Engineering Papers⌕ Search

Engineering topics

Northen, Trent R.

Publications and source records attributed to Northen, Trent R..

At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Lanthanide-chelator combinatorial barcoding

Disclosed herein are methods for tracking solutions, (e.g., reaction conditions in solutions). In some embodiments, the method comprises: contacting a first lanthanide-chelator complex to a first solution to generate a first barcoded solution, wherein the first lanthanide-chelator complex comprises a first lanthanide chelated by a first chelator; contacting a second lanthanide-chelator complex to a second solution to generate a second barcoded solution, wherein the second lanthanide-chelator complex comprises a second lanthanide chelated by a second chelator; mixing the first barcoded solution and the second barcoded solution to form one or more mixtures; and identifying the first lanthanide ions in the mass spectrum and the second lanthanide ions in the mass spectrum to track the condition of each of the one or more mixtures.

Duncombe, Todd↗
DOE OSTI2023

Quantitative Analysis of The High‐Yield Hydrolysis of Kelp by Laminarinase and Alginate Lyase

Abstract Kelp is an abundant, farmable biomass‐containing laminarin and alginate as major polysaccharides, providing an excellent model substrate to study their deconstruction by simple enzyme mixtures. Our previous study showed strong reactivity of the glycoside hydrolase family 55 during hydrolysis of purified laminarin, raising the question of its reactivity with intact kelp. In this study, we determined that a combination of a single glycoside hydrolase family 55 β‐1,3‐exoglucanase with a broad‐specificity alginate lyase from the polysaccharide lyase family 18 gives efficient hydrolysis of untreated kelp to a mixture of simple sugars, that is, glucose, gentiobiose, mannitol‐end glucose, and mannuronic and guluronic acids and their soluble oligomers. Quantitative assignments from nanostructure initiator mass spectrometry (NIMS) and 2D HSQC NMR spectroscopy and analysis of the reaction time‐course are provided. The data suggest that binary combinations of enzymes targeted to the unique polysaccharide composition of marine biomass are sufficient to deconstruct kelp into soluble sugars for microbial fermentation.

2D HSQC NMR↗
DOE OSTI2023

BLINK enables ultrafast tandem mass spectrometry cosine similarity scoring

Metabolomics has a long history of using cosine similarity to match experimental tandem mass spectra to databases for compound identification. Here we introduce the Blur-and-Link (BLINK) approach for scoring cosine similarity. By bypassing fragment alignment and simultaneously scoring all pairs of spectra using sparse matrix operations, BLINK is over 3000 times faster than MatchMS, a widely used loop-based alignment and scoring implementation. Using a similarity cutoff of 0.7, BLINK and MatchMS had practically equivalent identification agreement, and greater than 99% of their scores and matching ion counts were identical. This performance improvement can enable calculations to be performed that would typically be limited by time and available computational resources.

47 OTHER INSTRUMENTATION↗
DOE OSTI2023

Zn deficiency disrupts Cu and S homeostasis in Chlamydomonas resulting in over accumulation of Cu and Cysteine

Growth of Chlamydomonas reinhardtii in zinc (Zn) limited medium leads to disruption of copper (Cu) homeostasis, resulting in up to 40-fold Cu over-accumulation relative to its typical Cu quota. Here, we show that Chlamydomonas controls its Cu quota by balancing Cu import and export, which is disrupted in a Zn deficient cell, thus establishing a mechanistic connection between Cu and Zn homeostasis. Transcriptomics, proteomics and elemental profiling revealed that Zn-limited Chlamydomonas cells up-regulate a subset of genes encoding “first responder” proteins involved in sulfur (S) assimilation and consequently accumulate more intracellular S, which is incorporated into L-cysteine, γ-glutamylcysteine, and homocysteine. Most prominently, in the absence of Zn, free L-cysteine is increased ~80-fold, corresponding to ~2.8 × 10 9 molecules/cell. Interestingly, classic S-containing metal binding ligands like glutathione and phytochelatins do not increase. X-ray fluorescence microscopy showed foci of S accumulation in Zn-limited cells that co-localize with Cu, phosphorus and calcium, consistent with Cu-thiol complexes in the acidocalcisome, the site of Cu(I) accumulation. Notably, cells that have been previously starved for Cu do not accumulate S or Cys, causally connecting cysteine synthesis with Cu accumulation. We suggest that cysteine is an in vivo Cu(I) ligand, perhaps ancestral, that buffers cytosolic Cu.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format

