Publications by authors named "Tae Seok Moon"

48 Publications

An Improved CRISPR Interference Tool to Engineer .

ACS Synth Biol 2021 Mar 31. Epub 2021 Mar 31.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri 63130, United States.

is a nonmodel bacterium that is well suited for valorizing lignin. Despite recent advances in our systems-level understanding of its versatile metabolism, studies of its gene functions at a single gene level are still lagging. Elucidating gene functions in nonmodel organisms is challenging due to limited genetic engineering tools that are convenient to use. To address this issue, we developed a simple gene repression system based on CRISPR interference (CRISPRi). This gene repression system uses a T7 RNA polymerase system to express a small guide RNA, demonstrating improved repression compared to the previously demonstrated CRISPRi system (, the maximum repression efficiency improved from 58% to 85%). Additionally, our cloning strategy allows for building multiple CRISPRi plasmids in parallel without any PCR step, facilitating the engineering of this GC-rich organism. Using the improved CRISPRi system, we confirmed the annotated roles of four metabolic pathway genes, which had been identified by our previous transcriptomic analysis to be related to the consumption of benzoate, vanillate, catechol, and acetate. Furthermore, we showed our tool's utility by demonstrating the inducible accumulation of muconate that is a precursor of adipic acid, an important monomer for nylon production. While the maximum muconate yield obtained using our tool was 30% of the yield obtained using gene knockout, our tool showed its inducibility and partial repressibility. Our CRISPRi tool will be useful to facilitate functional studies of this nonmodel organism and engineer this promising microbial chassis for lignin valorization.
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http://dx.doi.org/10.1021/acssynbio.0c00591DOI Listing
March 2021

Engineering microbial diagnostics and therapeutics with smart control.

Curr Opin Biotechnol 2020 12 18;66:11-17. Epub 2020 Jun 18.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, United States; Division of Biology and Biomedical Sciences, Washington University in St. Louis, St. Louis, MO, United States. Electronic address:

Microbes have become an increasingly powerful chassis for developing diagnostic and therapeutic technologies. While many of the earlier engineering efforts used microbes that expressed relevant proteins constitutively, more microbes are being engineered to express them with region-selectivity and disease-responsiveness through biosensors. Such 'smart' microbes have been developed to diagnose and treat a wide range of disorders and diseases, including bacterial infections, cancers, inflammatory disorders, and metabolic disorders. In this review, we discuss synthetic biology technologies that have been applied to engineer microbes for biomedical applications, focusing on recent reports that demonstrate microbial sensing by using animal models or clinical samples. Advances in synthetic biology will enable engineered microbes to significantly improve the medical field.
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http://dx.doi.org/10.1016/j.copbio.2020.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744387PMC
December 2020

Tailoring microbes to upgrade lignin.

Curr Opin Chem Biol 2020 Dec 7;59:23-29. Epub 2020 May 7.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, 63130, USA. Electronic address:

Lignin depolymerization generates a mixture of numerous compounds that are difficult to separate cost-effectively. To address this heterogeneity issue, microbes have been employed to 'biologically funnel' a broad range of compounds present in depolymerized lignin into common central metabolites that can be converted into a single desirable product. Because the composition of depolymerized lignin varies significantly with the type of biomass and the depolymerization method, microbes should be selected and engineered by considering this compositional variation. An ideal microbe must efficiently metabolize all relevant lignin-derived compounds regardless of the compositional variation of feedstocks, but discovering or developing such a perfect microbe is very challenging. Instead, developing multiple tailored microbes to tolerate a given mixture of lignin-derived compounds and to convert most of these into a target product is more practical. This review summarizes recent progress toward the development of such microbes for lignin valorization and offers future directions.
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http://dx.doi.org/10.1016/j.cbpa.2020.04.001DOI Listing
December 2020

Analysis of RNA Interference (RNAi) Biopesticides: Double-Stranded RNA (dsRNA) Extraction from Agricultural Soils and Quantification by RT-qPCR.

Environ Sci Technol 2020 04 9;54(8):4893-4902. Epub 2020 Apr 9.

Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri 63130, United States.

Double-stranded RNA (dsRNA) molecules are used as a novel class of biopesticides. To enable assessments of the ecological risk associated with their release to receiving environments, we developed an approach to quantify dsRNA in agricultural soils using quantitative reverse transcription-polymerase chain reaction (RT-qPCR). To allow quantification of dsRNA adsorbed to particles, we also developed a protocol to transfer dsRNA from particles to the extraction buffer by changing particle surface charge and adding constituents to compete with dsRNA for adsorption sites. Our approach could quantify dsRNA amounts as low as 0.003 ng/g. This approach is the first available field-applicable approach able to quantify dsRNA biopesticides down to environmentally relevant concentrations. We applied this approach to investigate dsRNA dissipation (including dilution, degradation, and adsorption) in two agricultural soils. When we applied a low amount of dsRNA (1 ng/g) to the soils, we observed that a greater fraction of dsRNA was adsorbed to and extractable from soil particles in a silty clay loam soil than in a fine sandy loam soil. In both soils, dsRNA dissipated on the timescale of hours. Overall, these results demonstrate that our approach can be applied to assess the environmental fate of dsRNA biopesticides at concentrations relevant to their release to soils.
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http://dx.doi.org/10.1021/acs.est.9b07781DOI Listing
April 2020

Structural Determination of a New Peptidolipid Family from and the Pathogen by Multiple Stage Mass Spectrometry.

