John Ralph

John Ralph
University of Wisconsin–Madison | UW · Department of Biochemistry

Ph.D.

About

583
Publications
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Publications

Publications (583)
Article
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Morphological profiling is an omics-based approach for predicting intracellular targets of chemical compounds in which the dose-dependent morphological changes induced by the compound are systematically compared to the morphological changes in gene-deleted cells. In this study, we developed a reliable high-throughput (HT) platform for yeast morphol...
Article
Lignin is the main factor limiting the enzymatic conversion of lignocellulosic biomass into fermentable sugars. To reduce the recalcitrance engendered by the lignin polymer, the coumarin scopoletin was incorporated into the lignin polymer through the simultaneous expression of FERULOYL-CoA 6′-HYDROXYLASE 1 ( F6′H1 ) and COUMARIN SYNTHASE ( COSY ) i...
Article
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Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars mainly due to the presence of lignin. By engineering plants to partially replace traditional lignin monomers with alternative ones, lignin degradability and extractability can be enhanced. Previously, the alternative monomer curcumin has been successfully produced and...
Article
Lignin contributes substantially to the recalcitrance of biomass towards saccharification. To circumvent this problem, researchers have genetically altered lignin, although, in a number of cases, these efforts have resulted in an undesirable yield penalty. Recent findings have shown that by knocking out two subunits (MED5A and MED5B) of the transcr...
Article
Diferuloylputrescine has been found in a variety of plant species, and recent work has provided evidence of its covalent bonding into lignin. Results from nuclear magnetic resonance spectroscopy revealed the presence of bonding patterns consistent with homo-coupling of diferuloylputrescine and the possibility of cross-coupling with lignin. In the p...
Article
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To date, little is known about the evolution of fern genomes, with only two small genomes published from the heterosporous Salviniales. Here we assembled the genome of Alsophila spinulosa, known as the flying spider-monkey tree fern, onto 69 pseudochromosomes. The remarkable preservation of synteny, despite resulting from an ancient whole-genome du...
Article
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Renewed interests in the development of bioenergy, biochemicals, and biomaterials have elicited new strategies for engineering the lignin of biomass feedstock plants. This study shows, for the first time, that 3,4‐dihydroxybenzoate (DHB) is compatible with the radical coupling reactions that assemble polymeric lignin in plants. We introduced a bact...
Chapter
Lignin is largely produced by the oxidative radical coupling of the three monolignols, p-coumaryl, coniferyl, and sinapyl alcohols, which form the p-hydroxyphenyl, guaiacyl, and syringyl lignin units in the lignin polymer. However, it is increasingly appreciated that other “unconventional” phenolic compounds can also behave as lignin monomers parti...
Article
The complexity of lignin structure impedes efficient cell wall digestibility. Native lignin is composed of a mixture of three dominant monomers, coupled together through a variety of linkages. Work over the past few decades has demonstrated that lignin composition can be altered through a variety of mutational and transgenic approaches such that th...
Article
Plant BAHD acyltransferases perform a wide range of enzymatic tasks in primary and secondary metabolism. Acyl-CoA monolignol transferases, which couple a CoA substrate to a monolignol creating an ester linkage, represent a more recent class of such acyltransferases. The resulting conjugates may be used for plant defense but are also deployed as imp...
Article
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Plant NADPH-dependent cytochrome P450 reductase (CPR) is a multi-domain enzyme that donates electrons for hydroxylation reactions catalyzed by the Class II cytochrome P450 monooxygenases involved in the synthesis of many primary and secondary metabolites. These P450 enzymes include trans-cinnamate-4-hydroxylase (C4H), p-coumarate-3′-hydroxylase (C3...
Article
Lignin is a phenolic polymer deposited in the plant cell wall and is mainly polymerized from three canonical monomers (monolignols), i.e., p-coumaryl, coniferyl, and sinapyl alcohols. After polymerization, these alcohols form different lignin substructures. In dicotyledons, monolignols are biosynthesized from phenylalanine, an aromatic amino acid....
Article
This work shows the integration of reductive lignin depolymerization and microbial funneling with Novosphingobium aromaticivorans to produce 2-pyrone-4,6-dicarboxylic acid (PDC) in a lignocellulosic biorefinery.
Article
Full-text available
Hydroxycinnamoyl shikimates were reported in 2005 to be intermediates in monolignol biosynthesis. 3-Hydroxylation of p -coumarate, originally thought to occur via coumarate 3-hydroxylase (C3H) from p -coumaric acid or its CoA thioester, was revealed to be via the action of coumaroyl shikimate 3′-hydroxylase (C3′H) utilizing p -coumaroyl shikimate a...
