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  • 更新日期:2017-11-20
  • New Intracellular Shikimic Acid Biosensor for Monitoring Shikimate Synthesis in Corynebacterium glutamicum
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-15
    Chang Liu, Bo Zhang, Yi-Ming Liu, Ke-Qian Yang, Shuang-Jiang Liu
    更新日期:2017-11-15
  • Emergent Properties in Complex Synthetic Bacterial Promoters
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-14
    Lummy Maria Oliveira Monteiro, Letícia Magalhães Arruda, Rafael Silva-Rocha
    更新日期:2017-11-15
  • Split2 protein-ligation generates active IL-6-type Hyper-cytokines from inactive precursors.
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-14
    Jens Mark Moll, Melanie Wehmöller, Nils Christopher Frank, Lisa Homey, Paul Baran, Christoph Garbers, Larissa Lamertz, Jonathan H. Axelrod, Eithan Galun, Henning D. Mootz, Jürgen Scheller

    Trans-signaling of the major pro- and anti-inflammatory cytokines Interleukin (IL)-6 and IL-11 has the unique feature to virtually activate all cells of the body and is critically involved in chronic inflammation and regeneration. Hyper-IL-6 and Hyper-IL-11 are single chain designer trans-signaling cytokines, in which the cytokine and soluble receptor units are trapped in one complex via a flexible peptide linker. Albeit, Hyper-cytokines are essential tools to study trans-signaling in vitro and in vivo, the superior potency of these designer cytokines are accompanied by undesirable stress responses. To enable tailor-made generation of Hyper-cytokines, we developed inactive split-cytokine-precursors adapted for posttranslational reassembly by split-intein mediated protein trans-splicing (PTS). We identified cutting sites within IL-6 (E134/S135) and IL-11 (G116/S117) and obtained inactive split-Hyper-IL-6 and split-Hyper-IL-11 cytokine precursors. After fusion with split-inteins, PTS resulted in reconstitution of active Hyper-cytokines, which were efficiently secreted from transfected cells. Our strategy comprises the development of a background-free cytokine signaling system from reversibly inactivated precursor cytokines.

    更新日期:2017-11-15
  • Dynamic Control of Aptamer–Ligand Activity Using Strand Displacement Reactions
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-10
    Jonathan Lloyd, Claire H. Tran, Krishen Wadhwani, Christian Cuba Samaniego, Hari K. K. Subramanian, Elisa Franco
    更新日期:2017-11-11
  • Yield Improvement of the Anti-MRSA Antibiotics WAP-8294A by CRISPR/dCas9 Combined with Refactoring Self-protection Genes in Lysobacter enzymogenes OH11
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-10
    Lingjun Yu, Wei Su, Paul D Fey, Fengquan Liu, Liangcheng Du

    The cyclic lipodepsipeptides WAP-8294A are antibiotics with potent activity against methicillin-resistant Staphylococcus aureus (MRSA). One member of this family, WAP-8294A2 (Lotilibcin), was in clinical trials due to its high activity and distinct chemistry. However, WAP-8294A compounds are produced in a very low yield by Lysobacter and only under very stringent conditions. Improving WAP-8294A yield has become very critical for research and application of these anti-MRSA compounds. Here, we report a strategy to increase WAP-8294A production. We first used the CRISPR/dCas9 system to increase the expression of five co-transcribed genes (orf1-5) in the WAP gene cluster, by fusing the omega subunit of RNA polymerase with dCas9 that targets the operon’s promoter region. This led to the transcription of the genes increased by 5-48 folds in strain dCas9-ω3. We then refactored four putative self-protection genes (orf6, orf7, orf9 and orf10) by reorganizing them into an operon under the control of a strong Lysobacter promoter, PHSAF. The refactored operon was introduced into strain dCas9-ω3, and the transcription of the self-protection genes increased by 20-60 folds in the resultant engineered strains. The yield of the three main WAP-8294A compounds, WAP-8294A1, WAP-8294A2, and WAP-8294A4, increased by 6, 4, and 9 folds, respectively, in the engineered strains. The data also showed that the yield increase of WAP-8294A compounds was mainly due to the increase of the extracellular distribution. WAP-8294A2 exhibited potent (MIC 0.2-0.8 μg/ml) and specific activity against S. aureus among a battery of clinically relevant Gram-positive pathogens (54 isolates).

    更新日期:2017-11-11
  • Cell-free and in vivo characterization of Lux, Las, and Rpa quorum activation systems in E. coli
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-09
    Andrew Halleran, Richard M. Murray

    Synthetic biologists have turned towards quorum systems as a path for building sophisticated microbial consortia that exhibit group decision making. Currently, however, even the most complex consortium circuits rely on only one or two quorum sensing systems, greatly restricting the available design space. High-throughput characterization of available quorum sensing systems is useful for finding compatible sets of systems that are suitable for a defined circuit architecture. Recently, cell-free systems have gained popularity as a test-bed for rapid prototyping of genetic circuitry. We take advantage of the transcription-translation cell-free system to characterize three commonly used Lux-type quorum activators, Lux, Las, and Rpa. We then compare the cell-free characterization to results obtained in vivo. We find significant genetic crosstalk in both the Las and Rpa systems and substantial signal crosstalk in Lux activation. We show that cell-free characterization predicts crosstalk observed in vivo.