J Am Soc Mass Spectrom 2020 Mar 4;31(3):611-623. Epub 2020 Feb 4.

Institute for Cell Biology, University of Bonn, Ulrich-Haberland-Strasse 61a, 53121 Bonn, Germany.

The cell walls of the genus including the pathogenic bacterium () and biotechnologically important bacterium () contain an abundant eptidolipid (or termed lipopeptide) family whose structures have not been reported previously. Here, we describe a linear ion-trap multiple-stage mass spectrometric (LIT MS) approach with high resolution mass spectrometry (HRMS), in conjunction with NMR spectroscopy, chemical reactions, and GC/MS analysis to define the structures of these compounds. We employed LIT MS ( = 2-8) on the [M + Na] ion species to establish the peptide sequence, the identity of the fatty acyl substituent, and its location within the molecule, while NMR spectroscopy and GC/MS were used to recognize the Leu and Ile moieties. The major new lipopeptide found in is defined as CHCH(OH)CHCO-NHLeu-Ser-Leu-Ile-Thr-Ile-PheCOOH, where a β-OH fatty acyl (C-C) substituent is attached to the N-terminal of the LSLITIF peptide chain via a NH-CO bond. By contrast, the main peptidolipids found in belong to the cyclopeptidolipid family, which possesses the same peptide sequence and lipid chain, but the β-OH group of the fatty acyl moiety and the C-terminus of the peptide (i.e., the -COOH) are cyclized by an ester bond formation to a lactone, with a structure similar to iturin-A (Peypoux, F. et al. 1978, 17, 3992-3996). The antibiotic activity test of these new lipids did not reveal an activity against any of seven microorganisms tested.
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http://dx.doi.org/10.1021/jasms.9b00059DOI Listing
March 2020

Biosensing in Smart Engineered Probiotics.

Biotechnol J 2020 Oct 7;15(10):e1900319. Epub 2020 Jan 7.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, 63130, USA.

Engineered microbes are exciting alternatives to current diagnostics and therapeutics. Researchers have developed a wide range of genetic tools and parts to engineer probiotic and commensal microbes. Among these tools and parts, biosensors allow the microbes to sense and record or to sense and respond to chemical and environmental signals in the body, enabling them to report on health conditions of the animal host and/or deliver therapeutics in a controlled manner. This review focuses on how biosensing is applied to engineer "smart" microbes for in vivo diagnostic, therapeutic, and biocontainment goals. Hurdles that need to be overcome when transitioning from high-throughput in vitro systems to low-throughput in vivo animal models, new technologies that can be implemented to alleviate this experimental gap, and areas where future advancements can be made to maximize the utility of biosensing for medical applications are also discussed. As technologies for engineering microbes continue to be developed, these engineered organisms will be used to address many medical challenges.
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http://dx.doi.org/10.1002/biot.201900319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305048PMC
October 2020

Bioconversion of renewable feedstocks by Rhodococcus opacus.

Curr Opin Biotechnol 2020 08 30;64:10-16. Epub 2019 Sep 30.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130, United States. Electronic address:

The production of fuels and chemicals from renewable feedstocks is necessary for a fossil fuel independent economy. Lignin and other industrial wastes represent sustainable, non-food feedstocks that can be tapped for microbe-based bioproduction. Rhodococcus opacus is a gram-positive bacterium capable of catabolizing a broad range of feedstocks, and recent technological advances have further established its potential for lignin and industrial waste valorization. In the process of developing R. opacus as a platform for bioproduction, metabolic profiling has elucidated its native mechanisms of bioconversion, adaptive evolution has enhanced its tolerance towards inhibitory feedstocks, and genetic engineering has enabled it to produce novel products, such as wax esters, free fatty acids, and long chain hydrocarbons. Here, we present recent examples of broad feedstock utilization and value-added chemical production by R. opacus, demonstrating its potential as an industrially relevant strain.
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http://dx.doi.org/10.1016/j.copbio.2019.08.013DOI Listing
August 2020

Development of as a chassis for lignin valorization and bioproduction of high-value compounds.

Biotechnol Biofuels 2019 5;12:192. Epub 2019 Aug 5.

1The Edison Family Center for Genome Sciences and Systems Biology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 USA.

The current extraction and use of fossil fuels has been linked to extensive negative health and environmental outcomes. Lignocellulosic biomass-derived biofuels and bioproducts are being actively considered as renewable alternatives to the fuels, chemicals, and materials produced from fossil fuels. A major challenge limiting large-scale, economic deployment of second-generation biorefineries is the insufficient product yield, diversity, and value that current conversion technologies can extract from lignocellulose, in particular from the underutilized lignin fraction. PD630 is an oleaginous gram-positive bacterium with innate catabolic pathways and tolerance mechanisms for the inhibitory aromatic compounds found in depolymerized lignin, as well as native or engineered pathways for hexose and pentose sugars found in the carbohydrate fractions of biomass. As a result, holds potential as a biological chassis for the conversion of lignocellulosic biomass into biodiesel precursors and other value-added products. This review begins by examining the important role that lignin utilization will play in the future of biorefineries and by providing a concise survey of the current lignin conversion technologies. The genetic machinery and capabilities of that allow the bacterium to tolerate and metabolize aromatic compounds and depolymerized lignin are also discussed, along with a synopsis of the genetic toolbox and synthetic biology methods now available for engineering this organism. Finally, we summarize the different feedstocks that has been demonstrated to consume, and the high-value products that it has been shown to produce. Engineered will enable lignin valorization over the coming years, leading to cost-effective conversion of lignocellulose into fuels, chemicals, and materials.
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http://dx.doi.org/10.1186/s13068-019-1535-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683499PMC
August 2019

Construction of Genetic Logic Gates Based on the T7 RNA Polymerase Expression System in PD630.

