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At least 19 records

All sources NASA NTRS DOE OSTI

DOE OSTI2022

Identification and characterization of a set of monocot BAHD monolignol transferases

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 important “monomers” for lignification, in which they are incorporated into the growing lignin polymer chain. p-Coumaroyl-CoA monolignol transferases (PMTs) increase the production of monolignol p-coumarates, and feruloyl-CoA monolignol transferases (FMTs) catalyze the production of monolignol ferulate conjugates. We identified putative FMT and PMT enzymes in sorghum (Sorghum bicolor) and switchgrass (Panicum virgatum) and have compared their activities to those of known monolignol transferases. The putative FMT enzymes produced both monolignol ferulate and monolignol p-coumarate conjugates, whereas the putative PMT enzymes produced monolignol p-coumarate conjugates. Enzyme activity measurements revealed that the putative FMT enzymes are not as efficient as the rice (Oryza sativa) control OsFMT enzyme under the conditions tested, but the SbPMT enzyme is as active as the control OsPMT enzyme. These putative FMTs and PMTs were transformed into Arabidopsis (Arabidopsis thaliana) to test their activities and abilities to biosynthesize monolignol conjugates for lignification in planta. The presence of ferulates and p-coumarates on the lignin of these transformants indicated that the putative FMTs and PMTs act as functional feruloyl-CoA and p-coumaroyl-CoA monolignol transferases within plants.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Manipulation of Lignin Monomer Composition Combined with the Introduction of Monolignol Conjugate Biosynthesis Leads to Synergistic Changes in Lignin Structure

Abstract 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 the polymer is derived almost entirely from a single monomer. In this study, we investigated changes to lignin structure and digestibility in Arabidopsis thaliana in near-single-monolignol transgenics and mutants and determined whether novel monolignol conjugates, produced by a FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) or a p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT), could be integrated into these novel polymers to further improve saccharification efficiency. Monolignol conjugates, including a new conjugate of interest, p-coumaryl p-coumarate, were successfully integrated into high-H, high-G and high-S lignins in A. thaliana. Regardless of lignin composition, FMT- and PMT-expressing plants produced monolignol ferulates and monolignol p-coumarates, respectively, and incorporated them into their lignin. Through the production and incorporation of monolignol conjugates into near-single-monolignol lignins, we demonstrated that substrate availability, rather than monolignol transferase substrate preference, is the most important determining factor in the production of monolignol conjugates, and lignin composition helps dictate cell wall digestibility.

2D-HSQC-NMR↗

DOE OSTI2021

Coupling of Flavonoid Initiation Sites with Monolignols Studied by Density Functional Theory

Lignin recalcitrance presents a challenge for the development of a bioeconomy that employs lignocellulosic feedstocks. The efficiency of lignin deconstruction is improved by a reduction of molecular weight, and given the discovery that flavones serve as initiation sites in lignin biosynthesis, these molecular weight reductions could potentially be achieved with plant metabolic engineering to over-express flavonoids. Upon increasing the flavonoid content in lignin, the bond strengths and properties of flavonoid-monolignol linkages become increasingly important. To that end, the current work applies density functional theory calculations to elucidate the bond dissociation enthalpies (BDEs) of flavonoid-monolignol linkages, including dimers with oxidized monolignols. Specifically, the dimer bond strengths and monomer hydrogen abstraction energies for the flavonoids tricin, chrysoeriol, luteolin, apigenin, catechin, epicatechin, epigallocatechin, and epigallocatechin gallate are calculated, when coupled to seven natural and engineered monolignols. Results indicate that 4'-O-ß linkage strengths between flavonoids and monolignols are of comparable strength to inter-monolignol ß-O-4 linkages, with average flavonoid-monolignol BDEs of 70.7 kcal/mol relative to ~69.3 kcal/mol in analogous canonical monolignols. Epigallocatechin yielded the lowest 4'-O-ß bond strength of 52.3 kcal/mol when coupled to an oxidized monolignol, while the flavones overall produced lower average BDEs, relative to the flavanols. Substituents at the 3'-C and 5'-C positions on flavonoids affected the dimer linkage strengths to a greater extent than glycosylation or substituents further from the linkage. Erythro and threo stereochemistry across the flavonoid-monolignol linkage library exhibited only small energetic differences and no pronounced correlations. Taken together, the predictions from this work support the concept that higher concentrations of flavonoid initiation sites in lignin may afford linkage properties conducive to more facile lignin depolymerization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

