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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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102 records · Page 6
All sourcesNASA NTRSDOE OSTI
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

Telomere-to-telomere assemblies of chromosome 10 reveal complex adaptive variation of 3-ketoacyl-CoA-synthases in Populus trichocarpa likely driven by Helitrons

The model woody plant Populus trichocarpa displays an atypical alkene-diverse wax cuticle likely driven by copy number variation (CNV) of 3-ketoacyl-CoA synthases ( KCS ), which has been difficult to confirm with short-read assemblies. Long-read sequencing enables the development of telomere-to-telomere resources to detect cryptic variation, including CNVs, which are currently missed. Integrating this information can improve genomic prediction for breeding and provide insights into the evolutionary basis of important traits. Our analysis of 78 long-read haplotypes from chromosome 10 identified more than twice as many KCS genes as previously reported, and numerous intragenic non-synonymous substitutions. Random Forest predictive models highlighted the importance of Potri.010G079500 in producing very long chain alkenes; however, its absence did not predict previously reported alkene-deficient phenotypes. Instead, alkene levels are best predicted by the combinations of KCS copies. Additionally, amino acid substitutions clustered around ligand and donor binding pockets, suggesting they contribute to differing wax cuticle composition. Finally, each KCS gene and copy was linked to a Helitron transposon. A phylogenetic analysis suggests Helitrons are the evolutionary mechanism for generating KCS tandem arrays. Long-read generated telomere-to-telomere assemblies of P. trichocarpa chromosome 10 revealed large-effect loci critical to genetic studies that are unattainable from short-reads. This new resource produced novel insights into genome structure and function, and a novel mechanism for generating tandem gene duplication. Our results highlight that, given current challenges in annotation and assembly, detailed and focused long-read sequences are key to interpreting complex genomic regions that contain tandem copy number variants.

09 BIOMASS FUELS↗
DOE OSTI2024

Altering translation allows E. coli to overcome chemically stabilized G-quadruplexes

Genomic DNA from each sample was prepared using the Wizard Genomic DNA Purification Kit (Promega) and after, DNA was quantified using the QuantiFluor ONE dsDNA System (Promega). Genomic DNA underwent shearing to ~200 bp fragments via sonication and the gDNA fragments were prepared for sequencing using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). Bead-based size selection was used to select ~200 bp fragments and the fragments then underwent a splinkerette PCR using a Tn5-enriching forward primer and custom reverse primers for multiplexing. A final bead-based size selection was used to select for the correct length DNA. DNA was sequenced at the University of Michigan Advanced Genomics Core using Illumina sequencing with a custom read primer reading the last 10 nt of the transposon. PhiX174 DNA spike was added to the run to ensure sufficient sequence diversity on the flow cell. Then, a custom index read primer and standard Illumina primer were used to sequence the index reads and PhiX174, respectively.

Keck, James L.↗
NASA NTRS2000

A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin

Both physiological and genetic evidence indicate interconnections among plant responses to different hormones. We describe a pleiotropic recessive Arabidopsis transposon insertion mutation, designated hyponastic leaves (hyl1), that alters the plant's responses to several hormones. The mutant is characterized by shorter stature, delayed flowering, leaf hyponasty, reduced fertility, decreased rate of root growth, and an altered root gravitropic response. It also exhibits less sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid (ABA). The auxin transport inhibitor 2,3,5-triiodobenzoic acid normalizes the mutant phenotype somewhat, whereas another auxin transport inhibitor, N-(1-naph-thyl)phthalamic acid, exacerbates the phenotype. The gene, designated HYL1, encodes a 419-amino acid protein that contains two double-stranded RNA (dsRNA) binding motifs, a nuclear localization motif, and a C-terminal repeat structure suggestive of a protein-protein interaction domain. We present evidence that the HYL1 gene is ABA-regulated and encodes a nuclear dsRNA binding protein. We hypothesize that the HYL1 protein is a regulatory protein functioning at the transcriptional or post-transcriptional level.

NASA Discipline Plant Biology↗
NASA NTRSDate not supplied

Maize Rough Endosperm6 (rgh6) Encodes A Predicted Dead-Box RNA Helicase and Affects Mirna Processing in Endosperm Development

Maize rough endosperm (rgh) mutants have defective kernels with a rough, etched, or pitted endosperm surface. Molecular genetic analysis of this mutant class has identified multiple RNA processing proteins critical to endosperm development. Here, we report on the developmental and molecular function of the rgh6 locus. The rgh6 mutant was isolated from the UniformMu transposon tagging population. Mutant kernels have reduced endosperm size and defective embryos that develop in a more apical position than typical for defective embryos. TB translocation crosses revealed that rgh6 mutant endosperm inhibits normal embryo development. Positional cloning of the rgh6 locus found that it encodes a predicted DEAD-box RNA helicase. Consistent with a predicted function for RNA processing, transient expression of a RGH6-GFP fusion protein is localized to nucleolus and nuclear speckles in Nicotiana benthamiana leaves. Rgh6 transcripts are highly expressed in endosperm epidermal cell types such as the aleurone, basal endosperm cell layer, embryo surrounding region, and endosperm adjacent to scutellum. Markers of these cell types show increased levels in rgh6 mutant kernels. Mutant endosperm tissues have increased precursor microRNA (pre-miRNA) and decreased mature miRNA relative to normal sibling endosperm, indicating that rgh6 is required for miRNA processing. The transcript levels for most miRNA target genes accumulate to a higher level in rgh6 mutant tissue. These results suggest that miRNA processing and regulation of miRNA target genes are required for normal endosperm development.