ACS Synth Biol 2019 08 7;8(8):1921-1930. Epub 2019 Aug 7.

Department of Energy, Environmental and Chemical Engineering , Washington University in St. Louis , St. Louis , Missouri 63130 , United States.

PD630 () is a nonmodel, Gram-positive bacterium that holds promise as a biological catalyst for the conversion of lignocellulosic biomass to value-added products. In particular, it demonstrates both a high tolerance for and an ability to consume inhibitory lignin-derived aromatics, generates large quantities of lipids, exhibits a relatively rapid growth rate, and has a growing genetic toolbox for engineering. However, the availability of genetic parts for tunable, high-activity gene expression is still limited in . Furthermore, genetic logic circuits for sophisticated gene regulation have never been demonstrated in spp. To address these shortcomings, two inducible T7 RNA polymerase-based expression systems were implemented for the first time in and applied to the construction of AND and NAND genetic logic gates. Additionally, three isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible promoters were created by inserting LacI binding sites into newly characterized constitutive promoters. Furthermore, four novel aromatic sensors for 4-hydroxybenzoic acid, vanillic acid, sodium benzoate, and guaiacol were developed, expanding the gene expression toolbox. Finally, the T7 RNA polymerase platform was combined with a synthetic IPTG-inducible promoter to create an IMPLY logic gate. Overall, this work represents the first demonstration of a heterologous RNA polymerase system and synthetic genetic logic in , enabling complex and tunable gene regulation in this promising nonmodel host for bioproduction.
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http://dx.doi.org/10.1021/acssynbio.9b00213DOI Listing
August 2019

A concerted systems biology analysis of phenol metabolism in Rhodococcus opacus PD630.

Metab Eng 2019 09 2;55:120-130. Epub 2019 Jul 2.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, 63130, USA. Electronic address:

Rhodococcus opacus PD630 metabolizes aromatic substrates and naturally produces branched-chain lipids, which are advantageous traits for lignin valorization. To provide insights into its lignocellulose hydrolysate utilization, we performed C-pathway tracing, C-pulse-tracing, transcriptional profiling, biomass composition analysis, and metabolite profiling in conjunction with C-metabolic flux analysis (C-MFA) of phenol metabolism. We found that 1) phenol is metabolized mainly through the ortho-cleavage pathway; 2) phenol utilization requires a highly active TCA cycle; 3) NADPH is generated mainly via NADPH-dependent isocitrate dehydrogenase; 4) active cataplerotic fluxes increase plasticity in the TCA cycle; and 5) gluconeogenesis occurs partially through the reversed Entner-Doudoroff pathway (EDP). We also found that phenol-fed R. opacus PD630 generally has lower sugar phosphate concentrations (e.g., fructose 1,6-bisphosphatase) compared to metabolite pools in C-glucose-fed Escherichia coli (set as internal standards), while its TCA metabolites (e.g., malate, succinate, and α-ketoglutarate) accumulate intracellularly with measurable succinate secretion. In addition, we found that phenol utilization was inhibited by benzoate, while catabolite repressions by other tested carbon substrates (e.g., glucose and acetate) were absent in R. opacus PD630. Three adaptively-evolved strains display very different growth rates when fed with phenol as a sole carbon source, but they maintain a conserved flux network. These findings improve our understanding of R. opacus' metabolism for future lignin valorization.
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http://dx.doi.org/10.1016/j.ymben.2019.06.013DOI Listing
September 2019

Modulating Responses of Toehold Switches by an Inhibitory Hairpin.

ACS Synth Biol 2019 03 15;8(3):601-605. Epub 2019 Feb 15.

Department of Energy, Environmental and Chemical Engineering , Washington University in St. Louis , St. Louis , Missouri 63130 , United States.

The toehold switch consists of a cis-repressing switch RNA hairpin and a trans-acting trigger RNA. The binding of the trigger RNA to an unpaired toehold sequence of the switch hairpin allows for a branch migration process, exposing the start codon and ribosome binding site for translation initiation. In this work, we demonstrate that responses of toehold switches can be modulated by introducing an inhibitory hairpin that shortens the length of the unpaired toehold region. First, we investigated the effect of the toehold region length on output gene expression and showed that the second trigger RNA, which binds to the inhibitory hairpin, is necessary for output gene activation when the hairpin-to-hairpin spacing is short. Second, the apparent Hill coefficient was found generally to increase with decreasing hairpin-to-hairpin spacing or increasing hairpin number. This work expands the utility of toehold switches by providing a new way to modulate their response.
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http://dx.doi.org/10.1021/acssynbio.8b00488DOI Listing
March 2019

Lipid metabolism of phenol-tolerant strains for lignin bioconversion.

Biotechnol Biofuels 2018 28;11:339. Epub 2018 Dec 28.

1Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130 USA.

Background: Lignin is a recalcitrant aromatic polymer that is a potential feedstock for renewable fuel and chemical production. PD630 is a promising strain for the biological upgrading of lignin due to its ability to tolerate and utilize lignin-derived aromatic compounds. To enhance its aromatic tolerance, we recently applied adaptive evolution using phenol as a sole carbon source and characterized a phenol-adapted strain (evol40) and the wild-type (WT) strain by whole genome and RNA sequencing. While this effort increased our understanding of the aromatic tolerance, the tolerance mechanisms were not completely elucidated.

