Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “enzyme engineering”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 523 records · Page 29
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Xylanolytic metabolism is regulated by coordination of transcription factors XynR and XylR in extremely thermophilic Caldicellulosiruptorales

ABSTRACT Global transcription factors (TFs) control metabolic processes in bacteria to efficiently utilize available carbon. The orderCaldicellulosiruptoraleshas drawn interest due to the ability of its members to degrade components of lignocellulosic biomass. Regulatory reconstruction ofAnaerocellum (f. Caldicellulosiruptor) besciiidentified two major global transcription factors for xylan utilization, XynR and XylR, and the corresponding putative transcription factor binding sites. Recombinant versions of XynR (LacI family) and XylR (ROK family) were subjected to fluorescence polarization (FP) and biolayer interferometry (BLI) analysis to confirm the predicted binding sites. Four XynR sites and two XylR sites were validated, accounting for 20 of 26 genes regulated by XynR and six of seven genes regulated by XylR. Bioinformatic analysis of the individual genes controlled by the two regulators showed an inter-dependent scheme for xylan conversion; the transport of xylooligosaccharides (XOS) is dependent on XylR, while enzymes responsible for hydrolysis are controlled by both regulators. For xylose catabolism by the xylose isomerase-xylulose kinase pathway, regulation is also split, with XylR controlling xylose isomerase and XynR controlling xylokinase. The XynR/XylR regulator pair withinA. besciiis conserved in all sequenced species ofCaldicellulosiruptorales, suggesting similarities in regulating linear xylan conversion. In other xylanolytic thermophiles, XylR homologs control xylan degradation, compared to just 6 out of 26 genes forA. bescii. These results show that two separate regulatory schemes (dual repression) are coordinated byA. besciito effectively regulate the hemicellulose inventory and xylan catabolism. IMPORTANCE To take full advantage of extreme thermophiles as platform metabolic engineering microorganisms, the tools for genetic manipulation must be further developed, and strategies that exploit a better understanding of metabolic regulation need to be discerned.Anaerocellum bescii, the most studied of the extremely thermophilic fermentative anaerobic bacteria that can utilize microcrystalline cellulose, can degrade microcrystalline cellulose and hemicellulose and has been metabolically engineered to convert the resulting sugars to products such as ethanol and acetone. For xylan, in particular, two major global transcription factors (TFs), XynR and XylR, play a role in sugar metabolism, although their predicted regulatory interdependence from bioinformatics analysis has not been elucidated experimentally. Here, fluorescence polarization (FP) and biolayer interferometry (BLI) were used to explore this issue to support metabolic engineering efforts aimed at improving carbohydrate processing to industrial chemicals.

Biotechnology & Applied Microbiology↗
DOE OSTI2022

Understanding Proton Movement in [Fe-Fe] Hydrogenases

Nature uses specialized metalloenzymes to carry out small molecule activation reactions, including CO 2 fixation, O 2 activation, and proton reduction, with unparalleled efficiency, rates, and selectivity. The latter reactions are performed by hydrogenases, protein metallo-complexes that interconverts H 2 to protons and electrons (H 2 oxidation) and the reverse reaction (H 2 production) with incredibly low energy input and amazingly fast kinetics. Reproducing both the activity and efficiency of metalloenzymes in sustainable anthropogenic systems remains one of the “holy grails” of inorganic chemistry. However, identifying the precise molecular components responsible for these desirable properties has been challenging in the natural metalloenzymes, hindering efforts to develop analogous processes in synthetic compounds. Considering the inherent complexity of a metalloenzyme and the many interactions, both strong and weak, that contribute to the function of an enzyme, we have elected to model natural metalloenzymes on a biochemical platform. Towards this end, we have a developed structural, functional, and mechanistic mimic of the [Ni-Fe] hydrogenases within a robust protein scaffold, rubredoxin, to understand the influence of the secondary coordination environment on the metal center. This involved preparing a series of three rubredoxin constructs containing a single point mutation at Val positions and making the NMR chemical shift assignments for the paramagnetic (nickel-substituted) and non-paramagnetic (zinc-substituted) form. These physical studies were complemented with in silico molecular dynamic studies on proteins with [Fe-S] clusters to engineer new proton channels to test in vitro . To assist our search for new natural metalloprotein scaffolds within the vast number of sequenced genomes, we created a neural network-based program to identify proteins with specific metal-binding sites. These computational and physical studies with metalloenzymes provide direct insight into the fundamental chemical principles driving the natural systems and offer design principles for developing catalysts that utilize analogous principles.

