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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 OSTI2019

Modular design: Implementing proven engineering principles in biotechnology

Modular design resides at the foundation of contemporary engineering, enabling rapid, efficient, and reproducible construction and maintenance of complex systems across applications. Remarkably, modularity has recently been discovered as a governing principle in natural biological systems from genes to proteins to complex networks within a cell and organism communities. The convergent knowledge of natural and engineered modular systems provides a key to drive modern biotechnology to address emergent challenges associated with health, food, energy, and the environment. Here, we first present the theory and application of modular design in traditional engineering fields. We further discuss the significance and impact of modular architectures on systems biology and biotechnology. Next, we focus on the very recent theoretical and experimental advances in modular cell engineering that seeks to enable rapid and systematic development of microbial catalysts capable of efficiently synthesizing a large space of useful chemicals. We conclude with an outlook towards theoretical and practical opportunities for a more systematic and effective application of modular engineering in biotechnology.

59 BASIC BIOLOGICAL SCIENCES↗
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 OSTI2016

Expanding the modular ester fermentative pathways for combinatorial biosynthesis of esters from volatile organic acids

ABSTRACT Volatile organic acids are byproducts of fermentative metabolism, for example, anaerobic digestion of lignocellulosic biomass or organic wastes, and are often times undesired inhibiting cell growth and reducing directed formation of the desired products. Here, we devised a general framework for upgrading these volatile organic acids to high‐value esters that can be used as flavors, fragrances, solvents, and biofuels. This framework employs the acid‐to‐ester modules, consisting of an AAT (alcohol acyltransferase) plus ACT (acyl CoA transferase) submodule and an alcohol submodule, for co‐fermentation of sugars and organic acids to acyl CoAs and alcohols to form a combinatorial library of esters. By assembling these modules with the engineered Escherichia coli modular chassis cell, we developed microbial manufacturing platforms to perform the following functions: (i) rapid in vivo screening of novel AATs for their catalytic activities; (ii) expanding combinatorial biosynthesis of unique fermentative esters; and (iii) upgrading volatile organic acids to esters using single or mixed cell cultures. To demonstrate this framework, we screened for a set of five unique and divergent AATs from multiple species, and were able to determine their novel activities as well as produce a library of 12 out of the 13 expected esters from co‐fermentation of sugars and (C2‐C6) volatile organic acids. We envision the developed framework to be valuable for in vivo characterization of a repertoire of not‐well‐characterized natural AATs, expanding the combinatorial biosynthesis of fermentative esters, and upgrading volatile organic acids to high‐value esters. Biotechnol. Bioeng. 2016;113: 1764–1776. © 2016 Wiley Periodicals, Inc.

Layton, Donovan S.↗
DOE OSTI2019

Comparison of Multi-Objective Evolutionary Algorithms to Solve the Modular Cell Design Problem for Novel Biocatalysis

A large space of chemicals with broad industrial and consumer applications could be synthesized by engineered microbial biocatalysts. However, the current strain optimization process is prohibitively laborious and costly to produce one target chemical and often requires new engineering efforts to produce new molecules. To tackle this challenge, modular cell design based on a chassis strain that can be combined with different product synthesis pathway modules has recently been proposed. This approach seeks to minimize unexpected failure and avoid task repetition, leading to a more robust and faster strain engineering process. In our previous study, we mathematically formulated the modular cell design problem based on the multi-objective optimization framework. In this study, we evaluated a library of state-of-the-art multi-objective evolutionary algorithms (MOEAs) to identify the most effective method to solve the modular cell design problem. Using the best MOEA, we found better solutions for modular cells compatible with many product synthesis modules. Furthermore, the best performing algorithm could provide better and more diverse design options that might help increase the likelihood of successful experimental implementation. We identified key parameter configurations to overcome the difficulty associated with multi-objective optimization problems with many competing design objectives. Interestingly, we found that MOEA performance with a real application problem, e.g., the modular strain design problem, does not always correlate with artificial benchmarks. Overall, MOEAs provide powerful tools to solve the modular cell design problem for novel biocatalysis.

Garcia, Sergio (ORCID:000000023998986X)↗
DOE OSTI2017

Efficient and Extensible Quasi-Explicit Modular Nonlinear Multiscale Battery Model: GH-MSMD

Complex physics and long computation time hinder the adoption of computer aided engineering models in the design of large-format battery cells and systems. A modular, efficient battery simulation model -- the multiscale multidomain (MSMD) model -- was previously introduced to aid the scale-up of Li-ion material and electrode designs to complete cell and pack designs, capturing electrochemical interplay with 3-D electronic current pathways and thermal response. Here, this paper enhances the computational efficiency of the MSMD model using a separation of time-scales principle to decompose model field variables. The decomposition provides a quasi-explicit linkage between the multiple length-scale domains and thus reduces time-consuming nested iteration when solving model equations across multiple domains. In addition to particle-, electrode- and cell-length scales treated in the previous work, the present formulation extends to bus bar- and multi-cell module-length scales. We provide example simulations for several variants of GH electrode-domain models.

30 DIRECT ENERGY CONVERSION↗
DOE OSTI2018

Biochemical Production with Purified Cell-Free Systems

Bioprocesses offer a powerful set of opportunities for current chemical process innovation. Such conversions provide opportunities to employ process intensification strategies and the right characteristics to scale down and harness the power of modular process design, but strain engineering and the use of microbial systems for industrial biochemicals production remain unpredictable and expensive. Purified cell-free technologies, in which complex enzyme reaction cascades are used to catalyze bioconversions of interest, can be designed to mitigate these limitations. Such systems allow predictive kinetic modeling and full control of reaction conductions, enabling a dramatically increased design-built-test cycle iteration rate. When sufficiently robust engineered enzymes are used, opportunities to leverage harsh reaction conditions, long-term recycling strategies, and hybridization with chemical catalysis can be used to dramatically improve the value proposition of biochemicals production. As a result, further research is required to realize the potential of these approaches and to address the current platform-limiting challenges around cofactor cost and reuse.

