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1
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Barone M, D'Amico F, Fabbrini M, Rampelli S, Brigidi P, Turroni S. Over-feeding the gut microbiome: A scoping review on health implications and therapeutic perspectives. World J Gastroenterol 2021; 27:7041-7064. [PMID: 34887627 PMCID: PMC8613651 DOI: 10.3748/wjg.v27.i41.7041] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/29/2021] [Revised: 07/02/2021] [Accepted: 10/14/2021] [Indexed: 02/06/2023] Open
Abstract
The human gut microbiome has gained increasing attention over the past two decades. Several findings have shown that this complex and dynamic microbial ecosystem can contribute to the maintenance of host health or, when subject to imbalances, to the pathogenesis of various enteric and non-enteric diseases. This scoping review summarizes the current knowledge on how the gut microbiota and microbially-derived compounds affect host metabolism, especially in the context of obesity and related disorders. Examples of microbiome-based targeted intervention strategies that aim to restore and maintain an eubiotic layout are then discussed. Adjuvant therapeutic interventions to alleviate obesity and associated comorbidities are traditionally based on diet modulation and the supplementation of prebiotics, probiotics and synbiotics. However, these approaches have shown only moderate ability to induce sustained changes in the gut microbial ecosystem, making the development of innovative and tailored microbiome-based intervention strategies of utmost importance in clinical practice. In this regard, the administration of next-generation probiotics and engineered microbiomes has shown promising results, together with more radical intervention strategies based on the replacement of the dysbiotic ecosystem by means of fecal microbiota transplantation from healthy donors or with the introduction of synthetic communities specifically designed to achieve the desired therapeutic outcome. Finally, we provide a perspective for future translational investigations through the implementation of bioinformatics approaches, including machine and deep learning, to predict health risks and therapeutic outcomes.
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Affiliation(s)
- Monica Barone
- Microbiomics Unit, Department of Medical and Surgical Sciences, University of Bologna, Bologna 40138, Italy
- Unit of Microbiome Science and Biotechnology, Department of Pharmacy and Biotechnology, University of Bologna, Bologna 40126, Italy
| | - Federica D'Amico
- Microbiomics Unit, Department of Medical and Surgical Sciences, University of Bologna, Bologna 40138, Italy
- Unit of Microbiome Science and Biotechnology, Department of Pharmacy and Biotechnology, University of Bologna, Bologna 40126, Italy
| | - Marco Fabbrini
- Microbiomics Unit, Department of Medical and Surgical Sciences, University of Bologna, Bologna 40138, Italy
- Unit of Microbiome Science and Biotechnology, Department of Pharmacy and Biotechnology, University of Bologna, Bologna 40126, Italy
| | - Simone Rampelli
- Unit of Microbiome Science and Biotechnology, Department of Pharmacy and Biotechnology, University of Bologna, Bologna 40126, Italy
| | - Patrizia Brigidi
- Microbiomics Unit, Department of Medical and Surgical Sciences, University of Bologna, Bologna 40138, Italy
| | - Silvia Turroni
- Unit of Microbiome Science and Biotechnology, Department of Pharmacy and Biotechnology, University of Bologna, Bologna 40126, Italy
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2
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Vallejo D, Nikoomanzar A, Paegel BM, Chaput JC. Fluorescence-Activated Droplet Sorting for Single-Cell Directed Evolution. ACS Synth Biol 2019; 8:1430-1440. [PMID: 31120731 DOI: 10.1021/acssynbio.9b00103] [Citation(s) in RCA: 76] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Synthetic biology aims to improve human health and the environment by repurposing biological enzymes for use in practical applications. However, natural enzymes often function with suboptimal activity when engineered into biological pathways or challenged to recognize unnatural substrates. Overcoming this problem requires efficient directed evolution methods for discovering new enzyme variants that function with a desired activity. Here, we describe the construction, validation, and application of a fluorescence-activated droplet sorting (FADS) instrument that was established to evolve enzymes for synthesizing and modifying artificial genetic polymers (XNAs). The microfluidic system enables droplet sorting at ∼2-3 kHz using fluorescent sensors that are responsive to enzymatic activity. The ability to evolve nucleic acid enzymes with customized properties will uniquely drive emerging applications in synthetic biology, biotechnology, and healthcare.
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Affiliation(s)
| | | | - Brian M. Paegel
- Department of Chemistry, The Scripps Research Institute, Jupiter, Florida 33458, United States
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3
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Patel S, Panchasara H, Braddick D, Gohil N, Singh V. Synthetic small RNAs: Current status, challenges, and opportunities. J Cell Biochem 2018; 119:9619-9639. [DOI: 10.1002/jcb.27252] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2018] [Accepted: 06/20/2018] [Indexed: 12/25/2022]
Affiliation(s)
- Shreya Patel
- Department of Microbiology, Synthetic Biology Laboratory School of Biological Sciences and Biotechnology, Institute of Advanced Research, Koba Institutional Area Gandhinagar India
| | - Happy Panchasara
- Department of Microbiology, Synthetic Biology Laboratory School of Biological Sciences and Biotechnology, Institute of Advanced Research, Koba Institutional Area Gandhinagar India
| | | | - Nisarg Gohil
- Department of Microbiology, Synthetic Biology Laboratory School of Biological Sciences and Biotechnology, Institute of Advanced Research, Koba Institutional Area Gandhinagar India
| | - Vijai Singh
- Department of Microbiology, Synthetic Biology Laboratory School of Biological Sciences and Biotechnology, Institute of Advanced Research, Koba Institutional Area Gandhinagar India
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4
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张 晓. The Dynamical Modeling Studies of the Quorum Sensing Mechanism in Bacteria. Biophysics (Nagoya-shi) 2018. [DOI: 10.12677/biphy.2018.62002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
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5
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Sivasubramaniam D, Franks AE. Bioengineering microbial communities: Their potential to help, hinder and disgust. Bioengineered 2017; 7:137-44. [PMID: 27221461 PMCID: PMC4927200 DOI: 10.1080/21655979.2016.1187346] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The bioengineering of individual microbial organisms or microbial communities has great potential in agriculture, bioremediation and industry. Understanding community level drivers can improve community level functions to enhance desired outcomes in complex environments, whereas individual microbes can be reduced to a programmable biological unit for specific output goals. While understanding the bioengineering potential of both approaches leads to a wide range of potential uses, public acceptance of such technology may be the greatest hindrance to its application. Public perceptions and expectations of “naturalness,” as well as notions of disgust and dread, may delay the development of such technologies to their full benefit. We discuss these bioengineering approaches and draw on the psychological literature to suggest strategies that scientists can use to allay public concerns over the implementation of this technology.
