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At least 145 records · Page 8

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DOE OSTI2025

A scalable and autoclavable oxygen nanosensor platform for metabolic monitoring of Saccharomyces cerevisiae in a bioreactor and other in situ systems

Polymer-encapsulated dye nanoparticle sensors are a valuable approach to achieving in situ analyte measurements with luminescence; however, typical emulsion-based nanosensors are poorly suited for large-scale biological samples due to limitations of synthesis scalability and stability. Branched polyethylenimine (PEI) is a versatile polymer scaffold ideal for constructing nanoparticles with various covalently conjugated moieties due to their high density of reactive primary amines, high water solubility, and biological stability. In this work, we used branched polyethylenimine as a scaffold-based approach for making a stable and scalable ratiometric oxygen sensor. Pt (II) tetracarboxyporphine was used as an oxygen-sensing dye and coumarin 343 as a reference dye, all covalently linked to the PEI scaffold producing a product that could withstand sterilization procedures and easily be scaled. To minimize toxicity from the PEI scaffold, we conjugated it with 2000 MW PEG. The applicability of the sensors was demonstrated in a 200 mL Saccharomyces cerevisiae yeast culture, using orthogonal luminescent and electrochemical oxygen measurements to validate sensor response and measure the metabolic activity of the yeast in our culture. Further, this approach was able to match the sensitivity of our electrochemical measurements while improving upon drawbacks of other luminescent methods of oxygen detection, demonstrating effective monitoring for at least 20 h. Our scaffold-based approach is a modular and easily translatable technology that could be useful in various biotechnological applications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Biofoundries: Principles, Tools, and Applications

This chapter aims to provide a broad overview of biofoundries and introduces the principles, concepts, and case studies. We first outline the underlying principles of the Design-Build-Test-Learn (DBTL) framework and the role of automation, digital integration, and standardization. The chapter then explores core biofoundry technologies including robotic liquid handlers, high-throughput analytical instruments, and digital infrastructure for data management and workflow scheduling. Case studies spanning DNA assembly, protein engineering, metabolic engineering, and mammalian cell culture demonstrate the practical applications of the biofoundries. Economic and societal impacts are assessed alongside current limitations. We discuss the emerging trends including artificial intelligence integration and cloud-based distributed facilities to highlight its potential for biotechnology and the bioeconomy.

Singh, Nilmani↗

DOE OSTI2025

Review: Recent advances of ToF-SIMS for environmental analysis and imaging

Background: Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful surface analysis technique, initially developed and applied in inorganic materials and semiconductors. In past decades, ToF-SIMS has attracted more attention in its analysis capabilities of organic materials, with increased applications in biology, medical, and health development. It has also become a versatile and effective tool in environmental analysis due to its high mass resolution, mass accuracy, and depth profiling. Results: In this review, we first give an overview of the principle of ToF-SIMS and follow with recent ToF-SIMS applications in exemplary environmental study cases, including atmospheric aerosol, soil, water, plant, and organic solvent analysis. Moreover, sample preparation techniques are summarized in relation to corresponding environmental applications. Specifically, we call attention to ToF-SIMS investigations showcasing studies in surface chemical compositions, images, and depth profile analysis. These findings emphasize the important role of interfacial chemistry in environmental processes and provide valuable insights into dynamic processes, such as chemical transformation, particle formation, plant biology, and microbial inspired biotechnology development. The mass spectral imaging results acquired by ToF-SIMS offer a deeper understanding of intermediate stages and transient phases for environmental specimens. Significance: In situ and operando imaging offer new possibilities in studying phenomena in real time with high spatial resolution. Furthermore, it is anticipated that more research groups will use ToF-SIMS in environmental research given recent advances in measurement capabilities and surging needs in chemical mapping of complex analytes and systems.

