ACS Synthetic Biology最新文献_第3页

Reconstitution of Human Cytochrome P450 Activity using a Leishmania Cell-Free Protein Expression System. 利用利什曼原虫无细胞蛋白表达系统重组人细胞色素P450活性

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-14 DOI: 10.1021/acssynbio.5c00443

Wayne A Johnston, Raine E S Thomson, Juan Alfaro-Palma, Robert E Speight, Kirill Alexandrov, Elizabeth M J Gillam, James B Y Behrendorff

{"title":"Reconstitution of Human Cytochrome P450 Activity using a Leishmania Cell-Free Protein Expression System.","authors":"Wayne A Johnston, Raine E S Thomson, Juan Alfaro-Palma, Robert E Speight, Kirill Alexandrov, Elizabeth M J Gillam, James B Y Behrendorff","doi":"10.1021/acssynbio.5c00443","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00443","url":null,"abstract":"Cytochrome P450 enzymes (P450s) are ubiquitous in drug metabolism and natural product biosynthesis. Studying eukaryotic P450s has been limited by their dependence on membrane association and the requirement for partner reductases. Here, we demonstrate cell-free synthesis and assay of human P450s 3A4 and 2D6 using Leishmania tarentolae translational extract. These P450s were coexpressed with various NADPH-cytochrome P450 reductases (CPRs), and activity was assayed directly using unpurified reactions. P450s 3A4 and 2D6 showed distinct preferences in reductase coupling: P450 3A4 activity was greatest when coupled to the human CPR, whereas P450 2D6 performed better when coexpressed with CPRs from Arabidopsis thaliana. Inhibition assays with chloramphenicol, terbinafine, and erythromycin yielded results consistent with known P450-drug interactions. We conclude that Leishmania-based cell-free protein synthesis resolves previous challenges in eukaryotic P450 expression, allowing for rapid and convenient functional studies of unmodified eukaryotic P450 systems and offering a practical tool for drug metabolism studies and biocatalyst discovery.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145062893","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}

引用次数: 0

Strategies for Improved Docosahexaenoic Acid Synthesis in Microalgae. 微藻中改进二十二碳六烯酸合成的策略

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-14 DOI: 10.1021/acssynbio.5c00294

Kunal Dutta, Mariia S Ashikhmina, Ekaterina V Skorb, Sergey Shityakov

{"title":"Strategies for Improved Docosahexaenoic Acid Synthesis in Microalgae.","authors":"Kunal Dutta, Mariia S Ashikhmina, Ekaterina V Skorb, Sergey Shityakov","doi":"10.1021/acssynbio.5c00294","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00294","url":null,"abstract":"Docosahexaenoic acid (DHA) is an omega-3 fatty acid that is essential for optimal brain growth and development. Fish oil is the main dietary source of DHA. However, off-flavors and contamination with persistent organic pollutants are the main issues when DHA is sourced ethically from deep-sea fishes. Microalgae offer a sustainable, viable option for DHA production. However, optimization of the DHA yield using low-cost materials helps in reducing the fermentation cost. In this systematic review, we cover recent progress on enhanced DHA production during the last ten years, 2015-2025. We discuss how mutagenomics, genetic engineering, and numerous growth supplementations help in enhanced DHA production. ARTP mutagenesis significantly improves DHA yield up to 41.4 g/L. While overexpression/co-overexpression/manipulating selected genes linked to the central carbohydrate metabolism, lipid metabolism showed DHA yield up to 51.5 g/L. Furthermore, sustainable, low-cost carbon and nitrogen sources of fermentation media enhanced microalgal biomass and DHA yield. DHA yield was 20.7 g/L using maize starch hydrolysate as a carbon source and soybean meal hydrolysate as a nitrogen source. In addition, cane molasses as a nitrogen source along with overexpressed sucrose dehydrogenase in an adaptive laboratory evolution (ALE) optimized microalgal strain displayed a 162.86% increase in DHA yield (25.26 g/L). Differentially expressed genes (DEGs) revealed from transcriptomics are aligned with the metabolomics profile of DHA-producing microalgae. Enzymes linked to the central carbohydrate metabolism, fatty acid synthase (FAS), and polyketide synthase (PKS) pathways were upregulated along with high cellular demands of NADPH and acetyl-CoA. We believe this review may be useful for further advancement of high-yield DHA-producing microalgae.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145062940","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}

