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At least 397 records · Page 22

Metabolic dependencies govern microbial syntrophies during methanogenesis in an anaerobic digestion ecosystem

Methanogenesis, a biological process mediated by complex microbial communities, has attracted great attention due to its contribution to global warming and potential in biotechnological applications. The current study unveiled the core microbial methanogenic metabolisms in anaerobic vessel ecosystems by applying combined genome-centric metagenomics and metatranscriptomics. Here, we demonstrate that an enriched natural system, fueled only with acetate, could support a bacteria-dominated microbiota employing a multi-trophic methanogenic process. Moreover, significant changes, in terms of microbial structure and function, were recorded after the system was supplemented with additional H 2 . Methanosarcina thermophila, the predominant methanogen prior to H 2 addition, simultaneously performed acetoclastic, hydrogenotrophic, and methylotrophic methanogenesis. The methanogenic pattern changed after the addition of H 2 , which immediately stimulated Methanomicrobia-activity and was followed by a slow enrichment of Methanobacteria members. Interestingly, the essential genes involved in the Wood-Ljungdahl pathway were not expressed in bacterial members. The high expression of a glycine cleavage system indicated the activation of alternative metabolic pathways for acetate metabolism, which were reconstructed in the most abundant bacterial genomes. Moreover, as evidenced by predicted auxotrophies, we propose that specific microbes of the community were forming symbiotic relationships, thus reducing the biosynthetic burden of individual members. These results provide new information that will facilitate future microbial ecology studies of interspecies competition and symbiosis in methanogenic niches.

59 BASIC BIOLOGICAL SCIENCES↗

Roundup causes embryonic development failure and alters metabolic pathways and gut microbiota functionality in non-target species

Background: Research around the weedkiller Roundup is among the most contentious of the twenty-first century. Scientists have provided inconclusive evidence that the weedkiller causes cancer and other life-threatening diseases, while industry-paid research reports that the weedkiller has no adverse effect on humans or animals. Much of the controversial evidence on Roundup is rooted in the approach used to determine safe use of chemicals, defined by outdated toxicity tests. We apply a system biology approach to the biomedical and ecological model species Daphnia to quantify the impact of glyphosate and of its commercial formula, Roundup, on fitness, genome-wide transcription and gut microbiota, taking full advantage of clonal reproduction in Daphnia. We then apply machine learning-based statistical analysis to identify and prioritize correlations between genome-wide transcriptional and microbiota changes. Results: We demonstrate that chronic exposure to ecologically relevant concentrations of glyphosate and Roundup at the approved regulatory threshold for drinking water in the US induce embryonic developmental failure, induce significant DNA damage (genotoxicity), and interfere with signaling. Furthermore, chronic exposure to the weedkiller alters the gut microbiota functionality and composition interfering with carbon and fat metabolism, as well as homeostasis. Using the “Reactome,” we identify conserved pathways across the Tree of Life, which are potential targets for Roundup in other species, including liver metabolism, inflammation pathways, and collagen degradation, responsible for the repair of wounds and tissue remodeling. Conclusions: Our results show that chronic exposure to concentrations of Roundup and glyphosate at the approved regulatory threshold for drinking water causes embryonic development failure and alteration of key metabolic functions via direct effect on the host molecular processes and indirect effect on the gut microbiota. The ecological model species Daphnia occupies a central position in the food web of aquatic ecosystems, being the preferred food of small vertebrates and invertebrates as well as a grazer of algae and bacteria. The impact of the weedkiller on this keystone species has cascading effects on aquatic food webs, affecting their ability to deliver critical ecosystem services.