Aerosol↗

DOE OSTI2025

Understanding, inhibiting, and engineering membrane transporters with high-throughput mutational screens

Promiscuous membrane transporters play vital roles across domains of life, mediating the uptake and efflux of structurally and chemically diverse substrates. Although many transporter structures have been solved, the fundamental rules of polyspecific transport remain inscrutable. In recent years, high-throughput genetic screens have solidified as powerful tools for comprehensive, unbiased measurements of variant function and hypothesis generation, but have had infrequent application and limited impact in the transporter field. In this primer, we describe the principles of high-throughput screening methods available for studying polyspecific transporters and comment on the necessity and potential of high-throughput methods for deciphering these transporters in particular. We present several screening approaches which could provide a fundamental understanding of the molecular basis of function and promiscuity in transporters. Here, we further posit how this knowledge can be leveraged to design inhibitors that combat multidrug resistance and engineer transporters as needed tools for synthetic biology and biotechnology applications.

EPIs↗

DOE OSTI2025

Enzyme property prediction using artificial intelligence

Artificial intelligence (AI)-driven enzyme property prediction enables rapid discovery and engineering of enzymes for a wide range of biotechnological and therapeutic applications. Here, we first introduce the key components in AI model development, including enzyme datasets, protein representation methods, and model architectures. We then highlight a variety of AI tools developed for the prediction of enzyme properties and functional annotations, including enzyme structure, kinetic parameters, substrate specificity, thermostability, solubility, Enzyme Commission number, and Gene Ontology term. Moreover, we describe representative downstream applications enabled by these AI tools. Finally, we discuss some challenges and opportunities as well as future prospects.

Yuan, Le [University of Illinois at Urbana-Champai↗

DOE OSTI2025

Editorial overview: Unlocking the secrets of nongenetic plasticity, one cell at a time

Cellular noise, the non-genetic variability observed among isogenic cells, arises from factors such as growth conditions, aging, and stochastic gene expression, influencing cell stress-response, metabolism, morphology, and size. Here, such plasticity, while critical for adaptation, often goes unnoticed with traditional population-averaging biotechnologies that inevitably mask cell-specific variations and prompting the question, "What else might we be missing". However, recent breakthroughs in optical imaging, microfluidics, and omics, are beginning to uncover the complexity of cellular plasticity. This special is-sue highlights some of these breakthroughs, with key contributions including innovations in multimodal chemical imaging, label-free microscopy, spatial and temporal omics, and droplet-based microfluidics. Collectively, these cutting-edge tools provide unprecedent-ed insights into non-genetic cell-to-cell variability, enhancing our understanding of cellu-lar plasticity and its implications for health, energy, and ecology.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

ToF-SIMS spectral data analysis of Paenibacillus sp. 300A biofilms and planktonic cells

Analysis of bacterial biofilms is particularly challenging and important with diverse applications from systems biology to biotechnology. Among the variety of techniques that have been applied, time-of-flight secondary ion mass spectrometry (ToF-SIMS) has many promising features in studying the surface characteristics of biofilms. ToF-SIMS offers high spatial resolution and high mass accuracy, which permit surface sensitive analysis of biofilm components. Thus, ToF-SIMS provides a powerful solution to addressing the challenge of bacterial biofilm analysis. This dataset covers ToF-SIMS analysis of Paenibacillus sp. 300A (300A) isolated from the Hanford site in Richland, WA. The strain is known to have metal and sulfur reducing properties and can be used for bioremediation, wastewater treatment, bioengineering and technology development. There is a current need to identify small molecules and fragments produced from bacterial biofilms. Static ToF-SIMS spectra of 300A were obtained using an IONTOF TOF-SIMS V instrument equipped with a 25 keV Bi 3 + metal ion gun. Identified molecules and molecular fragments are compared against known biological databases and the reported peaks have at least 65 ppm mass accuracy. These molecules range from lipids and fatty acids to flavonoids, quinolones, and other naturally occurring organic compounds. It is anticipated that the spectral identification of key peaks will assist detection of metabolites, extracellular polymeric substance molecules like polysaccharides, and biologically relevant small molecules using ToF-SIMS in future surface and interface research of bacterial biofilms.

