Category Archives: SCIENCE

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

 

Naturally occurring transporter protein discovered which boosts rice yield by 50%

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

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.

Read the full article here.

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

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.

Read the full article here.

Spatial organization shapes the turnover of a bacterial transcriptome

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.

Read the full article here.

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

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.

Read the full article here.

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

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.

Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum

Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum

Background
Engineering lactic acid bacteria (LAB) is of growing importance for food and feed industry as well as for in vivo vaccination or the production of recombinant proteins in food grade organisms. Often, expression of a transgene is only desired at a certain time point or period, e.g. to minimize the metabolic burden for the host cell or to control the expression time span. For this purpose, inducible expression systems are preferred, though cost and availability of the inducing agent must be feasible. We selected the plasmid free strain Lactobacillus plantarum 3NSH for testing and characterization of novel inducible promoters/repressor systems. Their feasibility in recombinant protein production was evaluated. Expression of the reporter protein mCherry was monitored with the BioLector® micro-fermentation system.

Results
Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that PlacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. PxylA was inducible by xylose, yet showed basal expression under non-induced conditions. Growth on galactose (as compared to exponential growth phase on glucose) reduced basal mCherry expression at non-induced conditions. PlacSynth was inducible with TMG (methyl β-D-thiogalactopyranoside) and IPTG (isopropyl β-D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter PlacSynth was used for establishment of a dual plasmid expression system, based on T7 RNA polymerase driven expression in L. plantarum. Comparative Western blot supported BioLector® micro-fermentation measurements. Conclusively, overall expression levels were moderate (compared to a constitutive promoter).

Conclusions
We evaluated different inducible promoters, as well as an orthologous expression system, for controlled gene expression in L. plantarum. Furthermore, here we provide proof of concept for a T7 RNA polymerase based expression system for L. plantarum. Thereby we expanded the molecular toolbox for an industrial relevant and generally regarded as safe (GRAS) strain.

Read the full article here.

Enhancing full-length antibody production by signal peptide engineering

Enhancing full-length antibody production by signal peptide engineering

Background
Protein secretion to the periplasm of Escherichia coli offers an attractive route for producing heterologous proteins including antibodies. In this approach, a signal peptide is fused to the N-terminus of the heterologous protein. The signal peptide mediates translocation of the heterologous protein from the cytoplasm to the periplasm and is cleaved during the translocation process. It was previously shown that optimization of the translation initiation region (TIR) which overlaps with the nucleotide sequence of the signal sequence improves the production of heterologous proteins. Despite the progress, there is still room to improve yields using secretion as a means to produce protein complexes such as full-length monoclonal antibodies (mAbs).
Results
In this study we identified the inefficient secretion of heavy chain as the limitation for full-length mAb accumulation in the periplasm. To improve heavy chain secretion we investigated the effects of various signal peptides at controlled TIR strengths. The signal peptide of disulfide oxidoreductase (DsbA) mediated more efficient secretion of heavy chain than the other signal peptides tested. Mutagenesis studies demonstrated that at controlled translational levels, hydrophobicity of the hydrophobic core (H-region) of the signal peptide is a critical factor for heavy chain secretion and full-length mAb accumulation in the periplasm. Increasing the hydrophobicity of a signal peptide enhanced heavy chain secretion and periplasmic levels of assembled full-length mAbs, while decreasing the hydrophobicity had the opposite effect.
Conclusions
This study demonstrates that under similar translational strengths, the hydrophobicity of the signal peptide plays an important role in heavy chain secretion. Increasing the hydrophobicity of the H-region and controlling TIR strengths can serve as an approach to improve heavy chain secretion and full-length mAb production in E. coli.
Read the full article here.

Characterization of acetic acid-detoxifying Escherichia coli evolved under phosphate starvation conditions

Characterization of acetic acid-detoxifying Escherichia coli evolved under phosphate starvation conditions

Background
During prolonged incubation of Escherichia coli K-12 in batch culture under aerobic, phosphate (Pi) starvation conditions, excess glucose is converted into acetic acid, which may trigger cell death. Following serial cultures, we isolated five evolved strains in two populations that survived prolonged incubation.

Methods
We sequenced the genomes of the ancestral and evolved strains, and determined the effects of the genetic changes, tested alone and in combination, on characteristic phenotypes in pure and in mixed cultures.

Results
Evolved strains used two main strategies: (1) the constitutive expression of the Trk- and Kdp-dependent K+ transport systems, and (2) the inactivation of the ArcA global regulator. Both processes helped to maintain a residual activity of the tricarboxylic acid cycle, which decreased the production of acetic acid and eventually allowed its re-consumption. Evolved strains acquired a few additional genetic changes besides the trkH, kdpD and arcA mutations, which might increase the scavenging of organophosphates (phnE +, lapB, and rseP) and the resistance to oxidative (rsxC) and acetic acid stresses (e14−/icd +).

Conclusions
Evolved strains rapidly acquired mutations (phnE + lapB rpoS trkH and phnE + rseP kdpD) that were globally beneficial to growth on glucose and organophosphates, but detrimental to long-term viability. The spread of these mutant strains might give the ancestral strain time to accumulate up to five genetic changes (phnE + arcA rsxC crfC e14−/icd +), which allowed growth on glucose and organophosphates, and provided a long-term survival. The latter strain, which expressed several mechanisms of protection against endogenous and exogenous stresses, might provide a platform for producing toxic recombinant proteins and chemicals during prolonged incubation under aerobic, Pi starvation conditions.

Read the full article here.

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