Geranic Acid Formation, an Initial Reaction of Anaerobic Monoterpene Metabolism in Denitrifying Alcaligenes defragrans

Monoterpenes with an unsaturated hydrocarbon structure are mineralized anaerobically by the denitrifying β-proteobacterium Alcaligenes defragrans. Organic acids occurring in cells of A. defragrans and culture medium were characterized to identify potential products of the monoterpene activation reaction. Geranic acid (E,E-3,7-dimethyl-2,6-octadienoic acid) accumulated to 0.5 mM in cells grown on α-phellandrene under nitrate limitation. Cell suspensions of A. defragrans 65Phen synthesized geranic acid in the presence of β-myrcene, α-phellandrene, limonene, or α-pinene. Myrcene yielded the highest transformation rates. The alicyclic acid was consumed by cell suspensions during carbon limitation. Heat-labile substances present in cytosolic extracts catalyzed the formation of geranic acid from myrcene. These results indicated that a novel monoterpene degradation pathway must be present in A. defragrans.     

Geraniol and geranial dehydrogenases induced in anaerobic monoterpene degradation by Castellaniella defragrans

Castellaniella defragrans is a Betaproteobacterium capable of coupling the oxidation of monoterpenes with denitrification. Geraniol dehydrogenase (GeDH) activity was induced during growth with limonene in comparison to growth with acetate. The N-terminal sequence of the purified enzyme directed the cloning of the corresponding open reading frame (ORF), the first bacterial gene for a GeDH (geoA, for geraniol oxidation pathway). The C. defragrans geraniol dehydrogenase is a homodimeric enzyme that affiliates with the zinc-containing benzyl alcohol dehydrogenases in the superfamily of medium-chain-length dehydrogenases/reductases (MDR). The purified enzyme most efficiently catalyzes the oxidation of perillyl alcohol (k(cat)/K(m) = 2.02 × 10(6) M(-1) s(-1)), followed by geraniol (k(cat)/K(m) = 1.57 × 10(6) M(-1) s(-1)). Apparent K(m) values of <10 μM are consistent with an in vivo toxicity of geraniol above 5 μM. In the genetic vicinity of geoA is a putative aldehyde dehydrogenase that was named geoB and identified as a highly abundant protein during growth with phellandrene. Extracts of Escherichia coli expressing geoB demonstrated in vitro a geranial dehydrogenase (GaDH) activity. GaDH activity was independent of coenzyme A. The irreversible formation of geranic acid allows for a metabolic flux from β-myrcene via linalool, geraniol, and geranial to geranic acid.     

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.     

Metabolic engineering of geranic acid in maize to achieve fungal resistance is compromised by novel glycosylation patterns

Many terpenoids are known to have antifungal properties and overexpression of these compounds in crops is a potential tool in disease control. In this study, 15 different mono- and sesquiterpenoids were tested in vitro against two major pathogenic fungi of maize (Zea mays), Colletotrichum graminicola and Fusarium graminearum. Among all tested terpenoids, geranic acid showed very strong inhibitory activity against both fungi (MIC<46 μM). To evaluate the possibility of enhancing fungal resistance in maize by overexpressing geranic acid, we generated transgenic plants with the geraniol synthase gene cloned from Lippia dulcis under the control of a ubiquitin promoter. The volatile and non-volatile metabolite profiles of leaves from transgenic and control lines were compared. The headspaces collected from intact seedlings of transgenic and control plants were not significantly different, although detached leaves of transgenic plants emitted 5-fold more geranyl acetate compared to control plants. Non-targeted LC-MS profiling and LC-MS-MS identification of extracts from maize leaves revealed that the major significantly different non-volatile compounds were 2 geranic acid derivatives, a geraniol dihexose and 4 different types of hydroxyl-geranic acid-hexoses. A geranic acid glycoside was the most abundant, and identified by NMR as geranoyl-6-O-malonyl-β-d-glucopyranoside with an average concentration of 45μM. Fungal bioassays with C. graminicola and F. graminearum did not reveal an effect of these changes in secondary metabolite composition on plant resistance to either fungus. The results demonstrate that metabolic engineering of geraniol into geranic acid can rely on the existing default pathway, but branching glycosylation pathways must be controlled to achieve accumulation of the aglycones.     