09 BIOMASS FUELS↗
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 OSTI2016

Photosynthetic conversion of CO2 to farnesyl diphosphate-derived phytochemicals (amorpha-4,11-diene and squalene) by engineered cyanobacteria

Background: Metabolic engineering of cyanobacteria has enabled photosynthetic conversion of CO2 to value added chemicals as bio-solar cell factories. However, the production levels of isoprenoids in engineered cyanobacteria were quite low, compared to other microbial hosts. Therefore, modular optimization of multiple gene expressions for metabolic engineering of cyanobacteria is required for the production of farnesyl diphosphate-derived isoprenoids from CO2. Results: Here, we engineered Synechococcus elongatus PCC 7942 with modular metabolic pathways consisting of the methylerythritol phosphate pathway enzymes and the amorphadiene synthase for production of amorpha-4,11-diene, resulting in significantly increased levels (23-fold) of amorpha-4,11-diene (19.8 mg/L) in the best strain relative to a parental strain. Replacing amorphadiene synthase with squalene synthase led to the synthesis of a high amount of squalene (4.98 mg/L/OD730). Overexpression of farnesyl diphosphate synthase is the most critical factor for the significant production, whereas overexpression of 1-deoxy-d-xylulose 5-phosphate reductase is detrimental to the cell growth and the production. Additionally, the cyanobacterial growth inhibition was alleviated by expressing a terpene synthase in S. elongatus PCC 7942 strain with the optimized MEP pathway only (SeHL33). Conclusions: This is the first demonstration of photosynthetic production of amorpha-4,11-diene from CO2 in cyanobacteria and production of squalene in S. elongatus PCC 7942. Our optimized modular Over MEP strain (SeHL33) with either co-expression of ADS or SQS demonstrated the highest production levels of amorpha-4,11-diene and squalene, which could expand the list of farnesyl diphosphate-derived isoprenoids from CO2 as bio-solar cell factories.

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 OSTI2018

Synthetic Biology: Molecular Tools for Engineering Organisms

Synthetic biology is a biological engineering discipline based on abstracting living systems through the lens of physical engineering concepts. In particular, synthetic biology places an emphasis on the characterisation of simple parts that can be modularly assembled into configurations that give rise to complex, higher-order behaviours. Within the past two decades, this approach has enabled the development number of new molecular biology tools for modifying living systems in order to investigate fundamental processes or imbuing functions into cells that do not exist in nature. While specific synthetic biology applications span a huge range of seemingly unrelated disciplines (from biofuel producing microbes, to malaria-resistant mosquitoes, to living medical therapies), these distinct examples derive from reuse and rearrangement of relatively limited set of cell engineering technologies. The continuing development of these core molecular biology tools for controlling gene expression, protein activity and signalling networks can promote evermore ambitious biological engineering projects.

gene expression↗
DOE OSTI2017

Engineering the Bacterial Microcompartment Domain for Molecular Scaffolding Applications

As synthetic biology advances the intricacy of engineered biological systems, the importance of spatial organization within the cellular environment must not be marginalized. Increasingly, biological engineers are investigating means to control spatial organization within the cell, mimicking strategies used by natural pathways to increase flux and reduce cross-talk. A modular platform for constructing a diverse set of defined, programmable architectures would greatly assist in improving yields from introduced metabolic pathways and increasing insulation of other heterologous systems. Here, we review recent research on the shell proteins of bacterial microcompartments and discuss their potential application as “building blocks” for a range of customized intracellular scaffolds. As a result, we summarize the state of knowledge on the self-assembly of BMC shell proteins and discuss future avenues of research that will be important to realize the potential of BMC shell proteins as predictively assembling and programmable biological materials for bioengineering.

metabolic engineering↗
DOE OSTI2019

Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates

Abstract Isoprenoids are an attractive class of metabolites for enzymatic synthesis from renewable substrates. However, metabolic engineering of microorganisms for monoterpenoid production is limited by the need for time-consuming, and often non-intuitive, combinatorial tuning of biosynthetic pathway variations to meet design criteria. Towards alleviating this limitation, the goal of this work was to build a modular, cell-free platform for construction and testing of monoterpenoid pathways, using the fragrance and flavoring molecule limonene as a model. In this platform, multiple Escherichia coli lysates, each enriched with a single overexpressed pathway enzyme, are mixed to construct the full biosynthetic pathway. First, we show the ability to synthesize limonene from six enriched lysates with mevalonate substrate, an adenosine triphosphate (ATP) source, and cofactors. Next, we extend the pathway to use glucose as a substrate, which relies on native metabolism in the extract to convert glucose to acetyl-CoA along with three additional enzymes to convert acetyl-CoA to mevalonate. We find that the native E. coli farnesyl diphosphate synthase (IspA) is active in the lysate and diverts flux from the pathway intermediate geranyl pyrophospahte to farnesyl pyrophsophate and the byproduct farnesol. By adjusting the relative levels of cofactors NAD+, ATP and CoA, the system can synthesize 0.66 mM (90.2 mg l−1) limonene over 24 h, a productivity of 3.8 mg l−1 h−1. Our results highlight the flexibility of crude lysates to sustain complex metabolism and, by activating a glucose-to-limonene pathway with 9 heterologous enzymes encompassing 20 biosynthetic steps, expands an approach of using enzyme-enriched lysates for constructing, characterizing and prototyping enzymatic pathways.

59 BASIC BIOLOGICAL SCIENCES↗