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Engineering topics

Shih, Patrick M.

Publications and source records attributed to Shih, Patrick M..

At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Binary vector copy number engineering improves Agrobacterium -mediated transformation

The copy number of a plasmid is linked to its functionality, yet there have been few attempts to optimize higher-copy-number mutants for use across diverse origins of replication in different hosts. We use a high-throughput growth-coupled selection assay and a directed evolution approach to rapidly identify origin of replication mutations that influence copy number and screen for mutants that improve Agrobacterium-mediated transformation (AMT) efficiency. By introducing these mutations into binary vectors within the plasmid backbone used for AMT, we observe improved transient transformation of Nicotiana benthamiana in four diverse tested origins (pVS1, RK2, pSa and BBR1). For the best-performing origin, pVS1, we isolate higher-copy-number variants that increase stable transformation efficiencies by 60–100% in Arabidopsis thaliana and 390% in the oleaginous yeast Rhodosporidium toruloides. Our work provides an easily deployable framework to generate plasmid copy number variants that will enable greater precision in prokaryotic genetic engineering, in addition to improving AMT efficiency.

59 BASIC BIOLOGICAL SCIENCES↗
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

Design and Characterization of a Transcriptional Repression Toolkit for Plants

Regulation of gene expression is essential for all life. Tools to manipulate the gene expression level have therefore proven to be very valuable in efforts to engineer biological systems. However, there are few well-characterized genetic parts that reduce gene expression in plants, commonly known as transcriptional repressors. We characterized the repression activity of a library consisting of repression motifs from approximately 25% of the members of the largest known family of repressors. Combining sequence information with our trans-regulatory function data, we next generated a library of synthetic transcriptional repression motifs with function predicted in advance. After characterizing our synthetic library, we demonstrated not only that many of our synthetic constructs were functional as repressors but also that our advance predictions of repression strength were better than random guesses. Finally, we assessed the functionality of known transcriptional repression motifs from a wide range of eukaryotes. Our study represents the largest plant repressor motif library experimentally characterized to date, providing unique opportunities for tuning transcription in plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Expression of a mammalian RNA demethylase increases flower number and floral stem branching in Arabidopsis thaliana

Abstract RNA methylation plays a central regulatory role in plant biology and is a relatively new target for plant improvement efforts. In nearly all cases, perturbation of the RNA methylation machinery results in deleterious phenotypes. However, a recent landmark paper reported that transcriptome‐wide use of the human RNA demethylase FTO substantially increased the yield of rice and potatoes. Here, we have performed the first independent replication of those results and demonstrated broader transferability of the trait, finding increased flower and fruit count in the model species Arabidopsis thaliana . We also performed RNA‐seq of our FTO‐transgenic plants, which we analyzed in conjunction with previously published datasets to detect several previously unrecognized patterns in the functional and structural classification of the upregulated and downregulated genes. From these, we present mechanistic hypotheses to explain these surprising results with the goal of spurring more widespread interest in this promising new approach to plant engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structural and biochemical basis for regiospecificity of the flavonoid glycosyltransferase UGT95A1

Glycosylation is a predominant strategy plants use to fine-tune the properties of small molecule metabolites to affect their bioactivity, transport, and storage. It is also important in biotechnology and medicine as many glycosides are utilized in human health. Small molecule glycosylation is largely carried out by family 1 glycosyltransferases. Here, we report a structural and biochemical investigation of UGT95A1, a family 1 GT enzyme from Pilosella officinarum that exhibits a strong, unusual regiospecificity for the 3'-O position of flavonoid acceptor substrate luteolin. We obtained an apo crystal structure to help drive the analyses of a series of binding site mutants, revealing that while most residues are tolerant to mutations, key residues M145 and D464 are important for overall glycosylation activity. Interestingly, E347 is crucial for maintaining the strong preference for 3'-O glycosylation, while R462 can be mutated to increase regioselectivity. The structural determinants of regioselectivity were further confirmed in homologous enzymes. Our study also suggests that the enzyme contains large, highly dynamic, disordered regions. We showed that while most disordered regions of the protein have little to no implication in catalysis, the disordered regions conserved among investigated homologs are important to both the overall efficiency and regiospecificity of the enzyme. This report represents a comprehensive in-depth analysis of a family 1 GT enzyme with a unique substrate regiospecificity and may provide a basis for enzyme functional prediction and engineering.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Plants and methods for producing muconic acid

The present invention provides for a genetically modified plant or plant cell comprising a nucleic acid encoding one or more heterologous enzymes operatively linked a promoter, wherein one or more heterologous enzymes synthesizes muconic acid (MA).

