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Results for “lignin-derived compound characterization”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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18 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Energy-resolved mass spectrometry as a tool for identification of lignin depolymerization products

Lignin is the largest source of bio-based aromatic compounds in nature, and its valorization is essential to the sustainability of lignocellulosic biorefining. Characterizing lignin-derived compounds remains challenging due to the heterogeneity of this biopolymer. Tandem mass spectrometry is a promising tool for lignin structural analytics, as fragmentation patterns of model compounds can be extrapolated to identify characteristic moieties in complex samples. This work extends previous resonance excitation type collision-induced dissociation (CID) methods that identified lignin oligomers containing β-Ο-4, β-5, and β-β bonds, to also identify characteristics of 5-5, β-1, and 4-Ο-5 dimers, enabled by quadrupole time-of-flight (QTOF) CID with energy-resolved mass spectrometry (ERMS). Overall, beam type ERMS offers in-depth structural information and could ultimately contribute to tools for high-throughput lignin dimer identification.

09 BIOMASS FUELS↗
DOE OSTI2023

Biochemical Process Modeling and Simulation (BPMS)

The Biochemical Process Modeling and Simulation project aims to reduce the cost and time of research by applying theory, modeling, and simulation to the most relevant bottlenecks in the biochemical process. We use molecular modeling, quantum mechanics, metabolic modeling, fluid dynamics, and reaction-diffusion methods in close collaboration with pretreatment, hydrolysis, upgrading, and TEA. The project's outcomes are increased yields and efficiency of the biochemical process, added value to products, and reduced price of fuels by specifically targeting catalytic efficiency, reactor design, enzyme efficiency, and microbial design. We work closely with experimental projects to identify problems and iterate with experiments to find and refine solutions. By working with experimentalists, we decide on problems that can be solved with simulation that could otherwise not be solved or would take too long with experiment alone to reach BETO's targets. Over the years, we have produced solutions that have resulted in determining the most likely fatty-acid derivative for passive transport out of bacteria that upgrade biomass, and we have also designed enzyme mutations for enhanced lignin upgrading. Metabolic models have been developed to tune the activity of 2,3 butanediol production for the 2030 target. A computational method to deliver understanding of how complex omics data can be interpreted in the metabolic pathways of organisms used in the Agile Biofoundry. We have found methods to overcome specific barriers and continue to develop those methods. Our reactor studies have guided the design of both the microbes and reactors for aerobic and micro-aerobic production at all scales and have been instrumental in improving the accuracy of techno-economic analysis models. This project is essential in the process of selecting the final processes for 2030 SAF production targets. More specifically, recently, we have: 1) Predicted the strength of the basic structural interactions in commodity plastics to provide guidance for plastics upcycling strategies. 2) Developed computational tool to improve the characterization of lignin-derived compounds 3) Developed new methodologies to enable Machine Learning-based Directed Evolution for protein engineering. 4) Developed Machine Learning methods to predict protein promiscuity and mutations to further improve microbial and enzymatic driven processes and demonstrated the utility of ML approaches to engineering proteins from sparse experimental datasets. 5) Developed new methods to enable high-fidelity simulation of aerobic fermentation at industrial scale and resolving mismatch of time scales through subcycling/operator splitting 7) Identified the difficulty in preventing local high-oxygen conditions in industrial bubble columns, which leads to less-desirable acetoin production, suggesting future research directions in alternative reactor configurations (e.g loop reactors, shallow-channel reactors).

