A catecholic 9,10-seco steroid as a product of aerobic catabolism of cholic acid by a Pseudomonas sp

A mutant of the efficient bile acid-utilizing Pseudomonas putida ATCC 31752 was found to accumulate three major catabolites on aerobic growth on cholic acid. One of these catabolites was isolated and identified as 3,4,7,12 beta-tetrahydroxy-9,10-seco-1,3,5(10)-androstatriene-9,17-dione (2). This is the first catecholic 9,10-secosteroid isolated from the microbial degradation of bile acids or sterols and confirms the role of such secosteroids in the microbial degradative pathway for steroids.     

Effect of restricted aeration on catabolism of cholic acid by two Pseudomonas species.

Examination of some previously isolated bile acid-utilizing Pseudomonas strains showed that Pseudomonas sp. ATCC 31752, together with other fluorescent strains, can be assigned to Pseudomonas putida biotype B, whereas Pseudomonas sp. ATCC 31753, like most other nonfluorescent strains, is an unrecognized phenotype. A study was made of the growth of these two species at 25 degrees C and pH 7.0 in a fermentor with 2.5 g of sodium cholate liter-1 as sole carbon source, and the catabolism of the cholate and its products was followed by high-pressure liquid chromatographic and thin-layer chromatographic examination. At aeration rates of either 150 or 5 ml min-1 liter-1, growth of each species followed the same catabolic pathway. 7 alpha, 12 beta-Dihydroxy-1,4-androstadiene-3,17-dione was the major catabolite formed, with 0.3 g liter-1 being the maximum concentration that accumulated at the higher aeration rate, whereas 1.4 g liter-1 accumulated at the lower aeration rate, irrespective of the species used. The latter yield is sufficiently high to be of potential commercial value if such a catabolite were found to be economically useful for steroid drug manufacture. It is postulated that the rate-limiting step in cholic acid catabolism by these species at the lower aeration rate is 9 alpha-hydroxylation, a step requiring molecular oxygen, hence, the marked effect of oxygen limitation on catabolite accumulation. Another consequence of oxygen limitation is the production of a red pigment in the culture medium, which, however, does not affect catabolite recovery.     

Effect of restricted aeration on catabolism of cholic acid by two Pseudomonas species

Examination of some previously isolated bile acid-utilizing Pseudomonas strains showed that Pseudomonas sp. ATCC 31752, together with other fluorescent strains, can be assigned to Pseudomonas putida biotype B, whereas Pseudomonas sp. ATCC 31753, like most other nonfluorescent strains, is an unrecognized phenotype. A study was made of the growth of these two species at 25 degrees C and pH 7.0 in a fermentor with 2.5 g of sodium cholate liter-1 as sole carbon source, and the catabolism of the cholate and its products was followed by high-pressure liquid chromatographic and thin-layer chromatographic examination. At aeration rates of either 150 or 5 ml min-1 liter-1, growth of each species followed the same catabolic pathway. 7 alpha, 12 beta-Dihydroxy-1,4-androstadiene-3,17-dione was the major catabolite formed, with 0.3 g liter-1 being the maximum concentration that accumulated at the higher aeration rate, whereas 1.4 g liter-1 accumulated at the lower aeration rate, irrespective of the species used. The latter yield is sufficiently high to be of potential commercial value if such a catabolite were found to be economically useful for steroid drug manufacture. It is postulated that the rate-limiting step in cholic acid catabolism by these species at the lower aeration rate is 9 alpha-hydroxylation, a step requiring molecular oxygen, hence, the marked effect of oxygen limitation on catabolite accumulation. Another consequence of oxygen limitation is the production of a red pigment in the culture medium, which, however, does not affect catabolite recovery.     

Phenolic 9,10-secosteroids as products of the catabolism of bile acids by a Pseudomonas sp.

