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At least 991 records · Page 55
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Evolution and engineering of pathways for aromatic O -demethylation in Pseudomonas putida KT2440

In this study, biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However, aromatic compounds derived from lignin commonly contain methoxy groups, and O-demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic O-demethylation, but they are rarely compared in vivo to determine an optimal biocatalytic strategy. Here, two pathways for aromatic O-demethylation were compared in Pseudomonas putida KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in P. putida, and the strains were optimized via adaptive laboratory evolution (ALE) with vanillate as a model substrate. All evolved strains displayed improved growth phenotypes, with the evolved strains harboring the native VanAB pathway exhibiting growth rates ~1.8x faster than those harboring the heterologous LigM pathway. Enzyme kinetics and transcriptomics studies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, the reductase for vanillate O-demethylase, PP_3494, a global regulator of vanillate catabolism, and fghA, involved in formaldehyde detoxification. These three mutations were combined into a single strain, which exhibited approximately 5x faster vanillate consumption than the wild-type strain in the first 8 h of cultivation. Overall, this study illuminates the details of vanillate catabolism in the context of two distinct enzymatic mechanisms, yielding a platform strain for efficient O-demethylation of lignin-related aromatic compounds to value-added products.

09 BIOMASS FUELS↗
DOE OSTI2025

Discovery, characterization, and application of chromosomal integration sites for stable heterologous gene expression in Rhodotorula toruloides

Rhodotorula toruloides is a non-model, oleaginous yeast uniquely suited to produce acetyl-CoA-derived chemicals. However, the lack of well-characterized genomic integration sites has impeded the metabolic engineering of this organism. Here we report a set of computationally predicted and experimentally validated chromosomal integration sites in R. toruloides. We first implemented an in silico platform by integrating essential gene information and transcriptomic data to identify candidate sites that meet stringent criteria. We then conducted a full experimental characterization of these sites, assessing integration efficiency, gene expression levels, impact on cell growth, and long-term expression stability. Among the identified sites, 12 exhibited integration efficiencies of 50% or higher, making them sufficient for most metabolic engineering applications. Using selected high-efficiency sites, we achieved simultaneous double and triple integrations and efficiently integrated long functional pathways (up to 14.7 kb). Additionally, we developed a new inducible marker recycling system that allows multiple rounds of integration at our characterized sites. Here, we validated this system by performing five sequential rounds of GFP integration and three sequential rounds of MaFAR integration for fatty alcohol production, demonstrating, for the first time, precise gene copy number tuning in R. toruloides. These characterized integration sites should significantly advance metabolic engineering efforts and future genetic tool development in R. toruloides.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Important Roles of Thiols in Methylmercury Uptake and Translocation by Rice Plants

We report the bioaccumulation of the neurotoxin methylmercury (MeHg) in rice is a significant concern due to its potential risk to humans. Thiols have been known to affect MeHg bioavailability in microorganisms, but how thiols influence MeHg accumulation in rice plants remains unknown. Here, we investigated effects of common low-molecular-weight thiols, including cysteine (Cys), glutathione (GSH), and penicillamine (PEN), on MeHg uptake and translocation by rice plants. Results show that rice roots can rapidly take up MeHg, and this process is influenced by the types and concentrations of thiols in the system. The presence of Cys facilitated MeHg uptake by roots and translocation to shoots, while GSH could only promote MeHg uptake, but not translocation, by roots. Conversely, PEN significantly inhibited MeHg uptake and translocation to shoots. Using labeled 13 Cys assays, we also found that MeHg uptake was coupled with Cys accumulation in rice roots. Moreover, analyses of comparative transcriptomics revealed that key genes associated with metallothionein and SULTR transporter families may be involved in MeHg uptake. These findings provide new insights into the uptake and translocation of MeHg in rice plants and suggest potential roles of thiol attributes in affecting MeHg bioavailability and bioaccumulation in rice.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Phosphoproteome Response to Dithiothreitol Reveals Unique Versus Shared Features of Saccharomyces cerevisiae Stress Responses