Isotope ratio by HRGC-MS of citrus Junos tanaka (yuzu) essential oils: m/z 137/136 of terpene hydrocarbons

The isotope ratios of monoterpene hydrocarbons in Citrus junos Tanaka (yuzu) essential oils from different origins were determined by ordinary high-resolution gas chromatography-mass spectrometry (HRGC-MS). Both intensities of the molecular mass peaks (m/z 136) and of the isotope peaks (m/z 137) of monoterpene hydrocarbons were measured by single-ion monitoring with an MS analysis. The isotope ratios (m/z 137/136) of the ten monoterpene hydrocarbons commonly contained in citrus essential oils, alpha-pinene, beta-pinene, sabinene, myrcene, alpha-phellandrene, alpha-terpinene, limonene, gamma-terpinene, beta-phellandrene and terpinolene, were determined in yuzu samples of the highest commercial quality from 42 different production districts. Statistical treatment of these data by the t-test and sign test revealed significant differences of the isotope effects in each yuzu sample. It is suggested that this technique will be applicable for evaluating the quality, genuineness and origin of citrus fruits and their products. The isotope fingerprints were also demonstrated in several citrus fruits other than the yuzu samples.     

Composition of rugosa rose (Rosa rugosa thunb.) hydrolate according to the time of distillation

Rugosa rose (Rosa rugosa Thunb.) is one of the most common rose species in Poland. It has mild soil and climate requirements and is resistant to low temperatures. Rugosa rose hips are a valuable raw material used in food and pharmaceutical industries, while flowers and petals may be a source of fragrant products, such as essential oil and hydrolate. The main aim of this study was to verify usefulness of dried R. rugosa petals for essential oil and hydrolate production. We also assessed the use of rugosa rose petals remaining after oil distillation for hydrolate production.The R. rugosa dried petals immersed in water were subjected to simple distillation and five fractions of primary rose hydrolate were obtained. In parallel, essential oil from the second sample of petals was obtained by hydrodistillation in Clevenger-type apparatus. The distillation residue was used for obtaining four fractions of secondary hydrolate. The volatiles from hydrolate fractions were isolated by liquid–liquid extraction with diethyl ether. The essential oil and hydrolate volatiles were analyzed by GC–FID–MS.Hydrolate fractions contained similar amounts of volatiles (20–30 mg/L) with the exception of the first fraction of primary hydrolate (60 mg/L). β-Phenylethanol, citronellol, geraniol, and nerol were the main volatile constituents of primary hydrolate. β-Phenylethanol, citronellic acid, and geranic acid were the main volatile constituents of secondary hydrolate. The content of alcohols decreased, while the content of monoterpene esters (citronellyl, neryl, and geranyl acetate) as well as monoterpene acids (citronellic, neric, and geranic acid) increased in successive fractions of both hydrolates.The scent and composition of essential oil and hydrolate obtained from R. rugosa petals were similar to those of rose oil and rose water produced from damask rose (Rosa damascena Mill.). This proves, that rugosa rose distillation products may become an alternative to fragrant damask rose products.     

Geraniol biotransformation-pathway in spores of Penicillium digitatum

Spores of Penicillium digitatum ATCC 201167 transform geraniol, nerol, citral, and geranic acid into methylheptenone. Spore extracts of P. digitatum convert geraniol and nerol NAD+-dependently into citral. Spore extract also converts citral NAD+-dependently into geranic acid. Furthermore, a novel enzymatic activity, citral lyase, which cofactor-independently converts citral into methylheptenone and acetaldehyde, was detected. These result show that spores of P. digitatum convert geraniol via a novel biotransformation pathway. This is the first time a biotransformation pathway in fungal spores has been substantiated by biochemical studies. Geraniol and nerol are converted into citral by citrol dehydrogenase activity. The citral formed is subsequently deacetylated by citral lyase activity, forming methylheptenone. Moreover, citral is converted reversibly into geranic acid by citral dehydrogenase activity.     

