ACS Synthetic Biology最新文献

{"title":"Revolutionizing DNAzyme Biosensors: Pioneering Strategies, Advanced Immobilization, Versatile Applications, and Future Frontiers.","authors":"Adwaita Sr Nair, Arup Samanta, Saugata Hazra","doi":"10.1021/acssynbio.5c00074","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00074","url":null,"abstract":"<p><p>The hybridization chain reaction (HCR) is an amplification method recognized for detecting analytes present in trace quantities owing to its high specificity, sensitivity, and straightforward approach. Simultaneously, G-quadruplex DNAzymes augment HCR-based biosensors, serving as transducers due to their elevated catalytic activity and nonenzymatic methodology. The current review aims to provide readers with a critical overview of significant aspects of biosensors that utilize HCR for analyte detection amplification and G-quadruplex as transducers, focusing on the latest activities of global researchers. A bibliometric analysis was conducted to identify key research topics in HCR and G-quadruplex-based sensors. Further, the review aims to elucidate the advantages and disadvantages of each strategy employed for detection using this sort of sensor. Specific sections have been included to critically evaluate the potential of these biosensors across many scientific domains. In conclusion, the limitations of the existing sensor that restrict its applicability in real-time scenarios have been identified for subsequent enhancement. Plausible strategies that could enhance these sensors and emerging scientific fields that could benefit from HCR and G-quadruplex DNAzyme-based sensors have been elucidated.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144281670","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of Biosynthetic Precursors and Optimization of 7-Hydroxytropolone Bioproduction by <i>Pseudomonas sp.</i> PA14H7.","authors":"Euphrasie Munier-Lépinay, Coline Amaro-Lauer, Denis Faure, Anthony Quéro, David Mathiron, Mounia Khelifa, Sylvain Laclef, Serge Pilard","doi":"10.1021/acssynbio.4c00817","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00817","url":null,"abstract":"<p><p>Cell-free supernatant of the strain <i>Pseudomonas sp.</i> PA14H7 has previously shown interesting activity against Soft Rot <i>Pectobacteriaceae</i> (SRP), the bacterial pathogen responsible for blackleg and soft rot diseases in potatoes. A deeper understanding of its mode of action is essential to optimize its use as a biocontrol agent. We previously reported that <i>Pseudomonas sp.</i> PA14H7 produces a specialized metabolite, the 7-hydroxytropolone (7-HT), which acts as an iron chelator, limiting the growth of SRP. In this study, we have constructed a Δ<i>hts10</i> deletion mutant of <i>Pseudomonas sp.</i> PA14H7, encoding a putative acyl-CoA dehydrogenase corresponding to the ortholog of <i>orf10</i> in <i>Pseudomonas donghuensis</i>. We demonstrated that this mutant was deficient in 7-HT biosynthesis, confirming that this molecule is the metabolite responsible for the antagonist activity. After finding a minimum culture medium (MK) allowing the <i>Pseudomonas sp.</i> PA14H7 growth without 7-HT production, we investigated the biosynthetic pathway of this metabolite. We identified phenylalanine and phenylacetic acid as 7-HT precursors and demonstrated that the addition of 150 mg/L of phenylalanine to the MK medium enhanced the 7-HT bioproduction by <i>Pseudomonas sp.</i> PA14H7 up to 30 mg/L. These findings provide new insights into the biosynthesis and regulation of 7-HT, paving the way for the use of <i>Pseudomonas sp.</i> PA14H7 as a biocontrol agent.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144281669","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>In Vivo</i> Multicellular Feedback Control in Synthetic Microbial Consortia.","authors":"Davide Salzano, Barbara Shannon, Claire Grierson, Lucia Marucci, Nigel J Savery, Mario di Bernardo","doi":"10.1021/acssynbio.4c00862","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00862","url":null,"abstract":"<p><p>In this paper, we present a biomolecular control architecture able to guarantee stable and precise regulation of gene expression. Specifically, we engineer a microbial consortium comprising a cellular population, named <i>controllers</i>, that is tasked to regulate the expression of a gene in a second population, termed <i>targets</i>. Traditional biomolecular control strategies, while effective, are predominantly confined to single-cell applications, limiting their complexity and adaptability due to factors such as competition for limited cell resources and incompatible chemical reactions. Our approach overcomes these limitations by employing a distributed multicellular feedback loop between two strains of <i>Escherichia coli</i>, allowing for division of labor across the consortium. <i>In vivo</i> experiments demonstrate that this control system maintains precise and robust gene expression in the target population, even amid variations in consortium composition. Our study fills a critical gap in synthetic biology and paves the way for more complex and reliable applications in the field.