Biofilms↗

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↗

DOE OSTI2026

Exploring the binding properties and activities of ancestral expansins

Bacterial expansins are non-lytic proteins capable of loosening cellulose networks, offering promising applications in agriculture, biotechnology, and material science. Their ability to disrupt noncovalent interactions in biopolymer matrices such as cellulose and chitin positions them as valuable tools for upgrading abundant natural materials. However, their industrial use remains limited due to their relatively low wall-loosening activity compared to plant expansins. To address this limitation, we applied Ancestral Sequence Resurrection (ASR) to reconstruct and characterize ancient variants of the Bacillus subtilis expansin BsEXLX1. ASR is a powerful evolutionary tool that enables the inference and synthesis of ancestral proteins, allowing researchers to explore functional traits that may have been lost over time. This approach not only provides insights into protein evolution but also facilitates the design of proteins with enhanced properties, such as improved substrate affinity or structural stability. In this study, we combined biochemical and biophysical assays to evaluate the activity and binding behavior of ancestral expansins. Our results reveal that ancestral variants exhibit increased cellulose affinity, reduced binding to acidic polysaccharides, and greater salt resistance. Furthermore, these traits enhance their wall-loosening activity and demonstrate the utility of ASR in engineering surface-active proteins for industrial applications, particularly in biomass processing and cellulose modification.

09 BIOMASS FUELS↗

DOE OSTI2024

Structural and biochemical basis for regiospecificity of the flavonoid glycosyltransferase UGT95A1

Glycosylation is a predominant strategy plants use to fine-tune the properties of small molecule metabolites to affect their bioactivity, transport, and storage. It is also important in biotechnology and medicine as many glycosides are utilized in human health. Small molecule glycosylation is largely carried out by family 1 glycosyltransferases. Here, we report a structural and biochemical investigation of UGT95A1, a family 1 GT enzyme from Pilosella officinarum that exhibits a strong, unusual regiospecificity for the 3'-O position of flavonoid acceptor substrate luteolin. We obtained an apo crystal structure to help drive the analyses of a series of binding site mutants, revealing that while most residues are tolerant to mutations, key residues M145 and D464 are important for overall glycosylation activity. Interestingly, E347 is crucial for maintaining the strong preference for 3'-O glycosylation, while R462 can be mutated to increase regioselectivity. The structural determinants of regioselectivity were further confirmed in homologous enzymes. Our study also suggests that the enzyme contains large, highly dynamic, disordered regions. We showed that while most disordered regions of the protein have little to no implication in catalysis, the disordered regions conserved among investigated homologs are important to both the overall efficiency and regiospecificity of the enzyme. This report represents a comprehensive in-depth analysis of a family 1 GT enzyme with a unique substrate regiospecificity and may provide a basis for enzyme functional prediction and engineering.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2025

A distinct class of ferredoxin:NADP + oxidoreductase enzymes driving thermophilic ethanol production

Biofuel production from lignocellulosic biomass offers a transformative solution to reduce global fossil fuel dependency. Certain thermophilic anaerobes, including Clostridium thermocellum, show promise for renewable ethanol production due to their ability to break down plant material at high temperatures. However, achieving commercially viable ethanol yields has proven challenging despite extensive engineering efforts. Here, we characterized 27 ferredoxin:NADP + oxidoreductase (Fnor) enzymes for their enzyme activity, nicotinamide cofactor specificity, thermotolerance, and functional expression in C. thermocellum . We identified a subset of 10 of these enzymes as a novel class of Fnor enzymes suited for metabolic pathways aimed at high-titer ethanol production. When expressed in engineered C. thermocellum , these enzymes increased ethanol production up to 2.2-fold. These findings establish a novel ethanol pathway and provide insights into physiological roles and biotechnological applications of this new class of Fnor enzymes.

