Biotransformations of propenylbenzenes by an Arthrobacter sp. and its t-anethole blocked mutants

Propenylbenzenes are often used as starting materials in the chemical synthesis of aroma compounds and fine chemicals. In the present study, we demonstrate the ability of an Arthrobacter sp. to transform various structures of propenylbenzenes derived from essential oils to flavor, fragrance, and fine chemicals. Arthrobacter strain TA13 and its t-anethole blocked mutants (incapable of growing on t-anethole) converted isoeugenol to vanillin and vanillic acid; and safrole to hydroxychavicol. High conversion efficiencies were achieved in the biotransformations of isosafrole to piperonylic acid, and eugenol to a mixture of ferulic acid and vanillic acid. In addition, anisic acid was produced in high yields from t-anethole, anisyl alcohol, or anisaldehyde. The accumulation of the corresponding aromatic acids from the tested propenylbenzenes is due to the lack of m-demethylase activity in strain TA13 that prevents further cleavage of the benzene ring. Interestingly, in the transformation of eugenol (a 2-propenylbenzene) the side chain was initially oxidized to the corresponding cinamic acid derivative (ferulic acid) while the 1-propenylbenzenes gave substituted benzoic acids, suggesting two different chain shortening mechanisms.     

Papaverine degradation with papaverine mutants of a Nocardia sp

Nocardia mutants, capable of utilizing papaverine as a sole source of carbon and nitrogen, were induced with N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C. The isolated mutants were divided in four groups, depending on the accumulated papaverine metabolites: 4 = 2-(2-hydroxy-4,5-dimethoxyphenyl)ethanol; 5 = 3,4-dimethoxyphenylacetic acid; 6 = 2-hydroxy-4,5-dimethoxyphenylacetic acid; 10 = 3-(3,4-dimethoxybenzyl)-2-hydroxy-5,6-dimethoxy-1-indenone; 11 = 4-(3,4-dimethoxybenzyl)-6,7-dimethoxyisocoumarin. The degrading pathway of metabolites 5 and 6 was investigated. Studies of the cell wall and membrane fraction of the mutants and wild-type show two inducible, particle-bound proteins with the molecular mass of 29 000 and 60 000 Da involved in papaverine degradation.     

Degradation of naphthalene by thermophilic bacteria <Emphasis Type="Italic">via</Emphasis> a pathway,through protocatechuic acid

A number of thermophilic bacteria capable of utilizing naphthalene as a sole source of carbon were isolated from a high-temperature oilfield in Lithuania. These isolates were able to utilize several other aromatic compounds, such as anthracene, benzene, phenol, benzene-1, 3-diol, protocatechuic acid as well. Thermophilic isolate G27 ascribed to Geobacillus genus was found to have a high aromatic compound degrading capacity. Spectrophotometric determination of enzyme activities in cell-free extracts revealed that the last aromatic ring fission enzyme in naphthalene biotransformation by Geobacillus sp. G27 was inducible via protocatechuate 3, 4-dioxygenase; no protocatechuate 4, 5-dioxygenase, protocatechuate 2, 3-dioxygenase activities were detected. Intermediates such as o-phthalic and protocatechuic acids detected in culture supernatant confirmed that the metabolism of naphthalene by Geobacillus sp. G27 can proceed through protocatechuic acid via ortho-cleavage pathway and thus differs from the pathways known for mesophilic bacteria.     

Plasmid-mediated degradation of o- and p-phthalate by Pseudomonas fluorescens

A Pseudomonas fluorescens strain SKP3 capable of utilizing both phthalic acid and terephthalic acid as sole source of carbon and energy was isolated by enrichment technique. Phthalic acid, terephthalic acid and protocatechuic acid were easily oxidized by both phthalate-grown and glucose-grown cells without a lag period. Phthalic acid is metabolized through the ortho cleavage pathway and terephthalic acid through the meta cleavage pathway and the enzymes of the two pathways are constitutive in nature. A large plasmid of approximately 140kb in size was found to be involved in the degradation of phthalates. The catabolic plasmid pSKL was transferable to different hosts.     

