Category Archives: SCIENCE

Pseudomonas stutzeri as an alternative host for membrane proteins

Pseudomonas stutzeri as an alternative host for membrane proteins

Background

Studies on membrane proteins are often hampered by insufficient yields of the protein of interest. Several prokaryotic hosts have been tested for their applicability as production platform but still Escherichia coli by far is the one most commonly used. Nevertheless, it has been demonstrated that in some cases hosts other than E. coli are more appropriate for certain target proteins.

Results

Here we have developed an expression system for the heterologous production of membrane proteins using a single plasmid-based approach. The gammaproteobacterium Pseudomonas stutzeri was employed as a new production host. We investigated several basic microbiological features crucial for its handling in the laboratory. The organism belonging to bio-safety level one is a close relative of the human pathogen Pseudomonas aeruginosaPseudomonas stutzeri is comparable to E. coli regarding its growth and cultivation conditions. Several effective antibiotics were identified and a protocol for plasmid transformation was established. We present a workflow including cloning of the target proteins, small-scale screening for the best production conditions and finally large-scale production in the milligram range. The GFP folding assay was used for the rapid analysis of protein folding states. In summary, out of 36 heterologous target proteins, 20 were produced at high yields. Additionally, eight transporters derived from P. aeruginosa could be obtained with high yields. Upscaling of protein production and purification of a Gluconate:H+ Symporter (GntP) family transporter (STM2913) from Salmonella enterica to high purity was demonstrated.

Conclusions

Pseudomonas stutzeri is an alternative production host for membrane proteins with success rates comparable to E. coli. However, some proteins were produced with high yields in P. stutzeri but not in E. coliand vice versa. Therefore, P. stutzeri extends the spectrum of useful production hosts for membrane proteins and increases the success rate for highly produced proteins. Using the new pL2020 vector no additional cloning is required to test both hosts in parallel.

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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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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.

Synthetic Genomics team engineers Vmax™, an advantaged next-generation host organism for a wide range of biotechnology applications

Synthetic Genomics team engineers Vmax™, an advantaged next-generation host organism for a wide range of biotechnology applications

Optimized system has potential to replace the workhorse E. coli by increasing speed and efficiency of protein production and cloning

Researchers from Synthetic Genomics, Inc. (SGI) announced today the development and extensive engineering of Vibrio natriegens into a next-generation biotechnology host organism Vmax™. Looking to accelerate the pace of discovery and the path to sustainable solutions, the team set out to develop a novel bacterial host that will drastically reduce the amount of time scientists spend on each experiment and workflow and to enhance productivity of the resulting new host.

After screening for the fastest-growing strain and optimizing methods for introducing DNA into those cells at high efficiencies, the team developed genome engineering tools to improve the performance of Vmax™ for common biotech applications, namely, recombinant protein expression and molecular cloning. These breakthroughs build on expertise gleaned during the creation of the first synthetic cell and first minimal cell and again position SGI at the forefront of synthetic biology.

The paper describing this work is the first peer-reviewed publication of its kind and was published online today in Nature Methods by Matthew T. Weinstock, Eric D. Hesek, Christopher M. Wilson, and Daniel G. Gibson.

“This work provides a game-changing alternative to E. coli, the organism that has been a laboratory staple for decades, and again highlights the rapid and innovative synthetic biology expertise we’ve developed at SGI. We are in the process of designing and synthesizing new Vmax™ cells that operate at even higher efficiencies and productivity as we move toward a next-generation host for protein production,” said Daniel Gibson, Vice President, DNA Technologies, SGI.

Commenting on the origin of the research, Todd Peterson, Chief Technology Officer at SGI stated, “Despite the known drawbacks and shortcomings, scientists have been necessitated to use E. coli as a laboratory host primarily because there have been no suitable alternatives. We deployed our synthetic biology expertise to develop a new host strain that will drastically improve upon the traditional methods and tools.”

Typical cloning projects using E. coli competent cells span several days starting from the time a cloning process is initiated to the time plasmid DNA is prepared. Cloning strategies employing Vmax™ developed by the SGI team shorten that time to as little as one day.

The advancements described by the team set the stage for commercialization of these next-generation cells for cloning and protein expression by SGI in the coming months. Vmax™ is compatible with most kits, reagents, growth medium, vectors, and procedures already entrenched in laboratories. Making these cells commercially available will accelerate the pace of global biotechnological research, making a far-reaching and lasting impact toward genetic exploration and discovery worldwide.

About Synthetic Genomics Inc.
Synthetic Genomics Inc. (SGI), located in La Jolla, CA, is a leader in the fields of synthetic biology and synthetic genomics, advancing genomics to better life. SGI applies its intellectual property in this rapidly evolving field to design and build biological systems solving global sustainability challenges. SGI serves three end markets: research, bioproduction, and applied products. The company’s research offerings, commercialized through its subsidiary SGI-DNA, are revolutionizing science and medicine with next-generation genomic solutions, including the world’s first DNA printer. SGI applies its integrated synthetic biology capabilities to reinvent bio-based production by improving existing production systems and developing novel, optimized production hosts. SGI develops its applied products, typically in partnership with leading global organizations, across a variety of industries including sustainable bio-fuels, sustainable crops, nutritional supplements, vaccines, and transplantable organs.

About SGI-DNA
SGI-DNA, a wholly owned subsidiary of Synthetic Genomics, Inc (SGI), is responsible for all commercial aspects of SGI’s synthetic DNA business and focuses on strategic business relationships with both academic and commercial researchers. Building on the scientific advancements and breakthroughs from leading scientists such as J. Craig Venter, Ham Smith, Clyde Hutchison, Dan Gibson and their teams, SGI-DNA utilizes unique and proprietary DNA technologies to produce complex synthetic genes and reagents. SGI-DNA also offers the BioXp™ 3200 System, the world’s first DNA printer, in addition to a comprehensive suite of genomic services, including whole genome sequencing, library design, bioinformatics services, and reagent kits to enable synthetic biology.

 

http://www.syntheticgenomics.com

 

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