首页 > 最新文献

ACS Synthetic Biology最新文献

英文 中文
toward Practical Applications of Engineered Living Materials with Advanced Fabrication Techniques. 利用先进制造技术实现工程活体材料的实际应用。
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-07-13 DOI: 10.1021/acssynbio.4c00259
Chenjing Lu, Yaying Huang, Jian Cui, Junhua Wu, Chunping Jiang, Xiaosong Gu, Yi Cao, Sheng Yin

Engineered Living Materials (ELMs) are materials composed of or incorporating living cells as essential functional units. These materials can be created using bottom-up approaches, where engineered cells spontaneously form well-defined aggregates. Alternatively, top-down methods employ advanced materials science techniques to integrate cells with various kinds of materials, creating hybrids where cells and materials are intricately combined. ELMs blend synthetic biology with materials science, allowing for dynamic responses to environmental stimuli such as stress, pH, humidity, temperature, and light. These materials exhibit unique "living" properties, including self-healing, self-replication, and environmental adaptability, making them highly suitable for a wide range of applications in medicine, environmental conservation, and manufacturing. Their inherent biocompatibility and ability to undergo genetic modifications allow for customized functionalities and prolonged sustainability. This review highlights the transformative impact of ELMs over recent decades, particularly in healthcare and environmental protection. We discuss current preparation methods, including the use of endogenous and exogenous scaffolds, living assembly, 3D bioprinting, and electrospinning. Emphasis is placed on ongoing research and technological advancements necessary to enhance the safety, functionality, and practical applicability of ELMs in real-world contexts.

工程活体材料(ELM)是由活细胞组成或包含活细胞作为基本功能单元的材料。这些材料可以采用自下而上的方法制造,即工程细胞自发形成定义明确的聚集体。另外,自上而下的方法采用先进的材料科学技术,将细胞与各种材料整合在一起,创造出细胞与材料复杂结合的混合体。ELM 融合了合成生物学和材料科学,可对压力、pH 值、湿度、温度和光等环境刺激做出动态响应。这些材料表现出独特的 "活 "特性,包括自我修复、自我复制和环境适应能力,因此非常适合在医学、环境保护和制造领域广泛应用。这些材料固有的生物兼容性和基因修饰能力可实现定制功能和长期可持续性。本综述强调了 ELM 近几十年来的变革性影响,尤其是在医疗保健和环境保护方面。我们讨论了当前的制备方法,包括使用内源和外源支架、活体组装、三维生物打印和电纺丝。重点放在正在进行的研究和必要的技术进步上,以提高 ELMs 在现实世界中的安全性、功能性和实际应用性。
{"title":"toward Practical Applications of Engineered Living Materials with Advanced Fabrication Techniques.","authors":"Chenjing Lu, Yaying Huang, Jian Cui, Junhua Wu, Chunping Jiang, Xiaosong Gu, Yi Cao, Sheng Yin","doi":"10.1021/acssynbio.4c00259","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00259","url":null,"abstract":"<p><p>Engineered Living Materials (ELMs) are materials composed of or incorporating living cells as essential functional units. These materials can be created using bottom-up approaches, where engineered cells spontaneously form well-defined aggregates. Alternatively, top-down methods employ advanced materials science techniques to integrate cells with various kinds of materials, creating hybrids where cells and materials are intricately combined. ELMs blend synthetic biology with materials science, allowing for dynamic responses to environmental stimuli such as stress, pH, humidity, temperature, and light. These materials exhibit unique \"living\" properties, including self-healing, self-replication, and environmental adaptability, making them highly suitable for a wide range of applications in medicine, environmental conservation, and manufacturing. Their inherent biocompatibility and ability to undergo genetic modifications allow for customized functionalities and prolonged sustainability. This review highlights the transformative impact of ELMs over recent decades, particularly in healthcare and environmental protection. We discuss current preparation methods, including the use of endogenous and exogenous scaffolds, living assembly, 3D bioprinting, and electrospinning. Emphasis is placed on ongoing research and technological advancements necessary to enhance the safety, functionality, and practical applicability of ELMs in real-world contexts.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141602889","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
ShuffleAnalyzer: A Comprehensive Tool to Visualize DNA Shuffling. ShuffleAnalyzer:可视化 DNA 洗牌的综合工具
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-07-11 DOI: 10.1021/acssynbio.4c00251
Franz Schweiggert, Gregor Habeck, Patrick Most, Martin Busch, Jörg Schweiggert

DNA shuffling is a powerful technique for generating synthetic DNA via recombination of homologous parental sequences. Resulting chimeras are often incorporated into complex libraries for functionality screenings that identify novel variants with improved characteristics. To survey shuffling efficiency, subsequences of chimeras can be computationally assigned to their corresponding parental counterpart, yielding insight into frequency of recombination events, diversity of shuffling libraries and actual composition of final variants. Whereas tools for parental assignment exist, they do not provide direct visualization of the results, making the analysis time-consuming and cumbersome. Here we present ShuffleAnalyzer, a comprehensive, user-friendly, Python-based analysis tool that directly generates graphical outputs of parental assignments and is freely available under a BSD-3 license (https://github.com/joerg-swg/ShuffleAnalyzer/releases). Besides DNA shuffling, peptide insertions can be simultaneously analyzed and visualized, which makes ShuffleAnalyzer a highly valuable tool for integrated approaches often used in synthetic biology, such as AAV capsid engineering in gene therapy applications.