Degradation of phenanthrene via meta-cleavage of 2-hydroxy-1-naphthoic acid by Ochrobactrum sp. strain PWTJD

The present study describes the assimilation of phenanthrene by an aerobic bacterium, Ochrobactrum sp. strain PWTJD, isolated from municipal waste-contaminated soil sample utilizing phenanthrene as a sole source of carbon and energy. The isolate was identified as Ochrobactrum sp. based on the morphological, nutritional and biochemical characteristics as well as 16S rRNA gene sequence analysis. A combination of chromatographic analyses, oxygen uptake assay and enzymatic studies confirmed the degradation of phenanthrene by the strain PWTJD via 2-hydroxy-1-naphthoic acid, salicylic acid and catechol. The strain PWTJD could also utilize 2-hydroxy-1-naphthoic acid and salicylic acid, while the former was metabolized by a ferric-dependent meta-cleavage dioxygenase. In the lower pathway, salicylic acid was metabolized to catechol and was further degraded by catechol 2,3-dioxygenase to 2-hydroxymuconoaldehyde acid, ultimately leading to tricarboxylic acid cycle intermediates. This is the first report of the complete degradation of a polycyclic aromatic hydrocarbon molecule by Gram-negative Ochrobactrum sp. describing the involvement of the meta-cleavage pathway of 2-hydroxy-1-naphthoic acid in phenanthrene assimilation.     

Degradation of anthracene by Mycobacterium sp. strain LB501T proceeds via a novel pathway,through o-phthalic acid

Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.     

Dissimilation of ferulic acid byBacillus subtilis

Bacillus subtilis utilized ferulic acid and its intermediates vanillin, vanillic acid, and protocatechuic acid as sole carbon source. The enzymes of the ferulic acid degradative pathway such as deacetylase, vanillin oxidase, vanillate-o-demethylase, and protocatechuate 3,4-dioxygenase were inducible in nature. Concentration of the inducer profoundly influenced the induction of the enzymes involved in ferulic acid dissimilation.     

Degradation of lignin and lignin derivatives by Acinetobacter sp.

An Acinetobacter sp. utilized 2-methoxy-4-formylphenoxyacetic acid, dehydrodivanillyl alcohol, dehydrodiisoeugenol and conidendrin as sole carbon source. It also degraded ¹⁴C-labelled DHP lignin and teakwood lignin. Vanillic acid, protocatechuic acid and catechol were separated from 2-methoxy-4-formylphenoxyacetic acid grown cultures. Both protocatechuic acid and catechol were formed from dehydrodivanillyl alcohol, dehydrodiisoeugenol and conidendrin. On the dimeric lignin model substances this Acinetobacter sp. produced protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase.     

Degradation of Anthracene by Mycobacterium sp. Strain LB501T Proceeds via a Novel Pathway, through o-Phthalic Acid

Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.     

Degradation of p-benzyloxyphenol by Acinetobacter sp.

Abstract Acinetobacter sp. utilized p -benzyloxyphenol as sole carbon source and degraded it to p -hydroxybenzaldehyde, p -hydroxybenzoic acid, protocatechuic acid and catechol. The intermediates were identified by paper chromatography, TLC, IR, GC and HPLC. Acinetobacter sp. produced protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase during the degradation of p -benzoloxyphenol.     

Degradation of lawsone by Pseudomonas putida L2

From humus obtained from Stuttgart, a bacterium was isolated with lawsone (2-hydroxy-1,4-naphthoquinone) as selective source of carbon. This bacterium is capable of utilizing lawsone as sole source of carbon and energy. Morphological and physiological characteristics of the bacterium were examined and it was identified as a strain of Pseudomonas putida. The organism is referred to as Pseudomonas putida L2. The degradation of lawsone by Pseudomonas putida L2 was investigated. Salicylic acid and catechol were isolated and identified as metabolites. In lawsone-induced cells of Pseudomonas putida L2, salicylic acid is converted to catechol by salicylate 1-monooxygenase. Catechol 1,2-dioxygenase catalyses ortho-fission of catechol which is then metabolized via the beta-ketoadipate pathway. Formation of cis,cis-muconate and beta-ketoadipate was demonstrated by enzyme assays. Salicylate 1-monooxygenase and catechol 1,2-dioxygenase are induced sequentially. The enzymes of the beta-ketoadipate pathway are also inducible. Naphthoquinone hydroxylase, however, was demonstrated in induced and non-induced cells. This constitutive enzyme enables Pseudomonas putida L2 to degrade various 1,4-naphthoquinones in experiments with resting cells.     

