Biotransformation of isoeugenol to vanillin by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IE27 cells

The ability to produce vanillin and/or vanillic acid from isoeugenol was screened using resting cells of various bacteria. The vanillin- and/or vanillic-acid-producing activities were observed in strains belonging to the genera Achromobacter, Aeromonas, Agrobacerium, Alcaligenes, Arthrobacter, Bacillus, Micrococcus, Pseudomonas, Rhodobacter, and Rhodococcus. Strain IE27, a soil isolate showing the highest vanillin-producing activity, was identified as Pseudomonas putida. We optimized the culture and reaction conditions for vanillin production from isoeugenol using P. putida IE27 cells. The vanillin-producing activity was induced by adding isoeugenol to the culture medium but not vanillin or eugenol. Under the optimized reaction conditions, P. putida IE27 cells produced 16.1 g/l vanillin from 150 mM isoeugenol, with a molar conversion yield of 71% at 20 °C after a 24-h incubation in the presence of 10% (v/v) dimethyl sulfoxide.     

Metabolic characterization of green pods from Vanilla planifolia accessions grown in La Réunion

Large phenotypic variation has been observed between the cultivated vanillas since a single genetic source of Vanilla planifolia was spread to the Indian Ocean and the Indonesia in the 19th century. In order to differentiate the cultivated vanilla plants, genetic studies have been conducted in the past on the plants grown in various regions such as the French island, La Réunion. However, the genetic difference was not big enough to differentiate diverse accessions of V. planifolia. In this study, metabolomics, in which genetic variation could be amplified, was employed to delve into the variation between the cultivated vanilla plants. To obtain a broad view of the metabolome, nuclear magnetic resonance (NMR) spectroscopy was applied to the analysis of V. planifolia green pods. Principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) of the data showed that the accessions could be differentiated according to their glucovanillin and glucosides A and B contents. Furthermore, a correlation between the glucovanillin content and the pod length, number of flower and growth capacity of the accessions has been observed from the multivariate data analysis.     

Enhanced vanillin production from ferulic acid using adsorbent resin

High vanillin productivity was achieved in the batch biotransformation of ferulic acid by Streptomyces sp. strain V-1. Due to the toxicity of vanillin and the product inhibition, fed-batch biotransformation with high concentration of ferulic acid was unsuccessful. To solve this problem and improve the vanillin yield, a biotransformation strategy using adsorbent resin was investigated. Several macroporous adsorbent resins were chosen to adsorb vanillin in situ during the bioconversion. Resin DM11 was found to be the best, which adsorbed the most vanillin and the least ferulic acid. When 8% resin DM11 (wet w/v) was added to the biotransformation system, 45 g l⁻¹ ferulic acid could be added continually and 19.2 g l⁻¹ vanillin was obtained within 55 h, which was the highest vanillin yield by bioconversion until now. This yield was remarkable for exceeding the crystallization concentration of vanillin and therefore had far-reaching consequence in its downstream processing.     

Evolution of Novel O-methyltransferases from the Vanilla planifolia Caffeic Acid O-methyltransferase

The biosynthesis of many plant secondary compounds involves the methylation of one or more hydroxyl groups, catalyzed by O-methyltransferases (OMTs). Here, we report the characterization of two OMTs, Van OMT-2 and Van OMT-3, from the orchid Vanilla planifolia Andrews. These enzymes catalyze the methylation of a single outer hydroxyl group in substrates possessing a 1,2,3-trihydroxybenzene moiety, such as methyl gallate and myricetin. This is a substrate requirement not previously reported for any OMTs. Based on sequence analysis these enzymes are most similar to caffeic acid O-methyltransferases (COMTs), but they have negligible activity with typical COMT substrates. Seven of 12 conserved substrate-binding residues in COMTs are altered in Van OMT-2 and Van OMT-3. Phylogenetic analysis of the sequences suggests that Van OMT-2 and Van OMT-3 evolved from the V. planifolia COMT. These V. planifolia OMTs are new instances of COMT-like enzymes with novel substrate preferences.     

