Vanillate O-demethylase Research Articles

Evolution and engineering of pathways for aromatic O-demethylation in Pseudomonas putida KT2440

Biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However, aromatic compounds derived from lignin commonly contain methoxy groups, and O-demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic O-demethylation, but they are rarely compared in vivo to determine an optimal biocatalytic strategy. Here, two pathways for aromatic O-demethylation were compared in Pseudomonas putida KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in P. putida, and the strains were optimized via adaptive laboratory evolution (ALE) with vanillate as a model substrate. All evolved strains displayed improved growth phenotypes, with the evolved strains harboring the native VanAB pathway exhibiting growth rates ∼1.8x faster than those harboring the heterologous LigM pathway. Enzyme kinetics and transcriptomics studies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, the reductase for vanillate O-demethylase, PP_3494, a global regulator of vanillate catabolism, and fghA, involved in formaldehyde detoxification. These three mutations were combined into a single strain, which exhibited approximately 5x faster vanillate consumption than the wild-type strain in the first 8 h of cultivation. Overall, this study illuminates the details of vanillate catabolism in the context of two distinct enzymatic mechanisms, yielding a platform strain for efficient O-demethylation of lignin-related aromatic compounds to value-added products.

The catabolism of lignin-derived p-methoxylated aromatic compounds by Rhodococcus jostii RHA1.

Emergent strategies to valorize lignin, an abundant but underutilized aromatic biopolymer, include tandem processes that integrate chemical depolymerization and biological catalysis. To date, aromatic monomers from C-O bond cleavage of lignin have been converted to bioproducts, but the presence of recalcitrant C-C bonds in lignin limits the product yield. A promising chemocatalytic strategy that overcomes this limitation involves phenol methyl protection and autoxidation. Incorporating this into a tandem process requires microbial cell factories able to transform the p-methoxylated products in the resulting methylated lignin stream. In this study, we assessed the ability of Rhodococcus jostii RHA1 to catabolize the major aromatic products in a methylated lignin stream and elucidated the pathways responsible for this catabolism. RHA1 grew on a methylated pine lignin stream, catabolizing the major aromatic monomers: p-methoxybenzoate (p-MBA), veratrate, and veratraldehyde. Bioinformatic analyses suggested that a cytochrome P450, PbdA, and its cognate reductase, PbdB, are involved in p-MBA catabolism. Gene deletion studies established that both pbdA and pbdB are essential for growth on p-MBA and several derivatives. Furthermore, a deletion mutant of a candidate p-hydroxybenzoate (p-HBA) hydroxylase, ΔpobA, did not grow on p-HBA. Veratraldehyde and veratrate catabolism required both vanillin dehydrogenase (Vdh) and vanillate O-demethylase (VanAB), revealing previously unknown roles of these enzymes. Finally, a ΔpcaL strain grew on neither p-MBA nor veratrate, indicating they are catabolized through the β-ketoadipate pathway. This study expands our understanding of the bacterial catabolism of aromatic compounds and facilitates the development of biocatalysts for lignin valorization.IMPORTANCELignin, an abundant aromatic polymer found in plant biomass, is a promising renewable replacement for fossil fuels as a feedstock for the chemical industry. Strategies for upgrading lignin include processes that couple the catalytic fractionation of biomass and biocatalytic transformation of the resulting aromatic compounds with a microbial cell factory. Engineering microbial cell factories for this biocatalysis requires characterization of bacterial pathways involved in catabolizing lignin-derived aromatic compounds. This study identifies new pathways for lignin-derived aromatic degradation in Rhodococcus, a genus of bacteria well suited for biocatalysis. Additionally, we describe previously unknown activities of characterized enzymes on lignin-derived compounds, expanding their utility. This work advances the development of strategies to replace fossil fuel-based feedstocks with sustainable alternatives.