Molecular characterization of the genes pcaG and pcaH, encoding protocatechuate 3,4-dioxygenase, which are essential for vanillin catabolism in Pseudomonas sp. strain HR199

Pseudomonas sp. strain HR199 is able to utilize eugenol (4-allyl-2-methoxyphenol), vanillin (4-hydroxy-3-methoxybenzaldehyde), or protocatechuate as the sole carbon source for growth. Mutants of this strain which were impaired in the catabolism of vanillin but retained the ability to utilize eugenol or protocatechuate were obtained after nitrosoguanidine mutagenesis. One mutant (SK6169) was used as recipient of a Pseudomonas sp. strain HR199 genomic library in cosmid pVK100, and phenotypic complementation was achieved with a 5.8-kbp EcoRI fragment (E58). The amino acid sequences deduced from two corresponding open reading frames (ORF) identified on E58 revealed high degrees of homology to pcaG and pcaH, encoding the two subunits of protocatechuate 3,4-dioxygenase. Three additional ORF most probably encoded a 4-hydroxybenzoate 3-hydroxylase (PobA) and two putative regulatory proteins, which exhibited homology to PcaQ of Agrobacterium tumefaciens and PobR of Pseudomonas aeruginosa, respectively. Since mutant SK6169 was also complemented by a subfragment of E58 that harbored only pcaH, this mutant was most probably lacking a functional beta subunit of the protocatechuate 3, 4-dioxygenase. Since this mutant was still able to grow on protocatechuate and lacked protocatechuate 4,5-dioxygenase and protocatechuate 2,3-dioxygenase, the degradation had to be catalyzed by different enzymes. Two other mutants (SK6184 and SK6190), which were also impaired in the catabolism of vanillin, were not complemented by fragment E58. Since these mutants accumulated 3-carboxy muconolactone during cultivation on eugenol, they most probably exhibited a defect in a step of the catabolic pathway following the ortho cleavage. Moreover, in these mutants cyclization of 3-carboxymuconic acid seems to occur by a syn absolute stereochemical course, which is normally only observed for cis, cis-muconate lactonization in pseudomonads. In conclusion, vanillin is degraded through the ortho-cleavage pathway in Pseudomonas sp. strain HR199 whereas protocatechuate could also be metabolized via a different pathway in the mutants.     

An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments

The application of single-molecule fluorescence techniques to complex biological systems places demands on the performance of single fluorophores. We present an enzymatic oxygen scavenging system for improved dye stability in single-molecule experiments. We compared the previously described protocatechuic acid/protocatechuate-3,4-dioxygenase system to the currently employed glucose oxidase/catalase system. Under standardized conditions, we observed lower dissolved oxygen concentrations with the protocatechuic acid/protocatechuate-3,4-dioxygenase system. Furthermore, we observed increased initial lifetimes of single Cy3, Cy5, and Alexa488 fluorophores. We further tested the effects of chemical additives in this system. We found that biological reducing agents increase both the frequency and duration of blinking events of Cy5, an effect that scales with reducing potential. We observed increased stability of Cy3 and Alexa488 in the presence of the antioxidants ascorbic acid and n-propyl gallate. This new O₂-scavenging system should have wide application for single-molecule fluorescence experiments.     

Degradation of 4-chlorobiphenyl and 4-chlorobenzoic acid by the strain <Emphasis Type="Italic">Rhodococcus ruber</Emphasis> P25

The strain Rhodococcus ruber P25 utilizes 4-chlorobiphenyl (4CB) and 4-chlorobenzoic acid (4CBA) as sole carbon and energy sources. 4CB degradation by washed cells of strain P25 was accompanied by transient formation of 4CBA, followed by its utilization and release of equimolar amounts of chloride ions into the medium. The strain R. ruber P25 possessed active enzyme systems providing 4CBA degradation via the stages of formation of intermediates, para-hydroxybenzoate (PHBA) and protocatechuic acid (PCA), to compounds of the basic metabolism. The involvement of protocatechuate 4,5-dioxygenase in 4CBA degradation by rhodococci was revealed. It was established that the initial stage of 4CBA degradation (dehalogenation) in the strain R. ruber P25 was controlled by the fcbA and fcbB genes encoding 4-CBA-CoA ligase and 4-CBA-CoA dehalogenase, respectively. The genes encoding 4CBA dehalogenase components have not been previously detected and characterized in bacteria of the genus Rhodococcus.     