    更新日期:2017-11-09
  • Engineering an E. coli Near-Infrared Light Sensor
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-09
    Nicholas T. Ong, Evan J. Olson, Jeffrey J. Tabor
    更新日期:2017-11-09
  • An Autoregulated Fine-Tuning Strategy for Titer Improvement of Secondary Metabolites Using Native Promoters in Streptomyces
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-09
    Shanshan Li, Junyang Wang, Wensheng Xiang, Keqian Yang, Zilong Li, Weishan Wang
    更新日期:2017-11-09
  • Reconfigurable Analog Signal Processing by Living Cells
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-08
    Daniel D. Lewis, Michael Chavez, Kwan Lun Chiu, Cheemeng Tan

    Living cells are known for their capacity for versatile signal processing, particularly the ability to respond differently to the same stimuli using biochemical networks that integrate environmental signals and reconfigure their dynamic responses. However, the complexity of natural biological networks confounds the discovery of fundamental mechanisms behind versatile signaling. Here, we study one specific aspect of reconfigurable signal processing in which a minimal biological network integrates two signals, using one to reconfigure the network’s transfer function with respect to the other, producing an emergent switch between induction and repression. In contrast to known mechanisms, the new mechanism reconfigures transfer functions through genetic networks without extensive protein-protein interactions. These results provide a novel explanation for the versatility of genetic programs, and suggest a new mechanism of signal integration that may govern flexibility and plasticity of gene expression.

    更新日期:2017-11-08
  • Gene-Mediated Chemical Communication in Synthetic Protocell Communities
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-08
    T-Y. Dora Tang, Dario Cecchi, Giorgio Fracasso, Davide Accardi, Angelique Coutable-Pennarun, Sheref S. Mansy, Adam W. Perriman, J. L. Ross Anderson, Stephen Mann
    更新日期:2017-11-08
  • Control of Adipogenic Differentiation in Mesenchymal Stem Cells via Endogenous Gene Activation Using CRISPR-Cas9
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-07
    Yuichi Furuhata, Yuta Nihongaki, Moritoshi Sato, Keitaro Yoshimoto
    更新日期:2017-11-08
  • Targeted Gene Repression Using Novel Bifunctional Molecules to Harness Endogenous Histone Deacetylation Activity
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-07
    Kyle V. Butler, Anna M. Chiarella, Jian Jin, Nathaniel A. Hathaway
    更新日期:2017-11-08
  • Quantitatively Predictable Control of Cellular Protein Levels through Proteasomal Degradation
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-07
    Wenting Zhao, Lara Pferdehirt, Laura Segatori
    更新日期:2017-11-08
  • Muconic Acid Production via Alternative Pathways and a Synthetic “Metabolic Funnel”
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-02
    Brian Thompson, Shawn Pugh, Michael Machas, David R. Nielsen
    更新日期:2017-11-02
  • Engineering an E. coli near-infrared light sensor
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-01
    Nicholas Ting Xun Ong, Evan J. Olson, Jeffrey J. Tabor

    Optogenetics is a technology wherein researchers combine light and genetically-engineered photoreceptors to control biological processes with unrivaled precision. Near-infrared (NIR) wavelengths (> 700 nm) are desirable optogenetic inputs due to their low phototoxicity and spectral isolation from most photoproteins. The bacteriophytochrome photoreceptor 1 (BphP1), found in several purple photosynthetic bacteria, senses NIR light and activates transcription of photosystem promoters by binding to and inhibiting the transcriptional repressor PpsR2. Here, we examine the response of a library of output promoters to increasing levels of Rhodopseudomonas palustris PpsR2 expression, and identify that of Bradyrhizobium sp. BTAi1 crtE as the most strongly repressed in E. coli. Next, we optimize R. palustris bphP1 and ppsR2 expression in a strain engineered to produce the required chromophore biliverdin IXα in order to demonstrate NIR-activated transcription. Unlike a previously engineered bacterial NIR photoreceptor, our system does not require production of a second messenger, and exhibits rapid response dynamics. It is also the most red-shifted bacterial optogenetic tool yet reported by approximately 50 nm. Accordingly, our BphP1-PpsR2 system has numerous applications in bacterial optogenetics.

    更新日期:2017-11-02
  • Gene-mediated chemical communication in synthetic protocell communities
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-01
    T-Y Dora Tang, Dario Cecchi, Giorgio Fracasso, Davide Accardi, Angelique Coutable, Sheref S Mansy, Adam W. Perriman, John Leslie Ross Anderson, Stephen Mann

    A gene-directed chemical communication pathway between synthetic protocell signaling transmitters (lipid vesicles) and receivers (proteinosomes) was designed, built and tested using a bottom-up modular approach comprising small molecule transcriptional control, cell-free gene expression, porin-directed efflux, substrate signaling, and enzyme cascade-mediated processing.

