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John Ralph - One of the best experts on this subject based on the ideXlab platform.
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differences in the chemical structure of the lignins from sugarcane bagasse and straw
Biomass & Bioenergy, 2015Co-Authors: José C. Del Río, John Ralph, Ana Gutierrez, Angel T Martinez, Alessandro Guarino Lino, Jorge Luiz Colodette, Claudio F Lima, Jorge RencoretAbstract:Abstract Two major residues are produced by the sugarcane industry, the fibrous fraction following juice extraction (bagasse), and the harvest residue (straw). The structures of the lignins from these residues were studied by pyrolysis coupled to gas chromatography-mass spectrometry (Py-GC/MS), nuclear magnetic resonance (NMR), and derivatization followed by reductive cleavage (DFRC). Whereas the lignin from bagasse has a syringyl-rich p-hydroxyphenyl:guaiacyl:syringyl (H:G:S) molar composition of 2:38:60, the lignin from straw is guaiacyl-rich (H:G:S of 4:68:28). The compositional differences were also reflected in the relative abundances of the different interunit linkages. Bagasse lignin was primarily β–O–4′ alkyl-aryl ether substructures (representing 83% of NMR-measurable linkages), followed by minor amounts of β–5′ (phenylcoumarans, 6%) and other condensed substructures. The lignin from straw has lower levels of β-ethers (75%) but higher relative levels of phenylcoumarans (β–5′, 15%) and dibenzodioxocins (5–5/4–O–β, 3%), consistent with a lignin enriched in G-units. Both lignins are extensively acylated at the γ-hydroxyl of the lignin side-chain (42% and 36% acylation in bagasse and straw), predominantly with p-coumarates (preferentially on S-units) but also with acetates (preferentially on G-units) to a minor extent. Tetrahydrofuran structures diagnostically arising from β–β-coupling (dehydrodimerization) of sinapyl p-coumarate or its cross-coupling with sinapyl alcohol were found in both lignins, indicating that sinapyl p-coumarate acts as a monomer participating in lignification. The flavone tricin was also found in the lignins from sugarcane, as also occurs in other grasses.
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structural characterization of wheat straw lignin as revealed by analytical pyrolysis 2d nmr and reductive cleavage methods
Journal of Agricultural and Food Chemistry, 2012Co-Authors: José C. Del Río, John Ralph, Jorge Rencoret, Pepijn Prinsen, Angel T Martinez, Ana GutierrezAbstract:The structure of the lignin in wheat straw has been investigated by a combination of analytical pyrolysis, 2D-NMR, and derivatization followed by reductive cleavage (DFRC). It is a p-hydroxyphenyl-guaiacyl-syringyl lignin (with an H:G:S ratio of 6:64:30) associated with p-coumarates and ferulates. 2D-NMR indicated that the main substructures present are β-O-4′-ethers (∼75%), followed by phenylcoumarans (∼11%), with lower amounts of other typical units. A major new finding is that the flavone tricin is apparently incorporated into the lignins. NMR and DFRC indicated that the lignin is partially acylated (∼10%) at the γ-carbon, predominantly with acetates that preferentially acylate guaiacyl (12%) rather than syringyl (1%) units; in dicots, acetylation is predominantly on syringyl units. p-Coumarate esters were barely detectable (<1%) on monomer conjugates released by selectively cleaving β-ethers in DFRC, indicating that they might be preferentially involved in condensed or terminal structures.
