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Scheller, Henrik V.

Publications and source records attributed to Scheller, Henrik V..

At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Engineered reduction of S-adenosylmethionine alters lignin in sorghum

Abstract Background Lignin is an aromatic polymer deposited in secondary cell walls of higher plants to provide strength, rigidity, and hydrophobicity to vascular tissues. Due to its interconnections with cell wall polysaccharides, lignin plays important roles during plant growth and defense, but also has a negative impact on industrial processes aimed at obtaining monosaccharides from plant biomass. Engineering lignin offers a solution to this issue. For example, previous work showed that heterologous expression of a coliphage S -adenosylmethionine hydrolase (AdoMetase) was an effective approach to reduce lignin in the model plant Arabidopsis. The efficacy of this engineering strategy remains to be evaluated in bioenergy crops. Results We studied the impact of expressing AdoMetase on lignin synthesis in sorghum ( Sorghum bicolor L. Moench). Lignin content, monomer composition, and size, as well as biomass saccharification efficiency were determined in transgenic sorghum lines. The transcriptome and metabolome were analyzed in stems at three developmental stages. Plant growth and biomass composition was further evaluated under field conditions. Results evidenced that lignin was reduced by 18% in the best transgenic line, presumably due to reduced activity of the S -adenosylmethionine-dependent O -methyltransferases involved in lignin synthesis. The modified sorghum features altered lignin monomer composition and increased lignin molecular weights. The degree of methylation of glucuronic acid on xylan was reduced. These changes enabled a ~20% increase in glucose yield after biomass pretreatment and saccharification compared to wild type. RNA-seq and untargeted metabolomic analyses evidenced some pleiotropic effects associated with AdoMetase expression. The transgenic sorghum showed developmental delay and reduced biomass yields at harvest, especially under field growing conditions. Conclusions The expression of AdoMetase represents an effective lignin engineering approach in sorghum. However, considering that this strategy potentially impacts multiple S -adenosylmethionine-dependent methyltransferases, adequate promoters for fine-tuning AdoMetase expression will be needed to mitigate yield penalty.

Tian, Yang↗
DOE OSTI2024

EcoFAB 3.0: a sterile system for studying sorghum that replicates previous field and greenhouse observations

Introduction Studying plant-microbe interactions is one of the key elements in understanding the path to sustainable agricultural practices. These interactions play a crucial role in ensuring survival of healthy plants, soil and microbial communities. Many platforms have been developed over the years to isolate these highly complex interactions however, these are designed for small model plants. This creates a need for complementary devices for larger plants, such as sorghum. Methods This work introduces a novel platform, EcoFAB 3.0, which is designed to enable studying bioenergy plants such as sorghum for up to 4 weeks in a controlled sterile environment. Several other advantages of this platform such as dark root chambers and user-friendly assembly are also discussed in this work. Results and discussion EcoFAB 3.0 was found to replicate previous greenhouse and field observations when comparing an engineered sorghum line overproducing 4-hydroxybenzoic acid (4-HBA) and wildtype (variety BTx430). Consistent with greenhouse and field observations, it was found that the engineered line of sorghum grown in EcoFAB 3.0 had a higher 4-HBA content and a lower dry biomass.

Gupta, Kshitiz↗
DOE OSTI2024

Impact of Drought Stress on Sorghum bicolor Yield, Deconstruction, and Microbial Conversion Determined in a Feedstocks-to-Fuels Pipeline

Sorghum is an attractive feedstock for biobased fuel and chemical production because it is familiar to farmers, naturally drought tolerant, and versatile as a food, feed, and fuel crop. Although sorghum is a promising feedstock, particularly in regions that experience drought stress, little is known about how drought conditions impact the ease of conversion of sorghum to fuels and products. This study combines agronomic field trials with a high-throughput experimental pipeline to explore the field performance and liquid biofuel (bisabolene) yields resulting from three sorghum types (photosensitive forage sorghum, optimized grain sorghum, and drought-resistant grain sorghum) grown under pre- and postflowering water limitations in two different California locations. Multiple drought treatments are compared to the control, as the timing (preflowering versus postflowering) of drought stress elicits different survival strategies and corresponding impacts on yield and composition. Forage-type sorghum maintained the highest biomass yields across all irrigation conditions and locations. Glucose and xylose yields resulting from ionic liquid pretreatment and enzymatic saccharification were not significantly impacted by irrigation treatments but differed by location and genotype. However, Rhodosporidium toruloides grown on the resulting plant hydrolysates unexpectedly produced higher titers of bisabolene for drought-stressed sorghum samples regardless of genotype.