DNA 洗牌是一种通过同源亲本序列重组生成合成 DNA 的强大技术。由此产生的嵌合体通常被纳入复杂的文库中进行功能筛选,从而发现具有更好特性的新型变体。为了调查洗牌效率,可以通过计算将嵌合体的子序列分配给相应的亲本,从而了解重组事件的频率、洗牌文库的多样性以及最终变体的实际组成。虽然存在亲本分配工具,但这些工具不能提供结果的直接可视化,使得分析耗时且繁琐。在此,我们介绍基于 Python- 的 ShuffleAnalyzer,它是一种全面、用户友好的分析工具,可直接生成亲本分配的图形输出,并在 BSD-3 许可证下免费提供 (https://github.com/joerg-swg/ShuffleAnalyzer/releases)。除了 DNA 洗牌外,还可以同时分析和可视化肽插入,这使得 ShuffleAnalyzer 成为合成生物学中常用的集成方法的一个非常有价值的工具,例如基因治疗应用中的 AAV 胶囊工程。
{"title":"ShuffleAnalyzer: A Comprehensive Tool to Visualize DNA Shuffling.","authors":"Franz Schweiggert, Gregor Habeck, Patrick Most, Martin Busch, Jörg Schweiggert","doi":"10.1021/acssynbio.4c00251","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00251","url":null,"abstract":"<p><p>DNA shuffling is a powerful technique for generating synthetic DNA via recombination of homologous parental sequences. Resulting chimeras are often incorporated into complex libraries for functionality screenings that identify novel variants with improved characteristics. To survey shuffling efficiency, subsequences of chimeras can be computationally assigned to their corresponding parental counterpart, yielding insight into frequency of recombination events, diversity of shuffling libraries and actual composition of final variants. Whereas tools for parental assignment exist, they do not provide direct visualization of the results, making the analysis time-consuming and cumbersome. Here we present ShuffleAnalyzer, a comprehensive, user-friendly, Python-based analysis tool that directly generates graphical outputs of parental assignments and is freely available under a BSD-3 license (https://github.com/joerg-swg/ShuffleAnalyzer/releases). Besides DNA shuffling, peptide insertions can be simultaneously analyzed and visualized, which makes ShuffleAnalyzer a highly valuable tool for integrated approaches often used in synthetic biology, such as AAV capsid engineering in gene therapy applications.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141588903","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
Degron-Based bioPROTACs for Controlling Signaling in CAR T Cells. 用于控制 CAR T 细胞信号的 Degron-Based bioPROTACs。
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-07-11 DOI: 10.1021/acssynbio.4c00109
Matthew S Kim, Hersh K Bhargava, Gavin E Shavey, Wendell A Lim, Hana El-Samad, Andrew H Ng

Chimeric antigen receptor (CAR) T cells have made a tremendous impact in the clinic, but potent signaling through the CAR can be detrimental to treatment safety and efficacy. The use of protein degradation to control CAR signaling can address these issues in preclinical models. Existing strategies for regulating CAR stability rely on small molecules to induce systemic degradation. In contrast to small molecule regulation, genetic circuits offer a more precise method to control CAR signaling in an autonomous cell-by-cell fashion. Here, we describe a programmable protein degradation tool that adopts the framework of bioPROTACs, heterobifunctional proteins that are composed of a target recognition domain fused to a domain that recruits the endogenous ubiquitin proteasome system. We develop novel bioPROTACs that utilize a compact four-residue degron and demonstrate degradation of cytosolic and membrane protein targets using either a nanobody or synthetic leucine zipper as a protein binder. Our bioPROTACs exhibit potent degradation of CARs and can inhibit CAR signaling in primary human T cells. We demonstrate the utility of our bioPROTACs by constructing a genetic circuit to degrade the tyrosine kinase ZAP70 in response to recognition of a specific membrane-bound antigen. This circuit can disrupt CAR T cell signaling only in the presence of a specific cell population. These results suggest that bioPROTACs are powerful tools for expanding the CAR T cell engineering toolbox.

嵌合抗原受体(CAR)T细胞在临床上产生了巨大影响,但通过CAR发出的强信号可能不利于治疗的安全性和有效性。在临床前模型中,利用蛋白质降解来控制CAR信号传导可以解决这些问题。现有的 CAR 稳定性调控策略依赖于小分子诱导系统性降解。与小分子调控相比,基因回路提供了一种更精确的方法,可以逐个细胞自主控制 CAR 信号传导。在这里,我们描述了一种可编程的蛋白质降解工具,它采用了生物PROTACs的框架,生物PROTACs是由一个目标识别结构域与一个能招募内源性泛素蛋白酶体系统的结构域融合而成的异功能蛋白质。我们开发的新型 bioPROTACs 采用了紧凑的四残基降解子,并利用纳米抗体或合成亮氨酸拉链作为蛋白粘合剂,展示了对细胞膜和膜蛋白靶标的降解。我们的 bioPROTACs 能有效降解 CAR,并能抑制原代人类 T 细胞中的 CAR 信号转导。我们通过构建一个基因回路来降解酪氨酸激酶 ZAP70,以应对特定膜结合抗原的识别,从而证明了我们的 bioPROTACs 的实用性。只有在特定细胞群存在的情况下,该电路才能破坏 CAR T 细胞信号传导。这些结果表明,生物PROTAC是扩展CAR T细胞工程工具箱的有力工具。
{"title":"Degron-Based bioPROTACs for Controlling Signaling in CAR T Cells.","authors":"Matthew S Kim, Hersh K Bhargava, Gavin E Shavey, Wendell A Lim, Hana El-Samad, Andrew H Ng","doi":"10.1021/acssynbio.4c00109","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00109","url":null,"abstract":"<p><p>Chimeric antigen receptor (CAR) T cells have made a tremendous impact in the clinic, but potent signaling through the CAR can be detrimental to treatment safety and efficacy. The use of protein degradation to control CAR signaling can address these issues in preclinical models. Existing strategies for regulating CAR stability rely on small molecules to induce systemic degradation. In contrast to small molecule regulation, genetic circuits offer a more precise method to control CAR signaling in an autonomous cell-by-cell fashion. Here, we describe a programmable protein degradation tool that adopts the framework of bioPROTACs, heterobifunctional proteins that are composed of a target recognition domain fused to a domain that recruits the endogenous ubiquitin proteasome system. We develop novel bioPROTACs that utilize a compact four-residue degron and demonstrate degradation of cytosolic and membrane protein targets using either a nanobody or synthetic leucine zipper as a protein binder. Our bioPROTACs exhibit potent degradation of CARs and can inhibit CAR signaling in primary human T cells. We demonstrate the utility of our bioPROTACs by constructing a genetic circuit to degrade the tyrosine kinase ZAP70 in response to recognition of a specific membrane-bound antigen. This circuit can disrupt CAR T cell signaling only in the presence of a specific cell population. These results suggest that bioPROTACs are powerful tools for expanding the CAR T cell engineering toolbox.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141588902","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
Bioinformatic Prediction and High Throughput In Vivo Screening to Identify Cis-Regulatory Elements for the Development of Algal Synthetic Promoters. 通过生物信息学预测和高通量体内筛选确定用于开发藻类合成启动子的顺式调控元件。
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-07-10 DOI: 10.1021/acssynbio.4c00199
Y Torres-Tiji, H Sethuram, A Gupta, J McCauley, J-V Dutra-Molino, R Pathania, L Saxton, K Kang, N J Hillson, S P Mayfield