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Affiliation(s)
- Diane Sivasubramaniam
- a Department of Psychological Sciences , Swinburne University , Melbourne , Victoria , Australia
| | - Ashley E Franks
- b Department of Physiology , Anatomy and Microbiology, La Trobe University , Melbourne , Victoria , Australia
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6
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Roy R, Shilpa PP, Bagh S. A Systems Biology Analysis Unfolds the Molecular Pathways and Networks of Two Proteobacteria in Spaceflight and Simulated Microgravity Conditions. ASTROBIOLOGY 2016; 16:677-689. [PMID: 27623197 DOI: 10.1089/ast.2015.1420] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/06/2023]
Abstract
UNLABELLED Bacteria are important organisms for space missions due to their increased pathogenesis in microgravity that poses risks to the health of astronauts and for projected synthetic biology applications at the space station. We understand little about the effect, at the molecular systems level, of microgravity on bacteria, despite their significant incidence. In this study, we proposed a systems biology pipeline and performed an analysis on published gene expression data sets from multiple seminal studies on Pseudomonas aeruginosa and Salmonella enterica serovar Typhimurium under spaceflight and simulated microgravity conditions. By applying gene set enrichment analysis on the global gene expression data, we directly identified a large number of new, statistically significant cellular and metabolic pathways involved in response to microgravity. Alteration of metabolic pathways in microgravity has rarely been reported before, whereas in this analysis metabolic pathways are prevalent. Several of those pathways were found to be common across studies and species, indicating a common cellular response in microgravity. We clustered genes based on their expression patterns using consensus non-negative matrix factorization. The genes from different mathematically stable clusters showed protein-protein association networks with distinct biological functions, suggesting the plausible functional or regulatory network motifs in response to microgravity. The newly identified pathways and networks showed connection with increased survival of pathogens within macrophages, virulence, and antibiotic resistance in microgravity. Our work establishes a systems biology pipeline and provides an integrated insight into the effect of microgravity at the molecular systems level. KEY WORDS Systems biology-Microgravity-Pathways and networks-Bacteria. Astrobiology 16, 677-689.
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Affiliation(s)
- Raktim Roy
- 1 Department of Chemistry and Biochemistry, Presidency University , Kolkata, India
| | - P Phani Shilpa
- 1 Department of Chemistry and Biochemistry, Presidency University , Kolkata, India
| | - Sangram Bagh
- 2 Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics , Kolkata, India
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7
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Huang S, Lee AJ, Tsoi R, Wu F, Zhang Y, Leong KW, You L. Coupling spatial segregation with synthetic circuits to control bacterial survival. Mol Syst Biol 2016; 12:859. [PMID: 26925805 PMCID: PMC4770385 DOI: 10.15252/msb.20156567] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Engineered bacteria have great potential for medical and environmental applications. Fulfilling this potential requires controllability over engineered behaviors and scalability of the engineered systems. Here, we present a platform technology, microbial swarmbot, which employs spatial arrangement to control the growth dynamics of engineered bacteria. As a proof of principle, we demonstrated a safeguard strategy to prevent unintended bacterial proliferation. In particular, we adopted several synthetic gene circuits to program collective survival in Escherichia coli: the engineered bacteria could only survive when present at sufficiently high population densities. When encapsulated by permeable membranes, these bacteria can sense the local environment and respond accordingly. The cells inside the microbial swarmbot capsules will survive due to their high densities. Those escaping from a capsule, however, will be killed due to a decrease in their densities. We demonstrate that this design concept is modular and readily generalizable. Our work lays the foundation for engineering integrated and programmable control of hybrid biological–material systems for diverse applications.
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Affiliation(s)
- Shuqiang Huang
- Department of Biomedical Engineering, Duke University, Durham, NC, USA
| | - Anna Jisu Lee
- Department of Biomedical Engineering, Duke University, Durham, NC, USA
| | - Ryan Tsoi
- Department of Biomedical Engineering, Duke University, Durham, NC, USA
| | - Feilun Wu
- Department of Biomedical Engineering, Duke University, Durham, NC, USA
| | - Ying Zhang
- Department of Biomedical Engineering, Columbia University, New York, NY, USA
| | - Kam W Leong
- Department of Biomedical Engineering, Columbia University, New York, NY, USA
| | - Lingchong You
- Department of Biomedical Engineering, Duke University, Durham, NC, USA Center for Genomic and Computational Biology, Duke University, Durham, NC, USA
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8
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Andreozzi S, Chakrabarti A, Soh KC, Burgard A, Yang TH, Van Dien S, Miskovic L, Hatzimanikatis V. Identification of metabolic engineering targets for the enhancement of 1,4-butanediol production in recombinant E. coli using large-scale kinetic models. Metab Eng 2016; 35:148-159. [PMID: 26855240 DOI: 10.1016/j.ymben.2016.01.009] [Citation(s) in RCA: 60] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2015] [Revised: 12/16/2015] [Accepted: 01/29/2016] [Indexed: 11/24/2022]
Abstract
Rational metabolic engineering methods are increasingly employed in designing the commercially viable processes for the production of chemicals relevant to pharmaceutical, biotechnology, and food and beverage industries. With the growing availability of omics data and of methodologies capable to integrate the available data into models, mathematical modeling and computational analysis are becoming important in designing recombinant cellular organisms and optimizing cell performance with respect to desired criteria. In this contribution, we used the computational framework ORACLE (Optimization and Risk Analysis of Complex Living Entities) to analyze the physiology of recombinant Escherichia coli producing 1,4-butanediol (BDO) and to identify potential strategies for improved production of BDO. The framework allowed us to integrate data across multiple levels and to construct a population of large-scale kinetic models despite the lack of available information about kinetic properties of every enzyme in the metabolic pathways. We analyzed these models and we found that the enzymes that primarily control the fluxes leading to BDO production are part of central glycolysis, the lower branch of tricarboxylic acid (TCA) cycle and the novel BDO production route. Interestingly, among the enzymes between the glucose uptake and the BDO pathway, the enzymes belonging to the lower branch of TCA cycle have been identified as the most important for improving BDO production and yield. We also quantified the effects of changes of the target enzymes on other intracellular states like energy charge, cofactor levels, redox state, cellular growth, and byproduct formation. Independent earlier experiments on this strain confirmed that the computationally obtained conclusions are consistent with the experimentally tested designs, and the findings of the present studies can provide guidance for future work on strain improvement. Overall, these studies demonstrate the potential and effectiveness of ORACLE for the accelerated design of microbial cell factories.
