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At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Oxygenation influences xylose fermentation and gene expression in the yeast genera Spathaspora and Scheffersomyces

Abstract Background Cost-effective production of biofuels from lignocellulose requires the fermentation of d -xylose. Many yeast species within and closely related to the genera Spathaspora and Scheffersomyces (both of the order Serinales) natively assimilate and ferment xylose. Other species consume xylose inefficiently, leading to extracellular accumulation of xylitol. Xylitol excretion is thought to be due to the different cofactor requirements of the first two steps of xylose metabolism. Xylose reductase (XR) generally uses NADPH to reduce xylose to xylitol, while xylitol dehydrogenase (XDH) generally uses NAD + to oxidize xylitol to xylulose, creating an imbalanced redox pathway. This imbalance is thought to be particularly consequential in hypoxic or anoxic environments. Results We screened the growth of xylose-fermenting yeast species in high and moderate aeration and identified both ethanol producers and xylitol producers. Selected species were further characterized for their XR and XDH cofactor preferences by enzyme assays and gene expression patterns by RNA-Seq. Our data revealed that xylose metabolism is more redox balanced in some species, but it is strongly affected by oxygen levels. Under high aeration, most species switched from ethanol production to xylitol accumulation, despite the availability of ample oxygen to accept electrons from NADH. This switch was followed by decreases in enzyme activity and the expression of genes related to xylose metabolism, suggesting that bottlenecks in xylose fermentation are not always due to cofactor preferences. Finally, we expressed XYL genes from multiple Scheffersomyces species in a strain of Saccharomyces cerevisiae . Recombinant S. cerevisiae expressing XYL1 from Scheffersomyces xylosifermentans , which encodes an XR without a cofactor preference, showed improved anaerobic growth on xylose as the primary carbon source compared to S. cerevisiae strain expressing XYL genes from Scheffersomyces stipitis . Conclusion Collectively, our data do not support the hypothesis that xylitol accumulation occurs primarily due to differences in cofactor preferences between xylose reductase and xylitol dehydrogenase; instead, gene expression plays a major role in response to oxygen levels. We have also identified the yeast Sc. xylosifermentans as a potential source for genes that can be engineered into S. cerevisiae to improve xylose fermentation and biofuel production.

09 BIOMASS FUELS↗
DOE OSTI2023

FUN-PROSE: A deep learning approach to predict condition-specific gene expression in fungi

mRNA levels of all genes in a genome is a critical piece of information defining the overall state of the cell in a given environmental condition. Being able to reconstruct such condition-specific expression in fungal genomes is particularly important to metabolically engineer these organisms to produce desired chemicals in industrially scalable conditions. Most previous deep learning approaches focused on predicting the average expression levels of a gene based on its promoter sequence, ignoring its variation across different conditions. Here we present FUN-PROSE—a deep learning model trained to predict differential expression of individual genes across various conditions using their promoter sequences and expression levels of all transcription factors. We train and test our model on three fungal species and get the correlation between predicted and observed condition-specific gene expression as high as 0.85. We then interpret our model to extract promoter sequence motifs responsible for variable expression of individual genes. We also carried out input feature importance analysis to connect individual transcription factors to their gene targets. A sizeable fraction of both sequence motifs and TF-gene interactions learned by our model agree with previously known biological information, while the rest corresponds to either novel biological facts or indirect correlations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The quantitative genetics of gene expression in Mimulus guttatus

Gene expression can be influenced by genetic variants that are closely linked to the expressed gene (cis eQTLs) and variants in other parts of the genome (trans eQTLs). We created a multiparental mapping population by sampling genotypes from a single natural population of Mimulus guttatus and scored gene expression in the leaves of 1,588 plants. We find that nearly every measured gene exhibits cis regulatory variation (91% have FDR < 0.05). cis eQTLs are usually allelic series with three or more functionally distinct alleles. The cis locus explains about two thirds of the standing genetic variance (on average) but varies among genes and tends to be greatest when there is high indel variation in the upstream regulatory region and high nucleotide diversity in the coding sequence. Despite mapping over 10,000 trans eQTL / affected gene pairs, most of the genetic variance generated by trans acting loci remains unexplained. This implies a large reservoir of trans acting genes with subtle or diffuse effects. Mapped trans eQTLs show lower allelic diversity but much higher genetic dominance than cis eQTLs. Several analyses also indicate that trans eQTLs make a substantial contribution to the genetic correlations in expression among different genes. They may thus be essential determinants of “gene expression modules,” which has important implications for the evolution of gene expression and how it is studied by geneticists.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Spurious regulatory connections dictate the expression-fitness landscape of translation factors

