Shojiro Hishiyama

Structural Characterization of Modified Lignin in Transgenic Tobacco Plants in Which the Activity of 4-Coumarate:Coenzyme A Ligase Is Depressed.

Transgenic tobacco (Nicotiana tabacum L.) plants in which the activity of 4-coumarate:coenzyme A ligase is very low contain a novel lignin in their xylem. Details of changes in hydroxycinnamic acids bound to cell walls and in the structure of the novel lignin were identified by base hydrolysis, alkaline nitrobenzene oxidation, pyrolysis-gas chromatography, and 13C-nuclear magnetic resonance analysis. In the brownish tissue of the transgenic plants, the levels of three hydroxycinnamic acids, p-coumaric, ferulic, and sinapic, which were bound to cell walls, were apparently increased as a result of down-regulation of the expression of the gene for 4-coumarate:coenzyme A ligase. Some of these hydroxycinnamic acids were linked to cell walls via ester and ether linkages. The accumulation of hydroxycinnamic acids also induced an increase in the level of condensed units in the novel lignin of the brownish tissue. Our data indicate that the behavior of some of the incorporated hydroxycinnamic acids resembles lignin monomers in the brownish tissue, and their accumulation results in dramatic changes in the biosynthesis of lignin in transgenic plants.

Identification of Three Alcohol Dehydrogenase Genes Involved in the Stereospecific Catabolism of Arylglycerol-β-Aryl Ether by Sphingobium sp. Strain SYK-6

ABSTRACT Degradation of arylglycerol-β-aryl ether is the most important process in bacterial lignin catabolism. Sphingobium sp. strain SYK-6 degrades guaiacylglycerol-β-guaiacyl ether (GGE) to α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV), and then the ether linkage of MPHPV is cleaved to generate α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol. We have characterized three enantioselective glutathione S-transferase genes, including two genes that are involved in the ether cleavage of two enantiomers of MPHPV and one gene that is involved in the elimination of glutathione from a GS-HPV enantiomer. However, the first step in the degradation of four different GGE stereoisomers has not been characterized. In this study, three alcohol dehydrogenase genes, ligL, ligN, and ligO, which conferred GGE transformation activity in Escherichia coli, were isolated from SYK-6 and characterized, in addition to the previously cloned ligD gene. The levels of amino acid sequence identity of the four GGE dehydrogenases, which belong to the short-chain dehydrogenase/reductase family, ranged from 32% to 39%. Each gene was expressed in E. coli, and the stereospecificities of the gene products with the four GGE stereoisomers were determined by using chiral high-performance liquid chromatography with recently synthesized authentic enantiopure GGE stereoisomers. LigD and LigO converted (αR,βS)-GGE and (αR,βR)-GGE into (βS)-MPHPV and (βR)-MPHPV, respectively, while LigL and LigN transformed (αS,βR)-GGE and (αS,βS)-GGE to (βR)-MPHPV and (βS)-MPHPV, respectively. Disruption of the genes indicated that ligD is essential for the degradation of (αR,βS)-GGE and (αR,βR)-GGE and that both ligL and ligN contribute to the degradation of the two other GGE stereoisomers.

Characterization of the Third Glutathione S-Transferase Gene Involved in Enantioselective Cleavage of the β-Aryl Ether by Sphingobium sp. Strain SYK-6

The glutathione S-transferases, LigF and LigE, of Sphingobium sp. strain SYK-6 respectively play a role in cleavage of the β-aryl ether of (+)-(βS)-α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) and (−)-(βR)-MPHPV. The ligP gene, which showed 59% similarity to ligE at the amino acid level, was isolated from SYK-6. LigP produced in Escherichia coli revealed enantioselectivity for (−)-(βR)-MPHPV, and ligE and ligP alone contributed to the degradation of (−)-(βR)-MPHPV in SYK-6.

