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Results for “engineered E. coli”

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 OSTI2021

Dynamic control over feedback regulatory mechanisms improves NADPH flux and xylitol biosynthesis in engineered E. coli

We report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase. We utilize 2-stage dynamic metabolic control to compare two approaches to optimize xylitol biosynthesis, a stoichiometric approach, wherein competitive fluxes are decreased, and a regulatory approach wherein the levels of key regulatory metabolites are reduced. The stoichiometric and regulatory approaches lead to a 20-fold and 90-fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and an NADPH generation pathway, consisting of pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. Furthermore, these strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over the regulation of the membrane bound transhydrogenase as well as NADPH production through pyruvate ferredoxin oxidoreductase to broadly enable improved NADPH dependent bioconversions or production via NADPH dependent metabolic pathways.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Rare Earth Element Biomining from the Great Salt Lake Brine Using Engineered E. Coli

This data describes rare earth element adsorption onto E. coli cells engineered to express a lanthanide binding tag (LBT). We used a Great Salt Lake synthetic solution as the background matrix with Tb added to 1-10,000 ppb, concentrations much lower than the competing ions present. Our results showed that Tb binds to LBT, even in the presence of high concentrations of competing metals. We also tested REE adsorption at elevated temperatures (up to 100 degrees Celsius), and observed that Tb adsorption increases with temperature of to 70 degrees Celsius, and then remains constant until 100 degrees Celsius. Data analyses were performed using an ICP-MS at UCSC.

15 GEOTHERMAL ENERGY↗
DOE OSTI2018

Toward engineering E. coli with an autoregulatory system for lignin valorization

Significance Lignin valorization is critical for economic viability of future biorefineries but is hindered due to the challenges of engineered bio-chassis such as the slow kinetics of substrate uptake, aromatics toxicity, and cost. Here, an autoregulatory system involving a vanillin autoinducible promoter is demonstrated with an aromatics transporter in Escherichia coli that is induced by lignin-derived aromatics and simultaneously converted to value-added platform chemical with diverse applications. In addition to alleviating toxicity, the engineered E. coli strain eliminates the need for an external inducer such as isopropyl β- d -1-thiogalactopyranoside during fermentation, thereby significantly reducing the process cost. This study demonstrates an autoregulatory system for aromatics bioconversion and serves as a platform for future strain development for lignin valorization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Autonomous control of metabolic state by a quorum sensing (QS)-mediated regulator for bisabolene production in engineered E. coli

Inducible gene expression systems are widely used in microbial host strains for protein and commodity chemical production because of their extensive characterization and ease of use. However, some of these systems have disadvantages such as leaky expression, lack of dynamic control, and the prohibitively high costs of inducers associated with large-scale production. Quorum sensing (QS) systems in bacteria control gene expression in response to population density, and the LuxI/R system from Vibrio fischeri is a well-studied example. A QS system could be ideal for biofuel production strains as it is self-regulated and does not require the addition of inducer compounds, which reduce operational costs for inducer. In this study, a QS system was developed for inducer-free production of the biofuel compound bisabolene from engineered E. coli. Seven variants of the Sensor plasmid, which carry the luxI-luxR genes, and four variants of the Response plasmid, which carry bisabolene producing pathway genes under the control of the P luxI promoter, were designed for optimization of bisabolene production. Furthermore, a chromosome-integrated QS strain was engineered with the best combination of Sensor and Response plasmid and produced bisabolene at a titer of 1.1 g/L without addition of external inducers. This is a 44% improvement from our previous inducible system. The QS strain also displayed higher homogeneity in gene expression and isoprenoid production compared to an inducible-system strain.

09 BIOMASS FUELS↗
DOE OSTI2018

Improving formaldehyde consumption drives methanol assimilation in engineered E. coli

Abstract Due to volatile sugar prices, the food vs fuel debate, and recent increases in the supply of natural gas, methanol has emerged as a promising feedstock for the bio-based economy. However, attempts to engineer Escherichia coli to metabolize methanol have achieved limited success. Here, we provide a rigorous systematic analysis of several potential pathway bottlenecks. We show that regeneration of ribulose 5-phosphate in E. coli is insufficient to sustain methanol assimilation, and overcome this by activating the sedoheptulose bisphosphatase variant of the ribulose monophosphate pathway. By leveraging the kinetic isotope effect associated with deuterated methanol as a chemical probe, we further demonstrate that under these conditions overall pathway flux is kinetically limited by methanol dehydrogenase. Finally, we identify NADH as a potent kinetic inhibitor of this enzyme. These results provide direction for future engineering strategies to improve methanol utilization, and underscore the value of chemical biology methodologies in metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Two-stage dynamic deregulation of metabolism improves process robustness & scalability in engineered E. coli.