Loque, Dominique↗
DOE OSTI2024

Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides

Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates which support the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the approximately 200 structurally diverse HMOs at scale has proved difficult. Here we produce a diversity of HMOs by leveraging the robust carbohydrate anabolism of plants. This diversity includes high-value and complex HMOs, such as lacto-N-fucopentaose I. HMOs produced in transgenic plants provided strong bifidogenic properties, indicating their ability to serve as a prebiotic supplement with potential applications in adult and infant health. Technoeconomic analyses demonstrate that producing HMOs in plants provides a path to the large-scale production of specific HMOs at lower prices than microbial production platforms. Our work demonstrates the promise in leveraging plants for the low-cost and sustainable production of HMOs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Systematic identification of transcriptional activation domains from non-transcription factor proteins in plants and yeast

Transcription factors can promote gene expression through activation domains. Whole-genome screens have systematically mapped activation domains in transcription factors but not in non-transcription factor proteins (e.g., chromatin regulators and coactivators). To fill this knowledge gap, we employed the activation domain predictor PADDLE to analyze the proteomes of Arabidopsis thaliana and Saccharomyces cerevisiae. We screened 18,000 predicted activation domains from >800 non-transcription factor genes in both species, confirming that 89% of candidate proteins contain active fragments. Our work enables the annotation of hundreds of nuclear proteins as putative coactivators, many of which have never been ascribed any function in plants. Analysis of peptide sequence compositions reveals how the distribution of key amino acids dictates activity. Finally, we validated short, "universal" activation domains with comparable performance to state-of-the-art activation domains used for genome engineering. Our approach enables the genome-wide discovery and annotation of activation domains that can function across diverse eukaryotes.

59 BASIC BIOLOGICAL 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

Harnessing Plant Sugar Metabolism for Glycoengineering

Plants possess an innate ability to generate vast amounts of sugar and produce a range of sugar-derived compounds that can be utilized for applications in industry, health, and agriculture. Nucleotide sugars lie at the unique intersection of primary and specialized metabolism, enabling the biosynthesis of numerous molecules ranging from small glycosides to complex polysaccharides. Plants are tolerant to perturbations to their balance of nucleotide sugars, allowing for the overproduction of endogenous nucleotide sugars to push flux towards a particular product without necessitating the re-engineering of upstream pathways. Pathways to produce even non-native nucleotide sugars may be introduced to synthesize entirely novel products. Heterologously expressed glycosyltransferases capable of unique sugar chemistries can further widen the synthetic repertoire of a plant, and transporters can increase the amount of nucleotide sugars available to glycosyltransferases. In this opinion piece, we examine recent successes and potential future uses of engineered nucleotide sugar biosynthetic, transport, and utilization pathways to improve the production of target compounds. Additionally, we highlight current efforts to engineer glycosyltransferases. Ultimately, the robust nature of plant sugar biochemistry renders plants a powerful chassis for the production of target glycoconjugates and glycans.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Engineering custom morpho- and chemotypes of Populus for sustainable production of biofuels, bioproducts, and biomaterials

Humans have been modifying plant traits for thousands of years, first through selection (i.e., domestication) then modern breeding, and in the last 30 years, through biotechnology. These modifications have resulted in increased yield, more efficient agronomic practices, and enhanced quality traits. Precision knowledge of gene regulation and function through high-resolution single-cell omics technologies, coupled with the ability to engineer plant genomes at the DNA sequence, chromatin accessibility, and gene expression levels, can enable engineering of complex and complementary traits at the biosystem level. Populus spp., the primary genetic model system for woody perennials, are among the fastest growing trees in temperate zones and are important for both carbon sequestration and global carbon cycling. Ample genomic and transcriptomic resources for poplar are available including emerging single-cell omics datasets. To expand use of poplar outside of valorization of woody biomass, chassis with novel morphotypes in which stem branching and tree height are modified can be fabricated thereby leading to trees with altered leaf to wood ratios. These morphotypes can then be engineered into customized chemotypes that produce high value biofuels, bioproducts, and biomaterials not only in specific organs but also in a cell-type-specific manner. For example, the recent discovery of triterpene production in poplar leaf trichomes can be exploited using cell-type specific regulatory sequences to synthesize high value terpenes such as the jet fuel precursor bisabolene specifically in the trichomes. By spatially and temporally controlling expression, not only can pools of abundant precursors be exploited but engineered molecules can be sequestered in discrete cell structures in the leaf. The structural diversity of the hemicellulose xylan is a barrier to fully utilizing lignocellulose in biomaterial production and by leveraging cell-type-specific omics data, cell wall composition can be modified in a tailored and targeted specific manner to generate poplar wood with novel chemical features that are amenable for processing or advanced manufacturing. Precision engineering poplar as a multi-purpose sustainable feedstock highlights how genome engineering can be used to re-imagine a crop species.

