Biotransformation Experiments Research Articles

Off-flavours in water: hydroxyketones andβ-ionone derivatives as new odour compounds of freshwater cyanobacteria

Cyanobacteria essentially contribute to the off-flavours of drinking water and freshwater fish. They are primary nuisance microorganisms and their control in lakes and ponds used for the production of drinking water and fish farming is expensive. We describe new odour compounds of freshwater cyanobacteria that are fermentation products and norcarotenoids. These may contribute to the off-flavours frequently observed with cyanobacteria. The fermentation products were represented by 2,3-pentandione, 2,3-hexandione, 2-hydroxy-3-pentanone, 3-hydroxy-2-pentanone, 2-hydroxy-3-hexanone, 3-hydroxy-2-hexanone and 2,3-hexandiol. These compounds are important for odour quality in microbially-processed dairy food, caused by the metabolism of fungi and heterotrophic bacteria. Their cyanobacterial synthesis was shown by 13C-labelling, growth kinetics and biotransformation experiments. The labelled mass spectra of the hydroxyketones showed unequivocally the cyanobacterial rather than bacterial origin of the compounds. Norcarotenoids of the β-ionone-type (β-ionol, β-ionone-5,6-epoxide, dihydro-β-ionone, dihydro-β-ionol, tetrahydroionone, 4-oxo-β-ionone and 2,4,4-trimethyl-3-(3-oxobutyl)-2-cyclohexen-1-one) were another important group of compounds that were found in axenic cyanobacterial cultures and a monoxenic culture of Phormidium sp. The results implicate the presence of further oxygenases in cyanobacteria. Except for tetrahydroionone, the syntheses of the β-ionone derivatives were established by biotransformation experiments applying the precursor β-ionone. The norcarotenoid 6-methyl-5-hepten-2-one, which is regarded as a precursor compound of 1,3,3-trimethyl-2,7-dioxabicylo(2,2,1)heptane (TDH), was 13C-labelled, while TDH did not show any labelling. There is evidence that TDH arises from the analytical procedure and is an artefact rather than a biogenic compound produced by the cyanobacteria. Copyright © 2005 John Wiley & Sons, Ltd.

Hydroxylation of Ionized Aromatics Including Carboxylic Acid or Amine Using Recombinant Streptomyces lividans Cells Expressing Modified Biphenyl Dioxygenase Genes.

The bphA1(2072)A2A3A4 gene cluster codes for a shuffled biphenyl dioxygenase holoenzyme with broad substrate specificity. These bphA1(2072)A2A3A4 genes were expressed in the actinomycetes Streptomyces lividans using a thiostrepton-inducible promoter PtipA. Biotransformation experiments of various aromatics including carboxylic acid or amine in their molecular structure, such as 1-naphthoic acid, 2-(1-naphthyl)acetic acid, diphenylamine, and 1-benzyl-4-piperidone, were performed using the recombinant S. lividans cells. These ionized aromatics were converted to the corresponding 1,2-dihydrodiol, mono- or tri-hydroxy forms in 48 h. The structure of the converted products was determined by their EI-MS, 1H- and 13C NMR analysis, and several products were found to be novel compounds.

Hydroxylation of ionized aromatics including carboxylic acid or amine using recombinant Streptomyces lividans cells expressing modified biphenyl dioxygenase genes

The bphA1(2072) A2A3A4 gene cluster codes for a shuffled biphenyl dioxygenase holoenzyme with broad substrate specificity. These bphA1(2072) A2A3A4 genes were expressed in the actinomycetes Streptomyces lividans using a thiostrepton-inducible promoter P tipA . Biotransformation experiments of various aromatics including carboxylic acid or amine in their molecular structure, such as 1-naphthoic acid, 2-(1-naphthyl)acetic acid, diphenylamine, and 1-benzyl-4-piperidone, were performed using the recombinant S. lividans cells. These ionized aromatics were converted to the corresponding 1,2-dihydrodiol, mono- or tri-hydroxy forms in 48 h. The structure of the converted products was determined by their EI-MS, 1H- and 13C NMR analysis, and several products were found to be novel compounds.