Here, we report that two-stage dynamic control improves bioprocess robustness as a result of the dynamic deregulation of central metabolism. Dynamic control is implemented during stationary phase using combinations of CRISPR interference and controlled proteolysis to reduce levels of central metabolic enzymes. Reducing the levels of key enzymes alters metabolite pools resulting in deregulation of the metabolic network. Deregulated networks are less sensitive to environmental conditions improving process robustness. Process robustness in turn leads to predictable scalability, minimizing the need for traditional process optimization. We validate process robustness and scalability of strains and bioprocesses synthesizing the important industrial chemicals alanine, citramalate and xylitol. Predictive high throughput approaches that translate to larger scales are critical for metabolic engineering programs to truly take advantage of the rapidly increasing throughput and decreasing costs of synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Deciphering flux adjustments of engineered E. coli cells during fermentation with changing growth conditions

Microbial fermentation conditions are dynamic, due to transcriptional induction, nutrient consumption, or changes to incubation conditions. In this paper, 13 C-metabolic flux analysis was used to characterize two violacein-producing E. coli strains with vastly different productivities, and to profile their metabolic adjustments resulting from external perturbations during fermentation. The two strains were first grown at 37 °C in stage 1, and then the temperature was transitioned to 20 °C in stage 2 for the optimal expression of the violacein synthesis pathway. After induction, violacein production was minimal in stage 3, but accelerated in stage 4 (early production phase) and 5 (late production phase) in the high producing strain, reaching a final concentration of 1.5 mmol/L. On the contrary, ~0.02 mmol/L of violacein was obtained from the low producing strain. To have a snapshot of the temporal metabolic changes in each stage, we performed 13 C-MFA via isotopomer analysis of fast-turnover free metabolites. The results indicate strikingly stable flux ratios in the central metabolism throughout the early growth stages. In the late stages, however, the high producer rewired its flux distribution significantly, which featured an upregulated pentose phosphate pathway and TCA cycle, reflux from acetate utilization, negligible anabolic fluxes, and elevated maintenance loss, to compensate for nutrient depletion and drainage of some building blocks due to violacein overproduction. The low producer with stronger promoters shifted its relative fluxes in stage 5 by enhancing the flux through the TCA cycle and acetate overflow, while exhibiting a reduced biomass growth and a minimal flux towards violacein synthesis. Finally, interestingly, the addition of the violacein precursor (tryptophan) in the medium inhibited high producer but enhanced low producer's productivity, leading to hypotheses of unknown pathway regulations (such as metabolite channeling).

13C-MFA↗
DOE OSTI2016

CymA and Exogenous Flavins Improve Extracellular Electron Transfer and Couple It to Cell Growth in Mtr-Expressing Escherichia coli

Introducing extracellular electron transfer pathways into heterologous organisms offers the opportunity to explore fundamental biogeochemical processes and to biologically alter redox states of exogenous metals for various applications. While expression of the MtrCAB electron nanoconduit from Shewanella oneidensis MR-1 permits extracellular electron transfer in Escherichia coli, the low electron flux and absence of growth in these cells limits their practicality for such applications. In this paper, we investigate how the rate of electron transfer to extracellular Fe(III) and cell survival in engineered E. coli are affected by mimicking different features of the S. oneidensis pathway: the number of electron nanoconduits, the link between the quinol pool and MtrA, and the presence of flavin-dependent electron transfer. While increasing the number of pathways does not significantly improve the extracellular electron transfer rate or cell survival, using the native inner membrane component, CymA, significantly improves the reduction rate of extracellular acceptors and increases cell viability. Strikingly, introducing both CymA and riboflavin to Mtr-expressing E. coli also allowed these cells to couple metal reduction to growth, which is the first time an increase in biomass of an engineered E. coli has been observed under Fe 2 O 3 (s) reducing conditions. Overall and finally, this work provides engineered E. coli strains for modulating extracellular metal reduction and elucidates critical factors for engineering extracellular electron transfer in heterologous organisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Enhanced Silver Nanoparticle Synthesis by Escherichia Coli Transformed with Candida Albicans Metallothionein Gene

In this study, the metallothionein gene of Candida albicans (C. albicans) was assembled by polymerase chain reaction (PCR), inserted into pUC19 vector, and further transformed into Escherichia coli (E. coli) DH5α cells. The capacity of these recombinant E. coli DH5α cells to synthesize silver nanoparticles was examined. Our results demonstrated that the expression of C. albicans metallothionein in E. coli promoted the bacterial tolerance to metal ions and increased yield of silver nanoparticle synthesis. The compositional and morphological analysis of the silver nanoparticles revealed that silver nanoparticles synthesized by the engineered E. coli cells are around 20 nm in size, and spherical in shape. Importantly, the silver nanoparticles produced by the engineered cells were more homogeneous in shape and size than those produced by bacteria lack of the C. albicans metallothionein. Our study provided preliminary information for further development of the engineered E. coli as a platform for large-scale production of uniform nanoparticles for various applications in nanotechnology.