To cope with sudden changes in the external environment, the budding yeast Saccharomyces cerevisiae orchestrates a multifaceted response that spans many levels of physiology. Several studies have interrogated the transcriptome response to endoplasmic reticulum (ER) stress and the role of regulators such as the Ire1 kinase and Hac1 transcription factors. However, less is known about responses to ER stress at other levels of physiology. Here, we used quantitative phosphoproteomics and computational network inference to uncover the yeast phosphoproteome response to the reducing agent dithiothreitol (DTT) and the upstream signaling network that controls it. We profiled wild-type cells and mutants lacking IRE1 or MAPK kinases MKK1 and MKK2, before and at various times after DTT treatment. In addition to revealing downstream targets of these kinases, our inference approach predicted new regulators in the DTT response, including cell-cycle regulator Cdc28 and osmotic-response kinase Rck2, which we validated computationally. Our results also revealed similarities and surprising differences in responses to different stress conditions, especially in the response of protein kinase A targets. Furthermore, these results have implications for the breadth of signaling programs that can give rise to common stress response signatures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Quantitative Proteomics Analysis of Camelina sativa Seeds Overexpressing the AGG3 Gene to Identify the Proteomic Basis of Increased Yield and Stress Tolerance

Camelina sativa , a close relative of Arabidopsis , is an oilseed plant that is emerging as an important biofuel resource. The genome and transcriptome maps of Camelina have become available recently, but its proteome composition remained unexplored. In this work, a labeling LC-based quantitative proteomics approach was applied to decipher the Camelina seed proteome, which led to the identification of 1532 proteins. In addition, the effect of overexpression of the Arabidopsis G-protein γ subunit 3 ( AGG3 ) on the Camelina seed proteome was elucidated to identify the proteomic basis of its increased seed size and improved stress tolerance. The comparative analysis showed a significantly higher expression of proteins involved in primary and secondary metabolism, nucleic acid and protein metabolism, and abscisic acid related responses, corroborating the physiological effects of AGG3 overexpression. More importantly, the proteomic data suggested involvement of the AGG3 protein in the regulation of oxidative stress and heavy metal stress tolerance. These observations were confirmed by the physiological and biochemical characterization of AGG3 -overexpressing seeds, which exhibit a higher tolerance to exogenous cadmium in a glutathione-dependent manner. The activity of multiple redox-regulating enzymes is higher in seeds expressing enhanced levels of AGG3 . Overall, these data provide critical evidence for the role of redox regulation by the AGG3 protein in mediating important seed-related traits.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Generation of Pseudomonas putida KT2440 Strains with Efficient Utilization of Xylose and Galactose via Adaptive Laboratory Evolution

While Pseudomonas putida KT2440 has great potential for biomass-converting processes, its inability to utilize the biomass abundant sugars xylose and galactose has limited its applications. Here, in this study, we utilized Adaptive Laboratory Evolution (ALE) to optimize engineered KT2440 with heterologous expression of xylD encoding xylonate dehydratase from Caulobacter crescentus and galETKM encoding UDP-glucose 4-epimerase, galactose-1-phosphate uridylyltransferase, galactokinase, and galactose-1-epimerase from Escherichia coli K-12 MG1655. Poor starting strain growth (<0.1 h –1 or none) was evolutionarily optimized to rates of up to 0.25 h –1 on xylose and 0.52 h –1 on galactose. Whole-genome sequencing, transcriptomic analysis, and growth screens revealed significant roles of kguT encoding a 2-ketogluconate operon repressor and 2-ketogluconate transporter, and gtsABCD encoding an ATP-binding cassette (ABC) sugar transporting system in xylose and galactose growth conditions, respectively. Finally, we expressed the heterologous indigoidine production pathway in the evolved and unevolved engineered strains and successfully produced 3.2 g/L and 2.2 g/L from 10 g/L of either xylose or galactose in the evolved strains whereas the unevolved strains did not produce any detectable product. Thus, the generated KT2440 strains have the potential for broad application as optimized platform chassis to develop efficient microorganism-based biomass-utilizing bioprocesses.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Quorum-driven microbial consortium for Bioplastic production from agro-waste

