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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 811 records · Page 45
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Indirect Interspecies Regulation: Transcriptional and Physiological Responses of a Cyanobacterium to Heterotrophic Partnership

This study elucidates how a cyanobacterial primary producer acclimates to heterotrophic partnership by modulating the expression levels of key metabolic genes. Heterotrophic bacteria can indirectly regulate the physiology of the photoautotrophic primary producers, resulting in physiological changes identified here, such as increased intracellular ROS. Some of the interactions inferred from this model system represent putative principles of metabolic coupling in phototrophic-heterotrophic partnerships. The mechanisms by which microbes interact in communities remain poorly understood. Here, we interrogated specific interactions between photoautotrophic and heterotrophic members of a model consortium to infer mechanisms that mediate metabolic coupling and acclimation to partnership. This binary consortium was composed of a cyanobacterium, Thermosynechococcus elongatus BP-1, which supported growth of an obligate aerobic heterotroph, Meiothermus ruber strain A, by providing organic carbon, O 2 , and reduced nitrogen. Species-resolved transcriptomic analyses were used in combination with growth and photosynthesis kinetics to infer interactions and the environmental context under which they occur. We found that the efficiency of biomass production and resistance to stress induced by high levels of dissolved O 2 increased, beyond axenic performance, as a result of heterotrophic partnership. Coordinated transcriptional responses transcending both species were observed and used to infer specific interactions resulting from the synthesis and exchange of resources. The cyanobacterium responded to heterotrophic partnership by altering expression of core genes involved with photosynthesis, carbon uptake/fixation, vitamin synthesis, and scavenging of reactive oxygen species (ROS). IMPORTANCEThis study elucidates how a cyanobacterial primary producer acclimates to heterotrophic partnership by modulating the expression levels of key metabolic genes. Heterotrophic bacteria can indirectly regulate the physiology of the photoautotrophic primary producers, resulting in physiological changes identified here, such as increased intracellular ROS. Some of the interactions inferred from this model system represent putative principles of metabolic coupling in phototrophic-heterotrophic partnerships.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Genome-Wide Analysis of RNA Decay in the Cyanobacterium Synechococcus sp. Strain PCC 7002

ABSTRACT RNA degradation is an important process that influences the ultimate concentration of individual proteins inside cells. While the main enzymes that facilitate this process have been identified, global maps of RNA turnover are available for only a few species. Even in these cases, there are few sequence elements that are known to enhance or destabilize a native transcript; even fewer confer the same effect when added to a heterologous transcript. To address this knowledge gap, we assayed genome-wide RNA degradation in the cyanobacterium Synechococcus sp. strain PCC 7002 by collecting total RNA samples after stopping nascent transcription with rifampin. We quantified the abundance of each position in the transcriptome as a function of time using RNA-sequencing data and later analyzed the global mRNA decay map using machine learning principles. Half-lives, calculated on a per-ORF (open reading frame) basis, were extremely short, with a median half-life of only 0.97 min. Despite extremely rapid turnover of most mRNA, transcripts encoding proteins involved in photosynthesis were both highly expressed and highly stable. Upon inspection of these stable transcripts, we identified an enriched motif in the 3′ untranslated region (UTR) that had similarity to Rho-independent terminators. We built statistical models for half-life prediction and used them to systematically identify sequence motifs in both 5′ and 3′ UTRs that correlate with stabilized transcripts. We found that transcripts linked to a terminator containing a poly(U) tract had a longer half-life than both those without a poly(U) tract and those without a terminator. IMPORTANCE RNA degradation is an important process that affects the final concentration of individual mRNAs, affecting protein expression and cellular physiology. Studies of how RNA is degraded increase our knowledge of this fundamental process as well as enable the creation of genetic tools to manipulate RNA stability. By studying global transcript turnover, we searched for sequence elements that correlated with transcript (in)stability and used these sequences to guide tool design. This study probes global RNA turnover in a cyanobacterium, Synechococcus sp. strain PCC 7002, that both has a unique array of RNases that facilitate RNA degradation and is an industrially relevant strain that could be used to convert CO 2 and sunlight into useful products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A Gene Cluster That Encodes Histone Deacetylase Inhibitors Contributes to Bacterial Persistence and Antibiotic Tolerance in Burkholderia thailandensis