Sphingobium sp. V4, a bacterium degrading multiple allelochemical phenolic acids

BackgroundContinuous cropping challenges constrain the development of agriculture. Three main obstacles limit continuous cropping: autotoxicity of plant allelochemicals, deterioration of physicochemical characteristics of soil, and microflora imbalance. Plant-derived phenolic acids can cause autotoxicity, which is considered the main factor mediating continuous cropping obstacles. Reducing the phenolic acids in continuous cropping soils can decrease the autotoxicity of phenolic acids and ameliorate continuous cropping obstacles. Therefore, it is important to study the microbial resources that degrade allelochemical phenolic acids. Thus, the bacterial strain V4 that can degrade phenolic acids was isolated, identified, and genomically analyzed.ResultsStrain V4 isolated from strawberry soil using vanillic acid-mineral agar was identified as a Gram-negative short rod bacterium. Subsequent 16S rRNA phylogenetic analysis revealed that V4 clustered with members of the genus Sphingobium. The most closely related species were Sphingobium lactosutens DS20T (99% similarity) and Sphingobium abikonense NBRC 16140T (97.5% similarity). V4 also shared > 95% sequence similarity with other members of Sphingobium, so Sphingobium sp. V4 was named accordingly. Biochemical tests revealed that the biochemical characteristics of Sphingobium sp. V4 were similar to its most similar strains except for some properties. Sphingobium sp. V4 effectively degraded vanillic acid, ferulic acid, p-coumaric acid, p-hydroxybenzoic acid, and syringic acid. V4 grew best at the conditions of 30 °C, pH 6.0–7.0, and 0–0.05% NaCl. 500 mg/L vanillic acid was completely degraded by V4 within 24 h under the optimal conditions. Whole genome analysis showed that Sphingobium sp. V4 contained one chromosome and three plasmids. Two genes involved in vanillic acid degradation were found in the V4 genome: the gene encoding vanillate O-demethylase oxidoreductase VanB on the chromosome and the gene encoding vanillate monooxygenase on a large plasmid. The organization of vanillate catabolic genes differed from the adjacent organization of the genes, encoding vanillate o-demethylase VanA and VanB subunits, in Pseudomonas and Acinetobacter.ConclusionsThe isolated bacterium Sphingobium sp. V4 degraded multiple phenolic acids. Its properties and genome were further analyzed. The study provides support for further investigation and application of this phenolic acid-degrading microorganism to alleviate continuous cropping obstacles in agriculture.

Identification of a Phylogenetically Divergent Vanillate O-Demethylase from Rhodococcus ruber R1 Supporting Growth on Meta-Methoxylated Aromatic Acids.

Rieske-type two-component vanillate O-demethylases (VanODs) catalyze conversion of the lignin-derived monomer vanillate into protocatechuate in several bacterial species. Currently, VanODs have received attention because of the demand of effective lignin valorization technologies, since these enzymes own the potential to catalyze methoxy group demethylation of distinct lignin monomers. In this work, we identified a phylogenetically divergent VanOD from Rhodococcus ruber R1, only distantly related to previously described homologues and whose presence, along with a 3-hydroxybenzoate/gentisate pathway, correlated with the ability to grow on other meta-methoxylated aromatics, such as 3-methoxybenzoate and 5-methoxysalicylate. The complementation of catabolic abilities by heterologous expression in a host strain unable to grow on vanillate, and subsequent resting cell assays, suggest that the vanAB genes of R1 strain encode a proficient VanOD acting on different vanillate-like substrates; and also revealed that a methoxy group in the meta position and a carboxylic acid moiety in the aromatic ring are key for substrate recognition. Phylogenetic analysis of the oxygenase subunit of bacterial VanODs revealed three divergent groups constituted by homologues found in Proteobacteria (Type I), Actinobacteria (Type II), or Proteobacteria/Actinobacteria (Type III) in which the R1 VanOD is placed. These results suggest that VanOD from R1 strain, and its type III homologues, expand the range of methoxylated aromatics used as substrates by bacteria.

Transcriptome Profiling Combined With Activities of Antioxidant and Soil Enzymes Reveals an Ability of Pseudomonas sp. CFA to Mitigate p-Hydroxybenzoic and Ferulic Acid Stresses in Cucumber.