Article
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In sorghum (Sorghum bicolor) and other C4 grasses, brown midrib (bmr) mutants have long been associated with plants impaired in their ability to synthesize lignin. The brown midrib 30 (Bmr30) gene, identified using a bulk segregant analysis and next-generation sequencing, was determined to encode a chalcone isomerase (CHI). Two independent mutation...
Article
Poplar (Populus) lignin is naturally acylated with p-hydroxybenzoate ester moieties. However, the enzyme(s) involved in the biosynthesis of the monolignol-p-hydroxybenzoates have remained largely unknown. Here, we performed an in vitro screen of the Populus trichocarpa BAHD acyltransferase superfamily (116 genes) using a wheatgerm cell-free transla...
Article
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Lignin, a polyphenolic polymer, is a major chemical constituent of the cell walls of terrestrial plants. The biosynthesis of lignin is a highly plastic process, as highlighted by an increasing number of non-canonical monomers that have been successfully identified in an array of plants. Here, we engineered hybrid poplar (Populus alba x grandidentat...
Article
Full-text available
Ferulate-5-hydroxylase is a key enzyme involved in the conversion of the guaiacyl monolignol to the syringyl monolignol in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Stem rot caused by the fungus Sclerotinia sclerotiorum in Brassica napus causes severe losses in its produ...
Article
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Recent studies demonstrate that several polyphenolic compounds produced from beyond the canonical monolignol biosynthetic pathways can behave as lignin monomers, participating in radical coupling reactions and being incorporated into lignin polymers. Here, we show various classes of flavonoids, the chalconoid naringenin chalcone, the flavanones nar...
Article
Full-text available
Lignins are cell‐wall‐located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. Here, we explored the role of the two CSE homologs in poplar (Populus tremula x P. alba)....
Article
Plants have developed tissue‐specific defense strategies in response to various herbivores with different feeding habits. Although defense responses to leaf‐chewing insects have been well studied, little is known about stem‐specific responses, particularly in the pith, to stem‐boring herbivores. To understand the stem‐specific defense, we first con...
Article
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 se...
Article
Full-text available
To what degree can the lignin subunits in a monocot be derived from monolignol ferulate (ML‐FA) conjugates? This simple question comes with a complex set of variables. Three potential requirements for optimizing ML‐FA production are: 1) The presence of an active FERULOYL‐CoA MONOLIGNOL TRANSFERASE (FMT) enzyme throughout monolignol production; 2) S...
Article
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The purification of hydroxycinnamic acids [p-coumaric acid (pCA) and ferulic acid (FA)] from grass cell walls requires high-cost processes. Feedstocks with increased levels of one hydroxycinnamate in preference to the other are therefore highly desirable. We identified and conducted expression analysis for nine BAHD acyltransferase ScAts genes from...
Chapter
Lignin is a phenylpropanoid polymer derived essentially from the oxidative radical coupling of three p‐hydroxycinnamyl alcohols, the so‐called monolignols: p‐coumaryl, coniferyl, and sinapyl alcohols. Several other phenolic compounds, all originating from the shikimate‐derived monolignol biosynthetic pathway, have been found to behave as lignin mon...
Article
Full-text available
Pinoresinol is a high-value monolignol-derived lignan used in plant defense and with human health-supporting effects. The synthetic yield and isolation efficiency of racemic pinoresinol from coniferyl alcohol by conventional radical coupling methods is sub-optimal. In this work, a facile and efficient synthetic approach was developed to synthesize...
Article
Lignocellulose, the structural component of plant cells, is a major agricultural byproduct and the most abundant terrestrial source of biopolymers on Earth. The complex and insoluble nature of lignocellulose limits its conversion into value-added commodities, and currently, efficient transformation requires expensive pretreatments and high loadings...
Article
Full-text available
The monolignols, p-coumaryl, coniferyl, and sinapyl alcohol, arise from the general phenylpropanoid biosynthetic pathway. Increasingly, however, authentic lignin monomers derived from outside this process are being identified and found to be fully incorporated into the lignin polymer. Among them, hydroxystilbene glucosides, which are produced throu...
Article
Full-text available
Despite the scientific and economic importance of maize, little is known about its specialized metabolism. Here, five maize organs were profiled using different reversed-phase liquid chromatography-mass spectrometry methods. The resulting spectral metadata, combined with candidate substrate-product pair (CSPP) networks, allowed the structural chara...
Article
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Although measuring lignin contents is a routine operation for biomass compositional analysis in process development aiming at efficient utilization of woody biomass, it is still a challenging task requiring many...
