Biotransformation of limonene by bacteria,fungi, yeasts,and plants

The past 5 years have seen significant progress in the field of limonene biotransformation, especially with regard to the regiospecificity of microbial biocatalysts. Whereas earlier only regiospecific biocatalysts for the 1,2 position (limonene-1,2-diol) and the 8-position (alpha-terpineol) were available, recent reports describe microbial biocatalysts specifically hydroxylating the 3-position (isopiperitenol), 6-position (carveol and carvone), and 7-position (perillyl alcohol, perillylaaldehyde, and perillic acid). The present review also includes the considerable progress made in the characterization of plant P-450 limonene hydroxylases and the cloning of the encoding genes.     

Effects of R-(+)-limonene on submerged cultures of the terpene transforming basidiomycete Pleurotus sapidus.

The biotransformation of limonene by the basidiomycete Pleurotus sapidus yielded cis/trans-carveol and carvone as the main products. The transformation period was extended from 4 days after direct addition to 12 days by gas phase addition of the substrate. After 2 days of transformation, 97% of the substrate had accumulated in the mycelium, while only 3% were present in the culture medium. Substrate toxicity led to a decrease of dry matter. Adaptation of the precultures with small amounts of substrate doubled the concentration of carveol and increased the concentration of carvone by a factor of 3-4. Total product concentrations of > 100 mg l-1 were reached.     

不同化感水稻品种根系分泌物中萜类化合物的差异分析

以强化感水稻品种PI312777和弱化感水稻品种Lemont为供试材料,运用“循环法”收集供试水稻经土壤和石英沙介质后的根系分泌物,并经乙醚萃取,所获萃取物经气相色谱 质谱联用仪（GC-MS）测试,检索谱图库确定化合物.结果表明,乙醚萃取物中检测到含氧萜类化合物,如柠檬烯氧化物、柠檬烯二氧化物、香芹酮氧化物、香芹醇、雪松醇等,不同品种间含氧萜类化合物种类相似,但在不同介质中的存留方式和数量存在差异.文中还就不同培养方式和品种间萜类化合物的差异作了进一步讨论.     

Rhodococcus erythropolis DCL14 contains a novel degradation pathway for limonene

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (-)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1, 2-monooxygenase activity, a cofactor-independent limonene-1, 2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S, 4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R, 4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the beta-oxidation pathway.     

Development of a reaction system for the selective conversion of (−)-trans-carveol to (−)-carvone with whole cells of Rhodococcus erythropolis DCL14

The present article addresses the development of a microbial reaction system for the transformation of carveol to carvone, using whole cells of Rhodococcus erythropolis DCL14. This strain contains a NAD-dependent carveol dehydrogenase (CDH) when grown on limonene or on cyclohexanol. When a mixture of (−)-cis and (−)-trans-carveol is supplied, only (−)-trans-carveol is converted. Thus, besides (−)-carvone, pure (−)-cis-carveol can be obtained as product.

Initial experiments were performed batchwise using an aqueous system. (−)-Trans-carveol conversion rate gradually decreased during successive reutilisation batches. After the third reutilisation, activity was completely lost. Cells grown on cyclohexanol showed a slightly higher activity as compared to cells grown on (+)-limonene. A production of 4.3 μmol (−)-carvone formed per mg protein was achieved. A significant improvement with respect to initial reaction rate and productivity was obtained with aqueous–organic two-phase systems. Using a 5 to 1 buffer/iso-octane system, a 40% increase in the initial rate and a 16-fold increase of the production was observed. A further improvement resulted from increasing the volume of solvent (1 to 1 buffer/dodecane ratio). An initial reaction rate of 26 nmol/(min*mg protein) was observed, while production increased to 208 μmol (−)-carvone formed per mg protein. As in the single-phase system, reaction rate gradually decreased along the successive cell reutilisation batches. Addition of co-substrates for the regeneration of NAD did not prevent this decay. A simple downstream process was developed for the recovery of carvone and cis-carveol.     

Inhibitory effects of substrate and product on the carvone biotransformation activity of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The aqueous substrate and product toxicity thresholds in the microbial biotransformation of (-)-trans-carveol to the fragrance/flavor compound (R)-(-)-carvone by Rhodococcus erythropolis were determined. Above aqueous phase concentrations of approx. 500 mg carveol/l and 200-600 mg carvone/l, the biotransformation activity of the biocatalyst was inhibited. This biotransformation was undertaken in a single aqueous phase 3 l [corrected] reactor in which a total of 5 ml carveol (mixture of isomers) was added before the biotransformation rate decreased significantly. The carvone volumetric productivity was 31 mg/lh. Although the growth of the organism post-exposure was not affected, dramatic morphological changes in response to the accumulation of the inhibitory substrate and product were observed.     