During steady-state cell growth, individual enzymatic fluxes can be directly inferred from growth rate by mass conservation, but the inverse problem remains unsolved. Perturbing the flux and expression of a single enzyme could have pleiotropic effects that may or may not dominate the impact on cell fitness. Here, we quantitatively dissect the molecular and global responses to varied expression of translation termination factors (peptide release factors, RFs) in the bacterium Bacillus subtilis. While endogenous RF expression maximizes proliferation, deviations in expression lead to unexpected distal regulatory responses that dictate fitness reduction. Molecularly, RF depletion causes expression imbalance at specific operons, which activates master regulators and detrimentally overrides the transcriptome. Through these spurious connections, RF abundances are thus entrenched by focal points within the regulatory network, in one case located at a single stop codon. Such regulatory entrenchment suggests that predictive bottom-up models of expression-fitness landscapes will require near-exhaustive characterization of parts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Influence of Mild Chronic Stress and Social Isolation on Acute Ozone-Induced Alterations in Stress Biomarkers and Brain-Region-Specific Gene Expression in Male Wistar–Kyoto Rats

Individuals with psychosocial stress often experience an exaggerated response to air pollutants. Ozone (O 3 ) exposure has been associated with the activation of the neuroendocrine stress-response system. We hypothesized that preexistent mild chronic stress plus social isolation (CS), or social isolation (SI) alone, would exacerbate the acute effects of O 3 exposure on the circulating adrenal-derived stress hormones, and the expression of the genes regulating glucocorticoid stress signaling via an altered stress adaptation in a brain-region-specific manner. Male Wistar–Kyoto rats (5 weeks old) were socially isolated, plus were subjected to either CS (noise, confinement, fear, uncomfortable living, hectic activity, and single housing), SI (single housing only, restricted handling and no enrichment) or no stress (NS; double housing, frequent handling and enrichment provided) for 8 weeks. The rats were then exposed to either air or O 3 (0.8 ppm for 4 h), and the samples were collected immediately after. The indicators of sympathetic and hypothalamic–pituitary axis (HPA) activation (i.e., epinephrine, corticosterone, and lymphopenia) increased with O 3 exposure, but there were no effects from CS or SI, except for the depletion of serum BDNF. CS and SI revealed small changes in brain-region-specific glucocorticoid-signaling-associated markers of gene expression in the air-exposed rats (hypothalamic Nr3c1, Nr3c2 Hsp90aa1, Hspa4 and Cnr1 inhibition in SI; hippocampal HSP90aa1 increase in SI; and inhibition of the bed nucleus of the stria terminalis (BNST) Cnr1 in CS). Gene expression across all brain regions was altered by O 3 , reflective of glucocorticoid signaling effects, such as Fkbp5 in NS, CS and SI. The SI effects on Fkbp5 were greatest for SI in BNST. O 3 increased Cnr2 expression in the hypothalamus and olfactory bulbs of the NS and SI groups. O 3 , in all stress conditions, generally inhibited the expression of Nr3c1 in all brain regions, Nr3c2 in the hippocampus and hypothalamus and Bdnf in the hippocampus. SI, in general, showed slightly greater O 3 -induced changes when compared to NS and CS. Serum metabolomics revealed increased sphingomyelins in the air-exposed SI and O 3 -exposed NS, with underlying SI dampening some of the O 3 -induced changes. These results suggest a potential link between preexistent SI and acute O 3 -induced increases in the circulating adrenal-derived stress hormones and brain-region-specific gene expression changes in glucocorticoid signaling, which may partly underlie the stress dynamic in those with long-term SI.

gene expression↗
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 OSTI2025

Two deeply conserved non-coding sequences control PLETHORA1/2 expression and coordinate embryo and root development

