Membrane-Related Science

Royal Society Publishing has recently published a special issue of Interface Focus entitled “The artificial cell: biology-inspired compartmentalization of chemical function”, organised by Paul A Beales, Barbara Ciani and Stephen Mann.

This issue is based on a Royal Society Theo Murphy meeting held on the 26–27 February 2018. The articles reveal the rich diversity of research currently being undertaken in the field of artificial cell design and construction, and highlight the challenges that lie ahead.

The articles are FREE TO ACCESS here.

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University of York

CBMNet-funded scientists from the Universities of York and Oxford, along with industrial partner Unilever, have unravelled a key part of the molecular process by which armpit bacteria produce the most pungent component of the noxious smell we recognise as BO. The findings could result in more effective deodorants with targeted active ingredients, the researchers suggest. (read more…)

Antonella CaforioMelvin F. SiliakusMarten ExterkateSamta JainVarsha R. JumdeRuben L. H. AndringaServé W. M. KengenAdriaan J. MinnaardArnold J. M. Driessen and John van der Oost

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Translation Stress Positively Regulates MscL-Dependent Excretion of Cytoplasmic Proteins

ABSTRACT

The apparent mislocalization or excretion of cytoplasmic proteins is a commonly observed phenomenon in both bacteria and eukaryotes. However, reports on the mechanistic basis and the cellular function of this so-called “nonclassical protein secretion” are limited. Here we report that protein overexpression in recombinant cells and antibiotic-induced translation stress in wild-type Escherichia coli cells both lead to excretion of cytoplasmic protein (ECP). Condition-specific metabolomic and proteomic analyses, combined with genetic knockouts, indicate a role for both the large mechanosensitive channel (MscL) and the alternative ribosome rescue factor A (ArfA) in ECP. Collectively, the findings indicate that MscL-dependent protein excretion is positively regulated in response to both osmotic stress and arfA-mediated translational stress.

IMPORTANCE

Protein translocation is an essential feature of cellular organisms. Bacteria, like all single-cell organisms, interact with their environment by translocation of proteins across their cell membranes via dedicated secretion pathways. Proteins destined for secretion are directed toward the secretion pathways by the presence of specific signal peptides. This study demonstrates that under conditions of both osmotic stress and translation stress, E. coli cells undergo an excretion phenomenon whereby signal peptide-less proteins are translocated across both the inner and outer cell membranes into the extracellular environment. Confirming the presence of alternative translocation/excretion pathways and understanding their function and regulation are thus important for fundamental microbiology and biotechnology.

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Identification and utilization of two important transporters: SgvT1 and SgvT2, for griseoviridin and viridogrisein biosynthesis in Streptomyces griseoviridis

Background
Griseoviridin (GV) and viridogrisein (VG, also referred as etamycin), both biosynthesized by a distinct 105 kb biosynthetic gene cluster (BGC) in Streptomyces griseoviridis NRRL 2427, are a pair of synergistic streptogramin antibiotics and very important in treating infections of many multi-drug resistant microorganisms. Three transporter genes, sgvT1–T3 have been discovered within the 105 kb GV/VG BGC, but the function of these efflux transporters have not been identified.

Results
In the present study, we have identified the different roles of these three transporters, SgvT1, SgvT2 and SgvT3. SgvT1 is a major facilitator superfamily (MFS) transporter whereas SgvT2 appears to serve as the sole ATP-binding cassette (ABC) transporter within the GV/VG BGC. Both proteins are necessary for efficient GV/VG biosynthesis although SgvT1 plays an especially critical role by averting undesired intracellular GV/VG accumulation during biosynthesis. SgvT3 is an alternative MFS-based transporter that appears to serve as a compensatory transporter in GV/VG biosynthesis. We also have identified the γ-butyrolactone (GBL) signaling pathway as a central regulator of sgvT1–T3 expression. Above all, overexpression of sgvT1 and sgvT2 enhances transmembrane transport leading to steady production of GV/VG in titers ≈ 3-fold greater than seen for the wild-type producer and without any notable disturbances to GV/VG biosynthetic gene expression or antibiotic control.

Conclusions
Our results shows that SgvT1–T2 are essential and useful in GV/VG biosynthesis and our effort highlight a new and effective strategy by which to better exploit streptogramin-based natural products of which GV and VG are prime examples with clinical potential.

Read the full article here.

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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Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker’s yeast

Fungi are a valuable source of enzymatic diversity and therapeutic natural products including antibiotics. Here we engineer the baker’s yeast Saccharomyces cerevisiae to produce and secrete the antibiotic penicillin, a beta-lactam nonribosomal peptide, by taking genes from a filamentous fungus and directing their efficient expression and subcellular localization. Using synthetic biology tools combined with long-read DNA sequencing, we optimize productivity by 50-fold to produce bioactive yields that allow spent S. cerevisiae growth media to have antibacterial action against Streptococcus bacteria. This work demonstrates that S. cerevisiae can be engineered to perform the complex biosynthesis of multicellular fungi, opening up the possibility of using yeast to accelerate rational engineering of nonribosomal peptide antibiotics.

