scholarly journals Construction and optimization of a heterologous pathway for protocatechuate catabolism inEscherichia colienables rapid bioconversion of model lignin monomers

2017 ◽  
Author(s):  
Sonya M. Clarkson ◽  
Donna M. Kridelbaugh ◽  
James G. Elkins ◽  
Adam M. Guss ◽  
Joshua K. Michener
Keyword(s):  
Escherichia Coli ◽  
Sole Source ◽  
Biofuel Production ◽  
List Type ◽  
Cellulosic Biofuels ◽  
E Coli ◽  
Gene Pathway ◽  
Cellulosic Biofuel ◽  
Fuels And Chemicals ◽  
Heterologous Pathway

AbstractCellulosic biofuel production yields a substantial lignin byproduct stream that currently has few applications. Biological conversion of lignin compounds into chemicals and fuels has the potential to improve the economics of cellulosic biofuels, but few microbes are able both to catabolize lignin and generate valuable products. WhileEscherichia colihas been engineered to produce a variety of fuels and chemicals, it is incapable of catabolizing most aromatic compounds. Therefore, we have engineeredE. colito catabolize a model lignin monomer, protocatechuate, as the sole source of carbon and energy, via heterologous expression of a nine-gene pathway fromPseudomonas putidaKT2440. We next used experimental evolution to select for mutations that increased growth with PCA more than two-fold. Increasing the strength of a single ribosome binding site in the heterologous pathway was sufficient to recapitulate the increased growth. After optimization of the core pathway, we extended the pathway to enable catabolism of a second model compound, 4-hydroxybenzoate. These engineered strains will be useful platforms to discover, characterize, and optimize pathways for lignin bioconversions.HighlightsA heterologous pathway for PCA catabolism was transferred to Escherichia coli.Evolution identified a mutation that increased growth with PCA by 2.5-fold.Optimization plus further engineering allowed efficient catabolism of 4-HB

scholarly journals Construction and Optimization of a Heterologous Pathway for Protocatechuate Catabolism in Escherichia coli Enables Bioconversion of Model Aromatic Compounds

2017 ◽  
Vol 83 (18) ◽  
Author(s):  
Sonya M. Clarkson ◽  
Richard J. Giannone ◽  
Donna M. Kridelbaugh ◽  
James G. Elkins ◽  
Adam M. Guss ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Aromatic Compound ◽  
Aromatic Compounds ◽  
Sole Source ◽  
Rapid Identification ◽  
Content Type ◽  
E Coli ◽  
Aromatic Compound Degradation ◽  
Fuels And Chemicals ◽  
Heterologous Pathway

ABSTRACT The production of biofuels from lignocellulose yields a substantial lignin by-product stream that currently has few applications. Biological conversion of lignin-derived compounds into chemicals and fuels has the potential to improve the economics of lignocellulose-derived biofuels, but few microbes are able both to catabolize lignin-derived aromatic compounds and to generate valuable products. While Escherichia coli has been engineered to produce a variety of fuels and chemicals, it is incapable of catabolizing most aromatic compounds. Therefore, we engineered E. coli to catabolize protocatechuate, a common intermediate in lignin degradation, as the sole source of carbon and energy via heterologous expression of a nine-gene pathway from Pseudomonas putida KT2440. We next used experimental evolution to select for mutations that increased growth with protocatechuate more than 2-fold. Increasing the strength of a single ribosome binding site in the heterologous pathway was sufficient to recapitulate the increased growth. After optimization of the core pathway, we extended the pathway to enable catabolism of a second model compound, 4-hydroxybenzoate. These engineered strains will be useful platforms to discover, characterize, and optimize pathways for conversions of lignin-derived aromatics. IMPORTANCE Lignin is a challenging substrate for microbial catabolism due to its polymeric and heterogeneous chemical structure. Therefore, engineering microbes for improved catabolism of lignin-derived aromatic compounds will require the assembly of an entire network of catabolic reactions, including pathways from genetically intractable strains. Constructing defined pathways for aromatic compound degradation in a model host would allow rapid identification, characterization, and optimization of novel pathways. We constructed and optimized one such pathway in E. coli to enable catabolism of a model aromatic compound, protocatechuate, and then extended the pathway to a related compound, 4-hydroxybenzoate. This optimized strain can now be used as the basis for the characterization of novel pathways.