Use of <Emphasis Type="Italic">Pseudomonas mendocina</Emphasis>, or recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> cells expressing toluene-4-monooxygenase,and a cell-free tyrosinase for the synthesis of 4-fluorocatechol from fluorobenzene

The transformation of fluorobenzene (FB) by whole cell expressing toluene-4-monooxygenase (T4MO) resulted in the formation of various hydroxylated products. The predominant product was either 4-fluorophenol (4FP) or 4-fluorocatechol (4Fcat) depending on the ratio of biocatalyst to substrate concentration. The transformation of 1 mM FB by whole cells (1.5 mg CDW/ml) gave a 52% yield of 4Fcat as a single product. The yield of 4Fcat was improved 1.6-fold (80%) by adding 10 mM ascorbic acid to the biotransformations. A combination of two biocatalysts (whole cells expressing T4MO and cell free mushroom tyrosinase) also resulted in the transformation of FB (5 mM) to higher concentrations of 4Fcat (1.8 mM) compared to a whole cell biotransformation alone. However, mixed products were formed and the yield of 4Fcat from FB was lower using the two-step (tandem) method (27%) compared to the use of whole cells of P. mendocina KR1 alone (80%).     

Microbial transformation of ferulic acid to vanillic acid by <Emphasis Type="Italic">Streptomyces sannanensis</Emphasis> MTCC 6637

Streptomyces sannanensis MTCC 6637 was examined for its potentiality to transform ferulic acid into its corresponding hydroxybenzoate-derivatives. Cultures of S. sannanensis when grown on minimal medium containing ferulic acid as sole carbon source, vanillic acid accumulation was observed in the medium as the major biotransformed product along with transient formation of vanillin. A maximum amount of 400 mg/l vanillic acid accumulation was observed, when cultures were grown on 5 mM ferulic acid at 28°C. This accumulation of vanillic acid was found to be stable in the culture media for a long period of time, thus facilitating its recovery. Purification of vanillic acid was achieved by gel filtration chromatography using Sephadex™ LH-20 matrix. Catabolic route of ferulic acid biotransformation by S. sannanensis has also been demonstrated. The metabolic inhibitor experiment [by supplementation of 3,4 methylenedioxy-cinnamic acid (MDCA), a metabolic inhibitor of phenylpropanoid enzyme 4-hydroxycinnamoyl-CoA ligase (4-CL) along with ferulic acid] suggested that biotransformation of ferulic acid into vanillic acid mainly proceeds via CoA-dependent route. In vitro conversions of ferulic acid to vanillin, vanillic acid and vanillin to vanillic acid were also demonstrated with cell extract of S. sannanensis. Further degradation of vanillic acid to other intermediates such as, protocatechuic acid and guaiacol was not observed, which was also confirmed in vitro with cell extract.     

Synthesis of (R)-2-phenylpropanoic acid from its racemate through an isomerase-involving reaction by Nocardia diaphanozonaria

(R)-2-Phenylpropanoic acid was synthesized from the racemic acid through an isomerization reaction involving resting cells of Nocardia diaphanozonaria JCM3208. The isomerization activity of the cells was enhanced 25-fold by adding 5.5 mM racemic 2-phenylpropanoic acid to the culture medium. When 5 mM racemic 2-phenylpropanoic acid was included in the reaction mixture (4 ml) containing resting cells (100 mg dry cell wt) in 25 mM K₂HPO₄/KH₂PO₄ buffer (pH 7.0) at 30 °C for 8 h, 4.56 mM (R)-2-phenylpropanoic acid (95.8% e.e.) was formed with a 91% molar conversion yield.     

