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Synthetic Biology
Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications
May 22, 2017   PloS One
Christen M, Del Medico L, Christen H, Christen B
Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications
May 22, 2017
PloS One
Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.
Plasma and Serum Metabolite Association Networks: Comparability within and between Studies Using NMR and MS Profiling
May 18, 2017   Journal Of Proteome Research
Suarez-Diez M, Adam J, Adamski J, Chasapi SA, Luchinat C, Peters A, Prehn C, Santucci C, Spyridonidis A, Spyroulias GA, Tenori L, Wang-Sattler R, Saccenti E
Plasma and Serum Metabolite Association Networks: Comparability within and between Studies Using NMR and MS Profiling
May 18, 2017
Journal Of Proteome Research
Blood is one of the most used biofluids in metabolomics studies, and the serum and plasma fractions are routinely used as a proxy for blood itself. Here we investigated the association networks of an array of 29 metabolites identified and quantified via NMR in the plasma and serum samples of two cohorts of ∼1000 healthy blood donors each. A second study of 377 individuals was used to extract plasma and serum samples from the same individual on which a set of 122 metabolites were detected and quantified using FIA-MS/MS. Four different inference algorithms (ARANCE, CLR, CORR, and PCLRC) were used to obtain consensus networks. The plasma and serum networks obtained from different studies showed different topological properties with the serum network being more connected than the plasma network. On a global level, metabolite association networks from plasma and serum fractions obtained from the same blood sample of healthy people show similar topologies, and at a local level, some differences arise like in the case of amino acids.
Short single-stranded DNA degradation products augment the activation of Toll-like receptor 9
May 22, 2017   Nature Communications
Pohar J, Lainšček D, Ivičak-Kocjan K, Cajnko MM, Jerala R, Benčina M
Short single-stranded DNA degradation products augment the activation of Toll-like receptor 9
May 22, 2017
Nature Communications
Toll-like receptors encounter a diversity of degradation products in endosomes. TLR7 and TLR8 have been shown to be activated by RNA degradation products. Here we show that although TLR9 requires single-stranded DNA longer than 20 nucleotides for a robust response, TLR9 activation is augmented by CpG-containing oligodeoxyribonucleotides (sODNs) as short as 2 nucleotides, which, by themselves, do not induce activation in cell cultures, as well as in mice. sODNs also activate human TLR9 in combination with ODNs containing a single CpG motif that by themselves do not activate human TLR9. The specific sequence motif of sODN and colocalization of ODN and sODN suggest that the mechanism of activation involves binding of both ODN and sODN to TLR9. sODNs augment TLR9 activation by mammalian genomic DNA indicating the role of short DNA degradation products in the endosomes in response to infection or in autoimmune disease, particularly at limiting concentrations of ODNs.
Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA
May 12, 2017   Nucleic Acids Research
Lau YH, Stirling F, Kuo J, Karrenbelt MAP, Chan YA, Riesselman A, Horton CA, Schäfer E, Lips D, Weinstock MT, Gibson DG, Way JC, Silver PA
Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA
May 12, 2017
Nucleic Acids Research
The ability to rewrite large stretches of genomic DNA enables the creation of new organisms with customized functions. However, few methods currently exist for accumulating such widespread genomic changes in a single organism. In this study, we demonstrate a rapid approach for rewriting bacterial genomes with modified synthetic DNA. We recode 200 kb of the Salmonella typhimurium LT2 genome through a process we term SIRCAS (stepwise integration of rolling circle amplified segments), towards constructing an attenuated and genetically isolated bacterial chassis. The SIRCAS process involves direct iterative recombineering of 10-25 kb synthetic DNA constructs which are assembled in yeast and amplified by rolling circle amplification. Using SIRCAS, we create a Salmonella with 1557 synonymous leucine codon replacements across 176 genes, the largest number of cumulative recoding changes in a single bacterial strain to date. We demonstrate reproducibility over sixteen two-day cycles of integration and parallelization for hierarchical construction of a synthetic genome by conjugation. The resulting recoded strain grows at a similar rate to the wild-type strain and does not exhibit any major growth defects. This work is the first instance of synthetic bacterial recoding beyond the Escherichia coli genome, and reveals that Salmonella is remarkably amenable to genome-scale modification. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells
May 19, 2017   Nucleic Acids Research
Kawasaki S, Fujita Y, Nagaike T, Tomita K, Saito H
Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells
May 19, 2017
Nucleic Acids Research
Synthetic biology has great potential for future therapeutic applications including autonomous cell programming through the detection of protein signals and the production of desired outputs. Synthetic RNA devices are promising for this purpose. However, the number of available devices is limited due to the difficulty in the detection of endogenous proteins within a cell. Here, we show a strategy to construct synthetic mRNA devices that detect endogenous proteins in living cells, control translation and distinguish cell types. We engineered protein-binding aptamers that have increased stability in the secondary structures of their active conformation. The designed devices can efficiently respond to target proteins including human LIN28A and U1A proteins, while the original aptamers failed to do so. Moreover, mRNA delivery of an LIN28A-responsive device into human induced pluripotent stem cells (hiPSCs) revealed that we can distinguish living hiPSCs and differentiated cells by quantifying endogenous LIN28A protein expression level. Thus, our endogenous protein-driven RNA devices determine live-cell states and program mammalian cells based on intracellular protein information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
Translation initiation events on structured eukaryotic mRNAs generate gene expression noise
May 18, 2017   Nucleic Acids Research
Dacheux E, Malys N, Meng X, Ramachandran V, Mendes P, McCarthy JE
Translation initiation events on structured eukaryotic mRNAs generate gene expression noise
May 18, 2017
Nucleic Acids Research
Gene expression stochasticity plays a major role in biology, creating non-genetic cellular individuality and influencing multiple processes, including differentiation and stress responses. We have addressed the lack of knowledge about posttranscriptional contributions to noise by determining cell-to-cell variations in the abundance of mRNA and reporter protein in yeast. Two types of structural element, a stem-loop and a poly(G) motif, not only inhibit translation initiation when inserted into an mRNA 5΄ untranslated region, but also generate noise. The noise-enhancing effect of the stem-loop structure also remains operational when combined with an upstream open reading frame. This has broad significance, since these elements are known to modulate the expression of a diversity of eukaryotic genes. Our findings suggest a mechanism for posttranscriptional noise generation that will contribute to understanding of the generally poor correlation between protein-level stochasticity and transcriptional bursting. We propose that posttranscriptional stochasticity can be linked to cycles of folding/unfolding of a stem-loop structure, or to interconversion between higher-order structural conformations of a G-rich motif, and have created a correspondingly configured computational model that generates fits to the experimental data. Stochastic events occurring during the ribosomal scanning process can therefore feature alongside transcriptional bursting as a source of noise. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
The C-terminal peptide of Aquifex aeolicus riboflavin synthase directs encapsulation of native and foreign guests by a cage-forming lumazine synthase
May 18, 2017   The Journal Of Biological Chemistry
Azuma Y, Zschoche R, Hilvert D
The C-terminal peptide of Aquifex aeolicus riboflavin synthase directs encapsulation of native and foreign guests by a cage-forming lumazine synthase
May 18, 2017
The Journal Of Biological Chemistry
Encapsulation of specific enzymes in self-assembling protein cages is a hallmark of bacterial compartments that function as counterparts to eukaryotic organelles. The cage-forming enzyme lumazine synthase (LS) from Bacillus subtilis (BsLS), for example, encapsulates riboflavin synthase (BsRS), enabling channeling of lumazine from the site of its generation to the site of its conversion to vitamin B2. Elucidating the molecular mechanisms underlying the assembly of these supramolecular complexes could help inform new approaches for metabolic engineering, nanotechnology, and drug delivery. To that end, we investigated a thermostable LS from Aquifex aeolicus (AaLS) and found that it also forms cage complexes with the cognate riboflavin synthase (AaRS) when both proteins are co-produced in the cytosol of Escherichia coli. A 12 amino acid-long peptide at the C-terminus of AaRS serves as a specific localization sequence responsible for targeting the guest to the protein compartment. Sequence comparisons suggested that analogous peptide segments likely direct RS complexation by LS cages in other bacterial species. Covalent fusion of this peptide tag to heterologous guest molecules led to their internalization into AaLS assemblies both in vivo and in vitro, providing a firm foundation for creating tailored biomimetic nanocompartments for medical and biotechnological applications. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.