Microbial consortia have high relevance in natural environments. Here we present the production of polyhydroxyalkanoates (PHA) from agro-industrial residues by a synthetic interkingdom consortium formed by the saprotrophic fungus Ophiostoma piceae CECT 20146, which encodes a wide range of lignocellulolytic enzymes, and a natural PHA producer, Pseudomonas putida KT2440. Two agro-industrial residues were utilized: Brewer's Spent Grain (BSG) as a carbon/nitrogen source and biofilm scaffold and waste cooking oil (WCO) as a carbon source for PHA synthesis. Through biochemistry, microscopy, and omics analyses, it is shown that P. putida accumulates up to 40.2% of intracellular PHA when the quorum sensing molecule, farnesol (naturally produced by O. piceae) is added, thanks to the increased proliferation of P. putida cells. An interactive Shiny application has also been developed for an easy visualization and comprehension of all the transcriptomics and metabolomics data: https://jgf-bioinformatics.shinyapps.io/Visualization_app/. These results support the increased PHA production of the consortium by an induction of gene phaG, which redirects intermediaries of the fatty acid biosynthesis to PHA precursors, and the repression of the PHA depolymerase phaZ in P. putida. The trophic interaction between microorganisms seems to rely on the citric acid produced by O. piceae and the glycerol liberated from WCO, which can both be consumed by P. putida. Bioreactor scale-up experiments allowed a 3.3-fold increase in the PHA concentration in the consortium (6.7 g·L-1) without pretreatment or sterilization of the substrates, laying the groundwork for the implementation of an industrial consolidated bioprocess (CBP).

Bacteria↗
DOE OSTI2021

An Improved CRISPR Interference Tool to Engineer Rhodococcus opacus

Rhodococcus opacus is a non-model bacterium that is well suited for valorizing lignin. Despite recent advances in our systems-level understanding of its versatile metabolism, studies of its gene functions at a single gene level are still lagging. Elucidating gene functions in non-model organisms is challenging due to limited genetic engineering tools that are convenient to use. To address this issue, we developed a simple gene repression system based on CRISPR interference (CRISPRi). This gene repression system uses a T 7 RNA polymerase system to express a small guide RNA, demonstrating improved repression compared to the previously demonstrated CRISPRi system (i.e., the maximum repression efficiency improved from 58% to 85%). Additionally, our cloning strategy allows for building multiple CRISPRi plasmids in parallel without any PCR step, facilitating the engineering of this GC-rich organism. Using the improved CRISPRi system, we confirmed the annotated roles of four metabolic pathway genes, which had been identified by our previous transcriptomic analysis to be related to the consumption of benzoate, vanillate, catechol, and acetate. Furthermore, we showed our tool’s utility by demonstrating the inducible accumulation of muconate that is a precursor of adipic acid, an important monomer for nylon production. While the maximum muconate yield obtained using our tool was 30% of the yield obtained using gene knockout, our tool showed its inducibility and partial repressibility. In conclusion, our CRISPRi tool will be useful to facilitate functional studies of this non-model organism and engineer this promising microbial chassis for lignin valorization.

09 BIOMASS FUELS↗
DOE OSTI2018

Molecular Toolkit for Gene Expression Control and Genome Modification in Rhodococcus opacus PD630

Rhodococcus opacus PD630 is a non-model, gram-positive bacterium that possesses desirable traits for lignocellulosic biomass conversion. In particular, it has a relatively rapid growth rate, exhibits genetic tractability, produces high quantities of lipids, and can tolerate and consume toxic, lignin-derived aromatic compounds. Despite these unique, industrially relevant characteristics, R. opacus has been underutilized due to a lack of reliable genetic parts and engineering tools. In this work, we developed a molecular toolbox for reliable gene expression control and genome modification in R. opacus. To facilitate predictable gene expression, a constitutive promoter library spanning ~45-fold in output was constructed. To improve the characterization of available plasmids, the copy numbers of four heterologous and nine endogenous plasmids were determined using quantitative PCR. The molecular toolbox was further expanded by screening a previously unreported antibiotic resistance marker (HygR) and constructing a curable plasmid backbone for temporary gene expression (pB264). Furthermore, a system for genome modification was devised, and three neutral integration sites were identified using a novel combination of transcriptomic data, genomic architecture, and growth rate analysis. Finally, the first reported system for targeted, tunable gene repression in Rhodococcus was developed by utilizing CRISPR interference (CRISPRi). Overall, this work greatly expands the ability to manipulate and engineer R. opacus, making it a viable new chassis for bioproduction from renewable feedstocks.

09 BIOMASS FUELS↗
DOE OSTI2021

Spatially Resolved Mass Spectrometry at the Single Cell: Recent Innovations in Proteomics and Metabolomics