59 BASIC BIOLOGICAL SCIENCES↗

Data for Bioprocessing, Recovery, and Mass Balance of Vegetative Lipids from Metabolically Engineered ‘Oilcane’ Demonstrates Its Potential as an Alternative Feedstock for Drop-In Fuel Production

Plant oils are increasingly in demand as renewable feedstocks for biodiesel and biochemicals. Currently, oilseeds are the primary source of plant oils. Although the vegetative tissues of plants express lipid metabolism pathways, they do not hyper-accumulate lipids. Elevated synthesis, storage, and accumulation of lipids in vegetative tissues have been achieved by metabolic engineering of sugarcane to produce “oilcane.” This study evaluates the potential of oilcane as a renewable feedstock for the co-production of lipids and fermentable sugars. Oilcane was grown under favorable climatic and field conditions in Florida (FLOC) as well as during an abbreviated growing season, outside its typical growing region, in Illinois (ILOC). The potential lipid yield of 0.35 tons/ha was projected from the hyperaccumulation of fatty acids in the stored vegetative biomass of FLOC, which is approaching the lipid yield of soybean (0.44 tons/ha). Processing of the vegetative tissues of oilcane recovered 0.20 tons/ha, which represents the recovery of 55% of the total lipids from FLOC. Chemical-free hydrothermal bioprocessing of ILOC and FLOC bagasse and leaves at 180 °C for 10 min prevented the degeneration of in situ plant lipids. This allowed the recovery of lipids at the end of the bioprocess with a major fraction of lipids remaining in the biomass residues after pretreatment and saccharification. Improvements through refined biomass processing, crop management, and metabolic engineering are expected to boost lipid yields and make oilcane a prime feedstock for the production of biodiesel.

Conversion↗

Development and implementation of an in situ high-resolution isotopic microscope for measuring metabolic interactions in soil mesocosms (Final Report)

Many of our planet’s ecosystems rely on the activities of soil microbial communities. These microbes have significant and wide-ranging effects: they metabolize carbon and other nutrients, interact with plants and fungi, and perform other processes important to soil health. However, our ability to directly observe the enzymatic and metabolic activities of microbes within soil is currently limited, not only by the complexity of soil microbial communities themselves, but also by the lack of experimental tools to study them and their molecular interactions in situ. These challenges hinder our understanding of the lifesustaining processes of biomass decomposition and the manner in which it contributes to the movement of freed carbon within soil ecosystems. Here we aim to develop a novel ultrahigh-resolution isotopic microscope that combines complementary imaging modalities to gain insights into metabolic cycling in soil. This final report covers the portion of this work that was completed at the University of North Carolina at Chapel Hill from 2018 to 2019; the research is continuing through 2022 at the University of Massachusetts Chan Medical School.

59 BASIC BIOLOGICAL SCIENCES↗

Increasing bacterial tolerance and metabolism of the biofuel, n-butanol using community-level evolution and functional genomics

n-butanol is a strong biofuel candidate since it is more energy dense and less volatile than ethanol. The bottleneck for industrial production of biobutanol is the toxicity; most microbes cannot survive about 1.5% v/v. We propose to develop bioremediation capability along with the biofuel technology to mitigate future spills and their consequences. Two main approaches were used: community-level screening and functional genomics, both followed by directed evolution to increase biodegradation. For the first approach, a bacterial library from historic bioremediation projects was re-isolated and re-characterized, then mixed into artificial communities of ~20 strains (“master mixes”). These master mixes were screened for tolerance to butanol, and continuously exposed to increase tolerance. Promising communities were then tested for growth with butanol as the sole carbon source. Secondly, we looked for bacteria with alcohol dehydrogenase enzymes and looked to increase butanol metabolism. We found that the tolerance for n-butanol may be improved with repeated exposure, but it was difficult to switch from tolerance to metabolism. Bacterial community dynamics may be influenced by n-butanol concentration, and there was putative butanol metabolism found with both research approaches.