Article
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Canonical lignification occurs via the coupling of phenolic radicals, in which chain extension can occur only from phenolic ends of growing polymer chains. Radical coupling of catechyl monomers, including caffeyl and 5-hydroxyconiferyl alcohols, gives rise to benzodioxane units in the polymer. Anticipating that a catechol could oxidize to its o-ben...
Article
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Background Arabinoxylan in grass cell walls is acylated to varying extents by ferulate and p-coumarate at the 5-hydroxy position of arabinosyl residues branching off the xylan backbone. Some of these hydroxycinnamate units may then become involved in cell wall radical coupling reactions, resulting in ether and other linkages amongst themselves or t...
Article
Lignocellulosic biomass is an abundant byproduct from cereal crops that can potentially be valorized as a feedstock to produce biomaterials. Zea mays CINNAMYL ALCOHOL DEHYDROGENASE 2 (ZmCAD2) is involved in lignification and is a promising target to improve the cellulose‐to‐glucose conversion of maize stover. Here, we analyzed a field‐grown zmcad2...
Article
Following biomass fractionation of corn stover using γ-valerolactone (GVL) pretreatment, the resulting lignin stream was subjected to base hydrolysis reactions to achieve the isolation of p-coumaric acid (pCA) from p-coumarate esters present as pendent groups on the lignin. pCA was produced from 5-g-scale experiments in crude yields of 8.3 wt % wit...
Article
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The valorisation of the plant biopolymer lignin is now recognised as essential to enabling the economic viability of the lignocellulosic biorefining industry. In this context, the “lignin-first” biorefining approach, in which lignin valorisation is considered in the design phase, has demonstrated the fullest utilisation of lignocellulose. We define...
Article
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jats:title>Abstract Lignin causes lignocellulosic biomass recalcitrance to enzymatic hydrolysis. Engineered low-lignin plants have reduced recalcitrance but often exhibit yield penalties, offsetting their gains in fermentable sugar yield. Here, CRISPR/Cas9-generated CCR2 (−/*) line 12 poplars have one knockout CCR2 allele while the other contains a...
Article
Lignocellulosic biomass is mainly composed of polysaccharides and lignin. The complexity and diversity of the plant cell wall polymers makes it difficult to isolate the components in pure form for characterization. Many current approaches to analyzing lignocellulose structure, which involve sequential extraction and characterization of the resultin...
Article
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This article is a Commentary on Lui et al. (2020), 228: 269–284.
Article
The Cover Feature shows how authentic model compounds for biphenyl ether units in lignin cleave, but differently than commonly used simple models, under Pd/C hydrogenolysis conditions. More importantly, they and real lignins produce a novel and mechanistically revealing rearrangement monomer (highlighted), diagnostic for biphenyl ether structures i...
Article
This paper summarizes and compares computational results for the thermodynamics of bond dissociation, coupling, and rearomatization for a number of non-canonical lignin monomer-lignol combinations that have been found to occur experimentally. The non-canonical lignin monomers discussed are tricin, caffeyl alcohol and 5-hydroxyconiferyl alcohol, and...
Article
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Fatty acids play many important roles in cells and also in industrial processes. Furan fatty acids (FuFAs) are present in the lipids of some plant, fish, and microbial species and appear to function as second messengers in pathways that protect cells from membrane-damaging agents. We report here the results of chemical, genetic, and synthetic biolo...
Article
The Front Cover shows how a diverse team with expertise in many different fields works together in an integrated fashion to address complex problems. Only when the whole system, from field to the liquid fuels and co‐products, is assessed, can we identify the key parameters needed to design an economically viable biorefinery‐based economy. Cover art...
Article
Invited for this month's cover is the research team from the D.O.E. Great Lake Bioenergy Research Center (GLBRC) at the University of Wisconsin–Madison. The cover image shows how a diverse team with expertise in many different fields works together in an integrated fashion to address complex problems. Only when the whole system, from field to the l...
Article
Hydrogenolysis has emerged as one of the most effective means of converting polymeric lignin into monoaromatic fragments of value. Reported yields may be higher than for other methods and can exceed the theoretical yields estimated from measures of the content of lignin’s most readily cleaved alkyl‐aryl ether bonds in β‐ether units. The high yields...
Article
Full-text available
Lignin is conventionally defined as being formed by the oxidative polymerization of three main monolignols, p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, that are derived from the general phenylpropanoid biosynthetic pathway. Many other phenolic compounds that are also derived from the phenylpropanoid pathway are also known to perform...
Article
The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated 4-coumarate:CoA ligases (4CLs) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. Here we show that CRISPR-knockout of 4CL1 in p...