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

Co-expression of a β-d-xylosidase from Thermotoga maritima and a Family 10 xylanase from A. cellulolyticus significantly improves the xylan degrading activity of the Caldicellulosiruptor bescii exoproteome

Caldicellulosiruptor species are hyperthermophilic, Gram-positive, anaerobes and the most thermophilic cellulolytic bacteria so far described. They have been engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals and materials from plant biomass. Xylooligomers such as xylobiose and xylotriose that result from the breakdown of plant biomass more strongly inhibit cellulase activity than do glucose or cellobiose. High concentrations of xylobiose and xylotriose, are present in C. bescii fermentations after 90 h incubation and removal or breakdown of these types of xylooligomers is crucial to achieve high conversion of plant biomass to product. In previous studies the addition of exogenous β-d-xylosidase substantially improved the performance of glucanases and xylanases in vitro. β-d-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and β-d-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular β-d-xylosidase was identified in the C. bescii genome. Here we report that the co-expression of a thermal stable β-d-xylosidase from Thermotoga maritima and a xylanase from Acidothermus cellulolyticus in a C. bescii strain containing the A. cellulolyticus E1 endoglucanase significantly increased the activity of the exoproteome as well as growth on xylan substrates. The combination of these enzymes also resulted in increased growth on crystalline cellulose in the presence of exogeneous xylan.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Alpha ketoacid decarboxylases: Diversity, structures, reaction mechanisms, and applications for biomanufacturing of platform chemicals and fuels

In living cells, alpha-ketoacid decarboxylases (KDCs, EC 4.1.1.-) are a class of enzymes that convert alpha-ketoacids into aldehydes through decarboxylation. These aldehydes serve as either drop-in chemicals or precursors for the biosynthesis of alcohols, carboxylic acids, esters, and alkanes. These compounds play crucial roles in cellular metabolism and fitness and the bioeconomy, facilitating the sustainable and renewable biomanufacturing of platform chemicals and fuels. This review explores the diversity and classification of KDCs, detailing their structures, mechanisms, and functions. We highlight recent advancements in repurposing KDCs to enhance their efficiency and robustness for biomanufacturing. Additionally, we present modular KDC-dependent metabolic pathways for the microbial biosynthesis of aldehydes, alcohols, carboxylic acids, esters, and alkanes. Lastly, we discuss recent developments in the modular cell engineering technology that can potentially be applied to harness the diversity of KDC-dependent pathways for biomanufacturing platform chemicals and fuels.

09 BIOMASS FUELS↗
DOE OSTI2020

Small subunits can determine enzyme kinetics of tobacco Rubisco expressed in Escherichia coli

Rubisco catalyses the first step in carbon fixation and is a strategic target to improve photosynthetic efficiency. In plants, Rubisco is composed of eight large and eight small subunits and its biogenesis requires multiple chaperones. We optimised a system to produce tobacco Rubisco in Escherichia coli by co-expressing chaperones in auto-induction medium. We successfully assembled tobacco Rubisco in E. coli with each small subunit that is normally encoded by the nuclear genome. Even though each enzyme carries only a single type of small subunit in E. coli, the enzymes exhibit carboxylation kinetics very similar to that of the native Rubisco. Tobacco Rubisco assembled with a recently discovered trichome small subunit has a higher catalytic rate and a lower CO 2 affinity than those assembled with other small subunits. Finally, our E. coli expression system will allow probing of features of both subunits of Rubisco that affect its kinetic properties.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Unraveling metabolism underpinning biomass composition shift in Scenedesmus obliquus under simulated outdoor conditions using 13 C-fluxomics

To render the resulting biomass more attractive and amenable for utilization as the basis for low-carbon intensity bioproducts, single-celled algae need to be biochemically and metabolically poised to assimilate and store the delivered carbon in the fastest and most efficient manner. Accelerating biochemical carbon storage, as primarily carbohydrates or lipids, is critical to achieve the high carbon capture potential that is assigned to algae. To guide strain optimization and engineering for maximizing carbon capture and storage, it is essential to elucidate the link between carbon metabolism and biomass composition. Most published metabolomics work in algae remains largely restricted to ideal and simplified environmental conditions in model organisms, thereby limiting their translation to outdoor implementation. In this work, we utilize 13 C isotopic labeling to characterize distinct intracellular metabolic fluxes before, during, and after nitrogen depletion-induced compositional shifts in Scenedesmus obliquus UTEX 393. The results indicate that a transition to carbohydrates is characterized by diverting flux to starch instead of replenishing the Calvin cycle for CO 2 fixation whereas the subsequent transition to lipids is fueled by NADPH produced by upregulating the phosphoenolpyruvate carboxylase (PEPC)–malic enzyme (ME) cycle flux. Our work highlights bottlenecks to carbohydrate- and lipid-rich biomass and can guide implementable strategies to control the fate of fixed carbon in S. obliquus.

