Pub Date : 2024-06-21DOI: 10.1021/acssynbio.4c00292
Francis Hassard, Thomas P. Curtis, Gabriela C. Dotro, Peter Golyshin, Tony Gutierrez, Sonia Heaven, Louise Horsfall, Bruce Jefferson, Davey L. Jones, Natalio Krasnogor, Vinod Kumar, David J. Lea-Smith, Kristell Le Corre Pidou, Yongqiang Liu, Tao Lyu, Ronan R. McCarthy, Boyd McKew, Cindy Smith, Alexander Yakunin, Zhugen Yang, Yue Zhang and Frederic Coulon*,
{"title":"Scaling-up Engineering Biology for Enhanced Environmental Solutions","authors":"Francis Hassard, Thomas P. Curtis, Gabriela C. Dotro, Peter Golyshin, Tony Gutierrez, Sonia Heaven, Louise Horsfall, Bruce Jefferson, Davey L. Jones, Natalio Krasnogor, Vinod Kumar, David J. Lea-Smith, Kristell Le Corre Pidou, Yongqiang Liu, Tao Lyu, Ronan R. McCarthy, Boyd McKew, Cindy Smith, Alexander Yakunin, Zhugen Yang, Yue Zhang and Frederic Coulon*, ","doi":"10.1021/acssynbio.4c00292","DOIUrl":"10.1021/acssynbio.4c00292","url":null,"abstract":"","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.4c00292","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141430865","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}
Pub Date : 2024-06-21DOI: 10.1021/acssynbio.4c00196
Rachel L Huang, Delilah Jewel, Rachel E Kelemen, Quan Pham, Tarah J Yared, Shu Wang, Soumya Jyoti Singha Roy, Zeyi Huang, Samantha D Levinson, Bharathi Sundaresh, Suyen Espinoza Miranda, Tim van Opijnen, Abhishek Chatterjee
The Escherichia coli leucyl-tRNA synthetase (EcLeuRS)/tRNAEcLeu pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNAEcLeu. Here, we overcome this limitation by evolving tRNAEcLeu directly in mammalian cells by using a virus-assisted selection scheme. This directed evolution platform was optimized for higher throughput such that the entire acceptor stem of tRNAEcLeu could be simultaneously engineered, which resulted in the identification of several variants with remarkably improved efficiency for incorporating a wide range of ncAAs. The advantage of the evolved leucyl tRNAs was demonstrated by expressing ncAA mutants in mammalian cells that were challenging to express before using the wild-type tRNAEcLeu, by creating viral vectors that facilitated ncAA mutagenesis at a significantly lower dose and by creating more efficient mammalian cell lines stably expressing the ncAA-incorporation machinery.
{"title":"Directed Evolution of a Bacterial Leucyl tRNA in Mammalian Cells for Enhanced Noncanonical Amino Acid Mutagenesis.","authors":"Rachel L Huang, Delilah Jewel, Rachel E Kelemen, Quan Pham, Tarah J Yared, Shu Wang, Soumya Jyoti Singha Roy, Zeyi Huang, Samantha D Levinson, Bharathi Sundaresh, Suyen Espinoza Miranda, Tim van Opijnen, Abhishek Chatterjee","doi":"10.1021/acssynbio.4c00196","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00196","url":null,"abstract":"<p><p>The <i>Escherichia coli</i> leucyl-tRNA synthetase (EcLeuRS)/tRNA<sup>EcLeu</sup> pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNA<sup>EcLeu</sup>. Here, we overcome this limitation by evolving tRNA<sup>EcLeu</sup> directly in mammalian cells by using a virus-assisted selection scheme. This directed evolution platform was optimized for higher throughput such that the entire acceptor stem of tRNA<sup>EcLeu</sup> could be simultaneously engineered, which resulted in the identification of several variants with remarkably improved efficiency for incorporating a wide range of ncAAs. The advantage of the evolved leucyl tRNAs was demonstrated by expressing ncAA mutants in mammalian cells that were challenging to express before using the wild-type tRNA<sup>EcLeu</sup>, by creating viral vectors that facilitated ncAA mutagenesis at a significantly lower dose and by creating more efficient mammalian cell lines stably expressing the ncAA-incorporation machinery.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141430863","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}
The advancement in nanotechnology has completely revolutionized various fields, including pharmaceutical sciences, and streamlined the potential therapeutic of many diseases that endanger human life. The synthesis of green nanoparticles by biological processes is an aspect of the newly emerging scientific field known as "green nanotechnology". Due to their safe, eco-friendly, nontoxic nature, green synthesis tools are better suited to produce nanoparticles between 1 and 100 nm. Nanoformulation of different types of nanoparticles has been made possible by using green production techniques and commercially feasible novel precursors, such as seed extracts, algae, and fungi, that act as potent reducing, capping, and stabilizing agents. In addition to this, the biofunctionalization of nanoparticles has also broadened its horizon in the field of environmental remediation and various novel therapeutic innovations including wound healing, antimicrobial, anticancer, and nano biosensing. However, the major challenge pertaining to green nanotechnology is the agglomeration of nanoparticles that may alter the surface topology, which can affect biological physiology, thereby contributing to system toxicity. Therefore, a thorough grasp of nanoparticle toxicity and biocompatibility is required to harness the applications of nanotechnology in therapeutics.