09 BIOMASS FUELS↗

Identifying Hot Spots and Hot Moments of Metabolic Activity in Salt Marsh Sediments through BONCAT-FISH Microscale Mapping (Final Technical Report)

Understanding the biogeography and timing of microbial metabolic activity is a key priority for microbial ecologists. With such knowledge, the cumulative biogeochemical contributions of microbial communities become more predictable, and our ability to both understand the effects of environmental change on microbial activity and build synthetic communities with desired functions will increase substantially. In this project, we advanced this broad, ambitious goal in two substantial ways: we developed a multiplexed Fluorescence In Situ Hybridization (FISH) approach that links microbial identity with metabolic function, and we established a novel Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) technique that can resolve the timing of anabolic activity, providing new resolution of when microbial constituents are growing. We deployed both of these techniques in multiple environmental settings to demonstrate their versatility. At the Little Sippewissett Salt Marsh, we combined a novel dual-BONCAT technique with fluorescence activated cell sorting to determine which population of cells was metabolically active during the day and which was active during the night. We found that Methylobacterium was active during daylight hours, potentially feeding on carbon-rich molecules released from plant roots. Sulfur-cycling microbes dominate the population active during the dark night-time hours. Overall, the work conducted under the auspices of this project developed two promising new techniques for identifying the “hot spots” and “hot moments” of microbial activity in complex communities with taxonomic and functional resolution. We focused largely on a salt marsh sediment context, but are confident that microbial ecologists seeking to understand the microbial role in biogeochemical cycles in a wide range of settings will find our newly developed techniques useful in future work.

54 ENVIRONMENTAL SCIENCES↗

Symbiosis Under Stress: How Arbuscular Mycorrhizal Fungi and Sorghum Metabolism Shift Under Drought

As drought becomes more prevalent across the globe, causing billions of dollars in agricultural loss, the need to maintain crop health and productivity grows increasingly important. Out of the most important cereal crops, sorghum shows the greatest drought tolerance, and plant-microbiome interactions at the root region play a crucial role in this. A key microbial player is arbuscular mycorrhizal fungi (AMF), which deliver water and nutrients to plants in exchange for nutrients they cannot produce. Using sorghum as a model plant, we combine mass-spectrometry based proteomics and metabolomics to examine metabolic interactions between sorghum roots and AMF under drought stress. With AMF, sorghum downregulated lipid-related biological processes involving fatty acid biosynthesis and metabolism. Similarly, amino acid biosynthesis pathways were also suppressed; however, metabolite analysis revealed increased amino acid abundance related to the phenylpropanoid pathway. AMF also enhanced the upregulation of drought-protective osmolytes, such as mannitol and sorbitol, suggesting their key role in mediating sorghum’s response to drought stress. AMF hyphal biomass also had an increased abundance of key osmoprotectant amino acids, indicating similar mechanisms of drought tolerance between sorghum and AMF. Metabolomic evidence also suggested that carbohydrate exchange between sorghum and AMF shifted under stress, indicating an altered exudation pattern likely driven by drought response. Our results demonstrate the molecular mechanisms through which AMF modulate sorghum metabolism under drought conditions, highlighting their promising role in improving crop resilience. By identifying the molecular targets that can improve drought tolerance, we can begin engineering drought resistant agricultural biosystems.

60 APPLIED LIFE SCIENCES↗

KBase Narrative - Metabolic_reconstruction_ETDI_mangrove

Mangroves are complex and dynamic coastal ecosystems under frequent fluctuations in physicochemical conditions related to the tidal regime. The frequent variation in organic matter concentration, nutrients, and oxygen availability, among other factors, drives the microbial community composition, favoring syntrophic populations harboring a rich and diverse, stress-driven metabolism. Mangroves are known for their carbon sequestration capability, and their complex and integrated metabolic activity is essential to global biogeochemical cycling. Here, we present a metabolic reconstruction based on the genomic functional capability and flux profile between sympatric MAGs co-assembled from a tropical restored mangrove.

Laux, Marcele↗

Proteomic insights into the physiology and metabolism of oleaginous yeasts and filamentous fungi

