Interesting Membrane Related Science

Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

The exploitation of the CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) is becoming the method of choice for genome editing in E. coli. First proposed by Jiang and co-workers, the strategy has been subsequently fine-tuned by several authors who demonstrated, by using few selected loci, that the efficiency of mutagenesis (number of mutant colonies over total number of colonies analyzed) can be extremely high (up to 100%). However, from published data it is difficult to appreciate the robustness of the technology, defined as the number of successfully mutated loci over the total number of targeted loci. This information is particularly relevant in high-throughput genome editing, where repetition of experiments to rescue missing mutants would be impractical. This work describes a “brute force” validation activity, which culminated in the definition of a robust, simple and rapid protocol for single or multiple gene deletions.

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Microbial response to environmental stresses: from fundamental mechanisms to practical applications

Microbial response to environmental stresses: from fundamental mechanisms to practical applications

Environmental stresses are usually active during the process of microbial fermentation and have significant influence on microbial physiology. Microorganisms have developed a series of strategies to resist environmental stresses. For instance, they maintain the integrity and fluidity of cell membranes by modulating their structure and composition, and the permeability and activities of transporters are adjusted to control nutrient transport and ion exchange. Certain transcription factors are activated to enhance gene expression, and specific signal transduction pathways are induced to adapt to environmental changes. Besides, microbial cells also have well-established repair mechanisms that protect their macromolecules against damages inflicted by environmental stresses. Oxidative, hyperosmotic, thermal, acid, and organic solvent stresses are significant in microbial fermentation. In this review, we summarize the modus operandi by which these stresses act on cellular components, as well as the corresponding resistance mechanisms developed by microorganisms. Then, we discuss the applications of these stress resistance mechanisms on the production of industrially important chemicals. Finally, we prospect the application of systems biology and synthetic biology in the identification of resistant mechanisms and improvement of metabolic robustness of microorganisms in environmental stresses.

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Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli

Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli

Background

N-Butanol has favorable characteristics for use as either an alternative fuel or platform chemical. Bio-based n-butanol production using microbes is an emerging technology that requires further development. Although bio-industrial microbes such as Escherichia coli have been engineered to produce n-butanol, reactive oxygen species (ROS)-mediated toxicity may limit productivity. Previously, we show that outer-membrane-targeted tilapia metallothionein (OmpC-TMT) is more effective as an ROS scavenger than human and mouse metallothioneins to reduce oxidative stress in the host cell.

Results

The host strain (BUT1-DE) containing the clostridial n-butanol pathway displayed a decreased growth rate and limited n-butanol productivity, likely due to ROS accumulation. The clostridial n-butanol pathway was co-engineered with inducible OmpC-TMT in E. coli (BUT3-DE) for simultaneous ROS removal, and its effect on n-butanol productivity was examined. The ROS scavenging ability of cells overexpressing OmpC-TMT was examined and showed an approximately twofold increase in capacity. The modified strain improved n-butanol productivity to 320 mg/L, whereas the control strain produced only 95.1 mg/L. Transcriptomic analysis revealed three major KEGG pathways that were significantly differentially expressed in the BUT3-DE strain compared with their expression in the BUT1-DE strain, including genes involved in oxidative phosphorylation, fructose and mannose metabolism and glycolysis/gluconeogenesis.

Conclusions

These results indicate that OmpC-TMT can increase n-butanol production by scavenging ROS. The transcriptomic analysis suggested that n-butanol causes quinone malfunction, resulting in oxidative-phosphorylation-related nuo operon downregulation, which would diminish the ability to convert NADH to NAD+ and generate proton motive force. However, fructose and mannose metabolism-related genes (fucA, srlE and srlA) were upregulated, and glycolysis/gluconeogenesis-related genes (pfkB, pgm) were downregulated, which further assisted in regulating NADH/NAD+ redox and preventing additional ATP depletion. These results indicated that more NADH and ATP were required in the n-butanol synthetic pathway. Our study demonstrates a potential approach to increase the robustness of microorganisms and the production of toxic chemicals through the ability to reduce oxidative stress.

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Systems-level understanding of ethanol-induced stresses and adaptation in E. coli

Systems-level understanding of ethanol-induced stresses and adaptation in E. coli

Understanding ethanol-induced stresses and responses in biofuel-producing bacteria at systems level has significant implications in engineering more efficient biofuel producers. We present a computational study of transcriptomic and genomic data of both ethanol-stressed and ethanol-adapted E. coli cells with computationally predicated ethanol-binding proteins and experimentally identified ethanol tolerance genes. Our analysis suggests: (1) ethanol damages cell wall and membrane integrity, causing increased stresses, particularly reactive oxygen species, which damages DNA and reduces the O2 level; (2) decreased cross-membrane proton gradient from membrane damage, coupled with hypoxia, leads to reduced ATP production by aerobic respiration, driving cells to rely more on fatty acid oxidation, anaerobic respiration and fermentation for ATP production; (3) the reduced ATP generation results in substantially decreased synthesis of macromolecules; (4) ethanol can directly bind 213 proteins including transcription factors, altering their functions; (5) all these changes together induce multiple stress responses, reduced biosynthesis, cell viability and growth; and (6) ethanol-adapted E. coli cells restore the majority of these reduced activities through selection of specific genomic mutations and alteration of stress responses, ultimately restoring normal ATP production, macromolecule biosynthesis, and growth. These new insights into the energy and mass balance will inform design of more ethanol-tolerant strains.