引用次数: 0

A Novel De Novo Biosynthetic Pathway for Efficient Synthesis of 6'-Sialyllactose in Escherichia coli. 一种在大肠杆菌中高效合成6′-唾液基乳糖的新生物合成途径

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-11 DOI: 10.1021/acssynbio.5c00437

Manxiang Zhu, Hongzhi Xia, Xianhao Xu, Yanfeng Liu, Guocheng Du, Xueqin Lv, Long Liu, Shixiu Cui, Jianghua Li, Yingyue Li

{"title":"A Novel De Novo Biosynthetic Pathway for Efficient Synthesis of 6'-Sialyllactose in Escherichia coli.","authors":"Manxiang Zhu, Hongzhi Xia, Xianhao Xu, Yanfeng Liu, Guocheng Du, Xueqin Lv, Long Liu, Shixiu Cui, Jianghua Li, Yingyue Li","doi":"10.1021/acssynbio.5c00437","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00437","url":null,"abstract":"6'-Sialyllactose (6'-SL) is an essential human milk oligosaccharide (HMO) that plays a crucial role in infant development with significant industrial potential. In this study, a novel biosynthetic pathway was employed to produce 6'-SL through de novo synthesis from glycerol in E. coli. First, the key enzymes for 6'-SL biosynthesis were expressed to achieve a yield of 6'-SL of 9.72 mg/L in E. coli. Next, to further increase the yield of 6'-SL, lactose degradation was prevented and genes involved in competing pathways were knocked out. Afterwards, by eliminating carbon catabolite repression (CCR), together with previous genetic modifications, 1.92 g/L of 6'-SL was successfully produced. Finally, by alleviating metabolic pressure from growth production interference and knocking out GlcNAc degradation genes, the yield of 6'-SL reached 6.21 g/L, which was higher than previously reported shake-flask yields. Additionally, in a 3-L fermenter, a yield of 14.33 g/L of 6'-SL was successfully achieved. This study successfully developed a novel plasmid-free, high-yield 6'-SL strain, demonstrating the strong potential of the GlcNAc pathway for 6'-SL biosynthesis.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145038625","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}

引用次数: 0

Characterization of Fluorescent Reporters for Flow Cytometry-Based Single-Cell Studies in Saccharomyces cerevisiae 基于流式细胞术的酿酒酵母菌单细胞研究荧光报告的表征。

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-10 DOI: 10.1021/acssynbio.5c00432

Samuel Evans, Masahiro Tominaga, Zeyu Lu, Naga Chandra Bandari, Liam McDonnell, Chengqiang Wang, Robert E. Speight, Jun Ishii, Claudia E. Vickers and Bingyin Peng*,

{"title":"Characterization of Fluorescent Reporters for Flow Cytometry-Based Single-Cell Studies in Saccharomyces cerevisiae","authors":"Samuel Evans, Masahiro Tominaga, Zeyu Lu, Naga Chandra Bandari, Liam McDonnell, Chengqiang Wang, Robert E. Speight, Jun Ishii, Claudia E. Vickers and Bingyin Peng*, ","doi":"10.1021/acssynbio.5c00432","DOIUrl":"10.1021/acssynbio.5c00432","url":null,"abstract":"Fluorescent proteins (FPs) are commonly used as reporters to examine intracellular genetic, molecular, and biochemical status. Flow cytometry is a powerful technique for accurate quantification of single-cell fluorescent levels. Here, we characterize green, red, and blue FPs for use in yeast Saccharomyces cerevisiae. Fluorophore-containing FPs and fluorogen-activating FPs (YFAST and FrFAST) were characterized dynamically in batch cultivation. FPs with a low pKa, StayGold, E2-Crimson, mTagBFP2, and mScarlet-I3, showed relatively stable fluorescence in diauxic growth when they were expressed under the control of the TEF1 promoter. A pH sensor, based on the fusion of mNeonGreen and mTagBFP2, was used to investigate the dynamic change of intracellular pH in batch cultivation. High-concentration acetate (200 mM) interfered with intracellular pH dramatically, whereas low-concentration acetate (20 mM) could not. Using StayGold and mScarlet-I3, an Epac-based cAMP sensor was constructed, showing varied Förster resonance energy transfer (FRET) patterns in different growth phases in S. cerevisiae CEN.PK113–7D and EBY100 backgrounds. In summary, the change in intracellular pH can significantly affect the brightness of pH-sensitive FPs. It is important to use FPs with a low pKa, neutralize intracellular pH, or compensate pH impacts when FPs are used as reporters.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3804–3812"},"PeriodicalIF":3.9,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145028573","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}

