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All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

PET waste- and bio-derived imine vitrimers for shape-memory, intrinsic flame-retardant, and recyclable carbon fiber composites

Developing circular multifunctional vitrimers and carbon fiber–reinforced polymers (CFRPs) that are simultaneously recyclable, mechanically robust, and intrinsically flame retardant remains a major challenge. Here, in this study, we report multifunctional vitrimers and their carbon fiber–reinforced vitrimer (CFRV) composites, where the vitrimer design integrates closed-loop recyclability, enhanced interfacial adhesion, and intrinsic flame retardancy within a single materials platform. The vitrimer matrix is synthesized from post-consumer polyethylene terephthalate (PET) waste and a vanillin-derived phosphorus-containing crosslinker, forming an imine-based network. The resulting vitrimer resin exhibits high tensile strength, thermal healability, repeated reprocessability, programmable shape memory, and rapid chemical depolymerization under mild conditions. Amine-functionalized carbon fibers significantly improve fiber–matrix interfacial bonding, yielding CFRVs with tensile strengths up to 789 MPa and complete recovery of structurally intact fibers after chemical recycling. The phosphorus-rich aromatic network further imparts intrinsic flame retardancy, enabling self-extinguishing behavior without external additives. This work advances a materials design paradigm for next-generation multifunctional, sustainable vitrimers and CFRVs, while simultaneously addressing the recycling challenges associated with both plastic and CFRP waste.

Bio-derived crosslinker↗
DOE OSTI2025

MarK, a Novosphingobium aromaticivorans kinase required for catabolism of multiple aromatic monomers

The aromatic compounds used in a variety of industrial products are currently obtained from nonrenewable petroleum sources. Alternatively, the plant polymer lignin is an abundant renewable source of aromatics, and its depolymerization generates a variety of products that can include acetovanillone, a vanillin derivative containing an acetyl side chain. The Alphaproteobacterium Novosphingobium aromaticivorans DSM12444 can metabolize several chemically modified aromatics in deconstructed lignin, but not acetovanillone. In this work, adaptive laboratory evolution identified a single amino acid change in the previously uncharacterized gene product Saro_1862 that is necessary and sufficient for N. aromaticivorans growth with acetovanillone as a sole growth substrate, as well as other aromatic monomers not metabolized by wild-type cells. We show that a glutamate (E) to lysine (K) substitution at amino acid residue 16 of Saro_1862 results in a ~1600-fold increase in the rate of ATP-dependent acetovanillone phosphorylation. We also find that recombinant Saro_1862 E16K phosphorylates several other aromatic compounds in vitro , defining the first reported catalytic activity for the widespread UPF0261 protein domain contained in Saro_1862. Thus, we propose naming Saro_1862 MarK, for multiple aromatic kinase. A 1.57 Å crystal structure of MarK E16K predicts that the E16K substitution lies in a potential ATP binding site, suggesting how this amino acid change increased catalytic activity. A search for homologs of MarK and other proteins required for acetovanillone degradation predicts that this pathway for aromatic metabolism exists throughout the bacterial phylogeny.

Novosphingobium↗
DOE OSTI2022

A chiral alkali metal capped Ni 4 cubane complex: Synthesis, structure, magnetic and catalytic bromination studies

A new chiral NaO 2 Ni 4 O 2 N 2 cluster has been developed with a distorted capped cubane-type structure. The structure was determined by single crystal X-ray diffraction. The tetradentate Schiff-base ligand was synthesized by the condensation of o-vanillin and $\textit{D}$-(-)–threo-2-amino-1-(4-nitrophenyl)-1,3-propanediol. X-ray structure reveals that the complex possesses a Ni 4 cubane cluster where sodium ion is anchored on the face of the cubane structure. Continuous shape measurement studies revealed that all the Ni centres are in distorted octahedral geometry and sodium ion adopts the spherical square pyramidal geometry. Further structural exploration reveals that the complex shows local distortions of the coordination geometries at each metal site, and these distortions govern the magnetic anisotropy associated with each Ni II ion. For the present case, an elongation ($d_{str}$ > 0) of the octahedral coordination sphere results in a local easy-plane anisotropy, with a positive $D_{Ni(II)}$ parameter. Temperature dependant magnetization measurements showed a predominant ferromagnetic interaction amongst the four Ni centres. This complex is also catalytically active towards bromination of aromatic phenolic compounds. Initial catalytic activity was verified with the conversion of phenol red to bromophenol blue. Different phenolic substances show good conversion to their corresponding bromo derivatives. The complex showed good catalytic activity towards bromination of aromatic phenolic compounds, offering up to 99% conversion with 100% selectively to their corresponding bromo derivatives.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Influence of hydrocracking and ionic liquid pretreatments on composition and properties of Arabidopsis thaliana wild type and CAD mutant lignins