Algae biotechnology holds immense promise for revolutionizing the bioeconomy through the sustainable and scalable production of various bioproducts. However, their development has been hindered by the lack of advanced genetic tools. This study introduces a synthetic biology approach to develop such tools, focusing on the construction and testing of synthetic promoters. By analyzing conserved DNA motifs within the promoter regions of highly expressed genes across six different algal species, we identified cis-regulatory elements (CREs) associated with high transcriptional activity. Combining the algorithms POWRS, STREME, and PhyloGibbs, we predicted 1511 CREs and inserted them into a minimal synthetic promoter sequence in 1, 2, or 3 copies, resulting in 4533 distinct synthetic promoters. These promoters were evaluated in vivo for their capacity to drive the expression of a transgene in a high-throughput manner through next-generation sequencing post antibiotic selection and fluorescence-activated cell sorting. To validate our approach, we sequenced hundreds of transgenic lines showing high levels of GFP expression. Further, we individually tested 14 identified promoters, revealing substantial increases in GFP expression─up to nine times higher than the baseline synthetic promoter, with five matching or even surpassing the performance of the native AR1 promoter. As a result of this study, we identified a catalog of CREs that can now be used to build superior synthetic algal promoters. More importantly, here we present a validated pipeline to generate building blocks for innovative synthetic genetic tools applicable to any algal species with a sequenced genome and transcriptome data set.

藻类生物技术通过可持续、可扩展地生产各种生物产品,为彻底改变生物经济带来了巨大希望。然而,由于缺乏先进的遗传工具,藻类生物技术的发展一直受到阻碍。本研究介绍了一种合成生物学方法来开发此类工具,重点是合成启动子的构建和测试。通过分析六种不同藻类高表达基因启动子区域内的保守 DNA 主题,我们确定了与高转录活性相关的顺式调控元件(CRE)。结合 POWRS、STREME 和 PhyloGibbs 算法,我们预测了 1511 个 CRE,并将它们以 1、2 或 3 个拷贝的形式插入到最小合成启动子序列中,从而得到了 4533 个不同的合成启动子。通过抗生素选择后的下一代测序和荧光激活细胞分选,我们以高通量的方式评估了这些启动子在体内驱动转基因表达的能力。为了验证我们的方法,我们对数百个显示高水平 GFP 表达的转基因品系进行了测序。此外,我们还对 14 个确定的启动子进行了单独测试,结果显示 GFP 表达量大幅增加--比基线合成启动子高出九倍,其中五个启动子的性能与原生 AR1 启动子相当,甚至超过了后者。通过这项研究,我们确定了一个 CRE 目录,现在可以用它来构建优质的合成藻类启动子。更重要的是,我们在这里提出了一个经过验证的管道,用于生成创新合成基因工具的构件,适用于任何具有基因组和转录组测序数据集的藻类物种。
{"title":"Bioinformatic Prediction and High Throughput In Vivo Screening to Identify Cis-Regulatory Elements for the Development of Algal Synthetic Promoters.","authors":"Y Torres-Tiji, H Sethuram, A Gupta, J McCauley, J-V Dutra-Molino, R Pathania, L Saxton, K Kang, N J Hillson, S P Mayfield","doi":"10.1021/acssynbio.4c00199","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00199","url":null,"abstract":"<p><p>Algae biotechnology holds immense promise for revolutionizing the bioeconomy through the sustainable and scalable production of various bioproducts. However, their development has been hindered by the lack of advanced genetic tools. This study introduces a synthetic biology approach to develop such tools, focusing on the construction and testing of synthetic promoters. By analyzing conserved DNA motifs within the promoter regions of highly expressed genes across six different algal species, we identified cis-regulatory elements (CREs) associated with high transcriptional activity. Combining the algorithms POWRS, STREME, and PhyloGibbs, we predicted 1511 CREs and inserted them into a minimal synthetic promoter sequence in 1, 2, or 3 copies, resulting in 4533 distinct synthetic promoters. These promoters were evaluated in vivo for their capacity to drive the expression of a transgene in a high-throughput manner through next-generation sequencing post antibiotic selection and fluorescence-activated cell sorting. To validate our approach, we sequenced hundreds of transgenic lines showing high levels of GFP expression. Further, we individually tested 14 identified promoters, revealing substantial increases in GFP expression─up to nine times higher than the baseline synthetic promoter, with five matching or even surpassing the performance of the native AR1 promoter. As a result of this study, we identified a catalog of CREs that can now be used to build superior synthetic algal promoters. More importantly, here we present a validated pipeline to generate building blocks for innovative synthetic genetic tools applicable to any algal species with a sequenced genome and transcriptome data set.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578172","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
Cell-Free Translation Quantification via a Fluorescent Minihelix. 通过荧光小螺旋进行细胞自由翻译定量。
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-07-09 DOI: 10.1021/acssynbio.4c00266
Jessica A Willi, Ashty S Karim, Michael C Jewett

Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye. The fluorescent reporter helix is short enough to be encoded on a primer pair to tag any protein of interest via PCR. Both the tagged protein and the standalone reporter can be detected quantitatively in real time or at the end of cell-free expression reactions with standard 96/384-well plate readers, an RT-qPCR system, or gel electrophoresis without the need for staining. The fluorescent signal is stable and correlates linearly with the protein concentration, enabling product quantification. We modified the reporter to study cell-free expression dynamics and engineered ribosome activity. We anticipate that the fluorescent minihelix reporter will facilitate efforts in engineering in vitro transcription and translation systems.

无细胞基因表达系统应用广泛,包括制药、诊断和教育工具包。在这些系统中对非荧光蛋白进行精确定量仍然是一项挑战。为了应对这一挑战,我们报告了一种优化的四半胱氨酸小螺旋体的改造和使用情况,这种小螺旋体既可以作为融合蛋白,也可以作为带有 FlAsH 染料的独立报告物。荧光报告螺旋足够短,可以编码在引物对上,通过 PCR 标记任何感兴趣的蛋白质。标记的蛋白质和独立的报告基因都可以用标准的 96/384 孔平板阅读器、RT-qPCR 系统或凝胶电泳实时或在无细胞表达反应结束时进行定量检测,而无需染色。荧光信号稳定,与蛋白质浓度呈线性相关,可实现产品定量。我们对该报告器进行了改良,以研究无细胞表达动态和工程核糖体活性。我们预计荧光小螺旋报告器将有助于体外转录和翻译系统的工程化。
{"title":"Cell-Free Translation Quantification via a Fluorescent Minihelix.","authors":"Jessica A Willi, Ashty S Karim, Michael C Jewett","doi":"10.1021/acssynbio.4c00266","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00266","url":null,"abstract":"<p><p>Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye. The fluorescent reporter helix is short enough to be encoded on a primer pair to tag any protein of interest via PCR. Both the tagged protein and the standalone reporter can be detected quantitatively in real time or at the end of cell-free expression reactions with standard 96/384-well plate readers, an RT-qPCR system, or gel electrophoresis without the need for staining. The fluorescent signal is stable and correlates linearly with the protein concentration, enabling product quantification. We modified the reporter to study cell-free expression dynamics and engineered ribosome activity. We anticipate that the fluorescent minihelix reporter will facilitate efforts in engineering <i>in vitro</i> transcription and translation systems.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141557366","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
Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening. 利用高通量声学筛选实现声学报告基因的定向进化。
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-07-09 DOI: 10.1021/acssynbio.4c00283
Robert C Hurt, Zhiyang Jin, Mohamed Soufi, Katie K Wong, Daniel P Sawyer, Hao K Shen, Przemysław Dutka, Ramya Deshpande, Ruby Zhang, David R Mittelstein, Mikhail G Shapiro

A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 μm. Within the past decade, reporter genes for US have been introduced and engineered to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs). Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semiautomated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologues of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.

生物成像和合成生物学领域面临的一大挑战是如何以非侵入方式观察不透明样本(如活体动物)内天然细胞和工程细胞的功能。超声波(US)穿透深度达数厘米,空间分辨率约为 100 微米,是解决这一局限性的一项前景广阔的技术。过去十年间,人们引入并设计了超声报告基因,通过在共生细菌和哺乳动物细胞中异源表达,将细胞功能与超声信号联系起来。这些声学报告基因(ARGs)代表了一类新型的基因编码 US 对比剂,其基础是被称为气泡(GVs)的充满空气的蛋白质纳米结构。正如荧光蛋白被发现后,其光学特性通过定向进化得到了改进和多样化一样,我们在此描述了作为声学报告因子的 GVs 的进化过程。为了完成这项任务,我们在细菌培养物中对 ARGs 进行了高通量、半自动声学筛选,并利用它筛选出具有更强非线性 US 散射的变体库。从主要 GV 结构蛋白 GvpA/B 的两个同源物的扫描位点饱和文库开始,经过两轮进化,GV 变体的声学信号分别比母体蛋白强 5 倍和 14 倍。我们预计,这种方法和类似方法将有助于高通量蛋白质工程在声学生物分子的开发中发挥与荧光生物分子相同的作用。
{"title":"Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening.","authors":"Robert C Hurt, Zhiyang Jin, Mohamed Soufi, Katie K Wong, Daniel P Sawyer, Hao K Shen, Przemysław Dutka, Ramya Deshpande, Ruby Zhang, David R Mittelstein, Mikhail G Shapiro","doi":"10.1021/acssynbio.4c00283","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00283","url":null,"abstract":"<p><p>A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 μm. Within the past decade, reporter genes for US have been introduced and engineered to link cellular functions to US signals <i>via</i> heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs). Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semiautomated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologues of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141561924","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
Metabolic Profile of the Genome-Reduced Bacillus subtilis Strain IIG-Bs-27-39: An Attractive Chassis for Recombinant Protein Production. 基因组还原枯草芽孢杆菌菌株 IIG-Bs-27-39 的代谢概况:重组蛋白质生产的诱人底盘
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-07-09 DOI: 10.1021/acssynbio.4c00254
Rocío Aguilar Suárez, Michael Kohlstedt, Ayşegül Öktem, Jolanda Neef, Yuzheng Wu, Kaiya Ikeda, Ken-Ichi Yoshida, Josef Altenbuchner, Christoph Wittmann, Jan Maarten van Dijl