Conserved non-coding sequences (CNSs) are integral elements of transcriptional regulation. Transcriptional tuning of PLETHORA (PLT) genes that encode master regulators of plant development is vital for embryogenesis and meristematic function. However, how the expression of PLT genes is modulated through CNSs remains unclear. Through motif-based mining of upstream sequences in 120 angiosperm genomes, we identified 21 conserved and lineage-specific CNSs, two of which are unusually long, similar, and colinear within eudicots. Using Arabidopsis thaliana, we demonstrate that these two deeply conserved elements, which we named BOX1 and BOX2, control PLT1 and PLT2 expression. CRISPR mutants within these elements specifically reduced PLT expression levels, and reporter lines revealed that deletion of either or both BOXes altered and/or abrogated the PLT2 expression pattern in the root tip, affecting the ability to rescue the plt1 plt2 double mutant. We further show that the influence of these elements on expression patterns is already exerted during embryogenesis and functional in the context of the early embryo. Finally, we reveal the existence of a BOX-mediated autoregulatory feedback loop that, in large part, explains CNS influence on expression patterns. We thus uncover a transcriptional mechanism by which genes encoding master regulators of embryo and root meristem development are regulated.

PLETHORA↗
DOE OSTI2021

Protective effect of SIRT6 on cholesterol crystal-induced endothelial dysfunction via regulating ACE2 expression

Highlights: • ACE2 was involved in CC-induced endothelial dysfunction. • SIRT1 and SIRT6 contributed to regulating the protein expression of ACE2 in CC-stimulated endothelial cells. • SIRT6 regulated ACE2 with intranuclear p-ATF2, but not affecting p-ATF2 expression in the cytoplasm. Sirtuins are a family of highly conserved nicotinamide adenine dinucleotide (NAD{sup +})-dependent enzymes. Among the sirtuins, SIRT1 and SIRT6 participate in the regulation of endothelial functions and play significant roles in the physiological and pathological processes of cardiovascular diseases (CVD). Recently, our study found that minute cholesterol crystals (CC) can be endocytosed by endothelial cells and further impair endothelial functions. Since previous studies have reported that angiotensin-converting enzyme (ACE2) involves Angiotensin (Ang) II-induced inflammation in endothelial cells, this study was designed to investigate the role of SIRT1 and SIRT6 in CC-induced variation of ACE2 expression and the related mechanism between SIRT6 and ACE2. We found that ACE2 is involved in CC-induced endothelial dysfunction, which inhibits decreases in nitric oxide (NO) level and endothelial nitric oxide synthase (eNOS) activity and increases in inflammatory factors and adhesion molecules. Besides, SIRT1 and SIRT6 regulated the protein expression of ACE2 in CC-stimulated human umbilical vein endothelial cells (HUVECs). Moreover, bioinformatics analysis from the Enrichr database indicated that activating transcription factor 2 (ATF2), is highly correlated with genes that significantly upregulated after infection with the SIRT6 adenovirus vector. In CC-induced HUVECs, ACE2 expression was up-regulated in cells transfected with ATF2 siRNA. However, further mechanism studies revealed that overexpression of SIRT6 decreases the accumulation of p-ATF2 in the nucleus, but did not affect p-ATF2 expression in the cytoplasm. Taken together, these data indicated that SIRT6 regulates ACE2 might via inhibiting the accumulation of nucleus p-ATF2 in CC-induced endothelial dysfunction.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

RAN and YBX1 are required for cell proliferation and IL-4 expression and linked to poor prognosis in oral squamous cell carcinoma