09 BIOMASS FUELS↗
DOE OSTI2024

Biosynthesis of Strained Amino Acids by a PLP‐Dependent Enzyme through Cryptic Halogenation

Abstract Amino acids (AAs) are modular building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products, such as alkaloids and non‐ribosomal peptides. While the 20 main proteinogenic AAs display relatively limited side chain diversity, a wide range of non‐canonical amino acids (ncAAs) exist that are not used by the ribosome for protein synthesis, but contain a broad array of structural features and functional groups. In this communication, we report the discovery of the biosynthetic pathway for a new ncAA, pazamine, which contains a cyclopropane ring formed in two steps. In the first step, a chlorine is added onto the C 4 position of lysine by a radical halogenase, PazA. The cyclopropane ring is then formed in the next step by a pyridoxal‐5′‐phosphate‐dependent enzyme, PazB, via an S N 2‐like attack at C 4 to eliminate chloride. Genetic studies of this pathway in the native host, Pseudomonas azotoformans , show that pazamine potentially inhibits ethylene biosynthesis in growing plants based on alterations in the root phenotype of Arabidopsis thaliana seedlings. We further show that PazB can be utilized to make an alternative cyclobutane‐containing AA. These discoveries may lead to advances in biocatalytic production of specialty chemicals and agricultural biotechnology.

Sosa, Max B.↗
DOE OSTI2024

Unraveling plant–microbe symbioses using single-cell and spatial transcriptomics

Plant-microbe symbioses require intense interaction and genetic coordination to successfully establish in specific cell types of the host and symbiont. Traditional RNA-seq methodologies lack the cellular resolution to fully capture these complexities, but single-cell and spatial transcriptomics (ST) are now allowing scientists to probe symbiotic interactions at an unprecedented level of detail. Here, we discuss the advantages that novel spatial and single-cell transcriptomic technologies provide in studying plant-microbe endosymbioses and highlight key recent studies. Finally, we consider the remaining limitations of applying these approaches to symbiosis research, which are mainly related to the simultaneous capture of both plant and microbial transcripts within the same cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Defined synthetic microbial communities colonize and benefit field-grown sorghum

The rhizosphere constitutes a dynamic interface between plant hosts and their associated microbial communities. Despite the acknowledged potential for enhancing plant fitness by manipulating the rhizosphere, the engineering of the rhizosphere microbiome through inoculation has posed significant challenges. These challenges are thought to arise from the competitive microbial ecosystem where introduced microbes must survive, and the absence of adaptation to the specific metabolic and environmental demands of the rhizosphere. Here, in this study, we engineered a synthetic rhizosphere community (SRC1) with the anticipation that it would exhibit a selective advantage in colonizing the host Sorghum bicolor, thereby potentially fostering its growth. SRC1 was assembled from bacterial isolates identified either for their potential role in community cohesion through network analysis or for their ability to benefit from host-specific exudate compounds. The growth performance of SRC1 was assessed in vitro on solid media, in planta under gnotobiotic laboratory conditions, and in the field. Our findings reveal that SRC1 cohesion is most robust when cultivated in the presence of the plant host under laboratory conditions, with lineages being lost from the community when grown either in vitro or in a native field setting. We establish that SRC1 effectively promotes the growth of both above- and below-ground plant phenotypes in both laboratory and native field contexts. Furthermore, in laboratory conditions, these growth enhancements correlate with the transcriptional dampening of lignin biosynthesis in the host. Collectively, these results underscore the potential utility of synthetic microbial communities for modulating crop performance in controlled and native environments alike.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Advances in Engineering Nucleotide Sugar Metabolism for Natural Product Glycosylation in Saccharomyces cerevisiae

