ACS Synthetic Biology最新文献

{"title":"Genetic Entanglement Enables Ultrastable Biocontainment in the Mammalian Gut","authors":"Gary W. Foo,&nbsp;Aathavan S. Uruthirapathy,&nbsp;Claire Q. Zhang,&nbsp;Izabela Z. Batko,&nbsp;David E. Heinrichs and David R. Edgell*,&nbsp;","doi":"10.1021/acssynbio.5c00412","DOIUrl":"10.1021/acssynbio.5c00412","url":null,"abstract":"<p >Imbalances in the mammalian gut are associated with acute and chronic conditions, and using engineered probiotic strains to deliver synthetic constructs to treat them is a promising strategy. However, high rates of mutational escape and genetic instability <i>in vivo</i> limit the effectiveness of biocontainment circuits needed for safe and effective use. Here, we describe STALEMATE (<b>S</b>equence en<b>TA</b>ng<b>LE</b>d <b>M</b>ulti l<b>A</b>yered gene<b>T</b>ic buff<b>E</b>ring), a dual-layered failsafe biocontainment strategy that entangles genetic sequences to create pseudoessentiality and buffer against mutations. We entangled the colicin E9 immunity protein (Im9) with a thermoregulated meganuclease (TSM) by overlapping the reading frames. Mutations that disrupted this entanglement simultaneously inactivated both biocontainment layers, leading to cell death by the ColE9 nuclease and the elimination of escape mutants. By lengthening the entangled region, refining ColE9 expression, and optimizing the TSM sequence against IS<i>911</i> insertion, we achieved escape rates below 10^–10 as compared to rates of 10^–5 with the nonentangled TSM. The STALEMATE system contained plasmids in <i><i>E. coli</i></i> Nissle 1917 for over a week in the mouse gastrointestinal tract with nearly undetectable escape rates upon excretion. STALEMATE offers a modular and simple biocontainment approach to buffer against mutational inactivation in the mammalian gut without a requirement for engineered bacteria or exogenous signaling ligands.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3696–3708"},"PeriodicalIF":3.9,"publicationDate":"2025-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/acssynbio.5c00412","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145013474","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Directed Design of Anti-Idiotypic Nanobody with Antibacterial Activity against Foodborne Staphylococcus aureus and Its Innovative Application in Green Immunoassay","authors":"Chongxin Xu*,&nbsp;Yajun Qiu,&nbsp;Cheng Shen,&nbsp;Yajing Xie,&nbsp;Xin He,&nbsp;Xiaoming Sun,&nbsp;Ji Sun,&nbsp;Jianxing Shen and Yan Shen*,&nbsp;","doi":"10.1021/acssynbio.5c00404","DOIUrl":"10.1021/acssynbio.5c00404","url":null,"abstract":"<p >Anti-idiotypic antibodies (Anti-Ids) possess the properties to mimic the structure and biological activity of an antigen, which can be utilized for preventing and monitoring hazards. In this study, Nb4Mutant6-Anti-Id, which mimics the structure and antibacterial activity of vancomycin, was designed based on phage display antibody library screening and mutagenesis technology. The affinity of Nb4Mutant6-Anti-Id for the coated antigens of Van-pAbs F(ab)2 and inactivated <i>S. aureus</i> cells was 6.06 × 10¹⁰ and 2.97 × 10⁸ L/mol, respectively. Nb4Mutant6-Anti-Id maintained approximately 70% of its binding activity for the coated antigens that had undergone heat treatment at 65 °C, and its minimum inhibitory concentration (MIC) against foodborne <i>S. aureus</i> was 105 μg/mL. The limit of detection (LOD) of Nb4Mutant6-Anti-Id protein was 0.72 ng/mL when utilized as a “coated antigen” in a vancomycin-free indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for detecting vancomycin, which is a highly sensitive level (&lt;1.0 ng/mL). This study not only designed Nb4Mutant6-Anti-Id, which exhibits antibacterial activity comparable to that of vancomycin and offering potential for the environmentally friendly management of foodborne pathogens, but also has the potential to replace the vancomycin structure. This replacement enables the establishment of an antigen-free, highly sensitive IC-ELISA with good accuracy and stability for the green monitoring of vancomycin in agricultural environments and food.