Plate-based proteomic sample preparation offers a solution to the large sample throughput demands in the biotechnology field where hundreds or thousands of engineered microbes are constructed for testing is routine. Meanwhile, sample preparation methods that work efficiently on broader microbial groups are desirable for new applications of proteomics in other fields, such as microbial communities. Here, we detail a step-by-step protocol that consists of cell lysis in an alkaline chemical buffer (NaOH/SDS) followed by protein precipitation with high-ionic strength acetone in 96-well format. The protocol works for a broad range of microbes ( e . g ., Gram-negative bacteria, Gram-positive bacteria, non-filamentous fungi) and the resulting proteins are ready for tryptic digestion for bottom-up quantitative proteomic analysis without the need for desalting column cleanup. The yield of protein using this protocol increases linearly with respect to the amount of starting biomass from 0.5–2.0 OD*mL of cells. By using a bench-top automated liquid dispenser, a cost-effective and environmentally-friendly option to eliminating pipette tips and reducing reagent waste, the protocol takes approximately 30 minutes to extract protein from 96 samples. Tests on mock mixtures showed expected results that the biomass composition structure is in close agreement with the experimental design. Lastly, we applied the protocol for the composition analysis of a synthetic community of environmental isolates grown on two different media. This protocol has been developed to facilitate rapid, low-variance sample preparation of hundreds of samples and allow flexibility for future protocol development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A combinatorial droplet microfluidic device integrated with mass spectrometry for enzyme screening

Mass spectrometry (MS) enables detection of different chemical species with a very high specificity; however, it can be limited by its throughput. Integrating MS with microfluidics has a tremendous potential to improve throughput and accelerate biochemical research. In this work, we introduce Drop-NIMS, a combination of a passive droplet loading microfluidic device and a matrix-free MS laser desorption ionization technique called nanostructure-initiator mass spectrometry (NIMS). This platform combines different droplets at random to generate a combinatorial library of enzymatic reactions that are deposited directly on the NIMS surface without requiring additional sample handling. The enzyme reaction products are then detected with MS. Drop-NIMS was used to rapidly screen enzymatic reactions containing low (on the order of nL) volumes of glycoside reactants and glycoside hydrolase enzymes per reaction. MS “barcodes” (small compounds with unique masses) were added to the droplets to identify different combinations of substrates and enzymes created by the device. We assigned xylanase activities to several putative glycoside hydrolases, making them relevant to food and biofuel industrial applications. Overall, Drop-NIMS is simple to fabricate, assemble, and operate and it has potential to be used with many other small molecule metabolites.

47 OTHER INSTRUMENTATION↗
DOE OSTI2023

BONCAT-FACS-Seq reveals the active fraction of a biocrust community undergoing a wet-up event

Determining which microorganisms are active within soil communities remains a major technical endeavor in microbial ecology research. One promising method to accomplish this is coupling bioorthogonal non-canonical amino acid tagging (BONCAT) with fluorescence activated cell sorting (FACS) which sorts cells based on whether or not they are producing new proteins. Combined with shotgun metagenomic sequencing (Seq), we apply this method to profile the diversity and potential functional capabilities of both active and inactive microorganisms in a biocrust community after being resuscitated by a simulated rain event. We find that BONCAT-FACS-Seq is capable of discerning the pools of active and inactive microorganisms, especially within hours of applying the BONCAT probe. The active and inactive components of the biocrust community differed in species richness and composition at both 4 and 21 h after the wetting event. The active fraction of the biocrust community is marked by taxa commonly observed in other biocrust communities, many of which play important roles in species interactions and nutrient transformations. Among these, 11 families within the Firmicutes are enriched in the active fraction, supporting previous reports indicating that the Firmicutes are key early responders to biocrust wetting. We highlight the apparent inactivity of many Actinobacteria and Proteobacteria through 21 h after wetting, and note that members of the Chitinophagaceae, enriched in the active fraction, may play important ecological roles following wetting. Based on the enrichment of COGs in the active fraction, predation by phage and other bacterial members, as well as scavenging and recycling of labile nutrients, appear to be important ecological processes soon after wetting. To our knowledge, this is the first time BONCAT-FACS-Seq has been applied to biocrust samples, and therefore we discuss the potential advantages and shortcomings of coupling metagenomics to BONCAT to intact soil communities such as biocrust. In all, by pairing BONCAT-FACS and metagenomics, we are capable of highlighting the taxa and potential functions that typifies the microbes actively responding to a rain event.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Synthase-selected sorting approach identifies a beta-lactone synthase in a nudibranch symbiotic bacterium