Identification of three novel salicylate 1-hydroxylases involved in the phenanthrene degradation of Sphingobium sp. strain P2

Five sets of large and small subunits of terminal oxygenase ( ahdA1[ a– e] and ahdA2[ a– e]) and a single gene set encoding ferredoxin ( ahdA3) and ferredoxin reductase ( ahdA4) were found to be scattered through 15.8- and 14-kb DNA fragments of phenanthrene-degrading Sphingobium sp. strain P2. RT-PCR analysis indicated the inducible and specific expression of ahdA3, ahdA4, and three sets of genes for terminal oxygenase ( ahdA1[ c– e] and ahdA2[ c– e]) in this strain grown on phenanthrene. The biotransformation experiments with resting cells of Escherichia coli JM109 harboring recombinant ahd genes revealed that AhdA2cA1c, AhdA1dA2d, and AhdA1eA2e can all function as a salicylate 1-hydroxylase which converts salicylate, a metabolic intermediate of phenanthrene, to catechol in cooperation with the electron transport proteins AhdA3A4. The first two oxygenases exhibited a broad range of substrate specificities such that they also catalyzed the hydroxylation of methyl- and chloro-substituted salicylates to produce their corresponding substituted catechols.

An Interesting Backbone Rearrangement and Novel Derivatives from the Biotransformation of Trachyloban-19-oic Acid by Rhizopus stolonifer

A biotransformation experiment of trachyloban-19-oic acid (2) was carried out with Rhizopus stolonifer. After twenty days of reaction, four metabolites were extracted, isolated and characterized: two trachylobane type compounds, the 7b (3) and the 17 (5) hydroxyl derivatives, and two rearranged ent-kaur-11-en-19-oic acids, the 16b (4) and the 9a,16b (6) hydroxylated compounds. Products 4 and 5 were isolated as their methyl esters (4a and 5a) after esterification of a mixture containing their corresponding acids. The rearrangement of a trachylobane diterpene leading to ent-16b-hydroxy-kaur-11-ene derivatives gives support to the biogenetic proposal based on the occurrence of this rare group of kaurenes only in plants containing trachylobane representatives. By the best of our knowledge, this is the first report on the isolation of compounds 5 and 6.

Biotransformation of naphthalene and diaryl ethers by green microalgae.

The role of green microalgae in the biotransformation of naphthalene (a polycyclic aromatic hydrocarbon) and diaryl ethers was investigated using axenic cultures of Chlorella vulgaris and two environmental isolates, Scenedesmus SI1 and Ankistrodesmus SI2. Biotransformation experiments with dense cell cultures showed that these three green algae transformed toxic xenobiotics to more polar metabolites. Chlorella vulgaris metabolized naphthalene to 1-naphthol (0.36-0.65%). Ankistrodesmus SI2 biotransformed dibenzofuran to six metabolites (total over 7%), three of which (possibly four) were identified as monohydroxylated dibenzofurans, the remaining two may be dihydroxylated derivatives. Scenedesmus SI1 biotransformed dibenzo-p-dioxin to three metabolites, one of which was tentatively identified as 2-hydroxydibenzo-p-dioxin (approximately 3.8%), the remainder may be dihydroxylated derivatives. This is the first time that the biotransformation of diaryl ethers by green microalgae has been investigated.

Oxygenation reactions of various tricyclic fused aromatic compounds using Escherichia coli and Streptomyces lividans transformants carrying several arene dioxygenase genes.

Bioconversion (biotransformation) experiments on arenes (aromatic compounds), including various tricyclic fused aromatic compounds such as fluorene, dibenzofuran, dibenzothiophene, carbazole, acridene, and phenanthridine, were done using the cells of Escherichia coli transformants expressing several arene dioxygenase genes. E. coli carrying the phenanthrene dioxygenase (phdABCD) genes derived from the marine bacterium Nocardioides sp. strain KP7 converted all of these tricyclic aromatic compounds, while E. coli carrying the Pseudomonas putida F1 toluene dioxygenase (todC1C2BA) genes or the P. pseudoalcaligenes KF707 biphenyl dioxygenase (bphA1A2A3A4) genes was not able to convert these substrates. Surprisingly, E. coli carrying hybrid dioxygenase (todC1::bphA2A3A4) genes with a subunit substitution between the toluene and biphenyl dioxygenases was able to convert fluorene, dibenzofuran, and dibenzothiophene. The cells of a Streptomyces lividans transformant carrying the phenanthrene dioxygenase genes were also evaluated for bioconversion of various tricyclic fused aromatic compounds. The ability of this actinomycete in their conversion was similar to that of E. coli carrying the corresponding genes. Products converted from the aromatic compounds with these recombinant bacterial cells were purified by column chromatography on silica gel, and identified by their MS and 1H and 13C NMR analyses. Several products, e.g., 4-hydroxyfluorene converted from fluorene, and cis-1,2-dihydroxy-1,2-dihydrophenanthridine, cis-9,10-dihydroxy-9,10-dihydrophenanthridine, and 10-hydroxyphenanthridine, which were converted from phenanthridine, were novel compounds.