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Whole-cell biocatalysts by design

Whole-cell biocatalysts provide unique advantages and have been widely used for the efficient biosynthesis of value-added fine and bulk chemicals, as well as pharmaceutically active ingredients. What is more, advances in synthetic biology and metabolic engineering, together with the rapid development of molecular genetic tools, have brought about a renaissance of whole-cell biocatalysis. These rapid advancements mean that whole-cell biocatalysts can increasingly be rationally designed. Genes of heterologous enzymes or synthetic pathways are increasingly being introduced into microbial hosts, and depending on the complexity of the synthetic pathway or the target products, they can enable the production of value-added chemicals from cheap feedstock. Metabolic engineering and synthetic biology efforts aimed at optimizing the existing microbial cell factories concentrate on improving heterologous pathway flux, precursor supply, and cofactor balance, as well as other aspects of cellular metabolism, to enhance the efficiency of biocatalysts. In the present review, we take a critical look at recent developments in whole-cell biocatalysis, with an emphasis on strategies applied to designing and optimizing the organisms that are increasingly modified for efficient production of chemicals.

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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

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

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

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 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

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

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

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

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 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

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

 

In collaboration with researchers at Nanjing Agricultural University, Dr Tony Miller from the John Innes Centre has developed rice crops with an improved ability to manage their own pH levels, enabling them to take up significantly more nitrogen, iron and phosphorous from soil and increase yield by up to 54%.

Rice is a major crop, feeding almost 50% of the world’s population and has retained the ability to survive in changing environmental conditions. The crop is able to thrive in flooded paddy fields – where the soggy, anaerobic conditions favour the availability of ammonium – as well as in much drier, drained soil, where increased oxygen means more nitrate is available. nitrogen fertilizer is a major cost in growing many cereal crops and its overuse has a negative environmental impact.

The nitrogen that all plants need to grow is typically available in the form of nitrate or ammonium ions in the soil, which are taken up by the plant roots. For the plant, getting the right balance of nitrate and ammonium is very important: too much ammonium and plant cells become alkaline; too much nitrate and they become acidic. Either way, upsetting the pH balance means the plant’s enzymes do not work as well, affecting plant health and crop yield.

Together with the partners in Nanjing, China, Dr Miller’s team has been working out how rice plants can maintain pH under these changing environments.

Rice contains a gene called OsNRT2.3, which creates a protein involved in nitrate transport. This one gene makes two slightly different versions of the protein: OsNRT2.3a and OsNRT2.3b. Following tests to determine the role of both versions of the protein, Dr Miller’s team found that OsNRT2.3b is able to switch nitrate transport on or off, depending on the internal pH of the plant cell.

When this ‘b’ protein was overexpressed in rice plants they were better able to buffer themselves against pH changes in their environment. This enabled them to take up much more nitrogen, as well as more iron and phosphorus. These rice plants gave a much higher yield of rice grain (up to 54% more yield), and their nitrogen use efficiency increased by up to 40%.

Dr Miller said: “Now that we know this particular protein found in rice plants can greatly increase nitrogen efficiency and yields, we can begin to produce new varieties of rice and other crops. These findings bring us a significant step closer to being able to produce more of the world’s food with a lower environmental impact.”

This new technology has been patented by PBL, the John Innes Centre’s innovation management company, and has already been licensed to three different companies to develop new varieties of six different crop species.

This study, which will be published in the Proceedings of the National Academy of Sciences USA, was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and grants from the Chinese Government.

 

The paper “Overexpression of a pH-sensitive nitrate transporter in rice increases crop yields” has been published in the Proceedings of the National Academy of Science www.pnas.org/content/early/2016/06/01/1525184113.full

Full article from BBSRC http://www.bbsrc.ac.uk/news/food-security/2016/160628-pr-protein-discovered-boosts-rice-yield/?utm_source=MailingList&utm_medium=email&utm_campaign=BBSRC+News+-+July+2016

 

Development of an accurate kinetic model for the central carbon metabolism of Escherichia coli

Background

A kinetic model provides insights into the dynamic response of biological systems and predicts how their complex metabolic and gene regulatory networks generate particular functions. Of many biological systems,Escherichia coli metabolic pathways have been modeled extensively at the enzymatic and genetic levels, but existing models cannot accurately reproduce experimental behaviors in a batch culture, due to the inadequate estimation of a specific cell growth rate and a large number of unmeasured parameters.

Results

In this study, we developed a detailed kinetic model for the central carbon metabolism of E. coli in a batch culture, which includes the glycolytic pathway, tricarboxylic acid cycle, pentose phosphate pathway, Entner-Doudoroff pathway, anaplerotic pathway, glyoxylate shunt, oxidative phosphorylation, phosphotransferase system (Pts), non-Pts and metabolic gene regulations by four protein transcription factors: cAMP receptor, catabolite repressor/activator, pyruvate dehydrogenase complex repressor and isocitrate lyase regulator. The kinetic parameters were estimated by a constrained optimization method on a supercomputer. The model estimated a specific growth rate based on reaction kinetics and accurately reproduced the dynamics of wild-type E. coli and multiple genetic mutants in a batch culture.

