Pub Date : 2024-05-14DOI: 10.1021/acssynbio.3c00486
Jason Philippou, Guillermo Yáñez Feliú and Timothy J. Rudge*,
WebCM is a web platform that enables users to create, edit, run, and view individual-based simulations of multicellular microbial populations and communities on a remote compute server. WebCM builds upon the simulation software CellModeller in the back end and provides users with a web-browser-based modeling interface including model editing, execution, and playback. Multiple users can run and manage multiple simulations simultaneously, sharing the host hardware. Since it is based on CellModeller, it can utilize both GPU and CPU parallelization. The user interface provides real-time interactive 3D graphical representations for inspection of simulations at all time points, and the results can be downloaded for detailed offline analysis. It can be run on cloud computing services or on a local server, allowing collaboration within and between laboratories.
WebCM 是一个网络平台,使用户能够在远程计算服务器上创建、编辑、运行和查看基于个体的多细胞微生物种群和群落模拟。WebCM 的后端基于仿真软件 CellModeller,为用户提供基于网络浏览器的建模界面,包括模型编辑、执行和回放。多个用户可以共享主机硬件,同时运行和管理多个模拟。由于它基于 CellModeller,因此可以利用 GPU 和 CPU 并行化。用户界面提供实时交互式三维图形显示,用于检查所有时间点的模拟结果,并可下载结果进行详细的离线分析。它既可以在云计算服务上运行,也可以在本地服务器上运行,从而实现实验室内部和实验室之间的协作。
{"title":"WebCM: A Web-Based Platform for Multiuser Individual-Based Modeling of Multicellular Microbial Populations and Communities","authors":"Jason Philippou, Guillermo Yáñez Feliú and Timothy J. Rudge*, ","doi":"10.1021/acssynbio.3c00486","DOIUrl":"10.1021/acssynbio.3c00486","url":null,"abstract":"<p >WebCM is a web platform that enables users to create, edit, run, and view individual-based simulations of multicellular microbial populations and communities on a remote compute server. WebCM builds upon the simulation software CellModeller in the back end and provides users with a web-browser-based modeling interface including model editing, execution, and playback. Multiple users can run and manage multiple simulations simultaneously, sharing the host hardware. Since it is based on CellModeller, it can utilize both GPU and CPU parallelization. The user interface provides real-time interactive 3D graphical representations for inspection of simulations at all time points, and the results can be downloaded for detailed offline analysis. It can be run on cloud computing services or on a local server, allowing collaboration within and between laboratories.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.3c00486","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140920309","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 : 2024-05-11DOI: 10.1021/acssynbio.4c00030
Mengying Wang, Dongqi Jiang, Xinyao Lu, Hong Zong and Bin Zhuge*,
NAD is a redox coenzyme and is the center of energy metabolism. In metabolic engineering modifications, an insufficient NAD(H) supply often limits the accumulation of target products. In this study, Candida glycerinogenes was found to be able to supply NAD(H) in large fluxes, up to 7.6 times more than Saccharomyces cerevisiae in aerobic fermentation. Aerobic fermentation in a medium without amino nitrogen sources demonstrated that C. glycerinogenes NAD synthesis was not dependent on NAD precursors in the medium. Inhibition by antisense RNA and the detection of transcript levels indicated that the main NAD supply pathway is the de novo biosynthesis pathway. It was further demonstrated that NAD(H) supply was unaffected by changes in metabolic flow through C. glycerinogenes ΔGPD aerobic fermentation (80 g/L ethanol). In conclusion, the ability of C. glycerinogenes to supply NAD(H) in large fluxes provides a new approach to solving the NAD(H) supply problem in synthetic biology.