Biological systems are composed of heterogeneous populations of cells that intercommunicate to form a functional living tissue. Biological function varies greatly across populations of cells, as each single cell has a unique transcriptome, proteome, and metabolome that translates to functional differences within single species and across kingdoms. Over the past decade, substantial advancements in our ability to characterize omic profiles on a single cell level have occurred, including in multiple spectroscopic and mass spectrometry (MS)-based techniques. Of these technologies, spatially resolved mass spectrometry approaches, including mass spectrometry imaging (MSI), have shown the most progress for single cell proteomics and metabolomics. For example, reporter-based methods using heavy metal tags have allowed for targeted MS investigation of the proteome at the subcellular level, and development of technologies such as laser ablation electrospray ionization mass spectrometry (LAESI-MS) now mean that dynamic metabolomics can be performed in situ. In this Perspective, we showcase advancements in single cell spatial metabolomics and proteomics over the past decade and highlight important aspects related to high-throughput screening, data analysis, and more which are vital to the success of achieving proteomic and metabolomic profiling at the single cell scale. Finally, using this broad literature summary, we provide a perspective on how the next decade may unfold in the area of single cell MS-based proteomics and metabolomics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Characterization and Transcription of Arsenic Respiration and Resistance Genes During In Situ Uranium Bioremediation

The possibility of arsenic release and the potential role of Geobacter in arsenic biogeochemistry during in situ uranium bioremediation was investigated because increased availability of organic matter has been associated with substantial releases of arsenic in other subsurface environments. In a field experiment conducted at the Rifle, CO study site, groundwater arsenic concentrations increased when acetate was added. The number of transcripts from arrA, which codes for the alpha subunit of dissimilatory As(V) reductase, and acr3, which codes for the arsenic pump protein Acr3, were determined with quantitative RT-PCR. Most of the arrA (> 60%) and acr3-1 (> 90%) sequences that were recovered were most similar to Geobacter species, while the majority of acr3-2 (>50%) sequences were most closely related to Rhodoferax ferrireducens. Analysis of transcript abundance demonstrated that transcription of acr3-1 by the subsurface Geobacter community was correlated with arsenic concentrations in the groundwater. In contrast, Geobacter arrA transcript numbers lagged behind the major arsenic release and remained high even after arsenic concentrations declined. This suggested that factors other than As(V) availability regulated transcription of arrA in situ even though the presence of As(V) increased transcription of arrA in cultures of G. lovleyi, which was capable of As(V) reduction. These results demonstrate that subsurface Geobacter species can tightly regulate their physiological response to changes in groundwater arsenic concentrations. The transcriptomic approach developed here should be useful for the study of a diversity of other environments in which Geobacter species are considered to have an important influence on arsenic biogeochemistry.

arsenic resistance↗
DOE OSTI2013

Development of an ecophysiological model for Diplosphaera colotermitum TAV2, a termite hindgut Verrucomicrobium

Termite hindguts are populated by a dense and diverse community of microbial symbionts working in concert to transform lignocellulosic plant material and derived residues into acetate, to recycle and fix nitrogen, and to remove oxygen. Although much has been learned about the breadth of microbial diversity in the hindgut, the ecophysiological roles of its members is less understood. In this study, we present new information about the ecophysiology of microorganism Diplosphaera colotermitum strain TAV2, an autochthonous member of the Reticulitermes flavipes gut community. An integrated high-throughput approach was used to determine the transcriptomic and proteomic profiles of cells grown under hypoxia (2% O2) or atmospheric (20% O2) concentrations of oxygen. Our results revealed that genes and proteins associated with energy production and utilization, carbohydrate transport and metabolism, nitrogen fixation, and replication and recombination were upregulated under 2% O2. The metabolic map developed for TAV2 indicates that this microorganism may be involved in biological nitrogen fixation, amino-acid production, hemicellulose degradation and consumption of O2 in the termite hindgut. Variation of O2 concentration explained 55.9% of the variance in proteomic profiles, suggesting an adaptive evolution of TAV2 to the hypoxic periphery of the hindgut. Our findings advance the current understanding of microaerophilic microorganisms in the termite gut and expand our understanding of the ecological roles for members of the phylum Verrucomicrobia.

Environmental Molecular Sciences Laboratory↗
DOE OSTI2014

Alternatives to vitamin B 1 uptake revealed with discovery of riboswitches in multiple marine eukaryotic lineages

