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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Chapter 2: Genetics of Unstudied Thermophiles for Industry

Thermophilic organisms hold great potential for industry due to their numerous advantages in biotechnological applications such as higher reaction rate, higher substrate loading, decreased susceptibility to reaction contamination, energy savings in industrial fermentations, and ability to express thermostable proteins that can be utilized in many important industrial processes. Bioprospecting for thermophiles will continue to reveal new enzymatic and metabolic paradigms with industrial applicability. In order to translate these paradigms to production scale, routine methods for microbial genetic engineering are needed, yet remain to be developed in many newly isolated thermophiles. Major challenges and recent developments in the establishment of reliable genetic systems in thermophiles are discussed. Here, we use a hyperthermophilic, cellulolytic bacterium, Caldicellulosiruptor bescii, as a case study to demonstrate the development of a genetic system for an industrially useful thermophile, describing in detail methods for transformation, genetic tool utilization, and chromosomal modification using targeted gene deletion and insertion techniques.

93 SC - Biological and Environmental Research (BER↗
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 OSTI2021

Editorial: Multi-Omics Technologies for Optimizing Synthetic Biomanufacturing

Industrial manufacturing endures as an essential human activity yielding a variety of useful products; it plays a significant role in the global economy with huge impacts in everyday life. However, the manufacturing process requires consumption of various raw materials (especially petroleum derivatives), generates a variety of harmful waste products, causes pollution, and is energetically inefficient. Biological manufacturing from sustainable, affordable, and scalable feedstocks potentially enables the displacement of the entire portfolio of currently available products produced by industrial processes, enabling the manufacturing of renewable and eco-friendly products (Clomburg et al., 2017). Thus, successful development of a robust biomanufacturing strategy and technology platform, based on the latest advances in synthetic biology and chemical catalysis, will decrease both the cost and production time compared with previous manufacturing processes. Development of biomanufacturing processes using a synthetic biology platform requires the multidisciplinary efforts of science and engineering fields including molecular biology, microbiology, genetic engineering, informatics, metabolic modeling and chemical or process engineering (El Karoui et al., 2019).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Utilizing plant synthetic biology to accelerate plant-microbe interactions research

Plant-microbe interactions are critical to ecosystem resilience and substantially influence crop production. From the perspective of plant science, two important focus areas concerning plant-microbe interactions include: 1) understanding plant molecular mechanisms involved in plant-microbe interfaces and 2) engineering plants for increasing plant disease resistance or enhancing beneficial interactions with microbes to increase their resilience to biotic and abiotic stress conditions. Molecular biology and genetics approaches have been used to investigate the molecular mechanisms underlying plant responses to various beneficial and pathogenic microbes. While these approaches are valuable for elucidating the functions of individual genes and pathways, they fall short of unraveling the complex cross-talk across pathways or systems that plants employ to respond and adapt to environmental stresses. Also, genetic engineering of plants to increase disease resistance or enhance symbiosis with microbes has mainly been attempted or conducted through targeted manipulation of single genes/pathways of plants. Recent advancements in synthetic biology tool development are paving the way for multi-gene characterization and engineering in plants in relation to plant-microbe interactions. Here, we briefly summarize the current understanding of plant molecular pathways involved in plant interactions with beneficial and pathogenic microorganisms. Then, we highlight the progress in applying plant synthetic biology to elucidate the molecular basis of plant responses to microbes, enhance plant disease resistance, engineer synthetic symbiosis, and conduct in situ microbiome engineering. Lastly, we discuss the challenges, opportunities, and future directions for advancing plant-microbe interactions research using the capabilities of plant synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Biological funneling of phenolics from transgenic plants engineered to express the bacterial 3-dehydroshikimate dehydratase (qsuB) gene

The economic and environmental sustainability of lignocellulosic biomass biorefineries is predicated on generating biofuels and bioproducts from cell-wall polysaccharide and lignin polymers. Historical efforts in plant genetic engineering have focused on the development of strategies that facilitate biomass deconstruction, with more recently efforts including the synthesis of high-value chemicals in planta . One such genetic modification is the expression of the bacterial quinate and shikimate utilization B ( qsuB ) gene that increases the accumulation of protocatechuic acid in lignocellulosic biomass. Herein, we evaluated the effectiveness of an alkaline pretreatment process to extract phenolics directly from wild-type and QsuB-transgenic lines of Arabidopsis, poplar, and sorghum, and then upgrade them to the polyester precursor 2-pyrone-4,6-dicarboxylic acid (PDC) with an engineered strain of Novosphingobium aromaticivorans . Protocatechuic acid extracted from all QsuB transgenic lines was found to be mostly in the glycosylated form. Glycosylated protocatechuic acid and other plant-derived phenolics were effectively metabolized by N. aromaticivorans, and PDC production was greatest using extracts from an Arabidopsis QsuB transgenic line (∼5% w/w), followed by QsuB sorghum (∼1.1% w/w), and QsuB poplar (∼0.4% w/w) lines. The comparison of PDC production from wild-type and QsuB transgenic lines of Arabidopsis, poplar, and sorghum demonstrates the utility of a mild alkaline pretreatment to liberate phenolics from plant biomass that are either naturally present or that accumulate as a consequence of genetic engineering strategies. All QsuB transgenic lines outperformed their wild-type counterparts with respect to observed PDC yields. In addition, microbial funneling to PDC was effective even when most of the protocatechuic acid extracted was in glycosylated form, clearly demonstrating that this bacterium can metabolize these aromatic conjugates. These findings illustrate the benefits of combining plant and microbial engineering for bioproduct formation from phenolics in lignocellulosic biorefineries.

