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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 847 records · Page 47
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Drought adaptation in Arabidopsis thaliana by extensive genetic loss-of-function

Interdisciplinary syntheses are needed to scale up discovery of the environmental drivers and molecular basis of adaptation in nature. Here we integrated novel approaches using whole genome sequences, satellite remote sensing, and transgenic experiments to study natural loss-of-function alleles associated with drought histories in wild Arabidopsis thaliana. The genes we identified exhibit population genetic signatures of parallel molecular evolution, selection for loss-of-function, and shared associations with flowering time phenotypes in directions consistent with longstanding adaptive hypotheses seven times more often than expected by chance. We then confirmed predicted phenotypes experimentally in transgenic knockout lines. These findings reveal the importance of drought timing to explain the evolution of alternative drought tolerance strategies and further challenge popular assumptions about the adaptive value of genetic loss-of-function in nature. These results also motivate improved species-wide sequencing efforts to better identify loss-of-function variants and inspire new opportunities for engineering climate resilience in crops.

Life Sciences & Biomedicine - Other Topics↗
DOE OSTI2019

A high-capacity gene stacking toolkit for the oleaginous microalga, Nannochloropsis oceanica CCMP1779

Nannochloropsis oceanica CCMP1779 is an oleaginous heterokont microalga that is particularly amenable to synthetic biology approaches. The underlying complex genetic engineering often requires the expression of multiple transgenes (gene stacking). We integrated several techniques into a vector toolkit for multi-gene expression, including: multiple resistance marker genes, bidirectional promoters, and assembly of multiple expression cassettes into a single vector. We developed a series of gateway entry and destination vectors for assembly of two bidirectional promoter expression cassettes, which facilitates combinatorial high-capacity gene stacking. Several endogenous bidirectional promoters were demonstrated to be effective for driving transgene expression, with a dual-luciferase reporter system used to monitor transgene expression under different environmental conditions. The bidirectional promoter between the nitrate reductase and the nitrate transporter genes drives nitrogen source dependent transcription, enabling conditional transgene expression. Lethal concentrations of the antibiotics, Blasticidin, G418, and Nourseothricin, were determined and the corresponding marker genes used for genomic transformation selection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Immunogenicity and therapeutic targeting of a public neoantigen derived from mutated PIK3CA

Public neoantigens (NeoAgs) represent an elite class of shared cancer-specific epitopes derived from recurrently mutated driver genes. Here we describe a high-throughput platform combining single-cell transcriptomic and T cell receptor (TCR) sequencing to establish whether mutant PIK3CA, among the most frequently genomically altered driver oncogenes, generates an immunogenic public NeoAg. Using this strategy, we developed a panel of TCRs that recognize an endogenously processed neopeptide encompassing a common PIK3CA hotspot mutation restricted by the prevalent human leukocyte antigen (HLA)-A*03:01 allele. Mechanistically, immunogenicity to this public NeoAg arises from enhanced neopeptide/HLA complex stability caused by a preferred HLA anchor substitution. Structural studies indicated that the HLA-bound neopeptide presents a comparatively ‘featureless’ surface dominated by the peptide’s backbone. To bind this epitope with high specificity and affinity, we discovered that a lead TCR clinical candidate engages the neopeptide through an extended interface facilitated by an unusually long CDR3β loop. In patients with diverse malignancies, we observed NeoAg clonal conservation and spontaneous immunogenicity to the neoepitope. Finally, adoptive transfer of TCR-engineered T cells led to tumor regression in vivo in mice bearing PIK3CA-mutant tumors but not wild-type PIK3CA tumors. Together, these findings establish the immunogenicity and therapeutic potential of a mutant PIK3CA-derived public NeoAg.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Genetic and enzymatic characterization of Amy13E from Cellvibrio japonicus reclassifies it as a cyclodextrinase also capable of α-diglucoside degradation