BIOMASS FUELS↗
DOE OSTI2020

MassIVE MSV000085948 - Utilization of syringyl lignin-derived compounds and production of 2-pyrone-4,6-dicarboxylic acid by Pseudomonas putida KT2440: Lignin is an abundant aromatic polymer formed in plants by coupling reactions of p-coumaryl, coniferyl, and sinapyl alcohols. Pseudomonas putida KT2440 (P. putida KT2440), a robust soil bacterium, naturally catabolizes many aromatic compounds derived from lignin as carbon and energy sources and has been extensively engineered to valorize guaiacyl (G) and p-hydroxyphenyl (H) lignin-derived aromatic species to valuable chemicals. However, only a few studies report the capacity of Pseudomonads to grow on syringyl (S) lignin-derived compounds. In this work, we demonstrate that P. putida KT2440 catabolizes syringate in the presence of an auxiliary energy source. Overexpression of vanAB, encoding a native Rieske non-heme iron monooxygenase, enabled catabolism of syringate and syringaldehyde without an additional carbon and energy source. In vitro biochemical characterization of VanAB confirmed that this enzyme O-demethylates syringate to 3-O-methylgallate and, subsequently, 3-O-methylgallate to gallate, albeit 4- and 55-fold less efficiently than vanillate, respectively. Transcriptomics and proteomics experiments demonstrate that the galADBC transcripts and enzymes required for gallate catabolism are induced when vanAB is overexpressed during cultivation in syringate. Further, we show that that the endogenous protocatechuate-3,4-dioxygenase PcaHG ring-opens gallate to produce 2-pyrone-4,6-dicarboxylic acid (PDC), a promising bio-based chemical. Constitutive overexpression of vanAB and pcaHG along with galA deletion enabled PDC production from SA at a 70% (mol/mol) yield. Furthermore, leveraging the LigAB dioxygenase from Sphingobium sp. SYK-6 and VanAB O-demethylase from Pseudomonas sp. HR199 enabled simultaneous conversion of monomers with S-, G-, and H-type functionality to PDC at 82% (mol/mol) yield. Overall, this study elucidates a native pathway for catabolism of syringyl lignin-derived compounds in P. putida KT2440 and demonstrates the potential of this robust host for biological funneling of substrates derived from all three canonical monolignols.

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Beckham, Gregg↗
DOE OSTI2020

Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic–catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could pote

biological funneling↗
DOE OSTI2022

Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p -coumarate

We report Pseudomonas putida KT2440 is a well-studied bacterium for the conversion of lignin-derived aromatic compounds to bioproducts. The development of advanced genetic tools in P. putida has reduced the turnaround time for hypothesis testing and enabled the construction of strains capable of producing various products of interest. Here, we evaluate an inducible CRISPR-interference (CRISPRi) toolset on fluorescent, essential, and metabolic targets. Nuclease-deficient Cas9 (dCas9) expressed with the arabinose (8K)-inducible promoter was shown to be tightly regulated across various media conditions and when targeting essential genes. In addition to bulk growth data, single cell time lapse microscopy was conducted, which revealed intrinsic heterogeneity in knockdown rate within an isoclonal population. The dynamics of knockdown were studied across genomic targets in exponentially-growing cells, revealing a universal 1.75 ± 0.38 hour quiescent phase after induction where 1.5 ± 0.35 doublings occur before a phenotypic response is observed. To demonstrate application of this CRISPRi toolset, β-ketoadipate, a monomer for performance-advantaged nylon, was produced at a 4.39 ± 0.5 g/L and yield of 0.76 ± 0.10 mol/mol from p-coumarate, a hydroxycinnamic acid that can be derived from grasses. These cultivation metrics were achieved by using the higher strength IPTG (1K)-inducible promoter to knockdown the pcaIJ operon in the βKA pathway during early exponential phase. This allowed the majority of the carbon to be shunted into the desired product while eliminating the need for a supplemental carbon and energy source to support growth and maintenance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Laboratory evolution in Novosphingobium aromaticivorans enables rapid catabolism of a model lignin-derived aromatic dimer

Lignin contains a variety of interunit linkages, leading to a range of potential decomposition products that can be used as carbon and energy sources by microbes. β-O-4 linkages are the most common in native lignin, and associated catabolic pathways have been well characterized. However, the fate of the mono-aromatic intermediates that result from β-O-4 dimer cleavage has not been fully elucidated. Here, we used experimental evolution to identify mutant strains of Novosphingobium aromaticivorans with improved catabolism of a model aromatic dimer containing a β-O-4 linkage, guaiacylglycerol-β-guaiacyl ether (GGE). We identified several parallel causal mutations, including a single nucleotide polymorphism in the promoter of an uncharacterized gene that roughly doubled the growth yield with GGE. We characterized the associated enzyme and demonstrated that it oxidizes an intermediate in GGE catabolism, β-hydroxypropiovanillone, to vanilloyl acetaldehyde. Identification of this enzyme and its key role in GGE catabolism furthers our understanding of catabolic pathways for lignin-derived aromatic compounds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation

Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon–carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro -1,2-diguaiacylpropane-1,3-diol ( erythro -DGPD), a ring-opened lignin-derived β-1 dimer, was reported in Novosphingobium aromaticivorans . The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro -DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C 1 reaction product, and we further demonstrated that both enantiomers of erythro -DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.