Sensitization of Actinomyces naeslundii and Streptococcus sanguis in biofilms and suspensions to acid damage by fluoride and other weak acids

Fluoride and other weak acids, such as benzoate, indomethacin, salicylate and sorbate, were found to be sensitizers for acid killing of cells of Actinomyces naeslundii ATCC 19246 and Streptococcus sanguis NCTC 10904 in suspensions or in mono-organism biofilms on glass slides. These bacteria are among the more acid-sensitive organisms from dental plaque and were killed when acidified to pH values between 3.5 and 4.0. Biofilm cells were more resistant than cells in suspensions, especially in terms of the fraction of the initial population surviving acidification. The mechanism for sensitization to acid killing by fluoride and the other weak acids involved enhanced transmembrane transport of protons, reflected by increases in measured proton permeabilities of the cells. Thus, the weak acids thwarted the functions of F(H+)-ATPases in extruding protons and protecting cells against acid damage. Fluoride sensitization of biofilms or cells in suspensions to acid damage occurred rapidly. There was a delay in sensitization of biofilms by indomethacin and higher molecular weight acids which was interpreted in terms of diffusion limitation of sensitizer penetration. Overall, it seemed that weak-acid sensitization to acid killing is a general phenomenon that occurs not just for oral bacteria but also for organisms in food, soil, and other acidified environments.     

Degradation of the methyl substituted alkene, citronellol, by Pseudomonas aeruginosa, wild type and mutant strains

Pseudomonas aeruginosa W51D used citronellol (3,7-dimethyl-6-octen-1-ol) as sole source of carbon and oxidized it to citronellal, citronellic acid and finally to geranic acid. A mutant derived from this strain was isolated as unable to degrade citronellol and had diminished (93%) citronellal dehydrogenase activity.     

Catabolism of geraniol by cell suspension cultures of Citrus limon

The addition of geraniol to cell suspension cultures of Citrus limon resulted in the rapid formation of nerol, citronellol, geranic acid and citronellic acid. Concurrently, a transient accumulation of bound forms of branched chain fatty acids, and, with a few hours delay, of regular chain C2 to C12 fatty acids was elicited. A concerted action of combined alpha/beta-oxidation enzymes on the terpenic acids, followed by an enlarged acetyl CoA pool is suggested. Terpene catabolism in plants and in vitro plant cells is discussed.     

Microbial Transformation of Terpenoids. I. Indentification of Metabolites Produced by a Pseudomonad from Citronellal and Citral

A bacterium capable of utilizing citronellal or citral as the sole source of carbon and energy has been isolated from soil by the enrichment culture technique. It metabolizes citronellal to citronellic acid (65%), citronellol (0.6%), dihydrocitronellol (0.6%), menthol (0.75%), and 3,7-dimethyl-1,7-octane diol (1.7%). The metabolites of citral were geranic acid (62%), 6-methyl-5-heptanoic acid (0.5%), 3-methyl-2-butenoic acid (1%), and 1-hydroxy-3, 7-dimethyl-6-octen-2-one (0.75%).     

Fine-tuning the composition of the cranberry weevil (Coleoptera: Curculionidae) aggregation pheromone

The cranberry weevil Anthonomus musculus Say is a key pest of highbush blueberries (Vaccinium corymbosum L.) and cranberries (Vaccinium macrocarpon Aiton) in the northeastern United States. Previous studies have reported A. musculus adult attraction to traps baited with the aggregation pheromone of the pepper weevil Anthonomus eugenii Cano, likely because these two weevils share similar pheromone blends that differ only in two components. The A. musculus aggregation pheromone contains (Z)-2-(3,3-dimethylcyclohexylidene) ethanol (Z grandlure II), (Z)-(3,3-dimethylcyclohexylidene) acetaldehyde (grandlure III), (E)-(3,3-dimethylcyclohexylidene) acetaldehyde (grandlure IV) and (E)-3,7-dimethyl-2,6-octadien-1-ol (geraniol); whereas A. eugenii produces a pheromone blend that includes (E)-2-(3,3-dimethylcyclohexylidene) ethanol (E grandlure II) and (E)-3,7-dimethyl-2,6-octadienoic acid (geranic acid) in addition to the four A. musculus pheromone components. Here, we hypothesized that differences in pheromone composition between these two species influence A. musculus adult attraction to its aggregation pheromone. To test this, we studied the response of A. musculus to its pheromone blend with and without E grandlure II and geranic acid, a commercial A. eugenii pheromone lure and a no-lure control in highbush blueberry and cranberry fields in New Jersey and Massachusetts, respectively. Regardless of crop type, A. musculus adults were more attracted to their four-component pheromone blend and the blend plus geranic acid than the commercial A. eugenii pheromone and the no-lure controls. The A. musculus pheromone blend plus E grandlure II and the A. eugenii pheromone blend also captured more A. musculus adults than the no-lure control but not compared to the commercial A. eugenii pheromone. Further analysis showed that A. musculus adults are significantly (~27%) less attracted to their pheromone blend if it contains E grandlure II, although the addition of geranic acid did not affect their response. These findings may help guide future efforts towards the development of behaviour-based tools to monitor and manage A. musculus.     