Chemical Studies on the Essential Oils of Foeniculum vulgare

Twenty seven chemical constituents of oils from sweet leaves, flowers and fruits of Foeniculum vulgare Mill. are examined by GC and GC-MS with both different chromatographic columns. They are 1,1-diethoxyethane, α-thujene, α-pinene, camphene, sabinene, β-pinene, myrcene, α- phellandrene, p-cymene, limonene, cineole, γ-terpinene, fenchone, camphor, terpinen-4-ol, α-terpineol, estragole, verbenone, fenchol acetate, carveol, trans-fenchol acetate, carvone, anethole, anisaldehyde, trans-anethole, methoxyphenyl acetone and benzoic acid, 4-methoxy-, othylester. The limonene is 57.8% in the essential oil from leaves, 34.2% from flowers, 13.1% from fruits, The trans-anethole is 21.8% in the essential oil from leaves, 41.2% from flowers, 63.4% from fruits.     

Degradation of Pinene by Bacillus pallidus BR425

An aerobic thermophile has been isolated from an -pinene enrichment culture. The isolate, which was designated BR425, has been tentatively identified as Bacillus pallidus using 16S ribosomal RNA gene sequencing and organism morphology. Monophasic and biphasic incubations of BR425 cells with -pinene, -pinene, and limonene yielded a number of oxidized monoterpene metabolites with carveol as a common metabolite. A pinene degradation pathway with carveol and carvone as central metabolic intermediates is suggested.     

Preventing biofilm formation: promoting cell separation with terpenes

Both carveol and carvone were effective in dispersing Rhodococcus erythropolis cells that were being stimulated to aggregate by the presence of organic solvents. The two terpenes influenced the fatty acid composition of the cell membrane, decreasing the percentage of fatty acids with more than 16 carbon atoms, and thus cell hydrophobicity, and also the degree of saturation of the fatty acids. In the presence of 250 micromol of terpene, the volume of biofilm was reduced by one third in comparison with biofilms in the absence of terpenes. The percentage of aggregated cells was also found to depend on carvone concentration during the bioconversion of carveol to carvone, in a membrane reactor. The extent of cell aggregation decreased from 90% to 10% when carvone concentration reached ca. 48 mM in the organic phase.     

Chemical composition and antigenotoxic properties of Lippia alba essential oils

The present work evaluated the chemical composition and the DNA protective effect of the essential oils (EOs) from Lippia alba against bleomycin-induced genotoxicity. EO constituents were determined by Gas Chromatography/Mass Spectrometric (GC-MS) analysis. The major compounds encountered being citral (33% geranial and 25% neral), geraniol (7%) and trans-β-caryophyllene (7%) for L. alba specimen COL512077, and carvone (38%), limonene (33%) and bicyclosesquiphellandrene (8%) for the other, COL512078. The genotoxicity and antigenotoxicity of EO and the compounds citral, carvone and limonene, were assayed using the SOS Chromotest in Escherichia coli. The EOs were not genotoxic in the SOS chromotest, but one of the major compound (limonene) showed genotoxicity at doses between 97 and 1549 mM. Both EOs protected bacterial cells against bleomycin-induced genotoxicity. Antigenotoxicity in the two L. alba chemotypes was related to the major compounds, citral and carvone, respectively. The results were discussed in relation to the chemopreventive potential of L. alba EOs and its major compounds.     

Monoterpene biosynthesis pathway construction in Escherichia coli

Four genes encoding sequential steps for the biosynthesis of the spearmint monoterpene ketone (-)-carvone from the C(5) isoprenoid presursors isopentenyl diphosphate and dimethylallyl diphosphate were installed in Escherichia coli. Inducible overexpression of these genes in the bacterial host allowed production of nearly 5 mg/l of the pathway intermediate (-)-limonene, which was mostly excreted to the medium such that products of the downstream steps, (-)-carveol and (-)-carvone, were not detected. Assay of pathway enzymes and intermediates indicated that flux through the initial steps catalyzed by geranyl diphosphate synthase and limonene synthase was severely limited by the availability of C(5) isoprenoid precursors in the host. Feeding studies with (-)-limonene, to overcome the flux deficiency, demonstrated the functional capability of limonene-6-hydroxylase and carveol dehydrogenase to produce the end-product carvone; however, uptake and trafficking restrictions greatly compromised the efficiency of these conversions.     