Degradation of aniline by newly isolated,extremely aniline-tolerant <Emphasis Type="Italic">Delftia </Emphasis>sp. AN3

A bacterial strain, AN3, which was able to use aniline or acetanilide as sole carbon, nitrogen and energy sources was isolated from activated sludge and identified as Delftiasp. AN3. This strain was capable of growing on concentrations of aniline up to 53.8 mM (5000 mg/l). Substituted anilines such as N-methylaniline, N, N-dimethylaniline, 2-methylaniline, 4-methylaniline, 2-chloroaniline, 3-chloroaniline, o-aminoaniline, m-aminoaniline, p-aminoaniline, and sulfanilic acid did not support the growth of strain AN3. The optimal temperature and pH for growth and degradation of aniline were 30 degrees C and 7.0, respectively. The activities of aniline dioxygenase, catechol 2,3-dioxygenase and other enzymes involved in aniline degradation were determined, and results indicated that all of them were inducible. The K (m) and V (max) of aniline dioxygenase were 0.29 mM and 0.043 mmol/mg protein/min, respectively. The K (m) and V (max) of catechol 2, 3-dioxygenase for catechol were 0.016 mM and 0.015 mmol/mg protein/min, respectively. Based on the results obtained, a pathway for the degradation of aniline by Delftiasp. AN3 was proposed. The importance of the strain to the operation of municipal wastewater treatment plants is discussed.     

Degradation of (+)-catechin by Acinetobacter calcoaceticus MTC 127

Acinetobacter calcoaceticus MTC 127 was able to grow on catechin and protocatechuic acid (PCA) as sole carbon source. Cells induced with catechin oxidized catechin and PCA at rates higher than cells of uninduced cultures. Two aromatic compounds, PCA and phloroglucinol carboxylic acid (PGCA) were isolated from culture filtrate of cells grown in catechin and characterized by infrared spectrometry and high performance thin-layer chromatography. Moreover, A. calcoaceticus MTC 127 produced high levels of PCA compared to PGCA in the degradation of catechin. Based upon these results, a pathway for the degradation of (+)-catechin in A. calcoaceticus MTC 127 is proposed. Enzymes extracted from catechin-induced culture showed catechin oxygenase (cox) and protocatechuate 3,4-dioxygenase (pcd) activities. Catechin oxygenase was purified by column chromatography and SDS-PAGE analysis showed a single band with an apparent molecular weight of 47 kDa.     

Novel pathway for the degradation of 2-chloro-4-nitrobenzoic acid by Acinetobacter sp. strain RKJ12

The organism Acinetobacter sp. RKJ12 is capable of utilizing 2-chloro-4-nitrobenzoic acid (2C4NBA) as a sole source of carbon, nitrogen, and energy. In the degradation of 2C4NBA by strain RKJ12, various metabolites were isolated and identified by a combination of chromatographic, spectroscopic, and enzymatic activities, revealing a novel assimilation pathway involving both oxidative and reductive catabolic mechanisms. The metabolism of 2C4NBA was initiated by oxidative ortho dehalogenation, leading to the formation of 2-hydroxy-4-nitrobenzoic acid (2H4NBA), which subsequently was metabolized into 2,4-dihydroxybenzoic acid (2,4-DHBA) by a mono-oxygenase with the concomitant release of chloride and nitrite ions. Stoichiometric analysis indicated the consumption of 1 mol O(2) per conversion of 2C4NBA to 2,4-DHBA, ruling out the possibility of two oxidative reactions. Experiments with labeled H(2)(18)O and (18)O(2) indicated the involvement of mono-oxygenase-catalyzed initial hydrolytic dechlorination and oxidative denitration mechanisms. The further degradation of 2,4-DHBA then proceeds via reductive dehydroxylation involving the formation of salicylic acid. In the lower pathway, the organism transformed salicylic acid into catechol, which was mineralized by the ortho ring cleavage catechol-1,2-dioxygenase to cis, cis-muconic acid, ultimately forming tricarboxylic acid cycle intermediates. Furthermore, the studies carried out on a 2C4NBA(-) derivative and a 2C4NBA(+) transconjugant demonstrated that the catabolic genes for the 2C4NBA degradation pathway possibly reside on the ～55-kb transmissible plasmid present in RKJ12.     

