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At least 307 records · Page 17
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Genomic approaches to accelerate American chestnut restoration

More than a century after two introduced pathogens killed billions of American chestnut trees, introgression of resistance alleles from Chinese chestnuts has contributed to the recovery of self-sustaining populations. However, progress has been slow because of the complex genetic architecture of resistance. To better understand blight resistance, we compared reference genomes, gene expression responses, and stem metabolite profiles of the resistant Chinese and susceptible American chestnut species. To accelerate resistance breeding, we conducted large-scale phenotyping and genotyping in hybrids of these species. Simulation and inoculation experiments suggest that significant resistance gains are possible through selectively breeding trees with an average of 70 to 85% American chestnut ancestry. In conclusion, the resources developed in this work are foundational for breeding to create diverse restoration populations with sufficient disease resistance and competitive growth.

Westbrook, Jared W. [The American Chestnut Foundat↗
DOE OSTI2020

The Consortium for Advanced Sorghum Phenomics (CASP). Final report

The goal of CASP was to accelerate breeding of biomass sorghum [Sorghum bicolor (L.) Moench] by identifying genotypes exhibiting high yield under well-watered, pre- or post-drought and/or salinity-stress conditions. We did this by combining high-throughput, non-invasive drone phenotyping with genomics and molecular profiling. Field-based phenotyping utilized a multi-modal sensor suite of LiDAR, multispectral cameras, and thermal cameras mounted on a commercial drone to detect traits required for yield prediction and selection of drought and saline tolerant lines of sorghum. Traits of interest included plant height (PH), leaf area index (LAI), wet biomass (BMW), and biomass at 65% moisture (BM65) and were measured from emergence to harvest on a weekly basis over three growing seasons. The final output were measurements of traits on a plot-by-plot basis, identified by the plot ID used by the Proprietary data processing software enabled raw field data to be turned into plant traits and delivered to the PNNL and JGI within the same workday.

09 BIOMASS FUELS↗
DOE OSTI2023

Evolutionary consequences of nascent multicellular life cycles

A key step in the evolutionary transition to multicellularity is the origin of multicellular groups as biological individuals capable of adaptation. Comparative work, supported by theory, suggests clonal development should facilitate this transition, although this hypothesis has never been tested in a single model system. We evolved 20 replicate populations of otherwise isogenic clonally reproducing ‘snowflake’ yeast (Δace2/∆ace2) and aggregative ‘floc’ yeast (GAL1p::FLO1 /GAL1p::FLO1) with daily selection for rapid growth in liquid media, which favors faster cell division, followed by selection for rapid sedimentation, which favors larger multicellular groups. While both genotypes adapted to this regime, growing faster and having higher survival during the group-selection phase, there was a stark difference in evolutionary dynamics. Aggregative floc yeast obtained nearly all their increased fitness from faster growth, not improved group survival; indicating that selection acted primarily at the level of cells. In contrast, clonal snowflake yeast mainly benefited from higher group-dependent fitness, indicating a shift in the level of Darwinian individuality from cells to groups. Through genome sequencing and mathematical modeling, we show that the genetic bottlenecks in a clonal life cycle also drive much higher rates of genetic drift—a result with complex implications for this evolutionary transition. Our results highlight the central role that early multicellular life cycles play in the process of multicellular adaptation.

Life Sciences & Biomedicine - Other Topics↗
DOE OSTI2025

Inducible flippase-mediated metabolic engineering of Rhodosporidium toruloides for enhanced 3-hydroxypropionic acid production from corn stover hydrolysate

Rhodosporidium toruloides has gained increasing interests as a promising non-model host organism to produce a wide range of bioproducts from lignocellulosic biomass. Increasing the bioproduct titers, rates, and yields remains a challenge, largely due to a lack of robust and well-characterized genetic tools in this host. Here we developed an inducible flippase (FLP) and flippase recognition target (FRT) system that enables genetic manipulations without the need for additional selection markers. Synthetic inducible promoters were established, enabling regulation of FLP expression and efficient antibiotic marker removal. Leveraging this system, we engineered a strain to optimize 3-hydroxypropionic acid (3HP) production. Over four rounds of iterative genomic editing to resolve pathway bottlenecks, we achieved a 3HP titer of 69.4 g/L in fed-batch fermentation - the highest level reported in yeast from lignocellulosic hydrolysates. The engineered high 3HP producing strain offers a robust platform for sustainable bio-based chemical production from lignocellulosic feedstocks.

