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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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At least 595 records · Page 33
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Efficient production of oxidized terpenoids via engineering fusion proteins of terpene synthase and cytochrome P450

The functionalization of terpenes using cytochrome P450 enzymes is a versatile route to the production of useful derivatives that can be further converted to value-added products. Many terpenes are hydrophobic and volatile making their availability as a substrate for P450 enzymes significantly limited during microbial production. In this study, we developed a strategy to improve the accessibility of terpene molecules for the P450 reaction by linking terpene synthase and P450 together. As a model system, fusion proteins of 1,8-cineole synthase (CS) and P450 cin were investigated and it showed an improved hydroxylation of the monoterpenoid 1,8-cineole up to 5.4-fold. Structural analysis of the CS-P450 cin fusion proteins by SEC-SAXS indicated a dimer formation with preferred orientations of the active sites of the two domains. We also applied the enzyme fusion strategy to the oxidation of a sesquiterpene epi-isozizaene and the fusion enzymes significantly improved albaflavenol production in engineered E. coli. From the analysis of positive and negative examples of the fusion strategy, we proposed key factors in structure-based prediction and evaluation of fusion enzymes. Developing fusion enzymes for terpene synthase and P450 presents an efficient strategy toward oxidation of hydrophobic terpene compounds. This strategy could be widely applicable to improve the biosynthetic titer of the functionalized products from hydrophobic terpene intermediates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Photobiocatalysis for Abiological Transformations

Harnessing biocatalysts for novel abiological transformations is a longstanding goal of synthetic chemistry. Combining the merits of biocatalysis and photocatalysis allows for selective transformations fueled by visible light and offers many advantages including new reactivity, high enantioselectivity, greener syntheses, and high yields. Photoinduced electron or energy transfer enables synthetic methodologies that complement conventional two electron processes or offer orthogonal pathways for developing new reactions. Enzymes are well suited and can be tuned by directed evolution to exert control over radical intermediates thereby suppressing undesirable reactions and delivering high chemo- and stereoselectivities. Within the past decade, the combination of biocatalysis and photocatalysis was mainly focused on exploiting light-regenerated cofactors to increase native enzymatic activity. However, recent developments have demonstrated that the combination can unlock new-to-nature chemistry. Particularly, the discovery and application of new strategies are well poised to expand the applications of photobiocatalysis. In the past three years, our lab has been studying the combinations of photocatalysis and biocatalysis which can be applied to create new synthetic methodologies and solve challenges in synthetic organic chemistry. Our efforts have expanded the strategies for combining external photocatalysts with enzymes through the construction of a synergistic cooperative stereoconvergent reduction system consisting of photosensitized energy transfer and ene-reductase catalyzed alkene reduction. Additionally, our efforts have also extended the capability of cofactor-dependent photoenzymatic systems to include enantioselective bimolecular radical hydroalkylations of alkenes by irradiating electron donor-acceptor complexes comprised of enzymatic redox active cofactors and unnatural substrates. In this Account, we highlight strategies developed by our group and others for combining biocatalysis and photocatalysis with the aim of introducing nonnatural reactivity to enzymes. Presently, strategies applied to achieve this goal include the repurposing of newly discovered photoactive enzymes, the elucidation of new photoreactivity within cofactor-dependent enzymes, the combination of external photocatalysts with enzymes, and the construction of artificial photoenzymes. By demonstrating the successful application of these strategies for achieving selective new-to-nature transformations, we hope to spur interest in the expanding the scope of photobiocatalytic systems through the use and extension of these strategies and creation of new strategies. In addition, we hope to elucidate the intuition in synergizing the unique capabilities of biocatalysis and photocatalysis so that photobiocatalysis can be recognized as a potential solution to difficult challenges in synthetic organic chemistry.

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

Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber

The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa, and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 h in nylon bags within the rumen of cannulated dairy cows. Across a gene catalog covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of hemicelluloses, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo- and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8, and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryote-selected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks of in situ recalcitrant fiber deconstruction, and importantly, suggest that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.