The obligate aerobe, Pseudomonas putida ATCC 31752, efficiently utilises bile acids as a source of carbon and energy for growth and maintenance. When aeration is considerably restricted, a consequence to the catabolism of the bile acids in a fermentor is an accumulation of certain steroidal catabolites. Evidence is presented to show that among these are hydroxy-9,10-seco-1,3,5(10)-androstratriene-9,17-diones and those from four of the common bile acids, cholic, chenodeoxycholic, hyodeoxycholic and deoxycholic acids have been isolated and their structures determined. The product of catabolism of hyodeoxycholic acid appears to exist in a hemi-acetal form which readily forms an acetal during isolation procedures. All but one of these are described for the first time.     

Simultaneous degradation of atrazine and phenol by Pseudomonas sp. strain ADP: effects of toxicity and adaptation

The strain Pseudomonas sp. strain ADP is able to degrade atrazine as a sole nitrogen source and therefore needs a single source for both carbon and energy for growth. In addition to the typical C source for Pseudomonas, Na(2)-succinate, the strain can also grow with phenol as a carbon source. Phenol is oxidized to catechol by a multicomponent phenol hydroxylase. Catechol is degraded via the ortho pathway using catechol 1,2-dioxygenase. It was possible to stimulate the strain in order to degrade very high concentrations of phenol (1,000 mg/liter) and atrazine (150 mg/liter) simultaneously. With cyanuric acid, the major intermediate of atrazine degradation, as an N source, both the growth rate and the phenol degradation rate were similar to those measured with ammonia as an N source. With atrazine as an N source, the growth rate and the phenol degradation rate were reduced to approximately 35% of those obtained for cyanuric acid. This presents clear evidence that although the first three enzymes of the atrazine degradation pathway are constitutively present, either these enzymes or the uptake of atrazine is the bottleneck that diminishes the growth rate of Pseudomonas sp. strain ADP with atrazine as an N source. Whereas atrazine and cyanuric acid showed no significant toxic effect on the cells, phenol reduces growth and activates or induces typical membrane-adaptive responses known for the genus Pseudomonas. Therefore Pseudomonas sp. strain ADP is an ideal bacterium for the investigation of the regulatory interactions among several catabolic genes and stress response mechanisms during the simultaneous degradation of toxic phenolic compounds and a xenobiotic N source such as atrazine.     

Effects of dissolved organic carbon and second substrates on the biodegradation of organic compounds at low concentrations

Pseudomonas acidovorans and Pseudomonas sp. strain ANL but not Salmonella typhimurium grew in an inorganic salts solution. The growth of P. acidovorans in this solution was not enhanced by the addition of 2.0 micrograms of phenol per liter, but the phenol was mineralized. Mineralization of 2.0 micrograms of phenol per liter by P. acidovorans was delayed 16 h by 70 micrograms of acetate per liter, and the delay was lengthened by increasing acetate concentrations, whereas phenol and acetate were utilized simultaneously at concentrations of 2.0 and 13 micrograms/liter, respectively. Growth of Pseudomonas sp. in the inorganic salts solution was not affected by the addition of 3.0 micrograms each of glucose and aniline per liter, nor was mineralization of the two compounds detected during the initial period of growth. However, mineralization of both substrates by this organism occurred simultaneously during the latter phases of growth and after growth had ended at the expense of the uncharacterized dissolved organic compounds in the salts solution. In contrast, when Pseudomonas sp. was grown in the salts solution supplemented with 300 micrograms each of glucose and aniline, the sugar was mineralized first, and aniline was mineralized only after much of the glucose carbon was converted to CO2. S. typhimurium failed to multiply in the salts solution with 1.0 micrograms of glucose per liter. It grew slightly but mineralized little of the sugar at 5.0 micrograms/liter, but its population density rose at 10 micrograms of glucose per liter or higher. The hexose could be mineralized at 0.5 micrograms/liter, however, if the solution contained 5.0 mg of arabinose per liter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dissolved organic carbon and second substrates on the biodegradation of organic compounds at low concentrations.