Article
Full-text available
Lignin, an abundant renewable aromatic biopolymer, is an essential macromolecule in terrestrial vascular plants. Genetic modifications affecting monolignol biosynthesis, which produces lignin monomers, alter the metabolic flux through the pathway, and are becoming increasingly explored for improving the lignin and biomass quality and for producing...
Article
Selective and economic conversion of lignocellulosic biomass components, cellulose, hemicelluloses, and lignin, to bio‐based fuels and chemical, is the major goal of biorefineries but low yields and selectivities for fuel precurors such as sugars, furanics, and lignin‐derived monomers pose significant disadvantages in process economics. It is widel...
Article
Selective and economic conversion of lignocellulosic biomass components, cellulose, hemicelluloses, and lignin, to bio-based fuels and chemical, is the major goal of biorefineries but low yields and selectivities for fuel precurors such as sugars, furanics, and lignin-derived monomers pose significant disadvantages in process economics. It is widel...
Article
Full-text available
The hydroxycinnamic acids, p ‐coumaric acid ( p CA) and ferulic acid (FA), add diversity to the portfolio of products produced by grass‐fed lignocellulosic biorefineries. The level of lignin‐bound p CA in Zea mays was modified by alteration of PMT expression. The biomass was processed in a lab‐scale alkaline‐pretreatment biorefinery process and the...
Preprint
Full-text available
The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated 4-coumarate:CoA ligases ( 4CLs ) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. Here we show that CRISPR-knockout of 4CL1 in...
Article
Full-text available
Lignin is an abundant aromatic polymer found in plant secondary cell walls. In recent years, lignin has attracted renewed interest as a feedstock for bio-based chemicals via catalytic and biological approaches and has emerged as a target for genetic engineering to improve lignocellulose digestibility by altering its composition. In lignin biosynthe...
Article
Ferulate (FA) units esterified to grass arabinoxylans are involved in cross-linking cell wall polymers. In this work, this contention is strengthened by the identification of FA homo- and hetero-dimers esterified to methyl arabinofuranoside (MeAra) units after their release from the xylan by mild acidolysis in dioxane/methanol/HCl. Acidolysis of po...
Article
Phenolic endgroups on lignin molecules are important sites for continued lignification, their influence over lignin molecular properties, their ability to enhance lignin degradation, and their potential for derivatization to produce lignin materials. Here we use iodination to regioselectively tag, and HSQC-NMR to monitor, guaiacyl phenolic aromatic...
Article
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Pathogenic bacteria invade plant tissues and proliferate in the extracellular space. Plants have evolved the immune system to recognize and limit the growth of pathogens. Despite substantial progress in the study of plant immunity, the mechanism by which plants limit pathogen growth remains unclear. Here, we show that lignin accumulates in Arabidop...
Article
Plant‐based antifungal agents offer an alternative to synthetic fungicides in amenity turfgrass disease management. Poacic acid is a byproduct of the biofuel production process that has exhibited antifungal activity, and the objective of this research was to determine its ability to serve as an effective management tool for economically important t...
Article
Full-text available
Lignin is a phenylpropanoid polymer produced in the secondary cell walls of vascular plants. Although most eudicot and gymnosperm species generate lignins solely via polymerization of p-hydroxycinnamyl alcohols (monolignols), grasses additionally use a flavone, tricin, as a natural lignin monomer to generate tricin-incorporated lignin polymers in c...
Article
Recent experimental work has revealed that the hydroxystilbene piceatannol can function as a monomeric unit in the lignification of palm fruit endocarp tissues. Results indicated that piceatannol homo-couples and cross-couples with monolignols through radical reactions and is integrally incorporated into the lignin polymer. The current work reports...
Article
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The efficient utilization of crop waste is limited because of the plant cell wall recalcitrance. Solving this problem through sustainable processing, meanwhile producing value-added products, is crucial. Here we report a rapid lignocellulose fractionation method, applied to common reed, using milling under mild alkaline conditions without involving...
Article
DDQ oxidized lignins were used as substrates to assess the effectiveness of secondary depolymerization methods for the production of aromatics.
Article
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The manuscript initially failed to cite the work of Dr. Cong et al., which was the basis for this effort. We apologize for this error.
Article
Cellulose synthase A genes (CesAs) are responsible for cellulose biosynthesis in plant cell walls. In this study, functions of secondary wall cellulose synthases PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B were characterized during wood formation in Populus trichocarpa (Torr. & Gray). CesA RNAi knockdown transgenic plants exhibited stunted growth, narr...
Article
Lignin is a major impediment in the deconstruction of plant biomass to its soluble monomeric constituents. Modification of the lignin biosynthetic pathway has proven to be an effective means of reducing biomass recalcitrance but can often result in impaired growth. As one of the successful examples of “designer” biomass, “Zip-lignin” has been gener...