Hagen, Live↗
DOE OSTI2022

Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase

Abstract Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN + ) provide enhanced scalability for biomanufacturing. However, engineering enzymes to accept NCBs is difficult. Here, we establish a growth selection platform to evolve enzymes to utilize NMN + -based reducing power. This is based on an orthogonal, NMN + -dependent glycolytic pathway in Escherichia coli which can be coupled to any reciprocal enzyme to recycle the ensuing reduced NMN + . With a throughput of >10 6 variants per iteration, the growth selection discovers a Lactobacillus pentosus NADH oxidase variant with ~10-fold increase in NMNH catalytic efficiency and enhanced activity for other NCBs. Molecular modeling and experimental validation suggest that instead of directly contacting NCBs, the mutations optimize the enzyme’s global conformational dynamics to resemble the WT with the native cofactor bound. Restoring the enzyme’s access to catalytically competent conformation states via deep navigation of protein sequence space with high-throughput evolution provides a universal route to engineer NCB-dependent enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Cross-kingdom comparative genomics reveal the metabolic potential of fungi for lignin turnover in deadwood

Deadwood is a major carbon source in forests, and yet the fate of this carbon remains a gap in our understanding of global carbon cycling. Lignin, the most recalcitrant biopolymer in wood, is mainly decayed through extracellular enzymatic and chemical processes initiated by white-rot fungi. However, the intracellular conversion of lignin decay products has been overlooked in the fungal kingdom. Here we integrate comparative genomic and phylogenetic analyses to understand the distribution and evolution of enzymes responsible for modifying lignin-related aromatic compounds—such as decarboxylases, hydroxylases, dioxygenases and other downstream ring-cleavage enzymes—that funnel carbon to central metabolism across the bacterial and the fungal kingdoms. We demonstrate that specific fungal lineages conserve these enzyme families, and that the abilities to enzymatically depolymerize lignin and catabolize lignin-related aromatic compounds are not necessarily coupled. Our analyses also reveal an expanded substrate specificity of aromatic ring-cleavage enzymes during fungal evolution, as well as a clade of extracellular enzymes among them, broadening the spatial range of these biochemical capabilities. Together, our results highlight a large diversity of fungal enzymes and hosts that warrant further investigation for inclusion into carbon cycling models and biotechnological applications for the conversion of aromatic compounds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Stereoselective synthesis of a 4-α-glucoside of valienamine and its X-ray structure in complex with Streptomyces coelicolor GlgE1-V279S

Glycoside hydrolases (GH) are a large family of hydrolytic enzymes found in all domains of life. As such, they control a plethora of normal and pathogenic biological functions. Thus, understanding selective inhibition of GH enzymes at the atomic level can lead to the identification of new classes of therapeutics. In these studies, we identified a 4-α-glucoside of valienamine (8) as an inhibitor of Streptomyces coelicolor (Sco) GlgE1-V279S which belongs to the GH13 Carbohydrate Active EnZyme family. The results obtained from the dose–response experiments show that 8 at a concentration of 1000 µM reduced the enzyme activity of Sco GlgE1-V279S by 65%. The synthetic route to 8 and a closely related 4-α-glucoside of validamine (7) was achieved starting from readily available D-maltose. A key step in the synthesis was a chelation-controlled addition of vinylmagnesium bromide to a maltose-derived enone intermediate. X-ray structures of both 7 and 8 in complex with Sco GlgE1-V279S were solved to resolutions of 1.75 and 1.83 Å, respectively. Structural analysis revealed the valienamine derivative 8 binds the enzyme in an E 2 conformation for the cyclohexene fragment. Also, the cyclohexene fragment shows a new hydrogen-bonding contact from the pseudo-diaxial C(3)–OH to the catalytic nucleophile Asp 394 at the enzyme active site. Asp 394, in fact, forms a bidentate interaction with both the C(3)–OH and C(7)-OH of the inhibitor. In contrast, compound 7 disrupts the catalytic sidechain interaction network of Sco GlgE1-V279S via steric interactions resulting in a conformation change in Asp 394. These findings will have implications for the design other aminocarbasugar-based GH13-inhibitors and will be useful for identifying more potent and selective inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A pyridoxal 5'-phosphate-dependent Mannich cyclase

