Tea Leaves

We isolated and characterized a primeverosidase from fresh tea leaves (Camellia sinensis var. sinensis cv. Yabukita) as a main glycosidase involved in alcoholic aroma (geraniol, linalool, benzyl alcohol, 2-phenylethanol, linalool oxides etc.) formation from their aroma precursors (β-primeverosides: 6-O-β-D-xylopyranosyl-β-D-glucopyranosides) in tea leaves.     

Enantiospecific (S)-(+)-linalool formation from beta-myrcene by linalool dehydratase-isomerase

The linalool dehydratase-isomerase from Castellaniella defragrans strain 65Phen catalyzes in the thermodynamically unfavourable direction the hydration of betamyrcene to linalool and further the isomerization to geraniol, the initial steps in anaerobic beta-myrcene biodegradation. We have now investigated the stereochemistry of this reaction. (S)-(+)-Linalool is formed with an enantiomeric excess of at least 95.4%. (R)-(-)-Linalool was not detected. This indicates an introduction of the hydroxy group on the si-face of beta-myrcene.     

Tomato terpene synthases TPS5 and TPS39 account for a monoterpene linalool production in tomato fruits

Recombinant tomato terpene synthases, TPS5/37/39, catalyze the formation of linalool or nerolidol in vitro. However, little is known about their actual biological activities in tomato plants, especially in their fruits. Here, when all three TPSs were induced in tomato fruits by a chemical elicitor, geraniol, a significant linalool peak was detected in fruit tissues but not in control fruits. Considering the compartments of these TPS proteins and available substrates, the linalool peak induced by geraniol might be attributed to TPS5 and TPS37, both of them putatively localized in the plastids where high levels of monoterpene substrate geranyl diphosphate exist. In addition, application of geraniol also triggered jasmonic acid (JA)-related defense genes suggesting that the inducible TPSs might be correlated with JA-signaled defense responses.     

Chemotaxonomic study on Thymus villosus from Portugal

The composition of the essential oils of four populations of Thymus villosus subsp. lusitanicus (Boiss.) Coutinho from Portugal was investigated by GC and GC-MS. To study the chemical polymorphism the results obtained from GC analyses of the volatile oils from individual plants from four populations were submited to Principal Component and Cluster analyses. A comparision with the essential oil of T. villosus subsp. villosus, previously studied by us was done. Important differences with regard to the major constituents in these two taxa were found. Linalool, geranyl acetate, geraniol and terpinen-4-ol were the main components of the essential oils of T. villosus subsp. lusitanicus, whereas in the oil of T. villosus subsp. villosus p-cymene, myrcene and alpha-terpineol were the major ones. Although, both taxa showed chemical polymorphism, different types of essential oils were characterized in each one: linalool; linalool/ terpinen-4-ol/trans-sabinene hydrate; linalool/1,8-cineole; geranyl acetate/geraniol; geranyl acetate/geraniol/1,8-cineole in T. villosus subsp. lusitanicus and p-cymene/camphor/linalool; p-cymene/borneol; linalool/geraniol/geranyl acetate; alpha-terpineol/camphor/myrcene in T. villosus subsp. villosus. Thus, the two subspecies of T. villosus can be easely differenciated by the composition of their essential oils.     

Comparison of plant-based expression platforms for the heterologous production of geraniol

We compared the ability of different plant-based expression platforms to produce geraniol, a key metabolite in the monoterpenoid branch of the terpenoid indole alkaloid biosynthesis pathway. A geraniol synthase gene isolated from Valeriana officinalis (VoGES) was stably expressed in different tobacco systems. Intact plants were grown in vitro and in the greenhouse and were used to generate cell suspension and hairy root cultures. VoGES was also transiently expressed in N. benthamiana. The highest geraniol content was produced by intact transgenic plants grown in vitro (48 μg/g fresh weight, fw), followed by the transient expression system (27 μg/g fw), transgenic plants under hydroponic conditions in the greenhouse and cell suspension cultures (16 μg/g fw), and finally hairy root cultures (9 μg/g fw). Differences in biomass production and the duration of cultivation resulted in a spectrum of geraniol productivities. Cell suspension cultures achieved a geraniol production rate of 1.8 μg/g fresh biomass per day, whereas transient expression produced 5.9 μg/g fresh biomass per day (if cultivation prior to agroinfiltration is ignored) or 0.5 μg/g fresh biomass per day (if cultivation prior to agroinfiltration is included). The superior productivity, strict process control and simple handling procedures available for transgenic cell suspension cultures suggest that cells are the most promising system for further optimization and ultimately for the scaled-up production of geraniol.     