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Affiliation(s)
- Stefano Andreozzi
- Laboratory of Computational Systems Biotechnology (LCSB), Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland; Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland
| | - Anirikh Chakrabarti
- Laboratory of Computational Systems Biotechnology (LCSB), Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland; Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland
| | - Keng Cher Soh
- Laboratory of Computational Systems Biotechnology (LCSB), Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland; Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland
| | | | | | | | - Ljubisa Miskovic
- Laboratory of Computational Systems Biotechnology (LCSB), Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland; Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland
| | - Vassily Hatzimanikatis
- Laboratory of Computational Systems Biotechnology (LCSB), Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland; Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland.
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9
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Gibson TE, Bashan A, Cao HT, Weiss ST, Liu YY. On the Origins and Control of Community Types in the Human Microbiome. PLoS Comput Biol 2016; 12:e1004688. [PMID: 26866806 PMCID: PMC4750989 DOI: 10.1371/journal.pcbi.1004688] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2015] [Accepted: 12/01/2015] [Indexed: 01/12/2023] Open
Abstract
Microbiome-based stratification of healthy individuals into compositional categories, referred to as "enterotypes" or "community types", holds promise for drastically improving personalized medicine. Despite this potential, the existence of community types and the degree of their distinctness have been highly debated. Here we adopted a dynamic systems approach and found that heterogeneity in the interspecific interactions or the presence of strongly interacting species is sufficient to explain community types, independent of the topology of the underlying ecological network. By controlling the presence or absence of these strongly interacting species we can steer the microbial ecosystem to any desired community type. This open-loop control strategy still holds even when the community types are not distinct but appear as dense regions within a continuous gradient. This finding can be used to develop viable therapeutic strategies for shifting the microbial composition to a healthy configuration.
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Affiliation(s)
- Travis E. Gibson
- Channing Division of Network Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America
| | - Amir Bashan
- Channing Division of Network Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America
| | - Hong-Tai Cao
- Channing Division of Network Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America
- Department of Electrical Engineering, University of Southern California, Los Angeles, California, United States of America
- Chu Kochen Honors College, College of Electrical Engineering, Zhejiang University, Hangzhou, Zhejiang, China
| | - Scott T. Weiss
- Channing Division of Network Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America
| | - Yang-Yu Liu
- Channing Division of Network Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America
- Center for Cancer Systems Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America
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10
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Albers SC, Gallegos VA, Peebles CAM. Engineering of genetic control tools in Synechocystis sp. PCC 6803 using rational design techniques. J Biotechnol 2015; 216:36-46. [PMID: 26450561 DOI: 10.1016/j.jbiotec.2015.09.042] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2015] [Revised: 09/17/2015] [Accepted: 09/29/2015] [Indexed: 12/01/2022]
Abstract
Cyanobacteria show promise as photosynthetic microbial factories capable of harnessing sunlight and CO2 to produce valuable end products, but few genetic control tools have been characterized and utilized in these organisms. To develop a suite of control elements capable of gene control at a variety of expression strengths, a library of 10 promoter-constructs were developed and built via rational design techniques by adding individual nucleotides in a step-wise manner within the -10 and -35 cis-acting regions of the tac promoter. This suite produced a dynamic range of expression strength, exhibiting a 78 fold change between the lowest expressing promoter, Psca8- and the highest expressing promoter, Psca3-2 when tested within Synechocystis sp. PCC 6803. Additionally, this study details the construction of a chemically inducible construct for use in Synechocystis that is based on the tac repressor system most commonly used in Escherichia coli. This research demonstrates the construction of a highly expressed inducible promoter that is also capable of high levels of gene repression. Upon chemical induction with IPTG, this same mutant strain was capable of exhibiting an average 24X increase in GFP expression over that of the repressed state.
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Affiliation(s)
- Stevan C Albers
- Cell and Molecular Biology Graduate Program, Colorado State University, 1005 Campus Delivery, Fort Collins, CO 80523, USA.
| | - Victor A Gallegos
- Department of Biochemistry and Molecular Biology, Colorado State University, 1870 Campus Delivery, Fort Collins, CO 80523, USA; Department of Chemical and Biological Engineering, Colorado State University, 1370 Campus Delivery, Fort Collins, CO 80523, USA.
| | - Christie A M Peebles
- Cell and Molecular Biology Graduate Program, Colorado State University, 1005 Campus Delivery, Fort Collins, CO 80523, USA; Department of Chemical and Biological Engineering, Colorado State University, 1370 Campus Delivery, Fort Collins, CO 80523, USA.
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11
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Gupta SK, Shukla P. Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications. Crit Rev Biotechnol 2015; 36:1089-1098. [DOI: 10.3109/07388551.2015.1084264] [Citation(s) in RCA: 108] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Affiliation(s)
- Sanjeev K. Gupta
- Enzyme Technology and Protein Bioinformatics Laboratory, Department of Microbiology, Maharshi Dayanand University, Rohtak, Haryana, India and
- Advanced Biotech Lab (Centre for Research & Development), Ipca Laboratories Ltd., Kandivli (west), Mumbai, India
| | - Pratyoosh Shukla
- Enzyme Technology and Protein Bioinformatics Laboratory, Department of Microbiology, Maharshi Dayanand University, Rohtak, Haryana, India and
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12
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Synthetic biology and biomimetic chemistry as converging technologies fostering a new generation of smart biosensors. Biosens Bioelectron 2015; 74:1076-86. [PMID: 26277908 DOI: 10.1016/j.bios.2015.07.078] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2015] [Revised: 07/30/2015] [Accepted: 07/31/2015] [Indexed: 01/05/2023]
Abstract
Biosensors are powerful tunable systems able to switch between an ON/OFF status in response to an external stimulus. This extraordinary property could be engineered by adopting synthetic biology or biomimetic chemistry to obtain tailor-made biosensors having the desired requirements of robustness, sensitivity and detection range. Recent advances in both disciplines, in fact, allow to re-design the configuration of the sensing elements - either by modifying toggle switches and gene networks, or by producing synthetic entities mimicking key properties of natural molecules. The present review considered the role of synthetic biology in sustaining biosensor technology, reporting examples from the literature and reflecting on the features that make it a useful tool for designing and constructing engineered biological systems for sensing application. Besides, a section dedicated to bioinspired synthetic molecules as powerful tools to enhance biosensor potential is reported, and treated as an extension of the concept of biomimetic chemistry, where organic synthesis is used to generate artificial molecules that mimic natural molecules. Thus, the design of synthetic molecules, such as aptamers, biomimetics, molecular imprinting polymers, peptide nucleic acids, and ribozymes were encompassed as "products" of biomimetic chemistry.
