Engineering topics
Publications and source records attributed to Ralph, John.
Experimental Data for Comparison of microbial strains as candidate hosts and genetic reservoirs for the valorization of lignin streams
Cell walls in grasses contain arabinoxylan polysaccharides decorated with ferulate groups but the mechanism by which the ferulate is attached to arabinoxylans has long remained unknown. Here, a new study shows that ferulate is transferred to arabinoxylan from a 3,6-di-O-feruloyl sucrose intermediate formed in a sucrose ferulate cycle.
This database was designed to provide a coherent, single source of NMR data of lignin and other plant cell wall model compounds. The database exists as an Adobe pdf cross-platform file for viewing and printing. This is the latest public version of the Database, version 2024/08 updated from the 2009 version.
The phenolic polymer lignin is one of the primary chemical constituents of the plant secondary cell wall. Due to the inherent plasticity of lignin biosynthesis, several phenolic monomers have been shown to be incorporated into the polymer, as long as the monomer can undergo radicalization so it can participate in coupling reactions. In this study, we significantly enhance the level of incorporation of monolignol ferulate conjugates into the lignin polymer to improve the digestibility of lignocellulosic biomass. Overexpression of a rice Feruloyl-CoA Monolignol Transferase (FMT), OsFMT1, in hybrid poplar (Populus alba x grandidentata) produced transgenic trees clearly displaying increased cell wall-bound ester-linked ferulate, p-hydroxybenzoate, and p-coumarate, all of which are in the lignin cell wall fraction, as shown by NMR and DFRC. We also demonstrate the use of a novel UV–Vis spectroscopic technique to rapidly screen plants for the presence of both ferulate and p-hydroxybenzoate esters. Lastly we show, via saccharification assays, that the OsFMT1 transgenic poplars have significantly improved processing efficiency compared to wild-type and Angelica sinensis-FMT-expressing poplars. The findings demonstrate that OsFMT1 has a broad substrate specificity and a higher catalytic efficiency compared to the previously published FMT from Angelica sinensis (AsFMT). Importantly, enhanced wood processability makes OsFMT1 a promising gene to optimize the composition of lignocellulosic biomass.
The biosynthesis of cellulose, lignin, and hemicelluloses in plant secondary cell walls (SCWs) is regulated by a hierarchical transcriptional regulatory network. Here, this network features orthologous transcription factors shared between poplar and Arabidopsis, highlighting a foundational similarity in their genetic regulation. However, knowledge on the discrepant behavior of the transcriptional-level molecular regulatory mechanisms between poplar and Arabidopsis remains limited. In this study, we investigated the function of PagMYB128 during wood formation and found it had broader impacts on SCW formation compared to its Arabidopsis ortholog, AtMYB103. Transgenic poplar trees overexpressing PagMYB128 exhibited significantly enhanced xylem development, with fiber cells and vessels displaying thicker walls, and an increase in the levels of cellulose, lignin, and hemicelluloses in the wood. In contrast, plants with dominant repression of PagMYB128 demonstrated the opposite phenotypes. RNA sequencing and reverse transcription – quantitative polymerase chain reaction showed that PagMYB128 could activate SCW biosynthetic gene expression, and chromatin immunoprecipitation along with yeast one-hybrid, and effector–reporter assays showed this regulation was direct. Further analysis revealed that PagSND1 (SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN1) directly regulates PagMYB128 but not cell wall metabolic genes, highlighting the pivotal role of PagMYB128 in the SND1-driven regulatory network for wood development, thereby creating a feedforward loop in SCW biosynthesis.
Our ability to study and valorize the lignin fraction of biomass is hampered by the fundamental and still unmet challenge of precisely quantifying native lignin's structural features. Here, we developed a rapid elevated-temperature 1 H– 13 C Heteronuclear Single-Quantum Coherence Zero (HSQC 0 ) NMR method that enables this precise quantification of native lignin structural characteristics even with whole plant cell wall (WPCW) NMR spectroscopy, overcoming fast spin relaxation in the gel phase. We also formulated a Gaussian fitting algorithm to perform automatic and reliable spectral integration. By combining HSQC 0 measurements with yield measurements following depolymerisation, we can confirm the combinatorial nature of radical coupling reactions during biosynthesis leading to a random sequential organization of linkages within a largely linear lignin chain. Such analyses illustrate how this analytical method can greatly facilitate the study of native lignin structure, which can then be used for fundamental studies or to understand lignin depolymerization methods like reductive catalytic fractionation or aldehyde-assisted fractionation.
Our ability to study and valorize the lignin fraction of biomass is hampered by the fundamental and still unmet challenge of precisely quantifying native lignin's structural features. Here, we developed a rapid elevated-temperature 1 H– 13 C Heteronuclear Single-Quantum Coherence Zero (HSQC 0 ) NMR method that enables this precise quantification of native lignin structural characteristics even with whole plant cell wall (WPCW) NMR spectroscopy, overcoming fast spin relaxation in the gel phase. We also formulated a Gaussian fitting algorithm to perform automatic and reliable spectral integration. By combining HSQC 0 measurements with yield measurements following depolymerisation, we can confirm the combinatorial nature of radical coupling reactions during biosynthesis leading to a random sequential organization of linkages within a largely linear lignin chain. Such analyses illustrate how this analytical method can greatly facilitate the study of native lignin structure, which can then be used for fundamental studies or to understand lignin depolymerization methods like reductive catalytic fractionation or aldehyde-assisted fractionation.
