About

John Ralph is a Professor in the Department of Biological Systems Engineering at the University of Wisconsin–Madison. His contact information includes an email address at jralph@wisc.edu and a phone number (608) 890-2429. He is based at the 2113 WI Energy Institute Building, located at 1552 University Ave, Madison, WI 53726. The page does not provide additional details about his research focus, background, or key contributions.

Research topics

• Chemistry
• Organic chemistry
• Pulp and paper industry
• Biology
• Biochemistry
• Engineering
• Botany
• Environmental science
• Business
• Biotechnology
• Process engineering
• Biochemical engineering
• Waste management
• Food science
• Mathematics

Selected publications

• Introducing furanocoumarin biosynthetic genes in tomato results in coumarins accumulation and impacts growth

  Plant Science · 2026-03-18

  articleOpen access

  Over the past three decades, efforts to decipher plant metabolism have shed light on key enzymes driving specialized metabolite biosynthesis. Although only few pathways have been completely investigated to date, their characterization paves the way for exploring the potential effects of specialized metabolites on plant physiology. Among them is the linear furanocoumarin pathway, which was recently completed to produce up to psoralen. In this study, we report the first metabolic engineering of the linear furanocoumarin pathway to enable artificial psoralen production in tomato, through the integration of four genes coding for the enzymes: Umbelliferone Synthase ( Ps Diox), Demethylsuberosin Synthase ( Ps PT1), Marmesin Synthase ( Fc CYP76F112) and Psoralen Synthase ( Ps CYP71AJ3). Metabolic analyses confirmed the detection of small quantities of psoralen in the transgenic tomato line, but also highlighted a larger accumulation of coumarins and particularly scopoletin. Using morphophysiological and multi-omics analyses, we explorate how such metabolic modifications, could impact growth and affect plant physiology. • Coumarins impaired growth at low concentration in tomato. • The furanocoumarins pathway depend to the coumarins pathway regulation to be produced in plants. • Coumarins induces a phytotoxicity that the tomato can manage at low concentration. • Coumarins accumulation is linked to nitrate and consequently nitrogen uptakes. • Furanocoumarins could induce a potential lethal phytotoxicity in tomato.

  PublisherDOI

• Beyond Solanaceae: incorporation of feruloyltyramine and feruloyloctopamine into Cannabaceae lignins

  PLANT PHYSIOLOGY · 2026-04-10

  article

  The ferulic acid amides, feruloyltyramine and feruloyloctopamine, have been widely reported as integral constituents in the lignins in several species of Solanaceae in which they function as authentic lignin monomers. In the present study, we demonstrate that these ferulic acid amides are likewise incorporated into the lignins of species within Cannabaceae, including hemp (Cannabis sativa), hops (Humulus lupulus), and European nettle tree (Celtis australis). Structural analyses using derivatization followed by reductive cleavage (DFRC) and two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy revealed that these ferulic acid amides are incorporated via 4-O- and 8-O-ether linkages, as well as through 8-5' linkages forming phenylcoumaran structures. Examination of a broad phylogenetic range of plant families demonstrated the absence of these ferulic acid amides from the lignins of all families studied except Solanaceae and Cannabaceae. Given the distant phylogenetic relationship between Solanaceae and Cannabaceae, the recruitment of these ferulic acid amides as lignin monomers in both lineages likely constitutes a case for convergent evolution at the level of lignin biosynthetic pathways. The significance of these ferulic acid amides lies in their unique role as the sole nitrogen-containing phenolic compounds known to participate in lignin formation.

  PublisherDOI

• A biosynthetic gene cluster for three post-chorismate pathways in Arabidopsis

  Nature Plants · 2026-01-05 · 1 citations

  articleOpen access
  PublisherOA PDFDOI

• CRISPR/Cas9 editing of p-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE 1 in maize alters phenolic metabolism, lignin structure, and lignin-first biomass processing

  Trends in biotechnology · 2025-02-15 · 11 citations

  articleOpen access
  PublisherOA PDFDOI

• Correction to “Deep Eutectic Solvents for Efficient Fractionation of Lignocellulose to Produce Uncondensed Lignin and High-Quality Cellulose”

  ACS Sustainable Chemistry & Engineering · 2025-04-01 · 1 citations

  article
  PublisherDOI

• Deep Eutectic Solvents for Efficient Fractionation of Lignocellulose to Produce Uncondensed Lignin and High-Quality Cellulose