09 BIOMASS FUELS↗
DOE OSTI2025

A distinct subpopulation of membrane vesicles in Pseudomonas putida is enriched in enzymes for lignin catabolism

Bacterial membrane vesicles (MVs) mediate diverse microbial processes and are emerging as powerful biomedical tools, but MV population heterogeneity remains an open question. Here, we separate, enumerate, and characterize two MV populations from the soil bacterium Pseudomonas putida during growth with or without lignin-derived carbon, a major carbon source from plant cells in the rhizosphere. Small MVs (MV-S, diameter ~100 nm) were produced from all cultures, whereas large MVs (MV-L, diameter ~300 nm) were observed during the late stationary phase of lignin cultivations. MV-S contained selectively packaged proteins with diverse physiological functions, whereas the MV-L proteome was smaller and largely enriched in outer membrane proteins. Interestingly, enzymes known to mediate the catabolism of lignin-derived aromatic compounds were enriched in MV-S. Overall, this study highlights the need for careful consideration of MV populations in microbial systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

The Development of Catalysts for Upgrading of Pyrolysis Vapor for Refinery Feedstocks and Intermediates (CRADA Final Report)

Catalytic fast pyrolysis (CFP) is a versatile technology platform to convert biomass into fungible hydrocarbon transportation fuels and chemical co-products. Key technical barriers to reaching this goal include increasing the product yields and achieving the desired fuel properties for gasoline, diesel, and jet range fuels or blendstocks that would be suitable for introduction into existing refinery unit operations. Overcoming these barriers will require durable catalysts that are effective at upgrading and stabilizing biomass pyrolysis vapors. Towards these goals, this CRADA leveraged NREL experience as a leader in biomass pyrolysis research and Johnson Matthey's (JM) experience as a leader in the production of advanced catalytic materials. The scope spanned CFP catalyst development, characterization, multi-scale reaction testing, and computational modeling. CRADA benefits to DOE, Participant, and U.S. Taxpayer: Assists laboratory in achieving programmatic scope, Uses the laboratory’s core competencies. The purpose of this CRADA was to develop and deploy catalysts for biomass CFP to help achieve cost-competitive biofuels and bio-based products. This was accomplished through a close collaboration between biomass conversion researchers at NREL and catalyst development researchers at JM. Summary of Research Results: Focus Area 1. Foundational research on catalytic conversion and deactivation: Key interactions between pyrolysis vapors and heterogeneous catalysts were probed through catalyst characterization, model compound reaction testing, and atomistic-scale computational modeling. Catalyst development focused on multifunctional materials, which include zeolites, oxides, carbides, and nitrides. Computational modeling identified reaction mechanisms and elucidated surface chemistry to test hypotheses regarding mechanisms of deoxygenation, coupling, cracking, dehydration, coke formation, hydrogen transfer, and aromatic ring reactions. This information was used to design multifunctional catalysts to increase product yields, control product selectivity, and reduce deactivation during CFP and downstream processing steps. The results served to increase fundamental understanding of key catalyst attributes and durability features for the upgrading of biomass pyrolysis vapors. Model compound experiments confirmed the importance of metal-acid bifunctionality for the deoxygenation of lignin-derived phenolic species under hydrodeoxygenation conditions. This insight led to the development of catalysts such as Pt/TiO2 and Mo2C, which were confirmed as high-performing materials during subsequent bench-scale experiments using biomass-derived pyrolysis vapors. This focus area also led to the identification of important catalyst deactivation mechanisms associated with the deposition of inorganic contaminants such as potassium. The molecular-level insight from model compound experiments and computational modeling, shown in Figure 1, informed the development of regeneration procedures that have been shown to be effective for restoration of > 90% of initial catalyst activity. This understanding has subsequently been translated to other catalyst systems, including zeolite materials that can be operated without requirements for co-fed hydrogen.