Plant Sciences↗
NASA NTRS2024

Isolation, Characterization and Genetic Manipulation of Cold-Tolerant, Manganese-Oxidizing Pseudomonas sp. Strains

Manganese-oxidizing bacteria (MnOB) produce Mn oxide minerals that can be used by humans for bioremediation but the purpose for the bacterium is less clear. This study describes the isolation and characterization of cold-tolerant MnOB strains isolated from a compost pile in Morris, Minnesota, USA: Pseudomonas sp. MS-1 and DSV-1. The strains were preliminarily identified as members of species Pseudomonas psychrophila by 16S rRNA analysis and a multi-locus phylogenetic study using a database of 88 genomes from the Pseudomonas genus. However, the average nucleotide identity (ANI) between these strains and the P. psychrophila sp. CF149 type strain was less than 93%. Thus, the two strains are members of a novel species that diverged from P. psychrophila . DSV-1 and MS-1 are cold tolerant; both grow at 4C but grow faster at 24C. Unlike the mesophilic MnOB P. putida GB-1, both strains are capable of robustly oxidizing Mn at low temperatures. Both DSV-1 and MS-1 genomes contain homologs of several Mn oxidation genes found in P. putida GB-1 ( mnxG, mcoA, mnxS1, mnxS2 and mnxR ). Random mutagenesis by transposon insertion was successfully performed in both strains and identified genes involved in Mn oxidation that were similar to those found in P. putida GB-1. Our results show that MnOB can be isolated from compost, supporting a role for Mn oxidation in plant waste degradation. The novel isolates Pseudomonas spp. DSV-1 and MS-1 both can oxidize Mn at low temperature and likely employ similar mechanisms and regulation as P. putida GB-1.

manganese oxidation↗
DOE OSTI2023

ABF DFO with Technology Holding, Inc.

This Agile BioFoundry Directed Funding Opportunity project with Technology Holding and partners focuses on the development of both a strain of Pseudomonas putida KT2440 and a corresponding bioprocess to convert cellulosic sugars to beta-ketoadipic acid, which can be used in performance nylons and polyesters. Our approach follows the Design-Build-Test-Learn cycle wherein we have transferred learnings from muconic acid production in P. putida to develop a glucose and xylose-utilizing beta-ketoadipic acid production strain. This strain achieves 65 g/L of beta-ketoadipic acid at 0.7 g/L/hr and a C-mol yield of 0.40. We are currently on-boarding arabinose utilization as well. To identify non-intuitive strain modifications as well, we are deploying a beta-ketoadipic acid biosensor and building randomly barcoded transposon insertion sequencing (RB-TnSeq) libraries and gene over-expression libraries in beta-ketoadipic acid production strains. Moreover, we are using global metabolomics and other systems biology tools to identify off-target pathways. Lastly, we are scaling up beta-ketoadipic acid production to kg-scale production for Technology Holding to evaluate in performance polymers with their partners.

beta-ketoadipic acid↗
DOE OSTI2022

Giant Starship Elements Mobilize Accessory Genes in Fungal Genomes

Accessory genes are variably present among members of a species and are a reservoir of adaptive functions. In bacteria, differences in gene distributions among individuals largely result from mobile elements that acquire and disperse accessory genes as cargo. In contrast, the impact of cargo-carrying elements on eukaryotic evolution remains largely unknown. Here, we show that variation in genome content within multiple fungal species is facilitated by Starships, a newly discovered group of massive mobile elements that are 110 kb long on average, share conserved components, and carry diverse arrays of accessory genes. We identified hundreds of Starship-like regions across every major class of filamentous Ascomycetes, including 28 distinct Starships that range from 27 to 393 kb and last shared a common ancestor ca. 400 Ma. Using new long-read assemblies of the plant pathogen Macrophomina phaseolina, we characterize four additional Starships whose activities contribute to standing variation in genome structure and content. One of these elements, Voyager, inserts into 5S rDNA and contains a candidate virulence factor whose increasing copy number has contrasting associations with pathogenic and saprophytic growth, suggesting Voyager’s activity underlies an ecological trade-off. We propose that Starships are eukaryotic analogs of bacterial integrative and conjugative elements based on parallels between their conserved components and may therefore represent the first dedicated agents of active gene transfer in eukaryotes. Our results suggest that Starships have shaped the content and structure of fungal genomes for millions of years and reveal a new concerted route for evolution throughout an entire eukaryotic phylum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A High-Throughput Method for Identifying Novel Genes That Influence Metabolic Pathways Reveals New Iron and Heme Regulation in Pseudomonas aeruginosa

The ability to simultaneously and more directly correlate genes with metabolite levels on a global level would provide novel information for many biological platforms yet has thus far been challenging. Here, we describe a method to help address this problem, which we dub “Met-Seq” ( met abolite-coupled Tn seq uencing).

59 BASIC BIOLOGICAL SCIENCES↗