scholarly journals Cellulosic biofuel production using emulsified simultaneous saccharification and fermentation (eSSF) with conventional and thermotolerant yeasts

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Shannon M. Hoffman ◽  
Maria Alvarez ◽  
Gilad Alfassi ◽  
Dmitry M. Rein ◽  
Sergio Garcia-Echauri ◽  
...  
Keyword(s):  
Simultaneous Saccharification And Fermentation ◽  
Agricultural Waste ◽  
Biofuel Production ◽  
Cellulosic Biofuels ◽  
Processing Times ◽  
Cellulosic Biofuel ◽  
Thermotolerant Yeasts ◽  
Rapid Hydrolysis ◽  
Dedicated Energy Crops ◽  
Simultaneous Saccharification

Abstract Background Future expansion of corn-derived ethanol raises concerns of sustainability and competition with the food industry. Therefore, cellulosic biofuels derived from agricultural waste and dedicated energy crops are necessary. To date, slow and incomplete saccharification as well as high enzyme costs have hindered the economic viability of cellulosic biofuels, and while approaches like simultaneous saccharification and fermentation (SSF) and the use of thermotolerant microorganisms can enhance production, further improvements are needed. Cellulosic emulsions have been shown to enhance saccharification by increasing enzyme contact with cellulose fibers. In this study, we use these emulsions to develop an emulsified SSF (eSSF) process for rapid and efficient cellulosic biofuel production and make a direct three-way comparison of ethanol production between S. cerevisiae, O. polymorpha, and K. marxianus in glucose and cellulosic media at different temperatures. Results In this work, we show that cellulosic emulsions hydrolyze rapidly at temperatures tolerable to yeast, reaching up to 40-fold higher conversion in the first hour compared to microcrystalline cellulose (MCC). To evaluate suitable conditions for the eSSF process, we explored the upper temperature limits for the thermotolerant yeasts Kluyveromyces marxianus and Ogataea polymorpha, as well as Saccharomyces cerevisiae, and observed robust fermentation at up to 46, 50, and 42 °C for each yeast, respectively. We show that the eSSF process reaches high ethanol titers in short processing times, and produces close to theoretical yields at temperatures as low as 30 °C. Finally, we demonstrate the transferability of the eSSF technology to other products by producing the advanced biofuel isobutanol in a light-controlled eSSF using optogenetic regulators, resulting in up to fourfold higher titers relative to MCC SSF. Conclusions The eSSF process addresses the main challenges of cellulosic biofuel production by increasing saccharification rate at temperatures tolerable to yeast. The rapid hydrolysis of these emulsions at low temperatures permits fermentation using non-thermotolerant yeasts, short processing times, low enzyme loads, and makes it possible to extend the process to chemicals other than ethanol, such as isobutanol. This transferability establishes the eSSF process as a platform for the sustainable production of biofuels and chemicals as a whole.

scholarly journals The Evolution of Mass Cell Suicide in Bacterial Warfare

2020 ◽  
Author(s):  
Elisa T. Granato ◽  
Kevin R. Foster
Keyword(s):  
Escherichia Coli ◽  
Natural Selection ◽  
Reproductive Potential ◽  
Time Lapse ◽  
List Type ◽  
E Coli ◽  
Toxin Exposure ◽  
Small Benefit ◽  
Will Self ◽  
Cell Suicide