Discovery of pinoresinol reductase genes in sphingomonads

Bacterial genes for the degradation of major dilignols produced in lignifying xylem are expected to be useful tools for the structural modification of lignin in plants. For this purpose, we isolated pinZ involved in the conversion of pinoresinol from Sphingobium sp. strain SYK-6. pinZ showed 43-77% identity at amino acid level with bacterial NmrA-like proteins of unknown function, a subgroup of atypical short chain dehydrogenases/reductases, but revealed only 15-21% identity with plant pinoresinol/lariciresinol reductases. PinZ completely converted racemic pinoresinol to lariciresinol, showing a specific activity of 46±3 U/mg in the presence of NADPH at 30°C. In contrast, the activity for lariciresinol was negligible. This substrate preference is similar to a pinoresinol reductase, AtPrR1, of Arabidopsis thaliana; however, the specific activity of PinZ toward (±)-pinoresinol was significantly higher than that of AtPrR1. The role of pinZ and a pinZ ortholog of Novosphingobium aromaticivorans DSM 12444 were also characterized.

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

ABSTRACT 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 Km value of 63.5 μM 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.

Characterization of the catabolic pathway for a phenylcoumaran-type lignin-derived biaryl in Sphingobium sp. strain SYK-6

Sphingobium sp. strain SYK-6 is capable of degrading various lignin-derived biaryls. We determined the catabolic pathway of a phenylcoumaran-type compound, dehydrodiconiferyl alcohol (DCA) in SYK-6, and identified some of the DCA catabolism genes. In SYK-6 cells, the alcohol group of DCA was oxidized to the carboxyl group, first at the B-ring side chain and then at the A-ring side chain. The resultant metabolite was degraded to 5-formylferulate and vanillin through the decarboxylation and the Cα–Cβ cleavage of the A-ring side chain. Based on the DCA catabolic pathway, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) genes are thought to be involved in the conversion of DCA into an aldehyde intermediate (DCA-L) and the conversion of DCA-L into a carboxylic acid intermediate (DCA-C), respectively. SLG_05620 and SLG_24930, which belong to quinohemoprotein ADH and aryl ADH, respectively, were isolated as the genes responsible for the oxidation of DCA. In addition to these genes, multiple genes similar to SLG_05620 and SLG_24930 were found to confer DCA oxidation activities on Escherichia coli cells. In order to identify the DCA-L dehydrogenase genes, the DCA-L oxidation activities of the SYK-6 gene products of putative twenty-one ALDH genes were examined. Significant activities were observed in the four ALDH gene products, including the SLG_27910 product, which showed the highest activity. The disruption of SLG_27910 caused a decreased conversion of DCA-L, suggesting that SLG_27910 plays an important role in the DCA-L oxidation. In conclusion, no specific gene seems to be solely responsible for the conversion of DCA and DCA-L, however, the multiple genes encoding quinohemoprotein ADH and aryl ADH genes, and four ALDH genes are probably involved in the conversion processes.

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

ABSTRACT 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.

Successful expression of a novel bacterial gene for pinoresinol reductase and its effect on lignan biosynthesis in transgenic Arabidopsis thaliana

Pinoresinol reductase and pinoresinol/lariciresinol reductase play important roles in an early step of lignan biosynthesis in plants. The activities of both enzymes have also been detected in bacteria. In this study, pinZ, which was first isolated as a gene for bacterial pinoresinol reductase, was constitutively expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Higher reductive activity toward pinoresinol was detected in the resultant transgenic plants but not in wild-type plant. Principal component analysis of data from untargeted metabolome analyses of stem, root, and leaf extracts of the wild-type and two independent transgenic lines indicate that pinZ expression caused dynamic metabolic changes in stems, but not in roots and leaves. The metabolome data also suggest that expression of pinZ influenced the metabolisms of lignan and glucosinolates but not so much of neolignans such as guaiacylglycerol-8-O-4′-feruloyl ethers. In-depth quantitative analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) indicated that amounts of pinoresinol and its glucoside form were markedly reduced in the transgenic plant, whereas the amounts of glucoside form of secoisolariciresinol in transgenic roots, leaves, and stems increased. The detected levels of lariciresinol in the transgenic plant following β-glucosidase treatment also tended to be higher than those in the wild-type plant. Our findings indicate that overexpression of pinZ induces change in lignan compositions and has a major effect not only on lignan biosynthesis but also on biosynthesis of other primary and secondary metabolites.

