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Shanklin, John

Publications and source records attributed to Shanklin, John.

At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Genomics of turions from the Greater Duckweed reveal its pathways for dormancy and re‐emergence strategy

Summary Over 15 families of aquatic plants are known to use a strategy of developmental switching upon environmental stress to produce dormant propagules called turions. However, few molecular details for turion biology have been elucidated due to the difficulties in isolating high‐quality nucleic acids from this tissue. We successfully developed a new protocol to isolate high‐quality transcripts and carried out RNA‐seq analysis of mature turions from the Greater Duckweed Spirodela polyrhiza . Comparison of turion transcriptomes to that of fronds, the actively growing leaf‐like tissue, were carried out. Bioinformatic analysis of high confidence, differentially expressed transcripts between frond and mature turion tissues revealed major pathways related to stress tolerance, starch and lipid metabolism, and dormancy that are mobilized to reprogram frond meristems for turion differentiation. We identified the key genes that are likely to drive starch and lipid accumulation during turion formation, as well as those in pathways for starch and lipid utilization upon turion germination. Comparison of genome‐wide cytosine methylation levels also revealed evidence for epigenetic changes in the formation of turion tissues. Similarities between turions and seeds provide evidence that key regulators for seed maturation and germination were retooled for their function in turion biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis for enzymatic terminal C–H bond functionalization of alkanes

Alkane monooxygenase (AlkB) is a widely occurring integral membrane metalloenzyme that catalyzes the initial step in the functionalization of recalcitrant alkanes with high terminal selectivity. AlkB enables diverse microorganisms to use alkanes as their sole carbon and energy source. Here we present the 48.6-kDa cryo-electron microscopy structure of a natural fusion from Fontimonas thermophila between AlkB and its electron donor AlkG at 2.76 Å resolution. The AlkB portion contains six transmembrane helices with an alkane entry tunnel within its transmembrane domain. A dodecane substrate is oriented by hydrophobic tunnel-lining residues to present a terminal C–H bond toward a diiron active site. AlkG, an [Fe–4S] rubredoxin, docks via electrostatic interactions and sequentially transfers electrons to the diiron center. The archetypal structural complex presented reveals the basis for terminal C–H selectivity and functionalization within this broadly distributed evolutionary class of enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Microbiome differences in sugarcane and metabolically engineered oilcane accessions and their implications for bioenergy production

Abstract Oilcane is a metabolically engineered sugarcane ( Saccharum spp. hybrid) that hyper-accumulates lipids in its vegetable biomass to provide an advanced feedstock for biodiesel production. The potential impact of hyper-accumulation of lipids in vegetable biomass on microbiomes and the consequences of altered microbiomes on plant growth and lipid accumulation have not been explored so far. Here, we explore differences in the microbiome structure of different oilcane accessions and non-modified sugarcane. 16S SSU rRNA and ITS rRNA amplicon sequencing were performed to compare the characteristics of the microbiome structure from different plant compartments (leaf, stem, root, rhizosphere, and bulk soil) of four greenhouse-grown oilcane accessions and non-modified sugarcane. Significant differences were only observed in the bacterial microbiomes. In leaf and stem microbiomes, more than 90% of the entire microbiome of non-modified sugarcane and oilcane was dominated by similar core taxa. Taxa associated with Proteobacteria led to differences in the non-modified sugarcane and oilcane microbiome structure. While differences were observed between multiple accessions, accession 1566 was notable in that it was consistently observed to differ in its microbial membership than other accessions and had the lowest abundance of taxa associated with plant-growth-promoting bacteria. Accession 1566 is also unique among oilcane accessions in that it has the highest constitutive expression of the WRI 1 transgene. The WRI 1 transcription factor is known to contribute to significant changes in the global gene expression profile, impacting plant fatty acid biosynthesis and photomorphogenesis. This study reveals for the first time that genetically modified oilcanes associate with distinct microbiomes. Our findings suggest potential relationships between core taxa, biomass yield, and TAG in oilcane accessions and support further research on the relationship between plant genotypes and their microbiomes.