Method for Attaining Caraway Seed Oil Fractions with Different Composition

Caraway (Carum carvi L.) is a medicinal and aromatic plant; its seeds (fruits) are used as spice and they contain essential oils. We hypothesized that by collecting caraway oil at different time points during the extraction process, we could obtain oil fractions with distinct chemical composition. A hydrodistillation time (HDT) study was conducted to test the hypothesis. The caraway seed oil fractions were collected at eight different HDT (at 0 – 2, 2 – 7, 7 – 15, 15 – 30, 30 – 45, 45 – 75, 75 – 105, and 105 – 135 min). Additionally, a non‐stop HD for 135 min was conducted as a control. Most of the oil was eluted early in the HD process. The non‐stop HDT treatment yielded 2.76% oil by weight. Of the 24 essential oil constituents, limonene (77 – 19% of the total oil) and carvone (20 – 79%) were the major ones. Other constituents included myrcene (0.72 – 0.16%), trans‐carveol (0.07 – 0.39%), and β‐caryophyllene (0.07 – 0.24%). Caraway seed oil with higher concentration of limonene can be obtained by sampling oil fractions early in HD process; conversely, oil with high concentration of carvone can be obtained by excluding the fractions eluted early in the HD process. We demonstrated a method of obtaining caraway seed oil fractions with various and unique composition. These novel oil fractions with unique composition are not commercially available and could have much wider potential uses, and also target different markets compared to the typical caraway essential oil.     

Cell adaptation to solvent, substrate and product: a successful strategy to overcome product inhibition in a bioconversion system

Carvone has previously been found to highly inhibit its own production at concentrations above 50 mM during conversion of a diastereomeric mixture of (−)-carveol by whole cells of Rhodococcus erythropolis. Adaptation of the cells to the presence of increasing concentrations of carveol and carvone in n-dodecane prior to biotransformation proved successful in overcoming carvone inhibition. By adapting R. erythropolis cells for 197 h, an 8.3-fold increase in carvone production rate compared to non-adapted cells was achieved in an air-driven column reactor. After an incubation period of 268 h, a final carvone concentration of 1.03 M could be attained, together with high productivity [0.19 mg carvone h⁻¹ (ml organic phase)⁻¹] and high yield (0.96 g carvone g carveol⁻¹).     

Functional characterization of a catabolic plasmid from polychlorinated- biphenyl-degrading Rhodococcus sp. strain RHA1

Rhodococcus sp. strain RHA1, a potent polychlorinated-biphenyl (PCB)-degrading strain, contains three linear plasmids ranging in size from 330 to 1,100 kb. As part of a genome sequencing project, we report here the complete sequence and characterization of the smallest and least-well-characterized of the RHA1 plasmids, pRHL3. The plasmid is an actinomycete invertron, containing large terminal inverted repeats with a tightly associated protein and a predicted open reading frame (ORF) that is similar to that of a mycobacterial rep gene. The pRHL3 plasmid has 300 putative genes, almost 21% of which are predicted to have a catabolic function. Most of these are organized into three clusters. One of the catabolic clusters was predicted to include limonene degradation genes. Consistent with this prediction, RHA1 grew on limonene, carveol, or carvone as the sole carbon source. The plasmid carries three cytochrome P450-encoding (CYP) genes, a finding consistent with the high number of CYP genes found in other actinomycetes. Two of the CYP genes appear to belong to novel families; the third belongs to CYP family 116 but appears to belong to a novel class based on the predicted domain structure of its reductase. Analyses indicate that pRHL3 also contains four putative "genomic islands" (likely to have been acquired by horizontal transfer), insertion sequence elements, 19 transposase genes, and a duplication that spans two ORFs. One of the genomic islands appears to encode resistance to heavy metals. The plasmid does not appear to contain any housekeeping genes. However, each of the three catabolic clusters contains related genes that appear to be involved in glucose metabolism.     

Olfactory sensitivity for enantiomers and their racemic mixtures--a comparative study in CD-1 mice and spider monkeys

Using a conditioning paradigm, the olfactory sensitivity of six CD-1 mice for the enantiomers of carvone and of limonene as well as for their racemic mixtures was investigated. With all six stimuli, the animals significantly discriminated concentrations 相似文献   

Rhodococcus erythropolis DCL14 Contains a Novel Degradation Pathway for Limonene

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (−)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1,2-monooxygenase activity, a cofactor-independent limonene-1,2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S,4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R,4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the β-oxidation pathway.     

Biochemical and Histochemical Localization of Monoterpene Biosynthesis in the Glandular Trichomes of Spearmint (Mentha spicata)

The primary monoterpene accumulated in the glandular trichomes of spearmint (Mentha spicata) is the ketone (−)-carvone which is formed by cyclization of the C₁₀ isoprenoid intermediate geranyl pyrophosphate to the olefin (−)-limonene, hydroxylation to (−)-trans-carveol and subsequent dehydrogenation. Selective extraction of the contents of the glandular trichomes indicated that essentially all of the cyclase and hydroxylase activities resided in these structures, whereas only about 30% of the carveol dehydrogenase was located here with the remainder located in the rest of the leaf. This distribution of carveol dehydrogenase activity was confirmed by histochemical methods. Electrophoretic analysis of the partially purified carveol dehydrogenase from extracts of both the glands and the leaves following gland removal indicated the presence of a unique carveol dehydrogenase species in the glandular trichomes, suggesting that the other dehydrogenase found throughout the leaf probably utilizes carveol only as an adventitious substrate. These results demonstrate that carvone biosynthesis takes place exclusively in the glandular trichomes in which this natural product accumulates.     

