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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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192 records · Page 11
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

CRISPR-Cas9-mediated knockout of CYP79D1 and CYP79D2 in cassava attenuates toxic cyanogen production

Cassava (Manihot esculenta) is a starchy root crop that supports over a billion people in tropical and subtropical regions of the world. This staple, however, produces the neurotoxin cyanide and requires processing for safe consumption. Excessive consumption of insufficiently processed cassava, in combination with protein-poor diets, can have neurodegenerative impacts. This problem is further exacerbated by drought conditions which increase this toxin in the plant. To reduce cyanide levels in cassava, we used CRISPR-mediated mutagenesis to disrupt the cytochrome P450 genes CYP79D1 and CYP79D2 whose protein products catalyze the first step in cyanogenic glucoside biosynthesis. Knockout of both genes eliminated cyanide in leaves and storage roots of cassava accession 60444; the West African, farmer-preferred cultivar TME 419; and the improved variety TMS 91/02324. Although knockout of CYP79D2 alone resulted in significant reduction of cyanide, mutagenesis of CYP79D1 did not, indicating these paralogs have diverged in their function. The congruence of results across accessions indicates that our approach could readily be extended to other preferred or improved cultivars. This work demonstrates cassava genome editing for enhanced food safety and reduced processing burden, against the backdrop of a changing climate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Quantitative proteomics reveals extensive lysine ubiquitination and transcription factor stability states in Arabidopsis

Abstract Protein activity, abundance, and stability can be regulated by post-translational modification including ubiquitination. Ubiquitination is conserved among eukaryotes and plays a central role in modulating cellular function; yet, we lack comprehensive catalogs of proteins that are modified by ubiquitin in plants. In this study, we describe an antibody-based approach to enrich ubiquitinated peptides coupled with isobaric labeling to enable quantification of up to 18-multiplexed samples. This approach identified 17,940 ubiquitinated lysine sites arising from 6,453 proteins from Arabidopsis (Arabidopsis thaliana) primary roots, seedlings, and rosette leaves. Gene ontology analysis indicated that ubiquitinated proteins are associated with numerous biological processes including hormone signaling, plant defense, protein homeostasis, and metabolism. We determined ubiquitinated lysine residues that directly regulate the stability of three transcription factors, CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1 (CIB1), CIB1 LIKE PROTEIN 2 (CIL2), and SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) using in vivo degradation assays. Furthermore, codon mutation of CIB1 to create a K166R conversion to prevent ubiquitination, via CRISPR/Cas9-derived adenosine base editing, led to an early flowering phenotype and increased expression of FLOWERING LOCUS T (FT). These comprehensive site-level ubiquitinome profiles provide a wealth of data for future functional studies related to modulation of biological processes mediated by this post-translational modification in plants.

Biochemistry & Molecular Biology↗
DOE OSTI2020

The Link Between the Ecology of the Prokaryotic Rare Biosphere and Its Biotechnological Potential

Current research on the prokaryotic low abundance taxa, the prokaryotic rare biosphere, is growing, leading to a greater understanding of the mechanisms underlying organismal rarity and its relevance in ecology. From this emerging knowledge it is possible to envision innovative approaches in biotechnology applicable to several sectors. Bioremediation and bioprospecting are two of the most promising areas where such approaches could find feasible implementation, involving possible new solutions to the decontamination of polluted sites and to the discovery of novel gene variants and pathways based on the attributes of rare microbial communities. Bioremediation can be improved through the realization that diverse rare species can grow abundant and degrade different pollutants or possibly transfer useful genes. Further, most of the prokaryotic diversity found in virtually all environments belongs in the rare biosphere and remains uncultivatable, suggesting great bioprospecting potential within this vast and understudied genetic pool. This Mini Review argues that knowledge of the ecophysiology of rare prokaryotes can aid the development of future, efficient biotechnology-based processes, products and services. However, this promise may only be fulfilled through improvements in (and optimal blending of) advanced microbial culturing and physiology, metagenomics, genome annotation and editing, and synthetic biology, to name a few areas of relevance. In the future, it will be important to understand how activity profiles relate with abundance, as some rare taxa can remain rare and increase activity, whereas other taxa can grow abundant. The metabolic mechanisms behind those patterns can be useful in designing biotechnological processes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural basis for human ZBTB7A action at the fetal globin promoter

Elevated levels of fetal globin protect against β-hemoglobinopathies, such as sickle cell disease and β-thalassemia. Two zinc-finger (ZF) repressors, BCL11A and ZBTB7A/LRF, bind directly to the fetal globin promoter elements positioned at -115 and -200, respectively. Here, we describe X-ray structures of the ZBTB7A DNA-binding domain, consisting of four adjacent ZFs, in complex with the -200 sequence element, which contains two copies of four consecutive C:G base pairs. ZF1 and ZF2 recognize the 5' C:G quadruple, and ZF4 contacts the 3' C:G quadruple. Natural non-coding DNA mutations associated with hereditary persistence of fetal hemoglobin (HPFH) impair ZBTB7A DNA binding, with the most severe disruptions resulting from mutations in the base pairs recognized by ZF1 and ZF2. Our results firmly establish ZBTB7A/LRF as a key molecular regulator of fetal globin expression and inform genome-editing strategies that inhibit repressor binding and boost fetal globin expression to treat hemoglobinopathies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Strategies to identify and edit improvements in synthetic genome segments episomally

Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments at low copy number. However, all stages of whole-genome engineering have the potential to impose mutations on the synthetic genome that can reduce or eliminate the fitness of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability. This protocol was applied to recoding an Escherichia coli strain to replace seven codons with synonymous alternatives genome wide. Ten 44 402–47 179 bp de novo synthesized DNA segments contained in a BAC from the recoded strain were unable to complement deletion of the corresponding 33–61 wild-type genes using a single antibiotic resistance marker. Next-generation sequencing (NGS) was used to identify 1–7 non-recoding mutations in essential genes per segment, and MAGE in turn proved a useful strategy to repair these mutations on the recoded segment contained in the BAC when both the recoded and wild-type copies of the mutated genes had to exist by necessity during the repair process. Finally, two web-based tools were used to predict the impact of a subset of non-recoding missense mutations on strain fitness using protein structure and function calls.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Engineering Nannochloropsis oceanica for the production of diterpenoid compounds

Abstract Photosynthetic microalgae like Nannochloropsis hold enormous potential as sustainable, light‐driven biofactories for the production of high‐value natural products such as terpenoids. Nannochloropsis oceanica is distinguished as a particularly robust host with extensive genomic and transgenic resources available. Its capacity to grow in wastewater, brackish, and sea waters, coupled with advances in microalgal metabolic engineering, genome editing, and synthetic biology, provides an excellent opportunity. In the present work, we demonstrate how N. oceanica can be engineered to produce the diterpene casbene—an important intermediate in the biosynthesis of pharmacologically relevant macrocyclic diterpenoids. Casbene accumulated after stably expressing and targeting the casbene synthase from Daphne genkwa (DgTPS1) to the algal chloroplast. The engineered strains yielded production titers of up to 0.12 mg g −1 total dry cell weight (DCW) casbene. Heterologous overexpression and chloroplast targeting of two upstream rate‐limiting enzymes in the 2‐C‐methyl‐ d ‐erythritol 4‐phosphate pathway, Coleus forskohlii 1‐deoxy‐ d ‐xylulose‐5‐phosphate synthase and geranylgeranyl diphosphate synthase genes, further enhanced the yield of casbene to a titer up to 1.80 mg g −1 DCW. The results presented here form a basis for further development and production of complex plant diterpenoids in microalgae.

Du, Zhi‐Yan↗
DOE OSTI2022

Novel Mutant Alleles Reveal a Role of the Extra-Large G Protein in Rice Grain Filling, Panicle Architecture, Plant Growth, and Disease Resistance

Plant growth and grain filling are the key agronomical traits for grain weight and yield of rice. The continuous improvement in rice yield is required for a future sustainable global economy and food security. The heterotrimeric G protein complex containing a canonical α subunit (RGA1) couples extracellular signals perceived by receptors to modulate cell function including plant development and grain weight. We hypothesized that, besides RGA1, three atypical, extra-large GTP-binding protein (XLG) subunits also regulate panicle architecture, plant growth, development, grain weight, and disease resistance. Here, we identified a role of XLGs in agronomic traits and stress tolerance by genetically ablating all three rice XLGs individually and in combination using the CRISPR/Cas9 genome editing in rice. For this study, eight (three single, two double, and three triple) null mutants were selected. Three XLG proteins combinatorically regulate seed filling, because loss confers a decrease in grain weight from 14% with loss of one XLG and loss of three to 32% decrease in grain weight. Null mutations in XLG2 and XLG4 increase grain size. The mutants showed significantly reduced panicle length and number per plant including lesser number of grains per panicle compared to the controls. Loss-of-function of all individual XLGs contributed to 9% more aerial biomass compared to wild type (WT). The double mutant showed improved salinity tolerance. Moreover, loss of the XLG gene family confers hypersensitivity to pathogens. Our findings suggest that the non-canonical XLGs play important roles in regulating rice plant growth, grain filling, panicle phenotype, stress tolerance, and disease resistance. Genetic manipulation of XLG s has the potential to improve agronomic properties in rice.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus

Background: 2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material. Results: We work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus. First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 3 3 -factorial library includes all combinations of KmARO4, KmARO7, and KmPHA2, each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4 K221L and native KmPHA2, with the medium expression of feedback insensitive KmARO7 G141S , results in the highest increase in 2-PE biosynthesis, producing 684 ± 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 ± 6 mg/L. The best strain achieves 1943 ± 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures. Conclusions: The CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway.