{"title":"Sustainable Synthesis of Novel Green-Based Nanoparticles for Therapeutic Interventions and Environmental Remediation.","authors":"Swati Singh, Harshita Tiwari, Ashish Verma, Priyamvada Gupta, Amrit Chattopadhaya, Ananya Singh, Sanjana Singh, Brijesh Kumar, Abhijit Mandal, Rajiv Kumar, Ashok K Yadav, Hemant Kumar Gautam, Vibhav Gautam","doi":"10.1021/acssynbio.4c00206","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00206","url":null,"abstract":"<p><p>The advancement in nanotechnology has completely revolutionized various fields, including pharmaceutical sciences, and streamlined the potential therapeutic of many diseases that endanger human life. The synthesis of green nanoparticles by biological processes is an aspect of the newly emerging scientific field known as \"green nanotechnology\". Due to their safe, eco-friendly, nontoxic nature, green synthesis tools are better suited to produce nanoparticles between 1 and 100 nm. Nanoformulation of different types of nanoparticles has been made possible by using green production techniques and commercially feasible novel precursors, such as seed extracts, algae, and fungi, that act as potent reducing, capping, and stabilizing agents. In addition to this, the biofunctionalization of nanoparticles has also broadened its horizon in the field of environmental remediation and various novel therapeutic innovations including wound healing, antimicrobial, anticancer, and nano biosensing. However, the major challenge pertaining to green nanotechnology is the agglomeration of nanoparticles that may alter the surface topology, which can affect biological physiology, thereby contributing to system toxicity. Therefore, a thorough grasp of nanoparticle toxicity and biocompatibility is required to harness the applications of nanotechnology in therapeutics.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141425620","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}
Pub Date : 2024-06-19DOI: 10.1021/acssynbio.4c00239
Fu Wang, Zhenyuan Zang, Qian Zhao, Chunxiao Xiaoyang, Xiujuan Lei, Yingping Wang, Yiqiao Ma, Rongan Cao, Xixia Song, Lili Tang, Michael K Deyholos, Jian Zhang
Cannabis sativa L. is a multipurpose crop with high value for food, textiles, and other industries. Its secondary metabolites, including cannabidiol (CBD), have potential for broad application in medicine. With the CBD market expanding, traditional production may not be sufficient. Here we review the potential for the production of CBD using biotechnology. We describe the chemical and biological synthesis of cannabinoids, the associated enzymes, and the application of metabolic engineering, synthetic biology, and heterologous expression to increasing production of CBD.
{"title":"Advancement of Research Progress on Synthesis Mechanism of Cannabidiol (CBD).","authors":"Fu Wang, Zhenyuan Zang, Qian Zhao, Chunxiao Xiaoyang, Xiujuan Lei, Yingping Wang, Yiqiao Ma, Rongan Cao, Xixia Song, Lili Tang, Michael K Deyholos, Jian Zhang","doi":"10.1021/acssynbio.4c00239","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00239","url":null,"abstract":"<p><p><i>Cannabis sativa</i> L. is a multipurpose crop with high value for food, textiles, and other industries. Its secondary metabolites, including cannabidiol (CBD), have potential for broad application in medicine. With the CBD market expanding, traditional production may not be sufficient. Here we review the potential for the production of CBD using biotechnology. We describe the chemical and biological synthesis of cannabinoids, the associated enzymes, and the application of metabolic engineering, synthetic biology, and heterologous expression to increasing production of CBD.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141430862","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}
Pub Date : 2024-06-18DOI: 10.1021/acssynbio.4c00014
Hopen Yang, Wilfred Chen
BioPROTACs are heterobifunctional proteins designed for targeted protein degradation. While they offer a potential therapeutic avenue for modulating disease-related proteins, the current strategies are static in nature and lack the ability to modulate protein degradation dynamically. Here, we introduce a synthetic framework for dynamic fine-tuning of target protein levels using protease control switches. The idea is to utilize proteases as an interfacing layer between exogenous inputs and protein degradation by modulating the recruitment of target proteins to E3 ligase by separating the two binding domains on bioPROTACs. By decoupling the external inputs from the primary protease layer, new conditional degradation phenotypes can be readily adapted with minimal modifications to the design. We demonstrate the adaptability of this approach using two highly efficient "bioPROTAC" systems: AdPROM and IpaH9.8-based Ubiquibodies. Using the TEV protease as the transducer, we can interface small-molecule and optogenetic inputs for conditional targeted protein degradation. Our findings highlight the potential of bioPROTACs with protease-responsive linkers as a versatile tool for conditional targeted protein degradation.