Persister cells are genetically identical variants in a bacterial population that have phenotypically modified their physiology to survive environmental stress. In bacterial pathogens, persisters are able to survive antibiotic treatment and reinfect patients in a frustrating cycle of chronic infection. To better define core persistence mechanisms for therapeutics development, we performed transcriptomics analyses of Burkholderia thailandensis populations enriched for persisters via three methods: flow sorting for low proton motive force, meropenem treatment, and culture aging. Although the three persister-enriched populations generally displayed divergent gene expression profiles that reflect the multimechanistic nature of stress adaptations, there were several common gene pathways activated in two or all three populations. These include polyketide and nonribosomal peptide synthesis, Clp proteases, mobile elements, enzymes involved in lipid metabolism, and ATP binding cassette (ABC) transporter systems. In particular, identification of genes that encode polyketide synthases (PKSs) and fatty acid catabolism factors indicates that generation of secondary metabolites, natural products, and complex lipids could be part of the metabolic program that governs the persistence state. We also found that loss-of-function mutations in the PKS-encoding gene locus BTH_I2366, which plays a role in biosynthesis of histone deacetylase (HDAC) inhibitors, resulted in increased sensitivity to antibiotics targeting DNA replication. Furthermore, treatment of multiple bacterial pathogens with a fatty acid synthesis inhibitor, CP-640186, potentiated the efficacy of meropenem against the persister populations. Altogether, our results suggest that bacterial persisters may exhibit an outwardly dormant physiology but maintain active metabolic processes that are required to maintain persistence.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Sulfur Metabolites Play Key System-Level Roles in Modulating Denitrification

Competition between nitrate-reducing bacteria (NRB) and sulfate-reducing bacteria (SRB) for resources in anoxic environments is generally thought to be governed largely by thermodynamics. It is now recognized that intermediates of nitrogen and sulfur cycling (e.g., hydrogen sulfide, nitrite, etc.) can also directly impact NRB and SRB activities in freshwater, wastewater, and sediment and therefore may play important roles in competitive interactions. Here, through comparative transcriptomic and metabolomic analyses, we have uncovered mechanisms of hydrogen sulfide- and cysteine-mediated inhibition of nitrate respiratory growth for the NRB Intrasporangium calvum C5. Specifically, the systems analysis predicted that cysteine and hydrogen sulfide inhibit growth of I. calvum C5 by disrupting distinct steps across multiple pathways, including branched-chain amino acid (BCAA) biosynthesis, utilization of specific carbon sources, and cofactor metabolism. Here, we have validated these predictions by demonstrating that complementation with BCAAs and specific carbon sources relieves the growth inhibitory effects of cysteine and hydrogen sulfide. We discuss how these mechanistic insights give new context to the interplay and stratification of NRB and SRB in diverse environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Gene co-expression network analysis in Rhodobacter capsulatus and application to comparative expression analysis of Rhodobacter sphaeroides

The genus Rhodobacter contains purple nonsulfur bacteria found mostly in freshwater environments. Representative strains of two Rhodobacter species, R. capsulatus and R. sphaeroides, have had their genomes fully sequenced and both have been the subject of transcriptional profiling studies. Gene co-expression networks can be used to identify modules of genes with similar expression profiles. Functional analysis of gene modules can then associate co-expressed genes with biological pathways, and network statistics can determine the degree of module preservation in related networks. In this paper, we constructed an R. capsulatus gene co-expression network, performed functional analysis of identified gene modules, and investigated preservation of these modules in R. capsulatus proteomics data and in R. sphaeroides transcriptomics data. Results: The analysis identified 40 gene co-expression modules in R. capsulatus. Investigation of the module gene contents and expression profiles revealed patterns that were validated based on previous studies supporting the biological relevance of these modules. We identified two R. capsulatus gene modules preserved in the protein abundance data. We also identified several gene modules preserved between both Rhodobacter species, which indicate that these cellular processes are conserved between the species and are candidates for functional information transfer between species. Many gene modules were non-preserved, providing insight into processes that differentiate the two species. In addition, using Local Network Similarity (LNS), a recently proposed metric for expression divergence, we assessed the expression conservation of between-species pairs of orthologs, and within-species gene-protein expression profiles. Conclusions: Our analyses provide new sources of information for functional annotation in R. capsulatus because uncharacterized genes in modules are now connected with groups of genes that constitute a joint functional annotation. We identified R. capsulatus modules enriched with genes for ribosomal proteins, porphyrin and bacteriochlorophyll anabolism, and biosynthesis of secondary metabolites to be preserved in R. sphaeroides whereas modules related to RcGTA production and signalling showed lack of preservation in R. sphaeroides. In addition, we demonstrated that network statistics may also be applied within-species to identify congruence between mRNA expression and protein abundance data for which simple correlation measurements have previously had mixed results.