Pyridoxal 5'-phosphate (PLP)-dependent enzymes catalyse a diverse range of chemical transformations. Despite their extraordinary functional diversity, no PLP-dependent enzyme is known to catalyse Mannich-type reactions, an important carbon–carbon bond-forming reaction in synthetic organic chemistry. Here we report the discovery of a biosynthetic enzyme LolT, a PLP-dependent enzyme catalysing a stereoselective intramolecular Mannich reaction to construct the pyrrolizidine core scaffold in loline alkaloids. Importantly, its versatile catalytic activity is harnessed for stereoselective synthesis of a variety of conformationally constrained α,α-disubstituted α-amino acids, which bear vicinal quaternary–tertiary stereocentres and various aza(bi)cyclic backbones, such as indolizidine, quinolizidine, pyrrolidine and piperidine. Furthermore, crystallographic and mutagenesis analysis and computational studies together provided mechanistic insights and structural basis for understanding LolT’s catalytic activity and stereoselectivity. As a whole, this work expands the biocatalytic repertoire of carbon–carbon bond-forming enzymes and increases our knowledge of the catalytic versatility of PLP-dependent enzymes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Pyruvate, phosphate dikinase regulatory protein impacts light response of C4 photosynthesis in Setaria viridis

In C4 plants, the pyruvate (Pyr), phosphate dikinase regulatory protein (PDRP) regulates the activity of the C4 pathway enzyme Pyr, phosphate dikinase (PPDK) in a light-/dark-dependent manner. The importance of this regulatory action to C4 pathway function and overall C4 photosynthesis is unknown. To resolve this question, we assessed in vivo PPDK phospho-regulation and whole leaf photophysiology in a CRISPR–Cas9 PDRP knockout (KO) mutant of the NADP-ME C4 grass green millet (Setaria viridis). PDRP enzyme activity was undetectable in leaf extracts from PDRP KO lines. Likewise, PPDK phosphorylated at the PDRP-regulatory Thr residue was immunologically undetectable in leaf extracts. PPDK enzyme activity in rapid leaf extracts was constitutively high in the PDRP KO lines, irrespective of light or dark pretreatment of leaves. Gas exchange analysis of net CO2 assimilation revealed PDRP KO leaves had markedly slower light induction kinetics when leaves transition from dark to high-light or low-light to high-light. In the initial 30 min of the light induction phase, KO leaves had an ~15% lower net CO2 assimilation rate versus the wild-type (WT). Despite the impaired slower induction kinetics, we found growth and vigor of the KO lines to be visibly indistinguishable from the WT when grown in normal air and under standard growth chamber conditions. However, the PDRP KO plants grown under a fluctuating light regime exhibited a gradual multi-day decline in Fv/Fm, indicative of progressive photosystem II damage due to the absence of PDRP. Collectively, our results demonstrate that one of PDRP’s functions in C4 photosynthesis is to ensure optimal photosynthetic light induction kinetics during dynamic changes in incident light.

Plant Sciences↗
DOE OSTI2021

Crystal structures of a dodecameric multicopper oxidase from Marinithermus hydrothermalis

We report multicopper oxidases (MCOs) represent a diverse family of enzymes that catalyze the oxidation of either an organic or a metal substrate with concomitant reduction of dioxygen to water. These enzymes contain variable numbers of cupredoxin domains, two, three or six per subunit, and rely on four copper ions, a single type I copper and three additional copper ions organized in a trinuclear cluster (TNC), with one type II and two type III copper ions, to catalyze the reaction. Here, two crystal structures and the enzymatic characterization of Marinithermus hydrothermalis MCO, a two-domain enzyme, are reported. This enzyme decolorizes Congo Red dye at 70°C in the presence of high halide concentrations and may therefore be useful in the detoxification of industrial waste that contains dyes. In two distinct crystal structures, MhMCO forms the trimers seen in other two-domain MCOs, but differs from these enzymes in that four trimers interact to create a dodecamer. This dodecamer of MhMCO forms a closed ball-like structure and has implications for the sequestration of bound divalent metal ions as well as substrate accessibility. In each subunit of the dodecameric structures, a Trp residue, Trp351, located between the type I and TNC sites exists in two distinct conformations, consistent with a potential role in facilitating electron transfer in the enzyme.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Integrating continuous hypermutation with high–throughput screening for optimization of cis,cis –muconic acid production in yeast