09 BIOMASS FUELS↗
DOE OSTI2024

Co-hydrotreatment of Bio-oil and Waste Cooking Oil to Produce Transportation Fuels

This paper reports the co-hydrotreatment of the heavy bio-oil fraction with waste cooking oil (WCO) using NiMo/γ-Al 2 O 3 catalyst, followed by the distillation of resulting deoxygenated oil and the characterization of resulting fuel cuts. The heavy BTG bio-oil fraction was obtained by removing the very reactive light-oxygenated compounds via rotary evaporation, subsequently mixed with 1-butanol. The resulting oil was blended with WCO and subjected to a two-step co-hydrotreatment process. The first step, called “stabilization,” is aimed at saturating highly reactive hydrogen-deficient compounds. The second step, called “deoxygenation,” aimed to remove bio-oil oxygen, primarily as H 2 O. This study examined the impact of varying bio-oil concentrations (0, 10, 20, 30, 40 wt.% of WCO) on the upgraded oil's yield, composition, and fuel properties. The resulting hydrotreated oil was distilled into gasoline-range, kerosene-range, and diesel-range hydrocarbons at <150 °C, 150 to 250 °C, and 250 to 350 °C, respectively. The yield of the hydrotreated oil indicates that as the bio-oil concentration increases, the amounts of coke (0.7 to 2.4 %) and water (2 to 10 wt. %) increase while the organic layer yields decrease (80 to 63 %). The coke yield was comparable to the coke yield obtained when co-processing the pyrolytic lignin fraction. This suggests that coke is formed from both the sugar oligomers and the lignin-derived oligomers. The UV-fluorescence analysis on the hydrotreated oil shows that more polycondensed and conjugated ring compounds formed as the bio-oil concentration is increased. These compounds are precursors of coke. FTIR results showed that most raw materials were converted to biofuels after the hydrotreatment. To achieve less than 1 wt. % of coke yield, blends with up to 20 wt. % pyrolysis oil should be used. An increase in bio-oil concentration leads to a slight increase in gasoline yield and a decrease in kerosene and diesel yields. The identified carbon species found in the fuel cuts include n-paraffin, iso-paraffin, cycloparaffin, and aromatics. Further, the jet fuel cut (kerosene) was characterized by density, surface tension, and viscosity. Our product conforms to the standard specifications for sustainable aviation fuels (Jet A-1). Further research is suggested to fine-tune the operating parameters for achieving reduced coke yield and enhanced kerosene yield.

09 BIOMASS FUELS↗
DOE OSTI2022

Molecular Characterization of Water-Soluble Brown Carbon Chromophores in Snowpack from Northern Xinjiang, China

This study reports molecular-level characterization of brown carbon (BrC) attributed to water-soluble organic carbon in six snowpack samples collected from northern Xinjiang, China. Here, the molecular composition and light-absorbing properties of BrC chromophores were unraveled by application of high-performance liquid chromatography (HPLC) coupled to a photodiode array (PDA) detector and high-resolution mass spectrometry. The chromophores were classified into five major types, that is, (1) phenolic/lignin-derived compounds, (2) flavonoids, (3) nitroaromatics, (4) oxygenated aromatics, and (5) other chromophores. Identified chromophores account for ~23–64% of the total light absorption measured by the PDA detector in the wavelength range of 300–370 nm. In the representative samples from urban and remote areas, oxygenated aromatics and nitroaromatics dominate the absorption in the wavelengths below and above 320 nm, respectively. The highly polluted urban sample shows the most complex HPLC-PDA chromatogram, and more other chromophores contribute to the bulk absorption. Phenolic/ lignin-derived compounds are the most light-absorbing species in the soil-influenced sample. Chromophores in two remote samples exhibit ultraviolet–visible features distinct from other samples, which are attributed to flavonoids. Identification of individual chromophores and quantitative analysis of their optical properties are helpful for elucidating the roles of BrC in snow radiative balance and photochemistry.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Antimicrobial Properties of Corn Stover Lignin Fractions Derived from Catalytic Transfer Hydrogenolysis in Supercritical Ethanol with a Ru/C Catalyst