Comparative transcriptomics, Module preservation, ↗
DOE OSTI2018

Widespread signatures of selection for secreted peptidases in a fungal plant pathogen

Background: Fungal plant pathogens secrete a large arsenal of hydrolytic enzymes during the course of infection, including peptidases. Secreted peptidases have been extensively studied for their role as effectors. In this study, we combined transcriptomics, comparative genomics and evolutionary analyses to investigate all 39 secreted peptidases in the fungal wheat pathogen Zymoseptoria tritici and its close relatives Z. pseudotritici and Z. ardabiliae. Results: RNA-seq data revealed that a majority of the secreted peptidases displayed differential transcription during the course of Z. tritici infection, indicative of specialization for different stages in the life cycle. Evolutionary analyses detected widespread evidence of adaptive evolution acting on at least 28 of the peptidases. A few peptidases displayed lineage-specific rates of molecular evolution, suggesting altered selection pressure in Z. tritici following host specialization on domesticated wheat. The peptidases belonging to MEROPS families A1 and G1 emerged as a particularly interesting group that may play key roles in host-pathogen co-evolution, host adaptation and pathogenicity. Sister genes in the A1 and G1 families showed accelerated substitution rates after gene duplications. Conclusions: These results suggest widespread evolution of secreted peptidases leading to novel gene functions, consistent with predicted models of “escape from adaptive conflict” and “neo-functionalization”. Our analyses identified candidate genes worthy of functional analyses that may encode effector functions, for example by suppressing plant defenses during the biotrophic phase of infection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Alterations in tissue microRNA after heat stress in the conscious rat: potential biomarkers of organ-specific injury

Heat illness remains a significant cause of morbidity in susceptible populations. Recent research elucidating the cellular mechanism of heat stress leading to heat illness may provide information to develop better therapeutic interventions, risk assessment strategies, and early biomarkers of organ damage. microRNA (miRNA) are promising candidates for therapeutic targets and biomarkers for a variety of clinical conditions since there is the potential for high specificity for individual tissues and unique cellular functions. The objective of this study was to identify differentially expressed microRNAs and their putative mRNA targets in the heart, liver, kidney, and lung in rats at three time points: during heat stress (i.e., when core temperature reached 41.8 °C), or following a 24 or 48 h recovery period. Rats did not show histological evidence of tissue pathology until 48 h after heat stress, with 3 out of 6 rats showing cardiac inflammation and renal proteinosis at 48 h. The three rats with cardiac and renal pathology had 86, 7, 159, and 37 differentially expressed miRNA in the heart, liver, kidney, or lung, respectively compared to non-heat stressed control animals. During heat stress one differentially expressed miRNA was found in the liver and five in the lung, with no other modulated miRNA after 24 h or 48 h in animals with no evidence of organ injury. Pathway enrichment analysis revealed enrichment in functional pathways associated with heat stress, with the greatest effects observed in animals with histological evidence of cardiac and renal damage at 48 h. Inhibiting miR-21 in cultured cardiomyocytes increased the percent apoptotic cells five hours after heat stress from 70.9 ± 0.8 to 84.8 ± 2.2%. Global microRNA and transcriptomics analysis suggested that perturbed miRNA due to heat stress are involved in biological pathways related to organ injury, energy metabolism, the unfolded protein response, and cellular signaling. These miRNA may serve as biomarkers of organ injury and potential pharmacological targets for preventing heat illness or organ injury.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Poxvirus infection triggers remodeling of host m⁶A epitranscriptome and benefits from the m⁶A regulatory responses