3-hydroxypropionic acid↗
DOE OSTI2022

Tousled-like kinase 2 targets ASF1 histone chaperones through client mimicry

Tousled-like kinases (TLKs) are nuclear serine-threonine kinases essential for genome maintenance and proper cell division in animals and plants. A major function of TLKs is to phosphorylate the histone chaperone proteins ASF1a and ASF1b to facilitate DNA replication-coupled nucleosome assembly, but how TLKs selectively target these critical substrates is unknown. Here, we show that TLK2 selectivity towards ASF1 substrates is achieved in two ways. First, the TLK2 catalytic domain recognizes consensus phosphorylation site motifs in the ASF1 C-terminal tail. Second, a short sequence at the TLK2 N-terminus docks onto the ASF1a globular N-terminal domain in a manner that mimics its histone H3 client. Disrupting either catalytic or non-catalytic interactions through mutagenesis hampers ASF1 phosphorylation by TLK2 and cell growth. Our results suggest that the stringent selectivity of TLKs for ASF1 is enforced by an unusual interaction mode involving mutual recognition of a short sequence motifs by both kinase and substrate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

REC protein family expansion by the emergence of a new signaling pathway

This report presents multi-genome evidence that REC protein family expansion occurs when the emergence of new pathways gives rise to functional discordance. Specificity between residues in REC domain containing response regulators with paired histidine kinases is under negative purifying selection, constrained by the presence of other bacterial two-component systems signaling cascades that share sequence and structural identity. Presuming that the two-component systems can evolve by neutral amino acid changes (neutral drift) when purifying evolutionary constraints are relaxed, how might the REC protein family expand by amino acid changes when these constraints remain intact? Using an unsupervised machine learning approach to observe the sequence landscape of REC domains across long phylogenetic distances, we find that within-gene recombination, a subcategory of gene conversion, switched the effector domain and, consequently, the regulatory context of a duplicated response regulator from transcriptional regulation by σ54 to that by σ70. We determined that the recombined response regulator diverged from its parent by episodic diversifying selection and neutral drift. Functional experiments of the parent of recombined response regulators in a model Pseudomonas putida KT2440 model system revealed that the parent and recombined response regulators sense and respond to different carboxylic acids. Finally, a residue-switching experiment using structural predictions and functional characterization suggests that the new residues in the recombined regulator could form a new interaction interface and mediate condition-specific phosphotransfer. Overall, our study finds that genetic perturbations can create conditions of functional discordance, whereby the REC protein family can evolve by episodic diversifying selection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Engineered Microorganisms for Enhanced Rare Earth Element Bio-mining and Separations (Final Technical Report)

Rare earth elements (REE) are critical ingredients of sustainable energy technologies, but their extraction from ore and separation from one another pose formidable challenges. To solve the challenge of REE supply, we used advanced genomics, high-throughput screening with synthetic REE minerals, and synthetic biology to engineer two sets of exotic microbes to (1) extract REE from ores, spent cracking catalysts, coal ash and electronic waste with high efficiency and selectivity, and (2) to purify REE into single element batches, all under benign conditions without the need of harsh solvents and high temperatures. This work integrated our expertise in systems and synthetic biology (Buz Barstow); rare-earth geochemistry (Esteban Gazel) and mineral synthesis (Megan Holycross); and microsystems engineering (Mingming Wu) by first elucidating the set of rules that predict an organism’s phenotype and then applying them to solve this critical problem in sustainable energy. These new technologies could help to revitalize the US rare earth industry and provide a new source of these critical elements for future energy technologies. We have already had some big success in tech transfer. Two of our team members (postdoctoral fellow Alexa Schmitz and graduate student Sean Medin) were able to study the supply chain for REE in the United States, and identify an opportunity to commercialize our REE mineral-dissolution technology. Alexa and Sean recently founded REEgen, Inc., an REE biomining company. Dr. Schmitz was recently awarded a fellowship from the Activate Foundation to support the first two years of REEgen. Cornell showed its support for this technology and company, and Dr. Schmitz was awarded the Rising Women Innovator’s award. These two awards unlocked support from Cornell’s Praxis Incubator.

