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At least 415 records · Page 23
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Crash–and–dash: a new era in tree genome editing

In less than a decade since the first demonstrations of CRISPR genome editing of agronomic genes in several tree species (Zhou et al., 2015; Jia et al., 2016; Ren et al., 2016), this disruptive technology has been deployed for a growing number of traits in both basic and applied research. The precision and efficiency of CRISPR editing allow for the recovery of null mutants in the first generation (Zhou et al., 2015; Elorriaga et al., 2018; Muhr et al., 2018), which is a significant benefit for perennial trees with long generation times. The stability of editing outcomes over multiple years or clonal propagation cycles (Bewg et al., 2022; Chen et al., 2023; Goralogia et al., 2024) is another key advantage since most woody perennials are vegetatively propagated in commercial operations. However, in many countries, gene-edited trees with stably integrated T-DNA face the same regulatory hurdles as traditional transgenics, slowing field trial characterization and the integration of transgenesis with conventional breeding (Boerjan & Strauss, 2024). In an article recently published in New Phytologist, Hoengenaert et al. (2025; doi: 10.1111/nph.20415) demonstrate transgene-free editing in poplar (Populus tremula × alba) that shows promise for wide adoption. The CRISPR-edited, transgene-free canker-resistant citrus (Citrus sinensis) trees (Su et al., 2023) have recently been approved by USDA-APHIS and are exempt from regulation by the US Environmental Protection Agency (EPA) for commercial production. The work by Hoengenaert et al. (2025) suggests a similar path could be followed for purpose-grown plantations for bioenergy, bioproducts, and biomaterials.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Metabolic engineering of p –hydroxybenzoate in poplar lignin

Ester-linked p-hydroxybenzoate occurs naturally in poplar lignin as pendent groups that can be released by mild alkaline hydrolysis. These ‘clip-off’ phenolics can be separated from biomass and upgraded into diverse high-value bioproducts. We introduced a bacterial chorismate pyruvate lyase gene into transgenic poplar trees with the aim of producing more p-hydroxybenzoate from chorismate, itself a metabolic precursor to lignin. By driving heterologous expression specifically in the plastids of cells undergoing secondary wall formation, this strategy achieved a 50% increase in cell-wall-bound p-hydroxybenzoate in mature wood and nearly 10 times more in developing xylem relative to control trees. Comparable amounts also remained as soluble p-hydroxybenzoate-containing xylem metabolites, pointing to even greater engineering potential. Mass spectrometry imaging showed that the elevated p-hydroxybenzoylation was largely restricted to the cell walls of fibres. Finally, transgenic lines outperformed control trees in assays of saccharification potential. Here, this study highlights the biotech potential of cell-wall-bound phenolate esters and demonstrates the importance of substrate supply in lignin engineering.

4-hydroxybenzoic acid↗
DOE OSTI2022

Engineering triacylglycerol accumulation in duckweed ( Lemna japonica )

Summary Duckweeds are amongst the fastest growing of higher plants, making them attractive high‐biomass targets for biofuel feedstock production. Their fronds have high rates of fatty acid synthesis to meet the demand for new membranes, but triacylglycerols (TAG) only accumulate to very low levels. Here we report on the engineering of Lemna japonica for the synthesis and accumulation of TAG in its fronds. This was achieved by expression of an estradiol‐inducible cyan fluorescent protein‐ Arabidopsis WRINKLED1 fusion protein ( CFP‐AtWRI1 ), strong constitutive expression of a mouse diacylglycerol:acyl‐CoA acyltransferase2 ( MmDGAT ), and a sesame oleosin variant ( SiOLE(*) ). Individual expression of each gene increased TAG accumulation by 1‐ to 7‐fold relative to controls, while expression of pairs of these genes increased TAG by 7‐ to 45‐fold. In uninduced transgenics containing all three genes, TAG accumulation increased by 45‐fold to 3.6% of dry weight (DW) without severely impacting growth, and by 108‐fold to 8.7% of DW after incubation on medium containing 100 μ m estradiol for 4 days. TAG accumulation was accompanied by an increase in total fatty acids of up to three‐fold to approximately 15% of DW. Lipid droplets from fronds of all transgenic lines were visible by confocal microscopy of BODIPY‐stained fronds. At a conservative 12 tonnes (dry matter) per acre and 10% (DW) TAG, duckweed could produce 350 gallons of oil/acre/year, approximately seven‐fold the yield of soybean, and similar to that of oil palm. These findings provide the foundation for optimizing TAG accumulation in duckweed and present a new opportunity for producing biofuels and lipidic bioproducts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Creating yellow seed Camelina sativa with enhanced oil accumulation by CRISPR ‐mediated disruption of Transparent Testa 8