Here, vitamin B 1 (thiamine pyrophosphate, TPP) is essential to all life but scarce in ocean surface waters. In many bacteria and a few eukaryotic groups thiamine biosynthesis genes are controlled by metabolite-sensing mRNA-based gene regulators known as riboswitches. Using available genome sequences and transcriptomes generated from ecologically important marine phytoplankton, we identified 31 new eukaryotic riboswitches. These were found in alveolate, cryptophyte, haptophyte and rhizarian phytoplankton as well as taxa from two lineages previously known to have riboswitches (green algae and stramenopiles). The predicted secondary structures bear hallmarks of TPP-sensing riboswitches. Surprisingly, most of the identified riboswitches are affiliated with genes of unknown function, rather than characterized thiamine biosynthesis genes. Using qPCR and growth experiments involving two prasinophyte algae, we show that expression of these genes increases significantly under vitamin B 1 -deplete conditions relative to controls. Pathway analyses show that several algae harboring the uncharacterized genes lack one or more enzymes in the known TPP biosynthesis pathway. We demonstrate that one such alga, the major primary producer Emiliania huxleyi , grows on 4-amino-5-hydroxymethyl-2-methylpyrimidine (a thiamine precursor moiety) alone, although long thought dependent on exogenous sources of thiamine. Thus, overall, we have identified riboswitches in major eukaryotic lineages not known to undergo this form of gene regulation. In these phytoplankton groups, riboswitches are often affiliated with widespread thiamine-responsive genes with as yet uncertain roles in TPP pathways. Further, taxa with ‘incomplete’ TPP biosynthesis pathways do not necessarily require exogenous vitamin B 1 , making vitamin control of phytoplankton blooms more complex than the current paradigm suggests.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Impacts of chemical gradients on microbial community structure

Succession of redox processes is sometimes assumed to define a basic microbial community structure for ecosystems with oxygen gradients. In this paradigm, aerobic respiration, denitrification, fermentation and sulfate reduction proceed in a thermodynamically determined order, known as the ‘redox tower’. Here, we investigated whether redox sorting of microbial processes explains microbial community structure at low-oxygen concentrations. We subjected a diverse microbial community sampled from a coastal marine sediment to 100 days of tidal cycling in a laboratory chemostat. Oxygen gradients (both in space and time) led to the assembly of a microbial community dominated by populations that each performed aerobic and anaerobic metabolism in parallel. This was shown by metagenomics, transcriptomics, proteomics and stable isotope incubations. Effective oxygen consumption combined with the formation of microaggregates sustained the activity of oxygen-sensitive anaerobic enzymes, leading to braiding of unsorted redox processes, within and between populations. Analyses of available metagenomic data sets indicated that the same ecological strategies might also be successful in some natural ecosystems.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2012

Model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation

Macrophages are central players in the immune response, manifesting divergent phenotypes to control inflammation and innate immunity through the release of cytokines and other regulatory factor-dependent signaling pathways. In recent years, the focus on metabolism has been reemphasized as critical signaling and regulatory pathways of human pathophysiology, ranging from cancer to aging, often converge on metabolic responses. Here, we used genome-scale modeling and multi-omics (transcriptomics, proteomics, and metabolomics) analysis to assess metabolic features critical for macrophage functions. We constructed a genome-scale metabolic network for the RAW 264.7 cell line to determine metabolic modulators of macrophage activation. Metabolites well-known to be associated with immunoactivation (e.g., glucose and arginine) and immunosuppression (e.g., tryptophan and vitamin D3) were amongst the most critical effectors. Intracellular metabolic mechanisms linked to critical suppressive effectors were then assessed, identifying a suppressive role for de novo nucleotide synthesis. Finally, the underlying metabolic mechanisms of macrophage activation are identified by analyzing multi-omic data obtained from LPS-stimulated RAW cells in the context of our flux-based predictions. Our study demonstrates metabolism's role in regulating activation may be greater than previously anticipated and elucidates underlying metabolic connections between activation and metabolic effectors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas

Here we identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu + accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu + became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Joint mouse–human phenome-wide association to test gene function and disease risk

Phenome-wide association is a novel reverse genetic strategy to analyze genome-to-phenome relations in human clinical cohorts. Here we test this approach using a large murine population segregating for ~5 million sequence variants, and we compare our results to those extracted from a matched analysis of gene variants in a large human cohort. For the mouse cohort, we amassed a deep and broad open-access phenome consisting of ~4,500 metabolic, physiological, pharmacological and behavioural traits, and more than 90 independent expression quantitative trait locus (QTL), transcriptome, proteome, metagenome and metabolome data sets-by far the largest coherent phenome for any experimental cohort (www.genenetwork.org). Here, we tested downstream effects of subsets of variants and discovered several novel associations, including a missense mutation in fumarate hydratase that controls variation in the mitochondrial unfolded protein response in both mouse and Caenorhabditis elegans , and missense mutations in Col6a5 that underlies variation in bone mineral density in both mouse and human.

59 BASIC BIOLOGICAL SCIENCES↗