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.     

Screening of antibacterial activities of twenty-one oxygenated monoterpenes

Plant essential oils are widely used as fragrances and flavours in the cosmetic, perfume, drug and food industries. Oxygenated monoterpenes are widespread components of the essential oils, usually occurring in high amount. In this paper, the antibacterial activities of twenty-one oxygenated monoterpenes (borneol, borneol acetate, camphor, carvone, 1,8-cineole, citronellal, beta-citronellol, dihydrocarvone, fenchol, fenchone, geraniol acetate, isomenthol, limonene oxide, linalool, linalool acetate, nerol, nerol acetate, terpinen-4-ol, alpha-terpineol, menthol and menthone) and penicillin (standard antibiotic) were determined using a disc diffusion method (in vitro) against 63 bacterial strains, belonging to 37 different genera and 54 species (plant, food and clinic origins). The results showed that the oxygenated monoterpenes exhibited a variable degree of antibacterial activities. These compounds also inhibited the growth of bacterial strains by producing a weak zone of inhibition from 7 to 11 mm in diameter, depending on the susceptibility of the tested bacteria. Among the tested compounds, nerol, linalool alpha-terpineol, fenchol and terpinen-4-ol showed antibacterial activity at a broad spectrum. However, their antibacterial activities were lower than those of penicillin. In contrast to these compounds, camphor and 1,8-cineole exhibited no inhibition effects on the growth of all tested bacteria.     

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.     

Biotransformation of terpenes by fungi: Study of the pathways involved

The biotransformation of the pure terpene alcohols geraniol and nerol, the mixture of the alcohols, ‘citrol’, and the mixture of the aldehydes, citral, to 6-methyl-5-hepten-2-one by sporulated surface cultures of Penicillium digitatum was compared. It was found that citral was converted faster than the alcohols but gave a lower overall yield of ≈76%, whereas the pure alcohols and their mixture, ‘citrol’, gave a yield of ≈83%. It was also established that the bioconversion over prolonged periods was possible with an overall yield of 80–90% depending on the substrate used. The bioconversion of nerol to 6-methyl-5-hepten-2-one by a spore suspension was also shown. The pathways involved in the biotransformation of geraniol and citral to 6-methyl-5-hepten-2-one are also discussed.     

Syntheses and odor descriptions of cyclopropanated compounds, part 6: Analogs of aliphatic monoterpene dienols and non-branched alcohols

Analogs of aliphatic monoterpene dienols (geraniol, nerol, linalool, and lavandulol) and non-branched alcohols (norleaf alcohol, matsutake alcohol, etc.) bearing a cyclopropane ring were synthesized, and their odor characteristics were examined. Most of the analogs show odor properties different from their parent compounds.     

Physiology of deletion mutants in the anaerobic beta-myrcene degradation pathway in Castellaniella defragrans

ABSTRACT: BACKGROUND: Monoterpenes present a large and versatile group of unsaturated hydrocarbons of plant origin with widespread use in the fragrance as well as food industry. The anaerobic beta-myrcene degradation pathway in Castellaniella defragrans strain 65Phen differs from well known aerobic, monooxygenase-containing pathways. The initial enzyme linalool dehydratase-isomerase ldi/LDI catalyzes the hydration of beta-myrcene to (S)-(+)-linalool and its isomerization to geraniol. A high-affinity geraniol dehydrogenase geoA/GeDH and a geranial dehydrogenase geoB/GaDH contribute to the formation of geranic acid.A genetic system was for the first time applied for the betaproteobacterium to prove in vivo the relevance of the linalool dehydratase-isomerase and the geraniol dehydrogenase. In-frame deletion cassettes were introduced by conjugation and two homologous recombination events. RESULTS: Polar effects were absent in the in-frame deletion mutants C. defragrans Deltaldi and C. defragrans DeltageoA. The physiological characterization of the strains demonstrated a requirement of the linalool dehydratase-isomerase for growth on acyclic monoterpenes, but not on cyclic monoterpenes. The deletion of geoA resulted in a phenotype with hampered growth rate on monoterpenes as sole carbon and energy source as well as reduced biomass yields. Enzyme assays revealed the presence of a second geraniol dehydrogenase. The deletion mutants were in trans complemented with the broad-host range expression vector pBBR1MCS-4ldi and pBBR1MCS-2geoA, restoring in both cases the wild type phenotype. CONCLUSIONS: In-frame deletion mutants of genes in the anaerobic beta-myrcene degradation revealed novel insights in the in vivo function. The deletion of a high-affinity geraniol dehydrogenase hampered, but did not preclude growth on monoterpenes. A second geraniol dehydrogenase activity was present that contributes to the beta-myrcene degradation pathway. Growth on cyclic monoterpenes independent of the initial enzyme LDI suggests the presence of a second enzyme system activating unsaturated hydrocarbons.     