Stacking AsFMT overexpression with BdPMT loss of function enhances monolignol ferulate production in Brachypodium distachyon

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 as follows: (1) The presence of an active FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) enzyme throughout monolignol production; (2) Suppression or elimination of enzymatic pathways competing for monolignols and intermediates during lignin biosynthesis; and (3) Exclusion of alternative phenolic compounds that participate in lignification. A 16-fold increase in lignin-bound ML-FA incorporation was observed by introducing an AsFMT gene into Brachypodium distachyon. On its own, knocking out the native p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (BdPMT) pathway that competes for monolignols and the p-coumaroyl-CoA intermediate did not change ML-FA incorporation, nor did partial loss of CINNAMOYL-CoA REDUCTASE1 (CCR1) function, which reduced metabolic flux to monolignols. However, stacking AsFMT into the Bdpmt-1 mutant resulted in a 32-fold increase in ML-FA incorporation into lignin over the wild-type level.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Simultaneous suppression of lignin, tricin and wall‐bound phenolic biosynthesis via the expression of monolignol 4‐ O ‐methyltransferases in rice

Summary Grass lignocelluloses feature complex compositions and structures. In addition to the presence of conventional lignin units from monolignols, acylated monolignols and flavonoid tricin also incorporate into lignin polymer; moreover, hydroxycinnamates, particularly ferulate, cross‐link arabinoxylan chains with each other and/or with lignin polymers. These structural complexities make grass lignocellulosics difficult to optimize for effective agro‐industrial applications. In the present study, we assess the applications of two engineered monolignol 4‐ O ‐methyltransferases (MOMTs) in modifying rice lignocellulosic properties. Two MOMTs confer regiospecific para ‐methylation of monolignols but with different catalytic preferences. The expression of MOMTs in rice resulted in differential but drastic suppression of lignin deposition, showing more than 50% decrease in guaiacyl lignin and up to an 90% reduction in syringyl lignin in transgenic lines. Moreover, the levels of arabinoxylan‐bound ferulate were reduced by up to 50%, and the levels of tricin in lignin fraction were also substantially reduced. Concomitantly, up to 11 μmol/g of the methanol‐extractable 4‐ O ‐methylated ferulic acid and 5–7 μmol/g 4‐ O ‐methylated sinapic acid were accumulated in MOMT transgenic lines. Both MOMTs in vitro displayed discernible substrate promiscuity towards a range of phenolics in addition to the dominant substrate monolignols, which partially explains their broad effects on grass phenolic biosynthesis. The cell wall structural and compositional changes resulted in up to 30% increase in saccharification yield of the de‐starched rice straw biomass after diluted acid‐pretreatment. These results demonstrate an effective strategy to tailor complex grass cell walls to generate improved cellulosic feedstocks for the fermentable sugar‐based production of biofuel and bio‐chemicals.

4-O-methylated↗

DOE OSTI2021

p HBMT1, a BAHD-family monolignol acyltransferase, mediates lignin acylation in poplar

Abstract 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 translation system and found five enzymes capable of producing monolignol–p-hydroxybenzoates. We then compared the transcript abundance of the five corresponding genes with p-hydroxybenzoate concentrations using naturally occurring unrelated genotypes of P. trichocarpa and revealed a positive correlation between the expression of p-hydroxybenzoyl-CoA monolig-nol transferase (pHBMT1, Potri.001G448000) and p-hydroxybenzoate levels. To test whether pHBMT1 is responsible for the biosynthesis of monolignol–p-hydroxybenzoates, we overexpressed pHBMT1 in hybrid poplar (Populus alba × P. grandidentata) (35S::pHBMT1 and C4H::pHBMT1). Using three complementary analytical methods, we showed that there was an increase in soluble monolignol–p-hydroxybenzoates and cell-wall-bound monolignol–p-hydroxybenzoates in the poplar transgenics. As these pendent groups are ester-linked, saponification releases p-hydroxybenzoate, a precursor to parabens that are used in pharmaceuticals and cosmetics. This identified gene could therefore be used to engineer lignocellulosic biomass with increased value for emerging biorefinery strategies.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Data for Simultaneous Suppression of Lignin, Tricin and Wall-Bound Phenolic Biosynthesis via the Expression of Monolignol 4- O -Methyltransferases in Rice