Results: We hypothesize that the composition of lipids plays an important role in phenol tolerance. To test this hypothesis, we applied high-resolution mass spectrometry analysis to lipid samples obtained from the WT and evol40 strains grown in 1 g/L glucose (glucose), 0.75 g/L phenol (low phenol), or 1.5 g/L phenol (high phenol, evol40 only) as a sole carbon source. This analysis identified > 100 lipid species of mycolic acids, phosphatidylethanolamines (PEs), phosphatidylinositols (PIs), and triacylglycerols. In both strains, mycolic acids had fewer double bond numbers in phenol conditions than the glucose condition, and evol40 had significantly shorter mycolic acid chain lengths than the WT strain in phenol conditions. These results indicate that phenol adaptation affected mycolic acid membrane composition. In addition, the percentage of unsaturated phospholipids decreased for both strains in phenol conditions compared to the glucose condition. Moreover, the PI content increased for both strains in the low phenol condition compared to the glucose condition, and the PI content increased further for evol40 in the high phenol condition relative to the low phenol condition.

Conclusions: This work represents the first comprehensive lipidomic study on the membrane of grown using phenol as a sole carbon source. Our results suggest that the alteration of the mycolic acid and phospholipid membrane composition may be a strategy of for phenol tolerance.
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http://dx.doi.org/10.1186/s13068-018-1337-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309088PMC
December 2018

Establishing a Multivariate Model for Predictable Antisense RNA-Mediated Repression.

ACS Synth Biol 2019 01 18;8(1):45-56. Epub 2018 Dec 18.

Department of Energy, Environmental and Chemical Engineering , Washington University in St. Louis , St. Louis , Missouri 63130 , United States.

Recent advances in our understanding of RNA folding and functions have facilitated the use of regulatory RNAs such as synthetic antisense RNAs (asRNAs) to modulate gene expression. However, despite the simple and universal complementarity rule, predictable asRNA-mediated repression is still challenging due to the intrinsic complexity of native asRNA-mediated gene regulation. To address this issue, we present a multivariate model, based on the change in free energy of complex formation (Δ G) and percent mismatch of the target binding region, which can predict synthetic asRNA-mediated repression efficiency in diverse contexts. First, 69 asRNAs that bind to multiple target mRNAs were designed and tested to create the predictive model. Second, we showed that the same model is effective predicting repression of target genes in both plasmids and chromosomes. Third, using our model, we designed asRNAs that simultaneously modulated expression of a toxin and its antitoxin to demonstrate tunable control of cell growth. Fourth, we tested and validated the same model in two different biotechnologically important organisms: Escherichia coli Nissle 1917 and Bacillus subtilis 168. Last, multiple parameters, including target locations, the presence of an Hfq binding site, GC contents, and gene expression levels, were revisited to define the conditions under which the multivariate model should be used for accurate prediction. Together, 434 different strain-asRNA combinations were tested, validating the predictive model in a variety of contexts, including multiple target genes and organisms. The result presented in this study is an important step toward achieving predictable tunability of asRNA-mediated repression.
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http://dx.doi.org/10.1021/acssynbio.8b00227DOI Listing
January 2019

Synthetic Gene Regulation in Cyanobacteria.

Adv Exp Med Biol 2018;1080:317-355

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, USA.

Cyanobacteria are appealing hosts for green chemical synthesis due to their use of light and carbon dioxide. To optimize product yields and titers, specific and tunable regulation of the metabolic pathways is needed. Synthetic biology has increased and diversified the genetic tools available for biological process control. While early tool development focused on commonly used heterotrophs, there has been a recent expansion of tools for cyanobacteria. CRISPR-Cas9 has been used to edit the genome of cyanobacterial strains, while transcriptional regulation has been accomplished with CRISPR interference and RNA riboswitches. Promoter development has produced a significant number of transcriptional regulators, including those that respond to chemicals, environmental signals, and metabolic states. Trans-acting RNAs have been utilized for posttranscriptional and translational control. The regulation of translation initiation is beginning to be explored with ribosome binding sites and riboswitches, while protein degradation tags have been used to control expression levels. Devices built from multiple parts have also been developed to create more complex behaviors. These advances in development of synthetic cyanobacterial regulatory parts provide the groundwork for creation of new, even more sophisticated bioprocess control devices, bolstering the viability of cyanobacteria as sustainable biotechnology platforms.
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http://dx.doi.org/10.1007/978-981-13-0854-3_13DOI Listing
January 2019

Multi-omic elucidation of aromatic catabolism in adaptively evolved Rhodococcus opacus.

Metab Eng 2018 09 27;49:69-83. Epub 2018 Jul 27.