Highlights: • RAN and YBX1 are co-expressed and required for cell proliferation in OSCC cells. • RAN and YBX1 are positively associated with the immune infiltrates of CD4{sup +} Th2 cells. • RAN and YBX1 can promote IL-4 expression. • RAN is a direct target of YBX1. • RAN and YBX1 can serve as potential co-biomarkers for poor prognosis in OSCC. Oral squamous cell carcinoma (OSCC) is one of the most common malignancies in the world, with a high mortality rate. RAN is a member of the Ras GTPase family and is overexpressed in a range of cancers, however, the relationship between RAN and OSCC is rarely reported. In this study, we found that RAN is overexpressed in OSCC tissues. RAN inhibition retarded OSCC cell proliferation and led to apoptosis and cell cycle arrest. Knockdown of RAN inhibited tumor growth in vivo. Strikingly, we found that RAN and oncogene Y-box binding protein-1 (YBX1) are positively associated with the immune infiltrates of CD4{sup +} Th2 cells in multiple types of cancer, and can promote IL-4 expression. IL-4 treatment can partially rescue RAN knockdown-induced cell apoptosis in OSCC cells. Moreover, overexpression of RAN could rescue cell growth inhibition caused by knockdown of YBX1. Furthermore, patients with low expression of both RAN and YBX1 had better overall survival than others. Collectively, these findings indicate that RAN is a target of YBX1. RAN and YBX1 are required for cell proliferation and IL-4 expression. RAN and YBX1 are co-expressed and can serve as potential co-biomarkers for poor prognosis in OSCC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

CLEC12B suppresses lung cancer progression by inducing SHP-1 expression and inactivating the PI3K/AKT signaling pathway

Lung cancer is the leading cause of cancer mortality worldwide. CLEC12B, a C-type lectin-like receptor, is low-expressed in lung cancer tissues. However, the function of CLEC12B in lung cancer and its underlying mechanism remain unclear. Here, an obvious down-regulation of CLEC12B was observed in lung cancer cells compared with the normal lung epithelial cells. CLEC12B over-expression suppressed cell viability and cell cycle entry in lung cancer, along with the reduction of PCNA and cyclin D1 expressions, while silencing CLEC12B possessed the opposite effects. Over-expression of CLEC12B promoted lung cancer cell apoptosis, accompanied by decreased Bcl-2 and increased Bax, cleaved caspase-3 and cleaved caspase-9. Moreover, CLEC12B decreased phosphorylation of PI3K-p85 and AKT proteins. By contrast, CLEC12B knockdown activated the PI3K/AKT pathway. In vivo, CLEC12B inhibited tumor growth in lung cancer, which can be reversed by CLEC12B inhibition. Co-IP and immunofluorescence assays confirmed the interaction between CLEC12B and SHP-1, and CLEC12B over-expression increased SHP-1 level. Furthermore, knocking down SHP-1 abrogated the above biological phenotypes caused by CLEC12B elevation. Taken together, our findings demonstrate that CLEC12B serves as a tumor-suppressing gene in lung cancer through positively regulating SHP-1 expression, which may be mediated by the PI3K/AKT signaling pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

scRNA seq of an F1 cross of Marek’s disease resistant and susceptible chickens identifies allele specific expression signatures enriched in transcription modulators

Abstract Marek’s disease (MD), a T cell lymphoma disease in chickens, is caused by the Marek’s disease virus (MDV) found ubiquitously in the poultry industry. Genetically resistant Line 6 3 (L6) and susceptible Line 7 2 (L7) chickens have been instrumental to research on avian immune system response to MDV infection. In this study we characterized molecular signatures unique to splenic immune cell types across different genetic backgrounds 6 days after infection. Using three populations, L6, L7, and an F1 cross between L6xL7, we evaluated the immune cell transcriptome of responding cell types using single cell RNA sequencing. Several MDV genes were found expressed mainly in cytotoxic T cells while ICP4 and MEQ MDV genes were expressed across infected cell types. Using the F1 we quantified allele specific expression (ASE) of biallelic SNPs and found biased expression of parental alleles specific to immune cell subtypes. We identified 22 SNPs with ASE in response to MDV infection mapped to gene rich regions surrounding 59 genes of critical importance for chromatin remodeling and transcriptional regulation. Histone deacetylase genes (HDAC1 and HDAC8) had increased expression of L6 alleles, while small nuclear RNA genes (SNORA68 and SNORA72) expressed higher levels of L7 alleles with infection in T cell subsets. SNPs with ASE also mapped genes important for an adequate immune response including GNLY (cytotoxic activity) and PDIA3 (component of MHC class I peptide loading complex), and genes known to promote viral replication (MCM5 and EIF3M). These results show that functional variants associated with susceptibility to MD may have a bigger impact in subsets of immune cell types, and by characterizing the transcriptomes of these subtypes we can unravel molecular signatures specific to MD genomic resistance.