58 GEOSCIENCES↗
DOE OSTI2023

Mitochondrial genome diversity across the subphylum Saccharomycotina

Eukaryotic life depends on the functional elements encoded by both the nuclear genome and organellar genomes, such as those contained within the mitochondria. The content, size, and structure of the mitochondrial genome varies across organisms with potentially large implications for phenotypic variance and resulting evolutionary trajectories. Among yeasts in the subphylum Saccharomycotina, extensive differences have been observed in various species relative to the model yeast Saccharomyces cerevisiae, but mitochondrial genome sampling across many groups has been scarce, even as hundreds of nuclear genomes have become available.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Polysaccharide utilization loci in Bacteroides determine population fitness and community-level interactions

Polysaccharide utilization loci (PULs) are co-regulated bacterial genes that sense nutrients and enable glycan digestion. Human gut microbiome members, notably Bacteroides, contain numerous PULs that enable glycan utilization and shape ecological dynamics. To investigate the role of PULs on fitness and inter-species interactions, we develop a CRISPR-based genome editing tool to study 23 PULs in Bacteroides uniformis (BU). BU PULs show distinct glycan-degrading functions and transcriptional coordination that enables the population to adapt upon loss of other PULs. Exploiting a BU mutant barcoding strategy, we demonstrate that in vitro fitness and BU colonization in the murine gut are enhanced by deletion of specific PULs and modulated by glycan availability. PULs mediate glycan-dependent interactions with butyrate producers that depend on the degradation mechanism and glycan utilization ability of the butyrate producer. Thus, PULs determine community dynamics and butyrate production and provide a selective advantage or disadvantage depending on the nutritional landscape.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Improved high-throughput screening technique to rapidly isolate Chlamydomonas transformants expressing recombinant proteins

Abstract The single-celled eukaryotic green alga Chlamydomonas reinhardtii has long been a model system for developing genetic tools for algae, and is also considered a potential platform for the production of high-value recombinant proteins. Identifying transformants with high levels of recombinant protein expression has been a challenge in this organism, as random integration of transgenes into the nuclear genome leads to low frequency of cell lines with high gene expression. Here, we describe the design of an optimized vector for the expression of recombinant proteins in Chlamydomonas , that when transformed and screened using a dual antibiotic selection, followed by screening using fluorescence activated cell sorting (FACS), permits rapid identification and isolation of microalgal transformants with high expression of a recombinant protein. This process greatly reduces the time required for the screening process, and can produce large populations of recombinant algae transformants with between 60 and 100% of cells producing the recombinant protein of interest, in as little as 3 weeks, that can then be used for whole population sequencing or individual clone analysis. Utilizing this new vector and high-throughput screening (HTS) process resulted in an order of magnitude improvement over existing methods, which normally produced under 1% of algae transformants expressing the protein of interest. This process can be applied to other algal strains and recombinant proteins to enhance screening efficiency, thereby speeding up the discovery and development of algal-derived recombinant protein products. Key points • A protein expression vector using double-antibiotic resistance genes was designed • Double antibiotic selection causes fewer colonies with more positive for phenotype • Coupling the new vector with FACS improves microalgal screening efficiency > 60%

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Macroevolutionary diversity of traits and genomes in the model yeast genus Saccharomyces

Species is the fundamental unit to quantify biodiversity. In recent years, the model yeast Saccharomyces cerevisiae has seen an increased number of studies related to its geographical distribution, population structure, and phenotypic diversity. However, seven additional species from the same genus have been less thoroughly studied, which has limited our understanding of the macroevolutionary events leading to the diversification of this genus over the last 20 million years. Here, we show the geographies, hosts, substrates, and phylogenetic relationships for approximately 1,800 Saccharomyces strains, covering the complete genus with unprecedented breadth and depth. We generated and analyzed complete genome sequences of 163 strains and phenotyped 128 phylogenetically diverse strains. This dataset provides insights about genetic and phenotypic diversity within and between species and populations, quantifies reticulation and incomplete lineage sorting, and demonstrates how gene flow and selection have affected traits, such as galactose metabolism. These findings elevate the genus Saccharomyces as a model to understand biodiversity and evolution in microbial eukaryotes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The foxtail millet ( Setaria italica ) terpene synthase gene family

Terpenoid metabolism plays vital roles in stress defense and the environmental adaptation of monocot crops. Here, we describe the identification of the terpene synthase (TPS) gene family of the panicoid food and bioenergy model crop foxtail millet (Setaria italica). The diploid S. italica genome contains 32 TPS genes, 17 of which were biochemically characterized in this study. Unlike other thus far investigated grasses, S. italica contains TPSs producing all three ent-, (+)- and syn-copalyl pyrophosphate stereoisomers that naturally occur as central building blocks in the biosynthesis of distinct monocot diterpenoids. Conversion of these intermediates by the promiscuous TPS SiTPS8 yielded different diterpenoid scaffolds. Additionally, a cytochrome P450 monooxygenase (CYP99A17), which genomically clustered with SiTPS8, catalyzes the C19 hydroxylation of SiTPS8 products to generate the corresponding diterpene alcohols. The presence of syntenic orthologs to about 19% of the S. italica TPSs in related grasses supports a common ancestry of selected pathway branches. Among the identified enzyme products, abietadien-19-ol, syn-pimara-7,15-dien-19-ol and germacrene-D-4-ol were detectable in planta, and gene expression analysis of the biosynthetic TPSs showed distinct and, albeit moderately, inducible expression patterns in response to biotic and abiotic stress. In vitro growth-inhibiting activity of abietadien-19-ol and syn-pimara-7,15-dien-19-ol against Fusarium verticillioides and Fusarium subglutinans may indicate pathogen defensive functions, whereas the low antifungal efficacy of tested sesquiterpenoids supports other bioactivities. Together, these findings expand the known chemical space of monocot terpenoid metabolism to enable further investigations of terpenoid-mediated stress resilience in these agriculturally important species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