引用次数: 0

CRISPRi-Linked Multimodule Negative Feedback Loops to Address Winner-Take-All Resource Competition 与crispr相关的多模块负反馈循环解决赢者通吃的资源竞争

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-10 DOI: 10.1021/acssynbio.5c00394

Sadikshya Rijal, Kylie Standage-Beier, Rong Zhang, Austin Stone, Abdelrahman Youssef, Xiao Wang and Xiao-Jun Tian*,

{"title":"CRISPRi-Linked Multimodule Negative Feedback Loops to Address Winner-Take-All Resource Competition","authors":"Sadikshya Rijal, Kylie Standage-Beier, Rong Zhang, Austin Stone, Abdelrahman Youssef, Xiao Wang and Xiao-Jun Tian*, ","doi":"10.1021/acssynbio.5c00394","DOIUrl":"10.1021/acssynbio.5c00394","url":null,"abstract":"Cellular resource limitations create unintended interactions among synthetic gene circuit modules, compromising circuit modularity. This challenge is particularly pronounced in circuits with positive feedback, where uneven resource allocation can lead to Winner-Takes-All (WTA) behavior, favoring one module at the expense of others. In this study, we experimentally implemented a Negatively Competitive Regulatory (NCR) controller using CRISPR interference (CRISPRi) and evaluated its effectiveness in mitigating WTA behavior in two gene circuits: dual self-activation and cascading bistable switch. We chromosomally integrated a tunable dCas9 gene and designed module-specific gRNAs, with each module encoding its own gRNA to self-repress via competition for limited dCas9. This configuration introduces strong negative feedback to the more active module while reallocating resources to the less active one, promoting balanced module activation. Compared to the control group lacking dCas9-mediated repression, the NCR controller significantly increased module coactivation and suppressed WTA behavior. Our quantitative results demonstrate that NCR provides an effective strategy for regulating resource competition and improving the modularity of synthetic gene circuits.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3646–3654"},"PeriodicalIF":3.9,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145028567","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}

引用次数: 0

Controlling the Taxonomic Composition of Biological Information Storage in 16S rRNA 控制16S rRNA生物信息存储的分类组成。

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-09 DOI: 10.1021/acssynbio.5c00313

Kiara Reyes Gamas, Travis R. Seamons, Matthew J. Dysart, Lin Fang, James Chappell, Lauren B. Stadler* and Jonathan J. Silberg*,

{"title":"Controlling the Taxonomic Composition of Biological Information Storage in 16S rRNA","authors":"Kiara Reyes Gamas, Travis R. Seamons, Matthew J. Dysart, Lin Fang, James Chappell, Lauren B. Stadler* and Jonathan J. Silberg*, ","doi":"10.1021/acssynbio.5c00313","DOIUrl":"10.1021/acssynbio.5c00313","url":null,"abstract":"Microbes can be programmed to record participation in gene transfer by coding biological-recording devices into mobile DNA. Upon DNA uptake, these devices transcribe a catalytic RNA (cat-RNA) that binds to conserved sequences within ribosomal RNAs (rRNAs) and perform a trans-splicing reaction that adds a barcode to the rRNAs. Existing cat-RNA designs were generated to be broad-host range, providing no control over the organisms that were barcoded. To achieve control over the organisms barcoded by cat-RNA, we created a program called Ribodesigner that uses input sets of rRNA sequences to create designs with varying specificities. We show how this algorithm can be used to identify designs that enable kingdom-wide barcoding, or selective barcoding of specific taxonomic groups within a kingdom. We use Ribodesigner to create cat-RNA designs that target Pseudomonadales while avoiding Enterobacterales, and we compare the performance of one design to a cat-RNA that was previously found to be broad host range. When conjugated into a mixture of Escherichia coli and Pseudomonas putida, the new design presents increased selectivity compared to a broad host range cat-RNA. Ribodesigner is expected to aid in developing cat-RNAs that store information within user-defined sets of microbes in environmental communities for gene transfer studies.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3530–3542"},"PeriodicalIF":3.9,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145022430","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}