Converting lignin to value-added products at high yields provides an avenue for making ethanol biorefineries more profitable while reducing the carbon footprint of products generally derived from petroleum. In this study, corn stover lignin was depolymerized by catalytic transfer hydrogenolysis (CTH) in supercritical ethanol with a Ru/C catalyst. The lignin-derived bio-oil was then sequentially extracted utilizing hexane, petroleum ether, chloroform, and ethyl acetate as solvents in order of less polar to polar, and the subsequent bio-oils were characterized using GPC, GC/MS, and HSQC NMR. In this work, results show that the monomers in the bio-oil fractions contained primarily alkylated phenols, hydrogenated hydroxycinnamic acid derivatives, syringol and guaiacol-type lignins created from reductive cleavages of ether linkages, which were sequentially extracted into groups depending on the solvent polarity. The antimicrobial properties of the bio-oils were screened against Gram-positive (Bacillus subtilis, Lactobacillus amylovorus, and Staphylococcus epidermidis) and Gram-negative (Escherichia coli) bacteria and yeast (Saccharomyces cerevisiae) by examining microbial growth inhibition. Results show that CTH-derived bio-oils inhibited all tested organisms at concentrations less than 3 mg/mL. Total monomer concentration and the presence of specific monomers (i.e., syringyl propane) showed correlations to antimicrobial activity, likely due to cell death or membrane damage. This study provides insights into using sequential extraction to fractionate lignin-derived compounds and correlations between the properties of the extracted compounds and their antimicrobial activity.

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

In-depth understanding of molecular mechanisms of aldehyde toxicity to engineer robust Saccharomyces cerevisiae

Aldehydes are ubiquitous electrophilic compounds that ferment microorganisms including Saccharomyces cerevisiae encounter during the fermentation processes to produce food, fuels, chemicals, and pharmaceuticals. Aldehydes pose severe toxicity to the growth and metabolism of the S. cerevisiae through a variety of toxic molecular mechanisms, predominantly via damaging macromolecules and hampering the production of targeted compounds. Compounds with aldehyde functional groups are far more toxic to S. cerevisiae than all other functional classes, and toxic potency depends on physicochemical characteristics of aldehydes. The yeast synthetic biology community established a design–build–test–learn framework to develop S. cerevisiae cell factories to valorize the sustainable and renewable biomass, including the lignin-derived substrates. However, thermochemically pretreated biomass-derived substrate streams contain diverse aldehydes (e.g., glycolaldehyde and furfural), and biological conversions routes of lignocellulosic compounds consist of toxic aldehyde intermediates (e.g., formaldehyde and methylglyoxal), and some of the high-value targeted products have aldehyde functional group (e.g., vanillin and benzaldehyde). Numerous studies comprehensively characterized both single and additive effects of aldehyde toxicity via systems biology investigations, and novel molecular approaches have been discovered to overcome the aldehyde toxicity. Based on those novel approaches, researchers successfully developed synthetic yeast cell factories to convert lignocellulosic substrates to valuable products, including aldehyde compounds. Here, we highlight the salient relationship of physicochemical charac- teristics and molecular toxicity of aldehydes, the molecular detoxification and macromolecules protection mechanisms of aldehydes, and the advances of engineering robust S. cerevisiae against complex mixtures of aldehyde inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Discovery, characterization, and metabolic engineering of Rieske non-heme iron monooxygenases for guaiacol O-demethylation

Aryl-O-demethylation is a common rate-limiting step in the catabolism of lignin-related compounds, including guaiacol. In this work, we used randomly barcoded transposon insertion sequencing (RB-TnSeq) in the bacterium Novosphingobium aromaticivorans to identify a Rieske-type guaiacol O-demethylase, GdmA. Similarity searches identified GdmA homologs in other bacteria, along with candidate reductase partners, denoted GdmB. GdmAB combinations were biochemically characterized for activity with several lignin-related substrates. Structural and sequence comparisons of vanillate- and guaiacol-specific O-demethylase active sites revealed conserved hallmarks of substrate specificity. GdmAB combinations were also evaluated in Pseudomonas putida KT2440, which does not natively utilize guaiacol. GdmAB from Cupriavidus necator N-1 demonstrated the highest rate of guaiacol turnover in vitro and in engineered P. putida strains and notably higher catalytic efficiency than a cytochrome P450 system (GcoAB) and the vanillate Rieske-type O-demethylase from P. putida (VanAB). The GdmAB O-demethylases described here expand the suite of options for microbial conversion of a model lignin-derived substrate.