09 BIOMASS FUELS↗
DOE OSTI2022

How a Formate Dehydrogenase Responds to Oxygen: Unexpected O 2 Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters

The reversible two-electron interconversion of formate and CO 2 is catalyzed by both nonmetallo- and metallo-formate dehydrogenases (FDHs). The latter group comprises molybdenum- or tungsten-containing enzymes with the metal coordinated by two equivalents of a pyranopterin cofactor, a cysteinyl or selenocysteinyl (Sec) ligand supplied by the polypeptide, and a catalytically essential terminal sulfido ligand. In addition, these biocatalysts incorporate one or more [4Fe–4S] clusters for facilitating long-distance electron transfer. However, an interesting dichotomy arises when attempting to understand how the metallo-FDHs react with O 2 . Whereas existing scholarship portrays these enzymes as being unable to perform in air due to extreme O 2 lability of their metal centers, studies dating as far back as the 1930s emphasize that some of these systems exhibit formate oxidase (FOX) activity, coupling formate oxidation to O 2 reduction. Therefore, to reconcile these conflicting views, we explored context-dependent functional linkages between metallo-FDHs and their cognate electron acceptors within the same organism vis-à-vis catalysis under atmospheric O 2 . Here, we report the discovery and characterization of an O 2 -insensitive FDH2 from the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) that ligates tungsten, Sec, and four [4Fe–4S] clusters. By advancing a robust expression platform for its recombinant production, we eliminate both the requirement of nitrate or azide during purification and reductive activation with thiols and/or formate prior to catalysis. Because the distinctive spectral signatures of formate-reduced DvH-FDH2 remain invariant under anaerobic and aerobic conditions, we benchmarked the enzyme activity in air, identifying CO 2 as the catalytic product. Full reaction progress curve analysis discloses a high catalytic efficiency when probed with a high-potential artificial electron acceptor. Furthermore, we show that DvH-FDH2 enables near-stoichiometric hydrogen peroxide production without superoxide release to achieve O 2 insensitivity. Notably, simultaneous electron transfer to cytochrome c and O 2 reveals that metal-based electron bifurcation is operational in this system. Taken together, our work proves the co-occurrence of redox bifurcated FDH and FOX activities within a metalloenzyme scaffold. These findings set the stage for uncovering previously unknown O 2 -insensitive flavin-based electron bifurcation mechanisms, as well as for developing authentic formate/air biofuel cells, engineering O 2 -stable FDHs and biohybrid metallocatalysts, and discerning formate bioenergetics of gut microbiota.

13C NMR↗
DOE OSTI2022

Recovery of particulate methane monooxygenase structure and activity in a lipid bilayer

Bacterial methane oxidation using the enzyme particulate methane monooxygenase (pMMO) contributes to the removal of environmental methane, a potent greenhouse gas. Crystal structures determined using inactive, detergent-solubilized pMMO lack several conserved regions neighboring the proposed active site. We show that reconstituting pMMO in nanodiscs with lipids extracted from the native organism restores methane oxidation activity. Multiple nanodisc-embedded pMMO structures determined by cryo–electron microscopy to 2.14- to 2.46-angstrom resolution reveal the structure of pMMO in a lipid environment. The resulting model includes stabilizing lipids, regions of the PmoA and PmoC subunits not observed in prior structures, and a previously undetected copper-binding site in the PmoC subunit with an adjacent hydrophobic cavity. These structures provide a revised framework for understanding and engineering pMMO function.