AibA/AibB Induces an Intramolecular Decarboxylation in Isovalerate Biosynthesis by Myxococcus xanthus
May 16, 2017   Angewandte Chemie (International Ed. In English)
Bock T, Luxenburger E, Hoffmann J, Schütza V, Feiler C, Müller R, Blankenfeldt W
AibA/AibB Induces an Intramolecular Decarboxylation in Isovalerate Biosynthesis by Myxococcus xanthus
May 16, 2017
Angewandte Chemie (International Ed. In English)
Isovaleryl coenzyme A (IV-CoA) is an important precursor for iso-fatty acids and lipids. It acts in the development of myxobacteria, which can produce this compound from acetyl-CoA through alternative IV-CoA biosynthesis (aib). A central reaction of aib is catalyzed by AibA/AibB, which acts as a cofactor-free decarboxylase despite belonging to the family of CoA-transferases. We developed an efficient expression system for AibA/AibB that allowed the determination of high-resolution crystal structures in complex with different ligands. Through mutational studies, we show that an active-site cysteine previously proposed to be involved in decarboxylation is not required for activity. Instead, AibA/AibB seems to induce an intramolecular decarboxylation by binding its substrate in a hydrophobic cavity and forcing it into a bent conformation. Our study opens opportunities for synthetic biology studies, since AibA/AibB may be suitable for the production of isobutene, a precursor of biofuels and chemicals. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Engineering RGB color vision into Escherichia coli
May 22, 2017   Nature Chemical Biology Add nature.com free-link Cancel
Fernandez-Rodriguez J, Moser F, Song M, Voigt CA
Engineering RGB color vision into Escherichia coli
May 22, 2017
Nature Chemical Biology
Optogenetic tools use colored light to rapidly control gene expression in space and time. We designed a genetically encoded system that gives Escherichia coli the ability to distinguish between red, green, and blue (RGB) light and respond by changing gene expression. We use this system to produce 'color photographs' on bacterial culture plates by controlling pigment production and to redirect metabolic flux by expressing CRISPRi guide RNAs.
Synthetic biology: Return to sender
May 17, 2017   Nature Chemical Biology Add nature.com free-link Cancel
Miura G
Synthetic biology: Return to sender
May 17, 2017
Nature Chemical Biology
A conserved threonine prevents self-intoxication of enoyl-thioester reductases
May 15, 2017   Nature Chemical Biology Add nature.com free-link Cancel
Rosenthal RG, Vögeli B, Wagner T, Shima S, Erb TJ
A conserved threonine prevents self-intoxication of enoyl-thioester reductases
May 15, 2017
Nature Chemical Biology
Enzymes are highly specific biocatalysts, yet they can promote unwanted side reactions. Here we investigated the factors that direct catalysis in the enoyl-thioester reductase Etr1p. We show that a single conserved threonine is essential to suppress the formation of a side product that would otherwise act as a high-affinity inhibitor of the enzyme. Substitution of this threonine with isosteric valine increases side-product formation by more than six orders of magnitude, while decreasing turnover frequency by only one order of magnitude. Our results show that the promotion of wanted reactions and the suppression of unwanted side reactions operate independently at the active site of Etr1p, and that the active suppression of side reactions is highly conserved in the family of medium-chain dehydrogenases/reductases (MDRs). Our discovery emphasizes the fact that the active destabilization of competing transition states is an important factor during catalysis that has implications for the understanding and the de novo design of enzymes.