ABSTRACT Cyclodextrinases are carbohydrate-active enzymes involved in the linearization of circular amylose oligosaccharides. Primarily thought to function as part of starch metabolism, there have been previous reports of bacterial cyclodextrinases also having additional enzymatic activities on linear malto-oligosaccharides. This substrate class also includes environmentally rare α-diglucosides such as kojibiose (α−1,2), nigerose (α−1,3), and isomaltose (α−1,6), all of which have valuable properties as prebiotics or low-glycemic index sweeteners. Previous genome sequencing of threeCellvibrio japonicusstrains adapted to utilize these α-diglucosides identified multiple, but uncharacterized, mutations in each strain. One of the mutations identified was in theamy13Egene, which was annotated to encode a neopullulanase. In this report, we functionally characterized this gene and determined that it in fact encodes a cyclodextrinase with additional activities on α-diglucosides. Deletion analysis ofamy13Efound that this gene was essential for kojibiose and isomaltose metabolism inC. japonicus. Interestingly, a Δamy13Emutant was not deficient for cyclodextrin or pullulan utilization inC. japonicus; however, heterologous expression of the gene inE. coliwas sufficient for cyclodextrin-dependent growth. Biochemical analyses found thatCjAmy13E cleaved multiple substrates but preferred cyclodextrins and maltose, but had no activity on pullulan. Our characterization of theCjAmy13E cyclodextrinase is useful for refining functional enzyme predictions in related bacteria and for engineering enzymes for biotechnology or biomedical applications. IMPORTANCE Understanding the bacterial metabolism of cyclodextrins and rare α-diglucosides is increasingly important, as these sugars are becoming prevalent in the foods, supplements, and medicines humans consume that subsequently feed the human gut microbiome. Our analysis of a cyclomaltodextrinase with an expanded substrate range is significant because it broadens the potential applications of the GH13 family of carbohydrate active enzymes (CAZymes) in biotechnology and biomedicine. Specifically, this study provides a workflow for the discovery and characterization of novel activities in bacteria that possess a high number of CAZymes that otherwise would be missed due to complications with functional redundancy. Furthermore, this study provides a model from which predictions can be made why certain bacteria in crowded niches are able to robustly utilize rare carbon sources, possibly to gain a competitive growth advantage.

Biotechnology & Applied Microbiology↗
DOE OSTI2025

Multi‐Omics Analyses Reveal Divergent Molecular Mechanisms Underlying Plant Biomass Conversion by Five Fungi

Fungal plant biomass conversion (FPBC) is of great importance to the global carbon cycle and has been increasingly applied for the production of biofuel and biochemicals from lignocellulose. However, the comprehensive understanding of relevant molecular mechanisms in different fungi remains challenging. Here, we comparatively analyzed the transcriptome, proteome and metabolome profile of four ascomycetes and one basidiomycete fungi during their growth on two common agricultural feedstocks (soybean hulls and corn stover). We revealed strong time‐, substrate‐ and species‐specific responses at multi‐omics levels for the tested fungi, highlighting species‐specific carbon utilization approaches and evolutionary adaptation to environmental niches. Notably, a remarkable expressional diversity of lignocellulose degrading enzymes, sugar transporter and metabolic genes, as well as industrially relevant metabolites were identified across different fungi and cultivation conditions. The findings improves our understanding of complex molecular networks underlying FPBC and fungal ecological roles, offering novel insights that can guide future genetic engineering of fungi for valorization of agriculture waste into value‐added bioproducts.

CAZy↗
DOE OSTI2024

Computed entropy spectra for grain boundary segregation in polycrystals

Abstract Grain boundary solute segregation influences most bulk material properties, and understanding solute thermodynamics at grain boundaries is critical for engineering them. However, the vast grain boundary space in polycrystals is challenging to evaluate due to its size, especially for the intrinsically hard-to-compute segregation excess entropy. Here data science methods are used to generate a database of site-wise grain boundary segregation entropy spectra for 155 dilute binary alloys within the harmonic approximation. The spectral framework allows scale bridging between the calculated atomistic site-wise energy-entropy spectra and macroscopic segregation entropy estimates. The results affirm that macroscopic averaging is not sufficient: a spectral treatment of grain boundary segregation is needed to accurately model bulk temperature dependence of grain boundary solute segregation. The calculated spectral entropy database and thermodynamic framework can be applied for both understanding segregation experiments and alloy design exercises, paving the way to a finite-temperature grain boundary genome.

