Engineering Plant Synthetic Pathways for the Biosynthesis of Novel Antifungals
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59 BASIC BIOLOGICAL SCIENCES↗
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Results for “Genetic engineering”
Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.
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Quantum-inspired genetic algorithm for designing planar multilayer photonic structure
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36 MATERIALS SCIENCE↗
BRCore: An R Package Implementing Flexible Selection of Core Taxa Using Contribution to Bray-Curtis Dissimilarity and Neutral Model Fitting
BRCore provides a unified framework for identification and ecological interpretation of core microbiomes across time and space, enhancing robustness and reproducibility in microbiome data analysis. Is supplement to Software: https://github.com/germs-lab/BRCore/tree/v2.0.7 (URL)
Software↗
High-Throughput Microfluidic Electroporation (HTME): A Scalable, 384-Well Platform for Multiplexed Cell Engineering
Electroporation-mediated gene delivery is a cornerstone of synthetic biology, offering several advantages over other methods: higher efficiencies, broader applicability, and simpler sample preparation. Yet, electroporation protocols are often challenging to integrate into highly multiplexed workflows, owing to limitations in their scalability and tunability. These challenges ultimately increase the time and cost per transformation. As a result, rapidly screening genetic libraries, exploring combinatorial designs, or optimizing electroporation parameters requires extensive iterations, consuming large quantities of expensive custom-made DNA and cell lines or primary cells. To address these limitations, we have developed a High-Throughput Microfluidic Electroporation (HTME) platform that includes a 384-well electroporation plate (E-Plate) and control electronics capable of rapidly electroporating all wells in under a minute with individual control of each well. Fabricated using scalable and cost-effective printed-circuit-board (PCB) technology, the E-Plate significantly reduces consumable costs and reagent consumption by operating on nano to microliter volumes. Furthermore, individually addressable wells facilitate rapid exploration of large sets of experimental conditions to optimize electroporation for different cell types and plasmid concentrations/types. Use of the standard 384-well footprint makes the platform easily integrable into automated workflows, thereby enabling end-to-end automation. We demonstrate transformation of E. coli with pUC19 to validate the HTME's core functionality, achieving at least a single colony forming unit in more than 99% of wells and confirming the platform's ability to rapidly perform hundreds of electroporations with customizable conditions. This work highlights the HTME's potential to significantly accelerate synthetic biology Design-Build-Test-Learn (DBTL) cycles by mitigating the transformation/transfection bottleneck.
Gaillard, William R↗
A de novo matrix for macroscopic living materials from bacteria
Engineered living materials (ELMs) embed living cells in a biopolymer matrix to create materials with tailored functions. While bottom-up assembly of macroscopic ELMs with a de novo matrix would offer the greatest control over material properties, we lack the ability to genetically encode a protein matrix that leads to collective self-organization. Here we report growth of ELMs from Caulobacter crescentus cells that display and secrete a self-interacting protein. This protein formed a de novo matrix and assembled cells into centimeter-scale ELMs. Discovery of design and assembly principles allowed us to tune the composition, mechanical properties, and catalytic function of these ELMs. This work provides genetic tools, design and assembly rules, and a platform for growing ELMs with control over both matrix and cellular structure and function.
59 BASIC BIOLOGICAL SCIENCES↗
A microbial supply chain for production of the anti-cancer drug vinblastine
Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine. As MIAs are difficult to chemically synthesize, the world’s supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.
60 APPLIED LIFE SCIENCES↗
Functional characterization of diverse type I-F CRISPR-associated transposons
CRISPR-Cas systems generally provide adaptive immunity in prokaryotes through RNA-guided degradation of foreign genetic elements like bacteriophages and plasmids. Recently, however, transposon-encoded and nuclease-deficient CRISPR-Cas systems were characterized and shown to be co-opted by Tn7-like transposons for CRISPR RNA-guided DNA transposition. As a genome engineering tool, these CRISPR-Cas systems and their associated transposon proteins can be deployed for programmable, site-specific integration of sizable cargo DNA, circumventing the need for DNA cleavage and homology-directed repair involving endogenous repair machinery. Here, we selected a diverse set of type I-F3 CRISPR-associated transposon systems derived from Gammaproteobacteria, predicted all components essential for transposition activity, and deployed them for functionality testing within Escherichia coli. Our results demonstrate that these systems possess a significant range of integration efficiencies with regards to temperature, transposon size, and flexible PAM requirements. Additionally, our findings support the categorization of these systems into functional compatibility groups for efficient and orthogonal RNA-guided DNA integration. This work expands the CRISPR-based toolbox with new CRISPR RNA-guided DNA integrases that can be applied to complex and extensive genome engineering efforts.