Interlocked DNA topologies for nanotechnology
May 15, 2017   Current Opinion In Biotechnology
Valero J, Lohmann F, Famulok M
Interlocked DNA topologies for nanotechnology
May 15, 2017
Current Opinion In Biotechnology
Interlocked molecular architectures are well known in supramolecular chemistry and are widely used for various applications like sensors, molecular machines and logic gates. The use of DNA for constructing these interlocked structures has increased significantly within the current decade. Because of Watson-Crick base pairing rules, DNA is an excellent material for the self-assembly of well-defined interlocked nanoarchitectures. These DNA nanostructures exhibit sufficient stability, good solubility in aqueous media, biocompatibility, and can be easily combined with other biomolecules in bio-hybrid nano-assemblies. Therefore, the study of novel DNA-based interlocked systems is of interest for nanotechnology, synthetic biology, supramolecular chemistry, biotechnology, and for sensing purposes. Here we summarize recent developments and applications of interlocked supramolecular architectures made of DNA. Examples illustrating that these systems can be precisely controlled by switching on and off the molecular motion of its mechanically trapped components are discussed. Introducing different triggers into such systems creates molecular assemblies capable of performing logic gate operations and/or catalytic activity control. Interlocked DNA-based nanostructures thus represent promising frameworks for building increasingly complex and dynamic nanomachines with highly controllable functionality. Copyright © 2017 Elsevier Ltd. All rights reserved.
Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator
May 12, 2017   Scientific Reports
Hanko EKR, Minton NP, Malys N
Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator
May 12, 2017
Scientific Reports
3-hydroxypropionic acid (3-HP) is an important platform chemical used as a precursor for production of added-value compounds such as acrylic acid. Metabolically engineered yeast, Escherichia coli, cyanobacteria and other microorganisms have been developed for the biosynthesis of 3-HP. Attempts to overproduce this compound in recombinant Pseudomonas denitrificans revealed that 3-HP is consumed by this microorganism using the catabolic enzymes encoded by genes hpdH, hbdH and mmsA. 3-HP-inducible systems controlling the expression of these genes have been predicted in proteobacteria and actinobacteria. In this study, we identify and characterise 3-HP-inducible promoters and their corresponding LysR-type transcriptional regulators from Pseudomonas putida KT2440. A newly-developed modular reporter system proved possible to demonstrate that PpMmsR/P mmsA and PpHpdR/P hpdH are orthogonal and highly inducible by 3-HP in E. coli (12.3- and 23.3-fold, respectively) and Cupriavidus necator (51.5- and 516.6-fold, respectively). Bioinformatics and mutagenesis analyses revealed a conserved 40-nucleotide sequence in the hpdH promoter, which plays a key role in HpdR-mediated transcription activation. We investigate the kinetics and dynamics of the PpHpdR/P hpdH switchable system in response to 3-HP and show that it is also induced by both enantiomers of 3-hydroxybutyrate. These findings pave the way for use of the 3-HP-inducible system in synthetic biology and biotechnology applications.
Efficient modification of λ-DNA substrates for single-molecule studies
May 19, 2017   Scientific Reports
Kim Y, de la Torre A, Leal AA, Finkelstein IJ
Efficient modification of λ-DNA substrates for single-molecule studies
May 19, 2017
Scientific Reports
Single-molecule studies of protein-nucleic acid interactions frequently require site-specific modification of long DNA substrates. The bacteriophage λ is a convenient source of high quality long (48.5 kb) DNA. However, introducing specific sequences, tertiary structures, and chemical modifications into λ-DNA remains technically challenging. Most current approaches rely on multi-step ligations with low yields and incomplete products. Here, we describe a molecular toolkit for rapid preparation of modified λ-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into the λ-phage genome with 90-100% yield. Extrahelical structures and chemical modifications can be inserted at user-defined sites via an improved nicking enzyme-based strategy. As a proof-of-principle, we explore the interactions of S. cerevisiae Proliferating Cell Nuclear Antigen (yPCNA) with modified DNA sequences and structures incorporated within λ-DNA. Our results demonstrate that S. cerevisiae Replication Factor C (yRFC) can load yPCNA onto 5'-ssDNA flaps, (CAG)13 triplet repeats, and homoduplex DNA. However, yPCNA remains trapped on the (CAG)13 structure, confirming a proposed mechanism for triplet repeat expansion. We anticipate that this molecular toolbox will be broadly useful for other studies that require site-specific modification of long DNA substrates.