Continuous-cropping leads to obstacles in crop productivity by the accumulation of p-hydroxybenzoic acid (PHBA) and ferulic acid (FA). In this study, a strain CFA of Pseudomonas was shown to have a higher PHBA- and FA-degrading ability in media and soil and the mechanisms underlying this were explored. Optimal conditions for PHBA and FA degradation by CFA were 0.2 g/l of PHBA and FA, 37°C, and pH 6.56. Using transcriptome analysis, complete pathways that converted PHBA and FA to acetyl coenzyme A were proposed in CFA. When CFA was provided with PHBA and FA, we observed upregulation of genes in the pathways and detected intermediate metabolites including vanillin, vanillic acid, and protocatechuic acid. Moreover, 4-hydroxybenzoate 3-monooxygenase and vanillate O-demethylase were rate-limiting enzymes by gene overexpression. Knockouts of small non-coding RNA (sRNA) genes, including sRNA 11, sRNA 14, sRNA 20, and sRNA 60, improved the degradation of PHBA and FA. When applied to cucumber-planted soil supplemented with PHBA and FA, CFA decreased PHBA and FA in soil. Furthermore, a reduction of superoxide radical, hydrogen peroxide, and malondialdehyde in cucumber was observed by activating superoxide dismutase, catalase, glutathione peroxidase, ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase in seedlings, increasing the reduced glutathione and ascorbate in leaves, and inducing catalase, urease, and phosphatase in the rhizosphere. CFA has potential to mitigate PHBA and FA stresses in cucumber and alleviate continuous-cropping obstacles.

Isolation of a novel platform bacterium for lignin valorization and its application in glucose-free cis,cis-muconate production.

Microbial production of cis,cis-muconate (ccMA) from phenolic compounds obtained by chemical depolymerization of lignin is a promising approach to valorize lignin. Because microbial production requires a large amount of carbon and energy source, it is desirable to establish a ccMA-producing strain that utilizes lignin-derived phenols instead of general sources like glucose. We isolated Pseudomonas sp. strain NGC7 that grows well on various phenolic compounds derived from p-hydroxyphenyl, guaiacyl, and syringyl units of lignin. An NGC7 mutant of protocatechuate (PCA) 3,4-dioxygenase and ccMA cycloisomerase genes (NGC703) lost the ability to grow on vanillate and p-hydroxybenzoate but grew normally on syringate. Introduction of a plasmid carrying genes encoding PCA decarboxylase, flavin prenyltransferase, vanillate O-demethylase, and catechol 1,2-dioxygenase into NGC703 enabled production of 3.2g/L ccMA from vanillate with a yield of 75% while growing on syringate. This strain also produced ccMA from birch lignin-derived phenols. All these results indicate the utility of NGC7 in glucose-free ccMA production.

Transcriptome analysis of Pseudomonas sp. from subarctic tundra soil: pathway description and gene discovery for humic acids degradation.

Although humic acids (HA) are involved in many biological processes in soils and thus their ecological importance has received much attention, the degradative pathways and corresponding catalytic genes underlying the HA degradation by bacteria remain unclear. To unveil those uncertainties, we analyzed transcriptomes extracted from Pseudomonas sp. PAMC 26793 cells time-dependently induced in the presence of HA in a lab flask. Out of 6288 genes, 299 (microarray) and 585 (RNA-seq) were up-regulated by > 2.0-fold in HA-induced cells, compared with controls. A significant portion (9.7% in microarray and 24.1% in RNA-seq) of these genes are predicted to function in the transport and metabolism of small molecule compounds, which could result from microbial HA degradation. To further identify lignin (a surrogate for HA)-degradative genes, 6288 protein sequences were analyzed against carbohydrate-active enzyme database and a self-curated list of putative lignin degradative genes. Out of 19 genes predicted to function in lignin degradation, several genes encoding laccase, dye-decolorizing peroxidase, vanillate O-demethylase oxygenase and reductase, and biphenyl 2,3-dioxygenase were up-regulated > 2.0-fold in RNA-seq. This induction was further confirmed by qRT-PCR, validating the likely involvement of these genes in the degradation of HA.