Conclusions

This model overcame the intrinsic limitations of existing kinetic models in a batch culture, predicted the effects of multilayer regulations (allosteric effectors and gene expression) on central carbon metabolism and proposed rationally designed fast-growing cells based on understandings of molecular processes.

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Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables

Constructing microbial biocatalysts that produce biorenewables at economically viable yields and titers is often hampered by product toxicity. For production of short chain fatty acids, membrane damage is considered the primary mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering efforts in Escherichia coli to increase membrane integrity, with the goal of increasing fatty acid tolerance and production, have had mixed results. Herein, a novel approach was used to reconstruct the E. coli membrane by enabling production of a novel membrane component. Specifically, trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed, meaning that membrane polarization and rigidity were increased by TUFA incorporation. As a result, tolerance to exogenously added octanoic acid and production of octanoic acid were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene production, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviation of bio-product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness.

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Spatial organization shapes the turnover of a bacterial transcriptome

Spatial organization of the transcriptome has emerged as a powerful means for regulating the post-transcriptional fate of RNA in eukaryotes; however, whether prokaryotes use RNA spatial organization as a mechanism for post-transcriptional regulation remains unclear. Here we used super-resolution microscopy to image the E. coli transcriptome and observed a genome-wide spatial organization of RNA: mRNAs encoding inner-membrane proteins are enriched at the membrane, whereas mRNAs encoding outer-membrane, cytoplasmic and periplasmic proteins are distributed throughout the cytoplasm. Membrane enrichment is caused by co-translational insertion of signal peptides recognized by the signal-recognition particle. Time-resolved RNA-sequencing revealed that degradation rates of inner-membrane-protein mRNAs are on average greater that those of the other mRNAs and that this selective destabilization of inner-membrane-protein mRNAs is abolished by dissociating the RNA degradosome from the membrane. Together, these results demonstrate that the bacterial transcriptome is spatially organized and suggest that this organization shapes the post-transcriptional dynamics of mRNAs.

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Enhancing antibody folding and secretion by tailoring the Saccharomyces cerevisiae endoplasmic reticulum

Abstract

Background
The yeast Saccharomyces cerevisiae provides intriguing possibilities for synthetic biology and bioprocess applications, but its use is still constrained by cellular characteristics that limit the product yields. Considering the production of advanced biopharmaceuticals, a major hindrance lies in the yeast endoplasmic reticulum (ER), as it is not equipped for efficient and large scale folding of complex proteins, such as human antibodies.

Results
Following the example of professional secretory cells, we show that inducing an ER expansion in yeast by deleting the lipid-regulator gene OPI1 can improve the secretion capacity of full-length antibodies up to fourfold. Based on wild-type and ER-enlarged yeast strains, we conducted a screening of a folding factor overexpression library to identify proteins and their expression levels that enhance the secretion of antibodies. Out of six genes tested, addition of the peptidyl-prolyl isomerase CPR5 provided the most beneficial effect on specific product yield while PDI1, ERO1, KAR2, LHS1 and SIL1 had a mild or even negative effect to antibody secretion efficiency. Combining genes for ER enhancement did not induce any significant additional effect compared to addition of just one element. By combining the Δopi1 strain, with the enlarged ER, with CPR5 overexpression, we were able to boost the specific antibody product yield by a factor of 10 relative to the non-engineered strain.

Conclusions
Engineering protein folding in vivo is a major task for biopharmaceuticals production in yeast and needs to be optimized at several levels. By rational strain design and high-throughput screening applications we were able to increase the specific secreted antibody yields of S. cerevisiae up to 10-fold, providing a promising strain for further process optimization and platform development for antibody production.

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Genetic engineering of Synechocystis PCC6803 for the photoautotrophic production of the sweetener erythritol

Background
Erythritol is a polyol that is used in the food and beverage industry. Due to its non-caloric and non-cariogenic properties, the popularity of this sweetener is increasing. Large scale production of erythritol is currently based on conversion of glucose by selected fungi. In this study, we describe a biotechnological process to produce erythritol from light and CO2, using engineered Synechocystis sp. PCC6803.

Methods
By functionally expressing codon-optimized genes encoding the erythrose-4-phosphate phosphatase TM1254 and the erythrose reductase Gcy1p, or GLD1, this cyanobacterium can directly convert the Calvin cycle intermediate erythrose-4-phosphate into erythritol via a two-step process and release the polyol sugar in the extracellular medium. Further modifications targeted enzyme expression and pathway intermediates.

Conclusions
After several optimization steps, the best strain, SEP024, produced up to 2.1 mM (256 mg/l) erythritol, excreted in the medium.

Read the full article here.