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13
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Near-real-time analysis of the phenotypic responses of Escherichia coli to 1-butanol exposure using Raman Spectroscopy. J Bacteriol 2014; 196:3983-91. [PMID: 25157078 DOI: 10.1128/jb.01590-14] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Raman spectroscopy was used to study the time course of phenotypic responses of Escherichia coli (DH5α) to 1-butanol exposure (1.2% [vol/vol]). Raman spectroscopy is of interest for bacterial phenotyping because it can be performed (i) in near real time, (ii) with minimal sample preparation (label-free), and (iii) with minimal spectral interference from water. Traditional off-line analytical methodologies were applied to both 1-butanol-treated and control cells to draw correlations with Raman data. Here, distinct sets of Raman bands are presented that characterize phenotypic traits of E. coli with maximized correlation to off-line measurements. In addition, the observed time course phenotypic responses of E. coli to 1.2% (vol/vol) 1-butanol exposure included the following: (i) decreased saturated fatty acids levels, (ii) retention of unsaturated fatty acids and low levels of cyclopropane fatty acids, (iii) increased membrane fluidity following the initial response of increased rigidity, and (iv) no changes in total protein content or protein-derived amino acid composition. For most phenotypic traits, correlation coefficients between Raman spectroscopy and traditional off-line analytical approaches exceeded 0.75, and major trends were captured. The results suggest that near-real-time Raman spectroscopy is suitable for approximating metabolic and physiological phenotyping of bacterial cells subjected to toxic environmental conditions.
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14
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Wang Z, Wu X, Peng J, Hu Y, Fang B, Huang S. Artificially constructed quorum-sensing circuits are used for subtle control of bacterial population density. PLoS One 2014; 9:e104578. [PMID: 25119347 PMCID: PMC4132116 DOI: 10.1371/journal.pone.0104578] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2013] [Accepted: 07/15/2014] [Indexed: 01/31/2023] Open
Abstract
Vibrio fischeri is a typical quorum-sensing bacterium for which lux box, luxR, and luxI have been identified as the key elements involved in quorum sensing. To decode the quorum-sensing mechanism, an artificially constructed cell–cell communication system has been built. In brief, the system expresses several programmed cell-death BioBricks and quorum-sensing genes driven by the promoters lux pR and PlacO-1 in Escherichia coli cells. Their transformation and expression was confirmed by gel electrophoresis and sequencing. To evaluate its performance, viable cell numbers at various time periods were investigated. Our results showed that bacteria expressing killer proteins corresponding to ribosome binding site efficiency of 0.07, 0.3, 0.6, or 1.0 successfully sensed each other in a population-dependent manner and communicated with each other to subtly control their population density. This was also validated using a proposed simple mathematical model.
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Affiliation(s)
- Zhaoshou Wang
- Institute of Biochemical Engineering, Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China
- The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, China
| | - Xin Wu
- Institute of Biochemical Engineering, Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China
- The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, China
| | - Jianghai Peng
- Institute of Biochemical Engineering, Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China
| | - Yidan Hu
- Institute of Biochemical Engineering, Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China
- The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, China
| | - Baishan Fang
- Institute of Biochemical Engineering, Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China
- The Key Lab for Chemical Biology of Fujian Province, Xiamen University, Xiamen, China
- The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, China
- * E-mail:
| | - Shiyang Huang
- Institute of Biochemical Engineering, Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China
- The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, China
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15
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Elena C, Ravasi P, Castelli ME, Peirú S, Menzella HG. Expression of codon optimized genes in microbial systems: current industrial applications and perspectives. Front Microbiol 2014; 5:21. [PMID: 24550894 PMCID: PMC3912506 DOI: 10.3389/fmicb.2014.00021] [Citation(s) in RCA: 77] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2013] [Accepted: 01/14/2014] [Indexed: 11/24/2022] Open
Abstract
The efficient production of functional proteins in heterologous hosts is one of the major bases of modern biotechnology. Unfortunately, many genes are difficult to express outside their original context. Due to their apparent “silent” nature, synonymous codon substitutions have long been thought to be trivial. In recent years, this dogma has been refuted by evidence that codon replacement can have a significant impact on gene expression levels and protein folding. In the past decade, considerable advances in the speed and cost of gene synthesis have facilitated the complete redesign of entire gene sequences, dramatically improving the likelihood of high protein expression. This technology significantly impacts the economic feasibility of microbial-based biotechnological processes by, for example, increasing the volumetric productivities of recombinant proteins or facilitating the redesign of novel biosynthetic routes for the production of metabolites. This review discusses the current applications of this technology, particularly those regarding the production of small molecules and industrially relevant recombinant enzymes. Suggestions for future research and potential uses are provided as well.
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Affiliation(s)
- Claudia Elena
- Genetic Engineering and Fermentation Technology, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario-Conicet Rosario, Argentina
| | - Pablo Ravasi
- Genetic Engineering and Fermentation Technology, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario-Conicet Rosario, Argentina
| | - María E Castelli
- Genetic Engineering and Fermentation Technology, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario-Conicet Rosario, Argentina
| | - Salvador Peirú
- Genetic Engineering and Fermentation Technology, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario-Conicet Rosario, Argentina
| | - Hugo G Menzella
- Genetic Engineering and Fermentation Technology, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario-Conicet Rosario, Argentina
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16
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De Roy K, Marzorati M, Van den Abbeele P, Van de Wiele T, Boon N. Synthetic microbial ecosystems: an exciting tool to understand and apply microbial communities. Environ Microbiol 2013; 16:1472-81. [PMID: 24274586 DOI: 10.1111/1462-2920.12343] [Citation(s) in RCA: 179] [Impact Index Per Article: 14.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2013] [Accepted: 11/19/2013] [Indexed: 12/24/2022]
Abstract
Many microbial ecologists have described the composition of microbial communities in a plenitude of environments, which has greatly improved our basic understanding of microorganisms and ecosystems. However, the factors and processes that influence the behaviour and functionality of an ecosystem largely remain black boxes when using conventional approaches. Therefore, synthetic microbial ecology has gained a lot of interest in the last few years. Because of their reduced complexity and increased controllability, synthetic communities are often preferred over complex communities to examine ecological theories. They limit the factors that influence the microbial community to a minimum, allowing their management and identifying specific community responses. However, besides their use for basic research, synthetic ecosystems also found their way towards different applications, like industrial fermentation and bioremediation. Here, we review why and how synthetic microbial communities are applied for research purposes and for which applications they have been and could be successfully used.
