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At least 253 records · Page 14

Celestial Mapping System Videos

The Celestial Mapping System (CMS) is a software platform to generate virtual 3D globes for celestial bodies within our solar system. Multiple planetary data layers can be added to the virtual globe to provide visualization of high-resolution imagery and elevation data, which enables precise measurements, tools for analytical capabilities and a broad range of other functionalities to assist planetary scientists and mission planners. Third-party planetary data can be ingested into CMS with minimal effort. The present focus of CMS is on developing lunar mapping tools to provide features such as: 3D first person view with zoom and navigational capabilities, realistic terrain visualization based on LRO data, measurement tools, Apollo, CLPS and international mission landing site annotations, 3D Models, stereoscopic view, terrain profiling, line of sight analysis, sunlight shading and many more. The application has been developed to provide situational and domain awareness on the Lunar surface, planning capabilities for equipment placement and traverse path optimization.

Mapping↗

Machine Learning for Predicting Team Functioning in HERA Missions

Team functioning is integral to success in future long term space exploration missions. Proactively detecting declines in team functioning can mitigate conflict and ensure mission success. This project developed a speech-based artificial intelligence (AI) system that unobtrusively predicts degradation in team functioning, including performance and cohesion, in the Human Exploration Research Analog (HERA) Campaigns 4 and 5. The AI system conducted automated analysis of the prosodic (tone of voice) and linguistic (language content) components of speech, modeling interpersonal dynamics at both the turn-taking and day-wide levels. We investigated team functioning via observing structured interactions (i.e., multi-mission space exploration vehicle-extra vehicular activity [MMSEV-EVA], team interaction battery [TIB]) and unstructured interactions before the MMSEV-EVA task. We developed machine learning models to predict team functioning (objective task accuracy, self reported team efficacy and self reported team cohesion) by analyzing OpenSmile acoustic features, linguistic descriptors extracted via the linguistic inquiry and word count (LIWC) dictionary, and semantic embeddings. In the TIB, static models using logistic regression and random forests were not able to predict task accuracy, but predicted team efficacy and cohesion during both the decision making and relational tasks to a moderate level (60-70%). Majority voting on the individual turns to predict day long team efficacy further increased accuracies (70-80%). Finally, long short-term memory (LSTM) models showed the best performance across all variables (80-91%), including task performance. In the MMSEV-EVA, static models achieved an accuracy of 60% with majority voting, which increased to 80% through the incorporation of mission day as a variable, accounting for the learning effect. A key finding across both tasks was the "team-dependent" nature of these interactions; models achieved much higher accuracy when trained on prior days of the same team's data rather than attempting to generalize across entirely different teams, with even 1-2 days of prior data per team achieving 5-15% improvement over team-independent models. In addition, the incorporation of pre-task data from the same team also improves model performance, e.g., incorporating data from the decision-making task of the TIB, which preceded the relational task, improved the prediction of team efficacy and cohesion during the latter. We compared model performance when trained on machine-generated data compared to data that had been further corrected by human annotators. Overall, models trained on human-corrected data exhibited a modest improvement in performance, particularly when acoustic features were used. We found no significant correlation between word error rate (WER) and model accuracy (r(55) = -0.08, p = 0.51), but model’s accuracy was significantly higher for medium/high quality transcription (0.74 (SD = 0.48)) compared to the low-quality group (0.64 (SD = 0.36)) (t(63)=2.82, p = 0.006). Based on these, several design recommendation emerge, that could inform Standards at NASA. Models predicting team functioning should incorporate at least one to two days of historical interaction data, include brief pre-task discussions, and explicitly model temporal learning effects, especially for longer operational tasks. Minimum quality standards for automated speech-processing pipelines are needed, given the performance gains observed with manually corrected acoustic data. Finally, systems should leverage both acoustic features and language embeddings in complementary ways, with modality choices and fusion strategies tailored to mission context, task demands, and data quality requirements.