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.     

QTLs for muscat flavor and monoterpenic odorant content in grapevine (Vitis vinifera L.)

Little is known about the genetic determinism of muscat flavor in grape, although this trait is of major importance for table grape breeding. We therefore performed a search for QTLs (Quantitative Trait Loci) of both muscat score and berry content in the three main free monoterpene alcohols potentially involved, linalool, nerol and geraniol, based on two years of measures. Parental and consensus framework genetic maps of the cross MTP2687-85 (Olivette × Ribol) × Muscat of Hamburg were built after genotyping the 174 offspring for 139 well-scattered SSR markers. The female, male and consensus framework maps spanned 935, 1365 and 1267 cM, respectively. For QTL detection, simple and composite interval mapping were performed, as well as non parametric Kruskal–Wallis tests. QTLs for muscat score were found on linkage groups (LGs) 1, 5 and 7. For the three ln-transformed monoterpene contents, QTLs with major effects (explaining 17–55 % of total phenotypic variance) were found to be colocated on LG 5, on the male and consensus maps in both years. One additional QTL was found for linalool on LG 2, on female and consensus maps, as well as other colocated ones for nerol and geraniol on LG 13, on male and consensus maps. These additional QTLs had lower effects (9–25%). The contribution of these results to the knowledge of muscat aroma genetic determinism is discussed, as well as their potential usefulness for marker assisted breeding of new aromatic grape varieties.     

Characterization of geraniol synthase from the peltate glands of sweet basil

The monoterpene fraction of the lemon-scented sweet basil (Ocimum basilicum) cv Sweet Dani consists mostly of citral (a mixture of geranial and neral), with lower levels of geraniol and nerol. These compounds are stored in the peltate glands found on the leaf epidermis. Younger leaves, which have a higher density of such glands, also have a higher content of monoterpenes than older leaves. Geraniol synthase (GES) activity, generating geraniol from geranyl diphosphate, was shown to be localized exclusively or almost exclusively to glands. GES activity resides in a homodimeric protein that was purified to near homogeneity. Basil GES requires Mn2+ as a divalent metal cofactor for activity and produces only geraniol from geranyl diphosphate. Km values of 21 and 51 microM were obtained for geranyl diphosphate and Mn2+, respectively. In the presence of 18O-labeled water, GES catalyzed the formation of 18O-geraniol from geranyl diphosphate, indicating that the reaction mechanism of GES is similar to that of other monoterpene synthases and is different from the action of phosphatases. A GES cDNA was isolated based on analysis of a glandular trichome expressed sequence tag database, and the sequence of the protein encoded by this cDNA shows some similarity to sequences of other terpene synthases. The expression of the GES cDNA in Escherichia coli resulted in a protein with enzymatic activity essentially identical to that of plant-purified GES. RNA gel-blot analysis indicated that GES is expressed in glands but not in leaves of basil cv Sweet Dani, whose glands contain geraniol and citral, and not in glands or leaves of another basil variety that makes other monoterpenes but not geraniol or citral.     

A grapevine (Vitis vinifera L.) deoxy-d-xylulose synthase gene colocates with a major quantitative trait loci for terpenol content

Linalool, geraniol, nerol, citronellol and α-terpineol are isoprenoid molecules responsible for specific aromas found in grapes and wines. Total concentrations (free and bound forms) of these compounds were measured in the skins of mature berries during 2 successive years in two progenies obtained from Muscat Ottonel and Gewurztraminer selfings. Partial genetic maps based on microsatellite markers were constructed and several quantitative trait loci (QTLs) related to terpenol content were detected. A major QTL on linkage group (LG) 5 colocated with a deoxy-d-xylulose synthase gene, coding for the first enzyme of the plastidial isoprenoid biosynthesis pathway. The number of favourable alleles at this locus determined the level of terpenol synthesis. A second QTL, on LG 10, was found to determine the balance linalool versus geraniol and nerol in the Muscat self-progeny plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The 1-deoxy-d-xylulose 5-phosphate synthase gene co-localizes with a major QTL affecting monoterpene content in grapevine