Pseudomonas acidovorans and Pseudomonas sp. strain ANL but not Salmonella typhimurium grew in an inorganic salts solution. The growth of P. acidovorans in this solution was not enhanced by the addition of 2.0 micrograms of phenol per liter, but the phenol was mineralized. Mineralization of 2.0 micrograms of phenol per liter by P. acidovorans was delayed 16 h by 70 micrograms of acetate per liter, and the delay was lengthened by increasing acetate concentrations, whereas phenol and acetate were utilized simultaneously at concentrations of 2.0 and 13 micrograms/liter, respectively. Growth of Pseudomonas sp. in the inorganic salts solution was not affected by the addition of 3.0 micrograms each of glucose and aniline per liter, nor was mineralization of the two compounds detected during the initial period of growth. However, mineralization of both substrates by this organism occurred simultaneously during the latter phases of growth and after growth had ended at the expense of the uncharacterized dissolved organic compounds in the salts solution. In contrast, when Pseudomonas sp. was grown in the salts solution supplemented with 300 micrograms each of glucose and aniline, the sugar was mineralized first, and aniline was mineralized only after much of the glucose carbon was converted to CO2. S. typhimurium failed to multiply in the salts solution with 1.0 micrograms of glucose per liter. It grew slightly but mineralized little of the sugar at 5.0 micrograms/liter, but its population density rose at 10 micrograms of glucose per liter or higher. The hexose could be mineralized at 0.5 micrograms/liter, however, if the solution contained 5.0 mg of arabinose per liter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bile-Mediated Aminoglycoside Sensitivity in Lactobacillus Species Likely Results from Increased Membrane Permeability Attributable to Cholic Acid

Few studies have been conducted on antimicrobial resistance in lactobacilli, presumably because of their nonpathogenic nature as anaerobic commensals. We assessed resistance in 43 type strains and isolates representing 14 species by using agar disk diffusion and MIC analysis in MRS medium. Most noteworthy were two general phenotypes displayed by nearly every strain tested: (i) they were more susceptible (up to 256-fold in some cases) to the deconjugated bile acid cholic acid than to the conjugate taurocholic or taurodeoxycholic acid, and (ii) they became susceptible to aminoglycosides when assayed on agar medium containing 0.5% fractionated bovine bile (ox gall). Two-dimensional MIC analyses of one representative strain, Lactobacillus plantarum WCFS1, at increasing concentrations of ox gall (0 to 30.3 mg/ml) displayed corresponding decreases in resistance to all of the aminoglycosides tested and ethidium bromide. This effect was clinically relevant, with the gentamicin MIC decreasing from >1,000 to 4 μg/ml in just 3.8 mg of ox gall per ml. In uptake studies at pH 6.5, [G-³H]gentamicin accumulation increased over control levels when cells of this strain were exposed to bile acids or reserpine but not when they were exposed to carbonyl cyanide m-chlorophenylhydrazone. The effect was dramatic, particularly with cholic acid, increasing up to 18-fold, whereas only modest increases, 3- and 5-fold, could be achieved with taurocholic acid and ox gall, respectively. Since L. plantarum, particularly strain WCFS1, is known to encode bile salt hydrolase (deconjugation) activity, our data indicate that mainly cholic acid, but not taurocholic acid, effectively permeabilizes the membrane to aminoglycosides. However, at pHs approaching neutral conditions in the intestinal lumen, aminoglycoside resistance due to membrane impermeability may be complemented by a potential efflux mechanism.     

Production of extracellular β-1,3-/β-1,6-glucan by mono- and dikaryons ofSchizophyllum commune

From the dikaryotic mycelium ofSchizophyllum commune ATCC 38548 several monokaryotic strains were obtained by isolating the two types of monokaryotic protoplasts and their reversion to hyphal growth. The dikaryoticS. commune ATCC 38548 produced about 10 g/liter of extracellular β-1,3-/β-1,6-glucan (schizophyllan) after 96 h of growth, while the monokaryons excreted much less of this polysaccharide. During growth of strains of both types of monokaryons indigo and β-1,3-glucanase activities were excreted. Two selected monokaryons were mated with other monokaryoticS. commune strains and some of the dikaryotic mycelia obtained produced about 12 g/liter of extracellular β-1,3-/β-1,6-glucan after 120 h of cultivation.     