Glycosylation is a ubiquitous modification present across all of biology, affecting many things such as physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Nucleotide sugars are important precursors needed to study glycosylation and produce glycosylated products. Saccharomyces cerevisiae is a potentially powerful platform for producing glycosylated biomolecules, but it lacks nucleotide sugar diversity. Nucleotide sugar metabolism is complex, and understanding how to engineer it will be necessary to both access and study heterologous glycosylations found across biology. This review overviews the potential challenges with engineering nucleotide sugar metabolism in yeast from the salvage pathways that convert free sugars to their associated UDP-sugars to de novo synthesis where nucleotide sugars are interconverted through a complex metabolic network with governing feedback mechanisms. Finally, recent examples of engineering complex glycosylation of small molecules in S. cerevisiae are explored and assessed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Putative rhamnogalacturonan-II glycosyltransferase identified through callus gene editing bypasses embryo lethality

Rhamnogalacturonan II (RG-II) is a structurally complex and conserved domain of the pectin present in the primary cell walls of vascular plants. Borate crosslinking of RG-II is required for plants to grow and develop normally. Mutations that alter RG-II structure also affect crosslinking and are lethal or severely impair growth. Thus, few genes involved in RG-II synthesis have been identified. Here we developed a method to generate viable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in callus tissue via CRISPR/Cas9-mediated gene editing. We combined this with a candidate gene approach to characterize the male gametophyte defective 2 (MPG2) gene that encodes a putative family GT29 glycosyltransferase. Plants homozygous for this mutation do not survive. We showed that in the callus mutant cell walls, RG-II does not crosslink normally because it lacks 3-deoxy-D-manno-octulosonic acid (Kdo) and thus cannot form the α-L-Rhap-(1→5)-α-D-kdop-(1→ sidechain. We suggest that MGP2 encodes an inverting RG-II CMP-β-Kdo transferase (RCKT1). Our discovery provides further insight into the role of sidechains in RG-II dimerization. Our method also provides a viable strategy for further identifying proteins involved in the biosynthesis of RG-II.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A guayule C-repeat binding factor is highly activated in guayule under freezing temperature and enhances freezing tolerance when expressed in Arabidopsis thaliana

Natural Rubber (NR)-producing guayule (Parthenium argentatum Gray) has been developed as a new crop to diversify NR production. Guayule NR is mainly synthesized in its stem and is upregulated by cold temperatures. A guayule C-repeat binding factor 4 (PaCBF4) was highly expressed in cold-treated stem tissue, coinciding with active rubber biosynthesis and accumulation. Sequence alignments of PaCBF4 with other CBFs indicated that PaCBF4 contains DNA-binding domains responsible for regulating cold-regulated (COR) gene expression. Spatial gene expression profiling of PaCBF4 revealed that stems had the highest expression level among different organs examined. We further confirmed the function of PaCBF4 as regulator of cold-signaling processes by expressing it in the model plant Arabidopsis under a constitutive ubiquitin promoter from potato. Further, the resulting transgenic Arabidopsis lines expressing PaCBF4 turned on expression of a set of Arabidopsis COR genes under both room (24ºC) and cold (4ºC) temperatures, in contrast to the wild-type Arabidopsis that expressed these COR genes solely upon cold treatment. Furthermore, the transgenic plants displayed enhanced freezing tolerance at -5ºC, exhibiting a survival rate of 88–98% compared with 0% survival rate of wild-type plants. Our results suggest that PaCBF4 is a functional member of the guayule CBF gene family and plays a significant role in cold and freeze tolerance. Interestingly, overexpressing PaCBF4 in Arabidopsis did not affect the normal phenotype of the plant during vegetative and inflorescence growth, but the gene led to more undeveloped siliques after flowering.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Cynipid wasps systematically reprogram host metabolism and restructure cell walls in developing galls