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3671–3686"},"PeriodicalIF":3.9,"publicationDate":"2025-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145005615","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":"Thermodynamically Coupled Three-Enzyme Cascade Converts Styrene to Cinnamic Acid, l-Phenylalanine, and Phenylpyruvate via CO2 Fixation without External Energy Cofactors","authors":"Sunga Cho,&nbsp;Ye Chan Kim,&nbsp;Amol D. Pagar,&nbsp;Sangwoo Joo,&nbsp;Pritam Giri,&nbsp;Subin Yun,&nbsp;Geon-Woo Park,&nbsp;Young-Soo Keum and Hyungdon Yun*,&nbsp;","doi":"10.1021/acssynbio.5c00384","DOIUrl":"10.1021/acssynbio.5c00384","url":null,"abstract":"<p >We report the development of a cofactor-free CO₂ fixation platform based on a three-enzyme cascade comprising <i>Aspergillus niger</i> ferulic acid decarboxylase (AnFDC), <i>Anabaena variabilis</i> phenylalanine ammonia-lyase (AvPAL), and <i>Proteus mirabilis</i> <span>l</span>-amino acid deaminase (PmLAAD). Unlike canonical ATP- or NADPH-dependent CO₂ assimilation pathways, this system uses a prFMN-dependent carboxylation mechanism, enabling efficient CO₂ incorporation under ambient conditions without energy-intensive cofactors. Systematic screening identified AnFDC as the optimal decarboxylase for styrene carboxylation, while AvPAL and PmLAAD were selected for their superior catalytic efficiencies in the cascade. Optimization of prFMN biosynthesis (via UbiX/SccK coexpression), enzyme expression, and reaction conditions (notably, 1.5 M ammonium bicarbonate, pH 8.5) significantly enhanced cascade performance. Whole-cell microbial consortia with tailored cell ratios further alleviated kinetic bottlenecks, achieving a 3-fold improvement in phenylpyruvic acid production (6% conversion) from styrene and CO₂. The integrated cascade drives the CO₂ fixation with an overall equilibrium constant (K_eq′) of 4.3 × 10³⁰, converting low-cost styrene into high-value phenylpyruvic acid. Through enzyme screening and step-by-step optimization, we established an energy-independent system for CO₂ fixation. Our work challenges the cofactor dependence in biocatalytic carbon fixation for aromatic compounds and paves a novel way for sustainable, carbon-negative chemical manufacturing.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3624–3635"},"PeriodicalIF":3.9,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145005679","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":"Genome-Wide Simplification of the AcMNPV Genome Using Synthetic Biology","authors":"Yijia Guo,&nbsp;Hengrui Hu,&nbsp;Han Xiao,&nbsp;Xi Wang,&nbsp;Xiquan Ke,&nbsp;Jiang Li,&nbsp;Manli Wang* and Zhihong Hu*,&nbsp;","doi":"10.1021/acssynbio.5c00156","DOIUrl":"10.1021/acssynbio.5c00156","url":null,"abstract":"<p >Large-scale genome simplification represents a fundamental goal in synthetic biology. Baculoviruses, with their biphasic life cycle and inherent genomic plasticity, have emerged as ideal models for synthetic genome engineering. Although modified baculovirus genomes are widely used as expression vectors for robust recombinant protein production, many genomic regions are dispensable for in vitro budded virus (BV) production. In this study, guided by the synthetic biology “design-build-test-learn” framework, we systematically reduced the genome of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and obtained synthetic viruses capable of producing BVs. Building upon our previous work on whole-genome synthesis and partial genome reduction, we developed a strategy to rescue viruses by cotransfecting linearized genome fragments into host cells, thereby accelerating the iterative evaluation of genomic deletions. A total of 35 reduced genomes of varying sizes were synthesized, and the titers of the corresponding rescued viruses were measured. The most reduced functional genome, AcMNPV-Syn-mini, corresponds to the deletion of approximately 28 kb encompassing 39 nonessential genes. We analyze and discuss the gene organization and characteristics of this minimized genome. Our findings provide a foundation for the development of high-capacity baculoviral vectors and contribute to a deeper understanding of baculovirus functional genomics.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3414–3422"},"PeriodicalIF":3.9,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144999223","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":"Intelligent Design of Escherichia coli Terminators by Coupling Prediction and Generation Models","authors":"Jie Li,&nbsp;Lin-Feng Wu,&nbsp;Kai Liu and Bin-Guang Ma*,&nbsp;","doi":"10.1021/acssynbio.5c00429","DOIUrl":"10.1021/acssynbio.5c00429","url":null,"abstract":"<p >Terminators are specific nucleotide sequences located at the 3′ end of a gene and contain transcription termination information. As a fundamental genetic regulatory element, terminators play a crucial role in the design of gene circuits. Accurately characterizing terminator strength is essential for improving the precision of gene circuit designs. Experimental characterization of terminator strength is time-consuming and labor-intensive; therefore, there is a need to develop computational tools capable of accurately predicting terminator strength. Current prediction methods do not fully consider sequence or thermodynamic information related to terminators, lacking robust models for accurate prediction. Meanwhile, deep generative models have demonstrated tremendous potential in