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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Biosynthesis of bioprivileged, linear molecules via novel carboligase reactions

Over the award period, we made progress on the three aims. We screened twenty-five carboligases for activity coupling twenty-one possible -keto acids (Aim 1). The carboligases were selected across a diverse set of protein sequences. Using Q-Exactive UHPLC-MS, we tested a total of 210 coupled products per enzyme and generated a dataset of 5250 enzyme-substrate activity relationships. We identified multiple enzymes that had activity for synthesizing suberic acid and heptanoic acid (Aim 2). We built a random forest model for predicting the activity of each enzyme toward substrates on which it was not tested using the data from Aim 1. Finally, we evaluated growth defects that occurred due to expression of different carboligases in E. coli (Aim 3). We were able to identify specific metabolites and putative pathways that, when supplemented in the media, recovered the growth defect associated with the presence of specific carboligases. We are in the process of publishing two manuscript describing the methods for high-throughput screening of enzyme promiscuity, using machine learning to predict activity on untested substrates, and enzyme activity data we collected. This project has produced enabling data for biosynthesis of a range of new-to-nature compounds to support biomanufacturing.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Self-Assembling Cell-Free Systems for Scalable Bioconversion

This project focused on developing cell-free systems to directly express multi-enzyme catalysts and perform CO2 bioconversions for industrial chemical production. The use of cell-free expression (CFE) systems derived from bacterial lysates is emerging as a promising approach for biomanufacturing. CFEs are genetically programmable, permit the expression of toxic enzymes, and allow for rapid prototyping of metabolic pathways. Research Contributions: 1. Understanding the Area Investigated: This research advances the understanding of cell-free systems by demonstrating their capability to perform complex multi-enzyme reactions. By directly expressing multi-gene systems, CFEs avoid the high costs and inefficiencies associated with producing and purifying enzymes for multi-step pathways. 2. Technical Effectiveness and Economic Feasibility: The project successfully engineered a CFE-based multienzyme biocatalyst for the de novo synthesis of serine and glycine from CO2 equivalents (formate and bicarbonate) and ammonia. This method achieved a 30% conversion rate of formate into these industrially important amino acids. Additionally, an 8-gene CFE biocatalyst was developed to produce malate, conserving 43% of carbon that would otherwise be lost as CO2. This approach has the potential to reach higher carbon efficiency than microbial production. 3. Public Benefit: The cell-free production of chemicals like serine, glycine, and malate using electrochemically generated formate could significantly reduce CO2 emissions. For example, satisfying the global malate market with this method could avoid approximately 400,000 tons of CO2 emissions annually. This work demonstrates the potential of CFE systems to produce platform chemicals, contributing to environmental sustainability and reducing reliance on petrochemicals. Future Prospects: The CFE-based biocatalyst process could be extended to produce a variety of chemicals, including other industrial di-acids, aromatics, terpenes, alcohols, and polymers. This project showcases the capabilities of cell-free expression systems for prototyping carbon-conserving pathways and sustainably bioproducing platform chemicals, marking a significant step towards economically-viable industrial processes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Develop High-Throughput Workflows for Whole-Genome Sequencing and Insertion Site Screening (CRADA Final Report)

The engineering of microbes for biomanufacturing (e.g. of fuels, chemicals, materials) applications has advanced to a stage where researchers screen genetic libraries with millions of variations each for those with enhanced productivity. This screening, however, can be slow and expensive, as screening individual variants in a high-throughput yet cost-effective manner is challenging. In this project, we aimed to reduce by 3-fold costs associated with the sequencing aspects of the screening process (to determine which genetic variant is responsible for an observed change in productivity), while being able to process over 1,000 samples per batch.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Develop High-Throughput Workflows for Whole-Genome Sequencing and Insertion Site Screening