NAD 是一种氧化还原辅酶,是能量代谢的中心。在代谢工程改造中,NAD(H)供应不足往往会限制目标产物的积累。本研究发现,在有氧发酵过程中,甘油假丝酵母能够大量供应 NAD(H),其供应量是酿酒酵母的 7.6 倍。在不含氨基氮源的培养基中进行的有氧发酵表明,甘油酸酵母菌的 NAD 合成并不依赖于培养基中的 NAD 前体。反义 RNA 的抑制作用和转录本水平的检测表明,NAD 的主要供应途径是新生物合成途径。研究还进一步证明,通过 C. glycerinogenes ΔGPD 有氧发酵(80 克/升乙醇),NAD(H)供应不受代谢流量变化的影响。总之,C. glycerinogenes 提供大量 NAD(H) 的能力为解决合成生物学中的 NAD(H) 供应问题提供了一种新方法。
{"title":"Large Flux Supply of NAD(H) under Aerobic Conditions by Candida glycerinogenes","authors":"Mengying Wang, Dongqi Jiang, Xinyao Lu, Hong Zong and Bin Zhuge*, ","doi":"10.1021/acssynbio.4c00030","DOIUrl":"10.1021/acssynbio.4c00030","url":null,"abstract":"<p >NAD is a redox coenzyme and is the center of energy metabolism. In metabolic engineering modifications, an insufficient NAD(H) supply often limits the accumulation of target products. In this study, <i>Candida glycerinogenes</i> was found to be able to supply NAD(H) in large fluxes, up to 7.6 times more than <i>Saccharomyces cerevisiae</i> in aerobic fermentation. Aerobic fermentation in a medium without amino nitrogen sources demonstrated that <i>C. glycerinogenes</i> NAD synthesis was not dependent on NAD precursors in the medium. Inhibition by antisense RNA and the detection of transcript levels indicated that the main NAD supply pathway is the de novo biosynthesis pathway. It was further demonstrated that NAD(H) supply was unaffected by changes in metabolic flow through <i>C. glycerinogenes</i> Δ<i>GPD</i> aerobic fermentation (80 g/L ethanol). In conclusion, the ability of <i>C. glycerinogenes</i> to supply NAD(H) in large fluxes provides a new approach to solving the NAD(H) supply problem in synthetic biology.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-05-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140908407","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 : 2024-05-11DOI: 10.1021/acssynbio.4c00181
Neelu Batra, Mei-Juan Tu and Ai-Ming Yu*,
Synthetic biology constitutes a scientific domain focused on intentional redesign of organisms to confer novel functionalities or create new products through strategic engineering of their genetic makeup. Leveraging the inherent capabilities of nature, one may address challenges across diverse sectors including medicine. Inspired by this concept, we have developed an innovative bioengineering platform, enabling high-yield and large-scale production of biological small interfering RNA (BioRNA/siRNA) agents via bacterial fermentation. Herein, we show that with the use of a new tRNA fused pre-miRNA carrier, we can produce various forms of BioRNA/siRNA agents within living host cells. We report a high-level overexpression of nine target BioRNA/siRNA molecules at 100% success rate, yielding 3–10 mg of BioRNA/siRNA per 0.25 L of bacterial culture with high purity (>98%) and low endotoxin (<5 EU/μg RNA). Furthermore, we demonstrate that three representative BioRNA/siRNAs against GFP, BCL2, and PD-L1 are biologically active and can specifically and efficiently silence their respective targets with the potential to effectively produce downstream antiproliferation effects by PD-L1-siRNA. With these promising results, we aim to advance the field of synthetic biology by offering a novel platform to bioengineer functional siRNA agents for research and drug development.
{"title":"Molecular Engineering of Functional SiRNA Agents","authors":"Neelu Batra, Mei-Juan Tu and Ai-Ming Yu*, ","doi":"10.1021/acssynbio.4c00181","DOIUrl":"10.1021/acssynbio.4c00181","url":null,"abstract":"<p >Synthetic biology constitutes a scientific domain focused on intentional redesign of organisms to confer novel functionalities or create new products through strategic engineering of their genetic makeup. Leveraging the inherent capabilities of nature, one may address challenges across diverse sectors including medicine. Inspired by this concept, we have developed an innovative bioengineering platform, enabling high-yield and large-scale production of biological small interfering RNA (BioRNA/siRNA) agents via bacterial fermentation. Herein, we show that with the use of a new tRNA fused pre-miRNA carrier, we can produce various forms of BioRNA/siRNA agents within living host cells. We report a high-level overexpression of nine target BioRNA/siRNA molecules at 100% success rate, yielding 3–10 mg of BioRNA/siRNA per 0.25 L of bacterial culture with high purity (>98%) and low endotoxin (<5 EU/μg RNA). Furthermore, we demonstrate that three representative BioRNA/siRNAs against GFP, BCL2, and PD-L1 are biologically active and can specifically and efficiently silence their respective targets with the potential to effectively produce downstream antiproliferation effects by PD-L1-siRNA. With these promising results, we aim to advance the field of synthetic biology by offering a novel platform to bioengineer functional siRNA agents for research and drug development.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-05-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.4c00181","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140907571","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 : 2024-05-10DOI: 10.1021/acssynbio.3c00774
Hikari Baba, Tomohiro Fujita, Kosuke Mizuno, Mai Tambo and Satoshi Toda*,
The spatial sorting of cells into appropriate tissue compartments is essential for embryogenesis and tissue development. Spatial cell sorting is controlled by the interplay between cell surface affinity and intracellular mechanical properties. However, intracellular signaling that can sufficiently sort cell populations remains unexplored. In this study, we engineered chimeric cadherins by replacing the cadherin intracellular domain with cytoskeletal regulators to test their ability to induce spatial cell sorting. Using a fibroblast-based reconstitution system, we observed that Rac1 and RhoA activity in the cadherin tail induced outward and inward sorting, respectively. In particular, RhoA activity embedded cells toward the inside of E-cadherin-expressing spheroids and tumor spheroids, leading to tissue invagination. Despite the simplicity of chimeric cadherin design, our results indicate that differences in cadherin intracellular activities can determine the direction of spatial cell sorting, even when cell surface affinity is not different, and provide new molecular tools to engineer tissue architectures.