Fungi are vital to the bioeconomy, serving as key producers of food, beverages, biofuels, and medicines, while also acting as essential resource recyclers in ecosystem management. For nearly a century, oleaginous yeast and filamentous fungi have been explored for their proficiency in oleochemicals production and carbon storage. Lipogenesis is one of the most well-studied fungal processes, with substantial progress having been made through reductionist biochemical approaches; however, the physiology and metabolism of fungal systems operating under different conditions arise from the functions of thousands of proteins, for which very little is known outside of model yeast. In this review, we discuss how proteomics provides a valuable analytical approach to contextualize lipogenesis within a complex biological system, where lipid accumulation is fundamentally governed by changes in proteins of multiple pathways. In the past two decades, proteomics has been applied to study stress response to nutrient limitations, metabolism of various carbon and nitrogen sources, the lipid droplet hub of carbon storage, protein post-translational modifications and signaling pathways, as well as oleochemical biosynthesis, thereby advancing our understanding of the oleaginous phenotype. Over 40 studies are reviewed herein to evaluate the impact, critically assess the utility, and propose future applications of proteomics. In the coming years, large systems-level proteomics studies will lay a foundation for marrying modeling and metabolic engineering strategies to optimize oleochemicals production in oleaginous fungi.

Lipid production↗

Wounding rapidly alters transcription factor expression, hormonal signaling, and phenolic compound metabolism in harvested sugarbeet roots

Injuries sustained by sugarbeet (Beta vulgaris L.) roots during harvest and postharvest operations seriously reduce the yield of white sugar produced from stored roots. Although wound healing is critically important to reduce losses, knowledge of these processes is limited for this crop as well as for roots in other species. To better understand the metabolic signals and changes that occur in wounded roots, dynamic changes in gene expression were determined by RNA sequencing and the activity of products from key genes identified in this analysis were determined in the 0.25 to 24 h following injury. Nearly five thousand differentially expressed genes that contribute to a wide range of cellular and molecular functions were identified in wounded roots. Highly upregulated genes included transcription factor genes, as well as genes involved in ethylene and jasmonic acid (JA) biosynthesis and signaling and phenolic compound biosynthesis and polymerization. Enzyme activities for key genes in ethylene and phenolic compound biosynthesis and polymerization also increased due to wounding. Results indicate that wounding causes a major reallocation of metabolism in sugarbeet taproots. Although both ethylene and JA are likely involved in triggering wound responses, the greater and more sustained upregulation of ethylene biosynthesis and signaling genes relative to those of JA, suggest a preeminence of ethylene signaling in wounded sugarbeet roots. Changes in gene expression and enzymes involved in phenolic compound metabolism additionally indicate that barriers synthesized to seal off wounds, such as suberin or lignin, are initiated within the first 24 h after injury.

59 BASIC BIOLOGICAL SCIENCES↗

Relationship of Soil Microbiota to Seed Kernel Metabolism in Camellia oleifera Under Mulched

An experiment was conducted from 2016 to 2017 to assess the effect of kernel metabolism in development stages after organic mulching compared to control. Organic mulching significantly increased crop yields (higher 128% in 2016, higher 60% in 2017), oil content (the highest oil content was 27.6% higher than that of the control), and improved soil properties (SOC, SAN, AP, and AK). In this study, soil pH, SOC, AN, AP, and AK in 0–30 cm soil depth were measured. Results showed that the effect of mulching on soil pH was not significant at the harvesting stage. The greatest metabolic differences occurred during the period of high oil conversion (S2–S4), primarily involving 11 relevant metabolic pathways. This further verified that Camellia oleifera oil yield was improved after mulching. A total of 1,106 OTUs were detected by using 16S rRNA, and Venn diagram showed that there were 106 unique OTUs in control and 103 OTUs in the treatment, respectively. Correlation analysis showed that soil pH and soil temperature were two indicators with the most correlations with soil microbiota. The yield was significantly positively correlated with soil microbial Proteobacteria, Bacteroidetes, and soil nutrition indexes. Organic mulching improved the physicochemical properties of soils, caused differences in the relative abundance of dominant bacteria in soil bacteria, and improved the soil microbiological environment to promote plant growth, indicating that organic mulching is an effective measure to alleviate seasonal drought.