Science & Technology - Other Topics↗
DOE OSTI2025

Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation

Abstract Expanding the genetic toolkit for Leptospira spp. is a crucial step toward advancing our understanding of the biology and virulence of these atypical bacteria. Pathogenic Leptospiraare responsible for over 1 million human leptospirosis cases annually and significantly impact domestic animals. Bovine leptospirosis causes substantial financial losses due to abortion, stillbirths, and suboptimal reproductive performance. The advent of the CRISPR/Cas9 system has marked a turning point in genetic manipulation, with applications across multiple Leptospira species. However, incorporating controlled protein expression into existing genetic tools could further expand their utility. We developed and demonstrated the functionality of IPTG-inducible heterologous protein expression in Leptospira spp. This system was applied for regulated expression of dead Cas9 (dCas9) to generate knockdown mutants, and Cas9 to produce knockout mutants by inducing double-strand breaks (DSB) into desired targets. IPTG-induced dCas9 expression enabled validation of essential genes and non-coding RNAs. Additionally, IPTG-controlled Cas9 expression combined with a constitutive non-homologous end-joining (NHEJ) system allowed for successful recovery of knockout mutants, even in the absence of IPTG. These newly controlled protein expression systems will advance studies on the basic biology and virulence ofLeptospira, as well as facilitate knockout mutant generation for improved veterinary vaccines.

Science & Technology - Other Topics↗
DOE OSTI2025

Structural basis of differential gene expression at eQTLs loci from high-resolution ensemble models of 3D single-cell chromatin conformations

Abstract Motivation Techniques such as high-throughput chromosome conformation capture (Hi-C) have provided a wealth of information on nucleus organization and genome important for understanding gene expression regulation. Genome-Wide Association Studies have identified numerous loci associated with complex traits. Expression quantitative trait loci (eQTL) studies have further linked the genetic variants to alteration in expression levels of associated target genes across individuals. However, the functional roles of many eQTLs in noncoding regions remain unclear. Current joint analyses of Hi-C and eQTLs data lack advanced computational tools, limiting what can be learned from these data. Results We developed a computational method for simultaneous analysis of Hi-C and eQTL data, capable of identifying a small set of nonrandom interactions from all Hi-C interactions. Using these nonrandom interactions, we reconstructed large ensembles (×105) of high-resolution single-cell 3D chromatin conformations with thorough sampling, accurately replicating Hi-C measurements. Our results revealed many-body interactions in chromatin conformation at the single-cell level within eQTL loci, providing a detailed view of how 3D chromatin structures form the physical foundation for gene regulation, including how genetic variants of eQTLs affect the expression of associated eGenes. Furthermore, our method can deconvolve chromatin heterogeneity and investigate the spatial associations of eQTLs and eGenes at subpopulation level, revealing their regulatory impacts on gene expression. Together, ensemble modeling of thoroughly sampled single-cell chromatin conformations combined with eQTL data, helps decipher how 3D chromatin structures provide the physical basis for gene regulation, expression control, and aid in understanding the overall structure-function relationships of genome organization. Availability and implementation It is available at https://github.com/uic-liang-lab/3DChromFolding-eQTL-Loci.

Du, Lin (ORCID:0009000289869812)↗
DOE OSTI2022

Differential timing of gene expression and recruitment in independent origins of CAM in the Agavoideae (Asparagaceae)