the design of biological sequences and are expected to be applied to terminator sequence design. This study focuses on intelligent design of <i>Escherichia coli</i> terminators and primarily conducts the following research: (1) to construct an intrinsic terminator strength prediction model for <i>E. coli</i>, this study extracts sequence features and thermodynamic features from <i>E. coli</i> intrinsic terminators. Machine learning models based on the selected features achieved a prediction performance of <i>R</i>² = 0.72. (2) This study employs a generative adversarial network (GAN) to learn from intrinsic terminator sequence training data and generate terminator sequences. Evaluation reveals that the generated terminators exhibit similar data distributions to intrinsic terminators, demonstrating the reliability of GAN-generated terminator sequences. (3) This study uses the constructed terminator strength prediction model to screen for strong terminators from the generated set. Experimental verification shows that among the 18 selected terminators, 72% exhibit termination efficiencies greater than 90%, confirming the reliability of the intelligent design approach for <i>E. coli</i> terminators. In sum, this study constructs a terminator strength prediction model and a terminator generation model for <i>E. coli</i>, providing model support for terminator design in gene circuits. This enhances the modularity of biological component design and promotes the development of synthetic biology.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3744–3752"},"PeriodicalIF":3.9,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144990922","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":"CELLM: Bridging Natural Language Processing and Synthetic Genetic Circuit Design with AI","authors":"Lucas Abello Castillo*,&nbsp; and ,&nbsp;Martín Gutiérrez Pescarmona*,&nbsp;","doi":"10.1021/acssynbio.5c00391","DOIUrl":"10.1021/acssynbio.5c00391","url":null,"abstract":"<p >The complexity of the genetic circuit design limits accessibility and efficiency in synthetic biology. This study presents an integrated system that combines Cello software with large language models (DeepSeek-R1, Phi-4) and the LangChain framework in Python, which allows the creation, analysis, and optimization of genetic circuits using natural language instructions. <i>CELLM</i> automates the translation of textual descriptions into functional designs using Cello v2.1 as the basis for circuit synthesis and LLM for the interpretation of biological requirements and logical optimization. To the best of our knowledge, this work sets a precedent as the first system that integrates language models with synthetic biology design tools such as Cello, demonstrating that natural language processing can be translated into functional biological designs. This approach removes barriers by allowing researchers without bioengineering expertise to prototype genetic circuits using simple instructions.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3799–3803"},"PeriodicalIF":3.9,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144990894","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":"Correction to “Sustainable Production of Cyanidin-3-O-galactoside by Metabolic Engineered Escherichia coli from Catechin”","authors":"Zhen Zong,&nbsp;Lianghua Xie,&nbsp;Jiaqi Fu,&nbsp;Zhongyang Liu,&nbsp;Wen-Wen Zhou* and Wei Chen*,&nbsp;","doi":"10.1021/acssynbio.5c00579","DOIUrl":"10.1021/acssynbio.5c00579","url":null,"abstract":"","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3813–3814"},"PeriodicalIF":3.9,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144999240","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":"A Rapid and Modular Nanobody Assay for Plug-and-Play Antigen Detection","authors":"N. Rebecca Kang,&nbsp;Jisoo Im,&nbsp;John R. Biondo,&nbsp;Caitlin E. Sharpes,&nbsp;Katherine A. Rhea,&nbsp;Padric M. Garden,&nbsp;Juan J. Jaramillo Montezco,&nbsp;Alina Ringaci,&nbsp;Mark W. Grinstaff,&nbsp;Daniel A. Phillips,&nbsp;Aleksandr E. Miklos and Alexander A. Green*,&nbsp;","doi":"10.1021/acssynbio.5c00182","DOIUrl":"10.1021/acssynbio.5c00182","url":null,"abstract":"<p >Rapid and portable antigen detection is essential for managing infectious diseases and responding to toxic exposures, yet current methods face significant limitations. Highly sensitive platforms like the Enzyme-Linked Immunosorbent Assay (ELISA) are time- and cost-prohibitive for point-of-need detection, while portable options like lateral flow assays (LFAs) require systemic overhauls for new targets. Furthermore, the complex infrastructure, high production costs, and extended timelines for assay development constrain the manufacturing of traditional diagnostic platforms in low-resource settings. To address these