{"title":"Programming Spatial Cell Sorting by Engineering Cadherin Intracellular Activity","authors":"Hikari Baba, Tomohiro Fujita, Kosuke Mizuno, Mai Tambo and Satoshi Toda*, ","doi":"10.1021/acssynbio.3c00774","DOIUrl":"10.1021/acssynbio.3c00774","url":null,"abstract":"<p >The spatial sorting of cells into appropriate tissue compartments is essential for embryogenesis and tissue development. Spatial cell sorting is controlled by the interplay between cell surface affinity and intracellular mechanical properties. However, intracellular signaling that can sufficiently sort cell populations remains unexplored. In this study, we engineered chimeric cadherins by replacing the cadherin intracellular domain with cytoskeletal regulators to test their ability to induce spatial cell sorting. Using a fibroblast-based reconstitution system, we observed that Rac1 and RhoA activity in the cadherin tail induced outward and inward sorting, respectively. In particular, RhoA activity embedded cells toward the inside of E-cadherin-expressing spheroids and tumor spheroids, leading to tissue invagination. Despite the simplicity of chimeric cadherin design, our results indicate that differences in cadherin intracellular activities can determine the direction of spatial cell sorting, even when cell surface affinity is not different, and provide new molecular tools to engineer tissue architectures.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.3c00774","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140896620","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 : 2024-05-10DOI: 10.1021/acssynbio.4c00135
Kentarou Sakamoto*, Yuka Yamamoto, Hiroshi Inaba and Kazunori Matsuura*,
In-cell self-assembly of natural viral capsids is an event that can be visualized under transmission electron microscopy (TEM) observations. By mimicking the self-assembly of natural viral capsids, various artificial protein- and peptide-based nanocages were developed; however, few studies have reported the in-cell self-assembly of such nanocages. Our group developed a β-Annulus peptide that can form a nanocage called artificial viral capsid in vitro, but in-cell self-assembly of the capsid has not been achieved. Here, we designed an artificial viral capsid decorated with a fluorescent protein, StayGold, to visualize in-cell self-assembly. Fluorescence anisotropy measurements and fluorescence resonance energy transfer imaging, in addition to TEM observations of the cells and super-resolution microscopy, revealed that StayGold-conjugated β-Annulus peptides self-assembled into the StayGold-decorated artificial viral capsid in a cell. Using these techniques, we achieved the in-cell self-assembly of an artificial viral capsid.