Background - Nudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva. Results - We recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously. Conclusions - Altogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Decomposition decreases molecular diversity and ecosystem similarity of soil organic matter

Soil organic matter (SOM) is comprised of a diverse array of reactive carbon molecules, including hydrophilic and hydrophobic compounds, that impact rates of SOM formation and persistence. Despite clear importance to ecosystem science, little is known about broad-scale controls on SOM diversity and variability in soil. Here, we show that microbial decomposition drives significant variability in the molecular richness and diversity of SOM between soil horizons and across a continental-scale gradient in climate and ecosystem type (arid shrubs, coniferous, deciduous, and mixed forests, grasslands, and tundra sedges). The molecular dissimilarity of SOM was strongly influenced by ecosystem type (hydrophilic compounds: 17%, P < 0.001; hydrophobic compounds: 10% P < 0.001) and soil horizon (hydrophilic compounds: 17%, P < 0.001; hydrophobic compounds: 21%, P < 0.001), as assessed using metabolomic analysis of hydrophilic and hydrophobic metabolites. While the proportion of shared molecular features was significantly higher in the litter layer than subsoil C horizons across ecosystems (12 times and 4 times higher for hydrophilic and hydrophobic compounds, respectively), the proportion of site-specific molecular features nearly doubled from the litter layer to the subsoil horizon, suggesting greater differentiation of compounds after microbial decomposition within each ecosystem. Together, these results suggest that microbial decomposition of plant litter leads to a decrease in SOM α-molecular diversity, yet an increase in β-molecular diversity across ecosystems. The degree of microbial degradation, determined by the position in the soil profile, exerts a greater control on SOM molecular diversity than environmental factors, such as soil texture, moisture, and ecosystem type.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Dynamic Phaeodactylum tricornutum exometabolites shape surrounding bacterial communities

Summary Roles of different ecological classes of algal exometabolites in regulating microbial community composition are not well understood. Here, we identify exometabolites from the model diatom Phaeodactylum tricornutum and demonstrate their potential to influence bacterial abundances. We profiled exometabolites across a time course of axenic algal growth using liquid chromatography–tandem mass spectrometry. We then investigated growth of 12 bacterial isolates on individual‐identified exometabolites. Lastly, we compared responses of a P. tricornutum ‐adapted enrichment community to additions of two contrasting metabolites: selective growth substrate 4‐hydroxybenzoic acid and putative signaling/facilitator molecule lumichrome. We identified 50 P. tricornutum metabolites and found distinct temporal accumulation patterns. Two exometabolites (of 12 tested) supported growth of distinct subsets of bacterial isolates. While algal exudates and algal presence drove similar changes in community composition compared with controls, exogenous 4‐hydroxybenzoic acid addition promoted increased abundances of taxa that utilized it in isolation, and also revealed the importance of factors relating to algal presence in regulating community composition. This work demonstrates that secretion of selective bacterial growth substrates represents one mechanism by which algal exometabolites can influence bacterial community composition and illustrates how the algal exometabolome has the potential to modulate bacterial communities as a function of algal growth.

4-hydroxybenzoic acid↗
DOE OSTI2023

Complete integration of carbene-transfer chemistry into biosynthesis

Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here, in this paper, we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule—styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Rapid quantification of alcohol production in microorganisms based on nanostructure-initiator mass spectrometry (NIMS)

We described a mass spectrometry-based assay to rapidly quantify the production of primary alcohols directly from cell cultures. This novel assay used the combination of TEMPO-based oxidation chemistry and oxime ligation, followed by product analysis based on Nanostructure-Initiator Mass Spectrometry. This assay enables quantitative monitor both C5 to C18 alcohols as well as glucose and gluconate in the growth medium to support strain characterization and optimization. We find that this assay yields similar results to gas chromatography for isoprenol production but required much less acquisition time per sample. We applied this assay to gain new insights into P. Putida's utilization of alcohols and find that this strain largely could not grow on heptanol and octanol.