Acetogenesis coupled to the oxidation of aromatic aldehyde groups

Vanillin cultures of Clostridium formicoaceticum produced higher cell densities than did vanillate cultures. During growth at the expense of vanillin, vanillate was the predominat intermediate formed; 3,4-dihydroxybenzaldehyde was not a significantly detectable intermediate. Acetate and protocatechuate were both produced in equimolar ratio relative to vanillin consumption. 4-Hydroxybenzaldehyde was a growth-supportive aromatic compound for both C. formicoaceticum and Clostridium aceticum (doubling times approximated 5 h), was oxidized stoichiometrically to 4-hydroxybenzoate, and was not appreciably toxic at concentrations up to 15 mM. Acetate was (i) the major reduced end product detected concomitant to growth and to benzaldehyde oxidation and (ii) formed in close approximation to the following stoichiometry: 4 4-hydroxybenzaldehyde + 2CO₂+2H₂O4 4-hydroxybenzoate + CH₃COOH. We conclude that these two acetogens are capable of benzaldehyde-coupled acetogenesis and growth.     

Phenylpropanoid 2,3-dioxygenase involved in the cleavage of the ferulic acid side chain to form vanillin and glyoxylic acid in Vanilla planifolia

Enzyme catalyzing the cleavage of the phenylpropanoid side chain was partially purified by ion exchange and gel filtration column chromatography after (NH₄)₂SO₄ precipitation. Enzyme activities were dependent on the concentration of dithiothreitol (DTT) or glutathione (GSH) and activated by addition of 0.5 mM Fe²⁺. Enzyme activity for ferulic acid was as high as for 4-coumaric acid in the presence of GSH, suggesting that GSH acts as an endogenous reductant in vanillin biosynthesis. Analyses of the enzymatic reaction products with quantitative NMR (qNMR) indicated that an amount of glyoxylic acid (GA) proportional to vanillin was released from ferulic acid by the enzymatic reaction. These results suggest that phenylpropanoid 2,3-dioxygenase is involved in the cleavage of the ferulic acid side chain to form vanillin and GA in Vanilla planifolia.     

Improved vanillin production in baker's yeast through <Emphasis Type="Italic">in silico</Emphasis> design

Background  

Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid Vanilla planifolia. Currently vanillin is mostly produced via chemical synthesis. A de novo synthetic pathway for heterologous vanillin production from glucose has recently been implemented in baker's yeast, Saccharamyces cerevisiae. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis.     

Nutrient modifications for improved growth of Brassica nigra cell suspension cultures

Cell pellet yield of two Brassica nigra suspension cultures was stimulated by amino acid supplements in the growth medium. This could confound the interpretation of amino acid feeding studies involved in characterizing amino acid metabolism mutants. The nutritional requirements of one of the Brassica nigra suspension cultures growing in modified Murashige & Skoog medium were therefore reviewed. Sucrose at 2% w/v was growth limiting and amino or organic acid supplements stimulated growth rate and yield. Increasing sucrose to 6% and supplementing with 15 mM sodium succinate increased maximum cell pellet volume by 2.7 times and maximum dry weight by 2.8 times, stimulated cell enlargement and produced similar maximum numbers of cells per culture. The further addition of an amino acid supplement of 4 mM alanine, 4 mM glutamine and 1 mM glutamate produced no further improvement. The revised medium was more strongly buffered, supported cell growth for a longer period and permitted a 30-fold reduction in the minimum cell inoculum. Cells grown in the revised medium are 10-fold more resistant to growth inhibition by the tryptophan analogue 5MT. These advantages recommend the revised medium for amino acid feeding, mutant isolation and similar studies.     

Metabolism of isoeugenol via isoeugenol-diol by a newly isolated strain of Bacillus subtilis HS8

A bacterium designated as HS8 was newly isolated from soil based on its ability to degrade isoeugenol. The strain was identified as Bacillus subtilis according to its 16S rDNA sequence analysis and biochemical characteristics. The metabolic pathway for the degradation of isoeugenol was examined. Isoeugenol-diol, for the first time, was detected as an intermediate from isoeugenol to vanillin by a bacterial strain. Isoeugenol was converted to vanillin via isoeugenol-diol, and vanillin was then metabolized via vanillic acid to guaiacol by strain HS8. These metabolites, vanillin, vanillic acid, and guaiacol, are all valuable aromatic compounds in flavor production. At the same time, the bipolymerization of isoeugenol was observed, which produced dehydrodiisoeugenol and decreased the vanillin yield. High level of vanillic acid decarboxylase activity was detected in cell-free extract. These findings provided a detailed profile of isoeugenol metabolism by a B. subtilis strain for the first time, which would improve the production of valuable aromatic compounds by biotechnology.     