Physiological and phylogenetic diversity of bacteria growing on resin acids

Resin acids are tricyclic diterpenes which are synthesized by trees and are a major cause of toxicity of pulp mill effluents. Bacterial strains isolated from three different sources and which grow on resin acids were physiologically characterized. Eleven strains, representating distinct groups, were further characterized physiologically and phylogenetically. The isolates had distinct specificities for use, as growth substrates, of the different resin acids tested. The isolates also used fatty acids but were generally limited in use of other diverse substrates tested. According to their 16S rDNA sequences, the representative isolates are related to members of the genera, Sphingomonas, Zoogloea, Ralstonia, Burkholderia, Pseudomonas and Mycobacterium. Analysis of whole-cell fatty acid profiles generally supported those phylogenetic relationships. However, most of the isolated did not have high similarities to reference strains in the Microbial Identification System database of fatty acid profiles or in the Biolog database of substrate oxidation patterns. Described species of Sphingomonas, Zoolgoea, Burkholderia Pseudomonas, most closely related to the isolates we characterized, failed to grow on, or degrade, resin acids. We propose recognition of Zoogloea resiniphila sp. nov., Pseudomonas vancouverensis sp. nov., P. abietaniphila sp. nov. and P. multiresinivorans sp. nov.     

Role of dissolution rate and solubility in biodegradation of aromatic compounds.

Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp. able to grow on biphenyl were isolated from sewage. The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate. Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp. and Pseudomonas sp. In biphenyl-supplemented media, Flavobacterium sp. had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon. Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition. Pseudomonas sp. did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate. It was calculated that Pseudomonas sp. consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined. In a medium with anthracene as the sole added carbon source, Flavobacterium sp. converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing. Flavobacterium sp. and Beijerinckia sp. initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source. Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.     

Role of dissolution rate and solubility in biodegradation of aromatic compounds

Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp. able to grow on biphenyl were isolated from sewage. The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate. Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp. and Pseudomonas sp. In biphenyl-supplemented media, Flavobacterium sp. had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon. Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition. Pseudomonas sp. did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate. It was calculated that Pseudomonas sp. consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined. In a medium with anthracene as the sole added carbon source, Flavobacterium sp. converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing. Flavobacterium sp. and Beijerinckia sp. initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source. Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.     

Distribution of alkB genes within n-alkane-degrading bacteria

Fifty-four bacterial strains belonging to 37 species were tested for their ability to assimilate short chain and/or medium chain liquid n-alkanes. A gene probe derived from the alkB gene of Pseudomonas oleovorans ATCC 29347 was utilized in hybridization experiments. Results of Southern hybridization of PCR-amplificates were compared with those of colony hybridization and dot blot hybridization. Strongest signals were received only from Gram-negative bacteria growing solely with short n-alkanes (C10). Hybridization results with soil isolates growing with n-alkanes of different chain lengths suggested as well that alkB genes seem to be widespread only in solely short-chain n-alkane-degrading pseudomonads. PCR products of Rhodococcus sp., Nocardioides sp., Gordona sp. and Sphingomonas sp. growing additionally or solely with medium-chain n-alkane as hexadecane had only few sequence identity with alkB though hybridizing with the gene probe. The derived amino acid sequence of the alkB-amplificate of Pseudomonas aureofaciens showed high homology (95%) with AlkB from Ps. oleovorans. alkB gene disruptants were not able to grow with decane.     

Essential oil analysis and antimicrobial activity of eight Stachys species from Greece