Biotransformation of ferulic acid to protocatechuic acid by Corynebacterium glutamicum ATCC 21420 engineered to express vanillate O-demethylase

Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) is a lignin-derived phenolic compound abundant in plant biomass. The utilization of FA and its conversion to valuable compounds is desired. Protocatechuic acid (3,4-dihydroxybenzoic acid, PCA) is a precursor of polymers and plastics and a constituent of food. A microbial conversion system to produce PCA from FA was developed in this study using a PCA-producing strain of Corynebacterium glutamicum F (ATCC 21420). C. glutamicum strain F grown at 30 °C for 48 h utilized 2 mM each of FA and vanillic acid (4-hydroxy-3-methoxybenzoic acid, VA) to produce PCA, which was secreted into the medium. FA may be catabolized by C. glutamicum through proposed (I) non-β-oxidative, CoA-dependent or (II) β-oxidative, CoA-dependent phenylpropanoid pathways. The conversion of VA to PCA is the last step in each pathway. Therefore, the vanillate O-demethylase gene (vanAB) from Corynebacterium efficiens NBRC 100395 was expressed in C. glutamicum F (designated strain FVan) cultured at 30 °C in AF medium containing FA. Strain C. glutamicum FVan converted 4.57 ± 0.07 mM of FA into 2.87 ± 0.01 mM PCA after 48 h with yields of 62.8% (mol/mol), and 6.91 mM (1064 mg/L) of PCA was produced from 16.0 mM of FA after 12 h of fed-batch biotransformation. Genomic analysis of C. glutamicum ATCC 21420 revealed that the PCA-utilization genes (pca cluster) were conserved in strain ATCC 21420 and that mutations were present in the PCA importer gene pcaK.

Mass spectrometry and database search in the analysis of proteins from the fungus Pleurotus ostreatus

Tandem mass spectrometry is a method of choice for rapid analysis in proteomics. Identification and characterization of proteins from organisms with sequenced genomes is today a routine procedure as will be identification of proteins from organisms with unsequenced genomes with new developing tools. Here, we report the use of isotopic labeling with electrospray ionisation (ESI)-tandem mass spectrometry for de novo sequencing in combination with database search taking advantage of different programs for identification of fungal proteins. Using this approach we could identify the proteins of interest. Nevertheless, the identification of a novel protein responsible for the conversion of testosterone into androstenedione was still a difficult task, mostly due to the low homology of steroid transforming enzymes, especially those from microorganisms. Protein p27 was identified as the vanillate O-demethylase oxidoreductase, p33 and p36 as two isoenzymes of malate dehydrogenase, and p45 as citrate synthase. By rechecking the sequences using additional programs it could be shown that the protein p36 has a higher local homology to the steroid-transforming enzyme than to the malate dehydrogenase. Therefore, we assume that p36 is a pluripotent enzyme most probably responsible for the 17beta-hydroxysteroid dehydrogenase activity.

Isolation and characterization of a veratrol:corrinoid protein methyl transferase from Acetobacterium dehalogenans.

From 3-methoxyphenol-grown cells of Acetobacterium dehalogenans, an inducible enzyme was purified that mediated the transfer of the methyl groups of veratrol (1,2-dimethoxybenzene) to a corrinoid protein enriched from the same cells. In this reaction, veratrol was converted via 2-methoxyphenol to 1,2-dihydroxybenzene. The veratrol:corrinoid protein methyl transferase, designated MTIver, had an apparent molecular mass of about 32 kDa. With respect to the N-terminal amino acid sequence and other characteristics, MTIver is different from the vanillate:corrinoid protein methyl transferase (MTIvan) isolated earlier from the same bacterium. For the methyl transfer from veratrol to tetrahydrofolate, two additional protein fractions were required, one of which contained a corrinoid protein. This protein was not identical with the corrinoid protein of the vanillate O-demethylase system. However, the latter corrinoid protein could also serve as methyl acceptor for the veratrol:corrinoid protein methyl transferase. MTIver catalyzed the demethylation of veratrol, 3,4-dimethoxybenzoate, 2-methoxyphenol, and 3-methoxyphenol. Vanillate (3-methoxy-4-hydroxybenzoate), 2-methoxybenzoate, or 4-methoxybenzoate could not serve as substrates.