challenges, we describe the Rapid and Modular Nanobody Assay (RAMONA), a versatile antigen detection platform that leverages nanobody-coiled coil fusion proteins for modular integration with downstream readout methods. RAMONA merges the portability of LFAs with the benefits of nanobodies such as their smaller size, improved solubility, and compatibility with cell-free protein synthesis systems, enabling on-demand biomanufacturing and rapid adaptation for diverse targets. We demonstrate assay generalizability through the detection of three distinct protein targets, robustness across various temperatures and incubation periods, and compatibility with saliva samples and cell-free synthesis. Detection occurs in under 30 minutes, with results strongly and positively correlating to ELISA data while requiring minimal resources. Moreover, RAMONA supports multiplexed detection of three antigens simultaneously by using orthogonal capture probes. By overcoming several limitations of traditional immunoassays, RAMONA represents a significant advancement in rapid, adaptable, and field-deployable antigen detection technologies.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3423–3433"},"PeriodicalIF":3.9,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144935775","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":"Mutations in the Substrate-Binding Pocket of DiacylGlycerol Acyltransferase Alter the Fatty Acid Composition of Triacylglycerides in Yeast","authors":"Nazreen V. M. Abdul Muthaliff,&nbsp;Nur Eka Fitriani,&nbsp;Derek Smith,&nbsp;Jing Sen Ong,&nbsp;Lay Kien Yang,&nbsp;Coleen Toledo Busran,&nbsp;Aaron Thong,&nbsp;Prakash Arumugam and Naazneen Sofeo*,&nbsp;","doi":"10.1021/acssynbio.5c00143","DOIUrl":"10.1021/acssynbio.5c00143","url":null,"abstract":"<p >Triacylglycerols (TAGs) are the main components of food oils and fats. The fatty acid composition of TAGs varies for different oils and fats. Specific enzymes sequentially add three fatty acids to the glycerol backbone of TAGs. Diacylglycerol acyltransferase or DGAT adds the third and ultimate fatty acid to the glycerol backbone at the <i>sn-3</i> position. In this study, we characterized the substrate-binding pocket of enzyme DGAT1 from <i>Arabidopsis thaliana</i> through heterologous expression in the DGAT mutant of <i>Saccharomyces cerevisiae</i>. We performed site saturation mutagenesis on 10 amino acid residues in the catalytic site and examined their effects on the fatty acid profile of yeast cells. Our results indicate that mutations F373G, T240I, M289F, and V248I impact the yeast TAG profile either in terms of the total saturation level or the carbon chain length of the fatty acids, suggesting that they change the DGAT’s substrate preference. This offers insights into crucial amino acid residues in the DGAT binding pocket which can be engineered for fine tuning the lipid profile. In summary, we have harnessed the power of enzyme engineering to modify the fatty acyl makeup of triglycerides and created a sustainable platform for the production of customized alternative lipids.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3401–3413"},"PeriodicalIF":3.9,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144990915","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":"Diversifying Substrates and Reaction Conditions for Polymerase Strand Recycling","authors":"Yueyi Li,&nbsp;Arno Gundlach,&nbsp;Andrew Ellington and Julius B. Lucks*,&nbsp;","doi":"10.1021/acssynbio.5c00207","DOIUrl":"10.1021/acssynbio.5c00207","url":null,"abstract":"<p >Cell-free biosensing systems are being engineered as versatile and programmable diagnostic technologies. A core component of cell-free biosensors is programmable molecular circuits that improve biosensor speed, sensitivity, and specificity by performing molecular computations such as logic evaluation and signal amplification. In previous work, we developed one such circuit system called Polymerase Strand Recycling (PSR), which amplifies cell-free molecular circuits by using T7 RNA polymerase off-target transcription to recycle nucleic acid inputs. We showed that PSR circuits can be configured to detect RNA target inputs as well as be interfaced with allosteric transcription factor-based biosensors to amplify signals and enhance sensitivity. Here we expand the development of PSR circuit empirical design guidelines to generalize the platform for detecting a diverse set of microRNA inputs. We show that PSR circuit function can be enhanced through engineering T7 RNAP, and we present troubleshooting strategies to optimize PSR circuit performance.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3784–3790"},"PeriodicalIF":3.9,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144990947","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}