Monoterpene hydrocarbon biosynthesis by isolated leucoplasts of Citrofortunella mitis

A plastid vesicle preparation isolated from exocarpium of young Citrofortunella mitis (calamondin) fruits was able to synthesise monoterpene hydrocarbons when incubated with isopentenyl pyrophosphate. The electron-microscope comparison between this organelle fraction and the various plastid classes present in the peel tissues has shown the structural identity between these plastid vesicles and the leucoplasts of the epithelial cells lining the secretory pockets. The monoterpene biosynthesis required the presence of dimethylallyl pyrophosphate, Mn²⁺ or Mg²⁺ and was increased by addition of 2-mercaptoethanol. Evidence is provided that the leucoplast vesicles act as a complete system in which occur all the successive steps involved in monoterpene hydrocarbon elaboration from isopentenyl pyrophosphate.     

Morphology and anionic polymer content in the cell wall of a glycerol-requiring mutant ofBacillus subtilis

A glycerol-requiring mutant ofBacillus subtilis formed irregular spheres and showed disturbed septum formation, when subjected to growth limitation by the supply of glycerol. Under phosphate limitation the cells were also round and developed asymmetric septa. In magnesium-limited cultures the cells contained a thickened wall, as compared with that of the parent strain grown under the same conditions. Chemical analysis revealed the presence of teichoic acid as the major anionic polymer in the wall of the glycerol-, as well as the magnesium-limited cells of the glycerol-requiringB. subtilis mutant.Under phosphate limitation teichuronic acid was the only anionic polymer present in the wall. Thus, in this respect, there were no apparent differences between mutant organisms and the parent strain when grown under magnesium and phosphate limitation, respectively and the observed morphological deviations could not be correlated with an altered anionic polymer content of the wall.     

A study on the alkaloid production by resting cell suspensions of Catharanthus roseus in a continuous flow reactor

Summary During batch cultivation of Catharanthus roseus cell suspensions, alkaloids were found in the culture medium after growth had ceased. Resting cell suspensions with high alkaloid content were obtained by transferring the cells to a medium devoid of 2.4 D (2.4 dichlorophenoxy-acetic acid). A production system with continuous feeding was developped to study alkaloid production by these resting cell suspensions.     

Divergent pathways for δ-aminolevulinic acid synthesis in two species of Arthrobacter

Abstract Secretion of coproporphyrin III by suspensions of Arthrobacter photogonimos and A. globiformis facilitated analysis of the paths of synthesis of δ-aminolevulinic acid, the precursor of tetrapyrroles. Sensitivity of coproporphyrin accumulation to gabaculine and incorporation of ¹⁴C from [1-¹⁴C]glutamate indicated that suspensions of A. photogonimos synthesized δ-aminolevulinic acid from glutamate by the widespread C5 pathway. In contrast, A. globiformis cells switched from predominantly the C5 pathway for δ-aminolevulinic acid synthesis in early exponential phase cultures to δ-aminblevulinic acid synthase in stationary phase cultures.     

Citral dehydrogenase involved in geraniol oxidation pathway: purification,characterization and kinetic studies from <Emphasis Type="Italic">Persicaria minor</Emphasis> (<Emphasis Type="Italic">Polygonum minus</Emphasis> Huds.)