The Gram-positive bacterium Bacillus subtilis is extensively used in the industry for the secretory production of proteins with commercial value. To further improve its performance, this microbe has been the subject of extensive genome engineering efforts, especially the removal of large genomic regions that are dispensable or even counterproductive. Here, we present the genome-reduced B. subtilis strain IIG-Bs-27-39, which was obtained through systematic deletion of mobile genetic elements, as well as genes for extracellular proteases, sporulation, flagella formation, and antibiotic production. Different from previously characterized genome-reduced B. subtilis strains, the IIG-Bs-27-39 strain was still able to grow on minimal media. We used this feature to benchmark strain IIG-Bs-27-39 against its parental strain 168 with respect to heterologous protein production and metabolic parameters during bioreactor cultivation. The IIG-Bs-27-39 strain presented superior secretion of difficult-to-produce staphylococcal antigens, as well as higher specific growth rates and biomass yields. At the metabolic level, changes in byproduct formation and internal amino acid pools were observed, whereas energetic parameters such as the ATP yield, ATP/ADP levels, and adenylate energy charge were comparable between the two strains. Intriguingly, we observed a significant increase in the total cellular NADPH level during all tested conditions and increases in the NAD+ and NADP(H) pools during protein production. This indicates that the IIG-Bs-27-39 strain has more energy available for anabolic processes and protein production, thereby providing a link between strain physiology and production performance. On this basis, we conclude that the genome-reduced strain IIG-Bs-27-39 represents an attractive chassis for future biotechnological applications.

革兰氏阳性细菌枯草芽孢杆菌(Bacillus subtilis)在工业中被广泛用于分泌性生产具有商业价值的蛋白质。为了进一步提高其性能,这种微生物一直是大量基因组工程努力的对象,特别是去除可有可无甚至适得其反的大基因组区域。在这里,我们展示了基因组还原的枯草杆菌菌株 IIG-Bs-27-39,它是通过系统性地删除移动遗传元件以及胞外蛋白酶、孢子、鞭毛形成和抗生素生产基因而获得的。与之前的基因组还原枯草杆菌菌株不同,IIG-Bs-27-39 菌株仍能在最小培养基上生长。我们利用这一特点,将 IIG-Bs-27-39 菌株与其亲本菌株 168 在生物反应器培养过程中的异源蛋白产量和代谢参数进行了比较。IIG-Bs-27-39 菌株能更好地分泌难以生产的葡萄球菌抗原,并具有更高的特定生长率和生物量产量。在代谢层面,我们观察到副产品形成和内部氨基酸池发生了变化,而 ATP 产量、ATP/ADP 水平和腺苷酸能量电荷等能量参数在两株菌株之间具有可比性。有趣的是,在所有测试条件下,我们都观察到细胞 NADPH 总量显著增加,而在蛋白质生产过程中,NAD+ 和 NADP(H) 池也有所增加。这表明,IIG-Bs-27-39 菌株有更多的能量可用于合成代谢过程和蛋白质生产,从而提供了菌株生理机能与生产性能之间的联系。在此基础上,我们得出结论,基因组还原菌株 IIG-Bs-27-39 是未来生物技术应用的一个极具吸引力的底盘。
{"title":"Metabolic Profile of the Genome-Reduced <i>Bacillus subtilis</i> Strain IIG-Bs-27-39: An Attractive Chassis for Recombinant Protein Production.","authors":"Rocío Aguilar Suárez, Michael Kohlstedt, Ayşegül Öktem, Jolanda Neef, Yuzheng Wu, Kaiya Ikeda, Ken-Ichi Yoshida, Josef Altenbuchner, Christoph Wittmann, Jan Maarten van Dijl","doi":"10.1021/acssynbio.4c00254","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00254","url":null,"abstract":"<p><p>The Gram-positive bacterium <i>Bacillus subtilis</i> is extensively used in the industry for the secretory production of proteins with commercial value. To further improve its performance, this microbe has been the subject of extensive genome engineering efforts, especially the removal of large genomic regions that are dispensable or even counterproductive. Here, we present the genome-reduced <i>B. subtilis</i> strain IIG-Bs-27-39, which was obtained through systematic deletion of mobile genetic elements, as well as genes for extracellular proteases, sporulation, flagella formation, and antibiotic production. Different from previously characterized genome-reduced <i>B. subtilis</i> strains, the IIG-Bs-27-39 strain was still able to grow on minimal media. We used this feature to benchmark strain IIG-Bs-27-39 against its parental strain 168 with respect to heterologous protein production and metabolic parameters during bioreactor cultivation. The IIG-Bs-27-39 strain presented superior secretion of difficult-to-produce staphylococcal antigens, as well as higher specific growth rates and biomass yields. At the metabolic level, changes in byproduct formation and internal amino acid pools were observed, whereas energetic parameters such as the ATP yield, ATP/ADP levels, and adenylate energy charge were comparable between the two strains. Intriguingly, we observed a significant increase in the total cellular NADPH level during all tested conditions and increases in the NAD<sup>+</sup> and NADP(H) pools during protein production. This indicates that the IIG-Bs-27-39 strain has more energy available for anabolic processes and protein production, thereby providing a link between strain physiology and production performance. On this basis, we conclude that the genome-reduced strain IIG-Bs-27-39 represents an attractive chassis for future biotechnological applications.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141561925","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
AutoBioTech─A Versatile Biofoundry for Automated Strain Engineering. AutoBioTech--用于自动菌株工程的多功能生物设施。
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-07-08 DOI: 10.1021/acssynbio.4c00298
Tobias Michael Rosch, Julia Tenhaef, Tim Stoltmann, Till Redeker, Dominic Kösters, Niels Hollmann, Karin Krumbach, Wolfgang Wiechert, Michael Bott, Susana Matamouros, Jan Marienhagen, Stephan Noack