The volatile composition of eight Stachys species has been studied. The investigated taxa were St. alopecuros (L.) Bentham., St. scardica (Griseb.) Hayek, St. cretica L. ssp. cretica, St. germanica L. ssp. heldreichii (Boiss.) Hayek, St. recta L., St. spinulosa L., St. euboica Rech. and St. menthifolia Vis., growing wild in Greece. The essential oils were obtained by hydrodistillation in a modified Clevenger-type apparatus, and their analyses were performed by GC and GC-MS. Identification of the substances was made by comparison of mass spectra and retention indices with literature records. Sesquiterpene hydrocarbons were shown to be the main group of constituents of all taxa. Furthermore, the obtained essential oils were tested against the following six bacteria: Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 35210), Bacillus subtilis (ATCC 10907), Bacillus cereus (clinical isolates), Micrococcus flavus (ATCC 10240), Staphylococcus epidermidis (ATCC 2228), as well as against the following five fungi: Aspergillus niger (ATCC 6275), Penicillium ochrochloron (ATCC 9112), Epidermophyton floccosum (clinical isolates), Candida albicans (clinical isolates) and Trichophyton mentagrophytes (clinical isolates). The tested essential oils showed better activity against bacterial species than against fungi. Pseudomonas aeruginosa was the most resistant strain, as none of the essential oils was active against this strain. The essential oil of St. scardica has been proven most active against both bacteria and fungi.     

Bile-mediated aminoglycoside sensitivity in Lactobacillus species likely results from increased membrane permeability attributable to cholic acid

Few studies have been conducted on antimicrobial resistance in lactobacilli, presumably because of their nonpathogenic nature as anaerobic commensals. We assessed resistance in 43 type strains and isolates representing 14 species by using agar disk diffusion and MIC analysis in MRS medium. Most noteworthy were two general phenotypes displayed by nearly every strain tested: (i) they were more susceptible (up to 256-fold in some cases) to the deconjugated bile acid cholic acid than to the conjugate taurocholic or taurodeoxycholic acid, and (ii) they became susceptible to aminoglycosides when assayed on agar medium containing 0.5% fractionated bovine bile (ox gall). Two-dimensional MIC analyses of one representative strain, Lactobacillus plantarum WCFS1, at increasing concentrations of ox gall (0 to 30.3 mg/ml) displayed corresponding decreases in resistance to all of the aminoglycosides tested and ethidium bromide. This effect was clinically relevant, with the gentamicin MIC decreasing from >1,000 to 4 mug/ml in just 3.8 mg of ox gall per ml. In uptake studies at pH 6.5, [G-3H]gentamicin accumulation increased over control levels when cells of this strain were exposed to bile acids or reserpine but not when they were exposed to carbonyl cyanide m-chlorophenylhydrazone. The effect was dramatic, particularly with cholic acid, increasing up to 18-fold, whereas only modest increases, 3- and 5-fold, could be achieved with taurocholic acid and ox gall, respectively. Since L. plantarum, particularly strain WCFS1, is known to encode bile salt hydrolase (deconjugation) activity, our data indicate that mainly cholic acid, but not taurocholic acid, effectively permeabilizes the membrane to aminoglycosides. However, at pHs approaching neutral conditions in the intestinal lumen, aminoglycoside resistance due to membrane impermeability may be complemented by a potential efflux mechanism.     

Hydrolysis of Selected Organophosphorus Insecticides by Two Bacteria Isolated from Flooded Soil

Flavobacterium sp. ATCC 27551 hydrolysed both diethyl (parathion and diazinon) and dimethyl (methyl parathion and fenitrothion) phosphorothioates while Pseudomonas sp. ATCC 29353 hydrolysed only diethyl (parathion and diazinon) phosphorothioates. Glucose inhibited the hydrolysis of parathion by Pseudomonas sp., but not by Flavobacterium sp. Evidently, the Flavobacterium hydrolase differs from that of Pseudomonas sp. The Pseudomonas sp. converted 4-nitrophenol to 4-aminophenol in the presence of glucose and to nitrite in its absence; 4-nitrophenol was not metabolized by the Flavobacterium sp.     

