Characterization of the 5-carboxyvanillate decarboxylase gene and its role in lignin-related biphenyl catabolism in Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 degrades a lignin-related biphenyl compound, 5,5'-dehydrodivanillate (DDVA), to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by the DDVA O-demethylase (LigX), the ring cleavage oxygenase (LigZ), and the meta-cleavage compound hydrolase (LigY). In this study we examined the degradation step of 5CVA. 5CVA was transformed to vanillate, O-demethylated, and further degraded via the protocatechuate 4,5-cleavage pathway by this strain. A cosmid clone which conferred the 5CVA degradation activity to a host strain was isolated. In the 7.0-kb EcoRI fragment of the cosmid we found a 1,002-bp open reading frame responsible for the conversion of 5CVA to vanillate, and we designated it ligW. The gene product of ligW (LigW) catalyzed the decarboxylation of 5CVA to produce vanillate along with the specific incorporation of deuterium from deuterium oxide, indicating that LigW is a nonoxidative decarboxylase of 5CVA. LigW did not require any metal ions or cofactors for its activity. The decarboxylase activity was specific to 5CVA. Inhibition experiments with 5CVA analogs suggested that two carboxyl groups oriented meta to each other in 5CVA are important to the substrate recognition by LigW. Gene walking analysis indicated that the ligW gene was located on the 18-kb DNA region with other DDVA catabolic genes, including ligZ, ligY, and ligX.     

Aerobic and Anaerobic Catabolism of Vanillic Acid and Some Other Methoxy-Aromatic Compounds by Pseudomonas sp. Strain PN-1

Vanillic acid (4-hydroxy-3-methoxybenzoic acid) supported the anaerobic (nitrate respiration) but not the aerobic growth of Pseudomonas sp. strain PN-1. Cells grown anaerobically on vanillate oxidized vanillate, p-hydroxybenzoate, and protocatechuic acid (3,4-dihydroxybenzoic acid) with O₂ or nitrate. Veratric acid (3,4-dimethoxybenzoic acid) but not isovanillic acid (3-hydroxy-4-methoxybenzoic acid) induced cells for the oxic and anoxic utilization of vanillate, and protocatechuate was detected as an intermediate of vanillate breakdown under either condition. Aerobic catabolism of protocatechuate proceeded via 4,5-meta cleavage, whereas anaerobically it was probably dehydroxylated to benzoic acid. Formaldehyde was identified as a product of aerobic demethylation, indicating a monooxygenase mechanism, but was not detected during anaerobic demethylation. The aerobic and anaerobic systems had similar but not identical substrate specificities. Both utilized m-anisic acid (3-methoxybenzoic acid) and veratrate but not o- or p-anisate and isovanillate. Syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid), 3-O-methylgallic acid (3-methoxy-4,5-dihydroxybenzoic acid), and 3,5-dimethoxybenzoic acid were attacked under either condition, and formaldehyde was liberated from these substrates in the presence of O₂. The anaerobic demethylating system but not the aerobic enzyme was also active upon guaiacol (2-methoxyphenol), ferulic acid (3-[4-hydroxy-3-methoxyphenyl]-2-propenoic acid), 3,4,5-trimethoxycinnamic acid (3-[3,4,5-trimethoxyphenyl]-2-propenoic acid), and 3,4,5-trimethoxybenzoic acid. The broad specificity of the anaerobic demethylation system suggests that it probably is significant in the degradation of lignoaromatic molecules in anaerobic environments.     

Bioproduction of lauryl lactone and 4-vinyl guaiacol as value-added chemicals in two-phase biotransformation systems

Recombinant Escherichia coli whole-cell biocatalysts harboring either a Baeyer–Villiger monooxygenase or ferulic acid decarboxylase were employed in organic-aqueous two-phase bioreactor systems. The feasibility of the bioproduction of water-insoluble products, viz., lauryl lactone from cyclododecanone and 4-vinyl guaiacol from ferulic acid were examined. Using hexadecane as the organic phase, 10∼16 g of lauryl lactone were produced in a 3-l bioreactor that operated in a semicontinuous mode compared to 2.4 g of product in a batch mode. For the decarboxylation of ferulic acid, a new recombinant biocatalyst, ferulic acid decarboxylase derived from Bacillus pumilus, was constructed. Selected solvents as well as other parameters for in situ recovery of vinyl guaiacol were investigated. Up to 13.8 g vinyl guaiacol (purity of 98.4%) were obtained from 25 g of ferulic acid in a 2-l working volume bioreactor by using octane as organic phase. These selected examples highlight the superiority of the two-phase biotransformations systems over the conventional batch mode.     