PyPop: a mature open-source software pipeline for population genomics

Python for Population Genomics (PyPop) is a software package that processes genotype and allele data and performs large-scale population genetic analyses on highly polymorphic multi-locus genotype data. In particular, PyPop tests data conformity to Hardy-Weinberg equilibrium expectations, performs Ewens-Watterson tests for selection, estimates haplotype frequencies, measures linkage disequilibrium, and tests significance. Standardized means of performing these tests is key for contemporary studies of evolutionary biology and population genetics, and these tests are central to genetic studies of disease association as well. Here, we present PyPop 1.0.0, a new major release of the package, which implements new features using the more robust infrastructure of GitHub, and is distributed via the industry-standard Python Package Index. New features include implementation of the asymmetric linkage disequilibrium measures and, of particular interest to the immunogenetics research communities, support for modern nomenclature, including colon-delimited allele names, and improvements to meta-analysis features for aggregating outputs for multiple populations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Discovery of an ultraspecific triuret hydrolase (TrtA) establishes the triuret biodegradation pathway

Triuret (carbonyldiurea) is an impurity found in industrial urea fertilizer (<0.1% w/w) that is applied, worldwide, around 300 million pounds each year on agricultural lands. In addition to anthropogenic sources, endogenous triuret has been identified in amoeba and human urine, the latter being diagnostic for hypokalemia. The present study is the first to describe the metabolic breakdown of triuret, which funnels into biuret metabolism. We identified the gene responsible for triuret decomposition ( trtA ) in bacterial genomes, clustered with biuH , which encodes biuret hydrolase and has close protein sequence homology. TrtA is a member of the isochorismatase-like hydrolase (IHL) protein family, similarly to BiuH, and has a catalytic efficiency (k cat/ K M ) of 6 x 10 5 M –1 s –1 , a K M for triuret of 20 μM, and exquisite substrate specificity. Indeed, TrtA has four orders of magnitude less activity with biuret. Crystal structures of TrtA in apo and holo form were solved and compared with the BiuH structure. The high substrate selectivity was found to be conveyed by second shell residues around each active site. Mutagenesis of residues conserved in TrtA to the alternate consensus found in BiuHs revealed residues critical to triuret hydrolase activity but no single mutant evolved more biuret activity, and likely a combination of mutations is required to interconvert between TrtA, BiuH functions. TrtA-mediated triuret metabolism is relatively rare in recorded genomes (1–2%), but is largely found in plant-associated, nodulating, and endophytic bacteria. This study suggests functions for triuret hydrolase in certain eukaryotic intermediary processes and prokaryotic intermediary or biodegradative metabolism.

36 MATERIALS SCIENCE↗
DOE OSTI2022

Natural variation and improved genome annotation of the emerging biofuel crop field pennycress ( Thlaspi arvense )

The Brassicaceae family comprises more than 3,700 species with a diversity of phenotypic characteristics, including seed oil content and composition. Recently, the global interest in Thlaspi arvense L. (pennycress) has grown as the seed oil composition makes it a suitable source for biodiesel and aviation fuel production. However, many wild traits of this species need to be domesticated to make pennycress ideal for cultivation. Molecular breeding and engineering efforts require the availability of an accurate genome sequence of the species. Here, we describe pennycress genome annotation improvements, using a combination of long- and short-read transcriptome data obtained from RNA derived from embryos of 22 accessions, in addition to public genome and gene expression information. Our analysis identified 27,213 protein-coding genes, as well as on average 6,188 biallelic SNPs. In addition, we used the identified SNPs to evaluate the population structure of our accessions. The data from this analysis support that the accession Ames 32872, originally from Armenia, is highly divergent from the other accessions, while the accessions originating from Canada and the United States cluster together. When we evaluated the likely signatures of natural selection from alternative SNPs, we found 7 candidate genes under likely recent positive selection. These genes are enriched with functions related to amino acid metabolism and lipid biosynthesis and highlight possible future targets for crop improvement efforts in pennycress.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