Understanding how host gene regulation responds to viral infection is essential for developing effective antiviral strategies. Emerging evidence suggests that host transcripts undergo dynamic chemical modifications to counteract viral invasion. Conversely, viruses that rely on nuclear transcription exploit host RNA methyltransferases to enhance mRNA export and translation. Orthopoxviruses, however, complete their entire replication cycle within compartmentalized cytoplasmic “factories” utilizing enzymes encoded by their large double-stranded viral DNA genomes. The dynamic interplay between host and poxviral epitranscriptome remains poorly characterized. Using a temporally resolved model of Vaccinia virus (VV) infection, we investigated host-virus interactions through transcriptome and N6-methyladenosine (m⁶A) epitranscriptome whole genome sequencing. We found that host m⁶A modifications respond rapidly to VV infection, preceding the delayed transcriptional changes that emerge at later stages. Early m⁶A signatures included key innate immunity factors as well as host genes involved in transcriptional regulation, post-transcriptional modification, and protein ubiquitination. Functional assays validated two host factors with early m⁶A modification changes that are essential for VV infection: a m⁶A reader, YTHDF1, and a component of the SCF E3 ubiquitin ligase complex, FBXO31. The m⁶A gain on YTHDF1 enhanced its protein expression and promoted efficient VV replication. In addition, we identified previously unrecognized roles of FBXO31 and the SCF E3 ligase complex in supporting VV infection. Temporal profiling of the m⁶A epitranscriptome reveals how VV exploits host post-transcriptional regulatory pathways, specifically m⁶A RNA modification and protein ubiquitination. These findings highlight critical host factors co-opted during poxvirus infection and identify potential targets for therapeutic intervention.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Data for System Analysis of Lipomyces starkeyi During Growth on Various Plant‑Based Sugars

Oleaginous yeasts have received significant attention due to their substantial lipid storage capability. The accumulated lipids can be utilized directly or processed into various bioproducts and biofuels. Lipomyces starkeyi is an oleaginous yeast capable of using multiple plant-based sugars, such as glucose, xylose, and cellobiose. It is, however, a relatively unexplored yeast due to limited knowledge about its physiology. In this study, we have evaluated the growth of L. starkeyi on different sugars and performed transcriptomic and metabolomic analyses to understand the underlying mechanisms of sugar metabolism. Principal component analysis showed clear differences resulting from growth on different sugars. We have further reported various metabolic pathways activated during growth on these sugars. We also observed non-specific regulation in L. starkeyi and have updated the gene annotations for the NRRL Y-11557 strain. This analysis provides a foundation for understanding the metabolism of these plant-based sugars and potentially valuable information to guide the metabolic engineering of L. starkeyi to produce bioproducts and biofuels.

Conversion↗
DOE OSTI2025

Data for 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. 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 .

Conversion↗
DOE OSTI2014

Tissue-Specific RNA Expression Marks Distant-Acting Developmental Enhancers

Short non-coding transcripts can be transcribed from distant-acting transcriptional enhancer loci, but the prevalence of such enhancer RNAs (eRNAs) within the transcriptome, and the association of eRNA expression with tissue-specific enhancer activity in vivo remain poorly understood. Here, we investigated the expression dynamics of tissue-specific non-coding RNAs in embryonic mouse tissues via deep RNA sequencing. Overall, approximately 80% of validated in vivo enhancers show tissue-specific RNA expression that correlates with tissue-specific enhancer activity. Globally, we identified thousands of tissue-specifically transcribed non-coding regions (TSTRs) displaying various genomic hallmarks of bona fide enhancers. In transgenic mouse reporter assays, over half of tested TSTRs functioned as enhancers with reproducible activity in the predicted tissue. Together, our results demonstrate that tissue-specific eRNA expression is a common feature of in vivo enhancers, as well as a major source of extragenic transcription, and that eRNA expression signatures can be used to predict tissue-specific enhancers independent of known epigenomic enhancer marks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Comparison of Intact Arabidopsis thaliana Leaf Transcript Profiles during Treatment with Inhibitors of Mitochondrial Electron Transport and TCA Cycle

Plant mitochondria signal to the nucleus leading to altered transcription of nuclear genes by a process called mitochondrial retrograde regulation (MRR). MRR is implicated in metabolic homeostasis and responses to stress conditions. Mitochondrial reactive oxygen species (mtROS) are a MRR signaling component, but whether all MRR requires ROS is not established. Inhibition of the cytochrome respiratory pathway by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA), each of which initiates MRR, can increase ROS production in some plant cells. We found that for AA and MFA applied to leaves of soil-grown Arabidopsis thaliana plants, ROS production increased with AA, but not with MFA, allowing comparison of transcript profiles under different ROS conditions during MRR. Variation in transcript accumulation over time for eight nuclear encoded mitochondrial protein genes suggested operation of both common and distinct signaling pathways between the two treatments. Consequences of mitochondrial perturbations for the whole transcriptome were examined by microarray analyses. Expression of 1316 and 606 genes was altered by AA and MFA, respectively. A subset of genes was similarly affected by both treatments, including genes encoding photosynthesis-related proteins. MFA treatment resulted in more down-regulation. Functional gene category (MapMan) and cluster analyses showed that genes with expression levels affected by perturbation from AA or MFA inhibition were most similarly affected by biotic stresses such as pathogens. Overall, the data provide further evidence for the presence of mtROS-independent MRR signaling, and support the proposed involvement of MRR and mitochondrial function in plant responses to biotic stress.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Identification of a novel nematotoxic protein by challenging the model mushroom Coprinopsis cinerea with a fungivorous nematode