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structural characterization of the lignin in the cortex and pith of elephant grass pennisetum purpureum stems
Journal of Agricultural and Food Chemistry, 2012Co-Authors: Pepijn Prinsen, John Ralph, Jorge Rencoret, Angel T Martinez, Lidia Nieto, Jesus Jimenezbarbero, Ana GutierrezAbstract:The structure of the lignin in the cortex and pith of elephant grass (Pennisetum purpureum) stems was studied both in situ and in isolated milled “wood” lignins by several analytical methods. The presence of p-coumarate and ferulate in the cortex and pith, as well as in their isolated lignins, was revealed by pyrolysis in the presence of tetramethylammonium hydroxide, and by 2D NMR, and indicated that ferulate acylates the carbohydrates while p-coumarate acylates the lignin polymer. 2D NMR showed a predominance of alkyl aryl ether (β–O–4′) linkages (82% of total interunit linkages), with low amounts of “condensed” substructures, such as resinols (β–β′), phenylcoumarans (β–5′), and spirodienones (β–1′). Moreover, the NMR also indicated that these lignins are extensively acylated at the γ-carbon of the side chain. DFRC analyses confirmed that p-coumarate groups acylate the γ-OHs of these lignins, and predominantly on syringyl units.
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Suppression of 4-Coumarate-CoA Ligase in the Coniferous Gymnosperm Pinus radiata
Plant Physiology, 2008Co-Authors: Armin Wagner, Diane Steward, Gerald Koch, Kirk M Torr, Lorelle Phillips, Heather Flint, Uwe Schmitt, Lloyd Donaldson, John RalphAbstract:Severe suppression of 4-Coumarate-coenzyme A ligase (4CL) in the coniferous gymnosperm Pinus radiata substantially affected plant phenotype and resulted in dwarfed plants with a “bonsai tree-like” appearance. Microscopic analyses of stem sections from 2-year-old plants revealed substantial morphological changes in both wood and bark tissues. This included the formation of weakly lignified tracheids that displayed signs of collapse and the development of circumferential bands of axial parenchyma. Acetyl bromide-soluble lignin assays and proton nuclear magnetic resonance studies revealed lignin reductions of 36% to 50% in the most severely affected transgenic plants. Two-dimensional nuclear magnetic resonance and pyrolysis-gas chromatography-mass spectrometry studies indicated that lignin reductions were mainly due to depletion of guaiacyl but not p-hydroxyphenyl lignin. 4CL silencing also caused modifications in the lignin interunit linkage distribution, including elevated β-aryl ether (β-O-4 unit) and spirodienone (β-1) levels, accompanied by lower phenylcoumaran (β-5), resinol (β-β), and dibenzodioxocin (5-5/β-O-4) levels. A sharp depletion in the level of saturated (dihydroconiferyl alcohol) end groups was also observed. Severe suppression of 4CL also affected carbohydrate metabolism. Most obvious was an up to approximately 2-fold increase in galactose content in wood from transgenic plants due to increased compression wood formation. The molecular, anatomical, and analytical data verified that the isolated 4CL clone is associated with lignin biosynthesis and illustrated that 4CL silencing leads to complex, often surprising, physiological and morphological changes in P. radiata.
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p coumaroylated syringyl units in maize lignin implications for β ether cleavage by thioacidolysis
Phytochemistry, 1996Co-Authors: John H Grabber, Stéphane Quideau, John RalphAbstract:Abstract Recent NMR studies of lignin isolated from maize demonstrated that p -coumarate esters are attached exclusively to the γ-position of phenylpropane side chains. Thioacidolysis/desulphuration experiments have revealed that p -coumarate units are attached primarily ( ca 90%) to syringly moieties in maize lignin. In model studies with guaiacylglycerol and syringylglycerol-β-guaiacyl ethers, cleavage of β-ether linkages by thioacidolysis was reduced 40% by γ-acylation of phenylpropane side chains with p -coumarate. Our results indicate that γ - p -coumarate esters significantly reduce the yields of syringyl products recovered after thioacidolysis of grass lignins.
Jaime Barros - One of the best experts on this subject based on the ideXlab platform.