Directed evolution is a powerful method to optimize proteins and metabolic reactions towards user-defined goals. It usually involves subjecting genes or pathways to iterative rounds of mutagenesis, selection and amplification. While powerful, systematic searches through large sequence-spaces is a labour-intensive task, and can be further limited by a priori knowledge about the optimal initial search space, and/or limits in terms of screening throughput. Here, we demonstrate an integrated directed evolution workflow for metabolic pathway enzymes that continuously generate enzyme variants using the recently developed orthogonal replication system, OrthoRep and screens for optimal performance in high-throughput using a transcription factor-based biosensor. We demonstrate the strengths of this workflow by evolving a rate-limiting enzymatic reaction of the biosynthetic pathway for cis,cis-muconic acid (CCM), a precursor used for bioplastic and coatings, in Saccharomyces cerevisiae. After two weeks of simply iterating between passaging of cells to generate variant enzymes via OrthoRep and high-throughput sorting of best-performing variants using a transcription factor-based biosensor for CCM, we ultimately identified variant enzymes improving CCM titers > 13-fold compared with reference enzymes. Taken together, the combination of synthetic biology tools as adopted in this study is an efficient approach to debottleneck repetitive workflows associated with directed evolution of metabolic enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Effects of cold stress on metabolic regulation in the overwintering larvae of the Chinese white pine beetle, Dendroctonus armandi

Abstract The Chinese white pine beetle, Dendroctonus armandi Tsai & Li (Coleoptera: Curculionidae, Scolytinae), is considered the most destructive forest pest in the Qinling and Bashan Mountains of China. In recent years, winter temperature has dropped in these regions, and extremely low temperatures are hard to survive for insects. Cold hardiness becomes a crucial strategy because temperature change often leads to fluctuations in insect abundance, and the metabolism rate is a key index of resistance to cold in overwintering insects. Therefore, we investigated the relationship between the change in respiratory rate and the activity of metabolism‐related mitochondrial enzymes in D. armandi larvae under cold conditions. We found that the respiratory rate decreased, and it was matched with the activity of glutamate dehydrogenase, aconitase, and lipase during overwintering. Among the various test times under cold conditions, the respiratory rate also decreased with decreasing temperature and increased under very low temperatures. At all cold stress periods, glutamate dehydrogenase and lipase showed increased activity at higher temperatures and decreased activity under lower temperatures, but the activity of NAD‐malic enzyme, NADP‐malic enzyme, mitochondrial isocitrate dehydrogenase, and aconitase were contrary. Under all low temperatures, the activity of enzymes – except for NADP‐malic enzyme, glutamate dehydrogenase, and lipase – increased in short‐term cold stress and decreased in long‐term cold stress at 4, 0, −4, −6, −8, and −10 °C. However, at −2 °C, the activity of enzymes showed a decreasing trend in short‐term treatments and an increasing trend in long‐term treatments, except for mitochondrial isocitrate dehydrogenase. The results not only improve our understanding of the metabolic mechanism of cold adaptation in D. armandi , but also provide an important experimental basis for further study and biological pest control.