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144273653","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-Throughput Characterization of Tetracycline Repressor Function on Tetracycline Operator 2 Variants.","authors":"Alexa N Gormick, Adam M Zahm, Samuel R Himes, Kathleen E Rondem, Justin G English","doi":"10.1021/acssynbio.4c00809","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00809","url":null,"abstract":"<p><p>Chemogenetic regulators of transgene activity, such as the tetracycline-inducible system derived from the tetracycline resistance operon of the bacterial transposon Tn10, are critical and widely used systems in cellular engineering. The tetracycline-inducible system is prized for its selectivity, high affinity, inducibility, reversibility, and differential control of gene transcription. However, its optimization for binary on/off expression limits its application in systems biology and the modeling and construction of complex regulatory systems with intricate input/output paradigms. To overcome this limitation, we developed a high-throughput reporter system to investigate a saturated mutagenesis library of tetracycline resistance operator variants. Using this system, we mapped the functional interactions of Tet repressor DNA binding protein at single-nucleotide resolution in mammalian cells. Our comprehensive screen revealed a spectrum of variant effects, ranging from a nearly complete loss of repression to levels indistinguishable from the natural operator, validated through orthogonal assays. This comprehensive characterization of the sequence-specificity of a tetracycline resistance operator facilitates the construction of variably suppressive, inducible systems for dynamic and modular control over gene expression in mammalian cell culture.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144256631","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Robust Synthetic Biology Toolkit to Advance Carboxysome Study and Redesign.","authors":"Daniel S Trettel, Y Hoang, Anthony G Vecchiarelli, Cesar R Gonzalez-Esquer","doi":"10.1021/acssynbio.5c00144","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00144","url":null,"abstract":"<p><p>Carboxysomes are polyhedral protein organelles that microorganisms use to facilitate carbon dioxide assimilation. They are composed of a modular protein shell that envelops an enzymatic core mainly composed of physically coupled Rubisco and carbonic anhydrase. While the modular construction principles of carboxysomes make them attractive targets as customizable metabolic platforms, their size and complexity can be a hindrance. In this work, we design and validate a plasmid set, the pXpressome toolkit, in which α-carboxysomes are robustly expressed and remain intact and functional after purification. We tested this toolkit by introducing mutations that influence carboxysome structure and performance. We find that deletion of vertex-capping genes results in formation of larger carboxysomes, while deletion of facet forming genes produces smaller particles, suggesting that adjusting the ratio of these proteins can rationally affect morphology. Through a series of fluorescently labeled constructs, we observe that this toolkit leads to more uniform expression and better cell health than previously published carboxysome expression systems. Overall, the pXpressome toolkit facilitates the study and redesign of carboxysomes with robust performance and improved phenotype uniformity. The pXpressome toolkit will support efforts to remodel carboxysomes for enhanced carbon fixation or serve as a platform for other nanoencapsulation goals.