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Affiliation(s)
- Karen De Roy
- Laboratory of Microbial Ecology and Technology (LabMET), Coupure Links 653, 9000, Gent, Belgium
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Kogge W, Richter M. Synthetic biology and its alternatives. Descartes, Kant and the idea of engineering biological machines. STUDIES IN HISTORY AND PHILOSOPHY OF BIOLOGICAL AND BIOMEDICAL SCIENCES 2013; 44:181-189. [PMID: 23623436 DOI: 10.1016/j.shpsc.2013.03.013] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/02/2023]
Abstract
The engineering-based approach of synthetic biology is characterized by an assumption that 'engineering by design' enables the construction of 'living machines'. These 'machines', as biological machines, are expected to display certain properties of life, such as adapting to changing environments and acting in a situated way. This paper proposes that a tension exists between the expectations placed on biological artefacts and the notion of producing such systems by means of engineering; this tension makes it seem implausible that biological systems, especially those with properties characteristic of living beings, can in fact be produced using the specific methods of engineering. We do not claim that engineering techniques have nothing to contribute to the biotechnological construction of biological artefacts. However, drawing on Descartes's and Kant's thinking on the relationship between the organism and the machine, we show that it is considerably more plausible to assume that distinctively biological artefacts emerge within a paradigm different from the paradigm of the Cartesian machine that underlies the engineering approach. We close by calling for increased attention to be paid to approaches within molecular biology and chemistry that rest on conceptions different from those of synthetic biology's engineering paradigm.
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Affiliation(s)
- Werner Kogge
- Freie Universität Berlin, Institute of Philosophy, Habelschwerdter Allee 30, 14195 Berlin, Germany.
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Ienczak JL, Schmidell W, Aragão GMFD. High-cell-density culture strategies for polyhydroxyalkanoate production: a review. J Ind Microbiol Biotechnol 2013; 40:275-86. [PMID: 23455696 DOI: 10.1007/s10295-013-1236-z] [Citation(s) in RCA: 62] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2012] [Accepted: 01/25/2013] [Indexed: 11/30/2022]
Abstract
This article gives an overview of high-cell-density cultures for polyhydroxyalkanoate (PHA) production and their modes of operation for increasing productivity. High cell densities are very important in PHA production mainly because this polymer is an intracellular product accumulated in various microorganisms, so a high cellular content is needed for the polymer production. This review describes relevant results from fed-batch, repeated batch, and continuous modes of operation without and with cell recycle for the production of these polymers by microorganisms. Finally, recombinant microorganisms for PHA production, as well future directions for PHA production, are discussed.
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Affiliation(s)
- Jaciane Lutz Ienczak
- Brazilian Bioethanol Science and Technology Laboratory, CTBE/CNPEM, Campinas, SP, Brazil
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Korch SB, Stomel JM, León MA, Hamada MA, Stevenson CR, Simpson BW, Gujulla SK, Chaput JC. ATP sequestration by a synthetic ATP-binding protein leads to novel phenotypic changes in Escherichia coli. ACS Chem Biol 2013. [PMID: 23181457 DOI: 10.1021/cb3004786] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Artificial proteins that bind key metabolites with high affinity and specificity hold great promise as new tools in synthetic biology, but little has been done to create such molecules and examine their effects on living cells. Experiments of this kind have the potential to expand our understanding of cellular systems, as certain phenotypes may be physically realistic but not yet observed in nature. Here, we examine the physiology and morphology of a population of Escherichia coli as they respond to a genetically encoded, non-biological ATP-binding protein. Unlike natural ATP-dependent proteins, which transiently bind ATP during metabolic transformations, the synthetic protein DX depletes the concentration of intracellular ATP and ADP by a mechanism of protein-mediated ligand sequestration. The resulting ATP/ADP imbalance leads to an adaptive response in which a large population of bacilli cells transition to a filamentous state with dense lipid structures that segregate the cells into compartmentalized units. A wide range of biochemical and microscopy techniques extensively characterized these novel lipid structures, which we have termed endoliposomes. We show that endoliposomes adopt well-defined box-like structures that span the full width of the cell but exclude the synthetic protein DX. We further show that prolonged DX exposure causes a large fraction of the population to enter a viable-but-non-culturable state that is not easily reversed. Both phenotypes correlate with strong intracellular changes in ATP and ADP concentration. We suggest that artificial proteins, such as DX, could be used to control and regulate specific targets in metabolic pathways.
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Affiliation(s)
- Shaleen B. Korch
- Department
of Pharmacology, Midwestern University,
Glendale, Arizona 85308, United
States
| | | | | | - Matt A. Hamada
- Department
of Pharmacology, Midwestern University,
Glendale, Arizona 85308, United
States
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Abstract
Comparatively few studies have addressed directly the question of quantifying the benefits to be had from using molecular genetic markers in experimental breeding programmes (e.g. for improved crops and livestock), nor the question of which organisms should be mated with each other to best effect. We argue that this requires in silico modelling, an approach for which there is a large literature in the field of evolutionary computation (EC), but which has not really been applied in this way to experimental breeding programmes. EC seeks to optimise measurable outcomes (phenotypic fitnesses) by optimising in silico the mutation, recombination and selection regimes that are used. We review some of the approaches from EC, and compare experimentally, using a biologically relevant in silico landscape, some algorithms that have knowledge of where they are in the (genotypic) search space (G-algorithms) with some (albeit well-tuned ones) that do not (F-algorithms). For the present kinds of landscapes, F- and G-algorithms were broadly comparable in quality and effectiveness, although we recognise that the G-algorithms were not equipped with any ‘prior knowledge’ of epistatic pathway interactions. This use of algorithms based on machine learning has important implications for the optimisation of experimental breeding programmes in the post-genomic era when we shall potentially have access to the full genome sequence of every organism in a breeding population. The non-proprietary code that we have used is made freely available (via Supplementary information).
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Ravasi P, Peiru S, Gramajo H, Menzella HG. Design and testing of a synthetic biology framework for genetic engineering of Corynebacterium glutamicum. Microb Cell Fact 2012; 11:147. [PMID: 23134565 PMCID: PMC3539996 DOI: 10.1186/1475-2859-11-147] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2012] [Accepted: 11/03/2012] [Indexed: 12/31/2022] Open
Abstract
BACKGROUND Synthetic biology approaches can make a significant contribution to the advance of metabolic engineering by reducing the development time of recombinant organisms. However, most of synthetic biology tools have been developed for Escherichia coli. Here we provide a platform for rapid engineering of C. glutamicum, a microorganism of great industrial interest. This bacteria, used for decades for the fermentative production of amino acids, has recently been developed as a host for the production of several economically important compounds including metabolites and recombinant proteins because of its higher capacity of secretion compared to traditional bacterial hosts like E. coli. Thus, the development of modern molecular platforms may significantly contribute to establish C. glutamicum as a robust and versatile microbial factory. RESULTS A plasmid based platform named pTGR was created where all the genetic components are flanked by unique restriction sites to both facilitate the evaluation of regulatory sequences and the assembly of constructs for the expression of multiple genes. The approach was validated by using reporter genes to test promoters, ribosome binding sites, and for the assembly of dual gene operons and gene clusters containing two transcriptional units. Combinatorial assembly of promoter (tac, cspB and sod) and RBS (lacZ, cspB and sod) elements with different strengths conferred clear differential gene expression of two reporter genes, eGFP and mCherry, thus allowing transcriptional "fine-tuning"of multiple genes. In addition, the platform allowed the rapid assembly of operons and genes clusters for co-expression of heterologous genes, a feature that may assist metabolic pathway engineering. CONCLUSIONS We anticipate that the pTGR platform will contribute to explore the potential of novel parts to regulate gene expression, and to facilitate the assembly of genetic circuits for metabolic engineering of C. glutamicum. The standardization provided by this approach may provide a means to improve the productivity of biosynthetic pathways in microbial factories for the production of novel compounds.