The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering of microbes will not be sufficient to meet the ever-increasing demand for novel producer strains. Here we describe the AutoBioTech platform, a fully automated laboratory system with 14 devices to perform operations for strain construction without human interaction. Using modular workflows, this platform enables automated transformations of Escherichia coli with plasmids assembled via modular cloning. A CRISPR/Cas9 toolbox compatible with existing modular cloning frameworks allows automated and flexible genome editing of E. coli. In addition, novel workflows have been established for the fully automated transformation of the Gram-positive model organism Corynebacterium glutamicum by conjugation and electroporation, with the latter proving to be the more robust technique. Overall, the AutoBioTech platform excels at versatility due to the modularity of workflows and seamless transitions between modules. This will accelerate strain engineering of Gram-negative and Gram-positive bacteria.

近年来,从石化生产工艺向可再生替代工艺的过渡已不可避免,这引发了生物工厂的兴起。要满足对新型生产菌株日益增长的需求,人工微生物工程是远远不够的。我们在此介绍 AutoBioTech 平台,这是一个全自动实验室系统,配有 14 个设备,可在无需人工操作的情况下执行菌株构建操作。利用模块化工作流程,该平台可实现大肠杆菌与通过模块化克隆组装的质粒的自动转化。与现有模块化克隆框架兼容的 CRISPR/Cas9 工具箱可对大肠杆菌进行自动、灵活的基因组编辑。此外,还建立了新的工作流程,可通过共轭和电穿孔全自动转化革兰氏阳性模式生物谷氨酸棒杆菌,后者被证明是更稳健的技术。总体而言,AutoBioTech 平台因其工作流程的模块化和模块间的无缝转换而在多功能性方面表现出色。这将加速革兰氏阴性菌和革兰氏阳性菌的菌种工程。
{"title":"AutoBioTech─A Versatile Biofoundry for Automated Strain Engineering.","authors":"Tobias Michael Rosch, Julia Tenhaef, Tim Stoltmann, Till Redeker, Dominic Kösters, Niels Hollmann, Karin Krumbach, Wolfgang Wiechert, Michael Bott, Susana Matamouros, Jan Marienhagen, Stephan Noack","doi":"10.1021/acssynbio.4c00298","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00298","url":null,"abstract":"<p><p>The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering of microbes will not be sufficient to meet the ever-increasing demand for novel producer strains. Here we describe the AutoBioTech platform, a fully automated laboratory system with 14 devices to perform operations for strain construction without human interaction. Using modular workflows, this platform enables automated transformations of <i>Escherichia coli</i> with plasmids assembled via modular cloning. A CRISPR/Cas9 toolbox compatible with existing modular cloning frameworks allows automated and flexible genome editing of <i>E. coli</i>. In addition, novel workflows have been established for the fully automated transformation of the Gram-positive model organism <i>Corynebacterium glutamicum</i> by conjugation and electroporation, with the latter proving to be the more robust technique. Overall, the AutoBioTech platform excels at versatility due to the modularity of workflows and seamless transitions between modules. This will accelerate strain engineering of Gram-negative and Gram-positive bacteria.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141553549","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
Reliable Genomic Integration Sites in Pseudomonas putida Identified by Two-Dimensional Transcriptome Analysis. 通过二维转录组分析确定假单胞菌的可靠基因组整合位点
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-07-05 DOI: 10.1021/acssynbio.3c00747
Sebastian Köbbing, Thorsten Lechtenberg, Benedikt Wynands, Lars M Blank, Nick Wierckx

Genomic integration is commonly used to engineer stable production hosts. However, so far, for many microbial workhorses, only a few integration sites have been characterized, thereby restraining advanced strain engineering that requires multiple insertions. Here, we report on the identification of novel genomic integration sites, so-called landing pads, for Pseudomonas putida KT2440. We identified genomic regions with constant expression patterns under diverse experimental conditions by using RNA-Seq data. Homologous recombination constructs were designed to insert heterologous genes into intergenic sites in these regions, allowing condition-independent gene expression. Ten potential landing pads were characterized using four different msfGFP expression cassettes. An insulated probe sensor was used to study locus-dependent effects on recombinant gene expression, excluding genomic read-through of flanking promoters under changing cultivation conditions. While the reproducibility of expression in the landing pads was very high, the msfGFP signals varied strongly between the different landing pads, confirming a strong influence of the genomic context. To showcase that the identified landing pads are also suitable candidates for heterologous gene expression in other Pseudomonads, four equivalent landing pads were identified and characterized in Pseudomonas taiwanensis VLB120. This study shows that genomic "hot" and "cold" spots exist, causing strong promoter-independent variations in gene expression. This highlights that the genomic context is an additional parameter to consider when designing integrable genomic cassettes for tailored heterologous expression. The set of characterized genomic landing pads presented here further increases the genetic toolbox for deep metabolic engineering in Pseudomonads.