Umana, German E.↗
DOE OSTI2026

Improved Microalgal Carbon Utilization Efficiency via Integrated CO 2 Electro-Conversion to Formate and Microalgal Sequestration

This project developed a process to convert industrial carbon dioxide (CO 2 ) emissions into high-value, sustainable products through genetically engineered algae cultivation. While traditional microalgae cultivation depends on sparging CO 2 gas through water, this method is often inefficient because much of the gas escapes into the atmosphere before the algae can consume it. To overcome this challenge, the project designed an integrated system that first uses a CO 2 to formic acid electrolyzer to convert CO 2 into water-soluble formic acid/formate, then introduces formic acid/formate into the algae pond for cultivation, which allows the algae to access and utilize nearly all of the provided carbon, greatly increasing the efficiency of carbon utilization. The project team has successfully scaled up the CO 2 to formic acid electrolyzer from lab-scale to 1000 cm² and demonstrated industrially relevant current densities with the scaled-up electrolyzers using a CO 2 source that simulates industrial CO 2 waste.

42 ENGINEERING↗
DOE OSTI2023

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

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

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Heterologous expression of phosphite dehydrogenase in the chloroplast or nucleus enables phosphite utilization and genetic selection in Picochlorum spp.

Microalgae present a path to ameliorate problems associated with climate change via capture and reduction of CO2 to sustainable fuels and chemicals. Picochlorum is a genus of algae recently recognized for potential application in these regards due to its high productivity, thermotolerance, and halotolerance. Foundational genetic tools have recently been established in this genus. However, at present, genetic markers are limited, hindering genetic throughput and trait stacking approaches. To expand the suite of genetic tools and markers available for this genus, we sought to heterologously express the phosphite dehydrogenase (ptxD) gene from Pseudomonas stutzeri WM88 in both the nucleus and chloroplast of Picochlorum renovo and Picochlorum celeri. Additionally, the resultant strains allow for utilization of phosphite as a sole phosphorous source and as a nuclear and plastidial selection marker for genetic engineering. Growth analysis indicated comparable growth and composition when transgenic algae were grown in media containing phosphite as a sole phosphorus source, as compared to the conventionally used phosphate. Combined, these results expand the genetic toolbox available to the Picochlorum genus and present a potential crop protection and biocontainment strategy.

09 BIOMASS FUELS↗
DOE OSTI2026

Rapid T cell engineering to counter emerging threats (Full Technical Report)

Instead of genetic engineering, we sought to determine if a rapid method of membrane protein delivery could generate functional CAR-T cells in a rapid, safer, cost-effective manner. Using cell free protein synthesis, we generated CAR proteins embedded in a nanodisc, and thus effectively solubilized the protein. We demonstrated the first functional CAR proteins outside of a cellular context. We next tested uptake into immune cells and found extremely high uptake into T cells and multiple other populations of immune cells. We found some evidence of cytotoxicity in vitro conferred by the nanodisc delivered protein.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1996

An Archaea 5S rRNA analog is stably expressed in Escherichia coli

Mini-genes for 5S-like rRNA were constructed. These genes had a sequence which largely resembles that of the naturally occurring 5S rRNA of a bacterium, Halococcus morrhuae, which phylogenetically belongs to the Archaea. Plasmids carrying the mini-genes were transformed into Escherichia coli (Ec). Ribosomal incorporation was not a prerequisite for stable accumulation of the RNA product. However, only those constructs with a well-base-paired helix I accumulated RNA product. This result strongly implies that this aspect of the structure is likely to be an important condition for stabilizing 5S rRNA-like products. The results are consistent with our current understanding of 5S rRNA processing in Ec. When used in conjunction with rRNA probe technology, the resulting chimeric RNA may be useful as a monitoring tool for genetically engineered microorganisms or naturally occurring organisms that are released into the environment.