Laccase, cellulase and xylanase activities during growth ofPleurotus sajor-caju on sagohampas

Sagohampas, the fibrous pith residue left after starch extraction from sago palm, is abundant at sago-processing factories and can be used as a substrate for the production of laccase by solid substrate fermentation (SSF) withPleurotus sajorcaju, an edible mushroom. The fungus grown onhampas with an adjusted carbon : nitrogen ratio of 35:1, exhibited high laccase activity together with variable cellulase (0.3-2.8 U/g) and xylanase (0.9-10.1 U/g) activity. The maximum amount of laccase produced was approximately 17.7 U/g after 6 days of SSF using 4-week-old inoculum at a density of 10%. With the mature four-week inoculum, laccase activity increased 12-fold compared to that achieved with two-week-old inoculum. The optimum pH and temperature of the crude laccase were 6.0 and 50‡C, respectively. The apparent K_m and V_max values obtained were 0.073 mM and 0.962 U/min, respectively. The maximum laccase activity could be almost doubled after 6 days of fermentation by addition of 0.2 mM vanillin or ferulic acid; the cellulose to lignin ratio increased significantly during the 12 days of SSF, from 2.74 in the control to 3.3, when 0.2 mM of either vanillin or ferulic acid was added to the substrate.     

The differential effects of TCA-cycle acids on the growth of plant cells cultured in liquid media containing various nitrogen sources

Alfalfa (Medicago sativa L. cv. Canadian No. 1), tobacco (Nicotiana tabacum L. var. humilis) and wheat (triticum monococcum L.) cells were grown in a defined, liquid medium containing either ammonium sulfate, L-glutamine or potassium nitrate as the sole nitrogen source, and the effects of the tricarboxylic-acid (TCA) intermediates, citrate and -ketoglutarate (5, 10, 15 mM), on the growth (dry-weight increase) of these cells was observed. The three cell suspension cultures exhibited a different growth response to the TCA-cycle intermediate supplied, depending upon the concentration of the additive and the nitrogen source. Citrate (5 mM) greatly enhanced growth of alfalfa and wheat cells in an ammonium-based medium but was less effective at higher concentrations, and in the case of alfalfa cells markedly inhibited growth. Tobacco cell growth was inhibited by all citrate concentrations tested. In contrast, all concentrations of -ketoglutarate used stimulated the growth of all three cell cultures in an ammonium-based medium. Alfalfa and wheat cells grown in an L-glutamine-based medium were influenced by citrate in a manner similar to that in ammonium-based medium. The growth of tobacco cells was slightly enhanced by 5 mM citrate but inhibited by higher concentrations. -Ketoglutarate, at all concentrations tested, was stimulatory to the growth of the cells of all three species in a glutamine-based medium, except for alfalfa cells which were inhibited at 15 mM. Both TCA-cycle acids inhibited the growth of alfalfa and tobacco cells grown on a nitrate-based medium whereas the growth of wheat cells was almost unaffected.     

Use of bitter gourd (Momordica charantia) peroxidase together with redox mediators to decolorize disperse dyes

In this study, salt fractionated bitter gourd (Momordica charantia) peroxidase was used for the decolorization of water-insoluble disperse dyes; Disperse Red 17 and Disperse Brown 1. Effect of nine different redox mediators; bromophenol, 2,4-dichlorophenol, guaiacol, 1-hydroxybenzotriazole, m-cresol, quinol, syringaldehyde, violuric acid, and vanillin on decolorization of disperse dyes by bitter gourd peroxidase has been investigated. Among these redox mediators, 1-hydroxybenzotriazole was the most effective mediator for decolorization of both the dyes by peroxidase. Bitter gourd peroxidase (0.36 U/mL) could decolorize Disperse Red 17 maximally 90% in the presence of 0.1 mM 1-hydroxybenzotriazole while Disperse Brown 1 was decolorized 65% in the presence of 0.2 mM 1-hydroxybenzotriazole. Maximum decolorization of these dyes was obtained within 1 h of incubation at pH 3.0 and temperature 40°C. The application of such enzyme plus redox mediator systems may be extendable to other recalcitrant and water insoluble synthetic dyes using novel redox mediators and peroxidases from other new and cheaper sources.     