09 BIOMASS FUELS↗
DOE OSTI2023

Transcriptome architecture of the three main lineages of agrobacteria

ABSTRACT Agrobacteria are a diverse, polyphyletic group of prokaryotes with multipartite genomes capable of transferring DNA into the genomes of host plants, making them an essential tool in plant biotechnology. Despite their utility in plant transformation, genome-wide transcriptional regulation is not well understood across the three main lineages of agrobacteria. Transcription start sites (TSSs) are a necessary component of gene expression and regulation. In this study, we used differential RNA-seq and a TSS identification algorithm optimized on manually annotated TSS, then validated with existing TSS to identify thousands of TSS with nucleotide resolution for representatives of each lineage. We extend upon the 356 TSSs previously reported in Agrobacterium fabrum C58 by identifying 1,916 TSSs. In addition, we completed genomes and phenotyping of Rhizobium rhizogenes C16/80 and Allorhizobium vitis T60/94, identifying 2,650 and 2,432 TSSs, respectively. Parameter optimization was crucial for an accurate, high-resolution view of genome and transcriptional dynamics, highlighting the importance of algorithm optimization in genome-wide TSS identification and genomics at large. The optimized algorithm reduced the number of TSSs identified internal and antisense to the coding sequence on average by 90.5% and 91.9%, respectively. Comparison of TSS conservation between orthologs of the three lineages revealed differences in cell cycle regulation of ctrA as well as divergence of transcriptional regulation of chemotaxis-related genes when grown in conditions that simulate the plant environment. These results provide a framework to elucidate the mechanistic basis and evolution of pathology across the three main lineages of agrobacteria. IMPORTANCE Transcription start sites (TSSs) are fundamental for understanding gene expression and regulation. Agrobacteria, a group of prokaryotes with the ability to transfer DNA into the genomes of host plants, are widely used in plant biotechnology. However, the genome-wide transcriptional regulation of agrobacteria is not well understood, especially in less-studied lineages. Differential RNA-seq and an optimized algorithm enabled identification of thousands of TSSs with nucleotide resolution for representatives of each lineage. The results of this study provide a framework for elucidating the mechanistic basis and evolution of pathology across the three main lineages of agrobacteria. The optimized algorithm also highlights the importance of parameter optimization in genome-wide TSS identification and genomics at large.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The trans-regulatory landscape of gene networks in plants

The transcriptional effector domains of transcription factors play a key role in controlling gene expression; however, their functional nature is poorly understood, hampering our ability to explore this fundamental dimension of gene regulatory networks. To map the trans-regulatory landscape in a complex eukaryote, we systematically characterized the putative transcriptional effector domains of over 400 Arabidopsis thaliana transcription factors for their capacity to modulate transcription. We demonstrate that transcriptional effector activity can be integrated into gene regulatory networks capable of elucidating the functional dynamics underlying gene expression patterns. We further show how our characterized domains can enhance genome engineering efforts and reveal how plant transcriptional activators share regulatory features conserved across distantly related eukaryotes. Our results provide a framework to systematically characterize the regulatory role of transcription factors at a genome-scale in order to understand the transcriptional wiring of biological systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The pGinger Family of Expression Plasmids

Metabolic engineering and synthetic biology are predicated on the precise control of gene expression. As synthetic biology expands beyond model organisms, more tools will be required that function robustly in a wide range of bacterial hosts. The pGinger family of plasmids constitutes 43 plasmids that will enable both constitutive and inducible gene expression in a wide range of nonmodel Proteobacteria .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Carbon isotope fractionation by an ancestral rubisco suggests that biological proxies for CO 2 through geologic time should be reevaluated

The history of Earth’s carbon cycle reflects trends in atmospheric composition convolved with the evolution of photosynthesis. Fortunately, key parts of the carbon cycle have been recorded in the carbon isotope ratios of sedimentary rocks. The dominant model used to interpret this record as a proxy for ancient atmospheric CO 2 is based on carbon isotope fractionations of modern photoautotrophs, and longstanding questions remain about how their evolution might have impacted the record. Therefore, we measured both biomass (ε p ) and enzymatic (ε Rubisco ) carbon isotope fractionations of a cyanobacterial strain ( Synechococcus elongatus PCC 7942) solely expressing a putative ancestral Form 1B rubisco dating to ≫1 Ga. This strain, nicknamed ANC, grows in ambient pCO 2 and displays larger ε p values than WT, despite having a much smaller ε Rubisco (17.23 ± 0.61‰ vs. 25.18 ± 0.31‰, respectively). Surprisingly, ANC ε p exceeded ANC ε Rubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. Such models can be rectified by introducing additional isotopic fractionation associated with powered inorganic carbon uptake mechanisms present in Cyanobacteria, but this amendment hinders the ability to accurately estimate historical pCO 2 from geological data. Understanding the evolution of rubisco and the CO 2 concentrating mechanism is therefore critical for interpreting the carbon isotope record, and fluctuations in the record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO 2 .

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