    更新日期:2017-11-02
  • Emergent properties in complex synthetic bacterial promoters
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-01
    Lummy Maria Oliveira Monteiro, Letícia Magalhães Arruda, Rafael Silva-Rocha

    Regulation of gene expression in bacteria results from the interplay between transcriptional factors (TFs) at target promoters, and how the arrangement of binding sites determines the regulatory logic of promoters is not well known. Here, we generated and fully characterized a library of synthetic complex promoters for the global regulators, CRP and IHF, in Escherichia coli, formed by a weak -35/-10 consensus sequence preceded by four combinatorial binding sites for these TFs. We found that while cis-elements for CRP preferentially activate promoters when located immediately upstream of the promoter consensus, binding sites for IHF mainly function as UP elements and stimulate transcription in several different architectures in the absence of this protein. However, the combination of CRP- and IHF-binding sites resulted in emergent properties in these complex promoters, where the activity of combinatorial promoters cannot be predicted from the individual behavior of its components. Taken together, the results presented here add to the information on architecture-logic of complex promoters in bacteria.

    更新日期:2017-11-02
  • Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-11-01
    Sen Yang, Qingtao Liu, Yunfeng Zhang, Guocheng Du, Jian Chen, Zhen Kang
    更新日期:2017-11-01
  • An autoregulated fine-tuning strategy for titer improvement of secondary metabolites using native promoters in Streptomyces
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-31
    Shanshan Li, Junyang Wang, Wensheng Xiang, Keqian Yang, Zilong Li, Weishan Wang

    Streptomycetes are well-known producers of biologically active secondary metabolites. Various efforts have endeavored to increase productions of these metabolites, while few approaches could well coordinate the biosynthesis of secondary metabolites and other physiological events of their hosts. Here we develop a universal autoregulated strategy for fine-tuning the expression of secondary metabolites biosynthetic gene clusters (BGCs) in Streptomyces species. First, inducible promoters were used to control the expression of secondary metabolites BGCs. Then, the optimal induction condition was determined by response surface model in both dimensions of time and strength. Finally, native promoters with similar transcription profile to the inducible promoter under the optimal condition were identified based on time-course transcriptome analyses, and used to replace the inducible promoter following an elaborate replacement approach. The expression of actinorhodin (Act) and heterogeneous oxytetracycline (OTC) BGCs were optimized in Streptomyces coelicolor using this strategy. Compared to modulation via constitutive promoters, it could dramatically improve the titers of Act and OTC by 1.3- and 9.1-fold, respectively. The autoregulated fine-tuning strategy developed here opens a novel route for titer improvement of desired secondary metabolites in Streptomyces.

    更新日期:2017-11-01
  • New intracellular shikimic acid biosensor for monitoring shikimate synthesis in Corynebacterium glutamicum
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-31
    Chang Liu, Bo Zhang, Yi-Ming Liu, Keqian Yang, Shuang-Jiang Liu

    The quantitative monitoring of intracellular metabolites with in vivo biosensors provides an efficient means of identifying high-yield strains and observing product accumulation in real time. In this study, a shikimic acid (SA) biosensor was constructed from a LysR-type transcriptional regulator (ShiR) of Corynebacterium glutamicum. The SA biosensor specifically responded to the increase of intracellular SA concentration over a linear range of 19.5 ± 3.6 to 120.9 ± 1.2 fmole at the single-cell level. This new SA biosensor was successfully used to 1) monitor the SA production of different C. glutamicum strains; 2) develop a novel result-oriented high-throughput ribosome binding site screening and sorting strategy that was used for engineering high-yield shikimate-producing strains; and 3) engineer a whole-cell biosensor through the co-expression of the SA sensor and a shikimate transporter shiA gene in C. glutamicum RES167. This work demonstrated that a new intracellular SA biosensor is a valuable tool facilitating the fast development of microbial SA producer.

    更新日期:2017-11-01
  • 更新日期:2017-10-31
  • In Vitro Evolution of Unmodified 16S rRNA for Simple Ribosome Reconstitution
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-31
    Yoshiki Murase, Hiroki Nakanishi, Gakushi Tsuji, Takeshi Sunami, Norikazu Ichihashi
    更新日期:2017-10-31
  • A Modular Receptor Platform To Expand the Sensing Repertoire of Bacteria
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-30
    Hung-Ju Chang, Pauline Mayonove, Agustin Zavala, Angelique De Visch, Philippe Minard, Martin Cohen-Gonsaud, Jerome Bonnet
    更新日期:2017-10-31
  • Control of adipogenic differentiation in mesenchymal stem cells via endogenous gene activation using CRISPR-Cas9
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-27
    Yuichi Furuhata, Yuta Nihongaki, Moritoshi Sato, Keitaro Yoshimoto

    Mesenchymal stem cells (MSCs) are of interest in regenerative medicine owing to their multilineage differentiation and self-renewal properties. Understanding the in vivo differentiation process is necessary for clinical applications including cell therapy and transplantation. This remains challenging owing to the lack of induction methods that imitate the natural programming process. Endogenous gene regulation of tissue specific transcription factors is therefore desirable. In the present study, we demonstrated endogenous activation of adipogenic genes through the dCas9-based transcription system, and achieved efficient induction of different types of adipocyte-like cells from MSCs. Interestingly, the MSCs converted via single gene activation exhibited morphological and molecular properties of white adipocytes, while beige adipocyte-like cells were induced via multiplex gene activation of three specific transcription factors. These results reveal that the fate of MSCs can be effectively manipulated by direct activation of specific endogenous gene expression using a dCas9-based activator with reduced exogenous additives.