Bioconversion of ferulic acid into vanillic acid by means of a vanillate-negative mutant of Pseudomonas fluorescens strain BF13.

From a ferulic-acid-degrading Pseudomonas fluorescens strain (BF13), we have isolated a transposon mutant, which retained the ability to bioconvert ferulic acid into vanillic acid but lost the ability to further degrade the latter acid. The mutant, BF13-97, was very stable, and therefore it was suitable to be used as a biocatalyst for the preparative synthesis of vanillic acid from ferulic acid. By use of resting cells we determined the effect on the bioconversion rate of several parameters, such as the addition of nutritional factors, the concentration of the biomass, and the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtained with cells pregrown on M9 medium containing p-coumaric acid (0.1% [wt/vol]) as a sole carbon source and yeast extract (0.001% [wt/vol]) as a source of nutritional factors. Under these conditions, 1 mg (wet weight) of biomass produced 0.23 mg of vanillic acid per h. The genomic region of BF13-97 flanking the transposon's site of insertion was cloned and sequenced revealing two open reading frames of 1,062 (vanA) and 954 (vanB) bp, respectively. The van genes are organized in a cluster and encode the subunits of the vanillate-O-demethylase, which catalyzes the first step of the vanillate catabolism. Amino acid sequences deduced from vanA and vanB genes were shown to have high identity with known VanAs and VanBs from Pseudomonas and Acinetobacter spp. Highly conserved regions known to exist in class IA oxygenases were also found in the vanillate-O-demethylase components from P. fluorescens BF13. The terminal oxygenase VanA is characterized by a conserved Rieske-type [2Fe-2S](R) ligand center. The reductase VanB contains a plant-type ferredoxin [2Fe-2S](Fd), flavin mononucleotide, and NAD-ribose binding domains which are located in its C-terminal and N-terminal halves, respectively. Transfer of wild-type vanAB genes to BF13-97 complemented this mutant, which recovered its ability to grow on either vanillic or ferulic acid.

Vanillate O-demethylase from Pseudomonas species

Vanillate O-demethylase is an inducible enzyme that catalyzes the O-demethylation of vanillic acid to protocatechuic acid and formaldehyde. This chapter discusses the enzyme assay based on oxygen uptake measurements and spectrophotometric assay. The chapter also discusses identification of protocatechuic acid and formaldehyde as products of demethylase activity. Extracts of bacterial cells induced to oxidize vanillate also contain high levels of protocatechuate 3,4- or 4,5-dioxygenase activity. As O-demethylation is the rate-limiting step, and vanillate O-demethylase is sentitire to traditional enzyme purification techniques, protocatechuate is not readily isolated as the reaction product. However, bacterial extracts that rapidly oxidizes vanillate when supplemented with reduced pyridine nucleotide but that exhibit reduced ring-cleavage activity toward protocatchuate may be obtained using an ultracentrifugation procedure.

The separation of vanillate O-demethylase from protocatechuate 3,4-oxygenase by ultracentrifugation.

1. Protocatechuate 3,4-oxygenase in the soluble part of a cell-free extract of Pseudomonas fluorescens (strain T) sedimented more rapidly than vanillate O-demethylase under specified conditions in a preparative ultracentrifuge. 2. The supernatant from this process contained vanillate O-demethylase and formaldehyde dehydrogenase, and when supplemented with NADH oxidized vanillate with an uptake of 1 mole of oxygen/mole of substrate and accumulation of protocatechuate. 3. This uptake was decreased to 0.5mole/mole of substrate in the presence of semicarbazide as trapping agent for formaldehyde. 4. Reasons are presented for the process of methyl group removal from vanillate being oxidative demethylation.