Ye, Honglian↗

Cytochrome b 5 : A versatile electron carrier and regulator for plant metabolism

Cytochrome b 5 (CB5) is a small heme-binding protein, known as an electron donor delivering reducing power to the terminal enzymes involved in oxidative reactions. In plants, the CB5 protein family is substantially expanded both in its isoform numbers and cellular functions, compared to its yeast and mammalian counterparts. As an electron carrier, plant CB5 proteins function not only in fatty acid desaturation, hydroxylation and elongation, but also in the formation of specialized metabolites such as flavonoids, phenolic esters, and heteropolymer lignin. Furthermore, plant CB5s are found to interact with different non-catalytic proteins such as ethylene signaling regulator, cell death inhibitor, and sugar transporters, implicating their versatile regulatory roles in coordinating different metabolic and cellular processes, presumably in respect to the cellular redox status and/or carbon availability. Compared to the plentiful studies on biochemistry and cellular functions of mammalian CB5 proteins, the cellular and metabolic roles of plant CB5 proteins have received far less attention. This article summarizes the fragmentary information pertaining to the discovery of plant CB5 proteins, and discusses the conventional and peculiar functions that plant CB5s might play in different metabolic and cellular processes. Gaining comprehensive insight into the biological functions of CB5 proteins could offer effective biotechnological solutions to tailor plant chemodiversity and cellular responses to environment stimuli.

59 BASIC BIOLOGICAL SCIENCES↗

Stressing the importance of plant specialized metabolites: omics-based approaches for discovering specialized metabolism in plant stress responses

Plants produce a diverse range of specialized metabolites that play pivotal roles in mediating environmental interactions and stress adaptation. These unique chemical compounds also hold significant agricultural, medicinal, and industrial values. Despite the expanding knowledge of their functions in plant stress interactions, understanding the intricate biosynthetic pathways of these natural products remains challenging due to gene and pathway redundancy, multifunctionality of proteins, and the activity of enzymes with broad substrate specificity. In the past decade, substantial progress in genomics, transcriptomics, metabolomics, and proteomics has made the exploration of plant specialized metabolism more feasible than ever before. Notably, recent advances in integrative multi-omics and computational approaches, along with other technologies, are accelerating the discovery of plant specialized metabolism. In this review, we present a summary of the recent progress in the discovery of plant stress-related specialized metabolites. Emphasis is placed on the application of advanced omics-based approaches and other techniques in studying plant stress-related specialized metabolism. Additionally, we discuss the high-throughput methods for gene functional characterization. These advances hold great promise for harnessing the potential of specialized metabolites to enhance plant stress resilience in the future.

59 BASIC BIOLOGICAL SCIENCES↗

Transporter annotations are holding up progress in metabolic modeling

Mechanistic, constraint-based models of microbial isolates or communities are a staple in the metabolic analysis toolbox, but predictions about microbe-microbe and microbe-environment interactions are only as good as the accuracy of transporter annotations. A number of hurdles stand in the way of comprehensive functional assignments for membrane transporters. These include general or non-specific substrate assignments, ambiguity in the localization, directionality and reversibility of a transporter, and the many-to-many mapping of substrates, transporters and genes. In this perspective, we summarize progress in both experimental and computational approaches used to determine the function of transporters and consider paths forward that integrate both. Investment in accurate, high-throughput functional characterization is needed to train the next-generation of predictive tools toward genome-scale metabolic network reconstructions that better predict phenotypes and interactions. More reliable predictions in this domain will benefit fields ranging from personalized medicine to metabolic engineering to microbial ecology.

Casey, John↗

Metabolic Signaling Cascades Prompted by Glutaminolysis in Cancer

Aberrant glutamatergic signaling has been implicated in altered metabolic activity and the demand to synthesize biomass in several types of cancer including melanoma. In the last decade, there has been a significant contribution to our understanding of metabolic pathways. An increasing number of studies are now emphasizing the importance of glutamate functioning as a signaling molecule and a building block for cancer progression. To that end, our group has previously illustrated the role of glutamatergic signaling mediated by metabotropic glutamate receptor 1 (GRM1) in neoplastic transformation of melanocytes in vitro and spontaneous development of metastatic melanoma in vivo. Glutamate, the natural ligand of GRM1, is one of the most abundant amino acids in humans and the predominant excitatory neurotransmitter in the central nervous system. Elevated levels of glutaminolytic mitochondrial tricarboxylic acid (TCA) cycle intermediates, especially glutamate, have been reported in numerous cancer cells. Herein, we highlight and critically review metabolic bottlenecks that are prevalent during tumor evolution along with therapeutic implications of limiting glutamate bioavailability in tumors.