Muscat flavor is a relevant trait both in winemaking and in fresh grape consumption. From a chemical point of view, it is strongly related to the accumulation of monoterpenes in berries. However, knowledge of the genetic mechanisms underlying its regulation is still limited. The objective of this study was to dissect the genetic determinism of aroma in grapevine by applying the analysis of quantitative trait loci (QTL) and the candidate gene (CG) approach. Two F₁ segregating progenies were evaluated through high-resolution gas chromatography–mass spectrometry (HRGC–MS) for the amounts of individual monoterpenes over 3 and 2 years. In the Italia × Big Perlon cross 34 CGs, chosen according to gene ontology (GO) terms, were placed on a complete map and tested for linkage with QTLs for linalool, nerol and geraniol levels. Two CGs mapped within a QTL for linalool content on LG 10. A third one co-localized with a major QTL for the level of the three monoterpenes on LG 5; this gene encodes 1-deoxy-d-xylulose 5-phosphate synthase (DXS), which is the first enzyme in the plastidial pathway of terpene biosynthesis. Depending on these findings, we report the first in silico analysis of grapevine DXS genes based on the whole genome sequence. Further research on the functional significance of these associations might help to understand the genetic control of Muscat flavor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Battilana and L. Costantini equally contributed to the work.     

Volatiles from rhizomes of Rhodiola rosea L

Terpenes and aroma volatiles from rhizomes of Rhodiola rosea L. from Norway have been isolated by both steam distillation and headspace solid-phase micro-extraction coupled with gas chromatography and mass spectrometry analysis. The dried rhizomes contained 0.05% essential oil with the main chemical classes: monoterpene hydrocarbons (25.40%), monoterpene alcohols (23.61%) and straight chain aliphatic alcohols (37.54%). n-Decanol (30.38%), geraniol (12.49%) and 1,4-p-menthadien-7-ol (5.10%) were the most abundant volatiles detected in the essential oil, and a total of 86 compounds were identified in both the SD and HS-SPME samples. Geraniol was identified as the most important rose-like odour compound besides geranyl formate, geranyl acetate, benzyl alcohol and phenylethyl alcohol. Floral notes such as linalool and its oxides, nonanal, decanal, nerol and cinnamyl alcohol highlight the flowery scent of rose root rhizomes.     

Biosynthesis of geraniol and nerol and their β-d-glucosides in Perlargonium graveolens and Rosa dilecta

1. 3R-[2-(14)C]Mevalonate was incorporated into geranyl and neryl beta-d-glucosides in petals of Rosa dilecta in up to 10.6% yield, and the terpenoid part was specifically and equivalently labelled in the moieties derived from isopentenyl pyrophosphate and 3,3-dimethylallyl pyrophosphate. A similar labelling pattern, with incorporations of 0.06-0.1% was found for geraniol or nerol formed in leaves of Pelargonium graveolens The former results provide the best available evidence for the mevalonoid route to regular monoterpenes in higher plants. 2. Incorporation studies with 3RS-[2-(14)C,(4R)-4-(3)H(1)]-mevalonate and its (4S)-isomer showed that the pro-4R hydrogen atom of the precursor was retained and the pro-4S hydrogen atom was eliminated in both alcohols and both glucosides. These results suggest that the correlation of retention of the pro-4S hydrogen atom of mevalonate with formation of a cis-substituted double bond, such as has been found in certain higher terpenoids, does not apply to the biosynthesis of monoterpenes. It is proposed that either nerol is derived from isomerization of geraniol or the two alcohols are directly formed by different prenyltransferases. Possible mechanisms for these processes are discussed. 3. The experiments with [(14)C,(3)H]mevalonate also show that in these higher plants, as has been previously found in animal tissue and yeast, the pro-4S hydrogen atom of mevalonate was lost in the conversion of isopentenyl pyrophosphate into 3,3-dimethylallyl pyrophosphate.     

