Discovery of pinoresinol reductase genes in sphingomonads

Goto

Takahashi

et al. 2017

Sci Rep

Vanillin and syringaldehyde obtained from lignin are essential intermediates for the production of basic chemicals using microbial cell factories. However, in contrast to vanillin, the microbial conversion of syringaldehyde is poorly understood. Here, we identified an aromatic aldehyde dehydrogenase (ALDH) gene responsible for syringaldehyde catabolism from 20 putative ALDH genes of Sphingobium sp. strain SYK-6. All these genes were expressed in Escherichia coli, and nine gene products, including previously characterized BzaA, BzaB, and vanillin dehydrogenase (LigV), exhibited oxidation activities for syringaldehyde to produce syringate. Among these genes, SLG_28320 (desV) and ligV were most highly and constitutively transcribed in the SYK-6 cells. Disruption of desV in SYK-6 resulted in a significant reduction in growth on syringaldehyde and in syringaldehyde oxidation activity. Furthermore, a desV ligV double mutant almost completely lost its ability to grow on syringaldehyde. Purified DesV showed similar kcat/Km values for syringaldehyde (2100 s−1·mM−1) and vanillin (1700 s−1·mM−1), whereas LigV substantially preferred vanillin (8800 s−1·mM−1) over syringaldehyde (1.4 s−1·mM−1). These results clearly demonstrate that desV plays a major role in syringaldehyde catabolism. Phylogenetic analyses showed that DesV-like ALDHs formed a distinct phylogenetic cluster separated from the vanillin dehydrogenase cluster.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A bacterial aromatic aldehyde dehydrogenase critical for the efficient catabolism of syringaldehyde

Goto

Takahashi

et al. 2017

Sci Rep

“…When the OD 600 of the culture reached 0.5, DCA (2 mM) was added to the culture and then the culture was incubated for 2 h. Cells were collected by centrifugation (5,000 ϫ g for 10 min at 4°C) and washed twice with 50 mM Tris-HCl buffer (pH 7.5; buffer A). The cells were resuspended in the same buffer and sonicated by an ultrasonic disintegrator (UD201; Tomy Seiko Co.) (23). After the cell lysate was centrifuged at 19,000 ϫ g for 15 min at 4°C, the resulting supernatant was used as the cell extract.…”

Section: Methodsmentioning

confidence: 99%

“…strain SYK-6, is the best-characterized bacterial degrader of lignin-derived aromatic compounds (19). The catabolic pathways and catabolic genes in this strain for ␤-aryl ether (13)(14)(15), biphenyl (20)(21)(22), pinoresinol (23), ferulate (24,25), vanillin (26,27), and syringate (28) have been extensively characterized. However, the catabolic genes for a phenylcoumaran compound, dehydrodiconiferyl alco-hol (DCA), remain largely unknown.…”

mentioning

confidence: 99%

Membrane-Associated Glucose-Methanol-Choline Oxidoreductase Family Enzymes PhcC and PhcD Are Essential for Enantioselective Catabolism of Dehydrodiconiferyl Alcohol