    更新日期:2017-10-28
  • TARGETED GENE REPRESSION USING NOVEL BIFUNCTIONAL MOLECULES TO HARNESS ENDOGENOUS HISTONE DEACETYLATION ACTIVITY
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-26
    Kyle V Butler, Anna Marie Chiarella, Jian Jin, Nathaniel A Hathaway

    Epigenome editing is a powerful method that could give rise to new therapies for diseases driven by epigenetic dysregulation. To investigate and manipulate complex epigenetic interactions in live cells, we have developed a small-molecule platform for specifically inducing gene repression and histone deacetylation at a reporter gene. We synthesized bifunctional ligands, or chemical epigenetic modifiers (CEMs), that contain two functional groups: a FK506 derivative capable of binding to a Gal4-FKBP fusion transcription factor, and a histone deacetylase (HDAC) inhibitor that recruits HDAC-containing corepressor complexes. In our cell line, which contains a GFP reporter allele upstream of a Gal4 DNA binding array in the murine Oct4 locus, our lead CEM repressed GFP expression by 50%. We also show that CEM recruitment of deacetylation activity causes marked deacetylation. This cell engineering system directly modulates the chromatin environment of specific targets and has the potential to control disease-relevant gene regulation.

    更新日期:2017-10-27
  • Construction and Characterization of Broad-spectrum Promoters for Synthetic Biology
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-23
    Sen Yang, Qingtao Liu, Yunfeng Zhang, Guocheng Du, Jian Chen, Zhen Kang

    Characterization of genetic circuits and biosynthetic pathways in different hosts always requires promoter substitution and redesigning. Here, a strong, broad-spectrum promoter, Pbs, for Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae was constructed, and it was incorporated into the minimal E. coli–B. subtilis–S. cerevisiae shuttle plasmid pEBS (5.8 kb). By applying a random mutation strategy, three broad-spectrum promoters Pbs1, Pbs2, and Pbs3, with different strengths were generated and characterized. These broad-spectrum promoters will expand the synthetic biology toolbox for E. coli, B. subtilis, and S. cerevisiae.

    更新日期:2017-10-24
  • Quantitatively predictable control of cellular protein levels through proteasomal degradation
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-23
    Wenting Zhao, Lara Pferdehirt, Laura Segatori

    Protein function is typically studied and engineered by modulating protein levels within the complex cellular environment. To achieve fast, targeted, and predictable control of cellular protein levels without genetic manipulation of the target, we developed a technology for post-translational depletion based on a bifunctional molecule (NanoDeg) consisting of the antigen-binding fragment from the Camelidae species heavy-chain antibody (nanobody) fused to a degron signal that mediates degradation through the proteasome. We provide proof-of-principle demonstration of targeted degradation using a nanobody against the green fluorescent protein (GFP). Guided by predictive modeling, we show that customizing the NanoDeg rate of synthesis, rate of degradation, and mode of degradation enables quantitative and predictable control over the target’s levels. Integrating the GFP-specific NanoDeg within a genetic circuit based on stimulus-dependent GFP output results in enhanced dynamic range and resolution of the output signal. By providing predictable control over cellular proteins’ levels, the NanoDeg system could be readily used for a variety of systems level analyses of cellular protein function.

    更新日期:2017-10-24
  • Optogenetics manipulation enables to prevent biofilm formation of engineered Pseudomonas aeruginosa on surfaces
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-20
    Lu Pu, Shuai Yang, Aiguo Xia, Fan Jin

    Synthetic biologists have attempted to solve real-world problems, such as those of bacterial biofilms, that are involved in the pathogenesis of many clinical infections and difficult to eliminate. To address this, we employed a blue light responding system and integrated it into the chromosomes of Pseudomonas aeruginosa. With making rational adaption and improvement of the light-activated system, we provided a robust and convenient means to spatiotemporally control gene expression and manipulate biological processes with minimal perturbation in P. aeruginosa. It increased the light-induced gene expression up to 20-fold. Moreover, we deliberately introduced a functional protein gene PA2133 containing an EAL domain to degrade c-di-GMP into the modified system, and showed that the optimally engineered optogenetic tool inhibited the formation of P. aeruginosa biofilms through the induction of blue light, resulting in much sparser and thinner biofilms. Our approach establishes a methodology for leveraging the tools of synthetic biology to guide biofilm formation and engineer biofilm patterns with unprecedented spatiotemporal resolution. Furthermore, the findings suggest that the synthetic optogenetic system may provide a promising strategy that could be applied to control and fight biofilms.

    更新日期:2017-10-21
  • Muconic Acid Production via Alternative Pathways and a Synthetic ‘Metabolic Funnel’
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-20
    Brian Thompson, Shawn Pugh, Michael Machas, David R. Nielsen

    Muconic acid is a promising platform biochemical and precursor to adipic acid, which can be used to synthesize various plastics and polymers. In this study, the systematic construction and comparative evaluation of a modular network of non-natural pathways for muconic acid biosynthesis was investigated in Escherichia coli, including via three distinct and novel pathways proceeding via phenol as a common intermediate. However, poor recombinant activity and high promiscuity of phenol hydroxylase ultimately limited ‘phenol-dependent’ muconic acid production. A fourth pathway proceeding via p-hydroxybenzoate, protocatechuate, and catechol was accordingly developed, though with muconic acid titers by this route reaching just 819 mg/L, its performance lagged behind that of the established, ‘3-dehydroshikimiate-derived’ route. Finally, these two most promising pathways were co-expressed in parallel to create a synthetic ‘metabolic funnel’ that, by enabling maximal net precursor assimilation and flux while preserving native chorismate biosynthesis, nearly doubled muconic acid production to up to >3.1 g/L at a glucose yield of 158 mg/g while introducing only a single auxotrophy. This generalizable, ‘funneling’ strategy is expected to have broad applications in metabolic engineering for further enhancing production of muconic acid, as well as other important bioproducts of interest.