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

2024 IUFRO Tree Biotechnology Conference (Aug 4-8, 2024)

The 2024 IUFRO Tree Biotechnology Conference is the biennial meeting on genomics, molecular biology, and biotechnology of forest trees, associated with the IUFRO Working Party 2.04.06. This year's meeting was held in Annapolis, MD, USA from August 4th to 8th and was hosted by Yiping Qi (University of Maryland), Edward Eisenstein (University of Maryland), Gary Coleman (University of Maryland), and Heather Coleman (Syracuse University). The conference covered seven topics over the course of five days: 1) Biological and ecological insights from OMICS, 2) Advancing technologies for targeted trait manipulation and acceptability to diverse tree species, 3) Genes, development, and physiology, 4) Translating genomics and biotechnology to practice, 5) Trees in a changing world, 6) Genetic and phenotypic diversity for breeding and genomic selection, and 7) Biotechnology for biomaterials and bioeconomy. In addition to the sessions, there were two plenary sessions, provided by John Ralph (University of Wisconsin) and Tanja Pyrhäjärvi (University of Helsinki). The meeting celebrated the second awardees of the newly created IUFRO WG 2.04.06 Award: Excellence in Forest Molecular Biology and Genomics, which was presented to Chung-Jui (C.J.) Tsai (University of Georgia). Greg Goralogia (Oregon State University) was the recipient of the associated Early Career Award. The scientific presentations at the conference highlighted cutting-edge advancements in many facets of forest biotechnology research, including applications of genomic selection in forest genetics and breeding, the use of genetic editing, tree physiology, stress response, molecular breeding, wood development, "omics" technologies, and the social and economic impacts of genetically modified (GM) trees. Scientific take homes from the meeting include the power of NMR to dissect the composition of lignin, the genomic diversity of forest trees that has enormous potential for tree improvement and the integration of systems biology with climate and geographical data. The conference attracted a mix of students (25), postdoctoral fellows (32), and scientists from academia (66) and industry (18). In all, the conference was attended by 141 registered participants, representing 20 countries that participated in 23 invited lectures (including 6 'early-career' keynotes), 27 voluntary talks and 61 poster presentations. Support for the conference was drawn from a wide variety of Academia, Industry, and Government sources, and included financial support from several tree improvement companies. Overall, the conference was a great success, providing an exceptional mix of science and social activities in a relaxed and collegial atmosphere. More information about the meeting can be found at treebiotech.org. The next meeting will be held in Stellenbosch, South Africa, in 2026, hosted jointly by Zander Myburg, Dave Drew (University of Stellenbosch,) and Sanushka Naidoo (University of Pretoria, FABI).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes

Abstract RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and a TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12s. In eukaryotes, TnpB homologs occur as two distinct types, Fanzor1s and Fanzor2s. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, which revealed that both Fanzor1s and Fanzor2s stem from a single lineage of IS607 TnpBs with unusual active site arrangement. The widespread nature of Fanzors implies that the properties of this particular lineage of IS607 TnpBs were particularly suited to adaptation in eukaryotes. Biochemical analysis of an IS607 TnpB and Fanzor1s revealed common strategies employed by TnpBs and Fanzors to co-evolve with their cognate transposases. Collectively, our results provide a new model of sequential evolution from IS607 TnpBs to Fanzor2s, and Fanzor2s to Fanzor1s that details how genes of prokaryotic origin evolve to give rise to new protein families in eukaryotes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Artemether-Lumefantrine Treatment Selects Plasmodium falciparum Multidrug Resistance 1 ( pfmdr1 ) Increased Copy Number Among African Malaria Infections

Abstract Background Decreased efficacy of artemether-lumefantrine, the globally most used antimalarial, has recently emerged in Africa. Methods An efficacy trial was carried out based on directly observed artemether-lumefantrine therapy at Bengo, Northern Angola. One-hundred Plasmodium falciparum uncomplicated malaria patients (2–10 years old) were enrolled, hospitalized for the treatment period, and followed up for 42 days. Polymerase chain reaction (PCR) correction was performed with pfmsp1/2 plus glurp, with analysis considering 2 or 3 coincident markers. Infections were tested by quantitative PCR (qPCR) for pfmdr1 copy number (pfmdr1×N), a potential P. falciparum marker of lumefantrine resistance previously identified in the region. In vitro clone mixtures were built and used to determine the relation between qPCR copy number scores and actual intrainfection quantitative fractions of pfmdr1×N. Results We observed a significant posttreatment selection of gene amplification, suggesting a role in the parasite in vivo response to this drug. pfmdr1×2 qPCR scores of 1.3, 1.4, and 1.5 were determined to correspond to 15%, 25%, and 35% intrainfection rates. Patients carrying infections with a score ≥1.4 at baseline were linked to decreased artemether-lumefantrine day 42 efficacy (79% vs 97% single-copy pfmdr1). All infections were pfmdr1 N86 carriers and no pfk13 mutations were found. Conclusions Our study suggests pfmdr1×N as a marker of P. falciparum in vivo response to lumefantrine in Africa, while indicating patients carrying infections with a pretreatment pfmdr1×N score ≥1.4 before treatment are a group experiencing decreased artemether-lumefantrine performance.

Fançony, Claudia (ORCID:0000000344211769)↗
DOE OSTI2020

Comprehensive In Vivo Interrogation Reveals Phenotypic Impact of Human Enhancer Variants

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. In this paper, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.

59 BASIC BIOLOGICAL SCIENCES↗