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

Lignin-Derived Chemicals from Pulping Liquors: Cooperative Research and Development CRADA Number CRD-17-00714 (Final Report)

National Renewable Energy Laboratory (NREL) has developed a robust portfolio for lignin valorization using a “biological funneling” approach, which adopts a microbial process to convert the inherent heterogeneity of lignin-rich streams into single high-value products. In parallel, Sustainable Fiber Technologies (SFT) has developed a proprietary, high-pH process to convert non-woody biomass into valuable pulp for myriad biomaterial applications. Therefore, by combining the expertise of both SFT and NREL, this project aimed to valorize co-products from an industrial pulping process of non-woody feedstocks. A detailed analytical characterization of an SFT pulping stream was used to aid the development of a bench-scale separations strategy to isolate a mixture of low molecular weight compounds, and ultimately to aid in bioreactor cultivations used to produce polyhydroxyalkanoate (PHA) biopolymers at titers, rates, and yields necessary for moving towards commercialization.

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

Elucidation of Aromatic Catabolic Pathways in White-Rot Fungi

This project aims to investigate the hypothesis that white-rot fungi can simultaneously depolymerize lignin extracellularly and catabolize depolymerization products intracellularly as carbon and energy sources. Evaluating this hypothesis will provide deeper understanding of the role of white-rot fungi in facilitating carbon sequestration in Nature. Additionally, identifying the most promising fungal strains for lignin turnover and catabolism will catalyze future efforts in genetic tool development to enable metabolic engineering in white-rot fungi for lignin bioconversion to bioproducts. Lignin is the second most abundant plant-based biopolymer on Earth and represents up to 40% of the energy density of lignocellulosic biomass. Even though lignin is a massive natural carbon and energy reservoir, only a small group of basidiomycete fungi, namely white-rot fungi (WRF), have evolved the ability to efficiently depolymerize and mineralize lignin to CO2 and H2O. Considerable research efforts have been undertaken to understand how WRF depolymerize lignin but the biochemical reactions that convert lignin into CO2 have been largely neglected. In fact, it is unclear if WRF intracellularly catabolize lignin-derived aromatic compounds to utilize them as a carbon and energy source, or rather if lignin is depolymerized and mineralized extracellularly merely to facilitate access to cellulose and hemicellulose for use as a primary carbon source. To date, we have employed 13C-isotope labeling, systems biology approaches, and in vitro enzyme assays to definitively demonstrate that two WRF, Trametes versicolor and Gelatoporia (Ceriporiopsis) subvermispora, funnel carbon from lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways [1]. Specifically, 13C-isotopic labeling approaches showed that these WRF utilize poplar-derived aromatic compounds (e.g. 4-hydroxybenzoic acid (4-HBA)) as a carbon source. In silico genome analysis led us to hypothesize a complete catabolic pathway for 4-HBA and identify multiple homologous sequences for enzymes with putative oxidative decarboxylase, hydroxylase, and ring-opening dioxygenase activities, which are among the main biochemical reactions acting on aromatic compounds. Spatial and differential proteomic and metabolomic analyses supported the proposed catabolic pathways and showed alternative catabolic steps in T. versicolor that were not present in G. subvermispora. Based on the in silico, proteomics, and transcriptomics results, we down-selected enzymes for further in vitro characterization, and we have assigned a function to six fungal enzymes (including oxidative decarboxylases, hydroxylases, and ring-opening dioxygenases). Interestingly, even though we selected homologous enzyme pairs from both WRF with similar -omics trends, in a few cases only one of the studied fungi showed activity for the proposed substrate. Based on all the observations from this study [1], we hypothesized that 4-HBA preferentially undergoes oxidative decarboxylation to hydroquinone and subsequent hydroxylation to 1,2,4-benzenetriol in G. subvermispora before ring cleavage, whereas 4-HBA would preferentially undergo hydroxylation to protocatechuate and further oxidative decarboxylation to 1,2,4-benzenetriol in T. versicolor. Examining additional decarboxylases and hydroxylases as well as enzymes from other protein families that can perform the same or similar oxidative reactions, such as cytochromes P450 with aromatic hydroxylation activity, will also be key for elucidating enzyme preferences for specific substrates. Overall, this work forms the foundation of a new research area based on lignin catabolism by WRF, which could be further exploited to convert the undervalued biopolymer lignin into value-added compounds.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗
DOE OSTI2022