Shah, Raj (ORCID:0000000204400189)↗

Advances in S. cerevisiae Engineering for Xylose Fermentation and Biofuel Production: Balancing Growth, Metabolism, and Defense

Genetically engineering microorganisms to produce chemicals has changed the industrialized world. The budding yeast Saccharomyces cerevisiae is frequently used in industry due to its genetic tractability and unique metabolic capabilities. S. cerevisiae has been engineered to produce novel compounds from diverse sugars found in lignocellulosic biomass, including pentose sugars, like xylose, not recognized by the organism. Engineering high flux toward novel compounds has proved to be more challenging than anticipated since simply introducing pathway components is often not enough. Several studies show that the rewiring of upstream signaling is required to direct products toward pathways of interest, but doing so can diminish stress tolerance, which is important in industrial conditions. As an example of these challenges, we reviewed S. cerevisiae engineering efforts, enabling anaerobic xylose fermentation as a model system and showcasing the regulatory interplay’s controlling growth, metabolism, and stress defense. Enabling xylose fermentation in S. cerevisiae requires the introduction of several key metabolic enzymes but also regulatory rewiring of three signaling pathways at the intersection of the growth and stress defense responses: the RAS/PKA, Snf1, and high osmolarity glycerol (HOG) pathways. The current studies reviewed here suggest the modulation of global signaling pathways should be adopted into biorefinery microbial engineering pipelines to increase efficient product yields.

59 BASIC BIOLOGICAL SCIENCES↗

Evolution of a plant gene cluster in Solanaceae and emergence of metabolic diversity

Plants produce phylogenetically and spatially restricted, as well as structurally diverse specialized metabolites via multistep metabolic pathways. Hallmarks of specialized metabolic evolution include enzymatic promiscuity and recruitment of primary metabolic enzymes and examples of genomic clustering of pathway genes. Solanaceae glandular trichomes produce defensive acylsugars, with sidechains that vary in length across the family. We describe a tomato gene cluster on chromosome 7 involved in medium chain acylsugar accumulation due to trichome specific acyl-CoA synthetase and enoyl-CoA hydratase genes. This cluster co-localizes with a tomato steroidal alkaloid gene cluster and is syntenic to a chromosome 12 region containing another acylsugar pathway gene. We reconstructed the evolutionary events leading to this gene cluster and found that its phylogenetic distribution correlates with medium chain acylsugar accumulation across the Solanaceae. This work reveals insights into the dynamics behind gene cluster evolution and cell-type specific metabolite diversity.

59 BASIC BIOLOGICAL SCIENCES↗

Extracellular electron transfer increases fermentation in lactic acid bacteria via a hybrid metabolism

Energy conservation in microorganisms is classically categorized into respiration and fermentation; however, recent work shows some species can use mixed or alternative bioenergetic strategies. We explored the use of extracellular electron transfer for energy conservation in diverse lactic acid bacteria (LAB), microorganisms that mainly rely on fermentative metabolism and are important in food fermentations. The LAB Lactiplantibacillus plantarum uses extracellular electron transfer to increase its NAD + /NADH ratio, generate more ATP through substrate-level phosphorylation, and accumulate biomass more rapidly. This novel, hybrid metabolism is dependent on a type-II NADH dehydrogenase (Ndh2) and conditionally requires a flavin-binding extracellular lipoprotein (PplA) under laboratory conditions. It confers increased fermentation product yield, metabolic flux, and environmental acidification in laboratory media and during kale juice fermentation. The discovery of a single pathway that simultaneously blends features of fermentation and respiration in a primarily fermentative microorganism expands our knowledge of energy conservation and provides immediate biotechnology applications.

59 BASIC BIOLOGICAL SCIENCES↗