Science & Technology - Other Topics↗
DOE OSTI2022

Photoinduced chemomimetic biocatalysis for enantioselective intermolecular radical conjugate addition

Exploiting nature’s catalysts for non-natural transformations that are inaccessible to chemocatalysis is highly desirable but challenging. On the one hand, the widespread nicotinamide-dependent oxidoreductases have not been utilized for single-electron-transfer-induced bimolecular cross-couplings; on the other, the addition of catalytic asymmetric radical con- jugate to terminal alkenes remains a challenge owing to strong racemic background reaction and unselective termination of prochiral radical species. Here we report a chemomimetic biocatalysitic approach for construction of alpha-carbonyl stereocentres via an unnatural intermolecular conjugate addition of N-(acyloxy)phthalimides-derived radicals with acceptor-substituted terminal alkenes, by combination of visible-light excitation and nicotinamide-dependent ketoreductases (KREDs). Based on protein crystal structure, we engineered KREDs via a semi-rational mutagenesis strategy to improve reaction outcomes with a small and high-quality variants library. Here, mechanistic investigations combining wet experiments, crystallographic studies and computational simulations demonstrate that the repurposed biocatalyst can suppress racemic background reaction and unselected side reactions, yielding enantioselectivity that is challenging to achieve by chemocatalysis.

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

Evolutionary History of Plant Metabolism

Tremendous chemical diversity is the hallmark of plants and is supported by highly complex biochemical machinery. Plant metabolic enzymes originated and were transferred from eukaryotic and prokaryotic ancestors and further diversified by the unprecedented rates of gene duplication and functionalization experienced in land plants. Unlike microbes, which have frequent horizontal gene transfer events and multiple inputs of energy and organic carbon, land plants predominantly rely on organic carbon generated from CO 2 and have experienced very few, if any, gene transfers during their recent evolutionary history. As such, plant metabolic networks have evolved in a stepwise manner and on existing networks under various evolutionary constraints. This review aims to take a broader view of plant metabolic evolution and lay a framework to further explore evolutionary mechanisms of the complex metabolic network. Understanding the underlying metabolic and genetic constraints is also an empirical prerequisite for rational engineering and redesigning of plant metabolic pathways.

Plant Sciences↗
DOE OSTI2021

CRISPR-Cas9 editing of CAFFEOYL SHIKIMATE ESTERASE 1 and 2 shows their importance and partial redundancy in lignification in Populus tremula × P. alba

Lignins are cell wall-located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. Here, we explored the role of the two CSE homologs in poplar (Populus tremula × P. alba). Reporter lines showed that the expression conferred by both CSE1 and CSE2 promoters is similar. CRISPR-Cas9-generated cse1 and cse2 single mutants had a wild-type lignin level. Nevertheless, CSE1 and CSE2 are not completely redundant, as both single mutants accumulated caffeoyl shikimate. In contrast, the cse1 cse2 double mutants had a 35% reduction in lignin and associated growth penalty. The reduced-lignin content translated into a fourfold increase in cellulose-to-glucose conversion upon limited saccharification. Phenolic profiling of the double mutants revealed large metabolic shifts, including an accumulation of p-coumaroyl, 5-hydroxyferuloyl, feruloyl and sinapoyl shikimate, in addition to caffeoyl shikimate. This indicates that the CSEs have a broad substrate specificity, which was confirmed by in vitro enzyme kinetics. Taken together, our results suggest an alternative path within the phenylpropanoid pathway at the level of the hydroxycinnamoyl-shikimates, and show that CSE is a promising target to improve plants for the biorefinery.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Hydrolysis of lignocellulose by anaerobic fungi produces free sugars and organic acids for two–stage fine chemical production with Kluyveromyces marxianus

Development of the bioeconomy is driven by our ability to access the energy-rich carbon trapped in recalcitrant plant materials. Current strategies to release this carbon rely on expensive enzyme cocktails and physicochemical pretreatment, producing inhibitory compounds that hinder subsequent microbial bioproduction. Anaerobic fungi are an appealing solution as they hydrolyze crude, untreated biomass at ambient conditions into sugars that can be converted into value-added products by partner organisms. However, some carbon is lost to anaerobic fungal fermentation products. To improve efficiency and recapture this lost carbon, we built a two-stage bioprocessing system pairing the anaerobic fungus Piromyces indianae with the yeast Kluyveromyces marxianus, which grows on a wide range of sugars and fermentation products. In doing so we produce fine and commodity chemicals directly from untreated lignocellulose. P. indianae efficiently hydrolyzed substrates such as corn stover and poplar to generate sugars, fermentation acids, and ethanol, which K. marxianus consumed while producing 2.4 g/L ethyl acetate. An engineered strain of K. marxianus was also able to produce 550 mg/L 2-phenylethanol and 150 mg/L isoamyl alcohol from P. indianae hydrolyzed lignocellulosic biomass. Despite the use of crude untreated plant material, production yields were comparable to optimized rich yeast media due to the use of all available carbon including organic acids, which formed up to 97% of free carbon in the fungal hydrolysate. Furthermore, this work demonstrates that anaerobic fungal pretreatment of lignocellulose can sustain the production of fine chemicals at high efficiency by partnering organisms with broad substrate versatility.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Diverse events have transferred genes for edible seaweed digestion from marine to human gut bacteria