59 BASIC BIOLOGICAL SCIENCES↗
CRISPR-COPIES: an in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration
Abstract The CRISPR/Cas system has emerged as a powerful tool for genome editing in metabolic engineering and human gene therapy. However, locating the optimal site on the chromosome to integrate heterologous genes using the CRISPR/Cas system remains an open question. Selecting a suitable site for gene integration involves considering multiple complex criteria, including factors related to CRISPR/Cas-mediated integration, genetic stability, and gene expression. Consequently, identifying such sites on specific or different chromosomal locations typically requires extensive characterization efforts. To address these challenges, we have developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. This tool leverages ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search, and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. As a proof of concept, we utilized CRISPR-COPIES to characterize neutral integration sites in three diverse species: Saccharomyces cerevisiae, Cupriavidus necator, and HEK293T cells. In addition, we developed a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate that CRISPR-COPIES will serve as a valuable tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, enable rapid strain construction to produce valuable biochemicals, and support human gene and cell therapy applications.
59 BASIC BIOLOGICAL SCIENCES↗
A High-efficacy CRISPRi System for Gene Function Discovery in Zymomonas mobilis
Zymomonas mobilis is a promising biofuel producer due to its high alcohol tolerance and streamlined metabolism that efficiently converts sugar to ethanol. Z. mobilis genes are poorly characterized relative to those of model bacteria, hampering our ability to rationally engineer the genome with pathways capable of converting sugars from plant hydrolysates into valuable biofuels and bioproducts. Many of the unique properties that make Z. mobilis an attractive biofuel producer are controlled by essential genes; however, these genes cannot be manipulated using traditional genetic approaches (e.g., deletion or transposon insertion) because they are required for viability. CRISPR interference (CRISPRi) is a programmable gene knockdown system that can precisely control the timing and extent of gene repression, thus enabling targeting of essential genes. Here, we establish a stable, high-efficacy CRISPRi system in Z. mobilis that is capable of perturbing all genes—including essential genes. We show that Z. mobilis CRISPRi causes either strong knockdowns (>100-fold) using single guide RNA (sgRNA) spacers that perfectly match target genes or partial knockdowns using spacers with mismatches. We demonstrate the efficacy of Z. mobilis CRISPRi by targeting essential genes that are universally conserved in bacteria, are key to the efficient metabolism of Z. mobilis, or underlie alcohol tolerance. Our Z. mobilis CRISPRi system will enable comprehensive gene function discovery, opening a path to rational design of biofuel production strains with improved yields.
lignocellulosic hydrolysate↗
Multi-Objective Optimization of Uranium Target Assembly–3: A Comparison of Genetic and Traditional Methods
Commonly produced as a byproduct of uranium fission, 99 Mo is a key medical isotope that is in high demand in the United States. An international goal is to switch from medical isotope production technologies that require highly enriched uranium to medical isotope production technologies that require only low-enriched uranium. Niowave Inc. is contributing to this goal by developing an accelerator-driven subcritical assembly called the Uranium Target Assembly (UTA). This work compares the performance of Dakota’s Multi-Objective Genetic Algorithm (MOGA) against traditional sensitivity analysis in the neutronic optimization of the UTA-3 system. The design objectives are k-eigenvalue (k eff ) and natural uranium fission power, which are directly correlated with the amount of 99 Mo produced. Dakota:MOGA did not perform as well as human engineering ingenuity in optimization studies with high numbers of input parameters, such as fuel rod type selection and fuel rod placement. However, Dakota:MOGA did outperform traditional sensitivity analysis in optimization studies with fewer than 20 parameters and revealed the degree to which each parameter influences the optimal design space for k eff and natural uranium fission power (to a lesser extent). As the design model became more complex in the final stage of design, the computational resources required to calculate the design objective values in the Monte Carlo N-Particle transport code from selected input parameter combinations limited Dakota:MOGA’s performance, and, unfortunately, human intervention was required to discern the optimal design space. In conclusion, future work will attempt to reduce computational resource constraints by incorporating areduced-order neutronics model into the optimization cycle.