Lignin is the primary contributor to the high cost of biofuel-production from lignocellulosic biomass. Here, in order to study lignin removal and the release of aromatic monomers, we applied hydrocracking and ionic liquid pretreatments on Arabidopsis thaliana biomass from both wild type (WT) and a mutant (CAD cxd) defective in two cinnamyl alcohol dehydrogenase genes involved in the lignin biosynthetic pathway. For Arabidopsis WT, our results highlight that pretreatments reduce average molecular weight of lignin by about 65% and decrease the content of β-O-4 linkages between lignin monomers. For Arabidopsis CAD mutant, an opposite effect is evidenced. Fewer differences were observed on depolymerization and molecular structure of lignin, which indicates that (8-O-4), (8-5), and (8-8) linkages observed in CAD mutant make lignin more resilient to pretreatment than wild-type lignin. Finally, our study shows the potential of hydrocracking pretreatment technology for extracting valuable aldehyde monomers such as vanillin and syringaldehyde from biomass.

09 BIOMASS FUELS↗
DOE OSTI2021

Pathway discovery and engineering for cleavage of a β-1 lignin-derived biaryl compound

Lignin biosynthesis typically results in a polymer with several inter-monomer bond linkages, and the heterogeneity of linkages presents a challenge for depolymerization processes. While several enzyme classes have been shown to cleave common dimer linkages in lignin, the pathway of bacterial β-1 spirodienone linkage cleavage has not been elucidated. In this study, we identified a pathway for cleavage of 1,2-diguaiacylpropane-1,3-diol (DGPD), a β-1 linked biaryl representative of a ring-opened spirodienone linkage, in Novosphingobium aromaticivorans DSM12444. In vitro assays using cell lysates demonstrated that RS14230 (LsdE) converts DGPD to a lignostilbene intermediate, which the carotenoid oxygenase, LsdA, then converts to vanillin. A Pseudomonas putida KT2440 strain engineered with lsdEA expression catabolizes erythro-DGPD, but not threo-DGPD. We further engineered P. putida to convert DGPD to a product, cis,cis-muconic acid. Overall, this work demonstrates the potential to identify new enzymatic reactions in N. aromaticivorans and expands the biological funnel of P. putida for microbial lignin valorization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Impacts of Hydrogen Bond Donor Structures in Phenolic Aldehyde Deep Eutectic Solvents on Pretreatment Efficiency

As a green solvent for biomass processing, deep eutectic solvents (DESs) have shown effectiveness in biomass processing. Here, in this study, phenolic aldehydes with different numbers of methoxy groups, including 4-hydroxybenzaldehyde (HBA, no methoxy), vanillin (VA, monomethoxy), and syringaldehyde (SA, dimethoxy) were employed to synthesize DESs with choline chloride (ChCl). The presence of methoxy groups in the hydrogen bond donor structure affected DES properties, as well as biomass pretreatment performance. The high thermal stability of phenolic aldehyde DESs was shown with over 225 °C onset temperature. The hydrogen bond donor with one aldehyde and one hydroxyl group at the para position without a methoxy group (ChCl-HBA) showed the highest xylan removal and delignification, reaching 59.3 and 88.0%, respectively, leading to the highest enzymatic hydrolysis yield. Sonication after pretreatment further enhanced the hydrolysis yields, achieving 83.3% glucan conversion and 50.1% xylan conversion. In the lignin-rich fraction, the recovered lignin showed a low weight–average molecular weight under 2100 g/mol with a relatively uniform molecular weight dispersity below 1.5. This study provides insights into how the chemical structure of hydrogen bond donors in DESs affects biomass processing and paves the way for designing effective lignin-derived DES in future biorefinery processes.