Shrivatsa Mishra↗

Multi‐season analysis reveals hundreds of drought‐responsive genes in sorghum

Persistent drought affects global crop production and is becoming more severe in many parts of the world in recent decades. Deciphering how plants respond to drought will facilitate the development of flexible mitigation strategies. Sorghum bicolor L. Moench (sorghum), a major cereal crop and an emerging bioenergy crop, exhibits remarkable resilience to drought. To better understand the molecular traits that underlie sorghum's remarkable drought tolerance, we undertook a large-scale sorghum gene expression profiling effort, totaling nearly 1500 transcriptome profiles, across a 3-year field study with replicated plots in California's Central Valley. This study included time-resolved gene expression data from roots and leaves of two sorghum genotypes, BTx642 and RTx430, with different pre-flowering and post-flowering drought-tolerance adaptations under control and drought conditions. Quantification of genotype-specific drought tolerance effects was enabled by de novo sequencing, assembly, and annotation of both BTx642 and RTx430 genomes. These reference-quality genomes were used to construct a pangene set for characterizing conserved and genotype-specific expression. By integrating time-resolved transcriptomic responses to drought in the field across three consecutive years, we identified a set of 726 drought-responsive genes that responded similarly in all 3 years of our field study. Functional enrichment analysis identified abiotic stress, secondary cell wall-related processes and metabolism as particularly affected under both types of drought stress. We also found that some glyoxylate cycle pathway genes, including malate synthase and isocitrate lyase, are differentially regulated particularly during post-flowering drought stress, implicating this pathway as potentially important for drought responsiveness. This expansive dataset represents a unique resource for sorghum and drought research communities and provides a methodological framework for the integration of multi-faceted time-resolved transcriptomic datasets.

Cole, Benjamin [USDOE Joint Genome Institute (JGI)↗

Data and scripts associated with a manuscript investigating dissolved organic matter and microbial community linkages across seven globally distributed rivers

This data package is associated with the publication “Meta-metabolome ecology reveals that geochemistry and microbial functional potential are linked to organic matter development across seven rivers” submitted to Science of the Total Environment. This data package includes the data necessary to replicate the analyses presented within the manuscript to investigate dissolved organic matter (DOM) development across broad spatial distances and within divergent biomes. Specifically, we included the Fourier transform ion cyclotron mass spectrometry (FTICR-MS) data, geochemistry data, annotated metagenomic data, and results from ecological null modeling analyses in this data package. Additionally, we included the scripts necessary to generate the figures from the manuscript. Complete metagenomic data associated with this data package can be found at the National Center for Biotechnology (NCBI) under Bioproject PRJNA946291. This dataset consists of (1) four folders; (2) a file-level metadata (flmd) file; (3) a data dictionary (dd) file; (4) a factor sheet describing samples; and (5) a readme. The FTICR Data folder contains (1) the processed Fourier transform ion cyclotron mass spectrometry (FTICR-MS) data; (2) a transformation-weighted characteristics dendrogram generated from the FTICR-MS data; and (3) the script used to generate all FTICR-MS related figures. The Geochemical Data folder contains (1) the single geochemistry data file and (2) the R script responsible for generating associated figures. The Metagenomic Data folder contains (1) annotation information across different levels; (2) carbohydrate active enzyme (CAZyme) information from the dbCAN database (Yin et al., 2012); (3) phylogenetic tree data (FASTAs, alignments, and tree file); and (4) the scripts necessary to analyze all of these data and generate figures. The Null Modeling Data folder contains (1) data generated during null modeling for each river and all rivers combined and (2) the R scripts necessary to process the data. All files are .csv, .pdf, .tsv, .tre, .faa, .afa, .tree, or .R.