  ACS Sustainable Chemistry & Engineering · 2025-01-27 · 31 citations

  articleOpen access

  Simultaneously inhibiting lignin condensation and cellulose degradation remains a major challenge for achieving holistic valorization of lignocellulose. Here, we developed a deep eutectic solvent (DES), composed of l-cysteine (Cys) and lactic acid (LA), to fractionate both uncondensed lignin and high-quality cellulose from eucalyptus wood by leveraging the unique properties of Cys, i.e., highly nucleophilic groups (−SH) and hydrogen bond acceptor/donor groups (−NH2 and −COOH). The nucleophilic −SH in Cys effectively quenches the benzylic carbocations (Cα+ ions, formed at the benzylic sites of lignin) that lead to lignin condensation. This enables the high yield of uncondensed lignin (81%) with high retention of β–O–4 bonds (up to 90%). The separated uncondensed lignin is further depolymerized to prepare monophenols in a satisfactory 43% yield, equivalent to 73% of the theoretical yield. Moreover, the −NH2 and −COOH groups in Cys form extensive hydrogen bonds with the hydroxyl groups in cellulose, thus decreasing the interaction energy of DES on cellulose. As a result, the cellulose achieves an astonishing 99% retention and maintains a high degree of polymerization of 1160. The obtained high-quality cellulose is further conversed into cellulose nanofibers for strong and transparent films. This study provides new insights into the efficient separation of uncondensed lignin and high-quality cellulose from lignocellulose by a novel DES system.

  PublisherOA PDFDOI

• Enhancing Hydrogen Production from Bioenergy Crops via Photoreforming

  Journal of the American Chemical Society · 2025-08-08

  articleOpen access

  Photoreforming perennial bioenergy crops (willow, Miscanthus, and poplar) has the potential to produce H2 with reduced environmental impacts. To understand the compositional effects of the biomass on the average rate of H2 production over the first 30 min of reaction (rH2), the rH2 values of model biomass component (i.e., cellulose, hemicellulose, and lignin) mixtures were compared with those from the raw biomass. The higher cellulose or hemicellulose content in multicomponent mixtures resulted in higher rH2, whereas lignin reduced the hydrogen production rate. However, with raw biomass, the ratio of biomass components alone did not determine the rH2 via photoreforming, with rates of hydrogen production for different varieties of willow ranging between 1.9 μmol h–1 and 12.3 μmol h–1, 11.8 μmol h–1 for a poplar, and 6.8 μmol h–1 for a miscanthus biomass. In addition, comparable rH2 values of raw poplar and its extracted cellulose via an IonoSolv treatment indicated the possibility of using raw biomass materials without delignification for generating H2 via photoreforming. Importantly, rH2 was positively correlated with the interaction between water and the biomass, as assessed by NMR relaxation via an examination of the T1/T2 ratio. A stronger water-biomass interaction resulted in a higher rH2. Genetic modification of biomass has been suggested as a putative way to improve the rH2 of biomass with an enhanced interaction with water. This research enhances the understanding of factors influencing H2 production from lignocellulosic biomass by photoreforming and supports the breeding and management of perennial biomass crops to maximize H2 yields while minimizing land area requirements.

  PublisherOA PDFDOI

• Elucidation of a bacterial pathway for catabolism of the β–β-linked dilignol pinoresinol