SUMMARYBehaviours that reliably cause the death of an actor are typically strongly disfavoured by natural selection, and yet many bacteria undergo cell lysis to release anti-competitor toxins [1–4]. This behaviour is most easily explained if only a few cells die to release toxins and help their clonemates, but the number of cells that actually lyse during bacterial warfare is unknown. The challenge is that one cannot distinguish cells that have undergone programmed suicide from those that were simply killed by a competitor’s toxin. We developed a two-colour fluorescence reporter assay in Escherichia coli to overcome this problem. Surprisingly, this revealed conditions where nearly all cells undergo programmed lysis. Adding a DNA-damaging toxin (DNase colicin) to a focal strain causes it to engage in mass cell suicide where around 85% of cells lyse to release their own toxin. Time-lapse 3D confocal microscopy revealed that self-lysis occurs at even higher frequencies (~94%) at the interface between competing colonies. We sought to understand how such high levels of cell suicide could be favoured by natural selection. Exposing E. coli that do not perform lysis to the DNase colicin revealed that mass lysis only occurs when cells are going to die anyway from toxin exposure. From an evolutionary perspective, this renders the behaviour cost-free as these cells have zero reproductive potential. This explains how mass cell suicide can evolve, as any small benefit to surviving clonemates can lead to the strategy being favoured by natural selection. Our findings have strong parallels to the suicidal attacks of social insects [5–8], which are also performed by individuals with low reproductive potential, suggesting convergent evolution in these very different organisms.HIGHLIGHTSA novel assay can detect Escherichia coli undergoing cell suicide to release toxinsWe quantified the frequency of suicidal self-lysis during competitionsUnder some conditions, nearly all cells will self-lyse to release toxinsSelf-lysis makes evolutionary sense as cells will die anyway from competitors’ toxins

scholarly journals Escherichia coli Competence Gene Homologs Are Essential for Competitive Fitness and the Use of DNA as a Nutrient

2006 ◽  
Vol 188 (11) ◽  
pp. 3902-3910 ◽  
Author(s):  
Vyacheslav Palchevskiy ◽  
Steven E. Finkel
Keyword(s):  
Escherichia Coli ◽  
Sole Source ◽  
Extracellular Dna ◽  
Nutrient Source ◽  
Genetic Competence ◽  
Exogenous Dna ◽  
Competitive Fitness ◽  
E Coli ◽  
Widespread Phenomenon

ABSTRACT Natural genetic competence is the ability of cells to take up extracellular DNA and is an important mechanism for horizontal gene transfer. Another potential benefit of natural competence is that exogenous DNA can serve as a nutrient source for starving bacteria because the ability to “eat” DNA is necessary for competitive survival in environments containing limited nutrients. We show here that eight Escherichia coli genes, identified as homologs of com genes in Haemophilus influenzae and Neisseria gonorrhoeae, are necessary for the use of extracellular DNA as the sole source of carbon and energy. These genes also confer a competitive advantage to E. coli during long-term stationary-phase incubation. We also show that homologs of these genes are found throughout the proteobacteria, suggesting that the use of DNA as a nutrient may be a widespread phenomenon.

scholarly journals The Transcriptional Factors MurR and Catabolite Activator Protein Regulate N-Acetylmuramic Acid Catabolism in Escherichia coli

2008 ◽  
Vol 190 (20) ◽  
pp. 6598-6608 ◽  
Author(s):  
Tina Jaeger ◽  
Christoph Mayer
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Sole Source ◽  
Lag Phase ◽  
Face To Face ◽  
E Coli ◽  
Catabolite Activator Protein ◽  
Activator Protein ◽  
High Level ◽  
First Time