Humans harbor numerous species of colonic bacteria that digest fiber polysaccharides in commonly consumed terrestrial plants. More recently in history, regional populations have consumed edible macroalgae seaweeds containing unique polysaccharides. It remains unclear how extensively gut bacteria have adapted to digest these nutrients. Here, in this work, we show that the ability of gut bacteria to digest seaweed polysaccharides is more pervasive than previously appreciated. Enrichment-cultured Bacteroides harbor previously discovered genes for seaweed degradation, which have mobilized into several members of this genus. Additionally, other examples of marine bacteria-derived genes, and their mobile DNA elements, are involved in gut microbial degradation of seaweed polysaccharides, including genes in gut-resident Firmicutes. Collectively, these results uncover multiple separate events that have mobilized the genes encoding seaweed-degrading-enzymes into gut bacteria. This work further underscores the metabolic plasticity of the human gut microbiome and global exchange of genes in the context of dietary selective pressures.

42 ENGINEERING↗
DOE OSTI2024

Oxygenation influences xylose fermentation and gene expression in the yeast genera Spathaspora and Scheffersomyces

Abstract Background Cost-effective production of biofuels from lignocellulose requires the fermentation of d -xylose. Many yeast species within and closely related to the genera Spathaspora and Scheffersomyces (both of the order Serinales) natively assimilate and ferment xylose. Other species consume xylose inefficiently, leading to extracellular accumulation of xylitol. Xylitol excretion is thought to be due to the different cofactor requirements of the first two steps of xylose metabolism. Xylose reductase (XR) generally uses NADPH to reduce xylose to xylitol, while xylitol dehydrogenase (XDH) generally uses NAD + to oxidize xylitol to xylulose, creating an imbalanced redox pathway. This imbalance is thought to be particularly consequential in hypoxic or anoxic environments. Results We screened the growth of xylose-fermenting yeast species in high and moderate aeration and identified both ethanol producers and xylitol producers. Selected species were further characterized for their XR and XDH cofactor preferences by enzyme assays and gene expression patterns by RNA-Seq. Our data revealed that xylose metabolism is more redox balanced in some species, but it is strongly affected by oxygen levels. Under high aeration, most species switched from ethanol production to xylitol accumulation, despite the availability of ample oxygen to accept electrons from NADH. This switch was followed by decreases in enzyme activity and the expression of genes related to xylose metabolism, suggesting that bottlenecks in xylose fermentation are not always due to cofactor preferences. Finally, we expressed XYL genes from multiple Scheffersomyces species in a strain of Saccharomyces cerevisiae . Recombinant S. cerevisiae expressing XYL1 from Scheffersomyces xylosifermentans , which encodes an XR without a cofactor preference, showed improved anaerobic growth on xylose as the primary carbon source compared to S. cerevisiae strain expressing XYL genes from Scheffersomyces stipitis . Conclusion Collectively, our data do not support the hypothesis that xylitol accumulation occurs primarily due to differences in cofactor preferences between xylose reductase and xylitol dehydrogenase; instead, gene expression plays a major role in response to oxygen levels. We have also identified the yeast Sc. xylosifermentans as a potential source for genes that can be engineered into S. cerevisiae to improve xylose fermentation and biofuel production.