The engineering of microbes for biomanufacturing (e.g. of fuels, chemicals, materials) applications has advanced to a stage where researchers screen genetic libraries with millions of variations each for those with enhanced productivity. This screening, however, can be slow and expensive, as screening individual variants in a high-throughput yet cost-effective manner is challenging. In this project, we aimed to reduce by 3-fold costs associated with the sequencing aspects of the screening process (to determine which genetic variant is responsible for an observed change in productivity), while being able to process over 1,000 samples per batch.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Final Technical Report for DE-SC0022206

This project developed foundational genetic, genomic, and epigenetic tools for anaerobic fungi (Neocallimastigomycota), a group of microorganisms with exceptional natural abilities to deconstruct lignocellulosic biomass. Efficient biomass deconstruction remains a major barrier to economical production of renewable fuels, chemicals, and materials from agricultural and forestry residues. The project sought to enable mechanistic studies and future engineering of anaerobic fungi by improving genomic resources, establishing methods for gene expression, and investigating epigenetic regulation of biomass-degrading pathways. Major accomplishments included generation of the first chromosome-scale genome assemblies for multiple anaerobic fungal species, providing publicly available genomic resources that support both engineering and fundamental biological research. The project established the first reproducible system for heterologous gene expression in anaerobic fungi and identified genomic features and mobile genetic elements that may support future development of stable transformation technologies. In parallel, the project demonstrated direct conversion of untreated lignocellulosic biomass into fuels and specialty chemicals through a fungal-yeast bioprocess and identified anaerobic fungal enzymes with utility for metabolic engineering. The research also revealed that epigenetic regulation plays an important role in controlling fungal gene expression and enzyme production, identifying potential strategies for enhancing biomass degradation. Collectively, this work established anaerobic fungi as a tractable emerging platform for bioenergy and biomanufacturing research, generated valuable public resources, trained the next generation of researchers, and advanced DOE-BER goals related to predictive biology, sustainable bioprocessing, and the circular bioeconomy.

Solomon, Kevin [University of Delaware] (ORCID:000↗
DOE OSTI2025

An Innovative High Throughput Genome Releaser for Rapid and Efficient PCR Screening

High-throughput PCR screening is vital in synthetic biology and metabolic engineering as it allows researchers to rapidly analyze and detect numerous targeted genetic mutation in the genome. Current challenges for high-throughput PCR screening in synthetic biology include efficiently preparing genomic DNA, optimizing protocols for diverse sample types, managing contamination risks, and effectively analyzing the large volumes of data generated while ensuring consistent and accurate results. In this study, we present the development of a High Throughput Genome Releaser (HTGR), an innovative device addressing common challenges in screening PCR. This genome DNA releaser is designed based on a squash method for rapid, cost-effective, and efficient DNA release, optimized for subsequent PCR reactions. After experimenting with various synthetic materials, we selected a plastic that closely replicates the smooth surface and compression properties of microscope slides, ensuring reliable performance. We engineered a device featuring a 96-Well Plate and a shear applicator, operable both manually and automatically, and compatible with standard liquid-handling robot platform. This compatibility enhances ease of use in high-throughput PCR workflows. Additionally, we developed software to support its automatic functions. Our results demonstrated that the specially engineered 96-Well Plate and HTGR can effectively squash fungal spores , which release enough genome DNA for PCR screening. The genome releaser facilitates the preparation of PCR-amplifiable genomic DNA substrate from 96 samples within minutes, eliminates the need for extraction buffers, and is adaptable to a wide range of microorganisms and cells, which could significantly advance biomanufacturing processes.