    更新日期:2017-10-21
  • In vitro evolution of unmodified 16S rRNA for simple ribosome reconstitution
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-20
    Yoshiki Murase, Hiroki Nakanishi, Gakushi Tsuji, Takeshi Sunami, Norikazu Ichihashi

    One of the largest challenges in the synthesis of artificial cell that can reproduce is in vitro assembly of ribosomes from in vitro synthesized rRNAs and proteins. In this study, to circumvent the chemical modification of 16S rRNA for reconstitution of fully active 30S subunit, we performed artificial evolution of 16S rRNA, which forms the functional 30S subunit without chemical modifications. We first established an in vitro selection scheme by combining the integrated synthesis, assembly, and translation (iSAT) system with the liposome sorting technique. After 15 rounds of selection cycles, we found one point mutation (U1495C) near the 3′ terminus that significantly enhanced the reconstitution activity of the functional 30S subunit from unmodified 16S rRNA to approximately 57% of that from native modified 16S rRNA. The effect of the mutation did not depend on the reconstitution scheme, anti-SD sequences, or the target genes to be translated. The mutation we found in this study enabled reconstitution of the active 30S subunit without rRNA modification, and thus would be a useful tool for simple construction of self-reproducing ribosomes.

    更新日期:2017-10-21
  • Genetic Constructor: An Online DNA Design Platform
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-20
    Maxwell Bates, Joe Lachoff, Duncan Meech, Valentin Zulkower, Anaïs Moisy, Yisha Luo, Hille Tekotte, Cornelia Johanna Franziska Scheitz, Rupal Khilari, Florencio Mazzoldi, Deepak Chandran, Eli Groban
    更新日期:2017-10-20
  • The Tolerome: A Database of Transcriptome-Level Contributions to Diverse Escherichia coli Resistance and Tolerance Phenotypes
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-19
    Keesha E. Erickson, James D. Winkler, Danh T. Nguyen, Ryan T. Gill, Anushree Chatterjee
    更新日期:2017-10-19
  • Dynamic Blue Light-Inducible T7 RNA Polymerases (Opto-T7RNAPs) for Precise Spatiotemporal Gene Expression Control
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-18
    Armin Baumschlager, Stephanie K. Aoki, Mustafa Khammash
    更新日期:2017-10-19
  • Rational Design of an Anticalin-Type Sugar-Binding Protein Using a Genetically Encoded Boronate Side Chain
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-18
    Selvakumar Edwardraja, Andreas Eichinger, Ina Theobald, Carina Andrea Sommer, Andreas J. Reichert, Arne Skerra
    更新日期:2017-10-18
  • Dynamic control of aptamer-ligand activity using strand displacement reactions
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-13
    Jonathan Lloyd, Claire H. Tran, Krishen Wadhwani, Christian Cuba Samaniego, Hari K. K. Subramanian, Elisa Franco

    Nucleic acid aptamers are an expandable toolkit of sensors and regulators. To employ aptamer regulators within non-equilibrium molecular networks, the aptamer-ligand interactions should be tunable over time, so that functions within a given system can be activated or suppressed on demand. This is accomplished through complementary sequences to aptamers, which achieve programmable aptamer-ligand dissociation by displacing the aptamer from the ligand. We demonstrate the effectiveness of our simple approach on light-up aptamers as well as on aptamers inhibiting viral RNA polymerases, dynamically controlling the functionality of the aptamer-ligand complex. Mathematical models allow us to obtain estimates for the aptamer displacement kinetics. Our results suggest that aptamers-kleptamer pairs could be used to build dynamic nucleic acid networks with direct control over a variety of aptamer-controllable enzymes and their downstream pathways.

    更新日期:2017-10-14
  • Genetic Constructor: An online DNA design platform
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-11
    Maxwell Bates, Joe Lachoff, Duncan Meech, Valentin Zulkower, Anais Moisy, Yisha Luo, Hille Tekotte, Cornelia Johanna Franziska Scheitz, Rupal Khilari, Florencio Mazzoldi, Deepak Chandran, Eli S Groban

    Genetic Constructor is a cloud Computer Aided Design (CAD) application developed to support synthetic biologists from design intent through DNA fabrication and experiment iteration. The platform allows users to design, manage, and navigate complex DNA constructs and libraries, using a new visual language that focuses on functional parts abstracted from sequence. Features like combinatorial libraries and automated primer design allow the user to separate design from construction by focusing on functional intent, and design constraints aid iterative refinement of designs. A plugin architecture enables contributions from scientists and coders to leverage existing powerful software and connect to DNA foundries. The software is easily accessible and platform agnostic, free for academics, and available in an open-source community edition. Genetic Constructor seeks to democratize DNA design, manufacture, and access to tools and services from the synthetic biology community.