Changes in monoterpene composition of Mentha aquatica produced by gene substitution from a high limonene strain of M. Citrata

The dominant gene Lm that causes 60–90% limonene/cineole was substituted into M. aquatica by four convergent backcrosses. The natural strain of M. aquatica has 7·7% cineole, 4·9% limonene, traces of terpinolene and pulegone, 0·1% menthone, 0.2% menthol, and 66·4% menthofuran. The two modified hybrid strains with dominant gene Lm have 53·8 and 78·7% limonene/cineole and a total of only 1·0-3·8% 3-oxygenated compounds in contrast to a total of 66·7% found in the natural strain. The postulate is made that the Lm gene largely prevents either the conversion of a-terpineol → terpinolene or of limonene → isopiperitenone and that in these strains the recessive cc genotype largely but not completely prevents the conversion of limonene → carvone resulting in limonene accumulation. Mentha species almost invariably have either 2-oxygenated or 3-oxygenated compounds, not both. Close coupling phase linkage of the genes Lm and C explains why the self-pollinated progeny of M. spicata or M. crispa C-Lm/c-lm have a ratio of 3 carvone/dihydrocarvone: 1 pulegone/menthone rather than a ratio of 9 carvone : 3 limonene : 3 carvone and menthone: 1 menthone which would be expected if the genes Lm and C were independently inherited     

Demonstration that limonene is the first cyclic intermediate in the biosynthesis of oxygenated p-menthane monoterpenes in Mentha piperita and other Mentha species

The volatile oil of mature Mentha piperita (peppermint) leaves contains as major components the oxygenated p-menthane monoterpenes l-menthol (47%) and l-menthone (24%) as well as very low levels of the monoterpene olefins limonene (1%) and terpinolene (0.1%), which are considered to be probable precursors of the oxygenated derivatives. Immature leaves, which are actively synthesizing monoterpenes, produce an oil with comparatively higher levels of limonene (approximately 3%), and isolation of the pure olefin showed this compound to consist of approximately 80% of the l-(4S)-enantiomer and approximately 20% of the d-(4R)-enantiomer. The time course of incorporation of [U-14C]sucrose into the monoterpenes of M. piperita shoot tips was consistent with the initial formation of limonene and its subsequent conversion to menthone via pulegone. d,l-[9-3H]Limonene and [9,10-3H]terpinolene were prepared and tested directly as precursors of oxygenated p-menthane monoterpenes in M. piperita shoot tips. Limonene was readily incorporated into pulegone, menthone, and other oxygenated derivatives, whereas terpinolene was not appreciably incorporated into these compounds. Similarly, d,l-[9-3H]limonene was specifically incorporated into pulegone in Mentha pulegium and into the C-2-oxygenated derivative carvone in Mentha spicata, confirming the role of this olefin as the essential precursor of oxygenated p-menthane monoterpenes. Soluble enzyme preparations from the epidermis of immature M. piperita leaves converted the acyclic terpenoid precursor [1-3H]geranyl pyrophosphate to limonene as the major cyclic product, providing a further indication that this olefin plays a central role in the formation of oxygenated monoterpenes in Mentha. No free intermediates were detected in the cyclization of geranyl pyrophosphate to limonene, suggesting that the olefin is the first cyclic intermediate to arise in the pathway, and resolution of the biosynthetic limonene, by crystallization of the derived d- and l-carvoximes, indicated an enantiomer mixture nearly identical to that isolated from the leaf oil.     

Repellent Efficacy of Caraway and Grapefruit Oils for Sitophilus oryzae (Coleoptera: Curculionidae)

This study examined the repellent efficacy of six essential oils extracted from caraway, clary sage, grapefruit, strawberry, thyme white, ylangylang, and their related volatile constituents against the adult rice weevil, Sitophilus oryzae using an olfactometer. The caraway and grapefruit oil showed the highest repellent efficacy against the rice weevil at a dose of 10μl. Gas Chromatography-Mass Spectrophotometer analysis revealed caraway oil to be rich in carvone and limonene, and grapefruit oil to be rich in limonene, β-myrcene and α-pinene. When the monoterpene was mixed in equal parts with the caraway and grapefruit essential oils, carvone with limonene in caraway oil demonstrated the highest repellent efficacy (96.7%). Limonene with α-pinene and β-myrcene in grapefruit showed strong repellent efficacy (86.4%) with synergistic effects on the S. oryzae.A mixture of caraway and grapefruit oils, as well as carvone and limonene, can be potent repellents that may be useful for controlling S. oryzae.