ABSTRACT The MurNAc etherase MurQ of Escherichia coli is essential for the catabolism of the bacterial cell wall sugar N-acetylmuramic acid (MurNAc) obtained either from the environment or from the endogenous cell wall (i.e., recycling). High-level expression of murQ is required for growth on MurNAc as the sole source of carbon and energy, whereas constitutive low-level expression of murQ is sufficient for the recycling of peptidoglycan fragments continuously released from the cell wall during growth of the bacteria. Here we characterize for the first time the expression of murQ and its regulation by MurR, a member of the poorly characterized RpiR/AlsR family of transcriptional regulators. Deleting murR abolished the extensive lag phase observed for E. coli grown on MurNAc and enhanced murQ transcription some 20-fold. MurR forms a stable multimer (most likely a tetramer) and binds to two adjacent inverted repeats within an operator region. In this way MurR represses transcription from the murQ promoter and also interferes with its own transcription. MurNAc-6-phosphate, the substrate of MurQ, was identified as a specific inducer that weakens binding of MurR to the operator. Moreover, murQ transcription depends on the activation by cyclic AMP (cAMP)-catabolite activator protein (CAP) bound to a class I site upstream of the murQ promoter. murR and murQ are divergently orientated and expressed from nonoverlapping face-to-face (convergent) promoters, yielding transcripts that are complementary at their 5′ ends. As a consequence of this unusual promoter arrangement, cAMP-CAP also affects murR transcription, presumably by acting as a roadblock for RNA polymerase.

scholarly journals Growth of Escherichia coli Coexpressing Phosphotriesterase and Glycerophosphodiester Phosphodiesterase, Using Paraoxon as the Sole Phosphorus Source

2004 ◽  
Vol 70 (1) ◽  
pp. 404-412 ◽  
Author(s):  
Sean Yu McLoughlin ◽  
Colin Jackson ◽  
Jian-Wei Liu ◽  
David L. Ollis
Keyword(s):  
Escherichia Coli ◽  
Enterobacter Aerogenes ◽  
Sole Source ◽  
Limiting Factor ◽  
E Coli ◽  
Dimethyl Phosphate ◽  
Genes Encoding ◽  
Phosphorus Source ◽  
Hydrolysis Of ◽  
Methyl Phosphate

ABSTRACT Phosphotriesterases catalyze the hydrolytic detoxification of phosphotriester pesticides and chemical warfare nerve agents with various efficiencies. The directed evolution of phosphotriesterases to enhance the breakdown of poor substrates is desirable for the purposes of bioremediation. A limiting factor in the identification of phosphotriesterase mutants with increased activity is the ability to effectively screen large mutant libraries. To this end, we have investigated the possibility of coupling phosphotriesterase activity to cell growth by using methyl paraoxon as the sole phosphorus source. The catabolism of paraoxon to phosphate would occur via the stepwise enzymatic hydrolysis of paraoxon to dimethyl phosphate, methyl phosphate, and then phosphate. The Escherichia coli strain DH10B expressing the phosphotriesterase from Agrobacterium radiobacter P230 (OpdA) is unable to grow when paraoxon is used as the sole phosphorus source. Enterobacter aerogenes is an organism capable of growing when dimethyl phosphate is the sole phosphorus source. The enzyme responsible for hydrolyzing dimethyl phosphate has been previously characterized as a nonspecific phosphohydrolase. We isolated and characterized the genes encoding the phosphohydrolase operon. The operon was identified from a shotgun clone that enabled E. coli to grow when dimethyl phosphate is the sole phosphorus source. E. coli coexpressing the phosphohydrolase and OpdA grew when paraoxon was the sole phosphorus source. By constructing a short degradative pathway, we have enabled E. coli to use phosphotriesters as a sole source of phosphorus.

scholarly journals Escherichia coli Strains Engineered for Homofermentative Production of d-Lactic Acid from Glycerol

2010 ◽  
Vol 76 (13) ◽  
pp. 4327-4336 ◽  
Author(s):  
Suman Mazumdar ◽  
James M. Clomburg ◽  
Ramon Gonzalez
Keyword(s):  
Escherichia Coli ◽  
Lactic Acid ◽  
Lactate Dehydrogenase ◽  
Cell Mass ◽  
Lactate Production ◽  
Fumarate Reductase ◽  
Glycerol Metabolism ◽  
E Coli ◽  
Fuels And Chemicals ◽  
Microaerobic Conditions