Summary Camelina ( Camelina sativa L.), a hexaploid member of the Brassicaceae family, is an emerging oilseed crop being developed to meet the increasing demand for plant oils as biofuel feedstocks. In other Brassicas, high oil content can be associated with a yellow seed phenotype, which is unknown for camelina. We sought to create yellow seed camelina using CRISPR/Cas9 technology to disrupt its Transparent Testa 8 (TT8) transcription factor genes and to evaluate the resulting seed phenotype. We identified three TT8 genes, one in each of the three camelina subgenomes, and obtained independent CsTT8 lines containing frameshift edits. Disruption of TT8 caused seed coat colour to change from brown to yellow reflecting their reduced flavonoid accumulation of up to 44%, and the loss of a well‐organized seed coat mucilage layer. Transcriptomic analysis of CsTT8 ‐edited seeds revealed significantly increased expression of the lipid‐related transcription factors LEC1 , LEC2 , FUS3 , and WRI1 and their downstream fatty acid synthesis‐related targets. These changes caused metabolic remodelling with increased fatty acid synthesis rates and corresponding increases in total fatty acid (TFA) accumulation from 32.4% to as high as 38.0% of seed weight, and TAG yield by more than 21% without significant changes in starch or protein levels compared to parental line. These data highlight the effectiveness of CRISPR in creating novel enhanced‐oil germplasm in camelina. The resulting lines may directly contribute to future net‐zero carbon energy production or be combined with other traits to produce desired lipid‐derived bioproducts at high yields.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Variation on a theme: the structures and biosynthesis of specialized fatty acid natural products in plants

SUMMARY Plants are able to construct lineage‐specific natural products from a wide array of their core metabolic pathways. Considerable progress has been made toward documenting and understanding, for example, phenylpropanoid natural products derived from phosphoenolpyruvate via the shikimate pathway, terpenoid compounds built using isopentyl pyrophosphate, and alkaloids generated by the extensive modification of amino acids. By comparison, natural products derived from fatty acids have received little attention, except for unusual fatty acids in seed oils and jasmonate‐like oxylipins. However, scattered but numerous reports show that plants are able to generate many structurally diverse compounds from fatty acids, including some with highly elaborate and unique structural features that have novel bioproduct functionalities. Furthermore, although recent work has shed light on multiple new fatty acid natural product biosynthesis pathways and products in diverse plant species, these discoveries have not been reviewed. The aims of this work, therefore, are to (i) review and systematize our current knowledge of the structures and biosynthesis of fatty acid‐derived natural products that are not seed oils or jasmonate‐type oxylipins, specifically, polyacetylenic, very‐long‐chain, and aromatic fatty acid‐derived natural products, and (ii) suggest priorities for future investigative steps that will bring our knowledge of fatty acid‐derived natural products closer to the levels of knowledge that we have attained for other phytochemical classes.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Elongated galactan side chains mediate cellulose–pectin interactions in engineered Arabidopsis secondary cell walls

SUMMARY The plant secondary cell wall is a thickened matrix of polysaccharides and lignin deposited at the cessation of growth in some cells. It forms the majority of carbon in lignocellulosic biomass, and it is an abundant and renewable source for forage, fiber, materials, fuels, and bioproducts. The complex structure and arrangement of the cell wall polymers mean that the carbon is difficult to access in an economical and sustainable way. One solution is to alter the cell wall polymer structure so that it is more suited to downstream processing. However, it remains difficult to predict what the effects of this engineering will be on the assembly, architecture, and properties of the cell wall. Here, we make use of Arabidopsis plants expressing a suite of genes to increase pectic galactan chain length in the secondary cell wall. Using multi‐dimensional solid‐state nuclear magnetic resonance, we show that increasing galactan chain length enhances pectin–cellulose spatial contacts and increases cellulose crystallinity. We also found that the increased galactan content leads to fewer spatial contacts of cellulose with xyloglucan and the backbone of pectin. Hence, we propose that the elongated galactan side chains compete with xyloglucan and the pectic backbone for cellulose interactions. Due to the galactan topology, this may result in comparatively weak interactions and disrupt the cell wall architecture. Therefore, introduction of this strategy into trees or other bioenergy crops would benefit from cell‐specific expression strategies to avoid negative effects on plant growth.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Haplotype‐resolved genome assembly of Populus tremula × P. alba reveals aspen‐specific megabase satellite DNA