Chemistry↗
DOE OSTI2022

High-throughput identification of novel heat tolerance genes via genome-wide pooled mutant screens in the model green alga Chlamydomonas reinhardtii

Different high temperatures adversely affect crop and algal yields with various responses in photosynthetic cells. The list of genes required for thermotolerance remains elusive. Additionally, it is unclear how carbon source availability affects heat responses in plants and algae. Here, we utilized the insertional, indexed, genome-saturating mutant library of the unicellular, eukaryotic green alga Chlamydomonas reinhardtii to perform genome-wide, quantitative, pooled screens under moderate (35°C) or acute (40°C) high temperatures with or without organic carbon sources. We identified heat-sensitive mutants based on quantitative growth rates and identified putative heat tolerance genes (HTGs). By triangulating HTGs with heat-induced transcripts or proteins in wildtype cultures and MapMan functional annotations, we presented a high/medium-confidence list of 933 Chlamydomonas genes with putative roles in heat tolerance. Triangulated HTGs include those with known thermotolerance roles and novel genes with little or no functional annotation. About 50% of these high-confidence HTGs in Chlamydomonas have orthologs in green lineage organisms, including crop species. Arabidopsis thaliana mutants deficient in the ortholog of a high-confidence Chlamydomonas HTG were also heat sensitive. This work expands our knowledge of heat responses in photosynthetic cells and provides engineering targets to improve thermotolerance in algae and crops.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Metagenomes and metagenome-assembled genomes from microbial communities in a biological nutrient removal plant operated at Hamptons Road Sanitation District (HRSD) with high and low dissolved oxygen conditions

Aeration is a major cost at biological nutrient removal (BNR) plants. We report on microbial communities in a pilot-scale BNR system before and after a dissolved oxygen transition from 2.5 to 0.2 mg/L implemented over 18 months. Four PacBio metagenomes and 316 metagenome-assembled genomes are announced.

dissolved oxygen↗
DOE OSTI2023

Diverse electron carriers drive syntrophic interactions in an enriched anaerobic acetate-oxidizing consortium

Abstract In many anoxic environments, syntrophic acetate oxidation (SAO) is a key pathway mediating the conversion of acetate into methane through obligate cross-feeding interactions between SAO bacteria (SAOB) and methanogenic archaea. The SAO pathway is particularly important in engineered environments such as anaerobic digestion (AD) systems operating at thermophilic temperatures and/or with high ammonia. Despite the widespread importance of SAOB to the stability of the AD process, little is known about their in situ physiologies due to typically low biomass yields and resistance to isolation. Here, we performed a long-term (300-day) continuous enrichment of a thermophilic (55 °C) SAO community from a municipal AD system using acetate as the sole carbon source. Over 80% of the enriched bioreactor metagenome belonged to a three-member consortium, including an acetate-oxidizing bacterium affiliated with DTU068 encoding for carbon dioxide, hydrogen, and formate production, along with two methanogenic archaea affiliated with Methanothermobacter_A. Stable isotope probing was coupled with metaproteogenomics to quantify carbon flux into each community member during acetate conversion and inform metabolic reconstruction and genome-scale modeling. This effort revealed that the two Methanothermobacter_A species differed in their preferred electron donors, with one possessing the ability to grow on formate and the other only consuming hydrogen. A thermodynamic analysis suggested that the presence of the formate-consuming methanogen broadened the environmental conditions where ATP production from SAO was favorable. Collectively, these results highlight how flexibility in electron partitioning during SAO likely governs community structure and fitness through thermodynamic-driven mutualism, shedding valuable insights into the metabolic underpinnings of this key functional group within methanogenic ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Development of a high-productivity, halophilic, thermotolerant microalga Picochlorum renovo

Abstract Microalgae are promising biocatalysts for applications in sustainable fuel, food, and chemical production. Here, we describe culture collection screening, down-selection, and development of a high-productivity, halophilic, thermotolerant microalga, Picochlorum renovo . This microalga displays a rapid growth rate and high diel biomass productivity (34 g m −2 day −1 ), with a composition well-suited for downstream processing. P. renovo exhibits broad salinity tolerance (growth at 107.5 g L −1 salinity) and thermotolerance (growth up to 40 °C), beneficial traits for outdoor cultivation. We report complete genome sequencing and analysis, and genetic tool development suitable for expression of transgenes inserted into the nuclear or chloroplast genomes. We further evaluate mechanisms of halotolerance via comparative transcriptomics, identifying novel genes differentially regulated in response to high salinity cultivation. These findings will enable basic science inquiries into control mechanisms governing Picochlorum biology and lay the foundation for development of a microalga with industrially relevant traits as a model photobiology platform.