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4 coumarate 3 hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase
Nature Communications, 2019Co-Authors: Jaime Barros, Luhua Song, Maite Docampo Palacios, Feroza K. Choudhury, Luis Escamillatrevino, Juan C Serraniyarce, Nancy L Engle, Timothy J. TschaplinskiAbstract:Lignin biosynthesis is evolutionarily conserved among higher plants and features a critical 3-hydroxylation reaction involving phenolic esters. However, increasing evidence questions the involvement of a single pathway to lignin formation in vascular plants. Here we describe an enzyme catalyzing the direct 3-hydroxylation of 4-Coumarate to caffeate in lignin biosynthesis as a bifunctional peroxidase that oxidizes both ascorbate and 4-Coumarate at comparable rates. A combination of biochemical and genetic evidence in the model plants Brachypodium distachyon and Arabidopsis thaliana supports a role for this coumarate 3-hydroxylase (C3H) in the early steps of lignin biosynthesis. The subsequent efficient O-methylation of caffeate to ferulate in grasses is substantiated by in vivo biochemical assays. Our results identify C3H as the only non-membrane bound hydroxylase in the lignin pathway and revise the currently accepted models of lignin biosynthesis, suggesting new gene targets to improve forage and bioenergy crops.
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4-Coumarate 3-hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase
Nature Publishing Group, 2019Co-Authors: Jaime Barros, Luis Escamilla-trevino, Luhua Song, Xiaolan Rao, Juan Carlos Serrani-yarce, Maite Docampo Palacios, Nancy Engle, Feroza K. Choudhury, Timothy J. Tschaplinski, Barney J. VenablesAbstract:Lignin biosynthesis in higher plants relies upon a 3-hydroxylation reaction that can occur via shikimate esters of 4-Coumarate. Here, Barros et al. define an alternative biosynthetic pathway via cytosolic ascorbate peroxidase that can catalyze direct 3-hydroxylation of 4-Coumarate
Anthony R. Borneman - One of the best experts on this subject based on the ideXlab platform.
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heterologous production of raspberry ketone in the wine yeast saccharomyces cerevisiae via pathway engineering and synthetic enzyme fusion
Microbial Cell Factories, 2016Co-Authors: Natoiya D.r. Lloyd, Anthony R. Borneman, Isak S. PretoriusAbstract:Raspberry ketone is the primary aroma compound found in raspberries and naturally derived raspberry ketone is a valuable flavoring agent. The economic incentives for the production of raspberry ketone, combined with the very poor yields from plant tissue, therefore make this compound an excellent target for heterologous production in synthetically engineered microbial strains. A de novo pathway for the production of raspberry ketone was assembled using four heterologous genes, encoding phenylalanine/tyrosine ammonia lyase, cinnamate-4-hydroxlase, coumarate-CoA ligase and benzalacetone synthase, in an industrial strain of Saccharomyces cerevisiae. Synthetic protein fusions were also explored as a means of increasing yields of the final product. The highest raspberry ketone concentration achieved in minimal media exceeded 7.5 mg/L when strains were fed with 3 mM p-coumaric acid; or 2.8 mg/L for complete de novo synthesis, both of which utilized a coumarate-CoA ligase, benzalacetone synthase synthetic fusion protein that increased yields over fivefold compared to the native enzymes. In addition, this strain was shown to be able to produce significant amounts of raspberry ketone in wine, with a raspberry ketone titer of 3.5 mg/L achieved after aerobic fermentation of Chardonnay juice or 0.68 mg/L under anaerobic winemaking conditions. We have shown that it is possible to produce sensorially-relevant quantities of raspberry ketone in an industrial heterologous host. This paves the way for further pathway optimization to provide an economical alternative to raspberry ketone derived from plant sources.
Timothy J. Tschaplinski - One of the best experts on this subject based on the ideXlab platform.
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4 coumarate 3 hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase
Nature Communications, 2019Co-Authors: Jaime Barros, Luhua Song, Maite Docampo Palacios, Feroza K. Choudhury, Luis Escamillatrevino, Juan C Serraniyarce, Nancy L Engle, Timothy J. TschaplinskiAbstract:Lignin biosynthesis is evolutionarily conserved among higher plants and features a critical 3-hydroxylation reaction involving phenolic esters. However, increasing evidence questions the involvement of a single pathway to lignin formation in vascular plants. Here we describe an enzyme catalyzing the direct 3-hydroxylation of 4-Coumarate to caffeate in lignin biosynthesis as a bifunctional peroxidase that oxidizes both ascorbate and 4-Coumarate at comparable rates. A combination of biochemical and genetic evidence in the model plants Brachypodium distachyon and Arabidopsis thaliana supports a role for this coumarate 3-hydroxylase (C3H) in the early steps of lignin biosynthesis. The subsequent efficient O-methylation of caffeate to ferulate in grasses is substantiated by in vivo biochemical assays. Our results identify C3H as the only non-membrane bound hydroxylase in the lignin pathway and revise the currently accepted models of lignin biosynthesis, suggesting new gene targets to improve forage and bioenergy crops.