Fu, Danyang↗
DOE OSTI2022

The dimeric form of bacterial l ‐asparaginase YpAI is fully active

l ‐asparaginases from mesophilic bacteria (ASNases), including two enzymes very successfully used in the treatment of leukaemia, have been consistently described as homotetramers. On the contrary, structural studies show that homodimers of these enzymes should be sufficient to carry out the catalytic reaction. In this report, we investigated whether the type I Yersinia pestis asparaginase (YpAI) is active in a dimeric form or whether the tetrameric quaternary structure is critical for its activity. Using multiple biophysical techniques that investigate enzymatic properties and quaternary structure at either high or low protein concentration, we found that dimeric YpAI is fully active, suggesting that the tetrameric form of this subfamily of enzymes does not bear significant enzymatic relevance. In this process, we extensively characterized YpAI, showing that it is a cooperative enzyme, although the mechanism of allostery is still not definitely established. We showed that, like most type I ASNases, the substrate affinity of YpAI is low and this enzyme is very similar in terms of both the structure and enzymatic properties to homologous type I ASNase from Escherichia coli (EcAI). We extended these studies to more medically relevant type II ASNases, used as anti‐leukaemia drugs. We confirmed that type II ASNases are not allosteric, and that they might also be functional in a dimeric form. However, the determination of the accurate tetramer⇆dimer dissociation constants of these enzymes that most likely lie in the picomolar range is not possible with currently available biophysical techniques.

Biochemistry & Molecular Biology↗
DOE OSTI2020

NfoR: Chromate Reductase or Flavin Mononucleotide Reductase?

Soil bacteria can detoxify Cr(VI) ions by reduction. Within the last 2 decades, numerous reports of chromate reductase enzymes have been published. These reports describe catalytic reduction of chromate ions by specific enzymes. These enzymes each have sequence similarity to known redox-active flavoproteins. We investigated the enzyme NfoR from Staphylococcus aureus, which was reported to be upregulated in chromate-rich soils and to have chromate reductase activity (H. Han, Z. Ling, T. Zhou, R. Xu, et al., Sci Rep 7:15481, 2017, https://doi.org/10.1038/s41598-017-15588-y). We show that NfoR has structural similarity to known flavin mononucleotide (FMN) reductases and reduces FMN as a substrate. NfoR binds FMN with a dissociation constant of 0.4 μM. The enzyme then binds NADPH with a dissociation constant of 140 μM and reduces the flavin at a rate of 1,350 s -1 . Turnover of the enzyme is apparently limited by the rate of product release that occurs, with a net rate constant of 0.45 s -1 . The rate of product release limits the rate of observed chromate reduction, so the net rate of chromate reduction by NfoR is orders of magnitude lower than when this process occurs in solution. We propose that NfoR is an FMN reductase and that the criterion required to define chromate reduction as enzymatic has not been met. That NfoR expression is increased in the presence of chromate suggests that the survival adaption was to increase the net rate of chromate reduction by facile, adventitious redox processes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Phylogenetics-based identification and characterization of a superior 2,3-butanediol dehydrogenase for Zymomonas mobilis expression

Abstract Background Zymomonas mobilis has recently been shown to be capable of producing the valuable platform biochemical, 2,3-butanediol (2,3-BDO). Despite this capability, the production of high titers of 2,3-BDO is restricted by several physiological parameters. One such bottleneck involves the conversion of acetoin to 2,3-BDO, a step catalyzed by 2,3-butanediol dehydrogenase (Bdh). Several Bdh enzymes have been successfully expressed in Z. mobilis, although a highly active enzyme is yet to be identified for expression in this host. Here, we report the application of a phylogenetic approach to identify and characterize a superior Bdh, followed by validation of its structural attributes using a mutagenesis approach. Results Of the 11 distinct bdh genes that were expressed in Z. mobilis, crude extracts expressing Serratia marcescens Bdh ( Sm Bdh) were found to have the highest activity (8.89 µmol/min/mg), when compared to other Bdh enzymes (0.34–2.87 µmol/min/mg). The Sm Bdh crystal structure was determined through crystallization with cofactor (NAD + ) and substrate (acetoin) molecules bound in the active site. Active Sm Bdh was shown to be a tetramer with the active site populated by a Gln247 residue contributed by the diagonally opposite subunit. Sm Bdh showed a more extensive supporting hydrogen-bond network in comparison to the other well-studied Bdh enzymes, which enables improved substrate positioning and substrate specificity. This protein also contains a short α6 helix, which provides more efficient entry and exit of molecules from the active site, thereby contributing to enhanced substrate turnover. Extending the α6 helix to mimic the lower activity Enterobacter cloacae ( Ec Bdh) enzyme resulted in reduction of Sm Bdh function to nearly 3% of the total activity. In great contrast, reduction of the corresponding α6 helix of the Ec Bdh to mimic the Sm Bdh structure resulted in ~ 70% increase in its activity. Conclusions This study has demonstrated that Sm Bdh is superior to other Bdhs for expression in Z. mobilis for 2,3-BDO production. Sm Bdh possesses unique structural features that confer biochemical advantage to this protein. While coordinated active site formation is a unique structural characteristic of this tetrameric complex, the smaller α6 helix and extended hydrogen network contribute towards improved activity and substrate promiscuity of the enzyme.