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144245313","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Standardized Quorum Sensing Tools for Gram-Negative Bacteria.","authors":"Paula Múgica-Galán, Jesús Miró-Bueno, Ángeles Hueso-Gil, Pablo Japón, Ángel Goñi-Moreno","doi":"10.1021/acssynbio.5c00036","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00036","url":null,"abstract":"<p><p>Engineering synthetic consortia to perform distributed functions requires robust quorum sensing (QS) systems to facilitate communication between cells. However, the current QS toolbox lacks standardized implementations, which are particularly valuable for use in bacteria beyond the model species <i>Escherichia coli</i>. We developed a set of three QS systems encompassing both sender and receiver modules, constructed using backbones from the SEVA (Standard European Vector Architecture) plasmid collection. This increases versatility, allowing plasmid features like the origin of replication or antibiotic marker to be easily swapped. The systems were characterized using the synthetic biology chassis <i>Pseudomonas putida</i>. We first tested individual modules, then combined sender and receiver modules in the same host, and finally assessed the performance across separate cells to evaluate consortia dynamics. Alongside the QS set, we provide mathematical models and rate parameters to support the design efforts. Together, these tools advance the engineering of robust and predictable multicellular functions.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144232645","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthetic Phosphorylation Networks with Fluorescence and Luminescence Expansion.","authors":"Leah Davis, Evan J Hutt, Matthias Recktenwald, Samarth Patel, Madison Briggs, Madeline Dunsmore, Sebastián L Vega, Mary M Staehle, Peter A Galie, Nichole M Daringer","doi":"10.1021/acssynbio.4c00814","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00814","url":null,"abstract":"<p><p>Synthetic receptors have emerged as powerful tools for precisely modulating cellular function. However, existing synthetic receptor platforms rely mainly on transcription-mediated reporting processes that are incompatible with the rapid and real-time dynamics of cellular signaling events. To address this limitation, we present SPN-FLUX (synthetic phosphorylation networks with fluorescence and luminescence expansion), a fully post-translational platform that integrates synthetic phosphorylation networks with split fluorescent or luminescent proteins, enabling rapid and tunable reporting of cellular processes. SPN-FLUX is responsive to extracellular stimuli within 1 h, providing a robust alternative to transcription-based approaches. Using mammalian cells as a model, we showcase SPN-FLUX's versatility by designing a membrane-bound receptor that activates upon ligand-induced dimerization, as well as a constitutively active intracellular biosensor. We further validate SPN-FLUX's biosensing capabilities by examining its responsiveness to hypoxic conditions, showcasing the ability to detect environmental changes dynamically. The modularity and programmability of SPN-FLUX establish it as a powerful platform for advancing synthetic biology and biosensing, with broad applications in both biomedical research and environmental monitoring.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144232646","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering Microbial Consortium Biohybrid System to Efficiently Produce Electricity from Lignocellulose Biomass.","authors":"Junqi Zhang, Yuanxiu Li, Wenjing Lv, Zixuan You, Huan Yu, Baocai Zhang, Qijing Liu, Jing Zou, Tao Chen, Feng Li, Hao Song","doi":"10.1021/acssynbio.5c00178","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00178","url":null,"abstract":"<p><p>Converting lignocellulose into bioelectricity through a bioelectrocatalytic system (BES) has emerged as a promising approach to addressing environmental pollution and energy regeneration challenges. However, practical application of BES is significantly constrained by the fact that the electroactive biocatalyst <i>Shewanella oneidensis</i> lacks the essential metabolic pathways and enzymes required for utilizing lignocellulose for cell growth and power generation. Here, to realize clean electricity production from lignocellulose hydrolysate, an artificial microbial consortium comprising <i>S. oneidensis</i>, <i>Lactococcus lactis</i>, and <i>Bacillus subtilis</i> was developed. In this consortium, <i>L. lactis</i> is responsible for converting glucose into lactate; <i>B. subtilis</i> metabolizes glucose and xylose into riboflavin; and <i>S. oneidensis</i> then employs lactate as an electron donor and riboflavin as an electron shuttle to facilitate electricity generation. Subsequently, to increase substrate conversion efficiency of