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 α-terpineol. Linalool, α-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 α-terpineol and some unidentified compounds. With P. rugulosum the major bioconversion product from nerol and citral was linalool. The bioconversion of nerol to α-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.

Determination of the reaction yield during biotransformation of the volatile and chemically unstable compound beta-ionone by aspergillus niger

Biotransformation of beta-ionone by Aspergillus niger IFO 8541 was a complex system, due to the low precursor solubility in water (0.88 mol/m(3)) which gave a two-phase liquid system, its high volatility (vapor pressure 7.2 Pa), and its poor chemical stability. A dynamic model, considering transfers between organic, aqueous, and gas layers, was established. It also took into account the decrease in organic layer area exchange during a process, due to progressive precursor disappearance. Its solving needed separate determination of two parameters. The first-order kinetic constant for chemical degradation, K(1), was estimated from side experiments involving a model system at thermodynamic equilibrium; its value was found to be close to 4.17 x 10(-)(6) s(-)(1). The volumetric transfer coefficient between gas and aqueous phase was estimated by analogy with results obtained for oxygen transfer. Results obtained demonstrated high liquid-gas transfer rates, while liquid-liquid transfers were severely limited. These data were then used to estimate the true yield of a biotransformation experiment, which was found to be close to unity.

合成化学と生薬学の接点 天然活性化合物に対するモノクローナル抗体と生合成酵素

MAb against THCA was produced by fusing hybridoma with splenocytes immunized with THCA-BSA conjugate and mouse myeloma cell. The cross-reaction of anti-THCA MAb against other marihuana compounds was very wide. This ELISA was applied to the biotransformation experiments of marihuana compounds using plant tissue culture.Anti-ginsenoside Rb1 MAb cross-reacted with only limited to a few ginsenosides. Newly developed Western blotting used anti-ginsenoside Rb1 MAb surveyed only dammaran saponins. Immunoaffinity column made it possible to separate ginsenoside Rbl directly. Compact MAb, scFV against forskolin was prepared from a hybridoma by gene construction, and its expression in E. coli was discussed.Biosynthetic enzymes, THCA-, CBCA- and CBDA-synthase had been purified from the homogenate of fresh Cannabis plants, respectively. These characterizations were investigated and compared each other. It became evident that CBCA-synthase catalyzed enantiomeric cyclization of CBGA to CBCA.

Monoclonal antibodies against naturally occurring bioactive compounds.

The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determined by matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry. A hybridoma secreting monoclonal antibody (MAb) was produced by fusing splenocytes immunized with an antigen-BSA conjugate with HAT-sensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodies with 7-deacetyl forskolin was 5.6%. A very small cross-reaction appeared with other derivatives. The full measuring range of the assay extends from 5 ng to 5 mug/ml of forskolin. Immunoaffinity column chromatography using anti-forskolin MAbs appears to be far superior to previously published separation methods. The capacity of the immunoaffinity column as determined by ELISA is 9 mug/ml. Forskolin has been isolated directly from the crude extracts of tuberous roots and the callus culture of Coleus forskohlii. A MAb against tetrahydrocannabinolic acid (THCA) was produced. The cross-reaction of anti-THCA antibody against other cannabinoids was very wide. Many cannabinoids and a spiro-compound were reactive, but did not react with other phenolics. It became evident that this ELISA was able to be applied to the biotransformation experiments of cannabinoids in plant tissue culture system. Anti-ginsenoside Rb1 MAbs were produced. New western blotting method of determination for ginsenosides was established. Ginsenosides separated by silica gel TLC were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was treated with NaIO(4) solution followed by BSA, resulting in a ginsenoside-BSA conjugate. Immunostaining of ginsenosides was more sensitive compared to other staining. Immunostaining of ginsenosides in the fresh ginseng root was succeeded using anti-ginsenoside Rb1 (GRb1) MAb after blotting to PVDF membrane.