54 ENVIRONMENTAL SCIENCES↗

Software Construction and Analysis Tools for Future Space Missions

NASA and its international partners will increasingly depend on software-based systems to implement advanced functions for future space missions, such as Martian rovers that autonomously navigate long distances exploring geographic features formed by surface water early in the planet's history. The software-based functions for these missions will need to be robust and highly reliable, raising significant challenges in the context of recent Mars mission failures attributed to software faults. After reviewing these challenges, this paper describes tools that have been developed at NASA Ames that could contribute to meeting these challenges; 1) Program synthesis tools based on automated inference that generate documentation for manual review and annotations for automated certification. 2) Model-checking tools for concurrent object-oriented software that achieve memorability through synergy with program abstraction and static analysis tools.

Lowry, Michael R.↗

X-HAB 2020: AR Field Treks Summary and Conclusions

As part of the FY20 X-Hab Challenge, BLiSS sought to create an Augmented Reality (AR) toolkit to help with analog field trek operations under the supervision of the Solar System Exploration Research Virtual Institute (SSERVI). These treks are operational and technical demonstrations at space-like destinations on Earth to test current extra-vehicular activity (EVA) techniques. While BLiSS as an organization has experience studying operational tasks such as this, it has never developed AR software at this scale. For that reason, another team at the University was brought on to work in parallel. The Collaborative Lab for Advancing Work in Space (CLAWS) is a veteran group of the NASA Spacesuit User Interface Technologies for Students (SUITS) challenge in which Hololens displays for astronauts are created within a year. The operational and technological pairing was ideally suited for tackling this problem. The team divided its responsibilities so that BLiSS would handle the research required to shape the project. As this deliverable had an end user, it was decided that interviewing these field geologists and operations specialists would provide the best insight. These interviews paired with literature review would reveal niche applications for AR that remained within feasible bounds. These science-driven EVAs in unknown terrain require more flexible tools than the current generation of EVA assistants. Rather than focus on sequential instructions, there instead needs to be a broad toolkit that's only called upon in specific instances. This AR Toolkit for Lunar Astronauts and Scientists (ATLAS) became the development goal of the project: create a non-intrusive assembly of tools that could be accessed in AR on the field. The current ATLAS design makes use of a geospatially and temporally annotated eld note system called GeoNotes. This allows for data to be collected and coordinated in a way that's synchronized across time, space, and different users. A Mission Control Center (MCC) and Mobile Support Equipment (MSE) were all needed to transport the AR headset into the field with the user. A network infrastructure was designed and set up within the University to enable this functionality. The software is based on a Protocol-Module structure that allows for modular development of each capability. A Protocol Manager coordinates different protocols that make use of modules. Each module tackles a different individual task while the protocol puts each one to use. The protocol manager coordinates when these are called to be used. This software is hosted on a head-mounted display (HMD) with the MCC acting as support from afar. While the software would be unit-tested at each level and each hardware component verified, a final demonstration would serve to prove the system's capabilities: an analog field trek. The team would prepare to support a user in a remote location from the MCC back at the University. A local area near campus would be tested before going out to do sample field geology further away. This unfortunately became impossible with the arrival of COVID-19. Access to all of the facilities to complete the project as planned were shut down. Our team was scattered across the globe and forced to complete the rest virtually. Adjustments were made to produce a small virtual concept in Adobe XD in the meantime. Even digital surveys were created based on the NASA task-load index (TLX) originally intended for testing actual users. The goal shifted towards completing software and getting feedback on the user interfaces (UI) and user experiences (UX). This team has reformed in response to COVID and its focus has shifted to what can be done remotely. There is still an intention to finish the original deliverable described in this report. The work has been expanded beyond the original X-Hab challenge and has instead become its own research e ort to be continued afterwards. This report collects the processes and knowledge gained from a year of studying and working at this problem with two teams. It should preserve it for the time until the world returns to normal and work can resume. CLAWS will be taking over full responsibility from that point forward, eventually surpassing the original needs of the project. While this document captures the work done towards an eventual end, the CLAWS team has written their own proposal alongside it. It outlines a new future for ATLAS beyond X-Hab, BLiSS, and hopefully beyond COVID-19. This project began as a vague goal hoping to place a new technology into the unique setting of exploration science. The project has since comfortably taken root and will hopefully bloom over the next year.