Many insects have evolved the ability to manipulate plant growth to generate extraordinary structures called galls, in which insect larva can develop while being sheltered and feeding on the plant. In particular, cynipid (Hymenoptera: Cynipidae) wasps have evolved to form morphologically complex galls and generate an astonishing array of gall shapes, colors, and sizes. However, the biochemical basis underlying these remarkable cellular and developmental transformations remains poorly understood. A key determinant in plant cellular development is cell wall deposition that dictates the physical form and physiological function of newly developing cells, tissues, and organs. However, it is unclear to what degree cell walls are restructured to initiate and support the formation of new gall tissue. Here, we characterize the molecular alterations underlying gall development using a combination of metabolomic, histological, and biochemical techniques to elucidate how valley oak (Quercus lobata) leaf cells are reprogrammed to form galls. Strikingly, gall development involves an exceptionally coordinated spatial deposition of lignin and xylan to form de novo gall vasculature. Our results highlight how cynipid wasps can radically change the metabolite profile and restructure the cell wall to enable the formation of galls, providing insights into the mechanism of gall induction and the extent to which plants can be entirely reprogrammed to form unique structures and organs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Field performance of switchgrass plants engineered for reduced recalcitrance

Switchgrass ( Panicum virgatum L.) is a promising perennial bioenergy crop that achieves high yields with relatively low nutrient and energy inputs. Modification of cell wall composition for reduced recalcitrance can lower the costs of deconstructing biomass to fermentable sugars and other intermediates. We have engineered overexpression of OsAT10 , encoding a rice BAHD acyltransferase and QsuB , encoding dehydroshikimate dehydratase from Corynebacterium glutamicum , to enhance saccharification efficiency in switchgrass. These engineering strategies demonstrated low lignin content, low ferulic acid esters, and increased saccharification yield during greenhouse studies in switchgrass and other plant species. In this work, transgenic switchgrass plants overexpressing either OsAT10 or QsuB were tested in the field in Davis, California, USA for three growing seasons. No significant differences in the content of lignin and cell wall-bound p -coumaric acid or ferulic acid were detected in transgenic OsAT10 lines compared with the untransformed Alamo control variety. However, the transgenic overexpressing QsuB lines had increased biomass yield and slightly increased biomass saccharification properties compared to the control plants. This work demonstrates good performance of engineered plants in the field, and also shows that the cell wall changes in the greenhouse were not replicated in the field, emphasizing the need to validate engineered plants under relevant field conditions.

59 BASIC BIOLOGICAL 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

A Suite of Constitutive Promoters for Tuning Gene Expression in Plants

The need for convenient tools to express transgenes over a large dynamic range is pervasive throughout plant synthetic biology; however, current efforts are largely limited by the heavy reliance on a small set of strong promoters, precluding more nuanced and refined engineering endeavors in planta . To address this technical gap, we characterize a suite of constitutive promoters that span a wide range of transcriptional levels and develop a GoldenGate-based plasmid toolkit named PCONS, optimized for versatile cloning and rapid testing of transgene expression at varying strengths. We demonstrate how easy access to a stepwise gradient of expression levels can be used for optimizing synthetic transcriptional systems and the production of small molecules in planta . We also systematically investigate the potential of using PCONS as an internal standard in plant biology experimental design, establishing the best practices for signal normalization in experiments. Although our library has primarily been developed for optimizing expression in N. benthamiana , we demonstrate the translatability of our promoters across distantly related species using a multiplexed reporter assay with barcoded transcripts. In conclusion, our findings showcase the advantages of the PCONS library as an invaluable toolkit for plant synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural and biochemical insight into a modular β-1,4-galactan synthase in plants