在透射电子显微镜(TEM)观察下,可以看到天然病毒衣壳在细胞内的自组装。通过模仿天然病毒衣壳的自组装,人们开发出了各种基于蛋白质和肽的人工纳米笼;然而,很少有研究报道这种纳米笼的细胞内自组装。我们的研究小组开发了一种β-蒽多肽,它可以在体外形成一种称为人工病毒囊的纳米笼,但这种病毒囊在细胞内的自组装尚未实现。在这里,我们设计了一种用荧光蛋白StayGold装饰的人造病毒衣壳,以观察细胞内的自组装。除了细胞的 TEM 观察和超分辨率显微镜之外,荧光各向异性测量和荧光共振能量转移成像也揭示了 StayGold 共轭的 β-Annulus 肽在细胞内自组装成了 StayGold 装饰的人工病毒衣壳。利用这些技术,我们实现了人工病毒衣壳的细胞内自组装。
{"title":"Strategy toward In-Cell Self-Assembly of an Artificial Viral Capsid from a Fluorescent Protein-Modified β-Annulus Peptide","authors":"Kentarou Sakamoto*, Yuka Yamamoto, Hiroshi Inaba and Kazunori Matsuura*, ","doi":"10.1021/acssynbio.4c00135","DOIUrl":"10.1021/acssynbio.4c00135","url":null,"abstract":"<p >In-cell self-assembly of natural viral capsids is an event that can be visualized under transmission electron microscopy (TEM) observations. By mimicking the self-assembly of natural viral capsids, various artificial protein- and peptide-based nanocages were developed; however, few studies have reported the in-cell self-assembly of such nanocages. Our group developed a β-Annulus peptide that can form a nanocage called artificial viral capsid in vitro, but in-cell self-assembly of the capsid has not been achieved. Here, we designed an artificial viral capsid decorated with a fluorescent protein, StayGold, to visualize in-cell self-assembly. Fluorescence anisotropy measurements and fluorescence resonance energy transfer imaging, in addition to TEM observations of the cells and super-resolution microscopy, revealed that StayGold-conjugated β-Annulus peptides self-assembled into the StayGold-decorated artificial viral capsid in a cell. Using these techniques, we achieved the in-cell self-assembly of an artificial viral capsid.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140904039","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 : 2024-05-08DOI: 10.1021/acssynbio.4c00209
Gaku Sato, Saki Kinoshita, Takahiro G. Yamada, Satoshi Arai, Tetsuya Kitaguchi, Akira Funahashi, Nobuhide Doi and Kei Fujiwara*,
Inside cells, various biological systems work cooperatively for homeostasis and self-replication. These systems do not work independently as they compete for shared elements like ATP and NADH. However, it has been believed that such competition is not a problem in codependent biological systems such as the energy-supplying glycolysis and the energy-consuming translation system. In this study, we biochemically reconstituted the coupling system of glycolysis and translation using purified elements and found that the competition for ATP between glycolysis and protein synthesis interferes with their coupling. Both experiments and simulations revealed that this interference is derived from a metabolic tug-of-war between glycolysis and translation based on their reaction rates, which changes the threshold of the initial substrate concentration for the success coupling. By the metabolic tug-of-war, translation energized by strong glycolysis is facilitated by an exogenous ATPase, which normally inhibits translation. These findings provide chemical insights into the mechanism of competition among biological systems in living cells and provide a framework for the construction of synthetic metabolism in vitro.
在细胞内,各种生物系统协同工作,以实现平衡和自我复制。这些系统不能独立工作,因为它们要竞争 ATP 和 NADH 等共享元素。然而,人们一直认为这种竞争在相互依存的生物系统中不是问题,如能量供应的糖酵解和能量消耗的翻译系统。在这项研究中,我们利用纯化的元素以生物化学的方法重建了糖酵解和翻译的耦合系统,并发现糖酵解和蛋白质合成之间对 ATP 的竞争干扰了它们之间的耦合。实验和模拟都表明,这种干扰来自于糖酵解和翻译之间基于反应速率的代谢拉锯战,这种拉锯战改变了成功耦合的初始底物浓度阈值。在新陈代谢拉锯战中,通常会抑制翻译的外源 ATP 酶会促进强糖酵解所激发的翻译。这些发现从化学角度揭示了活细胞中生物系统之间的竞争机制,并为构建体外合成代谢提供了一个框架。
{"title":"Metabolic Tug-of-War between Glycolysis and Translation Revealed by Biochemical Reconstitution","authors":"Gaku Sato, Saki Kinoshita, Takahiro G. Yamada, Satoshi Arai, Tetsuya Kitaguchi, Akira Funahashi, Nobuhide Doi and Kei Fujiwara*, ","doi":"10.1021/acssynbio.4c00209","DOIUrl":"10.1021/acssynbio.4c00209","url":null,"abstract":"<p >Inside cells, various biological systems work cooperatively for homeostasis and self-replication. These systems do not work independently as they compete for shared elements like ATP and NADH. However, it has been believed that such competition is not a problem in codependent biological systems such as the energy-supplying glycolysis and the energy-consuming translation system. In this study, we biochemically reconstituted the coupling system of glycolysis and translation using purified elements and found that the competition for ATP between glycolysis and protein synthesis interferes with their coupling. Both experiments and simulations revealed that this interference is derived from a metabolic tug-of-war between glycolysis and translation based on their reaction rates, which changes the threshold of the initial substrate concentration for the success coupling. By the metabolic tug-of-war, translation energized by strong glycolysis is facilitated by an exogenous ATPase, which normally inhibits translation. These findings provide chemical insights into the mechanism of competition among biological systems in living cells and provide a framework for the construction of synthetic metabolism in vitro.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140890769","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 : 2024-05-08DOI: 10.1021/acssynbio.4c00062