Alex Sena↗

The Exometabolome of Xylella fastidiosa in Contact with Paraburkholderia phytofirmans Supernatant Reveals Changes in Nicotinamide, Amino Acids, Biotin, and Plant Hormones

Microbial competition within plant tissues affects invading pathogens’ fitness. Metabolomics is a great tool for studying their biochemical interactions by identifying accumulated metabolites. Xylella fastidiosa, a Gram-negative bacterium causing Pierce’s disease (PD) in grapevines, secretes various virulence factors including cell wall-degrading enzymes, adhesion proteins, and quorum-sensing molecules. These factors, along with outer membrane vesicles, contribute to its pathogenicity. Previous studies demonstrated that co-inoculating X. fastidiosa with the Paraburkholderia phytofirmans strain PsJN suppressed PD symptoms. Here, we further investigated the interaction between the phytopathogen and the endophyte by analyzing the exometabolome of wild-type X. fastidiosa and a diffusible signaling factor (DSF) mutant lacking quorum sensing, cultivated with 20% P. phytofirmans spent media. Liquid chromatography–mass spectrometry (LC-MS) and the Method for Metabolite Annotation and Gene Integration (MAGI) were used to detect and map metabolites to genomes, revealing a total of 121 metabolites, of which 25 were further investigated. These metabolites potentially relate to host adaptation, virulence, and pathogenicity. Notably, this study presents the first comprehensive profile of X. fastidiosa in the presence of a P. phytofirmans spent media. The results highlight that P. phytofirmans and the absence of functional quorum sensing affect the ratios of glutamine to glutamate (Gln:Glu) in X. fastidiosa. Additionally, two compounds with plant metabolism and growth properties, 2-aminoisobutyric acid and gibberellic acid, were downregulated when X. fastidiosa interacted with P. phytofirmans. These findings suggest that P. phytofirmans-mediated disease suppression involves modulation of the exometabolome of X. fastidiosa, impacting plant immunity.

60 APPLIED LIFE SCIENCES↗

Formal Safety Certification of Aerospace Software

In principle, formal methods offer many advantages for aerospace software development: they can help to achieve ultra-high reliability, and they can be used to provide evidence of the reliability claims which can then be subjected to external scrutiny. However, despite years of research and many advances in the underlying formalisms of specification, semantics, and logic, formal methods are not much used in practice. In our opinion this is related to three major shortcomings. First, the application of formal methods is still expensive because they are labor- and knowledge-intensive. Second, they are difficult to scale up to complex systems because they are based on deep mathematical insights about the behavior of the systems (t.e., they rely on the "heroic proof"). Third, the proofs can be difficult to interpret, and typically stand in isolation from the original code. In this paper, we describe a tool for formally demonstrating safety-relevant aspects of aerospace software, which largely circumvents these problems. We focus on safely properties because it has been observed that safety violations such as out-of-bounds memory accesses or use of uninitialized variables constitute the majority of the errors found in the aerospace domain. In our approach, safety means that the program will not violate a set of rules that can range for the simple memory access rules to high-level flight rules. These different safety properties are formalized as different safety policies in Hoare logic, which are then used by a verification condition generator along with the code and logical annotations in order to derive formal safety conditions; these are then proven using an automated theorem prover. Our certification system is currently integrated into a model-based code generation toolset that generates the annotations together with the code. However, this automated formal certification technology is not exclusively constrained to our code generator and could, in principle, also be integrated with other code generators such as RealTime Workshop or even applied to legacy code. Our approach circumvents the historical problems with formal methods by increasing the degree of automation on all levels. The restriction to safety policies (as opposed to arbitrary functional behavior) results in simpler proof problems that can generally be solved by fully automatic theorem proves. An automated linking mechanism between the safety conditions and the code provides some of the traceability mandated by process standards such as DO-178B. An automated explanation mechanism uses semantic markup added by the verification condition generator to produce natural-language explanations of the safety conditions and thus supports their interpretation in relation to the code. It shows an automatically generated certification browser that lets users inspect the (generated) code along with the safety conditions (including textual explanations), and uses hyperlinks to automate tracing between the two levels. Here, the explanations reflect the logical structure of the safety obligation but the mechanism can in principle be customized using different sets of domain concepts. The interface also provides some limited control over the certification process itself. Our long-term goal is a seamless integration of certification, code generation, and manual coding that results in a "certified pipeline" in which specifications are automatically transformed into executable code, together with the supporting artifacts necessary for achieving and demonstrating the high level of assurance needed in the aerospace domain.