The dung of herbivores, the natural habitat of the model mushroom Coprinopsis cinerea, is a nutrient-rich but also very competitive environment for a saprophytic fungus. Previously we showed that C. cinerea expresses constitutive, tissue-specific armories against antagonists such as animal predators and bacterial competitors. In order to dissect the inducible armories against such antagonists, we sequenced the poly(A)-positive transcriptome of C. cinerea vegetative mycelium upon challenge with fungivorous and bacterivorous nematodes, Gram-negative and Gram-positive bacteria and mechanical damage. As a response to the fungivorous nematode Aphelenchus avenae, C. cinerea was found to specifically induce the transcription of several genes encoding previously characterized nematotoxic lectins. In addition, a previously not characterized gene encoding a cytoplasmic protein with several predicted Ricin B-fold domains, was found to be strongly up-regulated under this condition. Functional analysis of the recombinant protein revealed a high toxicity towards the bacterivorous nematode Caenorhabditis elegans. Challenge of the mycelium with A. avenae also lead to the induction of several genes encoding putative antibacterial proteins. Some of these genes were also induced upon challenge of the mycelium with the bacteria Escherichia coli and Bacillus subtilis. Lastly, these results suggest that fungi have the ability to induce specific innate defense responses similar to plants and animals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Computational Resources for Biofuel Feedstock Species

While current production of ethanol as a biofuel relies on starch and sugar inputs, it is anticipated that sustainable production of ethanol for biofuel use will utilize lignocellulosic feedstocks. Candidate plant species to be used for lignocellulosic ethanol production include a large number of species within the Grass, Pine and Birch plant families. For these biofuel feedstock species, there are variable amounts of genome sequence resources available, ranging from complete genome sequences (e.g. sorghum, poplar) to transcriptome data sets (e.g. switchgrass, pine). These data sets are not only dispersed in location but also disparate in content. It will be essential to leverage and improve these genomic data sets for the improvement of biofuel feedstock production. The objectives of this project were to provide computational tools and resources for data-mining genome sequence/annotation and large-scale functional genomic datasets available for biofuel feedstock species. We have created a Bioenergy Feedstock Genomics Resource that provides a web-based portal or clearing house for genomic data for plant species relevant to biofuel feedstock production. Sequence data from a total of 54 plant species are included in the Bioenergy Feedstock Genomics Resource including model plant species that permit leveraging of knowledge across taxa to biofuel feedstock species. We have generated additional computational analyses of these data, including uniform annotation, to facilitate genomic approaches to improved biofuel feedstock production. These data have been centralized in the publicly available Bioenergy Feedstock Genomics Resource (http://bfgr.plantbiology.msu.edu/).

09 BIOMASS FUELS↗
DOE OSTI2020

The Brachypodium ENCODE Project – from sequence to function: Predicting physiological responses in grasses to facilitate engineering of biofuel crops

The overarching goal of this proposal was to gain deep knowledge of chromatin dynamics and gene networks related to stress responses in grasses. To accomplish this goal our Brachypodium ENCODE project (1) coupled drought-focused physiological analysis with multiple layers of ENCODE ‘omics’ profiling; (2) obtained fundamental information about the Brachypodium transcriptome, epigenome, chromatin dynamics, and gene regulatory networks; and (3) elucidated the regulatory dynamics of gene networks that can be manipulated to increase energy grass crop productivity.