Accelerator-driven systems↗
Metabolic Pathway Engineering
Modern microbial and enzyme engineering and their advancement are increasingly dependent on the marriage of a wide range of sophisticated technologies. For students entering the field of biotechnology, the outlook is indeed daunting. Expertise at levels beyond that of simple familiarity will be needed to conduct competitive research. It goes without saying that to be competitive, all new researchers in this field will need basic preparation in molecular biology, biochemistry, and genetics. Further, experience working with concepts and experimental tools in enzyme biochemistry and kinetics, gene editing, computational metabolic pathway modeling, experimental pathway flux analysis, and computational clustering tools to process complex data sets will be vital for success. We speculate that most biotechnology researchers in early career at this time will build teams of collaborators to address these disparate science fields rather than attempt to become experts in one lab.
BASIC BIOLOGICAL SCIENCES,BIOMASS FUELS↗
Longitudinal Multi-omics Reveal Phase-Dependent Viral Adaptive Strategies and Functional Potential During Formation of Algal-bacterial Granular Sludge
Virus-host interactions within microbial aggregates critically influence microbiome function and stability, yet how physicochemical stresses shape the interactive dynamics remains largely unexplored. Here, we investigated virus–host dynamics during the transition of algal-bacterial granular sludge (ABGS) from activated sludge under continuous hydraulic shear using integrated metagenomics and metatranscriptomics. Hydraulic stress initially reduced host a-diversity, which coincided with a marked increase in viral lysogenicity. During this host diversity bottleneck, viral microdiversity increased, and genes related to virion structure and DNA packaging were under positive selection (pN/pS >1). As host diversity recovered, viral microdiversity declined, while viral anti-defense systems (ADS) significantly increased in abundance. Lagged correlation analysis revealed a significant positive correlation between viral ADS and host defense systems (DS), suggesting an evolutionary arms race. Furthermore, active lysogenic infections were accompanied by enrichment of DS and auxiliary viral genes (AVGs) involved in genetic information processing and amino acid metabolism, potentially enhancing host fitness. Overall, our study unveils a phase-dependent co-evolutionary interplay between viruses and hosts during ABGS formation, providing insights into the development and maintenance of microbial structural and functional resilience in engineered ecosystems.
Qi, Huiyuan↗
Nano on micro: tuning microbial metabolisms by nano-based artificial mediators to enhance and expand production of biochemicals
Advances in synthetic biology and metabolic engineering across the past few decades have enabled the successful production of many novel chemicals. However, bioproduction of such chemicals is often limited by low yield and titer due to disrupted metabolic homeostasis. Finely tuning cellular metabolism to restore robust metabolic functions entails various genetic modifications, which is often not practical. Alternatively, artificial mediators capable of tailoring microbial metabolisms open a new avenue for restoring physiological functions. In this context, nanoparticle-based artificial mediators have been pursued to tune cellular metabolisms. They can not only enhance production of molecules from endogenous metabolism, but also expand bioproducts spectrum. Here, we reviewed recent advances toward the employment of nano-based artificial mediators for the tuning of cellular metabolism, with a focus on their positive effects on electron transfer and pathway flux. Perspectives for potential applications of artificial mediators for mediating microbial metabolisms in the future were also provided.