Non-NASA Center↗
DOE OSTI2022

Chromoanagenesis in plants: triggers, mechanisms, and potential impact

Chromoanagenesis is a single catastrophic event that involves, in most cases, localized chromosomal shattering and reorganization, resulting in a dramatically restructured chromosome. First discovered in cancer cells, it has since been observed in various other systems, including plants. In this review, we discuss the origin, characteristics, and potential mechanisms underlying chromoanagenesis in plants. Here, we report that multiple processes, including mutagenesis and genetic engineering, can trigger chromoanagenesis via a variety of mechanisms such as micronucleation, breakage–fusion–bridge (BFB) cycles, or chain-like translocations. The resulting rearranged chromosomes can be preserved during subsequent plant growth, and sometimes inherited to the next generation. Because of their high tolerance to genome restructuring, plants offer a unique system for investigating the evolutionary consequences and potential practical applications of chromoanagenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

CRISPR-Cas9/Cas12a systems for efficient genome editing and large genomic fragment deletions in Aspergillus niger

CRISPR technology has revolutionized fungal genetic engineering by accelerating the pace and expanding the feasible scope of experiments in this field. Among various CRISPR-Cas systems, Cas9 and Cas12a are widely used in genetic and metabolic engineering. In filamentous fungi, both Cas9 and Cas12a have been utilized as CRISPR nucleases. In this work we first compared efficacies and types of genetic edits for CRISPR-Cas9 and -Cas12a systems at the polyketide synthase (albA) gene locus in Aspergillus niger. By employing a tRNA-based gRNA polycistronic cassette, both Cas9 and Cas12a have demonstrated equally remarkable editing efficacy. Cas12a showed potential superiority over Cas9 protein when one gRNA was used for targeting, achieving an editing efficiency of 86.5% compared to 31.7% for Cas9. Moreover, when employing two gRNAs for targeting, both systems achieved up to 100% editing efficiency for single gene editing. In addition, the CRISPR-Cas9 system has been reported to induce large genomic deletions in various species. However, its use for engineering large chromosomal segments deletions in filamentous fungi still requires optimization. Here, we engineered Cas9 and -Cas12a-induced large genomic fragment deletions by targeting various genomic regions of A. niger ranging from 3.5 kb to 40 kb. Our findings demonstrate that targeted engineering of large chromosomal segments can be achieved, with deletions of up to 69.1% efficiency. Furthermore, by targeting a secondary metabolite gene cluster, we show that fragments over 100 kb can be efficiently and specifically deleted using the CRISPR-Cas9 or -Cas12a system. Overall, in this paper, we present an efficient multi-gRNA genome editing system utilizing Cas9 or Cas12a that enables highly efficient targeted editing of genes and large chromosomal regions in A. niger.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Microalgae as a future food source

One of the key challenges that we face in the 21st century is the need to feed an ever-increasing human population with increasingly limited natural resources. Even today it is estimated that roughly 1 out of 9 people in the world are undernourished, of which the most important factor is protein-energy malnutrition. By establishing microalgae as a new food and feed platform, we have the opportunity to increase the supply of these essential products to address global demands in a more efficient and environmentally sustainable way. Many types of algae are nutritionally complete foods, their yields outperform most plant crops, and there is a growing set of tools to develop improved strains of algae. Similar improvements were achieved in traditional crops through thousands of years of breeding and strain selection, whereas with the newest genetic engineering tools and advanced strain selection techniques, similar changes can be implemented in microalgae in just a few years. Here we describe different strategies that could be used to enhance the nutritional content, productivity, and organoleptic traits of algae to help drive development of this new crop. Clearly developing more efficient, sustainable, and nutritious foods and feed would be an enormous benefit for the planet, and algae represents an opportunity to develop a new crop that would complement traditional agriculture, and one that could potential result in a more efficient means to meet the world's food and feed supply.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Cyanobacterial Biofertilizer Production by Guanidine-Producing Enzymes

Cyanobacterial production of a biofertilizer shows promise as an environmentally benign alternative to conventional nitrogen fertilizers, reducing environmental and energy burdens through light-driven nitrogen and carbon fixation. One route to realizing the potential for a nitrogen-rich, slow-releasing biofertilizer involves the genetic engineering of cyanobacteria to produce guanidine. Recent advances have demonstrated enzymatic guanidine production in cyanobacteria, but an understanding of cyanobacterial guanidine metabolism is still limited. This Perspective highlights strategies and opportunities for cyanobacterial guanidine production in a Design−Build−Test−Learn cycle. Exploring new guanidine-producing enzymes via phylogenetics could expand candidate enzymes, while understanding the metabolism of substrates can identify constraints and opportunities in substrate utilization. Additionally, guanidine sensing and export are crucial areas of study to enable continuous fertilizer production and stable nitrogen flux. These strategies will guide the development of advanced nitrogen biofertilizer strategies for the agricultural sector.