09 BIOMASS FUELS↗
DOE OSTI2025

A multi-omic characterization of the physiological responses to salt stress in Scenedesmus obliquus UTEX393

Scenedesmus obliquus UTEX393 is a promising microalgal candidate for sustainable biomanufacturing but its limited halotolerance hinders large-scale cultivation in saline environments. To investigate the molecular basis of salt stress responses, we conducted a comprehensive multi-omic analysis integrating genomics, transcriptomics, proteomics, lipidomics, metabolomics, and DNA affinity purification sequencing (DAP-seq). An improved nuclear genome assembly and annotation yielded 19,017 gene models and a 97% BUSCO completeness score, enabling construction of a genome-scale metabolic model. Comparing 15 ppt salinity stress to 5 ppt control, growth and productivity were significantly reduced, accompanied by widespread transcriptomic and proteomic changes. Transcriptomic analysis revealed downregulation of photosynthetic machinery and energy conservation genes, and upregulation of stress-responsive elements such as expansins, flavodoxins, and osmoprotectants. Lipidomic profiling showed accumulation of triacylglycerols (TAGs) and degradation of galactosyl lipids, consistent with a shift toward lipid biosynthesis to mitigate redox imbalance. Depletion of key polar metabolites and branched-chain amino acids suggested a rerouting of central carbon metabolism under stress. DAP-seq identified key transcription factors, including LHY1 and SPL12, that target central metabolic enzymes involved in redox balancing, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and malate dehydrogenase (MDH). These findings establish a regulatory-metabolic framework linking redox stress to lipid accumulation and reveal potential engineering targets to enhance salt tolerance. Overall, the multi-omic analysis supports the “overflow” hypothesis, where impaired photosynthesis results in excess reducing equivalents being diverted into TAG synthesis and highlights transcriptional regulators as candidates for improving algal robustness in brackish environments.

09 BIOMASS FUELS↗
DOE OSTI2025

Tetranucleotide frequencies differentiate genomic boundaries and metabolic strategies across environmental microbiomes

Microbiomes are constrained by physicochemical conditions, nutrient regimes, and community interactions across diverse environments, yet genomic signatures of this adaptation remain unclear. Metagenome sequencing is a powerful technique to analyze genomic content in the context of natural environments, establishing concepts of microbial ecological trends. Here, we developed a data discovery tool-a tetranucleotide-informed metagenome stability diagram-that is publicly available in the integrated microbial genomes and microbiomes (IMG/M) platform for metagenome ecosystem analyses. We analyzed the tetranucleotide frequencies from quality-filtered and unassembled sequence data of over 12,000 metagenomes to assess ecosystem-specific microbial community composition and function. We found that tetranucleotide frequencies can differentiate communities across various natural environments and that specific functional and metabolic trends can be observed in this structuring. Our tool places metagenomes sampled from diverse environments into clusters and along gradients of tetranucleotide frequency similarity, suggesting microbiome community compositions specific to gradient conditions. Within the resulting metagenome clusters, we identify protein-coding gene identifiers that are most differentiated between ecosystem classifications. We plan for annual updates to the metagenome stability diagram in IMG/M with new data, allowing for refinement of the ecosystem classifications delineated here. This framework has the potential to inform future studies on microbiome engineering, bioremediation, and the prediction of microbial community responses to environmental change. IMPORTANCE: Microbes adapt to diverse environments influenced by factors like temperature, acidity, and nutrient availability. We developed a new tool to analyze and visualize the genetic makeup of over 12,000 microbial communities, revealing patterns linked to specific functions and metabolic processes. This tool groups similar microbial communities and identifies characteristic genes within environments. By continually updating this tool, we aim to advance our understanding of microbial ecology, enabling applications like microbial engineering, bioremediation, and predicting responses to environmental change.