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Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9

Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9 tadalafil generico

Abstract

Background
Microbial genome editing is a powerful tool to modify chromosome in way of deletion, insertion or replacement, which is one of the most important techniques in metabolic engineering research. The emergence of CRISPR/Cas9 technique inspires various genomic editing methods. viagra generic

Results
In this research, the goal of development of a fast and easy method for Escherichia coli genome editing with high efficiency is pursued. For this purpose, we designed modular plasmid assembly strategy, compared effects of different length of homologous arms for recombination, and tested different sets of recombinases. The final technique we developed only requires one plasmid construction and one transformation of practice to edit a genomic locus with 3 days and minimal lab work. In addition, the single temperature sensitive plasmid is easy to eliminate for another round of editing. Especially, process of the modularized editing plasmid construction only takes 4 h. cialis coupon 2017

Conclusion
In this study, we developed a fast and easy genome editing procedure based on CRISPR/Cas9 system that only required the work of one plasmid construction and one transformation, which allowed modification of a chromosome locus within 3 days and could be performed continuously for multiple loci. cialis 20mg prix en pharmacie

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Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system

Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system

Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a recently developed powerful tool for gene regulation. In Escherichia coli, the type I CRISPR system expressed endogenously shall be easy for internal regulation without causing metabolic burden in compared with the widely used type II system, which expressed dCas9 as an additional plasmid.

 

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The expression of glycerol facilitators from various yeast species improves growth on glycerol of Saccharomyces cerevisiae

The expression of glycerol facilitators from various yeast species improves growth on glycerol of Saccharomyces cerevisiae

Glycerol is an abundant by-product during biodiesel production and additionally has several assets compared to sugars when used as a carbon source for growing microorganisms in the context of biotechnological applications. However, most strains of the platform production organism Saccharomyces cerevisiae grow poorly in synthetic glycerol medium. It has been hypothesized that the uptake of glycerol could be a major bottleneck for the utilization of glycerol in S. cerevisiae. This species exclusively relies on an active transport system for glycerol uptake. This work demonstrates that the expression of predicted glycerol facilitators (Fps1 homologues) from superior glycerol-utilizing yeast species such as Pachysolen tannophilus, Komagatella pastoris, Yarrowia lipolytica and Cyberlindnera jadinii significantly improves the growth performance on glycerol of the previously selected glycerol-consuming S. cerevisiae wild-type strain (CBS 6412-13 A). The maximum specific growth rate increased from 0.13 up to 0.18 h−1and a biomass yield coefficient of 0.56 gDW/gglycerol was observed. These results pave the way for exploiting the assets of glycerol in the production of fuels, chemicals and pharmaceuticals based on baker’s yeast.

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Molecular genetic improvements of cyanobacteria to enhance the industrial potential of the microbe: A Review

Molecular genetic improvements of cyanobacteria to enhance the industrial potential of the microbe: A Review

The rapid increase in worldwide population coupled with the increasing demand for fossil fuels has led to an increased urgency to develop sustainable sources of energy and chemicals from renewable resources. Using microorganisms to produce high-value chemicals and next-generation biofuels is one sustainable option and is the focus of much current research. Cyanobacteria are ideal platform organisms for chemical and biofuel production because they can be genetically engineered to produce a broad range of products directly from CO2, H2O and sunlight, and require minimal nutrient inputs. The purpose of this review is to provide an overview on advances that have been or could be made to improve strains of cyanobacteria for industrial purposes. First, the benefits of using cyanobacteria as a platform for chemical and biofuel production are discussed. Next, an overview of cyanobacterial strain improvements by genetic engineering is provided. Finally, mutagenesis techniques to improve the industrial potential of cyanobacteria are described. Along with providing an overview on various areas of research that are currently being investigated to improve the industrial potential of cyanobacteria, this review aims to elucidate potential targets for future research involving cyanobacteria as an industrial microorganism. This article is protected by copyright. All rights reserved.

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Functional Membrane Microdomains Organize Signaling Networks in Bacteria

Functional Membrane Microdomains Organize Signaling Networks in Bacteria

Membrane organization is usually associated with the correct function of a number of cellular processes in eukaryotic cells as diverse as signal transduction, protein sorting, membrane trafficking, or pathogen invasion. It has been recently discovered that bacterial membranes are able to compartmentalize their signal transduction pathways in functional membrane microdomains (FMMs). In this review article, we discuss the biological significance of the existence of FMMs in bacteria and comment on possible beneficial roles that FMMs play on the harbored signal transduction cascades. Moreover, four different membrane-associated signal transduction cascades whose functions are linked to the integrity of FMMs are introduced, and the specific role that FMMs play in stabilizing and promoting interactions of their signaling components is discussed. Altogether, FMMs seem to play a relevant role in promoting more efficient activation of signal transduction cascades in bacterial cells and show that bacteria are more sophisticated organisms than previously appreciated.

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US researchers develop new ways of producing biofuels from E-coli

US researchers develop new ways of producing biofuels from E-coli
US researchers from Washington University have developed a new way to harness the chemical production properties of E.coli bacteria, making the production of certain biofuels from the bacteria more efficient. The new method, described in a paper published in the journal Metabolic Engineering, involves the development of two different protein pathways capable of chemically affecting the production of biofuel in E.coli. Previously, researchers have had difficulty producing branched-chain fatty acids (BCFA) as common bacteria like E.coli mostly produce straight-chain fatty acids (SCFA), which have inferior fuel properties. The new method could enable the E.coli bacteria to boost its BCFA production to 80% of all fuel products.