Involvement of linear plasmids in aerobic biodegradation of vinyl chloride

Pseudomonas putida strain AJ and Ochrobactrum strain TD were isolated from hazardous waste sites based on their ability to use vinyl chloride (VC) as the sole source of carbon and energy under aerobic conditions. Strains AJ and TD also use ethene and ethylene oxide as growth substrates. Strain AJ contained a linear megaplasmid (approximately 260 kb) when grown on VC or ethene, but it contained no circular plasmids. While strain AJ was growing on ethylene oxide, it was observed to contain a 100-kb linear plasmid, and its ability to use VC as a substrate was retained. The linear plasmids in strain AJ were cured, and the ability of strain AJ to consume VC, ethene, and ethylene oxide was lost following growth on a rich substrate (Luria-Bertani broth) through at least three transfers. Strain TD contained three linear plasmids, ranging in size from approximately 90 kb to 320 kb, when growing on VC or ethene. As with strain AJ, the linear plasmids in strain TD were cured following growth on Luria-Bertani broth and its ability to consume VC and ethene was lost. Further analysis of these linear plasmids may help reveal the pathway for VC biodegradation in strains AJ and TD and explain why this process occurs at many but not all sites where groundwater is contaminated with chloroethenes. Metabolism of VC and ethene by strains AJ and TD is initiated by an alkene monooxygenase. Their yields during growth on VC (0.15 to 0.20 mg of total suspended solids per mg of VC) are similar to the yields reported for other isolates (i.e., Mycobacterium sp., Nocardioides sp., and Pseudomonas sp.).     

Role and regulation of the ortho and meta pathways of catechol metabolism in pseudomonads metabolizing naphthalene and salicylate.

The enzymes of naphthalene metabolism are induced in Pseudomonas putida ATCC 17484, PpG7, NCIB 9816, and PG and in Pseudomonas sp. ATCC 17483 during growth on naphthalene or salicylate; 2-aminobenzoate is a gratuitous inducer of these enzymes. The meta-pathway enzymes of catechol metabolism are induced in ATCC 17483 and PPG7 during growth on naphthalene or salicylate or during growth in the presence of 2-aminobenzoate, but in ATCC 17484 and NCIB 9816 the ortho-pathway enzymes of catechol metabolism are induced during growth on naphthalene or salicylate. 2-Aminobenzoate does not induce any enzymes of catechol metabolism in the latter two organisms. In Pseudomonas PG the meta-pathway enzymes are present at high levels under all conditions of growth, but this organism and PpG7 can induce ortho-pathway enzymes during naphthalene or salicylate metabolism. Salicylate appears to be the inducer of the enzymes of naphthalene metabolism in all of the organisms studied and, where they are inducible, of the meta-pathway enzymes, but the properties of Pseudomonas PG suggest that separate, regulatory systems may exist.     

Highly efficient biotransformation of eugenol to ferulic acid and further conversion to vanillin in recombinant strains of Escherichia coli

The vaoA gene from Penicillium simplicissimum CBS 170.90, encoding vanillyl alcohol oxidase, which also catalyzes the conversion of eugenol to coniferyl alcohol, was expressed in Escherichia coli XL1-Blue under the control of the lac promoter, together with the genes calA and calB, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of Pseudomonas sp. strain HR199, respectively. Resting cells of the corresponding recombinant strain E. coli XL1-Blue(pSKvaomPcalAmcalB) converted eugenol to ferulic acid with a molar yield of 91% within 15 h on a 50-ml scale, reaching a ferulic acid concentration of 8.6 g liter(-1). This biotransformation was scaled up to a 30-liter fermentation volume. The maximum production rate for ferulic acid at that scale was 14.4 mmol per h per liter of culture. The maximum concentration of ferulic acid obtained was 14.7 g liter(-1) after a total fermentation time of 30 h, which corresponded to a molar yield of 93.3% with respect to the added amount of eugenol. In a two-step biotransformation, E. coli XL1-Blue(pSKvaomPcalAmcalB) was used to produce ferulic acid from eugenol and, subsequently, E. coli(pSKechE/Hfcs) was used to convert ferulic acid to vanillin (J. Overhage, H. Priefert, and A. Steinbüchel, Appl. Environ. Microbiol. 65:4837-4847, 1999). This process led to 0.3 g of vanillin liter(-1), besides 0.1 g of vanillyl alcohol and 4.6 g of ferulic acid liter(-1). The genes ehyAB, encoding eugenol hydroxylase of Pseudomonas sp. strain HR199, and azu, encoding the potential physiological electron acceptor of this enzyme, were shown to be unsuitable for establishing eugenol bioconversion in E. coli XL1-Blue.