Biotransformations of carboxylated aromatic compounds by the acetogen Clostridium thermoaceticum: generation of growth-supportive CO2 equivalents under CO2-limited conditions.

Clostridium thermoaceticum ATCC 39073 converted vanillate to catechol. Although carboxylated aromatic compounds which did not contain methoxyl groups were not by themselves growth supportive, protocatechuate and p-hydroxybenzoate (nonmethoxylated aromatic compounds) were converted to catechol and phenol, respectively, during carbon monoxide-dependent growth. Syringate is not subject to decarboxylation by C. thermoaceticum (Z. Wu, S. L. Daniel, and H. L. Drake, J. Bacteriol. 170:5705-5708, 1988), and sustained growth at the expense of syringate-derived methoxyl groups was dependent on supplemental CO2. In contrast, vanillate was growth supportive in the absence of supplemental CO2, and 14CO2 was the major 14C-labeled product during [carboxyl-14C]vanillate-dependent growth. Furthermore, the decarboxylation of protocatechuate and p-hydroxybenzoate supported methanol- and 1,2,3-trimethoxybenzene-dependent growth (CO2 is required for growth at the expense of these substrates) when supplemental CO2 was depleted from the growth medium, and the decarboxylation of protocatechuate was concomitant with improved cell yields of methanol cultures. These findings demonstrate that (i) C. thermoaceticum is competent in the decarboxylation of certain aromatic compounds and (ii) under certain conditions, decarboxylation may be integrated to the flow of carbon and energy during acetogenesis.     

Characterization of the 5-Carboxyvanillate Decarboxylase Gene and Its Role in Lignin-Related Biphenyl Catabolism in Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 degrades a lignin-related biphenyl compound, 5,5′-dehydrodivanillate (DDVA), to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by the DDVA O-demethylase (LigX), the ring cleavage oxygenase (LigZ), and the meta-cleavage compound hydrolase (LigY). In this study we examined the degradation step of 5CVA. 5CVA was transformed to vanillate, O-demethylated, and further degraded via the protocatechuate 4,5-cleavage pathway by this strain. A cosmid clone which conferred the 5CVA degradation activity to a host strain was isolated. In the 7.0-kb EcoRI fragment of the cosmid we found a 1,002-bp open reading frame responsible for the conversion of 5CVA to vanillate, and we designated it ligW. The gene product of ligW (LigW) catalyzed the decarboxylation of 5CVA to produce vanillate along with the specific incorporation of deuterium from deuterium oxide, indicating that LigW is a nonoxidative decarboxylase of 5CVA. LigW did not require any metal ions or cofactors for its activity. The decarboxylase activity was specific to 5CVA. Inhibition experiments with 5CVA analogs suggested that two carboxyl groups oriented meta to each other in 5CVA are important to the substrate recognition by LigW. Gene walking analysis indicated that the ligW gene was located on the 18-kb DNA region with other DDVA catabolic genes, including ligZ, ligY, and ligX.     

14C-most-probable-number method for enumeration of active heterotrophic microorganisms in natural waters.

A most-probable-number method using 14C-labeled substrates is described for the enumeration of aquatic populations of heterotrophic microorganisms. Natural populations of microorganisms are inoculated into dilution replicates prepared from the natural water from which the organisms originated. The natural water is supplemented with a 14C-labeled compound added so as to approximate a true environmental concentration. 14CO2 evolved by individual replicates is trapped in NaOH and counted by liquid scintillation techniques for use in scoring replicates as positive or negative. Positives (14CO2 evolution) are easily distinguished from negatives (no 14CO2 evolution). The results from a variety of environments using the 14CO2 procedure agreed well with previously described methods, in most instances. The 14C-most-probable-number method described here reduces handling procedures over previously described most-probable-number procedures using 14C-labeled substrates. It also appears to have advantages over other enumeration methods in its attempt to approximate natural conditions more closely.     

14C-most-probable-number method for enumeration of active heterotrophic microorganisms in natural waters.

A most-probable-number method using 14C-labeled substrates is described for the enumeration of aquatic populations of heterotrophic microorganisms. Natural populations of microorganisms are inoculated into dilution replicates prepared from the natural water from which the organisms originated. The natural water is supplemented with a 14C-labeled compound added so as to approximate a true environmental concentration. 14CO2 evolved by individual replicates is trapped in NaOH and counted by liquid scintillation techniques for use in scoring replicates as positive or negative. Positives (14CO2 evolution) are easily distinguished from negatives (no 14CO2 evolution). The results from a variety of environments using the 14CO2 procedure agreed well with previously described methods, in most instances. The 14C-most-probable-number method described here reduces handling procedures over previously described most-probable-number procedures using 14C-labeled substrates. It also appears to have advantages over other enumeration methods in its attempt to approximate natural conditions more closely.     