Airway Microbial Diversity is Inversely Associated with Mite-Sensitized Rhinitis and Asthma in Early Childhood
May 13, 2017   Scientific Reports
Chiu CY, Chan YL, Tsai YS, Chen SA, Wang CJ, Chen KF, Chung IF
Airway Microbial Diversity is Inversely Associated with Mite-Sensitized Rhinitis and Asthma in Early Childhood
May 13, 2017
Scientific Reports
Microbiota plays an important role in regulating immune responses associated with atopic diseases. We sought to evaluate relationships among airway microbiota, serum IgE levels, allergic sensitization and their relevance to rhinitis and asthma. Microbial characterization was performed using Illumina-based 16S rRNA gene sequencing of 87 throat swabs collected from children with asthma (n = 32) and rhinitis (n = 23), and from healthy controls (n = 32). Data analysis was performed using QIIME (Quantitative Insights Into Microbial Ecology) v1.8. Significantly higher abundance of Proteobacteria was found in children with rhinitis than in the healthy controls (20.1% vs. 16.1%, P = 0.009). Bacterial species richness (Chao1 index) and diversity (Shannon index) were significantly reduced in children with mite sensitization but not in those with food or IgE sensitization. Compared with healthy children without mite sensitization, the mite-sensitized children with rhinitis and asthma showed significantly lower Chao1 and Shannon indices. Moraxella and Leptotrichia species were significantly found in the interaction of mite sensitization with rhinitis and asthma respectively. Airway microbial diversity appears to be inversely associated with sensitization to house dust mites. A modulation between airway dysbiosis and responses to allergens may potentially cause susceptibility to rhinitis and asthma in early childhood.
Combined treatment with Metformin and 2-deoxy glucose induces detachment of viable MDA-MB-231 breast cancer cells in vitro
May 12, 2017   Scientific Reports
Bizjak M, Malavašič P, Dolinar K, Pohar J, Pirkmajer S, Pavlin M
Combined treatment with Metformin and 2-deoxy glucose induces detachment of viable MDA-MB-231 breast cancer cells in vitro
May 12, 2017
Scientific Reports
Triple naegative breast cancer has an increased rate of distant metastasis and consequently poor prognosis. To metastasize, breast cancer cells must detach from the main tumour mass and resist anoikis, a programmed cell death induced by lack of cell-extracellular matrix communication. Although cancer cells must detach to metastasize in vivo, the viability of floating cancer cells in vitro is rarely investigated. Here we show that co-treatment of anoikis-resistant MDA-MB-231 cells with metformin and 2-deoxy-D-glucose (2-DG) increased the percentage of floating cells, of which about 95% were viable. Floating cells resumed their proliferation once they were reseeded in the pharmacological compound-free medium. Similar effects on detachment were observed on anoikis-prone MCF-7 cells. Co-treatment of MDA-MB-231 cells with metformin and 2-DG induced a strong activation of AMP-activated protein kinase (AMPK), which was reduced by AMPK inhibitor compound C that prevented detachment of MDA-MB-231 cells. However, direct AMPK activators A-769662 and AICAR did not have any major effect on the percentage of floating MDA-MB-231 cells, indicating that AMPK activation is necessary but not sufficient for triggering detachment of cancer cells. Our results demonstrate that separate analysis of floating and attached cancer cells might be important for evaluation of anti-cancer agents.