基因组整合通常用于设计稳定的生产宿主。然而,迄今为止,对于许多微生物工作母机来说,只有少数整合位点被鉴定出来,从而限制了需要多次插入的高级菌株工程。在此,我们报告了对假单胞菌 KT2440 的新型基因组整合位点(即所谓的着陆垫)的鉴定。我们利用 RNA-Seq 数据确定了在不同实验条件下具有恒定表达模式的基因组区域。我们设计了同源重组构建物,以将异源基因插入这些区域的基因间位点,从而实现独立于条件的基因表达。使用四种不同的 msfGFP 表达盒对十个潜在的着陆点进行了鉴定。使用绝缘探针传感器研究了基因位点对重组基因表达的影响,排除了在不断变化的培养条件下侧翼启动子的基因组通读。虽然着陆垫表达的重现性非常高,但不同着陆垫之间的 msfGFP 信号差异很大,这证实了基因组环境的强烈影响。为了证明所鉴定的着陆垫也适合在其他假单胞菌中进行异源基因表达,研究人员在台湾假单胞菌 VLB120 中鉴定并表征了四个等效的着陆垫。这项研究表明,基因组中存在 "热点 "和 "冷点",导致基因表达的强烈变化与启动子无关。这突出表明,在设计可整合的基因组盒以实现定制的异源表达时,基因组环境是一个需要考虑的额外参数。本文介绍的这组特征基因组登陆垫进一步增加了假单胞菌深度代谢工程的基因工具箱。
{"title":"Reliable Genomic Integration Sites in <i>Pseudomonas putida</i> Identified by Two-Dimensional Transcriptome Analysis.","authors":"Sebastian Köbbing, Thorsten Lechtenberg, Benedikt Wynands, Lars M Blank, Nick Wierckx","doi":"10.1021/acssynbio.3c00747","DOIUrl":"https://doi.org/10.1021/acssynbio.3c00747","url":null,"abstract":"<p><p>Genomic integration is commonly used to engineer stable production hosts. However, so far, for many microbial workhorses, only a few integration sites have been characterized, thereby restraining advanced strain engineering that requires multiple insertions. Here, we report on the identification of novel genomic integration sites, so-called landing pads, for <i>Pseudomonas putida</i> KT2440. We identified genomic regions with constant expression patterns under diverse experimental conditions by using RNA-Seq data. Homologous recombination constructs were designed to insert heterologous genes into intergenic sites in these regions, allowing condition-independent gene expression. Ten potential landing pads were characterized using four different <i>msfGFP</i> expression cassettes. An insulated probe sensor was used to study locus-dependent effects on recombinant gene expression, excluding genomic read-through of flanking promoters under changing cultivation conditions. While the reproducibility of expression in the landing pads was very high, the msfGFP signals varied strongly between the different landing pads, confirming a strong influence of the genomic context. To showcase that the identified landing pads are also suitable candidates for heterologous gene expression in other Pseudomonads, four equivalent landing pads were identified and characterized in <i>Pseudomonas taiwanensis</i> VLB120. This study shows that genomic \"hot\" and \"cold\" spots exist, causing strong promoter-independent variations in gene expression. This highlights that the genomic context is an additional parameter to consider when designing integrable genomic cassettes for tailored heterologous expression. The set of characterized genomic landing pads presented here further increases the genetic toolbox for deep metabolic engineering in Pseudomonads.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141537141","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
Investigating and Engineering an 1,2-Propanediol-Responsive Transcription Factor-Based Biosensor. 基于转录因子的 1,2-丙二醇反应生物传感器的研究与工程设计
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-07-05 DOI: 10.1021/acssynbio.4c00237
Yuxi Teng, Xinyu Gong, Jianli Zhang, Ziad Obideen, Yajun Yan

Transcription factor (TF)-based biosensors have arisen as powerful tools in the advancement of metabolic engineering. However, with the emergence of numerous bioproduction targets, the variety of applicable TF-based biosensors remains severely limited. In this study, we investigated and engineered an 1,2-propanediol (1,2-PD)-responsive transcription activator, PocR, from Salmonella typhimurium to enrich the current biosensor repertoire. Heterologous characterization of PocR in E. coli revealed a significantly limited operational range and dynamic range, primarily attributed to the leaky binding between PocR and its corresponding promoters in the absence of the 1,2-PD inducer. Promiscuity characterization uncovered the minor responsiveness of PocR toward glycerol and 1,2-butanediol (1,2-BD). Using AlphaFold-predicted structure and protein mutagenesis, we preliminarily explored the underlying mechanism of PocR. Based on the investigated mechanism, we engineered a PcoR-F46R/G105D variant with an altered inducer specificity to glycerol, as well as a PocR-ARE (Q107A/S192R/A203E) variant with nearly a 4-fold higher dynamic range (6.7-fold activation) and a 20-fold wider operational range (0-20 mM 1,2-PD). Finally, we successfully converted PocR to a repressor through promoter engineering. Integrating the activation and repression functions established a versatile 1,2-PD-induced bifunctional regulation system based on PocR-ARE. Our work showcases the exploration and exploitation of an underexplored type of transcriptional activator capable of recruiting RNA polymerase. It also expands the biosensor toolbox by providing a 1,2-PD-responsive bifunctional regulator and glycerol-responsive activator.