the microbial consortium, three key genes <i>codY</i>, <i>ribA</i>, and <i>dld</i> encoding lactate dehydrogenase, GTP cyclohydrolase, and d-lactate dehydrogenase, were expressed in <i>L. lactis</i>, <i>B. subtilis</i>, and <i>S. oneidensis</i>, respectively, which accelerated glucose-to-lactate conversion, riboflavin synthesis, and lactate metabolism. Also, to accelerate the extracellular electron transfer (EET) capacity of the microbial consortium, the <i>cyc2</i> gene from <i>Acidithiobacillus ferrooxidans</i> encoding the outer membrane <i>c</i>-type cytochrome was further expressed in <i>S. oneidensis</i>. Finally, to further enhance the interfacial EET capability of the microbial consortium, a 3D microbiota biohybrid system <i>S</i>₇<i>L</i>₁<i>B</i>₁@CF&GO consisting of carbon felts and graphene oxide was developed to reduce the internal resistance of BES. The results showed that the artificial biohybrid system could obtain a maximum power density of ∼739.40 mW m^-2 using lignocellulosic hydrolysate as the carbon source. This system expands the range of carbon sources available to <i>S. oneidensis</i> for efficient power generation from the lignocellulosic hydrolysate.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144232644","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MOSAIC: A Highly Efficient, One-Step Recombineering Approach to Plasmid Editing and Diversification.","authors":"Marijn van den Brink, Timotheus Y Althuis, Christophe Danelon, Nico J Claassens","doi":"10.1021/acssynbio.4c00657","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00657","url":null,"abstract":"<p><p>The editing of plasmids and construction of plasmid libraries is paramount to the engineering of desired functionalities in synthetic biology. Typically, plasmids with targeted mutations are produced through time- and resource-consuming DNA amplification and/or cloning steps. In this study, we establish MOSAIC, a highly efficient protocol for the editing of plasmids and generation of combinatorial plasmid libraries. This quick protocol employs the efficient single-stranded DNA annealing protein (SSAP) CspRecT to incorporate (libraries of) DNA oligos harboring the desired mutations into a target plasmid in <i>Escherichia coli</i>. In addition to up to 90% single-target plasmid editing efficiency, we demonstrate that MOSAIC enables the generation of a combinatorial plasmid library spanning four different target regions on a plasmid, in a single transformation. Lastly, we integrated a user-friendly validation pipeline using Nanopore sequencing reads, requiring minimal computational experience. We anticipate that MOSAIC will provide researchers with a simple, rapid and resource-effective method to edit plasmids or generate large, diverse plasmid libraries for a wide range of <i>in vivo</i> or <i>in vitro</i> applications in molecular and synthetic biology.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144223705","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"SARS-CoV-2 Nsp1-Resistant Modified RNA for the Creation of Nsp1-Responsive Systems.","authors":"Malvin Leonardo Pardi, Kazuo Takayama, Hirohide Saito","doi":"10.1021/acssynbio.5c00075","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00075","url":null,"abstract":"<p><p>Modified RNA (modRNA) facilitates the introduction of complex synthetic genetic circuits into cells without the risk of genomic integration, opening up the implementation of synthetic circuits as therapeutics. However, the number of protein-RNA interfaces that are suitable for the construction of protein-responsive modRNA switches as well as the lack of protein-responsive exclusive selector systems stifles the development of RNA-based synthetic circuits. Here, we present the creation of a modRNA capable of resisting the effects of Nsp1 for the reliable expression of its coding sequence. Using both the subgenomic viral RNA 5'UTR and two modified nucleosides, we observed efficient exogenous protein expression even in Nsp1-transfected cells. To demonstrate its utility, we developed a barnase-barstar system capable of conditional transcript suppression in the presence of Nsp1. Altogether, the resistance to Nsp1-mediated translational suppression and the resulting Nsp1-sensing system we present in this study provide an invaluable opportunity to develop a new class of protein-sensing systems for the construction of more complex RNA-based genetic circuits.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144223706","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}