Plant-derived transcription factors for orthologous regulation of gene expression in the yeast Saccharomyces cerevisiae
May 22, 2017   ACS Synthetic Biology
Naseri G, Balazadeh S, Machens F, Kamranfar I, Messerschmidt K, Mueller-Roeber B
Plant-derived transcription factors for orthologous regulation of gene expression in the yeast Saccharomyces cerevisiae
May 22, 2017
ACS Synthetic Biology
Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects where tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harbouring cognate cis-regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver / reporter combinations varied over a wide spectrum, with EDLL being a considerable stronger transcription activation domain in yeast, than GAL4 and VP64 activation domains, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC - EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by episomal plasmids. Our library of TF - DNA-binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.
Meeting Report for Synthetic Biology for Natural Products 2017: The Interface of (Meta)Genomics, Machine Learning, and Natural Product Discovery
May 19, 2017   ACS Synthetic Biology
Smanski MJ, Mead D, Gustafsson C, Thomas MG
Modularly Constructed Synthetic Granzyme B Molecule Enables Interrogation of Intracellular Proteases for Targeted Cytotoxicity
May 16, 2017   ACS Synthetic Biology
Ho P, Ede C, Chen YY
Modularly Constructed Synthetic Granzyme B Molecule Enables Interrogation of Intracellular Proteases for Targeted Cytotoxicity
May 16, 2017
ACS Synthetic Biology
Targeted therapies promise to increase the safety and efficacy of treatments against diseases ranging from cancer to viral infections. However, the vast majority of targeted therapeutics relies on the recognition of extracellular biomarkers, which are rarely restricted to diseased cells and are thus prone to severe and sometimes-fatal off-target toxicities. In contrast, intracellular antigens present a diverse yet underutilized repertoire of disease markers. Here, we report a protein-based therapeutic platform-termed Cytoplasmic Oncoprotein VErifier and Response Trigger (COVERT)-which enables the interrogation of intracellular proteases to trigger targeted cytotoxicity. COVERT molecules consist of the cytotoxic protein granzyme B (GrB) fused to an inhibitory N-terminal peptide, which can be removed by researcher-specified proteases to activate GrB function. We demonstrate that fusion of a small ubiquitin-like modifier 1 (SUMO1) protein to GrB yields a SUMO-GrB molecule that is specifically activated by the cancer-associated sentrin-specific protease 1 (SENP1). SUMO-GrB selectively triggers apoptotic phenotypes in HEK293T cells that overexpress SENP1, and it is highly sensitive to different SENP1 levels across cell lines. We further demonstrate the rational design of additional COVERT molecules responsive to enterokinase (EK) and tobacco etch virus protease (TEVp), highlighting the COVERT platform's modularity and adaptability to diverse protease targets. As an initial step toward engineering COVERT-T cells for adoptive T-cell therapy, we verified that primary human T cells can express, package, traffic, and deliver engineered GrB molecules in response to antigen stimulation. Our findings set the foundation for future intracellular-antigen‒responsive therapeutics that can complement surface-targeted therapies.
A Novel and Efficient Method for Bacteria Genome Editing Employing both CRISPR/Cas9 and an Antibiotic Resistance Cassette
May 22, 2017   Frontiers In Microbiology
Zhang H, Cheng QX, Liu AM, Zhao GP, Wang J
A Novel and Efficient Method for Bacteria Genome Editing Employing both CRISPR/Cas9 and an Antibiotic Resistance Cassette
May 22, 2017
Frontiers In Microbiology
As Cas9-mediated cleavage requires both protospacer and protospacer adjacent motif (PAM) sequences, it is impossible to employ the CRISPR/Cas9 system to directly edit genomic sites without available PAM sequences nearby. Here, we optimized the CRISPR/Cas9 system and developed an innovative two-step strategy for efficient genome editing of any sites, which did not rely on the availability of PAM sequences. An antibiotic resistance cassette was employed as both a positive and a negative selection marker. By integrating the optimized two-plasmid CRISPR/Cas system and donor DNA, we achieved gene insertion and point mutation with high efficiency in Escherichia coli, and importantly, obtained clean mutants with no other unwanted mutations. Moreover, genome editing of essential genes was successfully achieved using this approach with a few modifications. Therefore, our newly developed method is PAM-independent and can be used to edit any genomic loci, and we hope this method can also be used for efficient genome editing in other organisms.
