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  • Articles  (111)
  • Synthetic Biology and Assembly Cloning  (70)
  • Biotechnology & Synthetic Biology  (41)
  • Oxford University Press  (111)
  • 11
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    Overexpression of two stress-responsive, small, non-coding RNAs, 6S and tmRNA, imparts butanol tolerance in Clostridium acetobutylicum (2016)
    Oxford University Press
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    Publication Date: 2016-04-08
    Description: While extensively studied in several model organisms, the role of small, non-coding RNAs in the stress response remains largely unexplored in Clostridium organisms. About 100 years after the first industrial Acetone–Butanol–Ethanol fermentation process, based on the Weizmann Clostridium acetobutylicum strain, strain tolerance to butanol remains a crucial factor limiting the economics of the process. Several studies have examined the response of this organism to metabolite stress, and several genes have been engaged to impart enhanced tolerance, but no sRNAs have yet been directly engaged in this task. We show that the two stress-responsive sRNAs, 6S and tmRNA, upon overexpression impart tolerance to butanol as assessed by viability assays under process-relevant conditions. 6S overexpression enhances cell densities as well as butanol titres. We discuss the likely mechanisms that these two sRNAs might engage in this tolerance phenotype. Our data support the continued exploration of sRNAs as a basis for engineering enhanced tolerance and enhanced solvent production, especially because sRNA-based strategies impose a minimal metabolic burden on the cells.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 12
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    Neofiscalin A and fiscalin C are potential novel indole alkaloid alternatives for the treatment of multidrug-resistant Gram-positive bacterial infections (2016)
    Oxford University Press
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    Publication Date: 2016-07-02
    Description: Ten indole alkaloids were obtained from the marine sponge-associated fungus Neosartorya siamensis KUFA 0017. We studied the antimicrobial properties of these and of three other compounds previously isolated from the soil fungus N. siamensis KUFC 6349. Only neofiscalin A showed antimicrobial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE); with a minimum inhibitory concentration (MIC) of 8 μg mL –1 against both strains. Another compound, fiscalin C, presented synergistic activity against MRSA when combined with oxacillin, although alone showed no antibacterial effect. Moreover, neofiscalin A, when present at sub-MICs, hampered the ability of both MRSA and VRE strains to form a biofilm. Additionally, the biofilm inhibitory concentration values of neofiscalin A against the MRSA and VRE isolates were 96 and 80 μg mL –1 , respectively. At a concentration of 200 μg mL –1 , neofiscalin A was able to reduce the metabolic activity of the biofilms by ~50%. One important fact is that our results also showed that neofiscalin A had no cytotoxicity against a human brain capillary endothelial cell line.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 13
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    The unconventional secretion of PepN is independent of a functional autophagy machinery in the filamentous fungus Aspergillus niger (2016)
    Oxford University Press
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    Publication Date: 2016-07-02
    Description: During unconventional protein secretion (UPS), proteins do not pass through the classical endoplasmic reticulum (ER)–Golgi-dependent pathway, but are transported to the cell membrane via alternative routes. One type of UPS is dependent on several autophagy-related (Atg) proteins in yeast and mammalian cells, but mechanisms for unconventional secretion are largely unknown for filamentous fungi. In this study, we investigated whether the autophagy machinery is used for UPS in the filamentous fungus Aspergillus niger . An aspartic protease, which we called PepN, was identified as being likely to be secreted unconventionally, as this protein is highly abundant in culture filtrates during carbon starvation while it lacks a conventional N-terminal secretion sequence. We analysed the presence of PepN in the culture filtrates of carbon starved wild-type, atg1 and atg8 deletion mutant strains by Western blot analysis and by secretome analysis using nanoLC-ESI-MS/MS (wild-type and atg8 deletion mutant). Besides the presence of carbohydrate-active enzymes and other types of proteases, PepN was abundantly found in culture filtrates of both wild-type and atg deletion strains, indicating that the secretion of PepN is independent of the autophagy machinery in A. niger and hence most likely occurs via a different mechanism.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 14
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    Blue light-mediated transcriptional activation and repression of gene expression in bacteria (2016)
    Oxford University Press
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    Publication Date: 2016-08-20