09 BIOMASS FUELS↗

DOE OSTI2025

Engineering Pseudomonas putida for production of 3-hydroxyacids using hybrid type I polyketide synthases

Engineered type I polyketide synthases (T1PKSs) are a potentially transformative platform for the biosynthesis of small molecules. Due to their modular nature, T1PKSs can be rationally designed to produce a wide range of bulk or specialty chemicals. While heterologous PKS expression is best studied in microbes of the genus Streptomyces, recent studies have focused on the exploration of non-native PKS hosts. The biotechnological production of chemicals in fast growing and industrial relevant hosts has numerous economic and logistic advantages. With its native ability to utilize alternative feedstocks, Pseudomonas putida has emerged as a promising workhorse for the sustainable production of small molecules. Here, we outline the assessment of P. putida as a host for the expression of engineered T1PKSs and production of 3-hydroxyacids. After establishing the functional expression of an engineered T1PKS, we successfully expanded and increased the pool of available acyl-CoAs needed for the synthesis of polyketides using transposon sequencing and protein degradation tagging. This work demonstrates the potential of T1PKSs in P. putida as a production platform for the sustainable biosynthesis of unnatural polyketides.

Schmidt, Matthias↗

DOE OSTI2024

Tapping the treasure trove of atypical phages

With advancements in genomics technologies, a vast diversity of ‘atypical’ phages, that is, with single-stranded DNA or RNA genomes, are being uncovered from different ecosystems. Though these efforts have revealed the existence and prevalence of these nonmodel phages, computational approaches often fail to associate these phages with their specific bacterial host(s), while the lack of methods to isolate these phages has limited our ability to characterize infectivity pathways and new gene function. In this review, we call for the development of generalizable experimental methods to better capture this understudied viral diversity via isolation and study them through gene-level characterization and engineering. Establishing a diverse set of new ‘atypical’ phage model systems has the potential to provide many new biotechnologies, including potential uses of these atypical phages in halting the spread of antibiotic resistance and engineering of microbial communities for beneficial outcomes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Comparative transcriptomics of CAZy enzymes in white- and brown-rot agaricomycetes: Evolutionary insights into lignocellulose degradation and the relevance of GH16 glycoside hydrolase functional divergence

White-rot and brown-rot fungi (WRF and BRF, respectively) decompose lignocellulose, the main structural component of plant biomass, through distinct mechanisms. This study examines the transcriptomic responses of three WRF species (Pleurotus ostreatus, Phanerochaete chrysosporium, and Heterobasidion irregulare) and two BRF species (Fomitopsis schrenkii and Rhodonia placenta) grown on poplar wood (W) and glucose (G) as sole carbon sources. RNA-seq analysis revealed upregulation of carbohydrate-active enzymes (CAZymes) linked to lignocellulose degradation. WRF displayed a broader enzymatic repertoire, whereas BRF employed a more selective approach. Among these responses, GH16 glycoside hydrolases were consistently upregulated across all species, including BRF. Since GH16 enzymes are involved in both plant hemicellulose modification and cell wall remodeling, their wood-induced expression may reflect multiple processes rather than a single conserved wood-decay mechanism. Structural and phylogenetic analyses revealed species-specific divergence, consistent with potential functional specialization. These findings broaden our understanding of fungal enzymatic strategies and highlight GH16 enzymes as candidates for further study. Beyond the immediate context of wood decay, this work has broader implications for fungal ecology, evolutionary biology, and biotechnological applications such as biomass conversion and sustainable bioenergy.