The Edison Family Center for Genome Sciences and Systems Biology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA; Department of Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO 63108, USA; Department of Biomedical Engineering, Washington University in St. Louis, St Louis, MO 63130, USA; Department of Molecular Microbiology, Washington University in St. Louis School of Medicine, St. Louis, MO 63108, USA. Electronic address:

Lignin utilization has been identified as a key factor in biorefinery profitability. However, lignin depolymerization generates heterogeneous aromatic mixtures that inhibit microbial growth and the conversion of lignocellulose to biochemicals. Rhodococcus opacus is a promising aromatic-catabolizing, oleaginous bacterium, but mechanisms for its aromatic tolerance and utilization remain undercharacterized. To better understand these mechanisms, we adaptively evolved R. opacus for improved utilization of 32 combinations of diverse aromatic compounds. Evolved R. opacus mutants showed up to 1900% growth improvement in the utilization of phenol, guaiacol, 4-hydroxybenzoate, vanillate, and benzoate compared to the wild-type strain. Whole genome sequencing revealed several redox-related genes with mutations shared across multiple adapted mutants. PVHG6, the mutant with the most improved growth on a mixture of multiple aromatic compounds, showed 56% lower superoxide dismutase activity than the wild-type strain, suggesting that redox reactions are important for aromatic tolerance and utilization. Comparative transcriptomics revealed by-product detoxification pathways and five aromatic funneling pathways that were upregulated in response to specific aromatic compounds. Gene knockout experiments confirmed the two degradation routes of the β-ketoadipate pathway for five aromatic compounds. These results provide an improved understanding of aromatic bioconversion and facilitate development of R. opacus as a biorefinery host.
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http://dx.doi.org/10.1016/j.ymben.2018.06.009DOI Listing
September 2018

Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630.

Sci Rep 2018 04 16;8(1):6019. Epub 2018 Apr 16.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri, 63130, USA.

Rhodococcus opacus PD630 is a gram-positive bacterium with promising attributes for the conversion of lignin into valuable fuels and chemicals. To develop an organism as a cellular factory, it is necessary to have a deep understanding of its metabolism and any heterologous pathways being expressed. For the purpose of quantifying gene transcription, reverse transcription quantitative PCR (RT-qPCR) is the gold standard due to its sensitivity and reproducibility. However, RT-qPCR requires the use of reference genes whose expression is stable across distinct growth or treatment conditions to normalize the results. Unfortunately, no in-depth analysis of stable reference genes has been conducted in Rhodococcus, inhibiting the utilization of RT-qPCR in R. opacus. In this work, ten candidate reference genes, chosen based on previously collected RNA sequencing data or literature, were examined under four distinct growth conditions using three mathematical programs (BestKeeper, Normfinder, and geNorm). Based on this analysis, the minimum number of reference genes required was found to be two, and two separate pairs of references genes were identified as optimal normalization factors for when ribosomal RNA is either present or depleted. This work represents the first validation of reference genes for Rhodococcus, providing a valuable starting point for future research.
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http://dx.doi.org/10.1038/s41598-018-24486-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902447PMC
April 2018

Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System.

ACS Synth Biol 2018 03 16;7(3):853-865. Epub 2018 Feb 16.

Department of Energy, Environmental and Chemical Engineering , Washington University in St. Louis , St. Louis , Missouri 63130 , United States.

Synthetic small RNA regulators have emerged as a versatile tool to predictably control bacterial gene expression. Owing to their simple design principles, small size, and highly orthogonal behavior, these engineered genetic parts have been incorporated into genetic circuits. However, efforts to achieve more sophisticated cellular functions using RNA regulators have been hindered by our limited ability to integrate different RNA regulators into complex circuits. Here, we present a combined RNA regulatory system in Escherichia coli that uses small transcription activating RNA (STAR) and antisense RNA (asRNA) to activate or deactivate target gene expression in a programmable manner. Specifically, we demonstrated that the activated target output by the STAR system can be deactivated by expressing two different types of asRNAs: one binds to and sequesters the STAR regulator, affecting the transcription process, while the other binds to the target mRNA, affecting the translation process. We improved deactivation efficiencies (up to 96%) by optimizing each type of asRNA and then integrating the two optimized asRNAs into a single circuit. Furthermore, we demonstrated that the combined STAR and asRNA system can control gene expression in a reversible way and can regulate expression of a gene in the genome. Lastly, we constructed and simultaneously tested two A AND NOT B logic gates in the same cell to show sophisticated multigene regulation by the combined system. Our approach establishes a methodology for integrating multiple RNA regulators to rationally control multiple genes.
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http://dx.doi.org/10.1021/acssynbio.7b00322DOI Listing
March 2018

Molecular Toolkit for Gene Expression Control and Genome Modification in Rhodococcus opacus PD630.

ACS Synth Biol 2018 02 5;7(2):727-738. Epub 2018 Feb 5.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis , St. Louis, Missouri 63130, United States.

Rhodococcus opacus PD630 is a non-model Gram-positive bacterium that possesses desirable traits for lignocellulosic biomass conversion. In particular, it has a relatively rapid growth rate, exhibits genetic tractability, produces high quantities of lipids, and can tolerate and consume toxic lignin-derived aromatic compounds. Despite these unique, industrially relevant characteristics, R. opacus has been underutilized because of a lack of reliable genetic parts and engineering tools. In this work, we developed a molecular toolbox for reliable gene expression control and genome modification in R. opacus. To facilitate predictable gene expression, a constitutive promoter library spanning ∼45-fold in output was constructed. To improve the characterization of available plasmids, the copy numbers of four heterologous and nine endogenous plasmids were determined using quantitative PCR. The molecular toolbox was further expanded by screening a previously unreported antibiotic resistance marker (HygR) and constructing a curable plasmid backbone for temporary gene expression (pB264). Furthermore, a system for genome modification was devised, and three neutral integration sites were identified using a novel combination of transcriptomic data, genomic architecture, and growth rate analysis. Finally, the first reported system for targeted, tunable gene repression in Rhodococcus was developed by utilizing CRISPR interference (CRISPRi). Overall, this work greatly expands the ability to manipulate and engineer R. opacus, making it a viable new chassis for bioproduction from renewable feedstocks.
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http://dx.doi.org/10.1021/acssynbio.7b00416DOI Listing
February 2018

Design rules of synthetic non-coding RNAs in bacteria.