SUMMARY Populus species play a foundational role in diverse ecosystems and are important renewable feedstocks for bioenergy and bioproducts. Hybrid aspen Populus tremula × P. alba INRA 717‐1B4 is a widely used transformation model in tree functional genomics and biotechnology research. As an outcrossing interspecific hybrid, its genome is riddled with sequence polymorphisms which present a challenge for sequence‐sensitive analyses. Here we report a telomere‐to‐telomere genome for this hybrid aspen with two chromosome‐scale, haplotype‐resolved assemblies. We performed a comprehensive analysis of the repetitive landscape and identified both tandem repeat array‐based and array‐less centromeres. Unexpectedly, the most abundant satellite repeats in both haplotypes lie outside of the centromeres, consist of a 147 bp monomer PtaM147, frequently span >1 megabases, and form heterochromatic knobs. PtaM147 repeats are detected exclusively in aspens (section Populus ) but PtaM147‐like sequences occur in LTR‐retrotransposons of closely related species, suggesting their origin from the retrotransposons. The genomic resource generated for this transformation model genotype has greatly improved the design and analysis of genome editing experiments that are highly sensitive to sequence polymorphisms. The work should motivate future hypothesis‐driven research to probe into the function of the abundant and aspen‐specific PtaM147 satellite DNA.

Zhou, Ran↗
DOE OSTI2024

Cell‐type‐specific transcriptomics uncovers spatial regulatory networks in bioenergy sorghum stems

SUMMARY Bioenergy sorghum is a low‐input, drought‐resilient, deep‐rooting annual crop that has high biomass yield potential enabling the sustainable production of biofuels, biopower, and bioproducts. Bioenergy sorghum's 4–5 m stems account for ~80% of the harvested biomass. Stems accumulate high levels of sucrose that could be used to synthesize bioethanol and useful biopolymers if information about cell‐type gene expression and regulation in stems was available to enable engineering. To obtain this information, laser capture microdissection was used to isolate and collect transcriptome profiles from five major cell types that are present in stems of the sweet sorghum Wray. Transcriptome analysis identified genes with cell‐type‐specific and cell‐preferred expression patterns that reflect the distinct metabolic, transport, and regulatory functions of each cell type. Analysis of cell‐type‐specific gene regulatory networks (GRNs) revealed that unique transcription factor families contribute to distinct regulatory landscapes, where regulation is organized through various modes and identifiable network motifs. Cell‐specific transcriptome data was combined with known secondary cell wall (SCW) networks to identify the GRNs that differentially activate SCW formation in vascular sclerenchyma and epidermal cells. The spatial transcriptomic dataset provides a valuable source of information about the function of different sorghum cell types and GRNs that will enable the engineering of bioenergy sorghum stems, and an interactive web application developed during this project will allow easy access and exploration of the data ( https://mc‐lab.shinyapps.io/lcm‐dataset/ ).

09 BIOMASS FUELS↗
DOE OSTI2026

Compilation and utilization of a sorghum transcriptome compendium for gene regulatory network analysis and crop trait engineering