09 BIOMASS FUELS↗
DOE OSTI2024

High-Resolution Tandem Mass Spectrometry-Based Analysis of Model Lignin–Iron Complexes: Novel Pipeline and Complex Structures

Understanding the chemical nature of soil organic carbon (SOC) with great potential to bind iron (Fe) minerals is critical for predicting the stability of SOC. Organic ligands of Fe are among the top candidates for SOCs able to strongly sorb on Fe minerals, but most of them are still molecularly uncharacterized. To shed insights into the chemical nature of organic ligands in soil and their fate, this study developed a protocol for identifying organic ligands using ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) and metabolomic tools. The protocol was used for investigating the Fe complexes formed by model compounds of lignin-derived organic ligands, namely, caffeic acid (CA), p-coumaric acid (CMA), vanillin (VNL), and cinnamic acid (CNA). Isotopologue analysis of 54/56 Fe was used to screen out the potential UHPLC-HRMS (m/z) features for complexes formed between organic ligands and Fe, with multiple features captured for CA, CMA, VNL, and CNA when 35/37 Cl isotopologue analysis was used as supplementary evidence for the complexes with Cl. MS/MS spectra, fragment analysis, and structure prediction with SIRIUS were used to annotate the structures of mono/bidentate mono/biligand complexes. The analysis determined the structures of monodentate and bidentate complexes of FeL x Cl y (L: organic ligand, x = 1–4, y = 0–3) formed by model compounds. The protocol developed in this study can be used to identify unknown organic ligands occurring in complex environmental samples and shed light on the molecular-level processes governing the stability of the SOC.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Oxidative Catalytic Fractionation of Lignocellulosic Biomass Using a Co-N-P-C Catalyst and One-Step Isolation of Aromatic Monomers via Centrifugal Partition Chromatography

Methods for catalytic fractionation of biomass provide a means to convert lignin directly into monomers while generating a high-quality cellulosic stream, contrasting conventional biomass pretreatment strategies that prioritize the cellulosic fraction. Here, a nonprecious-metal Co-N-P-C catalyst is identified for aerobic oxidative catalytic fractionation (OCF) of poplar feedstock in dimethyl carbonate that achieves significantly higher yields of aromatic monomers (24 wt %) relative to those obtained with a recently reported Co-N-C catalyst in acetone solvent (15 wt %). Mechanistic studies indicate that the acidic properties of the catalyst contribute to its improved performance by promoting extraction of lignin from insoluble polysaccharides. This OCF process is complemented by the development of a new centrifugal partition chromatography (CPC) method that supports isolation of all five major aromatic monomers (syringic acid, syringaldehyde, vanillic acid, vanillin, and para-hydroxybenzoic acid) in a single liquid–liquid extraction purification step. Furthermore, this OCF/CPC sequence has important implications for future lignin valorization efforts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Black Liquor Valorization by Using Marine Protist Thraustochytrium striatum and the Preliminary Metabolic Mechanism Study

Black liquor that contains various phenolic compounds from lignin solubilization has been the main byproduct of alkaline pretreatment of lignocellulosic biomass in the biorefinery. In this study, black liquor from alkaline pretreatment of corn stover was used as a sole carbon source for the cultivation of a marine protist, Thraustochytrium striatum. It was found that this strain can grow on black liquor and accumulate valuable products (e.g., fatty acids and carotenoids) simultaneously. Under optimal conditions (pH = 7 and NH 4 Cl = 2 g/L), the cell mass concentration reached 5.2 g/L with total aromatics decreased from 8.18 to 3.09 g/L within 7 day incubation. Fed-batch cultivation was adopted to increase the contents of total fatty acids and carotenoids to 13% and 0.24 mg/g dry cell mass, respectively. Although various compounds such as sugars and organic acids were detected in black liquor and consumed during microbial fermentation as carbon sources, lignin-derived compounds were identified as the major substrates for T. striatum fermentation. Of total aromatics consumed, monomers including p-coumaric acid, ferulic acid, vanillin, and syringaldehyde were observed to be consumed and converted while polymeric fragments were also depolymerized and degraded. Under optimal conditions, around 50% of the total aromatics was consumed. The dynamic changes of compounds in black liquor indicated that diverse metabolic processes were involved in black liquor degradation and utilization by T. striatum. A strong adaptation of T. striatum to a wide range of pH (3–9) was also observed during black liquor fermentation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Oxidative Catalytic Fractionation of Lignocellulosic Biomass under Non-alkaline Conditions

Biomass pretreatment methods are commonly used to isolate carbohydrates from biomass, but they often lead to modification, degradation, and/or low yields of lignin. Catalytic fractionation approaches provide a possible solution to these challenges by separating the polymeric sugar and lignin fractions in the presence of a catalyst that promotes cleavage of the lignin into aromatic monomers. In this work, we demonstrate an oxidative fractionation method conducted in the presence of a heterogeneous non-precious-metal Co-N-C catalyst and O 2 in acetone as the solvent. The process affords a 15 wt% yield of phenolic products bearing aldehydes (vanillin, syringaldehyde) and carboxylic acids (p-hydroxybenzoic acid, vanillic acid, syringic acid), complementing the alkylated phenols obtained from existing reductive catalytic fractionation methods. The oxygenated aromatics derived from this process have appealing features for use in polymer synthesis and/or biological funneling to value-added products, and the non-alkaline conditions associated with this process support preservation of the cellulose, which remains insoluble at reaction conditions and is recovered as a solid.