引用次数: 0

Engineering Noncanonical Cofactors To Expand Cellular Functions 工程非规范辅助因子扩展细胞功能。

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-09 DOI: 10.1021/acssynbio.5c00473

Samuel Lim, and , Scott Banta*,

引用次数: 0

Signal Peptide-Guided Delivery of a Mucin-Like Collagen Analogue for Periplasmic Barrier Reinforcement: A Platform for Enhancing Microbial Survival 信号肽引导的粘蛋白样胶原类似物的传递用于质周屏障增强：一个提高微生物存活的平台。

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-09 DOI: 10.1021/acssynbio.5c00607

Zilong Zhao, Weigang Yuwen, Wensha Zhu, Linlin Qu and Daidi Fan*,

{"title":"Signal Peptide-Guided Delivery of a Mucin-Like Collagen Analogue for Periplasmic Barrier Reinforcement: A Platform for Enhancing Microbial Survival","authors":"Zilong Zhao, Weigang Yuwen, Wensha Zhu, Linlin Qu and Daidi Fan*, ","doi":"10.1021/acssynbio.5c00607","DOIUrl":"10.1021/acssynbio.5c00607","url":null,"abstract":"The environmental resistance exhibited by microorganisms is concerned with their ability to withstand and adapt to an array of detrimental environmental conditions, with their survival and reproductive success being threatened. Within the realm of biotechnology, which emphasizes stress resistance, a critical role in bacterial adaptive strategies to environmental fluctuations is assumed to be in the periplasmic space. An innovative methodology to augment bacterial tolerance to stress by employing a mucin-mimetic collagen analogue, designated as S1552 (which is secreted into the periplasmic compartment), is introduced by this investigation. Exceptional protective barrier attributes are highly regarded in S1552. An appropriate signal peptide for the periplasmic expression of S1552 in Escherichia coli was identified by employing computational analysis (in silico). The fusion protein of S1552 and the DsbA signal peptide (DS1552) is efficiently translocated into the periplasmic space of E. coli. This approach not only fortifies the cellular barrier but also significantly enhances the bacterium’s resistance to a variety of stressors, including elevated salinity, high osmotic stress, extreme pH values, antibiotics, toxicants, and heavy metal ions. Meanwhile, this pioneering application has the potential to be extended to other bacteria with a periplasmic space, enhancing their viability and proliferation in harsh environments.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3767–3783"},"PeriodicalIF":3.9,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145028597","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}

引用次数: 0

High-Level Soluble Expression of Recombinant Human Bone Morphogenetic Protein-2 in Escherichia coli 重组人骨形态发生蛋白-2在大肠杆菌中的高可溶性表达。

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-09 DOI: 10.1021/acssynbio.5c00392