Yuan, Guoliang [BATTELLE (PACIFIC NW LAB)]↗
DOE OSTI2025

Developing a media formulation to sustain ex vivo chloroplast function

Chloroplasts are critical organelles in plants and algae responsible for accumulating biomass through photosynthetic carbon fixation and cellular maintenance through metabolism in the cell. Chloroplasts are increasingly appreciated for their role in biomanufacturing, as they can produce many useful molecules, and a deeper understanding of chloroplast regulation and function would provide more insight for the biotechnological applications of these organelles. However, traditional genetic approaches to manipulate chloroplasts are slow, and generation of transgenic organisms to study their function can take weeks to months, significantly delaying the pace of research. To develop chloroplasts themselves as a quicker and more defined platform, we isolated chloroplasts from the green algae, Chlamydomonas reinhardtii, and examined their photosynthetic function after extraction. Combined with a metabolic modeling approach using flux-balance analysis, we identified key metabolic reactions essential to chloroplast function and leveraged this information into reagents that can be used in a “chloroplast media” capable of maintaining chloroplast photosynthetic function over time ex vivo compared to buffer alone. We envision this could serve as a model platform to enable more rapid design-build-test-learn cycles to study and improve chloroplast function in combination with genetic modifications and potentially as a starting point for the bottom-up design of a synthetic organelle-containing cell.

Chlamydomonas reinhardtii↗
DOE OSTI2026

Advances in genetic tools for metabolic engineering of non-conventional yeasts

Non-conventional yeasts are emerging as powerful alternatives to Saccharomyces cerevisiae for metabolic engineering, owing to their innate stress tolerance, broad substrate utilization, and distinctive metabolic capabilities. These attributes position them as promising chassis for producing biofuels, pharmaceuticals, and specialty chemicals. This review synthesizes recent advances in genetic toolkits for four such species—Pichia kudriavzevii (Issatchenkia orientalis), Starmerella bombicola, Debaryomyces hansenii, and Pachysolen tannophilus—highlighting progress across plasmid architectures (episomal and integrative), identification of autonomously replicating sequences and centromeric elements, and the development of safe-harbor genomic loci. We summarize promoter and terminator libraries enabling tunable expression, the expansion of auxotrophic and antifungal selection markers with recycling strategies, and the rapid adaptation of CRISPR-based systems (Cas9 and Cas12a) with optimized guide RNA expression, multiplex editing, and approaches that enhance homologous recombination (e.g., KU70/80 disruption). We also review landing-pad platforms for modular, repeated integrations and transposon-based tools (e.g., piggyBac) that facilitate multigene pathway assembly. Collectively, these innovations are accelerating design-build-test-learn cycles and enabling precise, scalable engineering of non-conventional yeasts. Remaining challenges—including limited species-specific episomal systems, variable transformation efficiencies, genome-stability concerns, and alternative codon usage—define clear priorities for future toolkit development. Together, these advances and open needs chart a path toward robust, sustainable biomanufacturing using diverse non-conventional yeast chassis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

New Enzymes for Plastics Recycling from Biosensor-Guided Evolution and Machine Learning: Cooperative Research and Development Final Report, CRADA Number CRD-23-24335

The Agile BioFoundry (ABF) is a consortium of national laboratories dedicated to accelerating biomanufacturing and enabling the bioeconomy. The ABF operates a flexible biotechnology platform that can adjust to the needs of numerous government, academic, and industrial partners, thus enabling them to rapidly develop and optimize the production of a wide range of bioproducts. In this project, the ABF partnered with Birch Biosciences, Inc. to use ABF technology to assist in the development of high-performance enzymes that enable circular, sustainable recycling of poly(ethylene terephthalate) (PET) plastics. A primary goal of this project is to enable cost-effective, high yield, sustainable recycling of PET plastic packaging products.

09 BIOMASS FUELS↗
NASA NTRS2015

Effects of Polyhydroxybutyrate Production on Cell Division

Synthetic biological engineering can be utilized to aide the advancement of improved long-term space flight. The potential to use synthetic biology as a platform to biomanufacture desired equipment on demand using the three dimensional (3D) printer on the International Space Station (ISS) gives long-term NASA missions the flexibility to produce materials as needed on site. Polyhydroxybutyrates (PHBs) are biodegradable, have properties similar to plastics, and can be produced in Escherichia coli using genetic engineering. Using PHBs during space flight could assist mission success by providing a valuable source of biomaterials that can have many potential applications, particularly through 3D printing. It is well documented that during PHB production E. coli cells can become significantly elongated. The elongation of cells reduces the ability of the cells to divide and thus to produce PHB. I aim to better understand cell division during PHB production, through the design, building, and testing of synthetic biological circuits, and identify how to potentially increase yields of PHB with FtsZ overexpression, the gene responsible for cell division. Ultimately, an increase in the yield will allow more products to be created using the 3D printer on the ISS and beyond, thus aiding astronauts in their missions.