09 BIOMASS FUELS↗
DOE OSTI2021

A multiplexed nanostructure-initiator mass spectrometry (NIMS) assay for simultaneously detecting glycosyl hydrolase and lignin modifying enzyme activities

Lignocellulosic biomass is composed of three major biopolymers: cellulose, hemicellulose and lignin. Analytical tools capable of quickly detecting both glycan and lignin deconstruction are needed to support the development and characterization of efficient enzymes/enzyme cocktails. Previously we have described nanostructure-initiator mass spectrometry-based assays for the analysis of glycosyl hydrolase and most recently an assay for lignin modifying enzymes. Here we integrate these two assays into a single multiplexed assay against both classes of enzymes and use it to characterize crude commercial enzyme mixtures. Application of our multiplexed platform based on nanostructure-initiator mass spectrometry enabled us to characterize crude mixtures of laccase enzymes from fungi Agaricus bisporus (Ab) and Myceliopthora thermophila (Mt) revealing activity on both carbohydrate and aromatic substrates. Using time-series analysis we determined that crude laccase from Ab has the higher GH activity and that laccase from Mt has the higher activity against our lignin model compound. Inhibitor studies showed a significant reduction in Mt GH activity under low oxygen conditions and increased activities in the presence of vanillin (common GH inhibitor). Ultimately, this assay can help to discover mixtures of enzymes that could be incorporated into biomass pretreatments to deconstruct diverse components of lignocellulosic biomass.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Zirconium and hafnium polyhedral oligosilsesquioxane complexes – green homogeneous catalysts in the formation of bio-derived ethers via a MPV/etherification reaction cascade

The polyhedral oligosilsesquioxane complexes, {[(isobutyl) 7 Si 7 O 12 ]ZrOPr i ·(HOPr i )}2 (I), {[(cyclohexyl) 7 Si 7 O 12 ]ZrOPr i ·(HOPr i )} 2 (II), {[(isobutyl) 7 Si 7 O 12 ]HfOPr i ·(HOPr i )} 2 (III) and {[(cyclohexyl) 7 Si 7 O 12 ]HfOPr i ·(HOPr i )} 2 (IV), were synthesized in good yields from the reactions of M(OPr i ) 4 (M = Zr, Hf) with R-POSS(OH) 3 (R = isobutyl, cyclohexyl), resp. I–IV were characterized by 1 H, 13 C and 29 Si NMR spectroscopy and their dimeric solid-state structures were confirmed by X-ray analysis. I–IV catalyze the reductive etherification of 2-hydroxy- and 4-hydroxy and 2-methoxy and 4-methoxybenzaldehyde and vanillin to their respective isopropyl ethers in isopropanol as a “green” solvent and reagent. I–IV are durable and robust homogeneous catalysts operating at temperatures of 100–160 °C for days without significant loss of catalytic activity. Likewise, I–IV selectively catalyze the conversion of 5-hydroxymethylfurfural (HMF) into 2,5-bis(isopropoxymethyl)furane (BPMF), a potentially high-performance fuel additive. Similar results were achieved by using a combination of M(OPr i ) 4 and ligand R-POSS(OH) 3 as a catalyst system demonstrating the potential of this “in situ” approach for applications in biomass transformations. A tentative reaction mechanism for the reductive etherification of aldehydes catalysed by I–IV is proposed.