Fungal ecological strategies↗

DOE OSTI2025

XERICO as a target for engineering stress-resilient crops: Mechanisms, applications, and future directions

XERICO's capacity to enhance ABA-driven stress responses across diverse crops, its regulatory crosstalk with other hormonal pathways, and its compatibility with advanced genetic engineering tools highlight its central role in sustainable agriculture. Leveraging XERICO in crop improvement programs aligns with the urgent need to mitigate the impacts of climate-induced stress in agriculture, offering a pathway toward resilient and high-yielding crops. Here, by enabling crops to withstand drought and other environmental stresses, XERICO-based biotechnological approaches hold transformative potential for global food security and environmental sustainability.

09 BIOMASS FUELS↗

DOE OSTI2024

ToF-SIMS spectral analysis of Shewanella oneidensis MR-1 biofilms

Analysis of bacterial biofilms is particularly challenging and important with diverse applications from systems biology to biotechnology. Among the variety of techniques that have been applied, time-of-flight secondary ion mass spectrometry (ToF-SIMS) has many powerful features in studying the surface characteristics of biofilms. ToF-SIMS offers high spatial resolution, mass resolution, and mass accuracy, which permit surface sensitive analysis of biofilm components. Thus, ToF-SIMS provides a powerful solution to addressing the challenge of bacterial biofilm analysis. This dataset covers ToF-SIMS analysis of Shewanella oneidensis MR-1 isolated from freshwater lake sediment in New York state. The MR-1 strain is known to have metal and sulfur reducing properties and it can be used for bioremediation and wastewater treatment. There is a current need to identify small molecules and fragments produced from bacterial biofilms, especially those from extracellular polymeric substance (EPS). Static ToF-SIMS spectra of MR-1 were obtained using an IONTOF TOF.SIMS V instrument equipped with a 25 keV Bi$^+_3$ metal ion gun. Identified molecules and molecular fragments are compared against known biological databases and the reported peaks have at least 65 ppm mass accuracy. These molecules range from lipids, fatty acids, flavonoids, and quinolones to other naturally occurring organic compounds. It is anticipated that the mass spectral identification of key peaks will assist detection of metabolites, EPS molecules like polysaccharides, and biologically relevant small organic molecules using ToF-SIMS in future surface and interface research.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Artificial intelligence methods for protein structure and interaction prediction: Recent advances and challenges

Recent advances in artificial intelligence have introduced novel methods for high-accuracy prediction of protein tertiary structures, protein complex structures, and interactions between proteins and other biomolecules, such as small molecules and nucleic acids. Such advancements are accelerating biomedical research and the development of new protein design and bioengineering methods among many other important biotechnology applications. Here, in this review, we outline the recent advances in protein-centric biomolecular structure and interaction prediction, highlight some major challenges in the field, and discuss potential directions to address them.

Morehead, Alex [Lawrence Berkeley National Laborat↗

DOE OSTI2025

Revealing structure and shaping priorities in plant and fungal cell wall architecture via solid-state NMR

Plant and fungal cell walls are essential for growth, adaptation, and survival, with their intricate architectures dictating both resistance to stress and susceptibility to antifungal or biomass-degrading strategies. Understanding how these walls are built, remodeled, and function at the molecular level is therefore central to both clinical and biotechnological applications. Solid-state nuclear magnetic resonance (ssNMR) has emerged as a uniquely powerful tool for this purpose, as it reveals the structure, dynamics, and interactions of intact biopolymers without disrupting their native organization. Using this approach, recent studies have shown how structural polymorphism, polymer-polymer interactions, and species-specific remodeling govern mechanical integrity, drug resistance, and stress adaptation. Applications highlighted here include lignin-carbohydrate packing during plant stem maturation, fungal wall reorganization under treatment by wall-targeting antifungals such as echinocandin and nikkomycin, and the functional diversity of glucans, chitins, and mannans. Together, these insights uncover conserved principles of polymer assembly across kingdoms while informing new opportunities for antifungal development and biomass utilization. Ongoing advances in sensitivity and resolution are expected to broaden the reach of ssNMR and further accelerate its role in linking structural heterogeneity to biosynthetic complexity and biological function.

Antifungal↗