Development of ELISA-analysis methods for the quantification of bioactive natural products in plants, phytomedicines and in humans or similar

In the course of a program in developing new ELISA-methods for the quantification of bioactive natural products in plants, phytomedicines and animals in a μg and ng scale, monoclonal antibodies against various natural products of medicinal and analytical importance have been developed. The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determined by matrix-assisted laser desorption/ionization (MALDI) of mass spectrometry. A hybridoma secreting monoclonal antibodies (MAb) was produced by fusing splenocytes immunized with an antigen-BSA conjugate with HAT-sensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodies with 7-deacetyl-forskolin was 5.6%. A very small cross-reaction appeared with other derivatives. The full measuring range of the assay extends from 5 ng to 5 μg/ml of forskolin. Immunoaffinity column chromatography using anti-forskolin MAbs appears to be far superior to previously published separation methods. The capacity of the immunoaffinity column as determined by ELISA is 9 μg/ml. Forskolin has been isolated directly from the crude extracts of tuberous roots and the callus culture of Coleus forskohlii. A MAb against Δ(1)-tetrahydrocannabinolic acid (THCA) was produced. The cross-reaction of anti-Δ(1)-THCA antibody against other cannabinoids was very wide. Many cannabinoids and a spiro-compound were reactive, but did not react with other phenolics. It became evident that this ELISA method was able to be applied to the biotransformation experiments of cannabinoids in plant tissue culture system. Anti-solamargine MAbs were produced. A method of determination for solasodine glycosides by using TLC-immunostaining was established. Solasodine glycosides separated by silica gel TLC were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was treated with NaIO(4) solution followed by BSA, resulting in a solasodine glycoside-BSA conjugate. Immunostaining of solasodine glycosides was more sensitive compared to other staining. Immunostaining of ginsenosides in fresh ginseng root was successful using anti-ginsenoside Rb1 (G-Rb1) MAb after blotting to PVDF membrane.

Bacterial biotransformation of 3 α( S)-Strictosidine to the monoterpenoid indole alkaloid vallesiachotamine

3 α( S)-Strictosidine produced by heterologously expressed strictosidine synthase from Rauwolfia serpentina was used in biotransformation experiments with a series of 22 bacterial strains. All strains tested were found to deglucosylate and rearrange the alkaloid to vallesiachotamine, thereby providing an example of how gene technology and microbial biotransformation can be combined for the biotechnological production of alkaloidal natural products.

Enzyme specificity of 2-nitrotoluene 2,3-dioxygenase from Pseudomonas sp. strain JS42 is determined by the C-terminal region of the alpha subunit of the oxygenase component.

Biotransformations with recombinant Escherichia coli expressing the genes encoding 2-nitrotoluene 2,3-dioxygenase (2NTDO) from Pseudomonas sp. strain JS42 demonstrated that 2NTDO catalyzes the dihydroxylation and/or monohydroxylation of a wide range of aromatic compounds. Extremely high nucleotide and deduced amino acid sequence identity exists between the components from 2NTDO and the corresponding components from 2,4-dinitrotoluene dioxygenase (2,4-DNTDO) from Burkholderia sp. strain DNT (formerly Pseudomonas sp. strain DNT). However, comparisons of the substrates oxidized by these dioxygenases show that they differ in substrate specificity, regiospecificity, and the enantiomeric composition of their oxidation products. Hybrid dioxygenases were constructed with the genes encoding 2NTDO and 2,4-DNTDO. Biotransformation experiments with these hybrid dioxygenases showed that the C-terminal region of the large subunit of the oxygenase component (ISP alpha) was responsible for the enzyme specificity differences observed between 2NTDO and 2,4-DNTDO. The small subunit of the terminal oxygenase component (ISP beta) was shown to play no role in determining the specificities of these dioxygenases.

Biotransformations of protoberberines in cell cultures of Dicentra spectablis

A liquid chromatography-atmospheric pressure chemical ionization mass spectrometry procedure was applied to biotransformation experiments in cultured cells of Dicentra spectablis. Interconversions of tetrahydroberberine and berberine and those of cis- and trans-13-methyltetrahydroprotoberberines and 13-methylberberine were demonstrated. The corresponding α- N-metho salts having the B/C- cis ring-junction were produced from cis- and trans-13-methyltetrahydroprotoberberines. These findings show that the metabolism of protoberberines in D. spectablis proceeds analogously to that found in Cordyalis species.

Monoclonal antibody based enzyme immunoassay for marihuana (cannabinoid) compounds.

MAb against delta 1-THCA was produced by fusing hybridoma with splenocytes immunized with delta 1-THCA-BSA conjugate and hypoxanthine, aminopterine, thymidine-sensitive mouse myeloma cell line, P3-X63-Ag8-653. The cross-reaction of anti-delta 1-THCA antibody against other cannabinoids was very wide, thus many cannabinoids and a spiro-compound were reactive suggesting that 2'-hydroxyl, 6'-hydroxyl or 6'-O-alkyl, 4'-alkylbenzene ring moiety is necessary for its reactivity. It became evident that this ELISA was able to be applied to the biotransformation experiments of cannabinoids in plant tissue culture system. The metabolites of delta 6-THC such as two major metabolites, 7-oxo-delta 6-THC and 7-hydroxyl-delta 6-THC were also detectable by this ELISA.