New aspects of co-regulation of decarboxylation and demethylation activities in Nocardia

Vanillic acid at 0.2% concentration in the medium of Nocardia autotrophic DSM 43100 leads to cyclic production of guaiacol; protocatechuic and p-hydroxybenzoic acids as well as catechol appear at the same time in the medium instead of isovanillic acid, which accumulates at lower vanillic acid concentration. Transformation of catechol formed into guaiacol by methylation with formaldehyde, and successively into protocatechuic acid by carboxylation seems possible. Successive reactions of methylation/demethylation and carboxylation/decarboxylation result in cyclic production of guaiacol.     

Formation of 4-vinyl guaiacol as an intermediate in bioconversion of ferulic acid by Schizophyllum commune

In order to utilize phenolic compounds in unused biomass resources, the metabolic pathway of ferulic acid by way of a white-rot fungus, Schizophyllum commune, was investigated. Ferulic acid was immediately degraded, and the formation of 4-vinyl guaiacol was confirmed by GC-MS analysis. The metabolic test of ferulic acid and its degradation products indicated that S. commune converted ferulic acid into 4-vinyl guaiacol by decarboxylation. This was then oxidized to vanillin and vanillic acid. This result indicates that S. commune distinguished ferulic acid from lignins and metabolized it specifically.     

Enumeration of Acetogens by a Colorimetric Most-Probable-Number Assay

Anaerobic O demethylation by acetogenic bacteria often is the first step in the mineralization of methoxylated aromatic compounds in anoxic environments. In this reaction, an ether bond is cleaved and the resulting methyl group is metabolized via the acetyl coenzyme A pathway (acetogenesis). Anaerobic O demethylation was used to assess acetogen populations. Environmental samples were diluted in anaerobic medium containing a methoxylated aromatic substrate (vanillate) and titanium(III), and acetogen titers were estimated by the most-probable-number (MPN) method. Complex formation between Ti(III) and vicinal hydroxyl groups of the aromatic products of anaerobic O demethylation results in the development of a yellow color in the medium, which can be detected by eye and monitored spectrophotometrically. High-performance liquid chromatography analysis of the yellow MPN tubes showed that they contained the product of anaerobic O demethylation of vanillate (protocatechuate). This assay was used to enumerate O-demethylating acetogen populations in environmental samples.     

Effect of overexpression of Saccharomyces cerevisiae Pad1p on the resistance to phenylacrylic acids and lignocellulose hydrolysates under aerobic and oxygen-limited conditions

Lignocellulose hydrolysates, obtained by acid hydrolysis for production of bioethanol, contain, in addition to fermentable sugars, compounds that inhibit the fermenting micro-organism. One approach to alleviate the inhibition problem is to use genetic engineering to introduce increased tolerance. Phenylacrylic acid decarboxylase (Pad1p) catalyses a decarboxylation step, by which aromatic carboxylic acids are converted to the corresponding vinyl derivatives. Pad1p-overexpressing Saccharomyces cerevisiae was cultivated in synthetic medium in the presence of model compounds, ferulic acid [(2 E)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enoic acid] and cinnamic acid [(2 E)-3-phenylprop-2-enoic acid], as well as in a dilute acid hydrolysate of spruce to examine the resistance against fermentation inhibitors. Overexpression of S. cerevisiae phenylacrylic acid decarboxylase (Pad1p) resulted in an improved growth rate and ethanol productivity in the presence of ferulic acid, cinnamic acid, and in a dilute acid hydrolysate of spruce. Vinyl guaiacol (2-methoxy-4-vinylphenol) was identified as a major metabolite of ferulic acid, and dihydroferulic acid [3-(4-hydroxy-3-methoxyphenyl)propanoic acid] was detected under oxygen-limited conditions. Styrene (vinylbenzene) and dihydrocinnamic acid (3-phenylpropanoic acid) were identified as metabolites of cinnamic acid. Transformants overexpressing Pad1p had the ability to convert ferulic and cinnamic acid at a faster rate than a control transformant (PAD(C)) not overexpressing Pad1p. This enabled faster growth for Pad1p-overexpressing transformants under both aerobic and oxygen-limited conditions. Pad1p activity was also studied using non-growing cells. The overexpressing transformants showed approximately tenfold higher activity than PAD(C). The Pad1p overexpressing transformants also showed a 22-25% faster glucose consumption rate, a 40-45% faster mannose consumption rate, and a 24-29% faster ethanol production rate in the dilute acid hydrolysate of spruce.     