BioPROTACs 是一种专为靶向降解蛋白质而设计的异功能蛋白质。虽然它们为调节疾病相关蛋白提供了潜在的治疗途径,但目前的策略都是静态的,缺乏动态调节蛋白降解的能力。在这里,我们介绍一种合成框架,利用蛋白酶控制开关对目标蛋白水平进行动态微调。我们的想法是利用蛋白酶作为外源输入和蛋白质降解之间的界面层,通过分离生物PROTACs上的两个结合域来调节目标蛋白质对E3连接酶的招募。通过将外部输入与主要蛋白酶层分离,只需对设计进行最小限度的修改,就能随时调整新的条件降解表型。我们利用两个高效的 "bioPROTAC "系统展示了这种方法的适应性:AdPROM和基于IpaH9.8的Ubiquibodies。利用 TEV 蛋白酶作为转换器,我们可以将小分子和光遗传输入连接起来,实现有条件的靶向蛋白质降解。我们的研究结果凸显了带有蛋白酶响应连接体的生物PROTACs作为条件性靶向蛋白质降解的多功能工具的潜力。
{"title":"Protease-Responsive Toolkit for Conditional Targeted Protein Degradation.","authors":"Hopen Yang, Wilfred Chen","doi":"10.1021/acssynbio.4c00014","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00014","url":null,"abstract":"<p><p>BioPROTACs are heterobifunctional proteins designed for targeted protein degradation. While they offer a potential therapeutic avenue for modulating disease-related proteins, the current strategies are static in nature and lack the ability to modulate protein degradation dynamically. Here, we introduce a synthetic framework for dynamic fine-tuning of target protein levels using protease control switches. The idea is to utilize proteases as an interfacing layer between exogenous inputs and protein degradation by modulating the recruitment of target proteins to E3 ligase by separating the two binding domains on bioPROTACs. By decoupling the external inputs from the primary protease layer, new conditional degradation phenotypes can be readily adapted with minimal modifications to the design. We demonstrate the adaptability of this approach using two highly efficient \"bioPROTAC\" systems: AdPROM and IpaH9.8-based Ubiquibodies. Using the TEV protease as the transducer, we can interface small-molecule and optogenetic inputs for conditional targeted protein degradation. Our findings highlight the potential of bioPROTACs with protease-responsive linkers as a versatile tool for conditional targeted protein degradation.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141416530","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}
Pub Date : 2024-06-17DOI: 10.1021/acssynbio.4c00042
Joshua Cole, Rebecca Schulman
Synthetic biology is revolutionizing our approaches to biocomputing, diagnostics, and environmental monitoring through the use of designed genetic circuits that perform a function within a single cell. More complex functions can be performed by multiple cells that coordinate as they perform different subtasks. Cell-cell communication using molecular signals is particularly suited for aiding in this communication, but the number of molecules that can be used in different communication channels is limited. Here we investigate how proteases can limit the broadcast range of communicating cells. We find that adding barrierpepsin to Saccharomyces cerevisiae cells in two-dimensional multicellular networks that use α-factor signaling prevents cells beyond a specific radius from responding to α-factor signals. Such limiting of the broadcast range of cells could allow multiple cells to use the same signaling molecules to direct different communication processes and functions, provided that they are far enough from one another. These results suggest a means by which complex synthetic cellular networks using only a few signals for communication could be created by structuring a community of cells to create distinct broadcast environments.