Sorghum bicolor (Sorghum) is a drought and heat tolerant C4 grass crop used to produce grain, forage, biofuels, and other bioproducts. Genetic improvement of sorghum hybrid crops is aided by a large and diverse germplasm, sorghum's diploid inbreeding genetics, and a relatively small genome that has facilitated genomic research. Over the past 20 years, the sorghum research community characterized the cytogenetic and recombinant landscapes of sorghum's 10 chromosomes, sequenced and annotated the sorghum genome, and used that information to identify genes/alleles that modulate flowering time, plant height, seed shattering, and other important traits. More recently, >1000 RNA-seq transcriptome profiles were collected from 15 sorghum genotypes to help understand the genetic basis of variation in growth and development of sorghum stems, tillers, roots, and leaves, and the regulation of biosynthetic pathways that produce epicuticular wax, dhurrin, and RFOs, compounds that contribute to sorghum's resilience. Transcriptome studies were designed to identify differentially expressed genes that are co-expressed during development or in response to a treatment to enable construction of gene regulatory networks. Co-expression and network analysis identified transcription factors and their cognate binding sites in target gene promoters and signaling pathways that modulate gene regulatory networks providing gene editing targets for further trait optimization. RNA-seq data from >20 experiments targeting sorghum organs, tissues, cell types, developmental stages, and responses to environmental conditions (i.e., diel, day-length, shading, water-deficit, temperature) has been compiled in a sorghum transcriptome compendium. The goal of this resource paper is to describe compendium content, accessibility, and a compendium data analysis pipeline and to illustrate the types of information that can be derived from the compendium with a focus on the elucidation of gene regulatory networks useful for guiding the improvement of sorghum traits through gene editing.

RNA-seq↗
DOE OSTI2020

A High-efficacy CRISPRi System for Gene Function Discovery in Zymomonas mobilis

Zymomonas mobilis is a promising biofuel producer due to its high alcohol tolerance and streamlined metabolism that efficiently converts sugar to ethanol. Z. mobilis genes are poorly characterized relative to those of model bacteria, hampering our ability to rationally engineer the genome with pathways capable of converting sugars from plant hydrolysates into valuable biofuels and bioproducts. Many of the unique properties that make Z. mobilis an attractive biofuel producer are controlled by essential genes; however, these genes cannot be manipulated using traditional genetic approaches (e.g., deletion or transposon insertion) because they are required for viability. CRISPR interference (CRISPRi) is a programmable gene knockdown system that can precisely control the timing and extent of gene repression, thus enabling targeting of essential genes. Here, we establish a stable, high-efficacy CRISPRi system in Z. mobilis that is capable of perturbing all genes—including essential genes. We show that Z. mobilis CRISPRi causes either strong knockdowns (>100-fold) using single guide RNA (sgRNA) spacers that perfectly match target genes or partial knockdowns using spacers with mismatches. We demonstrate the efficacy of Z. mobilis CRISPRi by targeting essential genes that are universally conserved in bacteria, are key to the efficient metabolism of Z. mobilis, or underlie alcohol tolerance. Our Z. mobilis CRISPRi system will enable comprehensive gene function discovery, opening a path to rational design of biofuel production strains with improved yields.

lignocellulosic hydrolysate↗
DOE OSTI2025

Microfluidic droplets with amended culture media cultivate a greater diversity of soil microorganisms

ABSTRACT Uncultivated but abundant soil microorganisms have untapped potential for producing broad ranges of natural products, as well as for bioremediation. However, cultivating soil microorganisms while maintaining a broad microorganism diversity to enable phenotyping and functional analysis of as diverse individual isolates as possible remains challenging. In this study, we developed and tested the ability of several culture media formulations that contain defined soil metabolites or soil extracts to maintain microorganism diversity during culture. We also assessed their performance in microfluidic droplet cultivation where single-soil microorganism isolates were encapsulated and cultivated in picoliter-volume water-in-oil emulsion droplets to enable clonal growth needed for downstream functional analyses. Our results show that droplet cultivation with media supplemented by soil extract or soil metabolites enables the recovery of soil microorganisms with higher diversity (up to 1.5-fold higher richness) compared to bulk cultivation methods. Importantly, 1.7-fold more of less abundant (<1%) phyla and 11-fold more of unique genera were recovered, demonstrating the utility of this method for interrogating highly diverse soil microorganisms for broad ranges of applications. IMPORTANCE Although soil microorganisms hold a significant value in bioproduction and bioremediation, only a small fraction—less than 1%—can be cultured under specific media and cultivation conditions. This indicates that there are ample opportunities in harvesting the diverse environmental microorganisms if isolating and recovering these uncultured microorganisms are possible. This paper presents a new cultivation technique composed of isolating single-soil microorganism cell from anin situsoil microorganism community in microfluidic droplets and conducting in-droplet cultivation in media supplemented by soil extract or soil metabolites. This method enables the recovery of a broader diversity of the original microorganism community, laying the groundwork for a high-throughput phenotyping of these diverse microorganisms from their natural habitats.

