A TonB-dependent receptor constitutes the outer membrane transport system for a lignin-derived aromatic compound

TonB-dependent receptors (TBDRs) mediate substrate-specific transport across the outer membrane, utilizing energy derived from the proton motive force transmitted from the TonB−ExbB−ExbD complex located in the inner membrane (TonB system). Although a number of TonB systems involved in the uptake of siderophores, vitamin B12 and saccharides have been identified, their involvement in the uptake and catabolism of aromatic compounds was previously unknown. Here, we show that the outer membrane transport of a biphenyl compound derived from lignin is mediated by the TonB system in a Gram-negative bacterium capable of degrading lignin-derived aromatic compounds, Sphingobium sp. strain SYK-6. Furthermore, we found that overexpression of the corresponding TBDR gene enhanced the uptake of this biphenyl compound, contributing to the improved rate of platform chemical production. Our results will provide an important basis for establishing engineered strains optimized for use in lignin valorisation

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

Cellular and Genetic Regulation of Coniferaldehyde Incorporation in Lignin of Herbaceous and Woody Plants by Quantitative Wiesner Staining

Lignin accumulates in the cell walls of specialized cell types to enable plants to stand upright and conduct water and minerals, withstand abiotic stresses, and defend themselves against pathogens. These functions depend on specific lignin concentrations and subunit composition in different cell types and cell wall layers. However, the mechanisms controlling the accumulation of specific lignin subunits, such as coniferaldehyde, during the development of these different cell types are still poorly understood. We herein validated the Wiesner test (phloroglucinol/HCl) for the restrictive quantitative in situ analysis of coniferaldehyde incorporation in lignin. Using this optimized tool, we investigated the genetic control of coniferaldehyde incorporation in the different cell types of genetically-engineered herbaceous and woody plants with modified lignin content and/or composition. Our results demonstrate that the incorporation of coniferaldehyde in lignified cells is controlled by (a) autonomous biosynthetic routes for each cell type, combined with (b) distinct cell-to-cell cooperation between specific cell types, and (c) cell wall layer-specific accumulation capacity. This process tightly regulates coniferaldehyde residue accumulation in specific cell types to adapt their property and/or function to developmental and/or environmental changes

Improved chemical pulping and saccharification of a natural mulberry mutant deficient in cinnamyl alcohol dehydrogenase

Abstract Lignin content and its molecular structure influence various wood characteristics. In this study, the anatomical and physicochemical properties of wood derived from a naturally occurring mulberry mutant deficient in cinnamyl alcohol dehydrogenase (CAD), a key enzyme in lignin biosynthesis, were analyzed using conventional staining assays on stem sections, length and width measurements of xylem fiber cells, wood pulping and saccharification assays, and sugar compositional analysis of extractive-free wood powder. The present data indicate that the mutation in the CAD gene leads to improved wood delignification efficiency, increased pulp yield under alkaline pulping conditions, and enhanced saccharification efficiency following alkaline pretreatment. This study opens up new avenues for the multipurpose use of the mulberry CAD-deficient mutant as a raw material for biorefinery processes, in addition to its traditional use as a favored feed for silkworms.</jats:p

(+)-Larreatricin hydroxylase, an enantio-specific polyphenol oxidase from the creosote bush (Larrea tridentata)

An enantio-specific polyphenol oxidase (PPO) was purified approximately 1,700-fold to apparent homogeneity from the creosote bush (Larrea tridentata), and its encoding gene was cloned. The posttranslationally processed PPO ( approximately 43 kDa) has a central role in the biosynthesis of the creosote bush 8-8' linked lignans, whose representatives, such as nordihydroguaiaretic acid and its congeners, have potent antiviral, anticancer, and antioxidant properties. The PPO primarily engenders the enantio-specific conversion of (+)-larreatricin into (+)-3'-hydroxylarreatricin, with the regiochemistry of catalysis being unambiguously established by different NMR spectroscopic analyses; the corresponding (-)-enantiomer did not serve as a substrate. This enantio-specificity for a PPO, a representative of a widespread class of enzymes, provides additional insight into their actual physiological roles that hitherto have been difficult to determine

Membrane-associated glucose-methanol-choline oxidoreductase family enzymes PhcC and PhcD are essential for enantioselective catabolism of dehydrodiconiferyl alcohol

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