09 BIOMASS FUELS↗
DOE OSTI2023

CYCLIN‐DEPENDENT KINASE 8 positively regulates oil synthesis by activating WRINKLED1 transcription

Summary CYCLIN‐DEPENDENT KINASE 8 (CDK8), a component of the kinase module of the Mediator complex in Arabidopsis, is involved in many processes, including flowering, plant defense, drought, and energy stress responses. Here, we investigated cdk8 mutants and CDK8‐ overexpressing lines to evaluate whether CDK8 also plays a role in regulating lipid synthesis, an energy‐demanding anabolism. Quantitative lipid analysis demonstrated significant reductions in lipid synthesis rates and lipid accumulation in developing siliques and seedlings of cdk8 , and conversely, elevated lipid contents in wild‐type seed overexpressing CDK8 . Transactivation assays show that CDK8 is necessary for maximal transactivation of the master seed oil activator WRINKLED1 (WRI1) by the seed maturation transcription factor ABSCISIC ACID INSENSITIVE3, supporting a direct regulatory role of CDK8 in oil synthesis. Thermophoretic studies show GEMINIVIRUS REP INTERACTING KINASE1, an activating kinase of KIN10 (a catalytic subunit of SUCROSE NON‐FERMENTING1‐RELATED KINASE1), physically interacts with CDK8, resulting in its phosphorylation and degradation in the presence of KIN10. This work defines a mechanism whereby, once activated, KIN10 downregulates WRI1 expression and suppresses lipid synthesis via promoting the degradation of CDK8. The KIN10‐CDK8‐dependent regulation of lipid synthesis described herein is additional to our previously reported KIN10‐dependent phosphorylation and degradation of WRI1.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A toolkit for plant lipid engineering: surveying the efficacies of lipogenic factors for accumulating specialty lipids

Plants produce energy-dense lipids from carbohydrates using energy acquired via photosynthesis, making plant oils an economically and sustainably attractive feedstock for conversion to biofuels and value-added bioproducts. A growing number of strategies have been developed and optimized in model plants, oilseed crops and high-biomass crops to enhance the accumulation of storage lipids (mostly triacylglycerols, TAGs) for bioenergy applications and to produce specialty lipids with increased uses and value for chemical feedstock and nutritional applications. Most successful metabolic engineering strategies involve heterologous expression of lipogenic factors that outperform those from other sources or exhibit specialized functionality. In this review, we summarize recent progress in engineering the accumulation of triacylglycerols containing - specialized fatty acids in various plant species and tissues. We also provide an inventory of specific lipogenic factors derived from a wide variety of organisms, along with their reported efficacy in supporting the accumulation of desired lipids. A review of previously obtained results serves as a foundation to guide future efforts to optimize combinations of factors to achieve further enhancements to the production and accumulation of desired lipids in a variety of plant tissues and species.

59 BASIC BIOLOGICAL SCIENCES↗
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 OSTI2022

Metabolic engineering of energycane to hyperaccumulate lipids in vegetative biomass

Abstract Background The metabolic engineering of high-biomass crops for lipid production in their vegetative biomass has recently been proposed as a strategy to elevate energy density and lipid yields for biodiesel production. Energycane and sugarcane are highly polyploid, interspecific hybrids between Saccharum officinarum and Saccharum spontaneum that differ in the amount of ancestral contribution to their genomes. This results in greater biomass yield and persistence in energycane, which makes it the preferred target crop for biofuel production. Results Here, we report on the hyperaccumulation of triacylglycerol (TAG) in energycane following the overexpression of the lipogenic factors Diacylglycerol acyltransferase 1-2 ( DGAT 1-2) and Oleosin 1 ( OLE 1) in combination with RNAi suppression of SUGAR-DEPENDENT 1 ( SDP 1) and Trigalactosyl diacylglycerol 1 ( TGD 1). TAG accumulated up to 1.52% of leaf dry weight (DW,) a rate that was 30-fold that of non-modified energycane, in addition to almost doubling the total fatty acid content in leaves to 4.42% of its DW. Pearson’s correlation analysis showed that the accumulation of TAG had the highest correlation with the expression level of ZmDGAT 1-2, followed by the level of RNAi suppression for SDP 1. Conclusions This is the first report on the metabolic engineering of energycane and demonstrates that this resilient, high-biomass crop is an excellent target for the further optimization of the production of lipids from vegetative tissues.