09 BIOMASS FUELS↗
DOE OSTI2026

Leafweb: Leaf Gas Exchange and Pulse-Amplitude Modulated Fluorometry for C4 Species, June 2026 Release

This dataset contains leaf gas exchange and Pulse-Amplitude Modulated (PAM) fluorometry for 98 C4 species. The C4 photosynthetic pathway employs specialized CO2 concentration mechanisms and Kranz anatomy to enrich CO2 concentration around Rubisco, the enzyme that catalyzes carbon fixation in the Calvin-Benson cycle to suppress photorespiration and increase the use efficiencies of light, nitrogen, and water as compared to the C3 photosynthetic pathways. Large-scale C4 photosynthetic datasets are relatively scarce, which has affected C4 photosynthesis research. To improve C4 photosynthetic data availability, Leafweb organized an effort to systematically collect, compile, standardize, and organize measurements of leaf gas exchange and/or Pulse-Amplitude Modulated (PAM) fluorometry of C4 species. This derived a C4 photosynthetic dataset containing measurements made by independent researchers in multiple countries in various environments (field, garden, or greenhouse). It covers three biochemical subtypes – the nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME), nicotinamide adenine dinucleotide-malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PEP-CK) subtypes. This dataset is useful for using Artificial Intelligence / Machine Learning and mechanistic models to study C4 photosynthesis and compare across different biochemical subtypes. This dataset contains 3 compressed (*.zip) folders containing 1,892 data files in comma-separate values (*.csv) format. Additional metadata are provided: one data dictionary and a file-level metadata file in comma-separate values (*.csv) format and a user guide in PDF (*.pdf) format.

Zhou, Haoran [Tianjin University, China]↗
DOE OSTI2023

Defining the Minimal Set of Microbial Genes Required for Valorization of Lignin Biomass (Final Report)

Project Goals: Lignin is the second most abundant biopolymer on earth and represents a critically underutilized biomass resource for hydrocarbon feedstocks. Despite substantial effort, there is still no efficient process to convert lignin to useable carbon-based platform chemicals and materials. The goal of this project is identify a minimal set of microbial enzymes necessary for lignin breakdown and sufficient for the synthesis of valuable chemical intermediates from lignin isolated as a byproduct of lignocellulosic ethanol production. These genes will be then used to engineer functional whole cell biocatalysts for tunable lignin metabolism. To date, although a number of enzymes have been associated with lignin degradation, most have been tested in isolation (as individual enzymes) and on drastically different substrates -- often dyes that are not related to lignin. In contrast, lignin utilization in nature likely occurs by microbial consortia with multiple enzymes acting synergistically. We propose to examine two separate stages of lignin breakdown carried out by the microbes that do it best: (1) early breakdown of native polymeric lignin into soluble fragments by a set of sequenced wood-rotting fungal species, and (2) downstream metabolism of these soluble lignin fragments to useful chemical intermediates by a panel of sequenced soil saprophytes. Our approach involves testing sets of genes that will be assayed combinatorially in the context of a heterologous expression host. The resulting engineered strains will be systematically assayed using soluble lignin fragments, synthetic defined polymeric lignin, and finally lignin directly sourced from lignocellulosic processing streams. In addition to resulting in a functional whole cell biocatalyst for lignin utilization, we anticipate that this approach will allow us to address key unanswered questions about lignin metabolism in nature, including: (1) Why does the Trametes versicolor genome contain 25 different class II peroxidases? (2) What is the role of laccases in lignin metabolism? Why do some aggressive lignin degraders have many laccases (e.g. >7 in T. versicolor) while others have none (e.g. P. chrysosporium)? (3) How is peroxide provided in a controlled manner to drive peroxidase activity without causing the enzyme inhibition that is so often observed in vitro? (4) What strategies do microbial lignin degraders use to avoid the problem of repolymerization during active lignin degradation? and (5) Can microbial lignin metabolism be diverted for high level production of defined aromatics? A final critical question is whether combining key minimal sets of enzymes from a wide range of organisms will result in engineered strains capable of highly efficient, streamlined pathways for lignin utilization that can be tuned for a specific carbon output. This effort will leverage DOE investments in microbial genome sequencing, and secure a critical channel for lignin biomass utilization that will also help to render lignocellulosic a viable feedstock for the production of renewable liquid biofuels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Novel microbial based enzymatic CO2 fixation mechanisms: Conformational control of enzymatic reactivity