Grass lignocelluloses feature complex compositions and structures. In addition to the presence of conventional lignin units from monolignols, acylated monolignols and flavonoid tricin also incorporate into lignin polymer; moreover, hydroxycinnamates, particularly ferulate, cross-link arabinoxylan chains with each other and/or with lignin polymers. These structural complexities make grass lignocellulosics difficult to optimize for effective agro-industrial applications. In the present study, we assess the applications of two engineered monolignol 4- O -methyltransferases (MOMTs) in modifying rice lignocellulosic properties. Two MOMTs confer regiospecific para-methylation of monolignols but with different catalytic preferences. The expression of MOMTs in rice resulted in differential but drastic suppression of lignin deposition, showing more than 50% decrease in guaiacyl lignin and up to an 90% reduction in syringyl lignin in transgenic lines. Moreover, the levels of arabinoxylan-bound ferulate were reduced by up to 50%, and the levels of tricin in lignin fraction were also substantially reduced. Concomitantly, up to 11 μmol/g of the methanol-extractable 4- O -methylated ferulic acid and 5–7 μmol/g 4- O -methylated sinapic acid were accumulated in MOMT transgenic lines. Both MOMTs in vitro displayed discernible substrate promiscuity towards a range of phenolics in addition to the dominant substrate monolignols, which partially explains their broad effects on grass phenolic biosynthesis. The cell wall structural and compositional changes resulted in up to 30% increase in saccharification yield of the de-starched rice straw biomass after diluted acid-pretreatment. These results demonstrate an effective strategy to tailor complex grass cell walls to generate improved cellulosic feedstocks for the fermentable sugar-based production of biofuel and bio-chemicals.

Biomass Analytics↗

DOE OSTI2022

Biocatalytic system for comparatively assessing the functional association of monolignol cytochrome P450 monooxygenases with their redox partners

Lignin is a complex heterogenous polymer derived from oxidative radical polymerization of three monolignols, i.e., p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. These lignin monomeric precursors structurally differ in their methoxy groups of the benzene rings. In phenylpropanoid-monolignol biosynthetic pathway, the endoplasmic reticulum (ER)-resident cytochrome P450 monooxygenases, cinnamate 4-hydroxylase, coumaroyl ester 3´-hydroxylase and ferulate 5-hydroxylase, establish the key structural characteristics of monolignols. The catalysis of cytochrome P450 monooxygenase requires reducing power, which is supplied by the ER electron transfer chains, composed of cytochrome P450 oxidoreductase (CPR), cytochrome b5 reductase (CBR) and/or cytochrome b5 protein (CB5), from cofactor NADPH or NADH. While NADPH-dependent CPR serves as the typical electron donor for most P450 enzymes, in some cases, the CBR-CB5 or CPR-CB5 electron transfer system also transfers electrons to the terminal P450 enzymes. There are tremendous studies focusing on the discovery and characterization of cytochrome P450 monooxygenases. However, very limited attention has been paid to the versatility and the roles of electron transfer components in the P450 catalytic system. Due to the membrane-residence property of both P450 enzymes and electron transfer components, it is challenging to establish an effective experimental system to evaluate the functional association of P450s with their redox partners. This chapter describes a yeast cell biocatalytic system and the related experimental procedures for comparatively assessing the functional relationship of monolignol biosynthetic P450 enzymes and different redox partners in their catalysis.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Biomimetic oxidative copolymerization of hydroxystilbenes and monolignols