Biotransformation of acyclic monoterpenoids by Debaryomyces sp., Kluyveromyces sp., and Pichia sp. strains of environmental origin

Sixty yeast strains, which belong to 32 species of the genera Debaryomyces, Kluyveromyces, and Pichia, and which were isolated from plant-, soil- or insect-associated habitats, were screened for their ability to biotransform the acyclic monoterpenes geraniol and nerol. The aptitude to convert both compounds (from 2.6 to 30.6, and from 2.7 to 29.1%/g cell DW (=dry weight), resp.) was apparently a broad distributed character in such yeasts. Depending upon the substrate used, the production of linalool, alpha-terpineol, beta-myrcene, D-limonene, (E)-beta-ocimene, (Z)-beta-ocimene, or carene was observed. Linalool was the main product obtained from geraniol, whereas linalool and alpha-terpineol were the main products obtained through the conversion of nerol. Yet, differently from nerol, the aptitude to exhibit high bioconversion yields of geraniol to linalool was an apparently genus-related character, whereas the ability to produce other monoterpenes was a both genus- and habitat-related character. The possible pathways of bioconversion of geraniol or nerol to their derivatives were proposed/discussed.     

Efficacy of the botanical repellents geraniol,linalool, and citronella against mosquitoes

We determined the degree of personal protection provided by citronella, linalool, and geraniol in the form of commercially available candles or diffusers, both indoors and outdoors. Under the uniform conditions of the experiments, all substances repelled significantly more mosquitoes than the unprotected control. Furthermore, the repellents tested were more active when in the form of a continuous release diffuser than in candle form. All candles were 88g containing 5% of the active ingredient and all diffusers contained 20g of 100% active ingredient. Indoors, the repellency rate of citronella candles was only 14% while the repellency rate of citronella diffusers was 68%. The repellency of geraniol candles was 50% while the diffusers provided a repellency rate of 97%. No linalool candles were available for study but linalool diffusers repelled mosquitoes by 93%. Outdoors, citronella diffusers placed 6 m from mosquito traps repelled female mosquitoes by 22%, linalool repelled females by 58%, and geraniol repelled females by 75%. Trap catches were significantly reduced again when diffusers were placed 3 m from the traps. We concluded that geraniol had significantly more repellent activity than citronella or linalool in both indoor and outdoor settings.     

Volatile and Non-volatile Forms of Aroma Compounds in Tea Leaves and Their Changes due to Injury

Fresh tea leaves were homogenized in a chloroform-methanol mixture (1:1, v/v), and separated into chloroform-soluble and methanol-water-soluble fractions after addition of water. From the chloroform-soluble fraction, the volatile forms of the aroma compounds were obtained. The non-volatile forms of the aroma compounds were associated with the methanol-water-soluble fraction, and were converted to volatile forms by hydrolysis with dilute acid.

The amount of the aroma compounds in the free form, such as cis-3-pentenol, hexanol, cis-3-hexenol, trans-2-hexenol, linalool, linalool oxide (cis, 5-membered), linalool oxide (trans, 5-membered), linalool oxide (trans, 6-rnembered), linalool oxide (cis, 6-membered), nerol, geraniol, phenylmethanol, and 2-phenylethanol, markedly increased during black tea manufacture. However, those in the bound form, showed a slight decrease during the manufacture. The increases in the former were also brought about by maceration, or treatment of the tea leaves with monoiodoacetate or malonate.     