ABSTRACT Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the efficient conversion of glycerol to d-lactic acid (d-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for d-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of d-lactate from pyruvate (d-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (ΔpflB) and fumarate reductase (ΔfrdA) (strain LA01) and (ii) inactivation of fumarate reductase (ΔfrdA), phosphate acetyltransferase (Δpta), and alcohol/acetaldehyde dehydrogenase (ΔadhE) (strain LA02). A mutation that blocked the aerobic d-lactate dehydrogenase (Δdld) also was introduced in both LA01 and LA02 to prevent the utilization of d-lactate. The most efficient strain (LA02Δdld, with GlpK-GlpD overexpressed) produced 32 g/liter of d-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for d-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of d-lactate and is redox balanced, thus representing a viable metabolic pathway.

Effect of TiO2 Loading, Water Depth and Light Intensity on Photo-Disinfection Efficacy of Escherichia Coli O157:H7 Using TiO2 NP-Embedded Cellulose Acetate Film in Water

2021 ◽  
Vol 37 (1) ◽  
pp. 1-9
Author(s):  
Jing Xie ◽  
Yen-Con Hung
Keyword(s):  
Escherichia Coli ◽  
Light Intensity ◽  
Cellulose Acetate ◽  
Water Depth ◽  
Coefficient Of Determination ◽  
List Type ◽  
Water Safety ◽  
Operational Parameters ◽  
Bacterial Reduction ◽  
E Coli

HighlightsPhoto-disinfection using TiO2 NP-embedded CA film showed great potential.TiO2 loading, water depth, and light intensity affected TiO2 photo-disinfection.Bacterial reduction can be predicted based on water depth and light intensity.Durability of the TiO2 NP-embedded CA film has been demonstrated.Abstract. Photocatalysis disinfection has great potential for food and water safety applications. TiO2 NP-embedded cellulose acetate (CA) film has shown effectiveness in inactivating Escherichia coli (E. coli) O157:H7 in water. This study evaluated the effect of several operational parameters such as TiO2 load, light intensity, and water depth on photo-disinfection efficacy of TiO2 NP-embedded CA film. Effect of TiO2 load on photocatalysis disinfection efficacy was investigated by assessing the disinfection rate of the films at different TiO2 levels in inoculated water at a weak UV-A light intensity. The individual and interaction effects of light intensity and water depth on photocatalysis disinfection were evaluated using the response surface methodology (RSM). Based on a central composite design, 13 runs of experiments were conducted with the combination of the two factors at three levels. The effect of film size on photo-disinfection efficacy was also investigated. Results showed that CA film with a TiO2 load of 0.82 mg/cm2 yielded the highest disinfection rate. An empirical model was established to predict the effect of light intensity and water depth on bacterial reduction with a high coefficient of determination (R2 = 0.98). An increased water depth or a decreased light intensity reduced photo-inactivation efficacy. A 5 log reduction was achieved during a 3-h treatment at a light intensity of 1 mW/cm2 and water depth at 1.6 cm. The photo-disinfection efficacy of the film was not significantly (p>0.05) affected by film size. The CA film was able to maintain antimicrobial efficacy after four repeated uses. Results from this study demonstrated that UV-A assisted photo-disinfection using TiO2 NP-embedded CA film has great potential to inactivate pathogens in water. Keywords: Cellulose Acetate, E. coli O157:H7, Photocatalysis, TiO2 nanoparticles, Water Treatment.

scholarly journals Identification of a Phosphotransferase System of Escherichia coli Required for Growth on N-Acetylmuramic Acid