59 BASIC BIOLOGICAL SCIENCES↗
Engineered plants for the production of the antioxidants arbutin and gallate
The shikimate pathway is a crucial metabolic route for the biosynthesis of numerous valuable chemicals. In this study, we engineered the shikimate pathway in plants via expression of microbial enzymes to produce the two important antioxidants gallate and arbutin. The engineered pathways utilize the aromatics protocatechuate and 4-hydroxybenzoate as metabolic intermediates. Through transient expression in Nicotiana benthamiana leaves, we first identified biosynthetic routes for the production of gallate from either chorismate or 3-dehydroshikimate. Gallate production was then achieved in Arabidopsis using a genetic background that overproduces protocatechuate and via expression of a mutated version of the 4-hydroxybenzoate hydroxylase PobA from Pseudomonas sp. Arbutin production was obtained in Arabidopsis using a genetic background that overproduces 4-hydroxybenzoate and via expression of the monooxygenase MNX1 from Candida parapsilosis. The best Arabidopsis transgenic lines accumulated gallate and arbutin in the range of 0.25 and 0.93 dry weight % (dwt%), respectively. Using sorghum for large-scale in planta production, the titers of gallate and arbutin produced from the intermediate 4-hydroxybenzoate reached 0.58 dwt% and 0.50 dwt%, respectively, in mature transgenic plants, surpassing levels typically observed in plants that naturally produce these compounds. Gallate and arbutin were readily extracted from plant tissues using methanol solvent. Analysis of extractive-free biomass showed only trace amounts of gallate and its precursors 4-hydroxybenzoate and protocatechuate crosslinked to cell walls, suggesting that they mainly occur as soluble conjugated forms stored in the vacuole. This study presents alternative synthesis routes using plant hosts for the eco-friendly production of gallate and arbutin.
Antioxidant↗
Cryo-EM Visualization of Intermolecular π-Electron Interactions within π-Conjugated Peptidic Supramolecular Polymers
The self-assembly of “π-peptides” – molecules with π-electron cores substituted with two or more oligopeptide chains – brings organic electronic function into biologically relevant nanomaterials. π-Peptides assemble into fibrillar nanomaterials as driven by enthalpic peptide-based hydrogen bonding networks and pi-core-based quadrupolar interactions. A large body of spectroscopic, morphological and computational studies informs on the nature of the self-assembly process and the resulting nanostructures, but detailed structural information has remained elusive. Here, inspired by the recent use of cryogenic electron microscopy (cryo-EM) to provide high-resolution structures for synthetic peptide nanomaterials, we present here the use of cryo-EM to offer ca. 3 Å resolution of π-peptide nanomaterial assemblies, visualizing for the first time the nature of the intermolecular π-core electronic interactions responsible for energy transport through these supramolecular materials.
Group theory↗
Quantifying the local adaptive landscape of a nascent bacterial community
Abstract The fitness effects of all possible mutations available to an organism largely shape the dynamics of evolutionary adaptation. Yet, whether and how this adaptive landscape changes over evolutionary times, especially upon ecological diversification and changes in community composition, remains poorly understood. We sought to fill this gap by analyzing a stable community of two closely related ecotypes (“L” and “S”) shortly after they emerged within the E. coli Long-Term Evolution Experiment (LTEE). We engineered genome-wide barcoded transposon libraries to measure the invasion fitness effects of all possible gene knockouts in the coexisting strains as well as their ancestor, for many different, ecologically relevant conditions. We find consistent statistical patterns of fitness effect variation across both genetic background and community composition, despite the idiosyncratic behavior of individual knockouts. Additionally, fitness effects are correlated with evolutionary outcomes for a number of conditions, possibly revealing shifting patterns of adaptation. Together, our results reveal how ecological and epistatic effects combine to shape the adaptive landscape in a nascent ecological community.
59 BASIC BIOLOGICAL SCIENCES↗
Expanding the eukaryotic genetic code with a biosynthesized 21st amino acid
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Wu, Kuan-Lin↗
An extensible vector toolkit and parts library for advanced engineering of plant genomes
Abstract Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T‐DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site‐specific recombinases. Finally, we use a series of dual‐luciferase assays to measure contributions to expression from promoters, terminators, and from cross‐cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.
42 ENGINEERING↗