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Affiliation(s)
- Pablo Ravasi
- Genetic Engineering & Fermentation Technology. Instituto de Biología Celular y Molecular de Rosario-CONICET. Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario, 2000, República Argentina
- Geneg SRL, Cuba 4710, Buenos Aires, Argentina
| | - Salvador Peiru
- Genetic Engineering & Fermentation Technology. Instituto de Biología Celular y Molecular de Rosario-CONICET. Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario, 2000, República Argentina
| | - Hugo Gramajo
- Genetic Engineering & Fermentation Technology. Instituto de Biología Celular y Molecular de Rosario-CONICET. Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario, 2000, República Argentina
| | - Hugo G Menzella
- Genetic Engineering & Fermentation Technology. Instituto de Biología Celular y Molecular de Rosario-CONICET. Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario, 2000, República Argentina
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22
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Lanza AM, Cheng JK, Alper HS. Emerging synthetic biology tools for engineering mammalian cell systems and expediting cell line development. Curr Opin Chem Eng 2012. [DOI: 10.1016/j.coche.2012.09.005] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
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23
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Takano E, Bovenberg RAL, Breitling R. A turning point for natural product discovery--ESF-EMBO research conference: synthetic biology of antibiotic production. Mol Microbiol 2012; 83:884-93. [PMID: 22296491 DOI: 10.1111/j.1365-2958.2012.07984.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Synthetic Biology is in a critical phase of its development: it has finally reached the point where it can move from proof-of-principle studies to real-world applications. Secondary metabolite biosynthesis, especially the discovery and production of antibiotics, is a particularly relevant target area for such applications of synthetic biology. The first international conference to explore this subject was held in Spain in October 2011. In four sessions on General Synthetic Biology, Filamentous Fungal Systems, Actinomyces Systems, and Tools and Host Structures, scientists presented the most recent technological and scientific advances, and a final-day Forward Look Plenary Discussion identified future trends in the field.
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Affiliation(s)
- Eriko Takano
- Department of Microbial Physiology,Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborg 7, Groningen, The Netherlands.
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24
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25
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Zhu L, Zhu Y, Zhang Y, Li Y. Engineering the robustness of industrial microbes through synthetic biology. Trends Microbiol 2012; 20:94-101. [DOI: 10.1016/j.tim.2011.12.003] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2011] [Revised: 11/30/2011] [Accepted: 12/14/2011] [Indexed: 11/26/2022]
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Abstract
One important aim of synthetic biology is to develop a self-replicating biological system capable of performing useful tasks. A mathematical model of a synthetic organism would greatly enhance its value by providing a platform in which proposed modifications to the system could be rapidly prototyped and tested. Such a platform would allow the explicit connection of genomic sequence information to physiological predictions. As an initial step toward this aim, a minimal cell model (MCM) has been formulated. The MCM is defined as a model of a hypothetical cell with the minimum number of genes necessary to grow and divide in an optimally supportive culture environment. It is chemically detailed in terms of genes and gene products, as well as physiologically complete in terms of bacterial cell processes (e.g., DNA replication and cell division). A mathematical framework originally developed for modeling Escherichia coli has been used to build the platform MCM. A MCM with 241 product-coding genes (those which produce protein or stable RNA products) is presented. This gene set is genomically complete in that it codes for all the functions that a minimal chemoheterotrophic bacterium would require for sustained growth and division. With this model, the hypotheses behind a minimal gene set can be tested using a chemically detailed, dynamic, whole-cell modeling approach. Furthermore, the MCM can simulate the behavior of a whole cell that depends on the cell's (1) metabolic rates and chemical state, (2) genome in terms of expression of various genes, (3) environment both in terms of direct nutrient starvation and competitive inhibition leading to starvation, and (4) genomic sequence in terms of the chromosomal locations of genes.
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Affiliation(s)
- Michael L Shuler
- Department of Biomedical Engineering, Cornell University, Ithaca, NY, USA.
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27
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Solomon KV, Prather KLJ. The zero-sum game of pathway optimization: emerging paradigms for tuning gene expression. Biotechnol J 2011; 6:1064-70. [PMID: 21695787 DOI: 10.1002/biot.201100086] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2011] [Revised: 04/30/2011] [Accepted: 05/09/2011] [Indexed: 12/20/2022]
Abstract
With increasing price volatility and growing awareness of the lack of sustainability of traditional chemical synthesis, microbial chemical production has been tapped as a promising renewable alternative for the generation of diverse, stereospecific compounds. Nonetheless, many attempts to generate them are not yet economically viable. Due to the zero-sum nature of microbial resources, traditional strategies of pathway optimization are attaining minimal returns. This result is in part a consequence of the gross changes in host physiology resulting from such efforts and underscores the need for more precise and subtle forms of gene modulation. In this review, we describe alternative strategies and emerging paradigms to address this problem and highlight potential solutions from the emerging field of synthetic biology.
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Affiliation(s)
- Kevin V Solomon
- Department of Chemical Engineering, Synthetic Biology Engineering Research Center (SynBERC), Massachusetts Institute of Technology, Cambridge, MA, USA
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28
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Engineering microbial factories for synthesis of value-added products. J Ind Microbiol Biotechnol 2011; 38:873-90. [PMID: 21526386 DOI: 10.1007/s10295-011-0970-3] [Citation(s) in RCA: 150] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2011] [Accepted: 04/01/2011] [Indexed: 01/07/2023]
Abstract
Microorganisms have become an increasingly important platform for the production of drugs, chemicals, and biofuels from renewable resources. Advances in protein engineering, metabolic engineering, and synthetic biology enable redesigning microbial cellular networks and fine-tuning physiological capabilities, thus generating industrially viable strains for the production of natural and unnatural value-added compounds. In this review, we describe the recent progress on engineering microbial factories for synthesis of valued-added products including alkaloids, terpenoids, flavonoids, polyketides, non-ribosomal peptides, biofuels, and chemicals. Related topics on lignocellulose degradation, sugar utilization, and microbial tolerance improvement will also be discussed.