2004 ◽  
Vol 186 (8) ◽  
pp. 2385-2392 ◽  
Author(s):  
Ulrike Dahl ◽  
Tina Jaeger ◽  
Bao Trâm Nguyen ◽  
Julia M. Sattler ◽  
Christoph Mayer
Keyword(s):  
Escherichia Coli ◽  
Energy Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sole Source ◽  
Wild Type ◽  
Phosphotransferase System ◽  
E Coli ◽  
Glucose Transport System ◽  
Single Polypeptide

ABSTRACT We report here that wild-type Escherichia coli grows on N-acetylmuramic acid (MurNAc) as the sole source of carbon and energy. Analysis of mutants defective in N-acetylglucosamine (GlcNAc) catabolism revealed that the catabolic pathway for MurNAc merges into the GlcNAc pathway on the level of GlcNAc 6-phosphate. Furthermore, analysis of mutants defective in components of the phosphotransferase system (PTS) revealed that a PTS is essential for growth on MurNAc. However, neither the glucose-, mannose/glucosamine-, nor GlcNAc-specific PTS (PtsG, ManXYZ, and NagE, respectively) was found to be necessary. Instead, we identified a gene at 55 min on the E. coli chromosome that is responsible for MurNAc uptake and growth. It encodes a single polypeptide consisting of the EIIB and C domains of a so-far-uncharacterized PTS that was named murP. MurP lacks an EIIA domain and was found to require the activity of the crr-encoded enzyme IIA-glucose (EIIAGlc), a component of the major glucose transport system for growth on MurNAc. murP deletion mutants were unable to grow on MurNAc as the sole source of carbon; however, growth was rescued by providing murP in trans expressed from an isopropylthiogalactopyranoside-inducible plasmid. A functional His6 fusion of MurP was constructed, isolated from membranes, and identified as a polypeptide with an apparent molecular mass of 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Close homologs of MurP were identified in the genome of several bacteria, and we believe that these organisms might also be able to utilize MurNAc.

scholarly journals LAND-USE AND GREENHOUSE GAS IMPLICATIONS OF BIOFUELS: ROLE OF TECHNOLOGY AND POLICY

2012 ◽  
Vol 03 (03) ◽  
pp. 1250013 ◽  
Author(s):  
XIAOGUANG CHEN ◽  
HAIXIAO HUANG ◽  
MADHU KHANNA
Keyword(s):  
Land Use ◽  
Ghg Emissions ◽  
Carbon Price ◽  
Price Policy ◽  
Biofuel Production ◽  
Tax Credit ◽  
Cellulosic Biofuels ◽  
Cellulosic Biofuel ◽  
The U.S ◽  
Ghg Savings

This paper examines the changes in land use in the U.S. likely to be induced by biofuel and climate policies and the implications of these policies for greenhouse gas (GHG) emissions over the 2007–2022 period. The policies considered here include a modified Renewable Fuel Standard (RFS) by itself as well as combined with a cellulosic biofuel tax credit or a carbon price policy. We use a dynamic, spatial, multi-market equilibrium model, Biofuel and Environmental Policy Analysis Model (BEPAM), to endogenously determine the effects of these policies on cropland allocation, food and fuel prices, and the mix of first- and second-generation biofuels. We find that the RFS could be met by diverting 6% of cropland for biofuel production and would result in corn prices increasing by 16% in 2002 relative to the business-as-usual baseline. The reduction in GHG emissions in the U.S. due to the RFS is about 2%; these domestic GHG savings can be severely eroded by emissions due to indirect land-use changes and the increase in gasoline consumption in the rest of the world. Supplementing the RFS with a carbon price policy or a cellulosic biofuel tax credit induces a switch away from corn ethanol to cellulosic biofuels and achieves the mandated level of biofuel production with a smaller adverse impact on crop prices. These supplementary policies enhance the GHG savings achieved by the RFS alone, although through different mechanisms; greater production of cellulosic biofuels with the tax credit but larger reduction in fossil fuel consumption with a carbon tax.

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