Metabolism of Ferulic Acid by Paecilomyces variotii and Pestalotia palmarum

Ferulic acid metabolism was studied in cultures of two micromycetes producing different amounts of phenol oxidases. In cultures of the low phenol oxidase producer Paecilomyces variotii, ferulic acid was decarboxylated to 4-vinylguaiacol, which was converted to vanillin and then either oxidized to vanillic acid or reduced to vanillyl alcohol. Vanillic acid underwent simultaneously an oxidative decarboxylation to methoxyhydroquinone and a nonoxidative decarboxylation to guaiacol. Methoxyhydroquinone and guaiacol were demethylated to yield hydroxyquinol and catechol, respectively. Catechol was hydroxylated to pyrogallol. Degradation of ferulic acid by Paecilomyces variotii proceeded mainly via methoxyhydroquinone. The high phenol oxidase producer Pestalotia palmarum catabolized ferulic acid via 4-vinylguaiacol, vanillin, vanillyl alcohol, vanillic acid, and methoxyhydroquinone. However, the main reactions observed with this fungus involved polymerization reactions.     

Degradation of 3-O-methylgallate in Sphingomonas paucimobilis SYK-6 by pathways involving protocatechuate 4,5-dioxygenase

Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate and 3-O-methylgallate (3MGA), respectively. 3MGA is metabolized via multiple pathways involving 3MGA 3,4-dioxygenase, protocatechuate 4,5-dioxygenase (LigAB), and gallate dioxygenase whereas protocatechuate is degraded via the protocatechuate 4,5-cleavage pathway. Here the secondary role of LigAB in syringate metabolism is investigated. The reaction product of 3MGA catalyzed by His-tagged LigAB was identified as 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate (CHMOD) and 2-pyrone-4,6-dicarboxylate (PDC), indicating that 3MGA is transformed to CHMOD and PDC by both reactions catalyzed by DesZ and LigAB. Mutant analysis revealed that the 3MGA catabolic pathways involving LigAB are functional in SYK-6.     

Biocatalytic synthesis of vanillin

The conversions of vanillic acid and O-benzylvanillic acid to vanillin were examined by using whole cells and enzyme preparations of Nocardia sp. strain NRRL 5646. With growing cultures, vanillic acid was decarboxylated (69% yield) to guaiacol and reduced (11% yield) to vanillyl alcohol. In resting Nocardia cells in buffer, 4-O-benzylvanillic acid was converted to the corresponding alcohol product without decarboxylation. Purified Nocardia carboxylic acid reductase, an ATP and NADPH-dependent enzyme, quantitatively reduced vanillic acid to vanillin. Structures of metabolites were established by (1)H nuclear magnetic resonance and mass spectral analyses.     

Characterization of isocitrate dehydrogenase from the green sulfur bacterium Chlorobium limicola. A carbon dioxide-fixing enzyme in the reductive tricarboxylic acid cycle.

Isocitrate dehydrogenase (IDH) catalyzes the reversible conversion between isocitrate and 2-oxoglutarate accompanied by decarboxylation/carboxylation and oxidoreduction of NAD(P)+ cofactor. While this enzyme has been well studied as a catabolic enzyme in the tricarboxylic acid (TCA) cycle, here we have characterized NADP-dependent IDH from Chlorobium limicola, a green sulfur bacterium that fixes CO2 through the reductive tricarboxylic acid (RTCA) cycle, focusing on the CO2-fixation ability of the enzyme. The gene encoding Cl-IDH consisted of 2226 bp, corresponding to a polypeptide of 742 amino acid residues. The primary structure and the size of the recombinant protein indicated that Cl-IDH was a monomeric enzyme of 80 kDa distinct from the dimeric NADP-dependent IDHs predominantly found in bacteria or eukaryotic mitochondria. Apparent Michaelis constants for isocitrate (45 +/- 13 microm) and NADP+ (27 +/- 10 microm) were much smaller than those for 2-oxoglutarate (1.1 +/- 0.5 mm) and CO2 (1.3 +/- 0.3 mm). No significant differences in kinetic properties were observed between Cl-IDH and the dimeric, NADP-dependent IDH from Saccharomyces cerevisiae (Sc-IDH) at the optimum pH of each enzyme. However, in contrast to the 20% activity of Sc-IDH toward carboxylation as compared with that toward decarboxylation at pH 7.0, the activities of Cl-IDH for both directions were almost equivalent at this pH, suggesting a more favorable property of Cl-IDH than Sc-IDH as a CO2-fixation enzyme under physiological pH. Furthermore, we found that among various intermediates, oxaloacetate was a competitive inhibitor (K(i) = 0.35 +/- 0.04 mm) for 2-oxoglutarate in the carboxylation reaction by Cl-IDH, a feature not found in Sc-IDH.     