Kellom, Matthew↗
DOE OSTI2019

Construction of a Robust Non-Oxidative Glycolysis in Model Organisms for n-Butanol Production

The Embden-Meyerhoff-Parnas (EMP) pathway, commonly known as glycolysis, represents the fundamental biochemical infrastructure for sugar catabolism in almost all organisms, as it provides key components for biosynthesis, energy metabolism, and global regulation. EMP-based metabolism synthesizes three-carbon (C3) metabolites prior to two-carbon (C2) metabolites, and must emit one CO2 in the synthesis of the C2 building block, acetyl-CoA, a precursor for many industrially important products. Using rational design, genome editing, and evolution, here we replaced the native glycolytic pathways in Escherichia coli with the previously designed non-oxidative glycolysis (NOG), which bypasses initial C3 formation and directly generates stoichiometric amounts of C2 metabolites. The resulting strain, which contains 11 gene overexpressions, 10 gene deletions by design, and more than 50 genomic mutations (including 3 global regulators) through evolution, grows aerobically in glucose minimal medium, but can ferment anaerobically to products with nearly complete carbon conservation. We confirmed that the strain metabolizes glucose through NOG by 13C tracer experiments. This re-designed E. coli strain represents a different approach for carbon catabolism, and may serve as a useful platform for bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Identification of a pathway for electron uptake in Shewanella oneidensis

Extracellular electron transfer (EET) could enable electron uptake into microbial metabolism for the synthesis of complex, energy dense organic molecules from CO 2 and renewable electricity. Theoretically EET could do this with an efficiency comparable to H 2 -oxidation but without the need for a volatile intermediate and the problems it causes for scale up. However, significant gaps remain in understanding the mechanism and genetics of electron uptake. For example, studies of electron uptake in electroactive microbes have shown a role for the Mtr EET complex in the electroactive microbe Shewanella oneidensis MR-1, though there is substantial variation in the magnitude of effect deletion of these genes has depending on the terminal electron acceptor used. This speaks to the potential for previously uncharacterized and/or differentially utilized genes involved in electron uptake. To address this, we screened gene disruption mutants for 3667 genes, representing ≈99% of all nonessential genes, from the S. oneidensis whole genome knockout collection using a redox dye oxidation assay. Confirmation of electron uptake using electrochemical testing allowed us to identify five genes from S. oneidensis that are indispensable for electron uptake from a cathode. Knockout of each gene eliminates extracellular electron uptake, yet in four of the five cases produces no significant defect in electron donation to an anode. This result highlights both distinct electron uptake components and an electronic connection between aerobic and anaerobic electron transport chains that allow electrons from the reversible EET machinery to be coupled to different respiratory processes in S. oneidensis. Homologs to these genes across many different genera suggesting that electron uptake by EET coupled to respiration could be widespread. These gene discoveries provide a foundation for: studying this phenotype in exotic metal-oxidizing microbes, genetic optimization of electron uptake in S. oneidensis; and genetically engineering electron uptake into a highly tractable host like E. coli to complement recent advances in synthetic CO2 fixation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

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

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

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Artificial Intelligence and Machine Learning for Bioenergy Research: Opportunities and Challenges

