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Results for “modular cell 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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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Alpha ketoacid decarboxylases: Diversity, structures, reaction mechanisms, and applications for biomanufacturing of platform chemicals and fuels

In living cells, alpha-ketoacid decarboxylases (KDCs, EC 4.1.1.-) are a class of enzymes that convert alpha-ketoacids into aldehydes through decarboxylation. These aldehydes serve as either drop-in chemicals or precursors for the biosynthesis of alcohols, carboxylic acids, esters, and alkanes. These compounds play crucial roles in cellular metabolism and fitness and the bioeconomy, facilitating the sustainable and renewable biomanufacturing of platform chemicals and fuels. This review explores the diversity and classification of KDCs, detailing their structures, mechanisms, and functions. We highlight recent advancements in repurposing KDCs to enhance their efficiency and robustness for biomanufacturing. Additionally, we present modular KDC-dependent metabolic pathways for the microbial biosynthesis of aldehydes, alcohols, carboxylic acids, esters, and alkanes. Lastly, we discuss recent developments in the modular cell engineering technology that can potentially be applied to harness the diversity of KDC-dependent pathways for biomanufacturing platform chemicals and fuels.

09 BIOMASS FUELS↗
DOE OSTI2020

Towards renewable flavors, fragrances, and beyond

Esters demand a large space of unique molecules with broad range of applications as flavors, fragrances, pharmaceuticals, cosmetics, green solvents, and advanced biofuels. Global demand of natural esters in food, household cleaner, personal care, and perfume industries is increasing while the ester supply from natural sources has been limited. Development of novel microbial cell factories for ester production from renewable feedstocks can potentially provide an alternative and sustainable source of natural esters and hence help fulfill growing demand. In this work, we highlight recent advances in microbial production of esters and provide perspectives for improving its economic feasibility. As the field matures, microbial ester production platforms will enable renewable and sustainable production of flavors, fragrances, and open new market opportunities beyond what nature can offer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Designer membraneless organelles sequester native factors for control of cell behavior

Subcellular compartmentalization of macromolecules increases flux and prevents inhibitory interactions to control biochemical reactions. Inspired by this functionality, we sought to build designer compartments that function as hubs to regulate the flow of information through cellular control systems. Here we report a synthetic membraneless organelle platform to control endogenous cellular activities through sequestration and insulation of native proteins. We engineer and express a disordered protein scaffold to assemble micron size condensates and recruit endogenous clients via genomic tagging with high-affinity dimerization motifs. By relocalizing up to ninety percent of a targeted enzymes to synthetic condensates, we efficiently control cellular behaviors, including proliferation, division, and cytoskeletal organization. Further, we demonstrate multiple strategies for controlled cargo release from condensates to switch cells between functional states. These synthetic organelles offer a powerful and generalizable approach to modularly control cell decision-making in a variety of model systems with broad applications for cellular engineering.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Sneaking in SpyCatcher using cell penetrating peptides for in vivo imaging

Abstract In vivo imaging of protein complexes is a powerful method for understanding the underlying biological function of these key biomolecules. Though the engineering of small, high affinity nanobodies have become more prevalent, the off-rates of these tags may result in incomplete or partial labeling of proteins in live cells. The SpyCatcher003 and SpyTag split protein system allow for irreversible, covalent binding to a short target peptide unlike nanobody-affinity based probes. However, delivering these tags into a cell without disrupting its normal function is a key challenge. Cell penetrating peptides (CPPs) are short peptide sequences that facilitate the transduction of otherwise membrane-impermeable ‘cargo’ , such as proteins, into cells. Here we report on our efforts to engineer and characterize CPP-SpyCatcher003 fusions as modular imaging probes. We selected three CPPs, CUPID, Pentratin, and pVEC, to engineer fusion protein probes for superresolution microscopy, with the aim to eliminate prior permeabilization treatments that could introduce imaging artifacts. We find that fusing the CPP sequences to SpyCatcher003 resulted in dimer and multimer formation as determined by size exclusion chromatography, dynamic light scattering, and SDS resistant dimers on SDS-PAGE gels. By isolating and labeling the monomeric forms of the engineered protein, we show these constructs retained their ability to bind SpyTag and all three CPP sequences remain membrane active, as assessed by CD spectroscopy in the presence of SDS detergent. Using fluorescence and super resolution Lattice structured illumination microscopy (Lattice SIM) imaging we show that the CPPs did not enhance uptake of SpyCatcher by E. coli, however with Caulobacter crescentus cells, we show that Penetratin, and to a lesser degree CUPID, does enhance uptake. Our results demonstrate the ability of the CPP-SpyCatcher003 to label targets within living cells, providing the groundwork for using split protein systems for targeted in vivo imaging.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Controlling selectivity of modular microbial biosynthesis of butyryl-CoA-derived designer esters