Initial oxidative and subsequent conjugative metabolites produced during the metabolism of phenanthrene by fungi.

Three filamentous fungi were examined for the ability to biotransform phenanthrene to oxidative (phase I) and conjugative (phase II) metabolites. Phenanthrene metabolites were purified by high-performance liquid chromatography (HPLC) and identified by UV/visible absorption, mass, and 1H NMR spectra. Aspergillus niger ATCC 6275, Syncephalastrum racemosum UT-70, and Cunninghamella elegans ATCC 9245 initially transformed [9-(14)C]phenanthrene to produce metabolites at the 9,10-, 1,2-, and 3,4-positions. Subsequently, sulfate conjugates of phase I metabolites were formed by A. niger, S. racemosum, and C. elegans. Minor glucuronide conjugates of 9-phenanthrol and phenanthrene trans-9, 10-dihydrodiol were formed by S. racemosum and A. niger, respectively. In addition, C. elegans produced the glucose conjugates 1-phenanthryl beta-D-glucopyranoside and 2-hydroxy-1-phenanthryl beta-D-glucopyranoside, a novel metabolite. [9-(14)C]Phenanthrene metabolites were not detected in organic extracts from biotransformation experiments with the yeasts, Candida lipolytica 37-1, Candida tropicalis ATCC 32113, and Candida maltosa R-42.

13-hydroxylation of tetrahydroberberine in cell suspension cultures of some Corydalis species

Liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry was applied to biotransformation experiments in cultured cells of Corydalis ophiocarpa as well as C. ochotensis var. raddeana. Hydroxylation at C-13 of tetrahydroberberine was shown to take place in cell cultures as well as in C. ophiocarpa plants. N-Methylation of tetrahydroberberine occurred to form the α- N-metho salt incorporating the B/C- cis-quinolizidine system. Introduction of the C-13 methyl with berberine as substrate was confirmed to provide 13-methylberberine. In addition, the reversible oxidation-reduction of the C ring of protoberberines was demonstrated.

Diversity in steroidogenesis of symbiotic microorganisms from planthoppers

Ergosta-5,7,24(28)-trien-3β-ol (trienol)6 was isolated from the intracellular symbiotes (symbiotic microorganisms) of the planthoppers,Nilaparvata lugens andLaodelphax striatellus. The steroidogenic end product of the symbiotes was found to depend on the environmental conditions, i.e., although trienol6 was produced under symbiotic conditions, ergosterol4 was formed in aerobic culture. When the normal diet was replaced by a steroiddeficient artificial one, the ratio of 24-methylenecholesterol5 to total insect sterols was significantly increased. The above study offers further corroborating evidence for our assumption that the host insects require 24-methylenecholesterol5 as an alternate source for cholesterol1 under certain environmental conditions. In our previous biotransformation experiments, 24methylenecholesterol5 was shown to be an immediate precursor of cholesterol1. However, the complexity of the insects' vital functions hindered all subsequent attempted transformations into cholesterol1 of sitosterol2, trienol6, and ergosterol4, under injection conditions.

Identification of xyloside conjugates formed from anthracene by Rhizoctonia solani

Biotransformation experiments showed that a strain of Rhizoctonia solani was able to metabolize anthracene, a tricyclic aromatic hydrocarbon. The fungus was grown in a complex liquid medium containing [9-14C]anthracene; after 6 d, 98·8% of the anthracene had been converted to ethyl acetate-extractable metabolites. These compounds were separated by high-performance liquid chromatography (hplc) and detected by ultraviolet (uv) absorbance and liquid scintillation counting. The major metabolites were identified by their uv, mass, and nuclear magnetic resonance spectra. One of the principal metabolites was identified as trans-1,2-dihydroxy-1,2-dihydroanthracene (anthracene trans-1,2-dihydrodiol), which was shown by circular dichroism spectroscopy to be a mixture of two enantiomers. Chiral stationary phase hplc was used to resolve the trans-1,2-dihydrodiol into a (−)-1S,2S enantiomer (60%) and a (+)-1R,2R enantiomer (40%). The other principal metabolites were novel xyloside conjugates of anthracene: 1-O-(2-hydroxy-trans-1,2-dihydroanthryl)-β- d -xylopyranoside, 2-O-(1-hydroxy-trans-1,2-dihydroanthryl)-β- d -xylopyranoside, and 1-O-anthryl-β- d -xylopyranoside. Anthraquinone was found in the culture media but was also present in non-inoculated controls.