Crassulacean acid metabolism (CAM) photosynthesis has evolved repeatedly across the plant tree of life, however our understanding of the genetic convergence across independent origins remains hampered by the lack of comparative studies. Here, we explore gene expression profiles in eight species from the Agavoideae (Asparagaceae) encompassing three independent origins of CAM. Using comparative physiology and transcriptomics, we examined the variable modes of CAM in this subfamily and the changes in gene expression across time of day and between well watered and drought-stressed treatments. We further assessed gene expression and the molecular evolution of genes encoding phosphoenolpyruvate carboxylase (PPC), an enzyme required for primary carbon fixation in CAM. Most time-of-day expression profiles are largely conserved across all eight species and suggest that large perturbations to the central clock are not required for CAM evolution. By contrast, transcriptional response to drought is highly lineage specific. Yucca and Beschorneria have CAM-like expression of PPC2 , a copy of PPC that has never been shown to be recruited for CAM in angiosperms. Together the physiological and transcriptomic comparison of closely related C 3 and CAM species reveals similar gene expression profiles, with the notable exception of differential recruitment of carboxylase enzymes for CAM function.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Brachypodium SPEECHLESS2 Promoter Drives Expression of a Synthetic EPF to Reduce Stomatal Density in Sugarcane Without Pleiotropic Effects

Stomata are microscopic pores that regulate the exchange of CO 2 and water vapour, making them a major target for engineering plants with improved intrinsic water use efficiency (iWUE). Proof-of-concept studies have demonstrated the potential to increase iWUE by reducing stomatal density (SD) and stomatal conductance (g sw ) by ubiquitously expressing EPIDERMAL PATTERNING FACTOR (EPF) family genes. However, unwanted effects on leaf, stem and reproductive traits are often observed when EPFs are misexpressed in this fashion. We sought to test if these effects result from pleiotropy and to identify a targeted promoter that can circumvent the side effects while retaining the desired reduction in SD. A previously reported synthetic EPF (EPF syn ) was expressed in sugarcane ( Saccharum spp.) using two putatively tissue-specific promoters from Brachypodium distachyon (BdCESA7p and BdSPCH2p) and a ubiquitous control from Zea mays (ZmUBI4p). BdSPCH2p control reduced SD to statistically equivalent levels as ZmUBI4p on the abaxial (23%) and adaxial (23%) leaf surfaces. ZmUB4p and BdCESA7p induce expression in four tissue types often associated with pleiotropic effects in EPF-expressing low SD plants. Transgenic plants carrying either the BdCESA7p or ZmUBI4p EPF syn cassettes displayed leaf chlorosis, reduced leaf nitrogen and chlorophyll content, and altered stem architecture. However, transgenic events harboring the BdSPCH2p EPF syn cassette restricted EPF syn expression to the stomatal development zone and leaf nodal tissues and produced transgenic plants without the associated pleiotropic effects. These results represent an important step toward engineering low-SD crops since they show that targeted gene expression can engineer stomatal patterning without impairing agronomically important traits.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Data for Intron-Mediated Enhancement of DIACYLGLYCEROL ACYLTRANSFERASE1 Expression in Energycane Promotes a Step Change for Lipid Accumulation in Vegetative Tissues

Metabolic engineering for hyperaccumulation of lipids in vegetative tissues is a novel strategy for enhancing energy density and biofuel production from biomass crops. Energycane is a prime feedstock for this approach due to its high biomass production and resilience under marginal conditions. DIACYLGLYCEROL ACYLTRANSFERASE ( DGAT ) catalyzes the last and only committed step in the biosynthesis of triacylglycerol (TAG) and can be a rate-limiting enzyme for the production of TAG. In this study, we explored the effect of intron-mediated enhancement (IME) on the expression of DGAT1 and resulting accumulation of TAG and total fatty acid (TFA) in leaf and stem tissues of energycane. To maximize lipid accumulation these evaluations were carried out by co-expressing the lipogenic transcription factor WRINKLED1 ( WRI1 ) and the TAG protect factor oleosin ( OLE1 ). Including an intron in the codon-optimized TmDGAT1 elevated the accumulation of its transcript in leaves by seven times on average based on 5 transgenic lines for each construct. Plants with WRI1 (W), DGAT1 with intron (Di), and OLE1 (O) expression (WDiO) accumulated TAG up to a 3.85% of leaf dry weight (DW), a 192-fold increase compared to non-modified energycane (WT) and a 3.8-fold increase compared to the highest accumulation under the intron-less gene combination (WDO). This corresponded to TFA accumulation of up to 8.4% of leaf dry weight, a 2.8-fold or 6.1-fold increase compared to WDO or WT, respectively. Co-expression of WDiO resulted in stem accumulations of TAG up to 1.14% of DW or TFA up to 2.08% of DW that exceeded WT by 57-fold or 12-fold and WDO more than twofold, respectively. Constitutive expression of these lipogenic “push pull and protect” factors correlated with biomass reduction. Intron-mediated enhancement (IME) of the expression of DGAT resulted in a step change in lipid accumulation of energycane and confirmed that under our experimental conditions it is rate limiting for lipid accumulation. IME should be applied to other lipogenic factors and metabolic engineering strategies. The findings from this study may be valuable in developing a high biomass feedstock for commercial production of lipids and advanced biofuels.