Synthetic Biology↗
NASA NTRSDate not supplied

BioNutrients: Microbial on-demand production of short shelf-life micronutrients in space

Long duration deep space missions pose a significant challenge when considering adequate nutrition for human spaceflight. Many micronutrients supplied in pre-packaged foods for low Earth orbit flights have a short shelf life and will degrade over multi-year missions. One strategy to mitigate the risk of micronutrient malnutrition is to grow fresh foods to supplement a pre-packaged diet. The BioNutrients project aims to develop systems for growing edible microorganisms that are genetically engineered to produce short shelf life micronutrients. BioNutrients-1 (BN-1) is a five-year spaceflight project currently underway on the International Space Station (ISS) that tests long-term storage and production of two carotenoids, β-carotene and zeaxanthin, in genetically engineered yeast strains. BN-1 on-orbit and ground control activities indicate that the nutrient production system will be stable for at least two years in ambient storage conditions. BN-1 is also testing the effects of long-term ISS storage on viability and gene expression using “stasis packs” containing a series of microorganisms useful in food and biomanufacturing processes. Stasis pack analysis is distinguishing microorganisms with full viability after two years of storage from other species that lose viability rapidly. In parallel, BioNutrients-2 (BN-2) is being developed as a six-month flight experiment that will expand the range of foods produced on ISS to include yogurt, kefir, and probiotic yeast. BN-2 will improve upon hardware design by transitioning from a hard-shell polycarbonate bioreactor to a flexible fluorinated ethylene propylene (FEP) bag. This change reduces the mass and volume required to store production packs. The long-term goal of BioNutrients is to develop the capability for rapid and safe delivery of multiple micronutrients in single-use production packs. We expect synthetic biology approaches similar to BioNutrients will play an important role for astronaut health in future NASA missions to the Moon and Mars.

Synthetic Biology↗
NASA NTRSDate not supplied

LEIA: An Investigation of Radiation Risks to Biology at the Lunar South Pole

Radiation and reduced gravity pose biological risks to crewed deep space exploration. At the cellular level, radiation damage can be amplified by reduced gravity. Empirical evidence on cellular responses to beyond low Earth orbit (BLEO) environments is imperative to develop effective countermeasures for crew health and in-space biomanufacturing. The Lunar Explorer Instrument for Space Biology Applications (LEIA) project is developing an instrument suite to be delivered to the south polar region of the Moon by the Commercial Lunar Payload Services (CLPS) program. This presentation will provide an overview of the LEIA hardware, experiments, and mission timeline. The LEIA instruments include the BioSensor, the ARES charged particle detector, and the Mini-FND. The BioSensor is an autonomous light emitting diode (LED)-based spectrophotometer and microfluidic incubator. The BioSensor activates yeast cultures and can measure cell growth, metabolic activity, and carotenoid production. The ARES is a Timepix-based charged particle radiation detector that measures dose, dose rate, and linear energy transfer spectra. The Mini-FND is a fast neutron detector that measures albedo neutron flux and energy spectra. Combined, these instruments will be used for yeast genetics experiments to quantify growth, metabolism, and synthetic biology-enabled production of human nutrients, while taking real time measurements of biologically relevant radiation exposure on the lunar surface. These data will be used to test the importance of selected DNA damage repair and reactive oxygen species defense pathways in mitigating cellular damage from lunar surface radiation.