The integration of artificial intelligence and machine learning (AI/ML) with automated experimentation, genomics, biosystems design, and bioprocessing technologies is poised to revolutionize scientific investigation and, particularly, bioenergy research. To identify the opportunities and challenges in this emerging research area, the U.S. Department of Energy’s (DOE) Biological and Environmental Research program (BER) and Bioenergy Technologies Office (BETO) held a joint virtual workshop on AI/ML for Bioenergy Research (AMBER) on August 23–25, 2022. These interests have since been amplified in a September 2022 Executive Order, “Advancing Biotechnology and Biomanufacturing Innovation for a Sustainable, Safe, and Secure U.S. Bioeconomy,” to promote a whole-of government approach to biotechnology development (White House 2022). Approximately 50 scientists with various backgrounds and expertise from academia, industry, and DOE national laboratories met to discuss the opportunities and challenges of AI/ML for bioenergy research. Workshop participants were tasked with assessing the potential for AI/ML and laboratory automation to advance biological understanding and engineering in general. They particularly examined how integrating AI/ML tools with laboratory automation could accelerate biosystems design and optimize biomanufacturing. Discussions included the data and computational infrastructure needed to augment biosystems design applications and the expertise and workforce development efforts urgently required to shift integrated systems toward bioenergy research more broadly. Participants discussed many existing and future applications of AI/ML for biosystems design ranging from enzymes to plants and microbes, microbiomes, and bioprocess development. They also identified three key categories of scientific and technical opportunities and challenges: high-quality data, AI/ML algorithms, and laboratory automation. Several main takeaways emerged from the workshop: 1. Numerous AI/ML and automated experimentation applications exist for a variety of DOE mission needs in energy and the environment; 2. Exemplary research grand challenges for which AI/ML could provide solutions include: building microbes and microbial communities to specifications, developing closed-loop autonomous design and control for biosystems design, and advancing scale-up and automation; 3. Lack of sufficient high-quality, annotated data hinders the development of AI/ML applications; 4. New and improved AI/ML tools are needed, particularly those meeting the specific needs of the BER and BETO research communities; 5. Trade-offs in performance, cost, and reliability exist between deploying commercially available versus building custom-developed instrumentation and software for automated or autonomous experimentation; translation of manual to automated or autonomous methods is often a nontrivial endeavor; 6. Training a new generation of young scientists who can develop and apply AI/ML tools is needed to solve long-standing scientific challenges in bioenergy research. The integration of AI/ML tools and automated experimentation represents a new data-driven research paradigm complementary to the traditional hypothesis-driven research paradigm. This paradigm accelerates design and optimization of biological systems and processes for a variety of DOE mission needs in energy and the environment. The AMBER workshop broadly explored the potential of this new paradigm for bioenergy research, of particular interest to BER and BETO, and identified key challenges and opportunities that DOE can address in the coming years by leveraging its unique capabilities and resources.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Optimizing Cell-based Antimicrobials through Pooled Genomic Libraries

DNA synthesis and assembly technologies ushered in through synthetic biology have great promise for biomanufacturing, bioremediation, and the development of living therapeutics. Unfortunately, predicting sequence to function relationships, including for biosynthetic pathways expressed in a new host organism, is difficult and often requires many iterative cycles of design, construction, and testing. We are working to develop data-driven approaches to identify the genetic determinants of growth defects and productivity for the expression of a cell-based antimicrobial. We assayed the growth, pigment production, and antimicrobial activity of a collection of over 10,000 genetic mutants of the violacein biosynthetic pathway and sequenced the genetic variation of these mutants. Through this project, we have developed an innovative codebase to automate the determination of pigmentation and antimicrobial clearing diameter for tens of thousands of genetic mutants cultivated on agar dishes. Further, we have written DNA sequence analysis code to demultiplex & provide consensus sequences from high-throughput PacBio long-read circular consensus sequencing (CCS) datasets. From this foundation, we plan to map DNA sequence to function to predict an optimal genetic design to maximize antimicrobial activity while minimizing deleterious growth effects. The workflows and algorithms developed through this project can be broadly applied to other engineered functions in microbes, uncovering sequence to function relationships for complex phenotypes where function impacts fitness.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Separation of life stages within anaerobic fungi (Neocallimastigomycota) highlights differences in global transcription and metabolism

Anaerobic gut fungi of the phylum Neocallimastigomycota are microbes proficient in valorizing low-cost but difficult-to-breakdown lignocellulosic plant biomass. Characterization of different fungal life stages and how they contribute to biomass breakdown are critical for biotechnological applications, yet we lack foundational knowledge about the transcriptional, metabolic, and enzyme secretion behavior of different life stages of anaerobic gut fungi: zoospores, germlings, immature thalli, and mature zoosporangia. A Miracloth-based technique was developed to enrich cell pellets with zoospores - the free-swimming, flagellated, young life stage of anaerobic gut fungi. By contrast, fungal mats contained relatively more vegetative, encysted, mature sporangia that form films. Global gene expression profiles were compared from two sample types (zoospore-enriched cell pellets vs. mature mats) harvested from the anaerobic gut fungal strain Neocallimastix californiae G1. Despite cultures being grown on glucose, the fungal zoospore-enriched samples were transcriptionally primed to encounter plant matter substrate, as evidenced by upregulation of catabolic carbohydrate-active enzymes and putative carbohydrate transporters. Furthermore, we report significant differential gene expression for gene annotation groups, including putative secondary metabolites and transcription factors. Understanding global gene expression differences between the fungal zoospore-enriched cells and mature fungi aid in characterizing fungal development, unmasking gene function, and guiding cultivation conditions and engineering targets to promote enzyme secretion.

59 BASIC BIOLOGICAL SCIENCES↗