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29
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30
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Early Career Research Award Lecture. Structure, evolution and dynamics of transcriptional regulatory networks. Biochem Soc Trans 2011; 38:1155-78. [PMID: 20863280 DOI: 10.1042/bst0381155] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
The availability of entire genome sequences and the wealth of literature on gene regulation have enabled researchers to model an organism's transcriptional regulation system in the form of a network. In such a network, TFs (transcription factors) and TGs (target genes) are represented as nodes and regulatory interactions between TFs and TGs are represented as directed links. In the present review, I address the following topics pertaining to transcriptional regulatory networks. (i) Structure and organization: first, I introduce the concept of networks and discuss our understanding of the structure and organization of transcriptional networks. (ii) Evolution: I then describe the different mechanisms and forces that influence network evolution and shape network structure. (iii) Dynamics: I discuss studies that have integrated information on dynamics such as mRNA abundance or half-life, with data on transcriptional network in order to elucidate general principles of regulatory network dynamics. In particular, I discuss how cell-to-cell variability in the expression level of TFs could permit differential utilization of the same underlying network by distinct members of a genetically identical cell population. Finally, I conclude by discussing open questions for future research and highlighting the implications for evolution, development, disease and applications such as genetic engineering.
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31
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Synthetic biology: From the first synthetic cell to see its current situation and future development. ACTA ACUST UNITED AC 2011; 56:229-237. [PMID: 32214738 PMCID: PMC7088918 DOI: 10.1007/s11434-010-4304-z] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2010] [Accepted: 10/19/2010] [Indexed: 10/31/2022]
Abstract
Synthetic biology is an emerging field, which, since its birth, has shown great value and potential in many fields including medicine, energy, environment and agriculture. It is also important for the study of the origin and evolution of life. Since the publication of the first synthetic cell in May, 2010, synthetic biology again attracts high attention and leads to extensive discussions all over the world. There have been a number of researches and achievements on synthetic biology in the United States and European countries. While in China, so far there is no systematic research on synthetic biology. In order to promote the development of this new discipline in China, we organized this review to systematically introduce the concept and research content of synthetic biology, summarize the achievements, and investigate the current situation in both China and abroad. We also analyzed the opportunities and challenges in synthetic biology, and looked forward to the future development of synthetic biology, especially its future development in China.
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32
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An active intracellular device to prevent lethal disease outcomes in virus-infected bacterial cells. Biotechnol Bioeng 2010; 108:645-54. [DOI: 10.1002/bit.22969] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2010] [Revised: 09/10/2010] [Accepted: 09/27/2010] [Indexed: 01/26/2023]
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33
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Wang X, Sa N, Tian PF, Tan TW. Classifying DNA assembly protocols for devising cellular architectures. Biotechnol Adv 2010; 29:156-63. [PMID: 21034806 DOI: 10.1016/j.biotechadv.2010.10.004] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2010] [Revised: 10/09/2010] [Accepted: 10/17/2010] [Indexed: 11/26/2022]
Abstract
DNA assembly is one of the most fundamental techniques in synthetic biology. Efficient methods can turn traditional DNA cloning into time-saving and higher efficiency practice, which is a foundation to accomplish the dreams of synthetic biologists for devising cellular architectures, reprogramming cellular behaviors, or creating synthetic cells. In this review, typical strategies of DNA assembly are discussed with special emphasis on the assembly of long and multiple DNA fragments into intact plasmids or assembled compositions. Constructively, all reported strategies were categorized into in vivo and in vitro types, and protocols are presented in a functional and practice-oriented way in order to portray the general nature of DNA assembly applications. Significantly, a five-step blueprint is proposed for devising cell architectures that produce valuable chemicals.
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Affiliation(s)
- Xi Wang
- College of Life Science and Technology, Beijing University of Chemical Technology, 15 Beisanhuan East Road, Beijing 100029, PR China
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34
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Combining metabolic and protein engineering of a terpenoid biosynthetic pathway for overproduction and selectivity control. Proc Natl Acad Sci U S A 2010; 107:13654-9. [PMID: 20643967 DOI: 10.1073/pnas.1006138107] [Citation(s) in RCA: 256] [Impact Index Per Article: 17.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
A common strategy of metabolic engineering is to increase the endogenous supply of precursor metabolites to improve pathway productivity. The ability to further enhance heterologous production of a desired compound may be limited by the inherent capacity of the imported pathway to accommodate high precursor supply. Here, we present engineered diterpenoid biosynthesis as a case where insufficient downstream pathway capacity limits high-level levopimaradiene production in Escherichia coli. To increase levopimaradiene synthesis, we amplified the flux toward isopentenyl diphosphate and dimethylallyl diphosphate precursors and reprogrammed the rate-limiting downstream pathway by generating combinatorial mutations in geranylgeranyl diphosphate synthase and levopimaradiene synthase. The mutant library contained pathway variants that not only increased diterpenoid production but also tuned the selectivity toward levopimaradiene. The most productive pathway, combining precursor flux amplification and mutant synthases, conferred approximately 2,600-fold increase in levopimaradiene levels. A maximum titer of approximately 700 mg/L was subsequently obtained by cultivation in a bench-scale bioreactor. The present study highlights the importance of engineering proteins along with pathways as a key strategy in achieving microbial biosynthesis and overproduction of pharmaceutical and chemical products.
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35
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Foley PL, Shuler ML. Considerations for the design and construction of a synthetic platform cell for biotechnological applications. Biotechnol Bioeng 2010; 105:26-36. [PMID: 19816966 DOI: 10.1002/bit.22575] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
The design and construction of an artificial bacterial cell could revolutionize biotechnological processes and technologies. A functional platform cell that can be easily customized for a pre-defined task would be useful for applications from producing therapeutics to decontaminating waste streams. The platform cell must be robust and highly efficient. A biotechnological platform cell is related to the concept of a minimal cell, but several factors beyond those necessary for a minimal cell must be considered for a synthetic organism designed for biotechnological applications. Namely, a platform cell must exhibit robust cell reproduction, decreased genetic drift, a physically robust cell envelope, efficient and simplified transcription and translation controls, and predictable metabolic interactions. Achieving a biotechnological platform cell will benefit from insights acquired from a minimal cell, but an approach of minimizing an existing organism's genome may be a more practical experimental approach. Escherichia coli possess many of the desired characteristics of a platform cell and could serve as a useful model organism for the design and construction of a synthetic platform organism. In this article we review briefly the current state of research in this field and outline specific characteristics that will be important for a biotechnologically relevant synthetic cell that has a minimized genome and efficient regulatory structure.