Yeast↗
NASA NTRSDate not supplied

LEIA: An Investigation of Radiation Risks to Biology at the Lunar South Pole

Radiation and reduced gravity pose biological risks to crewed deep space exploration. At the cellular level, radiation damage can be amplified by reduced gravity. Empirical evidence on cellular responses to beyond low Earth orbit (BLEO) environments is imperative to develop effective countermeasures for crew health and in-space biomanufacturing. The Lunar Explorer Instrument for Space Biology Applications (LEIA) project is developing an instrument suite to be delivered to the south polar region of the Moon by the Commercial Lunar Payload Services (CLPS) program. This presentation will provide an overview of the LEIA hardware, experiments, and mission timeline. The LEIA instruments include the BioSensor, the ARES charged particle detector, and the Mini-FND. The BioSensor is an autonomous light emitting diode (LED)-based spectrophotometer and microfluidic incubator. The BioSensor activates yeast cultures and can measure cell growth, metabolic activity, and carotenoid production. The ARES is a Timepix-based charged particle radiation detector that measures dose, dose rate, and linear energy transfer spectra. The Mini-FND is a fast neutron detector that measures albedo neutron flux and energy spectra. Combined, these instruments will be used for yeast genetics experiments to quantify growth, metabolism, and synthetic biology-enabled production of human nutrients, while taking real time measurements of biologically relevant radiation exposure on the lunar surface. These data will be used to test the importance of selected DNA damage repair and reactive oxygen species defense pathways in mitigating cellular damage from lunar surface radiation.

Yeast↗
DOE OSTI2025

Smart culture medium optimization for recombinant protein production: Experimental, modeling, and AI/ML-driven strategies

Recombinant protein production (RPP) is central to biotechnology, where recombinant proteins are used as either end products or catalysts in the synthesis of chemicals, fuels, and materials. Among the major cost drivers, culture medium plays a pivotal role in determining protein yield and quality. This review presents a comprehensive perspective on the critical stages of “smart” culture medium optimization: planning, screening, modeling, optimization, and validation. In the planning stage, we examine the nutritional and energetic roles of medium components, including carbon, nitrogen, amino acids, salts, and trace metals, and their impacts on culture parameters such as pH, oxidative state, and osmolality. We highlight the variability in trace metal content due to water sources, culture vessels, and raw materials, which can substantially influence RPP. The screening stage covers Design of Experiments (DoE) approaches, assessing their theoretical basis, implementation, and limitations. For modeling, we describe methods that integrate experimental data to develop predictive models for smart medium formulation. Model-based optimization strategies can then be employed to select optimal media compositions for a given application. The validation stage aims to evaluate model predictions and provide feedback for model training and refinement. Finally, we survey mechanistic and artificial intelligence/machine learning (AI/ML)-driven models as integrated, transformational tools for predictive modeling of bioprocess conditions, nutrient availability, cellular metabolism, and protein quality, with the goal of optimizing culture media to enhance protein yields while reducing costs and environmental impact. We conclude by addressing the challenges of translating laboratory-scale medium optimization to industrial-scale settings and exploring future AI/ML-driven approaches that may overcome current bottlenecks and accelerate medium design for RPP. Overall, this review provides a unified framework for advancing smart medium design in RPP.

Artificial Intelligence/Machine Learning (AI/ML)↗
DOE OSTI2025

Workflow for evaluating enzyme immobilization and performance for continuous flow manufacturing