Hydroxystilbenes are a class of polyphenolic compounds that behave as lignin monomers participating in radical coupling reactions during the lignification. Here, we report the synthesis and characterization of various artificial copolymers of monolignols and hydroxystilbenes, as well as low-molecular-mass compounds, to obtain the mechanistic insights into their incorporation into the lignin polymer. Integrating the hydroxystilbenes, resveratrol and piceatannol, into monolignol polymerization in vitro, using horseradish peroxidase to generate phenolic radicals, produced synthetic lignins [dehydrogenation polymers (DHPs)]. Copolymerization of hydroxystilbenes with monolignols, especially sinapyl alcohol, by in vitro peroxidases notably improved the reactivity of monolignols and resulted in substantial yields of synthetic lignin polymers. The resulting DHPs were analyzed using two-dimensional NMR and 19 synthesized model compounds to confirm the presence of hydroxystilbene structures in the lignin polymer. The cross-coupled DHPs confirmed both resveratrol and piceatannol as authentic monomers participating in the oxidative radical coupling reactions during polymerization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

Feruloyl-CoA:monolignol transferases

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.

Ralph, John↗

DOE OSTI2022

P-coumaroyl-CoA:monolignol transferase

The invention relates to nucleic acids encoding a p-coumaroyl-CoA:monolignol transferase and to inhibitory nucleic acids adapted to inhibit the expression and/or translation of a p-coumaroyl-CoA:monolignol transferase RNA. Inhibition of p-coumaroyl-CoA:monolignol transferase in plants improves the incorporation of monolignol ferulates into the lignin of plants, giving rise to plant biomass that is more easily processed into useful products such as paper and biofuels.

Wilkerson, Curtis↗

DOE OSTI2023

Feruloyl-CoA:monolignol transferases

Ralph, John↗

DOE OSTI2020

Lignin Monomers from beyond the Canonical Monolignol Biosynthetic Pathway: Another Brick in the Wall

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 as genuine lignin monomers in many plants, as is the case of the monolignol ester conjugates, phenolic compounds arising from the truncated biosynthesis of monolignols, or ferulate esters. Recent investigations, however, have indicated that phenolic compounds arising from beyond the canonical phenylpropanoid pathway, namely flavonoids, hydroxystilbenes, and hydroxycinnamic amides, may also behave as authentic lignin monomers and are incorporated into the lignin in some plants, further challenging the traditional definition of lignin. This is the case of the flavone tricin that is incorporated into the lignin of grasses and other monocots, the hydroxystilbene piceatannol (together with resveratrol and isorhapontigenin, at lower levels) that has been found in the lignins of palm fruit shells, their respective O-glucosides (astringin, piceid, and isorhapontin) that are present in the lignin of Norway spruce bark, or the ferulic amides feruloyltyramine, incorporated into the lignin of tobacco and potato tubers, and diferuloylputrescine, which appears to be incorporated into maize kernel lignin. These valuable compounds are potentially available in high amounts and at low cost and may be obtained from the waste products from the processing of agricultural or forest biomass.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

Monolignol Benzoates Incorporate into the Lignin of Transgenic Populus trichocarpa Depleted in C3H and C4H

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 high-value commodity chemical feedstocks. Benzoate (BA) conjugates are important metabolites in plants that have not been adequately characterized as components of the lignin structure. In addition to finding trace levels of BA conjugates in wild-type (WT) Populus trichocarpa, these become significantly augmented in strategic triple transgenics. Genes for three key cytochrome P450 enzymes, two 4-hydroxylases (PtrC4H1 and PtrC4H2), and one 3-hydroxylase (PtrC3H3), were downregulated to produce the transgenic plants in which monolignol benzoate (ML-BA) conjugates were incorporated into their lignin at a level some 16-fold higher than in WT. The co-downregulation of PtrC4H1/PtrC4H2/PtrC3H3 genes may cause a rerouting or modification of the pathway due to the interaction of the benzoate biosynthetic pathway with the conventional pathway for monolignol biosynthesis. The total lignin content in the transgenics was decreased by 50%, and the lignin H-unit level was elevated some 70-fold. The wood from the transgenic trees showed reduced recalcitrance toward enzymatic saccharification, which has value for the conversion of plant biomass to bioenergy.