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Affiliation(s)
- P L Foley
- School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA
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36
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Na D, Kim TY, Lee SY. Construction and optimization of synthetic pathways in metabolic engineering. Curr Opin Microbiol 2010; 13:363-70. [PMID: 20219419 DOI: 10.1016/j.mib.2010.02.004] [Citation(s) in RCA: 88] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2010] [Accepted: 02/10/2010] [Indexed: 11/25/2022]
Abstract
Metabolic engineering has enabled us to develop strains suitable for their use as microbial factories of chemicals and materials from renewable sources. It has recently become more powerful with the advanced in synthetic biology, which is allowing us to create novel and fine-controlled metabolic and regulatory circuits maximizing metabolic fluxes to the desired products in the strain being developed. This enables us to engineer host microorganisms to enhance their innate metabolic capabilities or to gain new capabilities in the production of target compounds. Here we review recently constructed synthetic pathways that have been successfully applied for producing non-innate chemicals and also discuss recent approaches developed to increase the efficiency of synthetic pathways for achieving higher productivities of desired bioproducts.
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Affiliation(s)
- Dokyun Na
- Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Program), Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea
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37
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Alterovitz G, Muso T, Ramoni MF. The challenges of informatics in synthetic biology: from biomolecular networks to artificial organisms. Brief Bioinform 2009; 11:80-95. [PMID: 19906839 DOI: 10.1093/bib/bbp054] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
The field of synthetic biology holds an inspiring vision for the future; it integrates computational analysis, biological data and the systems engineering paradigm in the design of new biological machines and systems. These biological machines are built from basic biomolecular components analogous to electrical devices, and the information flow among these components requires the augmentation of biological insight with the power of a formal approach to information management. Here we review the informatics challenges in synthetic biology along three dimensions: in silico, in vitro and in vivo. First, we describe state of the art of the in silico support of synthetic biology, from the specific data exchange formats, to the most popular software platforms and algorithms. Next, we cast in vitro synthetic biology in terms of information flow, and discuss genetic fidelity in DNA manipulation, development strategies of biological parts and the regulation of biomolecular networks. Finally, we explore how the engineering chassis can manipulate biological circuitries in vivo to give rise to future artificial organisms.
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Affiliation(s)
- Gil Alterovitz
- Children's Hospital Informatics Program, Harvard/MITDivision of Health Sciences and Technology, USA
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38
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Crop improvement using small RNAs: applications and predictive ecological risk assessments. Trends Biotechnol 2009; 27:644-51. [DOI: 10.1016/j.tibtech.2009.08.005] [Citation(s) in RCA: 90] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2009] [Revised: 07/31/2009] [Accepted: 08/17/2009] [Indexed: 01/31/2023]
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39
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Stomel JM, Wilson JW, León MA, Stafford P, Chaput JC. A man-made ATP-binding protein evolved independent of nature causes abnormal growth in bacterial cells. PLoS One 2009; 4:e7385. [PMID: 19812699 PMCID: PMC2754611 DOI: 10.1371/journal.pone.0007385] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2009] [Accepted: 09/15/2009] [Indexed: 11/18/2022] Open
Abstract
Recent advances in de novo protein evolution have made it possible to create synthetic proteins from unbiased libraries that fold into stable tertiary structures with predefined functions. However, it is not known whether such proteins will be functional when expressed inside living cells or how a host organism would respond to an encounter with a non-biological protein. Here, we examine the physiology and morphology of Escherichia coli cells engineered to express a synthetic ATP-binding protein evolved entirely from non-biological origins. We show that this man-made protein disrupts the normal energetic balance of the cell by altering the levels of intracellular ATP. This disruption cascades into a series of events that ultimately limit reproductive competency by inhibiting cell division. We now describe a detailed investigation into the synthetic biology of this man-made protein in a living bacterial organism, and the effect that this protein has on normal cell physiology.
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Affiliation(s)
- Joshua M. Stomel
- Center for BioOptical Nanotechnology, Arizona State University, Tempe, Arizona, United States of America
- School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America
| | - James W. Wilson
- Center for Infectious Disease and Vaccinology, Arizona State University, Tempe, Arizona, United States of America
| | - Megan A. León
- Center for BioOptical Nanotechnology, Arizona State University, Tempe, Arizona, United States of America
| | - Phillip Stafford
- Center for Innovations in Medicine, The Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America
| | - John C. Chaput
- Center for BioOptical Nanotechnology, Arizona State University, Tempe, Arizona, United States of America
- Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona, United States of America
- * E-mail:
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Jung TS, Yeo HC, Reddy SG, Cho WS, Lee DY. WEbcoli: an interactive and asynchronous web application for in silico design and analysis of genome-scale E.coli model. Bioinformatics 2009; 25:2850-2. [PMID: 19689960 DOI: 10.1093/bioinformatics/btp496] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
SUMMARY WEbcoli is a WEb application for in silico designing, analyzing and engineering Escherichia coli metabolism. It is devised and implemented using advanced web technologies, thereby leading to enhanced usability and dynamic web accessibility. As a main feature, the WEbcoli system provides a user-friendly rich web interface, allowing users to virtually design and synthesize mutant strains derived from the genome-scale wild-type E.coli model and to customize pathways of interest through a graph editor. In addition, constraints-based flux analysis can be conducted for quantifying metabolic fluxes and charactering the physiological and metabolic states under various genetic and/or environmental conditions. AVAILABILITY WEbcoli is freely accessible at http://webcoli.org. CONTACT cheld@nus.edu.sg.
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Affiliation(s)
- Tae-Sung Jung
- Department of Information Industrial Engineering, CBITRC, Chungbuk National University, Cheongju Chungbuk 361-763, Korea
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Agapakis CM, Silver PA. Synthetic biology: exploring and exploiting genetic modularity through the design of novel biological networks. MOLECULAR BIOSYSTEMS 2009; 5:704-13. [PMID: 19562109 DOI: 10.1039/b901484e] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Synthetic biology has been used to describe many biological endeavors over the past thirty years--from designing enzymes and in vitro systems, to manipulating existing metabolisms and gene expression, to creating entirely synthetic replicating life forms. What separates the current incarnation of synthetic biology from the recombinant DNA technology or metabolic engineering of the past is an emphasis on principles from engineering such as modularity, standardization, and rigorously predictive models. As such, synthetic biology represents a new paradigm for learning about and using biological molecules and data, with applications in basic science, biotechnology, and medicine. This review covers the canonical examples as well as some recent advances in synthetic biology in terms of what we know and what we can learn about the networks underlying biology, and how this endeavor may shape our understanding of living systems.
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Expanding the repertoire of biofuel alternatives through metabolic pathway evolution. Proc Natl Acad Sci U S A 2009; 106:965-6. [PMID: 19164544 DOI: 10.1073/pnas.0811893106] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
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