Enzymes have shown promise in various industries due to their functional specificity, catalytic efficiency, and environmental sustainability. These biological catalysts can be a pivotal component of manufacturing pipelines like continuous flow chemistry. For this, there exists a need to robustly immobilize enzymes on solid supports and assess the effects of the solid supports on catalytic performance and stability. Here, we use an industrially relevant model enzyme, C. ensiformis (Jack bean) urease, to demonstrate immobilization and assess performance in the context of continuous flow manufacturing. Various immobilization strategies were screened focusing on immobilization efficiency, protocol simplicity, and urease biocatalyst kinetics. Based on this, CDI-agarose and NHS-agarose resins were identified as the best-performing immobilization strategies for urease. CDI-agarose-urease and NHS-agarose-urease were then scaled up and applied to a large-scale continuous flow reactor to evaluate product yields, operational stability, and long-term stability. These experiments identified differences in stability and performance depending on the immobilization method tested. This highlights the importance of screening immobilization methods and subsequent enzyme performance for each candidate biocatalyst used in manufacturing to promote optimal performance and stability. As such, this work provides a framework for evaluating enzyme biocatalyst immobilization approaches to improve performance and enable transition into industrial processes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Lignin valorization reshapes sustainable biomass refining

As the largest natural reservoir of aromatics, lignin offers significant potential for bioproduct manufacturing through advances in valorization technologies. However, the intrinsically complex structures of lignin pose significant challenges for its fractionization and downstream valorization. Overcoming challenges in lignin chemistry modification is crucial for achieving effective lignin valorization and establishing sustainable biorefinery industries. This review explores the potential of tailoring lignin reactivity to enable functional bioproduct manufacturing thereby contributing to profitable biorefining. The intrinsic characteristics of lignin are first summarized, highlighting their roles in both fractionization and valorization. The latest progress in lignin fractionation is then presented, emphasizing their potential to tailor lignin chemistry, reactivity, and processibility. Furthermore, advancements in lignin valorization are covered, recognizing that tailored lignin reactivity is key to defining bioproduct functionality. By examining these chemical mechanisms, this review sheds on the structure-function relationships between lignin and its derived products. To address the dilemma of lignin valorization and biorefineries, a promising synergistic biorefinery is proposed. This involves redesigning biomass fractionation strategies, tailoring lignin chemistry, and upgrading both carbohydrate and lignin streams across the entire biorefinery chain—from feedstock to application. Altogether, a deeper understanding of tailored lignin chemistry is crucial for decoding the reaction mechanisms in biomass processing. A synergistic biorefinery could harness lignin's intrinsic properties to improve product functionality and address key challenges, paving the way for cost-effective, sustainable biorefinery solutions.

09 BIOMASS FUELS↗
DOE OSTI2026

Engineered plants for the production of the antioxidants arbutin and gallate

The shikimate pathway is a crucial metabolic route for the biosynthesis of numerous valuable chemicals. In this study, we engineered the shikimate pathway in plants via expression of microbial enzymes to produce the two important antioxidants gallate and arbutin. The engineered pathways utilize the aromatics protocatechuate and 4-hydroxybenzoate as metabolic intermediates. Through transient expression in Nicotiana benthamiana leaves, we first identified biosynthetic routes for the production of gallate from either chorismate or 3-dehydroshikimate. Gallate production was then achieved in Arabidopsis using a genetic background that overproduces protocatechuate and via expression of a mutated version of the 4-hydroxybenzoate hydroxylase PobA from Pseudomonas sp. Arbutin production was obtained in Arabidopsis using a genetic background that overproduces 4-hydroxybenzoate and via expression of the monooxygenase MNX1 from Candida parapsilosis. The best Arabidopsis transgenic lines accumulated gallate and arbutin in the range of 0.25 and 0.93 dry weight % (dwt%), respectively. Using sorghum for large-scale in planta production, the titers of gallate and arbutin produced from the intermediate 4-hydroxybenzoate reached 0.58 dwt% and 0.50 dwt%, respectively, in mature transgenic plants, surpassing levels typically observed in plants that naturally produce these compounds. Gallate and arbutin were readily extracted from plant tissues using methanol solvent. Analysis of extractive-free biomass showed only trace amounts of gallate and its precursors 4-hydroxybenzoate and protocatechuate crosslinked to cell walls, suggesting that they mainly occur as soluble conjugated forms stored in the vacuole. This study presents alternative synthesis routes using plant hosts for the eco-friendly production of gallate and arbutin.

Antioxidant↗