Methods 2018 07 5;143:58-69. Epub 2018 Jan 5.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA. Electronic address:

One of the long-term goals of synthetic biology is to develop designable genetic parts with predictable behaviors that can be utilized to implement diverse cellular functions. The discovery of non-coding RNAs and their importance in cellular processing have rapidly attracted researchers' attention towards designing functional non-coding RNA molecules. These synthetic non-coding RNAs have simple design principles governed by Watson-Crick base pairing, but exhibit increasingly complex functions. Importantly, due to their specific and modular behaviors, synthetic non-coding RNAs have been widely adopted to modulate transcription and translation of target genes. In this review, we summarize various design rules and strategies employed to engineer synthetic non-coding RNAs. Specifically, we discuss how RNA molecules can be transformed into powerful regulators and utilized to control target gene expression. With the establishment of generalizable non-coding RNA design rules, the research community will shift its focus to RNA regulators from protein regulators.
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http://dx.doi.org/10.1016/j.ymeth.2018.01.001DOI Listing
July 2018

Development of Chemical and Metabolite Sensors for Rhodococcus opacus PD630.

ACS Synth Biol 2017 10 10;6(10):1973-1978. Epub 2017 Aug 10.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis , St. Louis, Missouri 63130, United States.

Rhodococcus opacus PD630 is a nonmodel, Gram-positive bacterium that possesses desirable traits for biomass conversion, including consumption capabilities for lignocellulose-based sugars and toxic lignin-derived aromatic compounds, significant triacylglycerol accumulation, relatively rapid growth rate, and genetic tractability. However, few genetic elements have been directly characterized in R. opacus, limiting its application for lignocellulose bioconversion. Here, we report the characterization and development of genetic tools for tunable gene expression in R. opacus, including: (1) six fluorescent reporters for quantifying promoter output, (2) three chemically inducible promoters for variable gene expression, and (3) two classes of metabolite sensors derived from native R. opacus promoters that detect nitrogen levels or aromatic compounds. Using these tools, we also provide insights into native aromatic consumption pathways in R. opacus. Overall, this work expands the ability to control and characterize gene expression in R. opacus for future lignocellulose-based fuel and chemical production.
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http://dx.doi.org/10.1021/acssynbio.7b00192DOI Listing
October 2017

Dynamics of sequestration-based gene regulatory cascades.

Nucleic Acids Res 2017 Jul;45(12):7515-7526

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, 63130, USA.

Gene regulatory cascades are ubiquitous in biology. Because regulatory cascades are integrated within complex networks, their quantitative analysis is challenging in native systems. Synthetic biologists have gained quantitative insights into the properties of regulatory cascades by building simple circuits, but sequestration-based regulatory cascades remain relatively unexplored. Particularly, it remains unclear how the cascade components collectively control the output dynamics. Here, we report the construction and quantitative analysis of the longest sequestration-based cascade in Escherichia coli. This cascade consists of four Pseudomonas aeruginosa protein regulators (ExsADCE) that sequester their partner. Our computational analysis showed that the output dynamics are controlled in a complex way by the concentration of the unbounded transcriptional activator ExsA. By systematically varying the cascade length and the synthesis rate of each regulator, we experimentally verified the computational prediction that ExsC plays a role in rapid circuit responses by sequestering the anti-activator ExsD, while ExsD increases response times by decreasing the free ExsA concentration. In contrast, when additional ExsD was introduced to the cascade via indirect negative feedback, the response time was significantly reduced. Sequestration-based regulatory cascades with negative feedback are often found in biology, and thus our finding provides insights into the dynamics of this recurring motif.
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http://dx.doi.org/10.1093/nar/gkx465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499576PMC
July 2017

Decoupling Resource-Coupled Gene Expression in Living Cells.

ACS Synth Biol 2017 08 4;6(8):1596-1604. Epub 2017 May 4.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis , St. Louis, Missouri 63130, United States.

Synthetic biology aspires to develop frameworks that enable the construction of complex and reliable gene networks with predictable functionalities. A key limitation is that increasing network complexity increases the demand for cellular resources, potentially causing resource-associated interference among noninteracting circuits. Although recent studies have shown the effects of resource competition on circuit behaviors, mechanisms that decouple such interference remain unclear. Here, we constructed three systems in Escherichia coli, each consisting of two independent circuit modules where the complexity of one module (Circuit 2) was systematically increased while the other (Circuit 1) remained identical. By varying the expression level of Circuit 1 and measuring its effect on the expression level of Circuit 2, we demonstrated computationally and experimentally that indirect coupling between these seemingly unconnected genetic circuits can occur in three different regulatory topologies. More importantly, we experimentally verified the computational prediction that negative feedback can significantly reduce resource-coupled interference in regulatory circuits. Our results reveal a design principle that enables cells to reliably multitask while tightly controlling cellular resources.
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http://dx.doi.org/10.1021/acssynbio.7b00119DOI Listing
August 2017

Cyanobacterial carbon metabolism: Fluxome plasticity and oxygen dependence.

Biotechnol Bioeng 2017 07 30;114(7):1593-1602. Epub 2017 Mar 30.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri 63130.