    更新日期:2017-10-12
  • Manipulating Cellular Activities Using an Ultrasound–Chemical Hybrid Tool
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-12
    Ching-Hsiang Fan, Yao-Shen Huang, Wei-En Huang, Albert Alexander Lee, Sheng-Yang Ho, Yu-Lin Kao, Cuei-Ling Wang, Yen-Ling Lian, Tasuku Ueno, Tsung-Shing Andrew Wang, Chih-Kuang Yeh, Yu-Chun Lin
    更新日期:2017-10-12
  • A Novel Tool for Microbial Genome Editing Using the Restriction-Modification System
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-12
    Hua Bai, Aihua Deng, Shuwen Liu, Di Cui, Qidi Qiu, Laiyou Wang, Zhao Yang, Jie Wu, Xiuling Shang, Yun Zhang, Tingyi Wen
    更新日期:2017-10-12
  • Design and Selection of a Synthetic Feedback Loop for Optimizing Biofuel Tolerance
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-12
    Yik Siu, Jesse Fenno, Jessica M. Lindle, Mary J. Dunlop
    更新日期:2017-10-12
  • Versatility of Prolyl Oligopeptidase B in Peptide Macrocyclization
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-12
    R. Michael Sgambelluri, Miranda O. Smith, Jonathan D. Walton
    更新日期:2017-10-12
  • Development of Transcription Factor-Based Designer Macrolide Biosensors for Metabolic Engineering and Synthetic Biology
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-12
    Christian M. Kasey, Mounir Zerrad, Yiwei Li, T. Ashton Cropp, Gavin J. Williams
    更新日期:2017-10-12
  • Development of a Terpenoid-Production Platform in Streptomyces reveromyceticus SN-593
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-11
    Ammara Khalid, Hiroshi Takagi, Suresh Panthee, Makoto Muroi, Joe Chappell, Hiroyuki Osada, Shunji Takahashi
    更新日期:2017-10-12
  • The Tolerome: A database of transcriptome-level contributions to diverse Escherichia coli resistance and tolerance phenotypes
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-10
    Keesha E Erickson, James D. Winkler, Danh Nyugen, Ryan T. Gill, Anushree Chatterjee

    Tolerance and resistance are complex biological phenotypes that are desirable bioengineering goals for those seeking to design industrial strains or prevent the spread of antibiotic resistance. Over decades of research, a wealth of information has been generated to attempt to decode a molecular basis for tolerance, but to fully achieve the goal of engineering tolerance, researchers must be able to easily learn from a variety of data sources. To this end, we here describe a resource designed to enable scrutiny of diverse tolerance phenotypes. We have curated hundreds of gene expression studies exploring the response of Escherichia coli to chemical and environmental perturbations, from antibiotics to biofuels and solvents and more. Overall, our efforts give rise to a database encompassing more than 56,000 gene expression changes across 89 different stress conditions. This resource is designed for compatibility with the Resistome database, which includes more than 5,000 strains with mutations conferring resistance or sensitivity but no transcriptomic data. Thus, the work here results in the first combined resource specialized to tolerance and resistance in E. coli that supports investigations across genomic, transcriptomic, and phenotypic levels. We leverage the database to identify promising bioengineering targets by searching globally across multiple stress conditions as well as by narrowing the focus to fewer conditions of interest, such as biofuel stress and antibiotic stress. We discuss some of the most frequently differentially expressed genes, present genes commonly differentially co-expressed, and predict which transcription factors and sigma factors most likely contribute to gene expression profiles in a wide array of conditions. We also compare profiles from sensitive and resistant strains, gaining knowledge of how responses differ per overrepresented gene ontology terms. Finally, we search for genes that are frequently differentially expressed but not mutated, with the expectation that these may present interesting targets for future engineering efforts. The curated data presented here is publicly available, and should be advantageous to those studying a variety of bacterial tolerance phenotypes.

    更新日期:2017-10-11
  • A Prototype for Modular Cell Engineering
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-10
    Brandon Wilbanks, Donovan Layton, Sergio Garcia, Cong Trinh

    When aiming to produce a target chemical at high yield, titer, and productivity, various combinations of genetic parts available to build the target pathway can generate a large number of strains for characterization. This engineering approach will become increasingly laborious and expensive when seeking to develop desirable strains for optimal production of a large space of biochemicals due to extensive screening. Our recent theoretical development of modular cell (MODCELL) design principles can offer a promising solution for rapid generation of optimal strains by coupling a modular cell and exchangeable production modules in a plug-and-play fashion. In this study, we experimentally validated some designed properties of MODCELL by demonstrating: i) a modular (chassis) cell is required to couple with a production module, a heterologous ethanol pathway, as a testbed, ii) degree of coupling between the modular cell and production modules can be modulated to enhance growth and product synthesis, iii) a modular cell can be used as a host to select an optimal pyruvate decarboxylase (PDC) of the ethanol production module and to help identify a hypothetical PDC protein, and iv) adaptive laboratory evolution based on growth selection of the modular cell can enhance growth and product synthesis rates. We envision that the MODCELL design provides a powerful prototype for modular cell engineering to rapidly create optimal strains for synthesis of a large space of biochemicals.