Denney, Ewen↗

Depth-resolved sagebrush root metabolomics, rhizosphere microbial communities, and geochemistry at the East River Watershed

This data set consists of results from soil nutrient profile, untargeted metabolomics, mass spec imaging, and amplicon sequencing. Data for soil nutrient profile includes common cations (Ca, Mg, Na, and K etc.) extracted from 3 digesting steps – ammonia acetate (for exchangeable cations), nitric acid (for acid dissolved fraction), and hydrofluoric acid/perchloric acid (HF/HClO4) for whole soil digestion. It also includes concentration of organic carbon, inorganic nitrogen (ammonia and nitrate) and phosphorus (Bray-1 P and nitric acid extract), and total nitrogen and phosphorus. Data for untargeted metabolomics includes metabolomic profile for root exudate/tissues and soil extracts from depths at surface soil to saprolite, that were measured using gas chromatography – mass spectrometry (GC-MS), and liquid chromatography – tandem mass spectrometry (LC-MS/MS). Data for mass spec imaging includes spatial distribution of metabolites that were detected and annotated with Fourier transformation ion cyclotron resonance mass spectrometer (FTICR-MS). Data for amplicon sequencing includes the base paired 16S and ITS ribosomal RNA sequences from Miseq Illumina sequencing. All samples were collected from 2 sampling campaign October 2022 and June 2023. Collectively, these datasets enable a mechanistic evaluation of how nutrient acquisition, especially nitrogen and phosphorus, differs between shallow roots operating in soil and deep roots functioning within the fractured bedrock zone. All files are provided as comma-separated values (CSV) fies (.csv) and (GZIP) file (.gz). The compressed .gz FASTQ files can be read directly in R using the dada2 package as part of the amplicon sequence analysis workflow. This work was supported by the Watershed Function Science Focus Area at Lawrence Berkeley National Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-05CH11231. This research was performed on a project award 60563 (https://dx.doi.org/10.46936/expl.proj.2022.60563/60008727) from the Environmental Molecular Sciences Laboratory, a DOE Office of Science User Facility sponsored by the Biological and Environmental Research program under Contract No. DE-AC05-76RL01830.

EARTH SCIENCE > AGRICULTURE > SOILS > CARBON↗

SFA-VirOmics

PNNL's Soil Microbiome Science Focus Area (SFA) is focused on understanding the basic biology underpinning how interactions among various soil microbial community members, across trophic levels, lead to the emergence of community functions. Moisture, in particular, drives microbial interactions and influences everything from cell function to substrate fate within soils. The group predicts this results in repeatable, predictable phenotypes. The sum of these phenotypes comprises the “soil metaphenome”. Understanding how the soil metaphenome shifts in response to moisture will provide a basis for modeling and predicting these shifts in reaction network responses. Visit the PNNL Soil Microbiome Science Focus Area Program homepage for more information. PNNL’s Soil Microbiome SFA Virome Dataset Annotation page is an extension to the PNNL Soil Microbiome SFA repository on DataHub allows for exploring and downloading integrated experimental omics dataset annotations, associated experimental metadata, pre- and post-processed viral data files, and other associated materials directly related to experimental viral project data.