Building a bio-based industry in the Middle East through harnessing the potential of the Red Sea biodiversity
May 21, 2017   Applied Microbiology And Biotechnology
Nielsen J, Archer J, Essack M, Bajic VB, Gojobori T, Mijakovic I
Building a bio-based industry in the Middle East through harnessing the potential of the Red Sea biodiversity
May 21, 2017
Applied Microbiology And Biotechnology
The incentive for developing microbial cell factories for production of fuels and chemicals comes from the ability of microbes to deliver these valuable compounds at a reduced cost and with a smaller environmental impact compared to the analogous chemical synthesis. Another crucial advantage of microbes is their great biological diversity, which offers a much larger "catalog" of molecules than the one obtainable by chemical synthesis. Adaptation to different environments is one of the important drives behind microbial diversity. We argue that the Red Sea, which is a rather unique marine niche, represents a remarkable source of biodiversity that can be geared towards economical and sustainable bioproduction processes in the local area and can be competitive in the international bio-based economy. Recent bioprospecting studies, conducted by the King Abdullah University of Science and Technology, have established important leads on the Red Sea biological potential, with newly isolated strains of Bacilli and Cyanobacteria. We argue that these two groups of local organisms are currently most promising in terms of developing cell factories, due to their ability to operate in saline conditions, thus reducing the cost of desalination and sterilization. The ability of Cyanobacteria to perform photosynthesis can be fully exploited in this particular environment with one of the highest levels of irradiation on the planet. We highlight the importance of new experimental and in silico methodologies needed to overcome the hurdles of developing efficient cell factories from the Red Sea isolates.
Harnessing plant metabolic diversity
May 20, 2017   Current Opinion In Chemical Biology
Owen C, Patron NJ, Huang A, Osbourn A
Harnessing plant metabolic diversity
May 20, 2017
Current Opinion In Chemical Biology
Advances in DNA sequencing and synthesis technologies in the twenty-first century are now making it possible to build large-scale pipelines for engineering plant natural product pathways into heterologous production species using synthetic biology approaches. The ability to decode the chemical potential of plants by sequencing their transcriptomes and/or genomes and to then use this information as an instruction manual to make drugs and other high-value chemicals is opening up new routes to harness the vast chemical diversity of the Plant Kingdom. Here we describe recent progress in methods for pathway discovery, DNA synthesis and assembly, and expression of engineered pathways in heterologous hosts. We also highlight the importance of standardization and the challenges associated with dataset integration in the drive to build a systematic framework for effective harnessing of plant metabolic diversity. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.