基于转录因子(TF)的生物传感器已成为促进代谢工程发展的有力工具。然而,随着众多生物生产目标的出现,适用的基于转录因子的生物传感器的种类仍然非常有限。在本研究中,我们研究并设计了鼠伤寒沙门氏菌中的 1,2-丙二醇(1,2-PD)反应型转录激活因子 PocR,以丰富当前的生物传感器种类。PocR 在大肠杆菌中的异源特性分析表明,其工作范围和动态范围明显有限,这主要是由于在没有 1,2-PD 诱导剂的情况下,PocR 与其相应启动子之间的结合存在漏洞。杂合性表征揭示了 PocR 对甘油和 1,2-丁二醇(1,2-BD)的小反应性。利用 AlphaFold 预测结构和蛋白质诱变,我们初步探索了 PocR 的基本机制。根据所研究的机制,我们设计了一个 PcoR-F46R/G105D 变体,其对甘油的诱导剂特异性发生了改变,同时还设计了一个 PocR-ARE (Q107A/S192R/A203E) 变体,其动态范围提高了近 4 倍(活化 6.7 倍),工作范围扩大了 20 倍(0-20 mM 1,2-PD)。最后,我们通过启动子工程成功地将 PocR 转化为抑制因子。激活和抑制功能的整合建立了一个基于 PocR-ARE 的多功能 1,2-PD-诱导双功能调控系统。我们的工作展示了对一种尚未充分开发的能够招募 RNA 聚合酶的转录激活因子的探索和利用。它还提供了一种 1,2-PD 响应型双功能调节器和甘油响应型激活剂,从而扩展了生物传感器工具箱。
{"title":"Investigating and Engineering an 1,2-Propanediol-Responsive Transcription Factor-Based Biosensor.","authors":"Yuxi Teng, Xinyu Gong, Jianli Zhang, Ziad Obideen, Yajun Yan","doi":"10.1021/acssynbio.4c00237","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00237","url":null,"abstract":"<p><p>Transcription factor (TF)-based biosensors have arisen as powerful tools in the advancement of metabolic engineering. However, with the emergence of numerous bioproduction targets, the variety of applicable TF-based biosensors remains severely limited. In this study, we investigated and engineered an 1,2-propanediol (1,2-PD)-responsive transcription activator, PocR, from <i>Salmonella typhimurium</i> to enrich the current biosensor repertoire. Heterologous characterization of PocR in <i>E. coli</i> revealed a significantly limited operational range and dynamic range, primarily attributed to the leaky binding between PocR and its corresponding promoters in the absence of the 1,2-PD inducer. Promiscuity characterization uncovered the minor responsiveness of PocR toward glycerol and 1,2-butanediol (1,2-BD). Using AlphaFold-predicted structure and protein mutagenesis, we preliminarily explored the underlying mechanism of PocR. Based on the investigated mechanism, we engineered a PcoR-F46R/G105D variant with an altered inducer specificity to glycerol, as well as a PocR-ARE (Q107A/S192R/A203E) variant with nearly a 4-fold higher dynamic range (6.7-fold activation) and a 20-fold wider operational range (0-20 mM 1,2-PD). Finally, we successfully converted PocR to a repressor through promoter engineering. Integrating the activation and repression functions established a versatile 1,2-PD-induced bifunctional regulation system based on PocR-ARE. Our work showcases the exploration and exploitation of an underexplored type of transcriptional activator capable of recruiting RNA polymerase. It also expands the biosensor toolbox by providing a 1,2-PD-responsive bifunctional regulator and glycerol-responsive activator.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141537140","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
期刊
ACS Synthetic Biology
全部 Acc. Chem. Res. ACS Applied Bio Materials ACS Appl. Electron. Mater. ACS Appl. Energy Mater. ACS Appl. Mater. Interfaces ACS Appl. Nano Mater. ACS Appl. Polym. Mater. ACS BIOMATER-SCI ENG ACS Catal. ACS Cent. Sci. ACS Chem. Biol. ACS Chemical Health & Safety ACS Chem. Neurosci. ACS Comb. Sci. ACS Earth Space Chem. ACS Energy Lett. ACS Infect. Dis. ACS Macro Lett. ACS Mater. Lett. ACS Med. Chem. Lett. ACS Nano ACS Omega ACS Photonics ACS Sens. ACS Sustainable Chem. Eng. ACS Synth. Biol. Anal. Chem. BIOCHEMISTRY-US Bioconjugate Chem. BIOMACROMOLECULES Chem. Res. Toxicol. Chem. Rev. Chem. Mater. CRYST GROWTH DES ENERG FUEL Environ. Sci. Technol. Environ. Sci. Technol. Lett. Eur. J. Inorg. Chem. IND ENG CHEM RES Inorg. Chem. J. Agric. Food. Chem. J. Chem. Eng. Data J. Chem. Educ. J. Chem. Inf. Model. J. Chem. Theory Comput. J. Med. Chem. J. Nat. Prod. J PROTEOME RES J. Am. Chem. Soc. LANGMUIR MACROMOLECULES Mol. Pharmaceutics Nano Lett. Org. Lett. ORG PROCESS RES DEV ORGANOMETALLICS J. Org. Chem. J. Phys. Chem. J. Phys. Chem. A J. Phys. Chem. B J. Phys. Chem. C J. Phys. Chem. Lett. Analyst Anal. Methods Biomater. Sci. Catal. Sci. Technol. Chem. Commun. Chem. Soc. Rev. CHEM EDUC RES PRACT CRYSTENGCOMM Dalton Trans. Energy Environ. Sci. ENVIRON SCI-NANO ENVIRON SCI-PROC IMP ENVIRON SCI-WAT RES Faraday Discuss. Food Funct. Green Chem. Inorg. Chem. Front. Integr. Biol. J. Anal. At. Spectrom. J. Mater. Chem. A J. Mater. Chem. B J. Mater. Chem. C Lab Chip Mater. Chem. Front. Mater. Horiz. MEDCHEMCOMM Metallomics Mol. Biosyst. Mol. Syst. Des. Eng. Nanoscale Nanoscale Horiz. Nat. Prod. Rep. New J. Chem. Org. Biomol. Chem. Org. Chem. Front. PHOTOCH PHOTOBIO SCI PCCP Polym. Chem.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1