Feedstock Production↗
DOE OSTI2021

Expression of a Bacterial Trehalose-6-phosphate Synthase otsA Increases Oil Accumulation in Plant Seeds and Vegetative Tissues

We previously demonstrated that exogenous trehalose 6-phosphate (T6P) treatment stabilized WRINKLED1 (WRI1), a master transcriptional regulator of fatty acid (FA) synthesis and increased total FA content in Brassica napus ( B. napus ) embryo suspension cell culture. Here, we explore Arabidopsis lines heterologously expressing the Escherichia coli T6P synthase (otsA) or T6P phosphatase (otsB) to refine our understanding regarding the role of T6P in regulating fatty acid synthesis both in seeds and vegetative tissues. Arabidopsis 35S: otsA transgenic seeds showed an increase of 13% in fatty acid content compared to those of wild type (WT), while seeds of 35: otsB transgenic seeds showed a reduction of 12% in fatty acid content compared to WT. Expression of otsB significantly reduced the level of WRI1 and expression of its target genes in developing seeds. Like Arabidopsis seeds constitutively expressing otsA, transient expression of otsA in Nicotiana benthamiana leaves resulted in strongly elevated levels of T6P. This was accompanied by an increase of 29% in de novo fatty acid synthesis rate, a 2.3-fold increase in triacylglycerol (TAG) and a 20% increase in total fatty acid content relative to empty vector (EV) controls. Taken together, these data support the heterologous expression of otsA as an approach to increasing TAG accumulation in plant seeds and vegetative tissues.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Genetic Modification of KNAT7 Transcription Factor Expression Enhances Saccharification and Reduces Recalcitrance of Woody Biomass in Poplars

The precise role of KNAT7 transcription factors (TFs) in regulating secondary cell wall (SCW) biosynthesis in poplars has remained unknown, while our understanding of KNAT7 functions in other plants is continuously evolving. To study the impact of genetic modifications of homologous and heterologous KNAT7 gene expression on SCW formation in transgenic poplars, we prepared poplar KNAT7 ( PtKNAT7 ) overexpression ( PtKNAT7 -OE) and antisense suppression ( PtKNAT7 -AS) vector constructs for the generation of transgenic poplar lines via Agrobacterium -mediated transformation. Since the overexpression of homologous genes can sometimes result in co-suppression, we also overexpressed Arabidopsis KNAT7 ( AtKNAT7-OE ) in transgenic poplars. In all these constructs, the expression of KNAT7 transgenes was driven by developing xylem (DX)-specific promoter, DX15. Compared to wild-type (WT) controls, many SCW biosynthesis genes downstream of KNAT7 were highly expressed in poplar PtKNAT7-OE and AtKNAT7-OE lines. Yet, no significant increase in lignin content of woody biomass of these transgenic lines was observed. PtKNAT7-AS lines, however, showed reduced expression of many SCW biosynthesis genes downstream of KNAT7 accompanied by a reduction in lignin content of wood compared to WT controls. Syringyl to Guaiacyl lignin (S/G) ratios were significantly increased in all three KNAT7 knockdown and overexpression transgenic lines than WT controls. These transgenic lines were essentially indistinguishable from WT controls in terms of their growth phenotype. Saccharification efficiency of woody biomass was significantly increased in all transgenic lines than WT controls. Overall, our results demonstrated that developing xylem-specific alteration of KNAT7 expression affects the expression of SCW biosynthesis genes, impacting at least the lignification process and improving saccharification efficiency, hence providing one of the powerful tools for improving bioethanol production from woody biomass of bioenergy crops and trees.

59 BASIC BIOLOGICAL SCIENCES↗