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

Tracing Carbon Metabolism with Stable Isotope Metabolomics Reveals the Legacy of Diverse Carbon Sources in Soil

Tracking the metabolic activity of whole soil communities can improve our understanding of the transformation and fate of carbon in soils. For this work, we used stable isotope metabolomics to trace 13 C from nine labeled carbon sources into the water-soluble metabolite pool of an agricultural soil over time. Soil was amended with a mixture of all nine sources, with one source isotopically labeled in each treatment. We compared changes in the 13 C enrichment of metabolites with respect to carbon source and time over a 48-day incubation and contrasted differences between soluble sources (glucose, xylose, amino acids, etc.) and insoluble sources (cellulose and palmitic acid). Whole soil metabolite profiles varied singularly by time, while the composition of 13 C-labeled metabolites differed primarily by carbon source (R 2 = 0.68) rather than time (R 2 = 0.07), with source-specific differences persisting throughout incubations. The 13 C labeling of metabolites from insoluble carbon sources occurred slower than that from soluble sources but yielded a higher average atom percent (atom%) 13 C in metabolite markers of biomass (amino acids and nucleic acids). The 13 C enrichment of metabolite markers of biomass stabilized between 5 and 15 atom% 13 C by the end of incubations. Temporal patterns in the 13 C enrichment of tricarboxylic acid cycle intermediates, nucleobases (uracil and thymine), and by-products of DNA salvage (allantoin) closely tracked microbial activity. Our results demonstrate that metabolite production in soils is driven by the carbon source supplied to the community and that the fate of carbon in metabolites do not generally converge over time as a result of ongoing microbial processing and recycling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Low-abundance populations distinguish microbiome performance in plant cell wall deconstruction

Abstract Background Plant cell walls are interwoven structures recalcitrant to degradation. Native and adapted microbiomes can be particularly effective at plant cell wall deconstruction. Although most understanding of biological cell wall deconstruction has been obtained from isolates, cultivated microbiomes that break down cell walls have emerged as new sources for biotechnologically relevant microbes and enzymes. These microbiomes provide a unique resource to identify key interacting functional microbial groups and to guide the design of specialized synthetic microbial communities. Results To establish a system assessing comparative microbiome performance, parallel microbiomes were cultivated on sorghum ( Sorghum bicolor L. Moench) from compost inocula. Biomass loss and biochemical assays indicated that these microbiomes diverged in their ability to deconstruct biomass. Network reconstructions from gene expression dynamics identified key groups and potential interactions within the adapted sorghum-degrading communities, including Actinotalea , Filomicrobium , and Gemmatimonadetes populations. Functional analysis demonstrated that the microbiomes proceeded through successive stages that are linked to enzymes that deconstruct plant cell wall polymers. The combination of network and functional analysis highlighted the importance of cellulose-degrading Actinobacteria in differentiating the performance of these microbiomes. Conclusions The two-tier cultivation of compost-derived microbiomes on sorghum led to the establishment of microbiomes for which community structure and performance could be assessed. The work reinforces the observation that subtle differences in community composition and the genomic content of strains may lead to significant differences in community performance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Diverse MarR bacterial regulators of auxin catabolism in the plant microbiome

Abstract Chemical signalling in the plant microbiome can have drastic effects on microbial community structure, and on host growth and development. Previously, we demonstrated that the auxin metabolic signal interference performed by the bacterial genus Variovorax via an auxin degradation locus was essential for maintaining stereotypic root development in an ecologically relevant bacterial synthetic community. Here, we dissect the Variovorax auxin degradation locus to define the genes iadDE as necessary and sufficient for indole-3-acetic acid (IAA) degradation and signal interference. We determine the crystal structures and binding properties of the operon’s MarR-family repressor with IAA and other auxins. Auxin degradation operons were identified across the bacterial tree of life and we define two distinct types on the basis of gene content and metabolic products: iac -like and iad -like. The structures of MarRs from representatives of each auxin degradation operon type establish that each has distinct IAA-binding pockets. Comparison of representative IAA-degrading strains from diverse bacterial genera colonizing Arabidopsis plants show that while all degrade IAA, only strains containing iad -like auxin-degrading operons interfere with auxin signalling in a complex synthetic community context. This suggests that iad -like operon-containing bacterial strains, including Variovorax species, play a key ecological role in modulating auxins in the plant microbiome.

59 BASIC BIOLOGICAL SCIENCES↗