Systematic identification of functional residues of Artemisia annua amorpha-4,11-diene synthase
May 20, 2017   The Biochemical Journal
Fang X, Li JX, Huang JQ, Xiao YL, Zhang P, Chen XY
Systematic identification of functional residues of Artemisia annua amorpha-4,11-diene synthase
May 20, 2017
The Biochemical Journal
Terpene synthases (TPSs) are responsible for the extremely diversified and complex structure of terpenoids. Amorpha-4,11-diene synthase (ADS) has a high (90%) fidelity in generating the sesquiterpene precursor for biosynthesis of artemisinin, an antimalarial drug, however, little is known about how active site residues of ADS involved in the carbocation rearrangement and cyclization reactions. Here, we identify seven residues that are key to most of the catalytic steps in ADS. By structural modeling and amino acid sequence alignments of ADS with two functionally relevant sesquiterpene synthases from Artemisia annua, we performed site-directed mutagenesis and found that a single substitution, T296V, impaired the ring closure activity almost completely, and tetra-substitutions (L374Y/L404V/L405I/G439S) led to an enzyme generating 80% monocyclic bisabolyl-type sesquiterpenes, whereas a double mutant (T399L/T447G) showed compromised activity in regioselective deprotonation to yield 34.7% and 37.7% normal and aberrant deprotonation products, respectively. Notably, Thr296, Leu374, Gly439, Thr399 and Thr447, which play a major role in directing the catalytic cascades, are located around conserved metal-binding motifs and function through impacting the folding of substrate/intermediate, implying that residues surrounding the two motifs could be valuable targets for engineering TPS activity. Using this knowledge, we increased amorpha-4,11-diene production substantially in a near-additive manner by engineering Thr399 and Thr447 for product releasing. Our results provide a new room for rational design of enzyme activity for synthetic biology. ©2017 The Author(s).
Insights into the prevalence and underlying causes of clonal variation through transcriptomic analysis in Pichia pastoris
May 23, 2017   Applied Microbiology And Biotechnology
Aw R, Barton GR, Leak DJ
Insights into the prevalence and underlying causes of clonal variation through transcriptomic analysis in Pichia pastoris
May 23, 2017
Applied Microbiology And Biotechnology
Clonal variation, wherein a range of specific productivities of secreted proteins are observed from supposedly identical transformants, is an accepted aspect of working with Pichia pastoris. It means that a significant number of transformants need to be tested to obtain a representative sample, and in commercial protein production, companies regularly screen thousands of transformants to select for the highest secretor. Here, we have undertaken a detailed investigation of this phenomenon by characterising clones transformed with the human serum albumin gene. The titers of nine clones, each containing a single copy of the human serum albumin gene (identified by qPCR), were measured and the clones grouped into three categories, namely, high-, mid- and low-level secretors. Transcriptomic analysis, using microarrays, showed that no regulatory patterns consistently correlated with titer, suggesting that the causes of clonal variation are varied. However, a number of physiological changes appeared to underlie the differences in titer, suggesting there is more than one biochemical signature for a high-secreting strain. An anomalous low-secreting strain displaying high transcript levels that appeared to be nutritionally starved further emphasises the complicated nature of clonal variation.
Enhanced Production of Gypenoside LXXV Using a Novel Ginsenoside-Transforming β-Glucosidase from Ginseng-Cultivating Soil Bacteria and Its Anti-Cancer Property
May 23, 2017   Molecules (Basel, Switzerland)
Cui CH, Kim DJ, Jung SC, Kim SC, Im WT
Enhanced Production of Gypenoside LXXV Using a Novel Ginsenoside-Transforming β-Glucosidase from Ginseng-Cultivating Soil Bacteria and Its Anti-Cancer Property
May 23, 2017
Molecules (Basel, Switzerland)
Minor ginsenosides, such as compound K, Rg₃(S), which can be produced by deglycosylation of ginsenosides Rb₁, showed strong anti-cancer effects. However, the anticancer effects of gypenoside LXXV, which is one of the deglycosylated shapes of ginsenoside Rb₁, is still unknown due to the rarity of its content in plants. Here, we cloned and characterized a novel ginsenoside-transforming β-glucosidase (BglG167b) derived from Microbacterium sp. Gsoil 167 which can efficiently hydrolyze gypenoside XVII into gypenoside LXXV, and applied it to the production of gypenoside LXXV at the gram-scale with high specificity. In addition, the anti-cancer activity of gypenoside LXXV was investigated against three cancer cell lines (HeLa, B16, and MDA-MB231) in vitro. Gypenoside LXXV significantly reduced cell viability, displaying an enhanced anti-cancer effect compared to gypenoside XVII and Rb₁. Taken together, this enzymatic method would be useful in the preparation of gypenoside LXXV for the functional food and pharmaceutical industries.

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