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4-Coumarate 3-hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase
Nature Publishing Group, 2019Co-Authors: Jaime Barros, Luis Escamilla-trevino, Luhua Song, Xiaolan Rao, Juan Carlos Serrani-yarce, Maite Docampo Palacios, Nancy Engle, Feroza K. Choudhury, Timothy J. Tschaplinski, Barney J. VenablesAbstract:Lignin biosynthesis in higher plants relies upon a 3-hydroxylation reaction that can occur via shikimate esters of 4-Coumarate. Here, Barros et al. define an alternative biosynthetic pathway via cytosolic ascorbate peroxidase that can catalyze direct 3-hydroxylation of 4-Coumarate
Ai-xia Cheng - One of the best experts on this subject based on the ideXlab platform.
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Cloning and Functional Characterization of Two 4-Coumarate: CoA Ligase Genes from Selaginella moellendorffii.
Molecules, 2018Co-Authors: Ping-ping Wang, Yi-feng Wu, Ai-xia ChengAbstract:Selaginella is an extant lycopodiophyte genus, which is representative of an ancient lineage of tracheophytes. The important evolutionary status makes it a valuable resource for the study of metabolic evolution in vascular plants. 4-Coumarate: CoA ligase (4CL) is the pivotal enzyme that controls the flow of carbon through the phenylpropanoid metabolic pathway into the specific lignin, flavonoid, and wall-bound phenolics biosynthesis pathways. Although 4CLs have been extensively characterized in other vascular plants, little is known of their functions in Selaginella. Here, we isolated two 4CL genes (Sm4CL1 and Sm4CL2) from Selaginella moellendorffii. Based on the enzymatic activities of the recombinant proteins, both of these genes encoded bona fide 4CLs. The 4CL isoforms in S. moellendorffii have different activities: Sm4CL2 was more active than Sm4CL1. The enzymatic properties and gene expression patterns indicated that the 4CL genes have been conserved in the evolution of vascular plants.
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Cloning and functional characterization of a 4-Coumarate CoA ligase from liverwort Plagiochasma appendiculatum.
Phytochemistry, 2015Co-Authors: Hai-na Yu, Rui-xue Xu, Ai-xia ChengAbstract:Abstract Plant phenylpropanoids represent a large group of secondary metabolites which have played an important role in terrestrial plant life, beginning with the evolution of land plants from primitive green algae. 4-Coumarate: coenzyme A ligase (4CL) is a provider of activated thioester substrates within the phenylpropanoid synthesis pathway. Although 4CLs have been extensively characterized in angiosperm, gymnosperm and moss species, little is known of their functions in liverworts. Here, a 4CL homolog (designated as Pa4CL1) was isolated from the liverwort species Plagiochasma appendiculatum . The full-length cDNA sequence of Pa4CL1 contains 1644 bp and is predicted to encode a protein with 547 amino acids. The gene products were 40–50% identical with 4CL sequences reported in public databases. The recombinant protein was heterologously expressed in Escherichia coli and exhibited a high level of 4CL activity, catalyzing formation of hydroxycinnamate-CoA thioesters by a two-step reaction mechanism from corresponding hydroxycinnamic acids. Kinetic analysis indicated that the most favorable substrate for Pa4CL1 is p -coumaric acid. The transcription of Pa4CL1 was induced when P. appendiculatum thallus was treated with either salicylic acid or methyl jasmonate.