Rhamnogalacturonan I (RGI) is a structurally complex pectic polysaccharide with a backbone of alternating rhamnose and galacturonic acid residues substituted with arabinan and galactan side chains. Galactan synthase 1 (GalS1) transfers galactose and arabinose to either extend or cap the ..beta..-1,4-galactan side chains of RGI, respectively. Here we report the structure of GalS1 from Populus trichocarpa, showing a modular protein consisting of an N-terminal domain that represents the founding member of a new family of carbohydrate-binding module, CBM95, and a C-terminal glycosyltransferase family 92 (GT92) catalytic domain that adopts a GT-A fold. GalS1 exists as a dimer in vitro, with stem domains interacting across the chains in a 'handshake' orientation that is essential for maintaining stability and activity. In addition to understanding the enzymatic mechanism of GalS1, here we gained insight into the donor and acceptor substrate binding sites using deep evolutionary analysis, molecular simulations and biochemical studies. Combining all the results, a mechanism for GalS1 catalysis and a new model for pectic galactan side-chain addition are proposed.

09 BIOMASS FUELS↗
DOE OSTI2022

Evaluation of engineered low-lignin poplar for conversion into advanced bioproducts

Lignocellulosic resources are promising feedstocks for the manufacture of bio-based products and bioenergy. However, the inherent recalcitrance of biomass to conversion into simple sugars currently hinders the deployment of advanced bioproducts at large scale. Lignin is a primary contributor to biomass recalcitrance as it protects cell wall polysaccharides from degradation and can inhibit hydrolytic enzymes via non-productive adsorption. Several engineering strategies have been designed to reduce lignin or modify its monomeric composition. For example, expression of bacterial 3-dehydroshikimate dehydratase (QsuB) in poplar trees resulted in a reduction in lignin due to redirection of metabolic flux toward 3,4-dihydroxybenzoate at the expense of lignin. This reduction was accompanied with remarkable changes in the pools of aromatic compounds that accumulate in the biomass. The impact of these modifications on downstream biomass deconstruction and conversion into advanced bioproducts was evaluated in the current study. Using ionic liquid pretreatment followed by enzymatic saccharification, biomass from engineered trees released more glucose and xylose compared to wild-type control trees under optimum conditions. Fermentation of the resulting hydrolysates using Rhodosporidium toruloides strains engineered to produce α-bisabolene, epi-isozizaene, and fatty alcohols showed no negative impact on cell growth and yielded higher titers of bioproducts (as much as + 58%) in the case of QsuB transgenics trees. Our data show that low-recalcitrant poplar biomass obtained with the QsuB technology has the potential to improve the production of advanced bioproducts.

09 BIOMASS FUELS↗
DOE OSTI2022

Low-abundance populations distinguish microbiome performance in plant cell wall deconstruction

Abstract Background Plant cell walls are interwoven structures recalcitrant to degradation. Native and adapted microbiomes can be particularly effective at plant cell wall deconstruction. Although most understanding of biological cell wall deconstruction has been obtained from isolates, cultivated microbiomes that break down cell walls have emerged as new sources for biotechnologically relevant microbes and enzymes. These microbiomes provide a unique resource to identify key interacting functional microbial groups and to guide the design of specialized synthetic microbial communities. Results To establish a system assessing comparative microbiome performance, parallel microbiomes were cultivated on sorghum ( Sorghum bicolor L. Moench) from compost inocula. Biomass loss and biochemical assays indicated that these microbiomes diverged in their ability to deconstruct biomass. Network reconstructions from gene expression dynamics identified key groups and potential interactions within the adapted sorghum-degrading communities, including Actinotalea , Filomicrobium , and Gemmatimonadetes populations. Functional analysis demonstrated that the microbiomes proceeded through successive stages that are linked to enzymes that deconstruct plant cell wall polymers. The combination of network and functional analysis highlighted the importance of cellulose-degrading Actinobacteria in differentiating the performance of these microbiomes. Conclusions The two-tier cultivation of compost-derived microbiomes on sorghum led to the establishment of microbiomes for which community structure and performance could be assessed. The work reinforces the observation that subtle differences in community composition and the genomic content of strains may lead to significant differences in community performance.

59 BASIC BIOLOGICAL SCIENCES↗