09 BIOMASS FUELS↗
DOE OSTI2021

Epigenetic Control of Drought Response in Sorghum (EPICON)

Genetic manipulation of crops to increase the presence of desirable traits has been critical to increasing agricultural productivity. These changes have primarily involved modification of the plant’s DNA sequence. However, there is increasing evidence that environmental responses are also mediated by epigenetics, which involves heritable changes without changes in DNA sequence. Epigenetic changes have been shown to play a major role in regulating plant responses to drought, an increasing problem worldwide due to climate change. In general, exposure of plants to water limitation triggers epigenetic changes, which include remodeling of chromatin, the network of DNA, RNA and various proteins making up chromosomes, and related changes in regulatory mechanisms. EPICON’s efforts focus on unraveling the role epigenetic signals play in acclimation to and recovery from drought through effects on individual transcription factors or transcriptional networks that direct entire metabolic pathways. To achieve this goal we will follow responses to water deprivation in sorghum, a widely cultivated cereal with recognized drought tolerance. In EPICON’s field trials, sorghum will be grown under controlled irrigation conditions. Leaf and root samples will be taken to perform molecular phenotyping to track changes in epigenetic, transcriptomic, metabolomic and proteomic footprints. Analysis of this data will provide a better understanding of the epigenetic processes related to drought tolerance, leading to our ultimate goal of identifying transcriptional regulators and pathways controlling drought resistance. The identified genetic targets and their regulatory pathways will be used in future efforts to improve growth of sorghum and other crops in the field and in marginal lands under water-limiting conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Systems Engineering of Rhodococcus opacus to Enable Production of Drop-in Fuels from Lignocellulose

Production of drop-in fuels from lignocellulose using Rhodococcus opacus PD630 (hereafter R. opacus) is a challenging goal. During the grant period we have pushed the field forward significantly in several areas of research. Towards the end goal of accelerating the adoption of R. opacus in biofuel production, during the grant period we have expanded the phenotypic characterization of R. opacus grown in single aromatic (model lignocellulosic) compounds or their mixtures, modeling the growth conditions in lignocellulosic biomass. Harnessing the power of adaptive evolution, we produced evolved R. opacus isolates with superior lignin valorization capabilities and identified differentially expressed genes and pathways after adaptation. We used next generation multi-omic techniques such as genomic, transcriptomic, and metabolomic analyses, to identify the catabolic pathways used by R. opacus to degrade aromatic compounds and funnel these degradation products into central metabolism, as well as the aromatic transport genes required for increased tolerance and utilization. Taking this information one step further, we identified endogenous transcription factors and regulatory mechanisms important for degradation of five model aromatic compounds. To accurately estimate R. opacus growth and consumption on model lignin compounds we pioneered the use of novel extraction procedures prior to GC-MS analysis. Alongside 13 C-metabolic flux analysis, we have elucidated the metabolic routes preferred by Rhodococcus opacus during aromatic compound degradation. Finally, we used in tandem lipidomics and high-resolution mass spectrometry to identify the modulation of mycolic acids and phospholipid membrane composition modification as a strategy for aromatic tolerance in R. opacus. Being a non-model organism, R. opacus lacks the breadth of tools and technical foundation which drive biofuel research in more well-understood microbes such as Escherichia coli. To reduce this burden for use, we designed and produced new tools for genomic manipulation and engineering in R. opacus. These engineering breakthroughs support efficient genomic editing, enabling gene overexpression, repression, and genetic alteration. Using these tools, we have generated synthetically engineered strains with increased lipogenesis and growth, both positive traits required for increased lignin valorization. Optimizing engineered strains for biofuel production from lignocellulose requires extremely sophisticated synthetic rewiring of metabolism. To facilitate systems-level reorganization of metabolism in R. opacus, we created a genome-scale model that accurately predicts metabolic flux and growth rates on the aromatic compound phenol. Lignin requires extensive pre-treatment before biological degradation by R. opacus. Towards an eventual goal of degrading real-world lignin, we developed new depolymerization processes to generate lignin breakdown products (LBP). We optimized LBP storage and composition analysis techniques, enabling accurate prediction of specific LBP compound integration into cell wall components. Overall, through the work funded by this grant we generated 20 manuscripts (17 published, 3 in review/preparation), methods for increased accuracy in metabolomics of aromatic compounds, multiple genetic tools for altering the R. opacus genome, genome scale models for predicting flux through metabolic pathways, as well as multi-omic data for community use. The work funded by this grant has increased the knowledge of aromatic degradation in bacteria and advanced our efforts to optimize R. opacus for lignin valorization.

09 BIOMASS FUELS↗