59 BASIC BIOLOGICAL SCIENCES↗

Global analysis of the yeast knockout phenome

Genome-wide phenotypic screens in the budding yeast Saccharomyces cerevisiae, enabled by its knockout collection, have produced the largest, richest, and most systematic phenotypic description of any organism. However, integrative analyses of this rich data source have been virtually impossible because of the lack of a central data repository and consistent metadata annotations. Here, we describe the aggregation, harmonization, and analysis of ~14,500 yeast knockout screens, which we call Yeast Phenome. Using this unique dataset, we characterized two unknown genes (YHR045W and YGL117W) and showed that tryptophan starvation is a by-product of many chemical treatments. Furthermore, we uncovered an exponential relationship between phenotypic similarity and intergenic distance, which suggests that gene positions in both yeast and human genomes are optimized for function.

59 BASIC BIOLOGICAL SCIENCES↗

Deep Green: Structural and Functional Genomic Characterization of Conserved Unannotated Green Lineage Proteins

Our overall objective is to improve and increase functional and structural predictions for a growing number of plant proteins of unknown structure and function (the Deep Green proteins), and to make our predictive data useful and accessible to the larger research community. The project is divided into five major objectives which include 1) Assembly and curation of Deep Green candidate protein sets; 2) in silico structural and functional predictions and network analyses; 3) assembly and validation of reverse genetic resources in Chlamydomonas reinhardtii (Chlamydomonas); 4) high throughput functional genomics characterization and prioritization in Chlamydomonas; and 5) structural validation of selected candidates and functional validation in two important reference plant species, Arabidopsis thaliana (Arabidopsis) and Setaria viridis (Setaria).

59 BASIC BIOLOGICAL SCIENCES↗

Realtime Knowledge Management (RKM): From an International Space Station (ISS) Point of View

We are developing automated methods to provide realtime access to spacecraft domain knowledge relevant a spacecraft's current operational state. The method is based upon analyzing state-transition signatures in the telemetry stream. A key insight is that documentation relevant to a specific failure mode or operational state is related to the structure and function of spacecraft systems. This means that diagnostic dependency and state models can provide a roadmap for effective documentation navigation and presentation. Diagnostic models consume the telemetry and derive a high-level state description of the spacecraft. Each potential spacecraft state description is matched against the predictions of models that were developed from information found in the pages and sections in the relevant International Space Station (ISS) documentation and reference materials. By annotating each model fragment with the domain knowledge sources from which it was derived we can develop a system that automatically selects those documents representing the domain knowledge encapsulated by the models that compute the current spacecraft state. In this manner, when the spacecraft state changes, the relevant documentation context and presentation will also change.

Robinson, Peter I.↗

Characterization of the biofilm landscape of Bacillus subtilis by spatial microproteomics

Bulk proteomics has been demonstrated to differentiate subpopulations within bacterial colonies, yet advanced analyses by mass spectrometry imaging (MSI) hold even greater promise for the future. This technology can enable high-throughput spatial phenotyping that can reshape biological discovery by providing visualization of components of various biomolecular mechanisms. With high mass resolving power and high spatial resolution analyses being routine, we can confidently enable intact protein imaging directly from samples with minimal preparation. Pairing those analyses with bulk experimental libraries can provide high confidence in annotations of post-translational modifications (PTMs) and truncations. Revealing PTM localization within the samples unlocks a direct window into unknown biology at the microscale. However, top-down proteomics (TDP) is not commonplace for microbial species, largely due to challenges in identifying detected peptides and proteins; considering the theoretical proteome of even the well-studied model bacterium Bacillus subtilis was only partially mapped recently. With little still known about the form and function of many of these proteins – let alone proteoforms, where PTMs and truncations of the same protein may possess unique physiological roles – there is a wealth of work to be done. Here we jointly apply TDP and MSI to describe the microscale spatial proteomic landscape within B. subtilis and further demonstrate the feasibility of detecting differentiated subpopulations through proteoforms across the biofilm landscape.