Evidence for geraniol as an obligatory precursor of isothujone

Isothujone (trans-thujan-3-one) was formed from MVA-[¹⁴C, ³H] in Tanacetum vulgare with retention of the pro-(4R) hydrogen of precursor, but with loss of the pro-(4S) hydrogen and of one hydrogen from C-5. Cell-free extracts could not sustain the formation of isothujone from MVA but yielded geraniol and nerol (3,7-dimethylocta-trans-2,6-dien-1-ol and its cis isomer) with retention of the pro-(4R) and loss of the pro-(4S) hydrogen in each case: no hydrogen was lost from C-5 of MVA in formation of geraniol, but one such atom was lost in the formation of nerol. These results support the sequence: geraniol → nerol → isothujone: in which the first two compounds (or their biogenetic equivalents) are interconverted by a redox process involving their derived aldehydes. They are not consistent with a direct pathway to nerol from C₅ intermediates or with routes involving cyclisation of linalol (3,7-dimethylocta-1,6-dien-3-ol) formed directly from the C₅ compounds or from geraniol. The cell-free preparations could not interconvert geraniol and nerol, their phosphates or pyrophosphates. This may be due to the inability of a prenyltransferase-isomerase multi-enzyme system to accept exogenously-supplied intermediates under these (in vitro) conditions.     

A homomeric geranyl diphosphate synthase-encoding gene from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> and its combinatorial optimization for production of geraniol in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Geranyl diphosphate (GPP), the unique precursor for all monoterpenoids, is biosynthesized from isopentenyl diphosphate and dimethylallyl diphosphate via the head-to-tail condensation reaction catalyzed by GPP synthase (GPPS). Herein a homomeric GPPS from Camptotheca acuminata, a camptothecin-producing plant, was obtained from 5′- and 3′-rapid amplification of cDNA ends and subsequent overlap extension and convenient PCR amplifications. The truncate CaGPPS was introduced to replace ispA of pBbA5c-MevT(CO)-MBIS(CO, ispA), a de novo biosynthetic construct for farnesyl diphosphate generation, and overexpressed in Escherichia coli, together with the truncate geraniol synthase-encoding gene from C. acuminata (tCaGES), to confirm CaGPPS-catalyzed reaction in vivo. A 24.0 ± 1.3 mg L^?1 of geraniol was produced in the recombinant E. coli. The production of GPP was also validated by the direct UPLC-HRMS^E analyses. The tCaGPPS and tCaGES genes with different copy numbers were introduced into E. coli to balance their catalytic potential for high-yield geraniol production. A 1.6-fold increase of geraniol production was obtained when four copies of tCaGPPS and one copy of tCaGES were introduced into E. coli. The following fermentation conditions optimization, including removal of organic layers and addition of new n-decane, led to a 74.6 ± 6.5 mg L^?1 of geraniol production. The present study suggested that the gene copy number optimization, i.e., the ratio of tCaGPPS and tCaGES, plays an important role in geraniol production in the recombinant E. coli. The removal and addition of organic solvent are very useful for sustainable high-yield production of geraniol in the recombinant E. coli in view of that the solubility of geraniol is limited in the fermentation broth and/or n-decane.     

Flavor of Black Tea

In comparing the aroma concentrates from various types of black tea by the use of gas chromatography (GLC), differences of aroma pattern were recognized among these black tea of Ceylon, India, Peru, Formosa and Japan.

One of the typical differences, by which the variety would be characterized, appeared in the proportion of linalool (include its oxides) to geraniol and phenylethanol. Furthermore the ratio of the total area of peaks before and after linalool seemed to have some relation with the variety of black tea.

Also, top note of black tea aroma was compared by head space vapor analyses.     

Kinetics of solvent-free geranyl acetate synthesis by Rhizopus oligosporus NRRL 5905 lipase immobilized on to cross-linked silica

The objective of the present work was to study the kinetics of the solvent-free synthesis of geranyl acetate by a novel lipase (activity 60 U g^?1) made by immobilization of lipase from Rhizopus oligosporous NRRL 5905 on to cross-linked silica gel. Transesterification was performed with vinyl acetate as the acyl donor. Vinyl acetate was used in large excess compared to geraniol, which made the reaction pseudo first order with respect to geraniol and the reaction rate followed Michaelis–Menten kinetics for a single substrate. To obtain the highest yield for geranyl acetate, various relevant physical parameters such as shaking speed, reaction time, enzyme concentration, initial water amount and reaction temperature that influence the activity of lipase were investigated. A maximum molar conversion of 67% was achieved after 48 h of reaction at 30°C, at an enzyme concentration of 25% w/v of reaction mixture. Substrate conversion remained constant for five successive cycles; thereafter the conversion dropped by only 11%. Using a pseudo first-order kinetic model for geranyl acetate synthesis in the absence of organic solvents, apparent K_m and V_max values were evaluated as 60 mM and 141 µmol g^?1 h^?1, respectively.