09 BIOMASS FUELS↗
DOE OSTI2021

Structural analysis and biochemical properties of laccase enzymes from two Pediococcus species

Prokaryotic laccases are emergent biocatalysts. However, they have not been broadly found and characterized in bacterial organisms, especially in lactic acid bacteria. Recently, a prokaryotic laccase from the lactic acid bacterium Pediococcus acidilactici 5930, which can degrade biogenic amines, was discovered. Thus, our study aimed to shed light on laccases from lactic acid bacteria focusing on two Pediococcus laccases, P. acidilactici 5930 and Pediococcus pentosaceus 4816, which have provided valuable information on their biochemical activities on redox mediators and biogenic amines. Both laccases are able to oxidize canonical substrates as ABTS, ferrocyanide and 2,6-DMP, and non-conventional substrates as biogenic amines. With ABTS as a substrate, they prefer an acidic environment and show sigmoidal kinetic activity, and are rather thermostable. Moreover, this study has provided the first structural view of two lactic acid bacteria laccases, revealing new structural features not seen before in other well-studied laccases, but which seem characteristic for this group of bacteria. We believe that understanding the role of laccases in lactic acid bacteria will have an impact on their biotechnological applications and provide a framework for the development of engineered lactic acid bacteria with enhanced properties.

36 MATERIALS SCIENCE↗
DOE OSTI2025

Crystal structure of a seven-substitution mutant of hydroxynitrile lyase from rubber tree

The α/β-hydrolase fold superfamily includes esterases and hydroxynitrile lyases which, despite catalyzing different reactions, share a Ser–His–Asp catalytic triad. We report a 1.99 Å resolution crystal structure of HNL6V, an engineered variant of hydroxynitrile lyase fromHevea brasiliensis(HbHNL) containing seven amino-acid substitutions (T11G, E79H, C81L, H103V, N104A, G176S and K236M). The structure reveals that HNL6V maintains the characteristic α/β-hydrolase fold while exhibiting systematic shifts in backbone and catalytic atom positions. Compared with wild-typeHbHNL, the C α positions in HNL6V differ by a mean of 0.2 ± 0.1 Å, representing a statistically significant displacement. Importantly, the catalytic triad and oxyanion-hole atoms have moved 0.2–0.8 Å closer to their corresponding positions in SABP2, although they remain 0.3–1.1 Å from fully achieving the configuration of SABP2. The substitutions also increase local flexibility, particularly in the lid domain covering the active site. This structural characterization demonstrates that targeted amino-acid substitutions can systematically shift catalytic geometries towards those of evolutionarily related enzymes.

Biochemistry & Molecular Biology↗
DOE OSTI2023

Non-canonical d-xylose and l-arabinose metabolism via d-arabitol in the oleaginous yeast Rhodosporidium toruloides

Abstract R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R. toruloides grows on several pentose sugars and alcohols, further engineering of the native pathway is required for efficient conversion of biomass-derived sugars to higher value bioproducts. A previous high-throughput study inferred that R. toruloides possesses a non-canonical l -arabinose and d -xylose metabolism proceeding through d -arabitol and d -ribulose. In this study, we present a combination of genetic and metabolite data that refine and extend that model. Chiral separations definitively illustrate that d -arabitol is the enantiomer that accumulates under pentose metabolism. Deletion of putative d -arabitol-2-dehydrogenase (RTO4_9990) results in > 75% conversion of d -xylose to d -arabitol, and is growth-complemented on pentoses by heterologous xylulose kinase expression. Deletion of putative d -ribulose kinase (RTO4_14368) arrests all growth on any pentose tested. Analysis of several pentose dehydrogenase mutants elucidates a complex pathway with multiple enzymes mediating multiple different reactions in differing combinations, from which we also inferred a putative l -ribulose utilization pathway. Our results suggest that we have identified enzymes responsible for the majority of pathway flux, with additional unknown enzymes providing accessory activity at multiple steps. Further biochemical characterization of the enzymes described here will enable a more complete and quantitative understanding of R. toruloides pentose metabolism. These findings add to a growing understanding of the diversity and complexity of microbial pentose metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Data Mining of Groundwater to Identify MAGs with Methane, Propane and Toluene Monooxygenases

Whole genome sequencing datasets, involving more than 600 groundwater samples, from nine countries, were analyzed to identify metagenome assembled genomes (MAGs) containing full operons for propane monooxygenase, soluble methane monooxygease, toluene monooxygenase and particulate ammonia/methane monooxygenase. The enzymes encoded by these genes are a focus of interest because of their ability to degrade common groundwater contaminants. Due to the large amount of data, sequence analyses involved more than 80 individual KBase narratives. The approach followed the KBase tutorial called "Metagenome-Assembled Genome Extraction from a Compost Microbiome Enrichment" The generated MAGs were exported from each individual narrative into separate summary KBase narratives for each monooxygenase. Three KBase narratives were generated for particulate ammonia/methane monooxygenase, due to the large number of MAGs identified.

59 BASIC BIOLOGICAL SCIENCES↗