{"title":"Limiting the Broadcast Range of a Secreting Cell during Intercellular Signaling Using Protease-Mediated Degradation.","authors":"Joshua Cole, Rebecca Schulman","doi":"10.1021/acssynbio.4c00042","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00042","url":null,"abstract":"<p><p>Synthetic biology is revolutionizing our approaches to biocomputing, diagnostics, and environmental monitoring through the use of designed genetic circuits that perform a function within a single cell. More complex functions can be performed by multiple cells that coordinate as they perform different subtasks. Cell-cell communication using molecular signals is particularly suited for aiding in this communication, but the number of molecules that can be used in different communication channels is limited. Here we investigate how proteases can limit the broadcast range of communicating cells. We find that adding barrierpepsin to <i>Saccharomyces cerevisiae</i> cells in two-dimensional multicellular networks that use α-factor signaling prevents cells beyond a specific radius from responding to α-factor signals. Such limiting of the broadcast range of cells could allow multiple cells to use the same signaling molecules to direct different communication processes and functions, provided that they are far enough from one another. These results suggest a means by which complex synthetic cellular networks using only a few signals for communication could be created by structuring a community of cells to create distinct broadcast environments.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141416529","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}
Pub Date : 2024-06-17DOI: 10.1021/acssynbio.3c00726
Samuel W Schaffter, Eli Kengmana, Joshua Fern, Shane R Byrne, Rebecca Schulman
Bacteriophage RNA polymerases, in particular T7 RNA polymerase (RNAP), are well-characterized and popular enzymes for many RNA applications in biotechnology both in vitro and in cellular settings. These monomeric polymerases are relatively inexpensive and have high transcription rates and processivity to quickly produce large quantities of RNA. T7 RNAP also has high promoter-specificity on double-stranded DNA (dsDNA) such that it only initiates transcription downstream of its 17-base promoter site on dsDNA templates. However, there are many promoter-independent T7 RNAP transcription reactions involving transcription initiation in regions of single-stranded DNA (ssDNA) that have been reported and characterized. These promoter-independent transcription reactions are important to consider when using T7 RNAP transcriptional systems for DNA nanotechnology and DNA computing applications, in which ssDNA domains often stabilize, organize, and functionalize DNA nanostructures and facilitate strand displacement reactions. Here we review the existing literature on promoter-independent transcription by bacteriophage RNA polymerases with a specific focus on T7 RNAP, and provide examples of how promoter-independent reactions can disrupt the functionality of DNA strand displacement circuit components and alter the stability and functionality of DNA-based materials. We then highlight design strategies for DNA nanotechnology applications that can mitigate the effects of promoter-independent T7 RNAP transcription. The design strategies we present should have an immediate impact by increasing the rate of success of using T7 RNAP for applications in DNA nanotechnology and DNA computing.
噬菌体 RNA 聚合酶,特别是 T7 RNA 聚合酶 (RNAP),是生物技术中许多 RNA 体外和细胞环境应用中的特性良好且常用的酶。这些单体聚合酶价格相对低廉,具有较高的转录速率和处理能力,可快速产生大量 RNA。T7 RNAP 在双链 DNA(dsDNA)上也具有高度的启动子特异性,因此它只能在其 17 个碱基的启动子位点下游的 dsDNA 模板上启动转录。不过,也有许多与启动子无关的 T7 RNAP 转录反应,涉及在单链 DNA(ssDNA)区域启动转录,这些反应已被报道和鉴定。在将 T7 RNAP 转录系统用于 DNA 纳米技术和 DNA 计算应用时,这些不依赖启动子的转录反应是必须考虑的因素,因为在这些应用中,ssDNA 域通常会稳定、组织和功能化 DNA 纳米结构,并促进链置换反应。在此,我们回顾了有关噬菌体 RNA 聚合酶启动子无关转录的现有文献,特别关注 T7 RNAP,并举例说明启动子无关反应如何破坏 DNA 链置换电路元件的功能,以及如何改变 DNA 基材料的稳定性和功能性。然后,我们重点介绍了可减轻启动子无关 T7 RNAP 转录影响的 DNA 纳米技术应用设计策略。我们介绍的设计策略将提高 T7 RNAP 在 DNA 纳米技术和 DNA 计算应用中的成功率,从而产生立竿见影的效果。
{"title":"Strategies to Reduce Promoter-Independent Transcription of DNA Nanostructures and Strand Displacement Complexes.","authors":"Samuel W Schaffter, Eli Kengmana, Joshua Fern, Shane R Byrne, Rebecca Schulman","doi":"10.1021/acssynbio.3c00726","DOIUrl":"https://doi.org/10.1021/acssynbio.3c00726","url":null,"abstract":"<p><p>Bacteriophage RNA polymerases, in particular T7 RNA polymerase (RNAP), are well-characterized and popular enzymes for many RNA applications in biotechnology both <i>in vitro</i> and in cellular settings. These monomeric polymerases are relatively inexpensive and have high transcription rates and processivity to quickly produce large quantities of RNA. T7 RNAP also has high promoter-specificity on double-stranded DNA (dsDNA) such that it only initiates transcription downstream of its 17-base promoter site on dsDNA templates. However, there are many promoter-independent T7 RNAP transcription reactions