Takahashi

Hirose

et al. 2015

Appl Environ Microbiol

e Sphingobium sp. strain SYK-6 is able to degrade various lignin-derived biaryls, including a phenylcoumaran-type compound, dehydrodiconiferyl alcohol (DCA). In SYK-6 cells, the alcohol group of the B-ring side chain of DCA is initially oxidized to the carboxyl group to generate 3-(2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl) acrylic acid (DCA-C). Next, the alcohol group of the A-ring side chain of DCA-C is oxidized to the carboxyl group, and then the resulting metabolite is catabolized through vanillin and 5-formylferulate. In this study, the genes involved in the conversion of DCA-C were identified and characterized. The DCA-C oxidation activities in SYK-6 were enhanced in the presence of flavin adenine dinucleotide and an artificial electron acceptor and were induced ca. 1.6-fold when the cells were grown with DCA. Based on these observations, SLG_09480 (phcC) and SLG_09500 (phcD), encoding glucose-methanol-choline oxidoreductase family proteins, were presumed to encode DCA-C oxidases. Analyses of phcC and phcD mutants indicated that PhcC and PhcD are essential for the conversion of (؉)-DCA-C and (؊)-DCA-C, respectively. When phcC and phcD were expressed in SYK-6 and Escherichia coli, the gene products were mainly observed in their membrane fractions. The membrane fractions of E. coli that expressed phcC and phcD catalyzed the specific conversion of DCA-C into the corresponding carboxyl derivatives. In the oxidation of DCA-C, PhcC and PhcD effectively utilized ubiquinone derivatives as electron acceptors. Furthermore, the transcription of a putative cytochrome c gene was significantly induced in SYK-6 grown with DCA. The DCA-C oxidation catalyzed by membrane-associated PhcC and PhcD appears to be coupled to the respiratory chain. L ignin, one of the major components of plant cell walls, is a complex phenolic polymer resulting from the oxidative combinatorial coupling of 4-hydroxycinnamyl alcohols (1). Although lignin has various intermolecular linkages between phenylpropane units and contains a number of asymmetric carbons, it is considered to be optically inactive, implying the racemic nature of the lignin backbone (2-4). In nature, lignin is initially decomposed by phenol oxidases such as lignin peroxidase, manganese peroxidase, versatile peroxidase, and laccase secreted by white rot fungi (5-7). Recently, dye-decolorizing peroxidases (Dyp) of Rhodococcus (8) and Amycolatopsis (9) and small laccase of Streptomyces (10) were characterized, and these enzymes have been implicated as being involved in lignin degradation. In addition, bacteria play key roles in the degradation and mineralization of low-molecular-weight aromatic compounds derived from lignin (11, 12). Since fragmented oligomers from lignin consist of stereoisomers that contain various types of intermolecular linkages between phenylpropane units, catabolic enzymes necessary for the conversion of such stereoisomers must have evolved in bacteria to fully utilize structurally and stereochemically complicated l...

“…The purity of the preparations was examined by sodium dodecyl sulfate-12% polyacrylamide gel (or 15% polyacrylamide gel for the analysis of LigXc) electrophoresis (SDS-PAGE). The molecular masses were determined by gel filtration chromatography using a Superdex200 10/300GL column (GE Healthcare) (54) and in vitro cross-linking (55). Native PAGE was performed using 7.5% polyacrylamide with a high-molecular-weight calibration kit for native electrophoresis (GE Healthcare).…”

Section: Construction Of Mutantsmentioning

confidence: 99%

Three-Component O -Demethylase System Essential for Catabolism of a Lignin-Derived Biphenyl Compound in Sphingobium sp. Strain SYK-6

Yoshikata

Suzuki

et al. 2014

Appl Environ Microbiol

c Sphingobium sp. strain SYK-6 is able to assimilate lignin-derived biaryls, including a biphenyl compound, 5,5=-dehydrodivanillate (DDVA). Previously, ligXa (SLG_07770), which is similar to the gene encoding oxygenase components of Rieske-type nonheme iron aromatic-ring-hydroxylating oxygenases, was identified to be essential for the conversion of DDVA; however, the genes encoding electron transfer components remained unknown. Disruption of putative electron transfer component genes scattered through the SYK-6 genome indicated that SLG_08500 and SLG_21200, which showed approximately 60% amino acid sequence identities with ferredoxin and ferredoxin reductase of dicamba O-demethylase, were essential for the normal growth of SYK-6 on DDVA. LigXa and the gene products of SLG_08500 (LigXc) and SLG_21200 (LigXd) were purified and were estimated to be a trimer, a monomer, and a monomer, respectively. LigXd contains FAD as the prosthetic group and showed much higher reductase activity toward 2,6-dichlorophenolindophenol with NADH than with NADPH. A mixture of purified LigXa, LigXc, and LigXd converted DDVA into 2,2=,3-trihydroxy-3=-methoxy-5,5=-dicarboxybiphenyl in the presence of NADH, indicating that DDVA O-demethylase is a three-component monooxygenase. This enzyme requires Fe(II) for its activity and is highly specific for DDVA, with a K m value of 63.5 M and k cat of 6.1 s ؊1 . Genome searches in six other sphingomonads revealed genes similar to ligXc and ligXd (>58% amino acid sequence identities) with a limited number of electron transfer component genes, yet a number of diverse oxygenase component genes were found. This fact implies that these few electron transfer components are able to interact with numerous oxygenase components and the conserved LigXc and LigXd orthologs are important in sphingomonads.