The broad, long-term goal of the research is to provide insight into the mechanism of novel carboxylation and electron bifurcation reactions as paradigms to study how enzyme utilized protein conformational change to protect reactive intermediates and/or control the pathway in a catalytic cycle. catalyzed by enzymes involved in bacterial alkene, ketone, and epoxide metabolism. We are examining two paradigms in which conformational change is a key mechanistic feature of 1) protecting reaction intermediates that are sensitive to breakdown in the presence of water and 2) directing electron flow in reactions that split electron pairs along two pathways in reactions that fine tune metabolic efficiency in microbial metabolism. Both paradigms are of key importance in defining the molecular determinants of reactions relevant to CO 2 capture and fuel upcycling. In the first paradigm the compounds acetone and bicarbonate are activated by the addition of phosphate creating reaction intermediates (phosphoenolacetone and carboxyphosphate) that are very sensitive to breakdown in the presence of water. We are studying the mechanism of how the enzyme protects these reactive intermediates from water by encapsulating them upon their formation within a hydrophobic protein channel leading to an enzyme active site approximately 40 Å away. In the second paradigm, we are examining how the direction of electron transfer reactions can be modulated through protein conformational change that alters either the difference driving force or distance or both between electron transfer pathways in enzymes that catalyze electron bifurcation. We are using a combination of enzyme kinetics, mass spectrometry, spectroscopy, and structural analysis through X-ray diffraction and scattering methods to characterize the mechanism of conformational changes along the catalytic path. Within the context of the mission of the Department of Energy and the core activities of the Energy Biosciences, the results obtained in the study will reveal new insights into the fundamentals of novel carboxylation / CO 2 capture chemical transformations and electron transfer reactions that equate to biological fuel upcycling.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Cell-free bioelectrocatalytic platform for carbon dioxide reduction (Final Technical Report)

The University of Minnesota (UMN) EcoSynBio Team aimed to develop a cell-free, enzyme-based platform for the electro- biocatalytic conversion of CO2 into formate as a platform chemical for further upgrading. This type of bio electrocatalytic process delivers a clean product stream without the need for extensive separation from the electrolyte as in electrochemical synthesis and microbial processes. The reduction reaction is catalyzed by metal-dependent formate dehydrogenases (mFDHs) that are capable of efficient electrocatalytic CO2 reduction without the need of costly co-factors. The development of an efficient, scalable electrobiocatalytic process with high total turnover numbers and viable space time yields, however, was not without its challenges. The UM team has developed a protein-based scaffolding system that facilitates enzyme stabilization and attachment to electrodes along with electron transfer. Yet, although FDHs are highly promising enzymes for cell-free, electrobiochemical CO2 reduction, they are also greatly understudied and especially for applications in electrocatalysis. The UM team used the best described mFDH from Clostridium as its benchmark system and spent significant time and effort in attempting to replicate published data and finally, redesigned a recombinant production system for proper metal co-factor incorporation. The UM team has also identified a small set of new enzyme homologs from extreme microorganisms with superior stabilities that have yielded initial structural data for further engineering. In addition, a new bioelectrocatalytic reactor system has been developed that can be 3D printed and used for enzyme attachment to electrodes. In summary the project has generated critical basic information for the further development of this class of enzymes for the electricity driven reduction of CO2 into formate as platform chemical for upgrading into various other chemicals, including fuels.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