involving transcription initiation in regions of single-stranded DNA (ssDNA) that have been reported and characterized. These promoter-independent transcription reactions are important to consider when using T7 RNAP transcriptional systems for DNA nanotechnology and DNA computing applications, in which ssDNA domains often stabilize, organize, and functionalize DNA nanostructures and facilitate strand displacement reactions. Here we review the existing literature on promoter-independent transcription by bacteriophage RNA polymerases with a specific focus on T7 RNAP, and provide examples of how promoter-independent reactions can disrupt the functionality of DNA strand displacement circuit components and alter the stability and functionality of DNA-based materials. We then highlight design strategies for DNA nanotechnology applications that can mitigate the effects of promoter-independent T7 RNAP transcription. The design strategies we present should have an immediate impact by increasing the rate of success of using T7 RNAP for applications in DNA nanotechnology and DNA computing.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141416531","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}
Synthetic viral nanostructures are useful as materials for analyzing the biological behavior of natural viruses and as vaccine materials. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus embedding a spike (S) protein involved in host cell infection. Although nanomaterials modified with an S protein without an envelope membrane have been developed, they are considered unsuitable for stability and functionality. We previously constructed an enveloped viral replica complexed with a cationic lipid bilayer and an anionic artificial viral capsid self-assembled from β-annulus peptides. In this study, we report the first example of an enveloped viral replica equipped with an S protein derived from SARS-CoV-2. Interestingly, even the S protein equipped on the enveloped viral replica bound strongly to the free angiotensin-converting enzyme 2 (ACE2) receptor as well as ACE2 localized on the cell membrane.
合成病毒纳米结构是分析天然病毒生物行为的有用材料,也可用作疫苗材料。严重急性呼吸系统综合征冠状病毒 2(SARS-CoV-2)是一种包膜病毒,内含参与宿主细胞感染的尖峰(S)蛋白。虽然已经开发出了用无包膜的 S 蛋白修饰的纳米材料,但它们被认为不适合稳定性和功能性。我们之前构建了一种与阳离子脂质双分子层复合的包膜病毒复制品,以及一种由 β-ulus肽自组装的阴离子人工病毒壳。在这项研究中,我们首次报道了一种装有源自 SARS-CoV-2 的 S 蛋白的包膜病毒复制品。有趣的是,即使是包膜病毒复制品上的 S 蛋白也能与游离的血管紧张素转换酶 2(ACE2)受体以及细胞膜上的 ACE2 紧密结合。
{"title":"Enveloped Viral Replica Equipped with Spike Protein Derived from SARS-CoV-2.","authors":"Hiroto Furukawa, Sosuke Nakamura, Ryosuke Mizuta, Kentarou Sakamoto, Hiroshi Inaba, Shin-Ichi Sawada, Yoshihiro Sasaki, Kazunari Akiyoshi, Kazunori Matsuura","doi":"10.1021/acssynbio.4c00165","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00165","url":null,"abstract":"<p><p>Synthetic viral nanostructures are useful as materials for analyzing the biological behavior of natural viruses and as vaccine materials. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus embedding a spike (S) protein involved in host cell infection. Although nanomaterials modified with an S protein without an envelope membrane have been developed, they are considered unsuitable for stability and functionality. We previously constructed an enveloped viral replica complexed with a cationic lipid bilayer and an anionic artificial viral capsid self-assembled from <i>β</i>-annulus peptides. In this study, we report the first example of an enveloped viral replica equipped with an S protein derived from SARS-CoV-2. Interestingly, even the S protein equipped on the enveloped viral replica bound strongly to the free angiotensin-converting enzyme 2 (ACE2) receptor as well as ACE2 localized on the cell membrane.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141416578","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}
Pub Date : 2024-06-14DOI: 10.1021/acssynbio.3c00736
Chester Pham, Peter J Stogios, Alexei Savchenko, Radhakrishnan Mahadevan
Transcription factor (TF)-based biosensors are useful synthetic biology tools for applications in a variety of areas of biotechnology. A major challenge of biosensor circuits is the limited repertoire of identified and well-characterized TFs for applications of interest, in addition to the challenge of optimizing selected biosensors. In this work, we implement the IclR family repressor TF TtgV from Pseudomonas putida DOT-T1E as an indole-derivative biosensor in Escherichia coli. We optimize the genetic circuit utilizing different components, providing insights into biosensor design and expanding on previous studies investigating this TF. We discover novel physiologically relevant ligands of TtgV, such as skatole. The broad specificity of TtgV makes it a useful target for directed evolution and protein engineering toward desired specificity. TtgV, as an indole-derivative biosensor, is a promising genetic component for the detection of compounds with biological activities relevant to health and the gut microbiome.