Substrate range and genetic analysis of Acinetobacter vanillate demethylase

An Acinetobacter sp. genetic screen was used to probe structure-function relationships in vanillate demethylase, a two-component monooxygenase. Mutants with null, leaky, and heat-sensitive phenotypes were isolated. Missense mutations tended to be clustered in specific regions, most of which make known contributions to catalytic activity. The vanillate analogs m-anisate, m-toluate, and 4-hydroxy-3,5-dimethylbenzoate are substrates of the enzyme and weakly inhibit the metabolism of vanillate by wild-type Acinetobacter bacteria. PCR mutagenesis of vanAB, followed by selection for strains unable to metabolize vanillate, yielded mutant organisms in which vanillate metabolism is more strongly inhibited by the vanillate analogs. Thus, the procedure opens for investigation amino acid residues that may contribute to the binding of either vanillate or its chemical analogs to wild-type and mutant vanillate demethylases. Selection of phenotypic revertants following PCR mutagenesis gave an indication of the extent to which amino acid substitutions can be tolerated at specified positions. In some cases, only true reversion to the original amino acid was observed. In other examples, a range of amino acid substitutions was tolerated. In one instance, phenotypic reversion failed to produce a protein with the original wild-type sequence. In this example, constraints favoring certain nucleotide substitutions appear to be imposed at the DNA level.     

Desulfurization of dibenzothiophene derivatives by whole cells of Rhodococcus erythropolis H-2

Abstract Transposon mutagenesis was performed to pursue the molecular basis of carbazole catabolic pathway in a carbazple-using bacterium, Pseudomonas sp. CA10. One mutant, TD2, was capable of using anthranilic acid but not carbazole as its sole source of carbon, nitrogen, and energy. Another isolated mutant, designated as TE1, was found to have the opposite ability as TD2. TD2 could not convert carbazole to any other compound under cometabolic conditions. On the other hand, TE1 accumulated catechol and cis,cis -muconate from carbazole. The clone containing Tn 5 -flanking region from TD2, showed the meta -cleavage activity for biphenyl-2,3-diol and analysis of the DNA sequence of this region suggests that the genes involved in the degradation of aromatic compounds are clustered. Our analysis of the DNA sequence of another clone from mutant TE1 showed that the Tn 5 -Mob can be inserted into the homologous catR gene, a gene that reportedly enpodes the positive regulatory protein of the catBC operon. These data suggests that carbazole catabolic pathway comprises at least two different gene clusters (upper pathway and lower pathway) in Pseudomonas sp. CA10.     

Transcriptional Regulation of the Vanillate Utilization Genes (vanABK Operon) of Corynebacterium glutamicum by VanR,a PadR-Like Repressor

Corynebacterium glutamicum is able to utilize vanillate, the product of lignin degradation, as the sole carbon source. The vanillate utilization components are encoded by the vanABK operon. The vanA and vanB genes encode the subunits of vanillate O-demethylase, converting vanillate to protocatechuate, while VanK is the specific vanillate transporter. The vanABK operon is regulated by a PadR-type repressor, VanR. Heterologous gene expression and variations of the vanR open reading frame revealed that the functional VanR contains 192 residues (21 kDa) and forms a dimer, as analyzed by size exclusion chromatography. In vivo, ferulate, vanillin, and vanillate induced P_vanABK in C. glutamicum, while only vanillate induced the activity of P_vanABK in Escherichia coli lacking the ferulate catabolic system. Differential scanning fluorimetry verified that vanillate is the only effector of VanR. Interaction between the P_vanABK DNA fragment and the VanR protein had an equilibrium dissociation constant (K_D) of 15.1 ± 1.7 nM. The VanR-DNA complex had a dissociation rate constant (K_d) of (267 ± 23) × 10⁻⁶ s⁻¹, with a half-life of 43.5 ± 3.6 min. DNase I footprinting localized the VanR binding site at P_vanABK, extending from +9 to +45 on the coding strand. Deletion of the nucleotides +18 to +27 inside the VanR binding site rendered P_vanABK constitutive. Fusion of the T7 promoter and the wild-type VanR operator, as well as its shortened versions, indicated that the inverted repeat AACTAACTAA(N₄)TTAGGTATTT is the specific VanR binding site. It is proposed that the VanR-DNA complex contains two VanR dimers at the VanR operator.