36 MATERIALS SCIENCE↗
DOE OSTI2017

Recovery of Rare Earth Elements from Low-Grade Feedstock Leachates Using Engineered Bacteria

The use of biomass for sorption of rare earth elements (REEs) for bioremediation and biomining applications has been the subject of many recent investigations. However, REE adsorption by bioengineered systems has been scarcely documented, and rarely tested with complex natural feedstocks. In this study, we engineered E. coli cells for enhanced cell surface-mediated extraction of REEs. Using Lpp-OmpA as an anchor, up to 16 copies of a Lanthanide binding tag (LBT) per OmpA protein were displayed on the E. coli cell surface, resulting in a 2-fold increase in Tb adsorption capacity compared to control cells. To test the efficacy of engineered E. coli cells to extract REEs from complex source materials, biosorption was performed with leachates from two mine tailings and two rare earth deposits. Although control E. coli cells exhibited preferential adsorption of REEs over most metals, functionalization of the cell surface with LBT yielded several notable advantages. First, the efficiency of REE adsorption from all tested leachates was enhanced with distribution coefficients for individual REEs increased by 2-10 fold. Second, although, Cu, Al, Ga and Pb were found to be the best competitors for REE binding to the E. coli cell surface, LBT-display enhanced the cell surface affinity for REEs over all non-REEs except Cu. Third, LBT-display systematically enhanced the affinity of the cell surface for REEs as a function of decreasing atomic radii, providing a means to separate high value HREEs such as Dy and Tb from more common LREEs such as La. Altogether, our results demonstrate that REE biosorption of high efficiency and selectivity can be achieved by engineering the native bacterial surface.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Genome engineering of E. coli for improved styrene production and polymerization

Microbial production of exogenous organic compounds is challenging as biosynthetic pathways are often complex and produce metabolites that are toxic to the hosts. Biogenic styrene is an example of this problem, which if addressed could result in a more sustainable supply of this important component of the plastics industry. In this study, we engineered Escherichia coli for the production of styrene. We systematically optimized the production capability by first screening different pathway expression levels in E. coli strains. We then further designed and constructed a transcription regulator library targeting 54 genes with 85,420 mutations, and tested this library for increased styrene resistance and production. A series of tolerant mutants not only exhibited improved styrene tolerance but also produced higher styrene concentrations compared to the parent strain. The best producing mutant, ST05 LexA_E45I, produced a 3.45-fold increase in styrene compared to the parent strain. The produced styrene was extracted via gas stripping into dodecane and used in a direct free radical synthesis of polystyrene.

09 BIOMASS FUELS↗
DOE OSTI2019

Recovery of Rare Earth Elements from Geothermal Fluids through Bacterial Cell Surface Adsorption

The increasing demand for rare earth elements (REEs) in the modern economy motivates the development of novel strategies for cost-effective REE recovery from nontraditional feedstocks. We previously engineered E. coli to express lanthanide binding tags on the cell surface, which increased the REE biosorption capacity and selectivity. Here we examined how REE adsorption by the engineered E. coli is affected by various geochemical factors relevant to geothermal fluids, including total dissolved solids (TDS), temperature, pH, and the presence of specific competing metals. REE biosorption is robust to TDS, with high REE recovery efficiency and selectivity observed with TDS as high as 165,000 ppm. Among several metals tested, U, Al, and Pb were found to be the most competitive, causing >25% reduction in REE biosorption when present at concentrations ~3- to 11-fold higher than the REEs. Optimal REE biosorption occurred between pH 5-6, and sorption capacity was reduced by ~65% at pH 2. REE recovery efficiency and selectivity increased as a function of temperature up to ~70 °C due to the thermodynamic properties of metal complexation on the bacterial surface. Ultimately, these data define the optimal and boundary conditions for biosorption and demonstrate its potential utility for selective REE recovery from geofluids.

58 GEOSCIENCES↗
DOE OSTI2020

Enhancing Photosynthetic Production of Glycogen-Rich Biomass for Use as a Fermentation Feedstock

Current sources of fermentation feedstocks, i.e., corn, sugar cane, or plant biomass, fall short of demand for liquid transportation fuels and commodity chemicals in the United States. Aquatic phototrophs including cyanobacteria have the potential to supplement the supply of current fermentable feedstocks. In our strategy, cells are engineered to accumulate storage molecules including glycogen, cellulose, and/or lipid oils that can be extracted from harvested biomass and fed to heterotrophic organisms engineered to produce desired chemical products. Here in this manuscript, we examine the production of glycogen in the model cyanobacteria, Synechococcus sp. strain PCC 7002, and subsequent conversion of cyanobacterial biomass by an engineered Escherichia coli to octanoic acid as a model product. In effort to maximize glycogen production, we explored the deletion of catabolic enzymes and overexpression of GlgC, an enzyme that catalyzes the first committed step toward glycogen synthesis. We found that deletion of glgP increased final glycogen titers when cells were grown in diurnal light. Overexpression of GlgC led to a temporal increase in glycogen content but not in an overall increase in final titer or content. The best strains were grown, harvested, and used to formulate media for growth of E. coli. The cyanobacterial media was able to support the growth of an engineered E. coli and produce octanoic acid at the same titer as common laboratory media.

09 BIOMASS FUELS↗