Plants emit semiochemicals as alarm signals upon attack by herbivores or insect pests. Complex insect-plant interaction through alarm pheromones can be manipulated to improve crop protection. Geraniol, citral and geranic acid are monoterpenoid compounds from plants and they play a role as semiochemical alarm pheromones. In plants, the oxidation of geraniol into geranic acid is catalyzed by two oxidoreductases, geraniol dehydrogenase and citral dehydrogenase. In this study, citral dehydrogenase isoenzymes from Persicaria minor (Polygonum minus) leaves were purified to homogeneity and characterized. Enzyme purification through Toyopearl GigaCap Q-650 M column chromatography at pH 7.5 produced two activity peaks, suggesting the existence of two citral dehydrogenase isoenzymes. Both isoenzymes were different in isoelectric point and kinetic parameters but similar in pH and optimal temperature as well as in substrate specificity. Findings from this study will provide a basic understanding for the development of recombinant production of these particular enzymes. Further studies on molecular structure involved could be exploited in transgenic plant as an integrated pest management strategy.     

Linalyl Acetate Is Metabolized by Pseudomonas incognita with the Acetoxy Group Intact

Metabolism of linalyl acetate by Pseudomonas incognita isolated by enrichment culture on the acyclic monoterpene alcohol linalool was studied. Biodegradation of linalyl acetate by this strain resulted in the formation of linalool, linalool-8-carboxylic acid, oleuropeic acid, and Δ⁵-4-acetoxy-4-methyl hexenoic acid. Cells adapted to linalyl acetate metabolized linalyl acetate-8-aldehyde to linalool-8-carboxylic acid, linalyl acetate-8-carboxylic acid, Δ⁵-4-acetoxy-4-methyl hexenoic acid, and geraniol-8-carboxylic acid. Resting cell suspensions previously grown with linalyl acetate oxidized linalyl acetate-8-aldehyde to linalyl acetate-8-carboxylic acid, Δ⁵-4-acetoxy-4-methyl hexenoic acid, and pyruvic acid. The crude cell-free extract (10,000 g of supernatant), obtained from the sonicate of linalyl acetate-grown cells, was shown to contain enzyme systems responsible for the formation of linalyl acetate-8-carboxylic acid and linalool-8-carboxylic acid from linalyl acetate. The same supernatant contained NAD-linked alcohol and aldehyde dehydrogenases involved in the formation of linalyl acetate-8-aldehyde and linalyl acetate-8-carboxylic acid, respectively. On the basis of various metabolites isolated from the culture medium, resting cell experiments, growth and manometric studies carried out with the isolated metabolites as well as related synthetic analogs, and the preliminary enzymatic studies performed with the cell-free extract, a probable pathway for the microbial degradation of linalyl acetate with the acetoxy group intact is suggested.     

Accumulation of docosahexaenoic acid-rich lipid in thraustochytrid <Emphasis Type="Italic">Aurantiochytrium</Emphasis> sp. strain T66: effects of N and P starvation and O<Subscript>2</Subscript> limitation

Aurantiochytrium sp. strain T66 was grown in batch bioreactor cultures in a defined glutamate- and glycerol-containing growth medium. Exponentially growing cells had a lipid content of 13% (w/w) of dry weight. A fattening of cells fed excess glycerol occurred in the post-exponential growth phase, after the medium was depleted of N or P. Lipid accumulation was also initiated by O(2) limitation (below 1% of saturation). N starvation per se, or in combination with O(2) limitation, gave the highest lipid content, i.e., 54% to 63% (w/w) of dry weight. The corresponding maximum culture density was 90 to 100 g/l dry biomass. The content of docosahexaenoic acid (22:6n-3) in N starved, well-oxygenated cells reached 29% (w/w) of total fatty acids but increased to 36% to 52% in O(2)-limited cells, depending on the time span of the limitation. O(2)-limited cells did not accumulate the monounsaturated fatty acids that were normally present. We inferred that the biological explanation is that O(2) limitation hindered the O(2)-dependent desaturase(s) and favored the O(2)-independent polyunsaturated fatty acid synthase. The highest overall volumetric productivity of docosahexaenoic acid observed was 93 mg/l/h. Additionally, we present a protocol for quantitative lipid extraction, involving heat and protease treatment of freeze-dried thraustochytrids.