    更新日期:2017-10-11
  • A Cell-Free Biosensor for Detecting Quorum Sensing Molecules in P. aeruginosa-Infected Respiratory Samples
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-05
    Ke Yan Wen, Loren Cameron, James Chappell, Kirsten Jensen, David J. Bell, Richard Kelwick, Margarita Kopniczky, Jane C. Davies, Alain Filloux, Paul S. Freemont
    更新日期:2017-10-05
  • Genetic Design via Combinatorial Constraint Specification
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-04
    Swapnil P. Bhatia, Michael J. Smanski, Christopher A. Voigt, Douglas M. Densmore
    更新日期:2017-10-04
  • Cloning and Transplantation of the Mesoplasma florum Genome
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-03
    Vincent Baby, Fabien Labroussaa, Joëlle Brodeur, Dominick Matteau, Géraldine Gourgues, Carole Lartigue, Sébastien Rodrigue
    更新日期:2017-10-04
  • A novel tool for microbial genome editing using the restriction-modification system
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-02
    Hua Bai, Aihua Deng, Shuwen Liu, Di Cui, Qidi Qiu, Laiyou Wang, Zhao Yang, Jie Wu, Xiuling Shang, Yun Zhang, Tingyi Wen

    Scarless genetic manipulation of genomes is an essential tool for biological research. The restriction-modification (R-M) system is a defense system in bacteria that protects against invading genomes based on its ability to distinguish foreign DNA from self DNA. Here, we designed an R-M system-mediated genome editing (RMGE) technique for scarless genetic manipulation in different microorganisms. For bacteria with Type IV REase, an RMGE technique using the inducible DNA methyltransferase gene, bceSIIM (RMGE-bceSIIM), as the counter-selection cassette was developed to edit the genome of Escherichia coli. For bacteria without Type IV REase, an RMGE technique based on a restriction endonuclease (RMGE-mcrA) was established in Bacillus subtilis. These techniques were successfully used for gene deletion and replacement with nearly 100% counter-selection efficiencies, which were higher and more stable compared to conventional methods. Furthermore, precise point mutation without limiting sites was achieved in E. coli using RMGE-bceSIIM to introduce a single base mutation of A128C into the rpsL gene. In addition, the RMGE-mcrA technique was applied to delete the CAN1 gene in Saccharomyces cerevisiae DAY414 with 100% counter-selection efficiency. The effectiveness of the RMGE technique in E. coli, B. subtilis, and S. cerevisiae suggests the potential universal usefulness of this technique for microbial genome manipulation.

    更新日期:2017-10-03
  • Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-02
    Eirik Adim Moreb, Benjamin Hoover, Adam Yaseen, Nisakorn Valyasevi, Zoe Roecker, Romel Menacho-Melgar, Michael D. Lynch
    更新日期:2017-10-03
  • Growing Membranes In Vitro by Continuous Phospholipid Biosynthesis from Free Fatty Acids
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-02
    Marten Exterkate, Antonella Caforio, Marc C. A. Stuart, Arnold J. M. Driessen
    更新日期:2017-10-03
  • Cell-Free Protein Synthesis Enhancement from Real-Time NMR Metabolite Kinetics: Redirecting Energy Fluxes in Hybrid RRL Systems
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-10-02
    Baptiste Panthu, Théophile Ohlmann, Johan Perrier, Uwe Schlattner, Pierre Jalinot, Bénédicte Elena-Herrmann, Gilles J. P. Rautureau
    更新日期:2017-10-02
  • Investigation of the Synergetic Effect of Xylose Metabolic Pathways on the Production of Glutaric Acid
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-29
    Jia Wang, Xiaolin Shen, Yuheng Lin, Zhenya Chen, Yaping Yang, Qipeng Yuan, Yajun Yan
    更新日期:2017-10-01
  • A modular receptor platform to expand the sensing repertoire of bacteria.
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-25
    Hung Ju Chang, Pauline Mayonove, Agustin Zavala, Angelique De Visch, Philippe Minard, Martin Cohen-Gonsaud, Bonnet Jerome

    Engineered bacteria promise to revolutionize diagnostics and therapeutics yet many applications are precluded by the limited number of detectable signals. Here we present a general framework to engineer synthetic receptors enabling bacterial cells to respond to novel ligands. These receptors are activated via ligand-induced dimerization of a single-domain antibody fused to monomeric DNA-Binding Domains (split-DBDs). Using E. coli as a model system, we engineer both transmembrane and cytosolic receptors using a VHH for ligand detection and demonstrate the scalability of our platform by using the DBDs of two different transcriptional regulators. We provide a method to optimize receptor behavior by finely tuning protein expression levels and optimizing interdomain linker regions. Finally, we show that these receptors can be connected to downstream synthetic gene circuits for further signal processing. The general nature of the split-DBD principle and the versatility of antibody-based detection should support the deployment of these receptors into various host to detect ligands for which no receptor is found in nature.

    更新日期:2017-09-26
  • Manipulating cellular activities using an ultrasound-chemical hybrid tool
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-25
    Ching-Hsiang Fan, Yao-Shen Huang, Wei-En Huang, Albert Alexander Lee, Sheng-Yang Ho, Yu-Lin Kao, Cuei-Ling Wang, Yen-Ling Lian, Tasuku Ueno, Tsung-Shing Andrew Wang, Chih-Kuang Yeh, YU-CHUN LIN

    We developed an ultrasound-chemical hybrid tool to precisely manipulate cellular activities. A focused ultrasound coupled with gas-filled microbubbles were used to rapidly trigger the influx of membrane-impermeable chemical dimerizers into living cells to regulate protein dimerization and location without inducing noticeable toxicity. With this system, we demonstrated the successful modulation of phospholipid metabolism triggered by a short pulse of ultrasound exposure. Our technique offers a powerful and versatile tool for using ultrasound to spatiotemporally manipulate the cellular physiology in living cells.