Degradation of 4-chlorobenzoic acid by an Arthrobacter species (author's transl)]

An Arthrobacter sp. growing on 4-Chlorobenzoic acid as its sole source of carbon excretes 4-hydroxygenzoic acid and protocatechuic acid into the culture medium. Protocatechuic acid is further attacked by "meta"-cleavage. During growth of the Arthrobacter sp. on benzoic acid cis-cis muconic acid can be isolated from the medium, suggesting the involvement of the "ortho"-cleavage pathway. The enzymes both for the "meta"- and the "ortho"-cleavage pathway are inducible.     

Degradation of coniferyl alcohol and other lignin-related aromatic compounds by Nocardia sp. DSM 1069

Nocardia sp. DSM 1069 was grown on mineral salt media with coniferyl alcohol, 4-methoxybenzoic acid, 3-methoxybenzoic acid or veratric acid as sole sources of carbon and energy. During incubation on coniferyl alcohol, the formation of coniferyl aldehyde, ferulic acid and quantitative accumulation of vanillic acid and proteocatechuic acid could be achieved with mutants. Washed cell suspensions of N. sp. grown on 4-methoxybenzoic acid, oxidized 4-hydroxybenzoic acid and protocatechuic acid. Cells grown on veratric acid, oxidized vanillic acid, isovanillic acid, and protocatechuic acid. Cell extracts were shown to cleave protocatechuic acid by ortho-fission.A mutant without protocatechuate 3,4-dioxygenase activity was not influenced in its growth on 3 methoxybenzoic acid. Cell free extracts of cells grown on 3-methoxybenzoic acid were shown to catalyze the oxidation of gentisic acid (2,5-dihydroxybenzoic acid). The resulting ring cleavage product was further metabolized by a glutathione dependent reaction.The specificity of the demethylation reactions has been investigated with a mutant unable to grow on vanillic acid. This mutant was not impaired in the degradation of isovanillic acid, 4-methoxy-, or 3-methoxybenzoic acid, whereas growth of this mutant on veratric acid (3,4-dimethoxybenzoic acid) was only half as much as that of the wild type. Concomitantly with growth on veratric acid this mutant accumulated vanillic acid with a yield of about 50%.A pathway for the catabolism of coniferyl alcohol, involving oxidation and shortening of the side chain, and of 4-methoxybenzoic acid and veratric acid with protocatechuic acid as intermediate is being proposed. A second one is proposed for the degradation of 3-methoxybenzoic acid with gentisic acid as intermediate.     

Metabolism of dibutylphthalate and phthalate by Micrococcus sp. strain 12B.

Micrococcus sp. strain 12B was isolated by enriching for growth with dibutylphthalate as the sole carbon and energy source. A pathway for the metabolism of dibutylphthalate and phthalate by micrococcus sp. strain 12B is proposed: dibutylphthalate leads to monobutylphthalate leads to phthalate leads to 3,4-dihydro-3,4-dihydroxyphthalate leads to 3,4-dihydroxyphthalate leads to protocatechuate (3,4-dihdroxybenzoate). Protocatechuate is metabolized both by the meta-cleavage pathway through 4-carboxy-2-hydroxymuconic semialdehyde and 4-carboxy-2-hydroxymuconate to pyruvate and oxaloacetate and by the ortho-cleavage pathway to beta-ketoadipate. Dibutylphthalate- and phthalate-grown cells readily oxidized dibutylphthalate, phthalate, 3,4-dihydroxyphthalate, and protocatechuate. Extracts of cells grown with dibutylphthalate or phthalate contained the 3,4-dihydroxyphthalate decarboxylase and the enzymes of the protocatechuater 4,5-meta-cleavage pathway. Extracts of dibutylphthalate-grown cells also contained the protocatechuate ortho-cleavage pathway enzymes. The dibutylphthalate-hydrolyzing esterase and 3,4-dihydroxyphthalate decarboxylase were constitutively synthesized; phthalate-3,4-dioxygenase (and possibly the "dihydrodiol" dehydrogenase) was inducible by phthalate or a metabolite occurring before protocatechuate in the pathway; two protocatechuate oxygenases and subsequent enzymes were inducible by protocatechuate or a subsequent metabolic product. During growth at 37 degrees C, strain 12B gave clones at high frequency that had lost the ability to grow with phthalate esters. One of these nonrevertible mutants, strain 12B-Cl, lacked all of the enzymes required for the metabolism of dibutylphthalate through the protocatechuate meta-cleavage pathway. Enzymes for the metabolism of protocatechuate by the ortho-cleavage pathway were present in this strain grown with p-hydroxybenzoate or protocatechuate.