Oxidation of isoeugenol by Nocardia iowensis

Isoeugenol is a starting material for both the synthetic and biotechnological production of vanillin and vanillic acid. Nocardia iowensis DSM 45197 (formerly Nocardia species NRRL 5646) resting cells catalyze the conversion of isoeugenol to vanillic acid, vanillin, vanillyl alcohol and guaiacol. The present study used a variety of chemical, microbial and enzymatic approaches to probe the pathways used by N. iowensis in the oxidation of isoeugenol to these products. Of three possible pathways considered, initial side-chain olefin epoxidation, epoxide hydrolysis to a vicinal diol, and diol cleavage to vanillin and subsequently further oxidation to vanillic acid appears as the most likely route. Isoeugenol was not oxidized to ferulic acid, a well-known microbial transformation precursor for vanillin and vanillic acid. ¹⁸O-Labeled oxygen (one atom) and water (two oxygen atoms) were incorporated into vanillic acid during the whole-cell biotransformation reaction with isoeugenol indicating the likely involvement of oxygenase and hydrolase systems in the bioconversion reaction. Vanillin was converted to singly labeled vanillic acid in the presence of H₂¹⁸O suggesting the presence of an aldehyde oxidase. Cell extracts achieved the conversion of isoeugenol to vanillic acid and vanillin without cofactors. Partial fractionation of two enzyme activities supported the presence of isoeugenol monooxygenase and vanillin oxidase activities in N. iowensis.     

Degradation of ferulic acid by a white rot fungus <Emphasis Type="Italic">Schizophyllum commune</Emphasis>

Summary A ubiquitous white rot fungus Schizophyllum commune was used for the first time to study the degradation of ferulic acid. Vanillic acid was observed as one of the major products of ferulic acid catabolism, with vanillin formed as an intermediate. Almost 99.9% ferulic acid with a initial concentration of 5 mM was consumed by this fungus after 16 days of incubation at 37 °C.     

Substitution of inosine for yeastolate in the culture medium forDrosophila cells

Summary The nutritional requirement ofDrosophila cells (GM₁ and GM₂) was studied. TC Yeastolate contained in the medium forDrosophila cell culture was found to be replaceable with adenosine or inosine without appreciable changes in the generation time of cells. The optimal concentration of either adenosine or inosine was 0.01 mM. Whereas adenosine manifested cell toxicity at concentrations higher than 0.1 mM, in the case of inosine, such an inhibitory effect was not observed up to and at the concentration of 1.0 mM. Further-more, the plating efficiency at cell densities as low as 2×10³ cells per cm² was raised from 0 to 10% by supplementing inosine (0.1 mM) for the TC Yeastolate. Therefore inosine is in practice more useful than adenosine. Experiments using radioactive nucleosides suggested that both adenosine and inosine were exclusively incorporated into RNA as adenosine-monophosphate.     

Substrate specificity of <Emphasis Type="Italic">Stenotrophomonas nitritireducens</Emphasis> in the hydroxylation of unsaturated fatty acid

An isolated bacterium that converted unsaturated fatty acids to hydroxy fatty acids was identified as Stenotrophomonas nitritireducens by API analysis, cellular fatty acids compositions, sequencing the full 16S ribosomal ribonucleic acid, and evaluating its nitrite reduction ability. S. nitritireducens has unique regio-specificity for C16 and C18 cis-9 unsaturated fatty acids. These fatty acids are converted to their 10-hydroxy fatty acids without detectable byproducts. Among the cis-9-unsaturated fatty acids, S. nitritireducens showed the highest specificity for linoleic acid. The cells converted 20 mM linoleic acid to 13.5 mM 10-hydroxy-12(Z)-octadecenoic acid at 30°C and pH 7.5 with a yield of 67.5% (mol/mol).