Short-chain esters have broad utility as flavors, fragrances, solvents, and biofuels. Controlling selectivity of ester microbial biosynthesis has been an outstanding metabolic engineering problem. In this study, we enabled the de novo fermentative microbial biosynthesis of butyryl-CoA-derived designer esters (e.g., butyl acetate, ethyl butyrate, butyl butyrate) in Escherichia coli with controllable selectivity. Using the modular design principles, we generated the butyryl-CoA-derived ester pathways as exchangeable production modules compatible with an engineered chassis cell for anaerobic production of designer esters. We designed these modules derived from an acyl-CoA submodule (e.g., acetyl-CoA, butyryl-CoA), an alcohol submodule (e.g., ethanol, butanol), a cofactor regeneration submodule (e.g., NADH), and an alcohol acetyltransferase (AAT) submodule (e.g., ATF1, SAAT) for rapid module construction and optimization by manipulating replication (e.g., plasmid copy number), transcription (e.g., promoters), translation (e.g., codon optimization), pathway enzymes, and pathway induction conditions. To further enhance production of designer esters with high selectivity, we systematically screened various strategies of protein solubilization using protein fusion tags and chaperones to improve the soluble expression of multiple pathway enzymes. Finally, our engineered ester-producing strains could achieve 19-fold increase in butyl acetate production (0.64 g/L, 96% selectivity), 6-fold increase in ethyl butyrate production (0.41 g/L, 86% selectivity), and 13-fold increase in butyl butyrate production (0.45 g/L, 54% selectivity) as compared to the initial strains. Altogether, this study presented a generalizable framework to engineer modular microbial platforms for anaerobic production of butyryl-CoA-derived designer esters from renewable feedstocks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Development of a Genome-Scale Metabolic Model of Clostridium thermocellum and Its Applications for Integration of Multi-Omics Datasets and Computational Strain Design

Solving environmental and social challenges such as climate change requires a shift from our current non-renewable manufacturing model to a sustainable bioeconomy. To lower carbon emissions in the production of fuels and chemicals, plant biomass feedstocks can replace petroleum using microorganisms as biocatalysts. The anaerobic thermophile Clostridium thermocellum is a promising bacterium for bioconversion due to its capability to efficiently degrade lignocellulosic biomass. However, the complex metabolism of C. thermocellum is not fully understood, hindering metabolic engineering to achieve high titers, rates, and yields of targeted molecules. In this study, we developed an updated genome-scale metabolic model of C. thermocellum that accounts for recent metabolic findings, has improved prediction accuracy, and is standard-conformant to ensure easy reproducibility. We illustrated two applications of the developed model. We first formulated a multi-omics integration protocol and used it to understand redox metabolism and potential bottlenecks in biofuel (e.g., ethanol) production in C. thermocellum. Second, we used the metabolic model to design modular cells for efficient production of alcohols and esters with broad applications as flavors, fragrances, solvents, and fuels. The proposed designs not only feature intuitive push-and-pull metabolic engineering strategies, but also present novel manipulations around important central metabolic branch-points. We anticipate the developed genome-scale metabolic model will provide a useful tool for system analysis of C. thermocellum metabolism to fundamentally understand its physiology and guide metabolic engineering strategies to rapidly generate modular production strains for effective biosynthesis of biofuels and biochemicals from lignocellulosic biomass.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

In vitro prototyping of limonene biosynthesis using cell-free protein synthesis

Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-build-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 h from 0.2 to 4.5 mM (23–610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. Finally, we anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Modular cell-free expression plasmids to accelerate biological design in cells

Industrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms—organisms that are easy to use like Escherichia coli—often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have desirable metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes, and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has matured as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institute’s DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression of functional enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for Clostridium autoethanogenum expression with efficiencies ranging from 68% to 90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

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↗
DOE OSTI2024

Light-inducible protein degradation in E. coli with the LOVdeg tag

Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light-controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVdeg, a tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVdeg by using it to tag a range of proteins, including the LacI repressor, CRISPRa activator, and the AcrB efflux pump. Additionally, we demonstrate the utility of pairing the LOVdeg tag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVdeg system. Finally, we use the LOVdeg tag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVdeg tag system and introduce a powerful new tool for bacterial optogenetics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Light-inducible protein degradation in E. coli with the LOVdeg tag

Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light-controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVdeg, a tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVdeg by using it to tag a range of proteins, including the LacI repressor, CRISPRa activator, and the AcrB efflux pump. Additionally, we demonstrate the utility of pairing the LOVdeg tag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVdeg system. Finally, we use the LOVdeg tag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVdeg tag system and introduce a powerful new tool for bacterial optogenetics.