Monoterpene’s multiple free radical scavenging capacity as compared with the radioprotective agent cysteamine and amifostine

Monoterpenes are major active components of lavender, thyme, and mint. The X-ray radioprotective activity of pure monoterpenes is attributed to their scavenging ability against active species, but so far no firm evidence has been demonstrated. The objective of this study is to quantitatively determine antioxidant abilities of monoterpenes and collate it with radioprotective activity. Using multiple free-radical scavenging (MULTIS) method, we have determined the scavenging abilities of monoterpenes (linalool, thymol, and menthol) against six active species. A previous study has shown that the monoterpene linalool is a radioprotector for cellular systems, therefore, its scavenging ability was compared with known radioprotective agents such as cysteamine and amifostine. Results indicated that the monoterpene menthol but not linalool is a potent scavenger of reactive oxygen species and its scavenging magnitude is comparable to cysteamine and amifostine. This paper is first to show a correlation between ROS scavenging ability and radioprotective action.     

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).     

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.     

Comparative phytotoxicity of four monoterpenes against Cassia occidentalis

The effect of four monoterpenes ‐ citronellol, citronellal, cineole and linalool ‐ on the germination, growth and physiology of Cassia occidentalis was investigated. All four monoterpenes reduced germination of C. occidentalis seeds but to varying extents. Citronellal and linalool completely inhibited germination beyond the concentrations of 55 and 110 μM, respectively, whereas in response to treatment of citronellol no germination was observed beyond 330 μM. Further, the growth of seedlings, measured in terms of seedling length and biomass, was also adversely affected. A reduction in chlorophyll content of the cotyledonary leaves of C. occidentalis was also noticed, indicating an adverse effect on photosynthesis. Likewise, respiratory ability of growing seeds was also impaired in response to all four monoterpenes, clearly indicating that monoterpenes affect energy metabolism. On the basis of overall phytotoxicity, potency of monoterpenes was in the order of citronellal > linalool > citronellol > cineole. The results from this study suggest that both citronellal and linalool possess strong phytotoxic potential and can thus serve as lead molecules for the synthesis of bioherbicides.     

Biotransformation of C13-norisoprenoids and monoterpenes by a cell suspension culture of cv. Gamay (Vitis vinifera)

A cell suspension culture of cv. Gamay was studied for its ability to metabolize two different C₁₃-norisoprenoidic volatiles, β-ionone and dehydrovomifoliol, together with monoterpenes, geraniol and linalool, biogenetically common pathways sharing compounds. β-Ionone was totally metabolized leading to fourteen norisoprenoidic volatiles oxygenated mainly at carbons 3 or 4 of the cyclohexane ring or reduced at side chain. The biotransformation of dehydrovomifoliol was at a lesser extent, giving rise to oxygenated and reduced derivatives. The norisoprenoidic metabolites were present both under free and glycosylated forms. Geraniol and linalool were also metabolized, leading to several free and glycosylated compounds. S. Mathieu, J. Wirth contributed equally to the work and should be considered joint first authors. A short part of this paper was published at the proceedings of the 10th Weurman Flavour Research Symposium, Flavour Research at the Dawn of the Twenty-first Century, J.-L.Le Quere, P.-X.Etievant, Editors; Lavoisier,2003/Intercept Ltd, 2003.     

Biosynthesis of monoterpenes by Ceratocystis moniliformis

Ceratocystis moniliformis produced and excreted monoterpenes when grown on potato-dextrose broth. Geraniol, nerol, citronellol, linalol, α-terpineol, geranial and neral were identified by GC-MS. Their production commenced with the depletion of nitrogen in the growth medium and their combined concentration peaked at about 50 μg/ml on the 5th day of growth. The pathway for the biosynthesis of the identified monoterpenes was studied by supplying the radioactive precursors mevalonic acid-[2-¹⁴C], l-leucine-[4,5-³H(N)], and acetate- [2-¹⁴C] to C. moniliformis. For each precursor, the extent of incorporation into the above monoterpenes and the distribution of radioactivity in geraniol was determined. It was concluded that monoterpenes were formed via the mevalonate pathway, previously established for higher terpenes in other organisms. This represents the first information available on the biosynthetic pathway for free monoterpenes in a microbial system.