09 BIOMASS FUELS↗
DOE OSTI2020

Non-catalytic oxidative depolymerization of lignin in perfluorodecalin to produce phenolic monomers

We demonstrate for the first time non-catalytic, oxidative cracking with molecular oxygen (O 2 ) to depolymerize native lignin into oxygenated phenolic monomers. Maximum monomer yield of 10.5 wt% was achieved at 250 °C after only 10 min of reaction and included vanillin, syringaldehyde, vanillic acid, and syringic acid. High rates of oxidation are attributed to the use of perfluorodecalin as solvent. Perfluorodecalin is a perfluorocarbon (PFC), characterized by their chemical stability and exceptionally high solubility for O 2 . Monomer yields were typically five-fold higher in perfluorodecalin compared to solvents more commonly employed in lignin conversion, such as methanol, butanol, acetonitrile, and ethyl acetate. Phenolic monomer production in perfluorodecalin favors high temperatures and short reaction times to prevent further oxidation of the produced monomers. Lignin oil obtained under oxidative conditions in perfluorodecalin showed lower molecular weight and smaller polydispersity compared to other solvents. Increasing the reaction time further decreased the molecular weight, while increasing reaction time in an inert atmosphere increased the molecular weight of the lignin oil. High concentrations of O 2 in perfluorodecalin not only increased lignin depolymerization but suppressed undesirable condensation reactions. Depolymerization is likely initiated by thermally induced homolytic cleavage of ether linkages in lignin to form phenoxy and carbon-based radicals. These radicals bind with O 2 as a radical scavenger and further react to form phenolic monomeric products rather than repolymerizing to large oligomers. The PFC process was scaled from 5 mL to 250 mL without any loss of yield. Because most organic compounds are not soluble in perfluorodecalin, recycling is easily achieved via liquid–liquid separation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Winter warming rapidly increases carbon degradation capacities of fungal communities in tundra soil: Potential consequences on carbon stability

We report high-latitude tundra ecosystems are increasingly affected by climate warming. As an important fraction of soil microorganisms, fungi play essential roles in carbon degradation, especially the old, chemically recalcitrant carbon. However, it remains obscure how fungi respond to climate warming and whether fungi, in turn, affect carbon stability of tundra. In a 2-year winter soil warming experiment of 2°C by snow fences, we investigated responses of fungal communities to warming in the active layer of an Alaskan tundra. Although fungal community composition, revealed by the 28S rRNA gene amplicon sequencing, remained unchanged (p > .05), fungal functional gene composition, revealed by a microarray named GeoChip, was altered (p < .05). Changes in functional gene composition were linked to winter soil temperature, thaw depth, soil moisture, and gross primary productivity (canonical correlation analysis, p < .05). Specifically, relative abundances of fungal genes encoding invertase, xylose reductase and vanillin dehydrogenase significantly increased (p < .05), indicating higher carbon degradation capacities of fungal communities under warming. Accordingly, we detected changes in fungal gene networks under warming, including higher average path distance, lower average clustering coefficient and lower percentage of negative links, indicating that warming potentially changed fungal interactions. Together, our study reveals higher carbon degradation capacities of fungal communities under short-term warming and highlights the potential impacts of fungal communities on tundra ecosystem respiration, and consequently future carbon stability of high-latitude tundra.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The Catabolism of Lignin-Derived p-Methoxylated Aromatic Compounds by Rhodococcus jostii RHA1

Emergent strategies to valorize lignin, an abundant but underutilized aromatic biopolymer, include tandem processes that integrate chemical depolymerization and biological catalysis. To date, aromatic monomers from C-O bond cleavage of lignin have been converted to bioproducts, but the presence of recalcitrant C-C bonds in lignin limits the product yield. A promising chemocatalytic strategy that overcomes this limitation involves phenol methyl protection and autoxidation. Incorporating this into a tandem process requires microbial cell factories able to transform the p-methoxylated products in the resulting methylated lignin stream. In this study, we assessed the ability of Rhodococcus jostii RHA1 to catabolize the major aromatic products in a methylated lignin stream and elucidated the pathways responsible for this catabolism. RHA1 grew on a methylated pine lignin stream, catabolizing the major aromatic monomers: p-methoxybenzoate (p-MBA), veratrate, and veratraldehyde. Bioinformatic analyses suggested that a cytochrome P450, PbdA, and its cognate reductase, PbdB, are involved in p-MBA catabolism. Gene deletion studies established that both pbdA and pbdB are essential for growth on p-MBA and several derivatives. Furthermore, a deletion mutant of a candidate p-hydroxybenzoate (p-HBA) hydroxylase, ..delta..pobA, did not grow on p-HBA. Veratraldehyde and veratrate catabolism required both vanillin dehydrogenase (Vdh) and vanillate O-demethylase (VanAB), revealing previously unknown roles of these enzymes. Finally, a ..delta..pcaL strain grew on neither p-MBA nor veratrate, indicating they are catabolized through the ..beta..-ketoadipate pathway. This study expands our understanding of the bacterial catabolism of aromatic compounds and facilitates the development of biocatalysts for lignin valorization.