Tague, Nathan (ORCID:0000000281146700)↗
DOE OSTI2019

Advanced Bottom‐Up Engineering of Living Architectures

Abstract Bottom‐up tissue engineering is a promising approach for designing modular biomimetic structures that aim to recapitulate the intricate hierarchy and biofunctionality of native human tissues. In recent years, this field has seen exciting progress driven by an increasing knowledge of biological systems and their rational deconstruction into key core components. Relevant advances in the bottom‐up assembly of unitary living blocks toward the creation of higher order bioarchitectures based on multicellular‐rich structures or multicomponent cell–biomaterial synergies are described. An up‐to‐date critical overview of long‐term existing and rapidly emerging technologies for integrative bottom‐up tissue engineering is provided, including discussion of their practical challenges and required advances. It is envisioned that a combination of cell–biomaterial constructs with bioadaptable features and biospecific 3D designs will contribute to the development of more robust and functional humanized tissues for therapies and disease models, as well as tools for fundamental biological studies.

Gaspar, Vítor M.↗
DOE OSTI2023

A modular vaccine platform enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. However, antigen display on OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. Here, we describe a universal approach for avidin-based vaccine antigen crosslinking (AvidVax) whereby biotinylated antigens are linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen-binding protein (SNAP) comprised of an outer membrane scaffold protein fused to a biotin-binding protein. We show that SNAP-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations are injected in mice, strong antigen-specific antibody responses are observed that depend on the physical coupling between the antigen and SNAP-OMV delivery vehicle. Overall, these results demonstrate AvidVax as a modular platform that enables rapid and simplified assembly of antigen-studded OMVs for application as vaccines against pathogenic threats.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Modular adjuvant-free pan-HLA-DR-immunotargeting subunit vaccine against SARS-CoV-2 elicits broad sarbecovirus-neutralizing antibody responses

Subunit vaccines typically require co-administration with an adjuvant to elicit protective immunity, adding development hurdles that can impede rapid pandemic responses. To circumvent the need for adjuvant in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit vaccine, we engineer a thermostable immunotargeting vaccine (ITV) that leverages the pan-HLA-DR monoclonal antibody 44H10 to deliver the viral spike protein receptor-binding domain (RBD) to antigen-presenting cells. X-ray crystallography shows that 44H10 binds to a conserved epitope on HLA-DR, providing the basis for its broad HLA-DR reactivity. Adjuvant-free ITV immunization in rabbits and ferrets induces robust anti-RBD antibody responses that neutralize SARS-CoV-2 variants of concern and protect recipients from SARS-CoV-2 challenge. We demonstrate that the modular nature of the ITV scaffold with respect to helper T cell epitopes and diverse RBD antigens facilitates broad sarbecovirus neutralization. Our findings support anti-HLA-DR immunotargeting as an effective means to induce strong antibody responses to subunit antigens without requiring an adjuvant.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Engineering phase-frustration-induced flat bands in an aza-triangulene covalent kagome lattice

π-Conjugated covalent organic frameworks provide a versatile molecular scaffold for the realization of designer quantum nanomaterials. Strong electron–electron correlation within these artificial lattices can give rise to exotic phases of matter. Their experimental realization, however, requires precise control over orbital symmetry, charge localization and band dispersion, all arising from the effective hybridization between molecular linkers and nodes. Here we present a modular strategy for constructing diatomic kagome lattices from aza-[3]triangulene nodes, in which a D 3h -symmetric ground state is stabilized through resonance contributions from a cumulenic linker. First-principles density functional theory and scanning tunnelling spectroscopy reveal that the hybridization of a sixfold-degenerate set of edge-localized Wannier functions in the unit cell gives rise to orbital-phase-frustration-induced non-trivial flat bands. These results establish a general design principle for engineering orbital interactions in organic lattices and open a pathway towards programmable covalent-organic-framework-based quantum materials with correlated electronic ground states.

conjugated polymers↗
DOE OSTI2024

A frugal CRISPR kit for equitable and accessible education in gene editing and synthetic biology

Equitable and accessible education in life sciences, bioengineering, and synthetic biology is crucial for training the next generation of scientists, fostering transparency in public decision-making, and ensuring biotechnology can benefit a wide-ranging population. As a groundbreaking technology for genome engineering, CRISPR has transformed research and therapeutics. However, hands-on exposure to this technology in educational settings remains limited due to the extensive resources required for CRISPR experiments. Here, we develop CRISPRkit, an affordable kit designed for gene editing and regulation in high school education. CRISPRkit eliminates the need for specialized equipment, prioritizes biosafety, and utilizes cost-effective reagents. By integrating CRISPRi gene regulation, colorful chromoproteins, cell-free transcription-translation systems, smartphone-based quantification, and an in-house automated algorithm (CRISPectra), our kit offers an inexpensive (~$2) and user-friendly approach to performing and analyzing CRISPR experiments, without the need for a traditional laboratory setup. Experiments conducted by high school students in classroom settings highlight the kit’s utility for reliable CRISPRkit experiments. Furthermore, CRISPRkit provides a modular and expandable platform for genome engineering, and we demonstrate its applications for controlling fluorescent proteins and metabolic pathways such as melanin production. We envision CRISPRkit will facilitate biotechnology education for communities of diverse socioeconomic and geographic backgrounds.

59 BASIC BIOLOGICAL SCIENCES↗