1-2↗
DOE OSTI2022

An expanded role for the transcription factor WRINKLED1 in the biosynthesis of triacylglycerols during seed development

The transcription factor WRINKLED1 ( WRI1 ) is known as a master regulator of fatty acid synthesis in developing oilseeds of Arabidopsis thaliana and other species. WRI1 is known to directly stimulate the expression of many fatty acid biosynthetic enzymes and a few targets in the lower part of the glycolytic pathway. However, it remains unclear to what extent and how the conversion of sugars into fatty acid biosynthetic precursors is controlled by WRI 1. To shortlist possible gene targets for future in-planta experimental validation, here we present a strategy that combines phylogenetic foot printing of cis-regulatory elements with additional layers of evidence. Upstream regions of protein-encoding genes in A. thaliana were searched for the previously described DNA-binding consensus for WRI1, the ASML1/WRI1 (AW)-box. For about 900 genes, AW-box sites were found to be conserved across orthologous upstream regions in 11 related species of the crucifer family. For 145 select potential target genes identified this way, affinity of upstream AW-box sequences to WRI1 was assayed by Microscale Thermophoresis. This allowed definition of a refined WRI1 DNA-binding consensus. We find that known WRI1 gene targets are predictable with good confidence when upstream AW-sites are phylogenetically conserved, specifically binding WRI1 in the in vitro assay, positioned in proximity to the transcriptional start site, and if the gene is co-expressed with WRI1 during seed development. When targets predicted in this way are mapped to central metabolism, a conserved regulatory blueprint emerges that infers concerted control of contiguous pathway sections in glycolysis and fatty acid biosynthesis by WRI1. Several of the newly predicted targets are in the upper glycolysis pathway and the pentose phosphate pathway. Of these, plastidic isoforms of fructokinase ( FRK 3) and of phosphoglucose isomerase ( PGI 1) are particularly corroborated by previously reported seed phenotypes of respective null mutations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Divergent evolution of extreme production of variant plant monounsaturated fatty acids

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia , closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Purple acid phosphatase2 stimulates a futile cycle of lipid synthesis and degradation, and mitigates the negative growth effects of triacylglycerol accumulation in vegetative tissues

Storage lipids (mostly triacylglycerols, TAGs) serve as an important energy and carbon reserve in plants and hyperaccumulation of TAG in vegetative tissues can cause negative effects on plant growth. Purple acid phosphatase2 (PAP2) was shown previously to affect carbon metabolism and boost plant growth. However, the effects of PAP2 on lipid metabolism remain unknown. Here, we demonstrated PAP2 as a factor that can stimulate a futile cycle of fatty acid (FA) synthesis and degradation and mitigate negative growth effects associated with TAG accumulation in vegetative tissues. Constitutive expression of PAP2 in Arabidopsis thaliana enhanced both lipid synthesis and degradation in leaves and led to substantial increase in seed oil yield. Suppressing lipid degradation in a PAP2-overexpressing line by disrupting sugar-dependent1 (SDP1), a predominant TAG lipase, significantly elevated vegetative TAG content and improved plant growth. Diverting FAs from membrane lipids to TAGs in PAP2-overexpressing plants by constitutively expressing phospholipid:diacylglycerol acyltransferase1 (PDAT1) increased TAG contents to up to 6% of dry weight in vegetative tissues without compromising biomass yield. Overall, these results highlight the potential of combining PAP2 with TAG-promoting factors to enhance carbon assimilation, FA synthesis and allocation to TAGs for optimized plant growth and storage lipid accumulation in vegetative tissues.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Data for Purple Acid Phosphatase2 Stimulates a Futile Cycle of Lipid Synthesis and Degradation, and Mitigates the Negative Growth Effects of Triacylglycerol Accumulation in Vegetative Tissues

Storage lipids (mostly triacylglycerols, TAGs) serve as an important energy and carbon reserve in plants, and hyperaccumulation of TAG in vegetative tissues can have negative effects on plant growth. Purple acid phosphatase2 (PAP2) was previously shown to affect carbon metabolism and boost plant growth. However, the effects of PAP2 on lipid metabolism remain unknown. Here, we demonstrated that PAP2 can stimulate a futile cycle of fatty acid (FA) synthesis and degradation, and mitigate negative growth effects associated with high accumulation of TAG in vegetative tissues. Constitutive expression of PAP2 in Arabidopsis thaliana enhanced both lipid synthesis and degradation in leaves and led to a substantial increase in seed oil yield. Suppressing lipid degradation in a PAP2-overexpressing line by disrupting sugar-dependent1 (SDP1), a predominant TAG lipase, significantly elevated vegetative TAG content and improved plant growth. Diverting FAs from membrane lipids to TAGs in PAP2-overexpressing plants by constitutively expressing phospholipid:diacylglycerol acyltransferase1 (PDAT1) greatly increased TAG content in vegetative tissues without compromising biomass yield. These results highlight the potential of combining PAP2 with TAG-promoting factors to enhance carbon assimilation, FA synthesis and allocation to TAGs for optimized plant growth and storage lipid accumulation in vegetative tissues.