Volatile products from thermal decomposition of lignocellulosic biomass have been well characterized, but the solid- and liquid-phase reactions during the early stages of decomposition are largely unknown. Here the initial solid-phase biomass thermal deconstruction reactions were analyzed in situ and with high particle heating rates, delineating how these processes occur. A variety of instrumentation was used to quantify the extent and relative rates of deconstruction, demonstrating that biopolymers resist the thermally energetic conditions to differing degrees, even when ensconced in biomass cell walls. Hemicellulose and the more frangible lignin components decompose and volatilize more readily than cellulose, which temporarily enriches biomass with cellulose. These chemical changes manifest in larger cell wall structural and mechanical property transformations. In all, this investigation concludes that these solid-phase reactions strongly influence the production rates of volatile species and will require additional study before these processes can be modeled precisely to improve yields of desired product.
As we work to transition the modern society that is based on non-renewable chemical feedstocks to a post-modern society built around renewable sources of energy, fuels, and chemicals, there is a need to identify the renewable resources and processes for converting them to platform chemicals. Herein, we explore a strategy for utilizing the p-hydroxybenzoate in biomass feedstocks (e.g., poplar and palm trees) and converting it into a portfolio of commodity chemicals. The targeted bio-derived product in the first processing stage is p-hydroxybenzamide produced from p-hydroxybenzoate esters found in the plant. In the second stage a continuous reaction process converts the p-hydroxybenzamide to p-aminophenol via the Hofmann rearrangement and recovers the unreacted p-hydroxybenzamide. In the third stage the p-aminophenol can be acetylated to form paracetamol, which is readily isolated by liquid/liquid extraction at >95 % purity and an overall p-hydroxybenzamide-to-paracetamol process yield of ~90 %. We explore how utilization of protecting groups alters the challenges in this process and expands the portfolio of possible products to include p-(methoxymethoxy)aniline and N-acetyl-p-(methoxymethoxy)aniline. These target compounds could become value-added renewably-sourced platform chemicals that could be used to produce biodegradable plastics, pigments, and pharmaceuticals.
There are several methods to isolate near-native lignins, including milled-wood lignin, enzymatic lignin, cellulolytic enzyme lignin, and enzymatic mild-acidolysis lignin. Which one is the most representative of the native lignin? Herein, near-native lignins were isolated from different plant groups and structurally analyzed to determine how well these lignins represented their native lignin counterparts. Analytical methods were applied to understand the molecular weight, monomer composition, and distribution of interunit linkages in the structure of the lignins. The results indicated that either enzymatic lignin or cellulolytic enzyme lignin may be used to represent native lignin in softwoods and hardwoods. None of the lignins, however, appeared to represent native lignins in grasses (monocot plants) because of substantial syringyl/guaiacyl differences. Furthermore, complicating the understanding of grass lignin structure, large amounts of hydroxycinnamates acylate their polysaccharides and, when released, are often conflated with actual lignin monomers.
The invention relates to feruloyl-CoA:monolignol transferase enzymes and nucleic acids encoding the feruloyl-CoA:monolignol transferase enzymes. The enzymes and/or the nucleic acids enable incorporation of monolignol ferulates into the lignin of plants. The monolignol ferulates include, for example, p-coumaryl ferulate, coniferyl ferulate, and/or sinapyl ferulate.
The invention is directed to p-coumaroyl-CoA:monolignol transferase enzymes, nucleic acids encoding p-coumaroyl-CoA:monolignol transferase enzymes, and inhibitory nucleic acids adapted to inhibit the expression and/or translation of p-coumaroyl-CoA:monolignol transferase RNA; expression cassettes, plant cells, and plants that have or encode such nucleic acids and enzymes; and methods of making and using such nucleic acids, enzymes, expression cassettes, cells, and plants.
Whole genome sequencing of a pinoresinol-degrading bacterium
Novosphingobium rhizosphaerae strain LY chromosome, complete genome
Novosphingobium rhizosphaerae strain LY plasmid pLY1, complete sequence
Novosphingobium rhizosphaerae strain LY plasmid pLY2, complete sequence
Bergen Cable Technology (BCT) has introduced a new product they refer to as 'safety cable'. This product is intended as a replacement for lockwire when installed per Aerospace Standard (AS) 4536 (included in Appendix D of this document). Installation of safety cable is reportedly faster and more uniform than lockwire. NASA/GSFC proposes to use this safety cable in Shuttle Small Payloads Project (SSPP) applications on upcoming Shuttle missions. To assure that BCT safety cable will provide positive locking of fasteners equivalent to lockwire, the SSPP will conduct vibration and pull tests of the safety cable.