09 BIOMASS FUELS↗
NASA NTRSDate not supplied

Bionutrients: Microbial Production of on-Demand Nutrients on the International Space Station

Providing adequate nutrition to crew members is essential as deep-space missions cannot rely on consistent Earth-based resupply. The current NASA pre-packaged food system is designed for low-earth-orbit missions with a stated shelf-life of two years and notable vitamin degradation over time. One strategy to mitigate nutrient loss is to implement bioregenerative food sources to supplement the pre-packaged food system. The BioNutrients project is designed to provide targeted production of short shelf-life nutrients in a single-use production pack. BioNutrients-1 includes two strains of edible yeast that have been genetically engineered to produce carotenoids, β-carotene, and zeaxanthin. This five-year mission on the International Space Station (ISS), launched in 2019, tests the long-term storage and nutrient production of the microorganisms of interest. Additional microorganisms are also stored in stasis packs to determine the effects of long-duration storage on the ISS. These organisms may be useful for production of fermented foods, pharmaceuticals, or biomanufacturing processes. Improving on the BioNutrients-1 project, BioNutrients-2 has optimized the production pack hardware by reducing the overall mass and volume of the system. Furthermore, BioNutrients-2 has expanded the projects scope by investigating novel products and microbial food sources. BioNutrients-2 is a six-month mission, launched in 2022, which tests production of fresh foods such as yogurt and kefir. The BioNutrients missions seek to provide advances for in-space biomanufacturing by addressing safe and reliable production of high-value nutrients and on-demand foods for future exploration efforts.

BioNutrients↗
NASA NTRSDate not supplied

Bionutrients: Microbial Production of on-Demand Nutrients on the International Space Station

Providing adequate nutrition to crew members is essential as deep-space missions cannot rely on consistent Earth-based resupply. The current NASA pre-packaged food system is designed for low-earth-orbit missions with a stated shelf-life of two years and notable vitamin degradation over time. One strategy to mitigate nutrient loss is to implement bioregenerative food sources to supplement the pre-packaged food system. The BioNutrients project is designed to provide targeted production of short shelf-life nutrients in a single-use production pack. BioNutrients-1 includes two strains of edible yeast that have been genetically engineered to produce carotenoids, β-carotene, and zeaxanthin. This five-year mission on the International Space Station (ISS), launched in 2019, tests the long-term storage and nutrient production of the microorganisms of interest. Additional microorganisms are also stored in stasis packs to determine the effects of long-duration storage on the ISS. These organisms may be useful for production of fermented foods, pharmaceuticals, or biomanufacturing processes. Improving on the BioNutrients-1 project, BioNutrients-2 has optimized the production pack hardware by reducing the overall mass and volume of the system. Furthermore, BioNutrients-2 has expanded the projects scope by investigating novel products and microbial food sources. BioNutrients-2 is a six-month mission, launched in 2022, which tests production of fresh foods such as yogurt and kefir. The BioNutrients missions seek to provide advances for in-space biomanufacturing by addressing safe and reliable production of high-value nutrients and on-demand foods for future exploration efforts.

BioNutrients↗
DOE OSTI2023

Differential ability of three bee species to move genes via pollen

Since the release of genetically engineered (GE) crops, there has been increased concern about the introduction of GE genes into non-GE fields of a crop and their spread to feral or wild cross-compatible relatives. More recently, attention has been given to the differential impact of distinct pollinators on gene flow, with the goal of developing isolation distances associated with specific managed pollinators. To examine the differential impact of bee species on gene movement, we quantified the relationship between the probability of getting a GE seed in a pod, and the order in which a flower was visited, or the cumulative distance traveled by a bee in a foraging bout. We refer to these relationships as ‘seed curves’ and compare these seeds curves among three bee species. The experiments used Medicago sativa L. plants carrying three copies of the glyphosate resistance (GR) allele as pollen donors (M. sativa is a tetraploid), such that each pollen grain carried the GR allele, and conventional plants as pollen recipients. Different foraging metrics, including the number of GR seeds produced over a foraging bout, were also quantified and contrasted among bee species. The lowest number of GR seeds set per foraging bout, and the GR seeds set at the shortest distances, were produced following leafcutting bee visits. In contrast, GR seeds were found at the longest distances following bumble bee visits. Values for honey bees were intermediate. The ranking of bee species based on seed curves correlated well with field-based gene flow estimates. Thus, differential seed curves of bee species, which describe patterns of seed production within foraging bouts, translated into distinct abilities of bee species to move genes at a landscape level. Bee behavior at a local scale (foraging bout) helps predict gene flow and the spread of GE genes at the landscape scale.

59 BASIC BIOLOGICAL SCIENCES↗