    更新日期:2017-09-26
  • Investigation of the synergetic effect of xylose metabolic pathways on the production of glutaric acid
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-25
    Jia Wang, Xiaolin Shen, Yuheng Lin, Zhenya Chen, Yaping Yang, Qipeng Yuan, Yajun Yan

    Efficient utilization of lignocellulose is pivotal for economically converting renewable feedstocks into value-added products. Xylose is the second most abundant sugar in lignocellulose, but it is quite challenging to ferment xylose as efficiently as glucose by microorganisms. Here, we investigated the metabolic potential of three xylose catabolic pathways (isomerase, Weimberg and Dahms pathways) and illustrated the synergetic effect between isomerase pathway and Weimberg pathway for the synthesis of chemicals derived from 2-ketoglutarate and acetyl-CoA. When using glutaric acid as the target product, employment of such synergetic pathways in combination resulted in an increased glutaric acid titer (602 mg/L) compared with using each pathway alone (104 or 209 mg/L) and this titer even outcompetes that obtained from glucose catabolic pathway for glutaric acid synthesis (420 mg/L). This work validates a novel and powerful strategy for xylose metabolic utilization to overcome the inefficiency of using single xylose metabolic pathway for the synthesis of TCA cycle derived chemicals.

    更新日期:2017-09-26
  • Rational Design of an Anticalin-Type Sugar-Binding Protein Using a Genetically Encoded Boronate Side Chain
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-22
    Selvakumar Edwardraja, Andreas Eichinger, Ina Theobald, Carina Andrea Sommer, Andreas J. Reichert, Arne Skerra

    The molecular recognition of carbohydrates plays a fundamental role in many biological processes. However, the development of carbohydrate-binding reagents for biomedical research and use poses a challenge due to the generally poor affinity of proteins towards sugars in aqueous solution. Here, we describe the effective molecular recognition of pyranose monosaccharides (in particular, galactose and mannose) by a rationally designed protein receptor based on the human lipocalin scaffold (Anticalin). Complexation relies on reversible covalent cis-diol boronate diester formation with a genetically encoded L-boronophenylalanine (Bpa) residue which was incorporated as a non-natural amino acid at a sterically permissive position in the binding site of the Anticalin, as confirmed by X-ray crystallography. Com-pared with the metal-ion and/or avidity-dependent oligovalent lectins that prevail in nature, our approach offers a novel and promising route to generate tight sugar-binding reagents both as research reagents and for biomedical applications.

    更新日期:2017-09-23
  • Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-21
    S. M. Nayeemul Bari, Forrest C. Walker, Katie Cater, Barbaros Aslan, Asma Hatoum-Aslan
    更新日期:2017-09-21
  • Growing membranes in vitro by continuous phospholipid biosynthesis from free fatty acids
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-18
    Marten Exterkate, Antonella Caforio, Marc C. A. Stuart, Arnold J.M. Driessen

    One of the key aspects that defines a cell as a living entity is its ability to self-reproduce. In this process, membrane biogenesis is an essential element. Here, we developed an in vitro phospholipid biosynthesis pathway based on a cascade of eight enzymes, starting from simple fatty acid building blocks and glycerol 3-phosphate. The reconstituted system yields multiple phospholipid species that vary in acyl-chain and polar head group compositions. Due to the high fidelity and versatility, complete conversion of the fatty acid substrates into multiple phospholipid species is achieved simultaneously, leading to membrane expansion as a first step towards a synthetic minimal cell.

    更新日期:2017-09-19
  • Managing the SOS Response for Enhanced CRISPR-Cas-based Recombineering in E. coli Through Transient Inhibition of Host RecA Activity
    ACS Synth. Biol. (IF 5.382) Pub Date : 2017-09-15
    Eirik Adim Moreb, Benjamin Hoover, Adam Yaseen, Nisakorn Valyasevi, Zoe Roecker, Romel Menacho-Melgar, Michael Lynch

    Phage-derived “recombineering” methods are utilized for bacterial genome editing. Recombineering results in a heterogeneous population of modified and unmodified chromosomes and therefore selection methods, such as CRISPR-Cas9, are required to select for edited clones. Cells can evade CRISPR-Cas-induced cell death through recA-mediated induction of the SOS response. The SOS response increases RecA dependent repair as well mutation rates through the induction of the umuDC error prone polymerase. As a result, CRISPR-Cas selection is more efficient in recA mutants. We report an approach to inhibiting the SOS response and RecA activity through the expression of a mutant dominant negative form of RecA, which incorporates into wild type RecA filaments and inhibits activity. Using a plasmid-based system in which Cas9 and recA mutants are co-expressed, we can achieve increased efficiency and consistency of CRISPR-Cas9-mediated selection and recombineering in E. coli, while reducing the induction of the SOS response. To date, this approach has been shown to be independent of recA genotype and host strain lineage. Using this system, we demonstrate increased CRISPR-Cas selection efficacy with over 10,000 guides covering the E. coli chromosome. The use of dominant negative RecA or homologs may be of broad use in bacterial CRISPR-Cas-based genome editing where the SOS pathways are present.

    更新日期:2017-09-15
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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