bacterial biofilms↗

Interpreting the Lipidome: Bioinformatic Approaches to Embrace the Complexity

Background Improvements in mass spectrometry (MS) technologies coupled with bioinformatics developments have allowed considerable advancement in the measurement and interpretation of lipidomics data in recent years. Since research areas employing lipidomics are rapidly increasing, there is a great need for bioinformatic tools that capture and utilize the complexity of the data. Currently, the diversity and complexity within the lipidome is often concealed by summing over or averaging individual lipids up to (sub)class-based descriptors, losing valuable information about biological function and interactions with other distinct lipids molecules, proteins and/or metabolites. Aim of review To address this gap in knowledge, novel bioinformatics methods are needed to improve identification, quantification, integration and interpretation of lipidomics data. The purpose of this mini-review is to summarize exemplary methods to explore the complexity of the lipidome. Key scientific concepts of review Here we describe six approaches that capture three core focus areas for lipidomics: (1) lipidome annotation including a resolvable database identifier, (2) interpretation via pathway- and enrichment-based methods, and (3) understanding complex interactions to emphasize specific steps in the analytical process and highlight challenges in analyses associated with the complexity of lipidome data.

Kyle, Jennifer E.↗

Genome analyses reveal population structure and a purple stigma color gene candidate in finger millet

Finger millet is a key food security crop widely grown in eastern Africa, India and Nepal. Long considered a ‘poor man’s crop’, finger millet has regained attention over the past decade for its climate resilience and the nutritional qualities of its grain. To bring finger millet breeding into the 21 st century, here we present the assembly and annotation of a chromosome-scale reference genome. We show that this ~1.3 million years old allotetraploid has a high level of homoeologous gene retention and lacks subgenome dominance. Population structure is mainly driven by the differential presence of large wild segments in the pericentromeric regions of several chromosomes. Trait mapping, followed by variant analysis of gene candidates, reveals that loss of purple coloration of anthers and stigma is associated with loss-of-function mutations in the finger millet orthologs of the maize R1/B1 and Arabidopsis GL3/EGL3 anthocyanin regulatory genes. Proanthocyanidin production in seed is not affected by these gene knockouts.

59 BASIC BIOLOGICAL SCIENCES↗

A large-scale screening campaign of putative carbohydrate-active enzymes reveals a novel xylanase from anaerobic gut fungi

The genomes of anaerobic gut fungi (AGF) encode a diverse array of carbohydrate-active enzymes (CAZymes), yet exceedingly few of these enzymes have been experimentally validated or expressed in heterologous systems. Here, we developed a predictive bioinformatic pipeline to annotate novel putative CAZymes from anaerobic fungi and validate their activity through large-scale heterologous expression in Escherichia coli. A total of 173 fungal proteins from Piromyces finnis associated with biomass degradation were synthesized and expressed in E. coli, and 9.8% were soluble with expression levels exceeding 5% of the total proteome using high-throughput proteomic screening. Among these 17 heterologously expressed proteins, analysis with AlphaFold and FoldSeek predicted 13 multi-functional proteins containing catalytic domains fused with repetitive fungal dockerins, and half of the substrate predictions were experimentally validated. One promising enzyme, celsome_012, exhibited robust and specific activity against beechwood xylan at 37°C and pH 6.4, with titers that were also fivefold higher than those of other recombinant proteins screened here. Both Michaelis-Menten kinetics and the linearized Lineweaver-Burk equation yielded consistent values for K m , and its activation energy was estimated at 51.9 kJ/mol based on the Arrhenius model. This work supports the industrial translation of anaerobic fungal CAZymes due to their robust lignocellulolytic activity and provides a framework for prioritizing AGF proteins for efficient E. coli heterologous expression.

59 BASIC BIOLOGICAL SCIENCES↗