Biomass Analytics↗
DOE OSTI2022

Hepatitis C virus NS3/4A inhibitors and other drug-like compounds as covalent binders of SARS-CoV-2 main protease

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (M pro also known as 3CL pro ) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the M pro Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the M pro as potential COVID-19 therapeutics for further testing and possibly clinical trials.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Regioselectivity mechanism of the Thunbergia alata Δ 6 -16:0-acyl carrier protein desaturase

Plant plastidial acyl–acyl carrier protein (ACP) desaturases are a soluble class of diiron-containing enzymes that are distinct from the diiron-containing integral membrane desaturases found in plants and other organisms. The archetype of this class is the stearoyl-ACP desaturase which converts stearoyl-ACP into oleoyl (18:1Δ 9 cis )-ACP. Several variants expressing distinct regioselectivity have been described including a Δ 6 -16:0-ACP desaturase from black-eyed Susan vine ( Thunbergia alata ). We solved a crystal structure of the T. alata desaturase at 2.05 Å resolution. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position C6 and C7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ 6 desaturase to Δ 9 regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. Furthermore, to validate the in-silico predictions, we synthesized two variants of the T. alata desaturase and analyzed their reaction products using gas chromatography-coupled mass spectrometry. Assay results confirmed that mutants designed to convert T. alata Δ 6 to Δ 9 selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Δ 9 to Δ 6 selectivity lost some of their Δ9 desaturation ability and gained the ability to desaturate at the Δ 6 position. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A consensus-based ensemble approach to improve transcriptome assembly

Systems-level analyses, such as differential gene expression analysis, co-expression analysis, and metabolic pathway reconstruction, depend on the accuracy of the transcriptome. Multiple tools exist to perform transcriptome assembly from RNAseq data. However, assembling high quality transcriptomes is still not a trivial problem. This is especially the case for non-model organisms where adequate reference genomes are often not available. Different methods produce different transcriptome models and there is no easy way to determine which are more accurate. Furthermore, having alternative-splicing events exacerbates such difficult assembly problems. While benchmarking transcriptome assemblies is critical, this is also not trivial due to the general lack of true reference transcriptomes. In this study, we first provide a pipeline to generate a set of the simulated benchmark transcriptome and corresponding RNAseq data. Using the simulated benchmarking datasets, we compared the performance of various transcriptome assembly approaches including both de novo and genome-guided methods. The results showed that the assembly performance deteriorates significantly when alternative transcripts (isoforms) exist or for genome-guided methods when the reference is not available from the same genome. To improve the transcriptome assembly performance, leveraging the overlapping predictions between different assemblies, we present a new consensus-based ensemble transcriptome assembly approach, ConSemble. Without using a reference genome, ConSemble using four de novo assemblers achieved an accuracy up to twice as high as any de novo assemblers we compared. When a reference genome is available, ConSemble using four genome-guided assemblies removed many incorrectly assembled contigs with minimal impact on correctly assembled contigs, achieving higher precision and accuracy than individual genome-guided methods. Furthermore, ConSemble using de novo assemblers matched or exceeded the best performing genome-guided assemblers even when the transcriptomes included isoforms. We thus demonstrated that the ConSemble consensus strategy both for de novo and genome-guided assemblers can improve transcriptome assembly. The RNAseq simulation pipeline, the benchmark transcriptome datasets, and the script to perform the ConSemble assembly are all freely available from: http://bioinfolab.unl.edu/emlab/consemble/.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

AlphaFold Protein Structure Database for Sequence-Independent Molecular Replacement

Crystallographic phasing recovers the phase information that is lost during a diffraction experiment. Molecular replacement is a commonly used phasing method for crystal structures in the protein data bank. In one form it uses a protein sequence to search a structure database to find suitable templates for phasing. However, sequence information is not always available, such as when proteins are crystallized with unknown binding partner proteins or when the crystal is of a contaminant. The recent development of AlphaFold published the predicted protein structures for every protein from twenty distinct species. In this work, we tested whether AlphaFold-predicted E. coli protein structures were accurate enough to enable sequence-independent phasing of diffraction data from two crystallization contaminants of unknown sequence. Using each of more than 4000 predicted structures as a search model, robust molecular replacement solutions were obtained, which allowed the identification and structure determination of YncE and YadF. Our results demonstrate the general utility of the AlphaFold-predicted structure database with respect to sequence-independent crystallographic phasing.

59 BASIC BIOLOGICAL SCIENCES↗