Youming Cui, Yanqiu Liang, Bin Zhang, Quanfeng Liang, Yi Wang and Wei Luo*,

{"title":"High-Level Soluble Expression of Recombinant Human Bone Morphogenetic Protein-2 in Escherichia coli","authors":"Youming Cui, Yanqiu Liang, Bin Zhang, Quanfeng Liang, Yi Wang and Wei Luo*, ","doi":"10.1021/acssynbio.5c00392","DOIUrl":"10.1021/acssynbio.5c00392","url":null,"abstract":"Human Bone Morphogenetic Protein-2 (hBMP-2) serves as a critical regulator in bone and cartilage formation; however, its industrial application is hindered by its inherent tendency to form inclusion bodies in prokaryotic expression systems. To address this issue, we established a recombinant hBMP-2 (rhBMP-2) expression system using the pCold II plasmid and the SHuffle T7 strain. We explored several strategies to enhance the solubility of rhBMP-2, including coexpression with molecular chaperones, vesicle-mediated secretory expression, fusion expression with synthetic intrinsically disordered proteins (SynIDPs), and fusion expression with small-molecule peptide tags. Our results showed that coexpression with the molecular chaperone pGro7 significantly improved the solubility of rhBMP-2. Fusion with SynIDPs led to complete solubility of rhBMP-2; however, the protein was expressed exclusively in the monomeric form. Among the tested small-molecule peptide tags, GB1 was the most effective, achieving fully soluble rhBMP-2 expression. Western blot analysis confirmed the coexistence of monomeric and dimeric forms of rhBMP-2. Subsequent purification of rhBMP-2 through metal chelate chromatography resulted in an expression level of 109.7 ± 5.0 mg·L–1. In summary, we successfully demonstrated fully soluble expression of rhBMP-2 in Escherichia coli, providing a valuable foundation for its industrial-scale production.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3687–3695"},"PeriodicalIF":3.9,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145022499","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}

引用次数: 0

Engineering of a Complex of the DNase Domain of Colicin E9 with the Immunity Protein Im9 Activated by the Protease pS273R of African Swine Fever Virus 非洲猪瘟病毒蛋白酶pS273R活化免疫蛋白Im9与Colicin E9 dna酶结构域复合物的工程设计

IF 3.9 2区生物学

ACS Synthetic Biology Pub Date : 2025-09-09 DOI: 10.1021/acssynbio.5c00398

Danil S. Kalinin, Oleg R. Latypov, Bogdan S. Melnik, Tatiana N. Melnik, Michael G. Shlyapnikov, Maria A. Gorshkova, Eva N. Titova, Sergey G. Mayorov, Artem F. Stetoi, Alexander V. Efimov, Andrey V. Kajava, Rustam H. Ziganshin, Marina Y. Zemskova, Alexey N. Fedorov and Igor E. Granovsky*,

{"title":"Engineering of a Complex of the DNase Domain of Colicin E9 with the Immunity Protein Im9 Activated by the Protease pS273R of African Swine Fever Virus","authors":"Danil S. Kalinin, Oleg R. Latypov, Bogdan S. Melnik, Tatiana N. Melnik, Michael G. Shlyapnikov, Maria A. Gorshkova, Eva N. Titova, Sergey G. Mayorov, Artem F. Stetoi, Alexander V. Efimov, Andrey V. Kajava, Rustam H. Ziganshin, Marina Y. Zemskova, Alexey N. Fedorov and Igor E. Granovsky*, ","doi":"10.1021/acssynbio.5c00398","DOIUrl":"10.1021/acssynbio.5c00398","url":null,"abstract":"African swine fever virus (ASFV) is a large DNA virus that causes a highly lethal disease in pigs and currently has no effective vaccines or antiviral treatments available. We designed a protein switch that combines the DNase domain of colicin E9 (DNase E9) and its inhibitor Im9 with the viral protease cleavage site. The complex is only destroyed in the presence of an ASFV pS273R protease, which releases DNase activity. Several Im9 variants were constructed by inserting the pS273R protease cleavage sequence into different exposed loops. From these, we identified an optimized variant (Im9–1.4) that remains highly stable and tightly bound to DNase E9, suppressing its activity in the absence of protease. Exposure to the ASFV protease results in cleavage of Im9–1.4, rendering it unable to inhibit DNase E9 activity. In vitro assays confirmed that the DNase E9/Im9–1.4 complex becomes catalytically active upon proteolytic digestion with pS273R protease. This virus-triggered ‘kill switch’ is designed to render pig cells nonpermissive to ASFV by aborting infection via viral DNA degradation. Our study offers a generalizable synthetic biology strategy that uses virus-encoded proteases to trigger dormant effectors, exemplified by this protease-sensing DNase. This synthetic restriction system might be used to develop ASFV-resistant pigs.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 9","pages":"3655–3670"},"PeriodicalIF":3.9,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145028538","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}

引用次数: 0