Synechocystis sp. strain PCC 6803 has been widely used as a photo-biorefinery chassis. Based on its genome annotation, this species contains a complete TCA cycle, an Embden-Meyerhof-Parnas pathway (EMPP), an oxidative pentose phosphate pathway (OPPP), and an Entner-Doudoroff pathway (EDP). To evaluate how Synechocystis 6803 catabolizes glucose under heterotrophic conditions, we performed C metabolic flux analysis, metabolite pool size analysis, gene knockouts, and heterologous expressions. The results revealed a cyclic mode of flux through the OPPP. Small, but non-zero, fluxes were observed through the TCA cycle and the malic shunt. Independent knockouts of 6-phosphogluconate dehydrogenase (gnd) and malic enzyme (me) corroborated these results, as neither mutant could grow under dark heterotrophic conditions. Our data also indicate that Synechocystis 6803 metabolism relies upon oxidative phosphorylation to generate ATP from NADPH under dark or insufficient light conditions. The pool sizes of intermediates in the TCA cycle, particularly acetyl-CoA, were found to be several fold lower in Synechocystis 6803 (compared to E. coli metabolite pool sizes), while its sugar phosphate intermediates were several-fold higher. Moreover, negligible flux was detected through the native, or heterologous, EDP in the wild type or Δgnd strains under heterotrophic conditions. Comparing photoautotrophic, photomixotrophic, and heterotrophic conditions, the Calvin cycle, OPPP, and EMPP in Synechocystis 6803 possess the ability to regulate their fluxes under various growth conditions (plastic), whereas its TCA cycle always maintains at low levels (rigid). This work also demonstrates how genetic profiles do not always reflect actual metabolic flux through native or heterologous pathways. Biotechnol. Bioeng. 2017;114: 1593-1602. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/bit.26287DOI Listing
July 2017

Enabling complex genetic circuits to respond to extrinsic environmental signals.

Biotechnol Bioeng 2017 07 20;114(7):1626-1631. Epub 2017 Mar 20.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis 63130, One Brookings Dr., Box 1180, Missouri.

Genetic circuits have the potential to improve a broad range of metabolic engineering processes and address a variety of medical and environmental challenges. However, in order to engineer genetic circuits that can meet the needs of these real-world applications, genetic sensors that respond to relevant extrinsic and intrinsic signals must be implemented in complex genetic circuits. In this work, we construct the first AND and NAND gates that respond to temperature and pH, two signals that have relevance in a variety of real-world applications. A previously identified pH-responsive promoter and a temperature-responsive promoter were extracted from the E. coli genome, characterized, and modified to suit the needs of the genetic circuits. These promoters were combined with components of the type III secretion system in Salmonella typhimurium and used to construct a set of AND gates with up to 23-fold change. Next, an antisense RNA was integrated into the circuit architecture to invert the logic of the AND gate and generate a set of NAND gates with up to 1168-fold change. These circuits provide the first demonstration of complex pH- and temperature-responsive genetic circuits, and lay the groundwork for the use of similar circuits in real-world applications. Biotechnol. Bioeng. 2017;114: 1626-1631. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/bit.26279DOI Listing
July 2017

Physical, chemical, and metabolic state sensors expand the synthetic biology toolbox for Synechocystis sp. PCC 6803.

Biotechnol Bioeng 2017 07 9;114(7):1561-1569. Epub 2017 Mar 9.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri.

Many under-developed organisms possess important traits that can boost the effectiveness and sustainability of microbial biotechnology. Photoautotrophic cyanobacteria can utilize the energy captured from light to fix carbon dioxide for their metabolic needs while living in environments not suited for growing crops. Various value-added compounds have been produced by cyanobacteria in the laboratory; yet, the products' titers and yields are often not industrially relevant and lag behind what have been accomplished in heterotrophic microbes. Genetic tools for biological process control are needed to take advantage of cyanobacteria's beneficial qualities, as tool development also lags behind what has been created in common heterotrophic hosts. To address this problem, we developed a suite of sensors that regulate transcription in the model cyanobacterium Synechocystis sp. PCC 6803 in response to metabolically relevant signals, including light and the cell's nitrogen status, and a family of sensors that respond to the inexpensive chemical, l-arabinose. Increasing the number of available tools enables more complex and precise control of gene expression. Expanding the synthetic biology toolbox for this cyanobacterium also improves our ability to utilize this important under-developed organism in biotechnology. Biotechnol. Bioeng. 2017;114: 1561-1569. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/bit.26275DOI Listing
July 2017

Development of Design Rules for Reliable Antisense RNA Behavior in E. coli.

ACS Synth Biol 2016 12 2;5(12):1441-1454. Epub 2016 Aug 2.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis , St. Louis, Missouri 63130, United States.