aromatic biopolymers↗
DOE OSTI2024

Catabolism of β-5 linked aromatics by Novosphingobium aromaticivorans

ABSTRACT Aromatic compounds are an important source of commodity chemicals traditionally produced from fossil fuels. Aromatics derived from plant lignin can potentially be converted into commodity chemicals through depolymerization followed by microbial funneling of monomers and low molecular weight oligomers. This study investigates the catabolism of the β-5 linked aromatic dimer dehydrodiconiferyl alcohol (DC-A) by the bacterium Novosphingobium aromaticivorans . We used genome-wide screens to identify candidate genes involved in DC-A catabolism. Subsequent in vivo and in vitro analyses of these candidate genes elucidated a catabolic pathway composed of four required gene products and several partially redundant dehydrogenases that convert DC-A to aromatic monomers that can be funneled into the central aromatic metabolic pathway of N. aromaticivorans . Specifically, a newly identified γ-formaldehyde lyase, PcfL, opens the phenylcoumaran ring to form a stilbene and formaldehyde. A lignostilbene dioxygenase, LsdD, then cleaves the stilbene to generate the aromatic monomers vanillin and 5-formylferulate (5-FF). We also showed that the aldehyde dehydrogenase FerD oxidizes 5-FF before it is decarboxylated by LigW, yielding ferulic acid. We found that some enzymes involved in the β-5 catabolism pathway can act on multiple substrates and that some steps in the pathway can be mediated by multiple enzymes, providing new insights into the robust flexibility of aromatic catabolism in N. aromaticivorans . A comparative genomic analysis predicted that the newly discovered β-5 aromatic catabolic pathway is common within the order Sphingomonadales. IMPORTANCE In the transition to a circular bioeconomy, the plant polymer lignin holds promise as a renewable source of industrially important aromatic chemicals. However, since lignin contains aromatic subunits joined by various chemical linkages, producing single chemical products from this polymer can be challenging. One strategy to overcome this challenge is using microbes to funnel a mixture of lignin-derived aromatics into target chemical products. This approach requires strategies to cleave the major inter-unit linkages of lignin to release monomers for funneling into valuable products. In this study, we report newly discovered aspects of a pathway by which the Novosphingobium aromaticivorans DSM12444 catabolizes aromatics joined by the second most common inter-unit linkage in lignin, the β-5 linkage. This work advances our knowledge of aromatic catabolic pathways, laying the groundwork for future metabolic engineering of this and other microbes for optimized conversion of lignin into products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

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

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.

Novosphingobium↗
DOE OSTI2022

Understanding of bacterial lignin extracellular degradation mechanisms by Pseudomonas putida KT2440 via secretomic analysis

Abstract Background Bacterial lignin degradation is believed to be primarily achieved by a secreted enzyme system. Effects of such extracellular enzyme systems on lignin structural changes and degradation pathways are still not clearly understood, which remains as a bottleneck in the bacterial lignin bioconversion process. Results This study investigated lignin degradation using an isolated secretome secreted by Pseudomonas putida KT2440 that grew on glucose as the only carbon source. Enzyme assays revealed that the secretome harbored oxidase and peroxidase/Mn 2+ -peroxidase capacity and reached the highest activity at 120 h of the fermentation time. The degradation rate of alkali lignin was found to be only 8.1% by oxidases, but increased to 14.5% with the activation of peroxidase/Mn 2+ -peroxidase. Gas chromatography–mass spectrometry (GC–MS) and two-dimensional 1 H– 13 C heteronuclear single-quantum coherence (HSQC) NMR analysis revealed that the oxidases exhibited strong C–C bond ( β-β , β -5, and β -1) cleavage. The activation of peroxidases enhanced lignin degradation by stimulating C–O bond ( β -O-4) cleavage, resulting in increased yields of aromatic monomers and dimers. Further mass spectrometry-based quantitative proteomics measurements comprehensively identified different groups of enzymes particularly oxidoreductases in P. putida secretome, including reductases, peroxidases, monooxygenases, dioxygenases, oxidases, and dehydrogenases, potentially contributed to the lignin degradation process. Conclusions Overall, we discovered that bacterial extracellular degradation of alkali lignin to vanillin, vanillic acid, and other lignin-derived aromatics involved a series of oxidative cleavage, catalyzed by active DyP-type peroxidase, multicopper oxidase, and other accessory enzymes. These results will guide further metabolic engineering design to improve the efficiency of lignin bioconversion. Graphical Abstract

09 BIOMASS FUELS↗