In–context promoter bashing of the Sorghum bicolor gene models functionally annotated as bundle sheath cell preferred expressing phosphoenolpyruvate carboxykinase and alanine aminotransferase

In-context promoter bashing via genome editing is a route to identify and characterize critical regulatory regions that govern expression of genes of interest. The outcomes of in-context promoter bashing can be used to inform editing strategies to modulate the expression of selected gene models in a desired fashion. Here, we employed in-context promoter bashing to characterize the proximal upstream regulatory regions of sorghum genes encoding phosphoenolpyruvate carboxykinase bundle sheath (SbPEPCK.BS, SbiTx430.01G455400) and alanine aminotransferase bundle sheath (SbAlaAT.BS, SbiTx430.02G006600), two proteins involved in the PCK C 4 pathway. Characterized germinal edits within the targeted regions upstream of these two genes ranged in size from 138 up to 1790 bp. A 138 bp within the SbPEPCK.BS upstream region and a 1643 bp element within the SbAlaAT.BS upstream region were determined to be important for maintenance of transcription levels. No change in development or various physiological parameters was observed in characterized lineages carrying promoter edits. However, significant changes in seed reserves and a reduction in 100-seed weight were consistently observed, under both greenhouse and field environments, in plants carrying an edit in the promoter of SbPEPCK.BS gene, which were significantly reduced in transcript accumulation for this gene.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Supplementary table, figures and DNA sequences of sorghum gene models SbiRTx430.01G455400 and SbiRTx.02G006600 that feature primers, gRNAs and indels created

In-context promoter bashing via genome editing is a route to identify and characterize critical regulatory regions that govern expression of genes of interest. The outcomes of in-context promoter bashing can be used to inform editing strategies to modulate the expression of selected gene models in a desired fashion. Here we employed in-context promoter bashing to characterize the proximal upstream regulatory regions of sorghum genes encoding phosphoenolpyruvate carboxykinase (Sb.PEPCK.BS, SbiTx430.01G455400) and alanine aminotransferase (SbiTx430.02G006600, SbAlaAT.BS), two proteins involved in the PCK C4 pathway. Characterized germinal edits within the targeted regions upstream of these two genes ranged in size from 138 bp up to 1790 bp. A 138 bp within the Sb.PEPCK.BS upstream region and a 1643 bp element within the Sb.AlaAT.BS upstream region were determined to be important for maintenance of transcription levels. No change in development or various physiological parameters was observed in characterized lineages carrying promoter edits. However, significant changes in seed reserves and a reduction in 100 seed weight were consistently observed, under both greenhouse and field environments, in plants carrying an edit in the promoter of Sb.PEPCK.BS gene were significantly reduced in transcript accumulation for this gene.

Quach, Truyen [Center for Plant Science Innovation↗
DOE OSTI2025

A haplotype-resolved reference genome for Eucalyptus grandis

Eucalyptus grandis is a hardwood tree used worldwide as pure species or hybrid partner to breed fast-growing plantation forestry crops that serve as feedstocks of timber and lignocellulosic biomass for pulp, paper, biomaterials, and biorefinery products. The current v2.0 genome reference for the species served as the first reference for the genus and has helped drive the development of molecular breeding tools for eucalypts. Using PacBio HiFi long reads and Omni-C proximity ligation sequencing, we produced an improved, haplotype-phased assembly (v4.0) for TAG0014, an early-generation selection of E. grandis. The 2 haplotypes are 571 Mbp (HAP1) and 552 Mbp (HAP2) in size and consist of 37 and 46 contigs scaffolded onto 11 chromosomes (contig N50 of 28.9 and 16.7 Mbp), respectively. These haplotype assemblies are 70-90 Mbp smaller than the diploid v2.0 assembly but capture all except one of the 22 telomeres, suggesting that substantial redundant sequence was included in the previous assembly. A total of 35,929 (HAP1) and 35,583 (HAP2) gene models were annotated, of which 438 and 472 contain long introns (>10 kbp) in gene models previously (v2.0) identified as multiple smaller genes. These and other improvements have increased gene annotation completeness levels from 93.8 to 99.4% in the v4.0 assembly. We found that 6,493 and 6,346 genes are within tandem duplicate arrays (HAP1 and HAP2, respectively, 18.4 and 17.8% of the total) and >43.8% of the haplotype assemblies consists of repeat elements. Analysis of synteny between the haplotypes and the E. grandis v2.0 reference genome revealed extensive regions of collinearity, but also some major rearrangements, and provided a preview of population and pangenome variation in the species.

Lötter, Anneri↗