  mBio · 2025-09-24 · 3 citations

  articleOpen access

  ABSTRACT Monolignol-derived dimers containing β–β linkages are synthesized by vascular plants and can be released during lignin depolymerization. In this work, we isolated a bacterium, Novosphingobium rhizosphaerae LY, that grows with the β–β lignan (+)-pinoresinol as a sole growth substrate. Sequence analysis suggested that this strain encodes a broad range of pathways for assimilation of aromatic monomers as well as one enzyme implicated in pinoresinol catabolism but lacks other known pathways for aromatic dimer catabolism. We constructed a genome-wide barcoded transposon library and identified genes required for pinoresinol catabolism. Using feeding studies, compound isolation, targeted synthesis, and analysis of purified enzymes, we elucidated the biochemical intermediates and reaction pathway involved in pinoresinol catabolism. We demonstrated that the first enzymatic reaction is the reductive cleavage of a furan ring in (±)-pinoresinol with retention of configuration to yield lariciresinol. We additionally confirmed that the final pathway enzyme, PinU, is related to lignostilbene dioxygenases and oxidatively cleaves a diguaiacylbutadiene intermediate to yield vanillin and coniferaldehyde. Finally, based on the enzyme characterization, we demonstrated that the strain can grow with a second β–β lignan, (–)-syringaresinol, as a sole growth substrate. In combination, these results demonstrate a new biocatalytic route for transforming a widely occurring group of plant phenylpropanoid natural products. IMPORTANCE Plants synthesize a variety of aromatic phenylpropanoid compounds containing β–β linkages, including lignin, a major structural polymeric component of the vascular plant cell wall, and lignans, biochemically related secondary metabolites with a wide range of bioactivities. Although microbial catabolic pathways have been described for dimeric phenylpropanoids featuring other interunit linkages, relatively little is known about pathways for catabolism of β–β-linked compounds. In this work, we isolated a Novosphingobium strain capable of degrading the β–β-linked lignan (+)-pinoresinol and elucidated the catabolic pathway. Understanding how bacteria catabolize β–β-linked compounds provides a basis for new biocatalytic transformations of lignans and oligolignols and has the potential to improve bacterial lignin valorization.

  PublisherDOI

• Genetically Engineered Poplar Wood Effectively Enhances the Efficiency of Deep Eutectic Solvent‐Mediated One‐Pot Processing

  ChemSusChem · 2025-07-08 · 3 citations

  articleOpen access

  Although lignocellulosic biomass is a renewable resource with the potential to replace fossil-derived fuels and chemicals, its recalcitrance, largely due to lignin, limits its utilization. Recent advancements in genetic engineering have produced transgenic trees with reduced lignin content and/or modified lignin structure without compromising growth traits. Here, three engineered poplar varieties are evaluated as feedstocks using a biocompatible one-pot deep eutectic solvent-mediated process that integrates biomass fractionation and enzymatic saccharification within a single reactor, eliminating water washing and reconditioning. All transgenic poplars exhibit higher fermentable sugar yields than wild-type (WT) trees. Notably, QsuB poplar, incorporating 3,4-dihydroxybenzoate in lignin, achieves the highest glucose conversion yield of 91.3% (vs. 73.0% from WT). AT5 and MdCHS3 poplars, incorporating ferulate esters and naringenin, also demonstrate improved glucose yields (86.7 and 84.7%, respectively), confirming reduced biomass recalcitrance. Additionally, residual lignins are valorized via hydrogenolysis into phenolic compounds, with comparable alkylphenol production across all lines. These findings demonstrate that the transgenic poplar lines not only serve as superior feedstocks for sugar conversion but also provide a rich resource for phenolic compound production, enhancing the operational and economic viability of integrated biorefinery processes.

  PublisherOA PDFDOI

• <i>p</i>-Coumaroylated Lignins Are Natively Produced in Three Rosales Families

  ACS Omega · 2025-02-04 · 3 citations

  articleOpen access

  Carbon-rich plant cell walls contain biopolymers that, with some processing, could replace fossil fuels as a major component of the current petrochemical production. To realize this, biorefineries need to be paired with biomass that during the deconstruction and fractionation processes transforms into the desired products. One component of interest is p-coumarate that, in some species, can account for up to 1% of the biomass’ dry weight. When p-coumarate is present in eudicot cell walls, it is mostly part of the suberin (bark and root), acylates the γ-hydroxy group of the lignin, in part of the tannins, or is a metabolite. The current understanding of eudicot plant cell wall composition is that the lignin is sometimes acylated with acetate and rarely with hydroxycinnamates (p-coumarate or ferulate). This study identified a clear division in the Rosales in which three families produce p-coumaroylated lignins whereas the other six families showed no evidence of the trait.

  PublisherDOI

Recent grants

• NIH Grant P41RR002301

  NIH · $6.0M · 2015

Frequent coauthors

• Fachuang Lu

  University of Wisconsin–Madison

  606 shared

• Hoon Kim

  Great Lakes Bioenergy Research Center

  491 shared

• Wout Boerjan

  Ghent University

  390 shared

• Kris Morreel

  Research Institute for Chromatography

  248 shared

• Ronald D. Hatfield

  U.S. Dairy Forage Research Center

  221 shared

• Steven D. Karlen

  University of Wisconsin–Madison

  199 shared

• Geert Goeminne

  178 shared

• Jane M. Marita

  Agricultural Research Service

  164 shared