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      Engineering and modification of microbial chassis for systems and synthetic biology

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          Abstract

          Engineering and modifying synthetic microbial chassis is one of the best ways not only to unravel the fundamental principles of life but also to enhance applications in the health, medicine, agricultural, veterinary, and food industries. The two primary strategies for constructing a microbial chassis are the top-down approach (genome reduction) and the bottom-up approach (genome synthesis). Research programs on this topic have been funded in several countries. The ‘Minimum genome factory’ (MGF) project was launched in 2001 in Japan with the goal of constructing microorganisms with smaller genomes for industrial use. One of the best examples of the results of this project is E. coli MGF-01, which has a reduced-genome size and exhibits better growth and higher threonine production characteristics than the parental strain [1]. The ‘cell factory’ project was carried out from 1998 to 2002 in the Fifth Framework Program of the EU (European Union), which tried to comprehensively understand microorganisms used in the application field. One of the outstanding results of this project was the elucidation of proteins secreted by Bacillus subtilis, which was summarized as the ‘secretome’ [2]. The GTL (Genomes to Life) program began in 2002 in the United States. In this program, researchers aimed to create artificial cells both in silico and in vitro, such as the successful design and synthesis of a minimal bacterial genome by John Craig Venter's group [3]. This review provides an update on recent advances in engineering, modification and application of synthetic microbial chassis, with particular emphasis on the value of learning about chassis as a way to better understand life and improve applications.

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          Efficient In Vivo Genome Editing Using RNA-Guided Nucleases

          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those obtained using ZFNs and TALENs for the same genes. RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results demonstrate that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNA-guided nucleases for genome editing in a wide range of organisms.
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            Synthetic biology: applications come of age

            Key Points Early synthetic biology designs, namely the genetic toggle switch and repressilator, showed that regulatory components can be characterized and assembled to bring about complex, electronics-inspired behaviours in living systems (for example, memory storage and timekeeping). Through the characterization and assembly of genetic parts and biological building blocks, many more devices have been constructed, including switches, memory elements, oscillators, pulse generators, digital logic gates, filters and communication modules. Advances in the field are now allowing expansion beyond small gene networks to the realm of larger biological programs, which hold promise for a wide range of applications, including biosensing, therapeutics and the production of biofuels, pharmaceuticals and biomaterials. Synthetic biosensing circuits consist of sensitive elements that bind analytes and transducer modules that mobilize cellular responses. Balancing these two modules involves engineering modularity and specificity into the various circuits. Biosensor sensitive elements include environment-responsive promoters (transcriptional), RNA aptamers (translational) and protein receptors (post-translational). Biosensor transducer modules include engineered gene networks (transcriptional), non-coding regulatory RNAs (translational) and protein signal-transduction circuits (post-translational). The contributions of synthetic biology to therapeutics include: engineered networks and organisms for disease-mechanism elucidation, drug-target identification, drug-discovery platforms, therapeutic treatment, therapeutic delivery, and drug production and access. In the microbial production of biofuels and pharmaceuticals, synthetic biology has supplemented traditional genetic and metabolic engineering efforts by aiding the construction of optimized biosynthetic pathways. Optimizing metabolic flux through biosynthetic pathways is traditionally accomplished by driving the expression of pathway enzymes with strong, inducible promoters. New synthetic approaches include the rapid diversification of various pathway components, the rational and model-guided assembly of pathway components, and hybrid solutions.
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              Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome.

              We have synthesized a 582,970-base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted "watermarks" at intergenic sites known to tolerate transposon insertions. Overlapping "cassettes" of 5 to 7 kilobases (kb), assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4 genome"), which were all cloned as bacterial artificial chromosomes in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments.
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                Author and article information

                Contributors
                Journal
                Synth Syst Biotechnol
                Synth Syst Biotechnol
                Synthetic and Systems Biotechnology
                KeAi Publishing
                2405-805X
                11 December 2018
                March 2019
                11 December 2018
                : 4
                : 1
                : 25-33
                Affiliations
                [a ]Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sciences, Zhongnan Hospital, Wuhan University, Wuhan, 430071, China
                [b ]Taihe Hospital, Hubei University of Medicine, Shiyan, 442000, Hubei, China
                Author notes
                []Corresponding author. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sciences, Zhongnan Hospital, Wuhan University, Wuhan, 430071, China. shichen@ 123456whu.edu.cn
                Article
                S2405-805X(18)30041-3
                10.1016/j.synbio.2018.12.001
                6290258
                30560208
                e0cec5dd-188d-4b49-af66-fcf26b762b55
                © 2018 Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 31 August 2018
                : 14 November 2018
                : 5 December 2018
                Categories
                Article

                microbial chassis,systems biology,synthetic biology
                microbial chassis, systems biology, synthetic biology

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