Introduction of alkali-labile units into lignin in transgenic plants by genetic engineering

Background Lignin is one of major components of plant secondary cell wall. In plant cell wall, it is synthesized via radical coupling of precursors such as p-coumaryl, coniferyl, and sinapyl alcohols. In early stage of the lignification, 8-O-4’, 8-8’ and 8-5’ dimers are thought to be synthesized mainly from the precursors in the wall. A gramnegative bacterium, Shingobium sp. strain SYK-6 (hereafter refer to as SYK-6) is able to catabolize a wide variety of phenolic compounds including the lignin precursors by its unique enzymatic system. One of catabolic enzymes, LigD, catalyzes oxidation at alpha (benzyl) position of 8-O-4’ dimers and forms carbonyl group at the position (Figure 1). This oxidation is the first step of catabolic pathway of 8-O-4’ dimers in SYK-6. When we express LigD polypeptide in the cell wall of transgenic plants, the oxidative dimers will be expected to be generated and then incorporated into lignin polymer. In some past studies, it has been shown that the presence of carbonyl groups at the alpha position of aryl propane units in lignin greatly speeds up the rate of cleavage of beta-aryl ether linkages during kraft pulping condition [1,2]. In order to contribute to efficient and sustainable production of kraft pulp and the other biomass-derived products such as bioethanol, we introduced the ligD gene into Arabidopsis and hybrid aspen and tried to generate transgenic plants whose lignin can be easy to remove from hollocellulose fraction under alkaline conditions. Method Because of codon usage is significantly different between genes in plants and SYK-6, we chemically synthesized open reading frame (ORF) of the ligD gene for improving its expression in the transgenic plants. After addition of nucleotide sequence for apoplast-targeting signal peptide to the synthesized ligD ORF, it was introduced into Arabidopsis thaliana, tobacco BY-2 and hybrid aspen under the control of cauliflower mosaic virus 35S promoter. LigD expression in the transgenic plants was monitored by Western blot analysis and enzymatic activity with crude extract prepared from each transgenic line. Preliminary analysis of lignin structure by 2D-NMR and nitrobenzene oxidation was also performed.

Two novel decarboxylase genes play a key role in the stereospecific catabolism of dehydrodiconiferyl alcohol in Sphingobium sp. strain SYK-6: Novel decarboxylases for phenylcoumaran catabolism

Sphingobium sp. strain SYK-6 is able to use a phenylcoumaran-type biaryl, dehydrodiconiferyl alcohol (DCA), as a sole source of carbon and energy. In SYK-6 cells, the alcohol group of the B-ring side chain of DCA was first oxidized to the carboxyl group, and then the alcohol group of the A-ring side chain was oxidized to generate 5-(2-carboxyvinyl)-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylate (DCA-CC). We identified phcF, phcG and phcH, which conferred the ability to convert DCA-CC into 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl)acrylate (DCA-S) in a host strain. These genes exhibited no significant sequence similarity with known enzyme genes, whereas phcF and phcG, which contain a DUF3237 domain of unknown function, showed 32% amino acid sequence identity with each other. The DCA-CC conversion activities were markedly decreased by disruption of phcF and phcG, indicating that phcF and phcG play dominant roles in the conversion of DCA-CC. Purified PhcF and PhcG catalysed the decarboxylation of the A-ring side chain of DCA-CC, producing DCA-S, and showed enantiospecificity towards (+)- and (-)-DCA-CC respectively. PhcF and PhcG formed homotrimers, and their Km for DCA-CC were determined to be 84 μM and 103 μM, and Vmax were 307 μmol⋅min-1 ⋅mg-1 and 137 μmol⋅min-1 ⋅mg-1 respectively. In conclusion, PhcF and PhcG are enantiospecific decarboxylases involved in phenylcoumaran catabolism.