Lignin Utilization

Valorization of lignin has massive potential economic and sustainability benefits for the lignocellulosic biorefinery. However, challenges remain to realize lignin conversion to coproducts, especially related to selective, high yield depolymerization to monomers and quantitative analytics on lignin, the latter of which is critical for accurate process modeling. Towards these goals, the Lignin Utilization project focuses on catalytic lignin deconstruction chemistries for C-O and C-C bond cleavage, development of analytical chemistry techniques to quantitatively characterize lignin, and syntheses of requisite model compounds for understanding lignin transformations. This work is done with and supports multiple BETO projects, including the Biological Lignin Valorization (BLV) project, the Separations Consortium, and others. Outcomes of Lignin Utilization for analytics and synthesis include 1) development of new mass spectrometry (MS) methods to characterize lignin dimers/oligomers in process streams, 2) deployment of a computational-experimental tool (with Biochemical Process Modeling and Simulation) to identify lignin-derived compounds with high fidelity from MS, and 3) delivery of >40 unique compounds. From a catalysis perspective, we have developed new oxidative approaches to cleave C-O and C-C bonds and produce >50% bio-available aromatic monomers for biological funneling for the BLV project and have developed recoverable bases for base-catalyzed deconstruction of lignin.

BIOMASS FUELS↗
DOE OSTI2023

Characterizing and Engineering Trehalose Biosynthesis Pathways to Improve Acetate Tolerance in Pseudomonas putida KT2440

Alkaline pretreatment can depolymerize lignin into a diverse mixture of monomers and smaller compounds, enabling bioconversion of these compounds to value-added chemicals in engineered bacterial hosts such as Pseudomonas putida KT2440. Acetate and salts comprise approximately 8% and 32% of the lignin stream, respectively. High concentrations of these components can cause cell death in P. putida, so lignin-derived streams must be fed in lower amounts to decrease stress. Investigation of the mechanisms of bacterial tolerance to chemicals in lignin-rich substrates is therefore required to overcome this obstacle in lignin bioconversion. Previous work has shown that trehalose, an endogenously produced disaccharide, is involved in tolerance to heat, cold, and osmotic stress, as well as implicated in acid stress in other bacteria. As such, trehalose biosynthesis presents a useful target to increase acetate and osmotic tolerance in P. putida. This work investigates the native TreSA, TreSB, and TreY/TreZ trehalose biosynthesis pathways in this bacterial strain as well as the OtsAB pathway of Sphingobium sp. SYK-6. Overexpression of the OtsAB pathway in P. putida increased tolerance to acetate relative to wild-type. Additionally, functional knockouts of treSA, treSB, treY, and treZ in P. putida showed that interruption of any trehalose biosynthesis pathway, and, in the case of TreY/TreZ, interruption of the pathway at any point, significantly hindered growth in acetate as well as on glucose and LB broth. Each trehalose biosynthesis pathway was then engineered for overexpression in P. putida to determine whether overexpression of these genes conferred additional tolerance to acetate. The TreSA, TreSB, OtsA, and OtsB enzymes were also expressed and purified to determine the reaction rates and kinetics of these pathways. The hypothetical glycoside hydrolase family 15 protein, encoded in the gene located in between otsB and otsA in the Sphingobium operon, was purified as well to investigate its potential involvement in the OtsAB pathway. The findings of this work illuminated the mechanisms of trehalose biosynthesis in P. putida and identified genetic targets to improve strain tolerance to common components of alkaline-pretreated lignin streams. These findings could ultimately improve yields of desired products from lignin in engineered strains of P. putida.

alkaline pretreatment↗