    Description: Light-regulated modules offer unprecedented new ways to control cellular behavior in precise spatial and temporal resolution. The availability of such tools may dramatically accelerate the progression of synthetic biology applications. Nonetheless, current optogenetic toolbox of prokaryotes has potential issues such as lack of rapid and switchable control, less portable, low dynamic expression and limited parts. To address these shortcomings, we have engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using the blue light dependent DNA-binding protein EL222. We demonstrated that by modulating the dosage of light pulses or intensity we could control the level of gene expression precisely. We show that both light-inducible and repressible system can function in parallel with high spatial precision in a single cell and can be switched stably between ON- and OFF-states by repetitive pulses of blue light. In addition, the light-inducible and repressible expression kinetics were quantitatively analysed using a mathematical model. We further apply the system, for the first time, to optogenetically synchronize two receiver cells performing different logic behaviors over time using blue light as a molecular clock signal. Overall, our modular approach layers a transformative platform for next-generation light-controllable synthetic biology systems in prokaryotes.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 15
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    CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA (2016)
    Oxford University Press
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    Publication Date: 2016-08-20
    Description: Current DNA assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized DNAs (e.g. synthetic genomes). We have developed a new method designated ‘CasHRA ( Cas 9-facilitated H omologous R ecombination A ssembly)’ that directly uses large circular DNAs in a one-step in vivo assembly process. The large circular DNAs are co-introduced into Saccharomyces cerevisiae by protoplast fusion, and they are cleaved by RNA-guided Cas9 nuclease to release the linear DNA segments for subsequent assembly by the endogenous homologous recombination system. The CasHRA method allows efficient assembly of multiple large DNA segments in vivo ; thus, this approach should be useful in the last stage of genome construction. As a proof of concept, we combined CasHRA with an upstream assembly method (Gibson procedure of genome assembly) and successfully constructed a 1.03 Mb MGE-syn1.0 ( M inimal G enome of Escherichia coli ) that contained 449 essential genes and 267 important growth genes. We expect that CasHRA will be widely used in megabase-sized genome constructions.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 16
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    Effects of MreB paralogs on poly-{gamma}-glutamic acid synthesis and cell morphology in Bacillus amyloliquefaciens (2016)
    Oxford University Press
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    Publication Date: 2016-08-27
    Description: Actin-like MreB paralogs play important roles in cell shape maintenance, cell wall synthesis and the regulation of the D,L-endopeptidases, CwlO and LytE. The gram-positive bacteria, Bacillus amyloliquefaciens LL3, is a poly--glutamic acid (-PGA) producing strain that contains three MreB paralogs: MreB, Mbl and MreBH. In B. amyloliquefaciens , CwlO and LytE can degrade -PGA. In this study, we aimed to test the hypothesis that modulating transcript levels of MreB paralogs would alter the synthesis and degradation of -PGA. The results showed that overexpression or inhibition of MreB, Mbl or MreBH had distinct effects on cell morphology and the molecular weight of the -PGA products. In fermentation medium, cells of mreB inhibition mutant were 50.2% longer than LL3, and the -PGA titer increased by 55.7%. However, changing the expression level of mbl showed only slight effects on the morphology, -PGA molecular weight and titer. In the mreBH inhibition mutant, -PGA production and its molecular weight increased by 56.7% and 19.4%, respectively. These results confirmed our hypothesis that suppressing the expression of MreB paralogs might reduce -PGA degradation, and that improving the cell size could strengthen -PGA synthesis. This is the first report of enhanced -PGA production via suppression of actin-like MreB paralogs.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 17
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    Robust, tunable genetic memory from protein sequestration combined with positive feedback (2015)
    Oxford University Press
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    Publication Date: 2015-10-15