Biotechnology & Applied Microbiology↗
DOE OSTI2024

The Catabolism of Lignin-Derived p-Methoxylated Aromatic Compounds by Rhodococcus jostii RHA1

Emergent strategies to valorize lignin, an abundant but underutilized aromatic biopolymer, include tandem processes that integrate chemical depolymerization and biological catalysis. To date, aromatic monomers from C-O bond cleavage of lignin have been converted to bioproducts, but the presence of recalcitrant C-C bonds in lignin limits the product yield. A promising chemocatalytic strategy that overcomes this limitation involves phenol methyl protection and autoxidation. Incorporating this into a tandem process requires microbial cell factories able to transform the p-methoxylated products in the resulting methylated lignin stream. In this study, we assessed the ability of Rhodococcus jostii RHA1 to catabolize the major aromatic products in a methylated lignin stream and elucidated the pathways responsible for this catabolism. RHA1 grew on a methylated pine lignin stream, catabolizing the major aromatic monomers: p-methoxybenzoate (p-MBA), veratrate, and veratraldehyde. Bioinformatic analyses suggested that a cytochrome P450, PbdA, and its cognate reductase, PbdB, are involved in p-MBA catabolism. Gene deletion studies established that both pbdA and pbdB are essential for growth on p-MBA and several derivatives. Furthermore, a deletion mutant of a candidate p-hydroxybenzoate (p-HBA) hydroxylase, ..delta..pobA, did not grow on p-HBA. Veratraldehyde and veratrate catabolism required both vanillin dehydrogenase (Vdh) and vanillate O-demethylase (VanAB), revealing previously unknown roles of these enzymes. Finally, a ..delta..pcaL strain grew on neither p-MBA nor veratrate, indicating they are catabolized through the ..beta..-ketoadipate pathway. This study expands our understanding of the bacterial catabolism of aromatic compounds and facilitates the development of biocatalysts for lignin valorization.

aromatic biopolymers↗
DOE OSTI2023

The genetics of aerotolerant growth in an alphaproteobacterium with a naturally reduced genome

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living alphaproteobacterium Zymomonas mobilis has a naturally reduced genome containing fewer than 2,000 protein-coding genes. Despite its small genome, Z. mobilis thrives in diverse conditions including the presence or absence of atmospheric oxygen. However, insufficient characterization of essential and conditionally essential genes has limited broader adoption of Z. mobilis as a model alphaproteobacterium. Here, we use genome-scale CRISPRi-seq (clustered regularly interspaced short palindromic repeats interference sequencing) to systematically identify and characterize Z. mobilis genes that are conditionally essential for aerotolerant or anaerobic growth or are generally essential across both conditions. Comparative genomics revealed that the essentiality of most “generally essential” genes was shared between Z. mobilis and other Alphaproteobacteria, validating Z. mobilis as a reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, recJ, was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F1FO ATP synthase and Rhodobacter nitrogen fixation (Rnf) respiratory complex are required for the anaerobic growth of Z. mobilis. Combining CRISPRi partial knockdowns with metabolomics and membrane potential measurements, we determined that the ATP synthase generates membrane potential that is consumed by Rnf to power downstream processes. Rnf knockdown strains accumulated isoprenoid biosynthesis intermediates, suggesting a key role for Rnf in powering essential biosynthetic reactions. Our work establishes Z. mobilis as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs Z. mobilis genome manipulation for optimized production of valuable isoprenoid-based bioproducts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Annotated Genome Sequence of the High-Biomass-Producing Yellow-Green Alga Tribonema minus

Here, we report the annotated genome sequence for a heterokont alga from the class Xanthophyceae. This high-biomass-producing strain, Tribonema minus UTEX B 3156, was isolated from a wastewater treatment plant in California. It is stable in outdoor raceway ponds and is a promising industrial feedstock for biofuels and bioproducts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Near-complete genome sequence of Lipomyces tetrasporous NRRL Y-64009, an oleaginous yeast capable of growing on lignocellulosic hydrolysates

ABSTRACT Lipomyces tetrasporous is an oleaginous yeast that can utilize a variety of plant-based sugars. It accumulates lipids during growth on lignocellulosic biomass hydrolysates. We present the annotated genome sequence of L. tetrasporous NRRL Y-64009 to aid in its development as a platform organism for producing lipids and lipid-based bioproducts.

59 BASIC BIOLOGICAL SCIENCES↗