2D NMR↗

DOE OSTI2021

Monolignol acyltransferase for lignin p-hydroxybenzoylation in Populus

Plant lignification exhibits significant plasticity. Lignin in many species including Populus spp. has long been known decorated with p-hydroxybenzoates. However, the molecular basis for such structural modification remains undetermined. Here we report the identification and characterization of a Populus BAHD family acyltransferase that catalyzes monolignol p-hydroxybenzoylation, thus controlling the formation of p-hydroxybenzoylated lignin structures. We reveal that Populus acyltransferase PHBMT1 kinetically preferentially utilizes p-hydroxybenzoyl-CoA to acylate syringyl lignin monomer sinapyl alcohol in vitro. Consistently, disrupting PHBMT1 in Populus via CRISPR/Cas9 gene editing nearly completely depletes p-hydroxybenzoates of stem lignin; conversely, overexpression of PHBMT1 enhances stem lignin p-hydroxybenzoylation, suggesting PHBMT1 functions as a prime monolignol p-hydroxybenzoyltransferase in planta. Altering lignin p-hydroxybenzoylation significantly changes lignin solvent dissolution rate, indictive of its structural significance on lignin physiochemical properties. Identification of monolignol p-hydroxybenzoyltransferase offers a valuable tool for tailoring lignin structure and physiochemical properties and for engineering the industrially important platform chemical in woody biomass.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Disruption of p -coumaroyl-CoA:monolignol transferases in rice drastically alters lignin composition

Grasses are abundant feedstocks that can supply lignocellulosic biomass for production of cell-wall-derived chemicals. In grass cell walls, lignin is acylated with p-coumarate. These p-coumarate decorations arise from the incorporation of monolignol p-coumarate conjugates during lignification. A previous biochemical study identified a rice (Oryza sativa) BAHD acyltransferase (AT) with p-coumaroyl-CoA:monolignol transferase (PMT) activity in vitro. In this study, we determined that that enzyme, which we name OsPMT1 (also known as OsAT4), and the closely related OsPMT2 (OsAT3) harbor similar catalytic activity toward monolignols. We generated rice mutants deficient in either or both OsPMT1 and OsPMT2 by CRISPR/Cas9-mediated mutagenesis and subjected the mutants’ cell walls to analysis using chemical and nuclear magnetic resonance methods. Our results demonstrated that OsPMT1 and OsPMT2 both function in lignin p-coumaroylation in the major vegetative tissues of rice. Notably, lignin-bound p-coumarate units were undetectable in the ospmt1 ospmt2-2 double-knockout mutant. Further, in-depth structural analysis of purified lignins from the ospmt1 ospmt2-2 mutant compared with control lignins from wild-type rice revealed stark changes in polymer structures, including alterations in syringyl/guaiacyl aromatic unit ratios and inter-monomeric linkage patterns, and increased molecular weights. Furthermore, our results provide insights into lignin polymerization in grasses that will be useful for the optimization of bioengineering approaches for the effective use of biomass in biorefineries.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Synthesis of hydroxycinnamoyl shikimates and their role in monolignol biosynthesis

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 (C 3 H) from p-coumaric acid or its CoA thioester, was revealed to be via the action of coumaroyl shikimate 3'-hydroxylase (C 3 'H) utilizing p-coumaroyl shikimate as the substrate, itself derived from p-coumaroyl-CoA via hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyltransferase (HCT). The same HCT was conjectured to convert the product, caffeoyl shikimate, to caffeoyl-CoA to continue on the pathway starting with its 3-O-methylation. At least in some plants, however, a more recently discovered caffeoyl shikimate esterase (CSE) enzyme hydrolyzes caffeoyl shikimate to caffeic acid from which it must again produce its CoA thioester to continue on the monolignol biosynthetic pathway. HCT and CSE are therefore monolignol biosynthetic pathway enzymes that have provided new opportunities to misregulate lignification. To facilitate studies into the action and substrate specificity of C 3 H/C 3 'H, HCT, and CSE enzymes, as well as for metabolite authentication and for enzyme characterization, including kinetics, a source of authentic substrates and products was required. A synthetic scheme starting from commercially available shikimic acid and the four key hydroxycinnamic acids (p-coumaric, caffeic, ferulic, and sinapic acid) has been developed to provide this set of hydroxycinnamoyl shikimates for researchers.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

P-coumaroyl-CoA:monolignol transferases

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.

Ralph, John↗