    Description: Natural regulatory networks contain many interacting components that allow for fine-tuning of switching and memory properties. Building simple bistable switches, synthetic biologists have learned the design principles of complex natural regulatory networks. However, most switches constructed so far are so simple (e.g. comprising two regulators) that they are functional only within a limited parameter range. Here, we report the construction of robust, tunable bistable switches in Escherichia coli using three heterologous protein regulators (ExsADC) that are sequestered into an inactive complex through a partner swapping mechanism. On the basis of mathematical modeling, we accurately predict and experimentally verify that the hysteretic region can be fine-tuned by controlling the interactions of the ExsADC regulatory cascade using the third member ExsC as a tuning knob. Additionally, we confirm that a dual-positive feedback switch can markedly increase the hysteretic region, compared to its single-positive feedback counterpart. The dual-positive feedback switch displays bistability over a 10 6 -fold range of inducer concentrations, to our knowledge, the largest range reported so far. This work demonstrates the successful interlocking of sequestration-based ultrasensitivity and positive feedback, a design principle that can be applied to the construction of robust, tunable, and predictable genetic programs to achieve increasingly sophisticated biological behaviors.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 18
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    Industrial production of acetone and butanol by fermentation--100 years later (2016)
    Oxford University Press
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    Publication Date: 2016-06-08
    Description: Microbial production of acetone and butanol was one of the first large-scale industrial fermentation processes of global importance. During the first part of the 20th century, it was indeed the second largest fermentation process, superseded in importance only by the ethanol fermentation. After a rapid decline after the 1950s, acetone-butanol-ethanol (ABE) fermentation has recently gained renewed interest in the context of biorefinery approaches for the production of fuels and chemicals from renewable resources. The availability of new methods and knowledge opens many new doors for industrial microbiology, and a comprehensive view on this process is worthwhile due to the new interest. This thematic issue of FEMS Microbiology Letters, dedicated to the 100th anniversary of the first industrial exploitation of Chaim Weizmann's ABE fermentation process, covers the main aspects of old and new developments, thereby outlining a model development in biotechnology. All major aspects of industrial microbiology are exemplified by this single process. This includes new technologies, such as the latest developments in metabolic engineering, the exploitation of biodiversity and discoveries of new regulatory systems such as for microbial stress tolerance, as well as technological aspects, such as bio- and down-stream processing.
    Keywords: Biotechnology & Synthetic Biology
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    Topics: Biology
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  • 19
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    A new lactobacilli in vivo expression system for the production and delivery of heterologous proteins at mucosal surfaces (2016)
    Oxford University Press
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    Publication Date: 2016-06-02
    Description: Food-grade lactic acid bacteria, such as lactobacilli, represent good candidates for the development of mucosal vectors. Indeed, they are generally recognized as safe microorganisms and some strains display beneficial effects (probiotics). In this study, we described a new lactobacilli in vivo expression (LIVE) system for the production and delivery of therapeutic molecules at mucosal surfaces. The versatility and functionality of this system was successfully validated in several lactobacilli species; furthermore, we assessed in vivo LIVE system in two different mouse models of human pathologies: (i) a model of therapy against intestinal inflammation (inflammatory bowel diseases) and (ii) a model of vaccination against dental caries. We demonstrated that Lactobacillus gasseri expressing the anti-inflammatory cytokine IL-10 under LIVE system efficiently delivered the recombinant protein at mucosal surfaces and display anti-inflammatory effects. In the vaccination model against caries, LIVE system allowed the heterologous expression of Streptococcus mutans antigen GbpB by L. gasseri , leading to a stimulation of the host immune response.
    Keywords: Biotechnology & Synthetic Biology
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    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 20
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    Bioreactors and in situ product recovery techniques for acetone-butanol-ethanol fermentation (2016)
    Oxford University Press
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    Publication Date: 2016-06-02
    Description: The microbial fermentation process is one of the sustainable and environment-friendly ways to produce 1-butanol and other bio-based chemicals. The success of the fermentation process greatly relies on the choice of bioreactors and the separation methods. In this review, the history and the performance of bioreactors for the acetone–butanol–ethanol (ABE) fermentation is discussed. The subject is then focused on in situ product recovery (ISPR) techniques, particularly for the integrated extraction-gas stripping. The usefulness of this promising hybrid ISPR device is acknowledged by its incorporation with batch, fed-batch and continuous processes to improve the performance of ABE fermentation.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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