{"title":"Design and Characterization of a Generalist Biosensor for Indole Derivatives.","authors":"Chester Pham, Peter J Stogios, Alexei Savchenko, Radhakrishnan Mahadevan","doi":"10.1021/acssynbio.3c00736","DOIUrl":"https://doi.org/10.1021/acssynbio.3c00736","url":null,"abstract":"<p><p>Transcription factor (TF)-based biosensors are useful synthetic biology tools for applications in a variety of areas of biotechnology. A major challenge of biosensor circuits is the limited repertoire of identified and well-characterized TFs for applications of interest, in addition to the challenge of optimizing selected biosensors. In this work, we implement the IclR family repressor TF TtgV from <i>Pseudomonas putida</i> DOT-T1E as an indole-derivative biosensor in <i>Escherichia coli</i>. We optimize the genetic circuit utilizing different components, providing insights into biosensor design and expanding on previous studies investigating this TF. We discover novel physiologically relevant ligands of TtgV, such as skatole. The broad specificity of TtgV makes it a useful target for directed evolution and protein engineering toward desired specificity. TtgV, as an indole-derivative biosensor, is a promising genetic component for the detection of compounds with biological activities relevant to health and the gut microbiome.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141320087","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}
The budding yeast Kluyveromyces lactis has emerged as a promising microbial chassis in industrial biotechnology. However, a lack of efficient molecular genetic manipulation tools and strategies has hindered the development of K. lactis as a biomanufacturing platform. In this study, we developed and applied a CRISPR/Cas9-based genome editing method to K. lactis. Single-gene editing efficiency was increased to 80% by disrupting the nonhomologous end-joining-related gene KU80 and performing a series of process optimizations. Subsequently, the CRISPR/Cas9 system was explored based on different sgRNA delivery modes for simultaneous multigene editing. With the aid of the color indicator, the editing efficiencies of two and three genes reached 73.3 and 36%, respectively, in the KlΔKU80 strain. Furthermore, the CRISPR/Cas9 system was used for multisite integration to enhance lactase production and combinatorial knockout of TMED10 and HSP90 to characterize the extracellular secretion of lactase in K. lactis. Generally, genome editing is a powerful tool for constructing K. lactis cell factories for protein and chemical production.
{"title":"CRISPR/Cas9-Based Genome Editing for Protein Expression and Secretion in <i>Kluyveromyces lactis</i>.","authors":"Lingtong Liao, Xiuru Shen, Zhiyu Shen, Guocheng Du, Jianghua Li, Guoqiang Zhang","doi":"10.1021/acssynbio.4c00157","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00157","url":null,"abstract":"<p><p>The budding yeast <i>Kluyveromyces lactis</i> has emerged as a promising microbial chassis in industrial biotechnology. However, a lack of efficient molecular genetic manipulation tools and strategies has hindered the development of <i>K. lactis</i> as a biomanufacturing platform. In this study, we developed and applied a CRISPR/Cas9-based genome editing method to <i>K. lactis</i>. Single-gene editing efficiency was increased to 80% by disrupting the nonhomologous end-joining-related gene <i>KU80</i> and performing a series of process optimizations. Subsequently, the CRISPR/Cas9 system was explored based on different sgRNA delivery modes for simultaneous multigene editing. With the aid of the color indicator, the editing efficiencies of two and three genes reached 73.3 and 36%, respectively, in the <i>Kl</i>Δ<i>KU80</i> strain. Furthermore, the CRISPR/Cas9 system was used for multisite integration to enhance lactase production and combinatorial knockout of <i>TMED10</i> and <i>HSP90</i> to characterize the extracellular secretion of lactase in <i>K. lactis</i>. Generally, genome editing is a powerful tool for constructing <i>K. lactis</i> cell factories for protein and chemical production.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141315999","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}