A key driver of synthetic biology is the development of designable genetic parts with predictable behaviors that can be quickly implemented in complex genetic systems. However, the intrinsic complexity of gene regulation can make the rational design of genetic parts challenging. This challenge is apparent in the design of antisense RNA (asRNA) regulators. Though asRNAs are well-known regulators, the literature governing their design is conflicting and leaves the synthetic biology community without clear asRNA design rules. The goal of this study is to perform a comprehensive experimental characterization and statistical analysis of 121 unique asRNA regulators in order to resolve the conflicts that currently exist in the literature. asRNAs usually consist of two regions, the Hfq binding site and the target binding region (TBR). First, the behaviors of several high-performing Hfq binding sites were compared, in terms of their ability to improve repression efficiencies and their orthogonality. Next, a large-scale analysis of TBR design parameters identified asRNA length, the thermodynamics of asRNA-mRNA complex formation, and the percent of target mismatch as key parameters for TBR design. These parameters were used to develop simple asRNA design rules. Finally, these design rules were applied to construct both a simple and a complex genetic circuit containing different asRNAs, and predictable behavior was observed in both circuits. The results presented in this study will drive synthetic biology forward by providing useful design guidelines for the construction of asRNA regulators with predictable behaviors.
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http://dx.doi.org/10.1021/acssynbio.6b00036DOI Listing
December 2016

Diurnal Regulation of Cellular Processes in the Cyanobacterium Synechocystis sp. Strain PCC 6803: Insights from Transcriptomic, Fluxomic, and Physiological Analyses.

mBio 2016 05 3;7(3). Epub 2016 May 3.

Department of Biology, Washington University, St. Louis, Missouri, USA

Unlabelled: Synechocystis sp. strain PCC 6803 is the most widely studied model cyanobacterium, with a well-developed omics level knowledgebase. Like the lifestyles of other cyanobacteria, that of Synechocystis PCC 6803 is tuned to diurnal changes in light intensity. In this study, we analyzed the expression patterns of all of the genes of this cyanobacterium over two consecutive diurnal periods. Using stringent criteria, we determined that the transcript levels of nearly 40% of the genes in Synechocystis PCC 6803 show robust diurnal oscillating behavior, with a majority of the transcripts being upregulated during the early light period. Such transcripts corresponded to a wide array of cellular processes, such as light harvesting, photosynthetic light and dark reactions, and central carbon metabolism. In contrast, transcripts of membrane transporters for transition metals involved in the photosynthetic electron transport chain (e.g., iron, manganese, and copper) were significantly upregulated during the late dark period. Thus, the pattern of global gene expression led to the development of two distinct transcriptional networks of coregulated oscillatory genes. These networks help describe how Synechocystis PCC 6803 regulates its metabolism toward the end of the dark period in anticipation of efficient photosynthesis during the early light period. Furthermore, in silico flux prediction of important cellular processes and experimental measurements of cellular ATP, NADP(H), and glycogen levels showed how this diurnal behavior influences its metabolic characteristics. In particular, NADPH/NADP(+) showed a strong correlation with the majority of the genes whose expression peaks in the light. We conclude that this ratio is a key endogenous determinant of the diurnal behavior of this cyanobacterium.

Importance: Cyanobacteria are photosynthetic microbes that use energy from sunlight and CO2 as feedstock. Certain cyanobacterial strains are amenable to facile genetic manipulation, thus enabling synthetic biology and metabolic engineering applications. Such strains are being developed as a chassis for the sustainable production of food, feed, and fuel. To this end, a holistic knowledge of cyanobacterial physiology and its correlation with gene expression patterns under the diurnal cycle is warranted. In this report, a genomewide transcriptional analysis of Synechocystis PCC 6803, the most widely studied model cyanobacterium, sheds light on the global coordination of cellular processes during diurnal periods. Furthermore, we found that, in addition to light, the redox level of NADP(H) is an important endogenous regulator of diurnal entrainment of Synechocystis PCC 6803.
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http://dx.doi.org/10.1128/mBio.00464-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4959675PMC
May 2016

Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.

Nucleic Acids Res 2016 Mar 2;44(5):2462-73. Epub 2016 Feb 2.

Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, USA

A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.
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http://dx.doi.org/10.1093/nar/gkw056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797300PMC
March 2016

Comparative transcriptomics elucidates adaptive phenol tolerance and utilization in lipid-accumulating Rhodococcus opacus PD630.

Nucleic Acids Res 2016 Mar 2;44(5):2240-54. Epub 2016 Feb 2.

Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA

Lignin-derived (e.g. phenolic) compounds can compromise the bioconversion of lignocellulosic biomass to fuels and chemicals due to their toxicity and recalcitrance. The lipid-accumulating bacterium Rhodococcus opacus PD630 has recently emerged as a promising microbial host for lignocellulose conversion to value-added products due to its natural ability to tolerate and utilize phenolics. To gain a better understanding of its phenolic tolerance and utilization mechanisms, we adaptively evolved R. opacus over 40 passages using phenol as its sole carbon source (up to 373% growth improvement over wild-type), and extensively characterized two strains from passages 33 and 40. The two adapted strains showed higher phenol consumption rates (∼20 mg/l/h) and ∼2-fold higher lipid production from phenol than the wild-type strain. Whole-genome sequencing and comparative transcriptomics identified highly-upregulated degradation pathways and putative transporters for phenol in both adapted strains, highlighting the important linkage between mechanisms of regulated phenol uptake, utilization, and evolved tolerance. Our study shows that the R. opacus mutants are likely to use their transporters to import phenol rather than export them, suggesting a new aromatic tolerance mechanism. The identified tolerance genes and pathways are promising candidates for future metabolic engineering in R. opacus for improved lignin conversion to lipid-based products.
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http://dx.doi.org/10.1093/nar/gkw055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797299PMC
March 2016

Robust, tunable genetic memory from protein sequestration combined with positive feedback.

Nucleic Acids Res 2015 Oct 17;43(18):9086-94. Epub 2015 Sep 17.

Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA

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.
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http://dx.doi.org/10.1093/nar/gkv936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4605329PMC
October 2015