Désirée Körner, Niklas M. Schäfer, Antonio Lagares Jr., Lukas Birmes, Niels N. Oehlmann, Holly Addison, Sebastian Pöhl, Martin Thanbichler, Johannes G. Rebelein, Jörn Petersen and Anke Becker*,
Members of the alphaproteobacterial order Rhodobacterales are metabolically diverse and highly abundant in the ocean. They are becoming increasingly interesting for marine biotechnology, due to their ecological adaptability, wealth of versatile low-copy-number plasmids, and their ability to produce secondary metabolites. However, molecular tools for engineering strains of this bacterial lineage are limited. Here, we expand the genetic toolbox by establishing standardized, modular repABC-based plasmid vectors of four well-characterized compatibility groups from the Roseobacter group applicable in the Rhodobacterales, and likely in further alphaproteobacterial orders (Hyphomicrobiales, Rhodospirillales, Caulobacterales). We confirmed replication of these newly constructed pABC vectors in two members of Rhodobacterales, namely, Dinoroseobacter shibae DFL 12 and Rhodobacter capsulatus B10S, as well as in two members of the alphaproteobacterial order Hyphomicrobiales (synonym: Rhizobiales; Ensifer meliloti 2011 and “Agrobacterium fabrum” C58). Maintenance of the pABC vectors in the biotechnologically valuable orders Rhodobacterales and Hyphomicrobiales facilitates the shuttling of genetic constructs between alphaproteobacterial genera and orders. Additionally, plasmid replication was verified in one member of Rhodospirillales (Rhodospirillum rubrum S1) as well as in one member of Caulobacterales (Caulobacter vibrioides CB15N). The modular construction of pABC vectors and the usage of four compatible replication systems, which allows their coexistence in a host cell, are advantageous features for future implementations of newly designed synthetic pathways. The vector applicability was demonstrated by functional complementation of a nitrogenase mutant phenotype by two complementary pABC-based plasmids in R. capsulatus.
{"title":"Modular Low-Copy-Number Plasmid Vectors for Rhodobacterales with Extended Host Range in Alphaproteobacteria","authors":"Désirée Körner, Niklas M. Schäfer, Antonio Lagares Jr., Lukas Birmes, Niels N. Oehlmann, Holly Addison, Sebastian Pöhl, Martin Thanbichler, Johannes G. Rebelein, Jörn Petersen and Anke Becker*, ","doi":"10.1021/acssynbio.4c00062","DOIUrl":"10.1021/acssynbio.4c00062","url":null,"abstract":"<p >Members of the alphaproteobacterial order Rhodobacterales are metabolically diverse and highly abundant in the ocean. They are becoming increasingly interesting for marine biotechnology, due to their ecological adaptability, wealth of versatile low-copy-number plasmids, and their ability to produce secondary metabolites. However, molecular tools for engineering strains of this bacterial lineage are limited. Here, we expand the genetic toolbox by establishing standardized, modular <i>repABC</i>-based plasmid vectors of four well-characterized compatibility groups from the Roseobacter group applicable in the Rhodobacterales, and likely in further alphaproteobacterial orders (Hyphomicrobiales, Rhodospirillales, Caulobacterales). We confirmed replication of these newly constructed pABC vectors in two members of Rhodobacterales, namely, <i>Dinoroseobacter shibae</i> DFL 12 and <i>Rhodobacter capsulatus</i> B10S, as well as in two members of the alphaproteobacterial order Hyphomicrobiales (synonym: Rhizobiales; <i>Ensifer meliloti</i> 2011 and “<i>Agrobacterium fabrum</i>” C58). Maintenance of the pABC vectors in the biotechnologically valuable orders Rhodobacterales and Hyphomicrobiales facilitates the shuttling of genetic constructs between alphaproteobacterial genera and orders. Additionally, plasmid replication was verified in one member of Rhodospirillales (<i>Rhodospirillum rubrum</i> S1) as well as in one member of Caulobacterales (<i>Caulobacter vibrioides</i> CB15N). The modular construction of pABC vectors and the usage of four compatible replication systems, which allows their coexistence in a host cell, are advantageous features for future implementations of newly designed synthetic pathways. The vector applicability was demonstrated by functional complementation of a nitrogenase mutant phenotype by two complementary pABC-based plasmids in <i>R. capsulatus</i>.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140890727","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 : 2024-05-02DOI: 10.1021/acssynbio.3c00684
Fengjie Zhao, Christina M. Niman, Ghazaleh Ostovar, Marko S. Chavez, Joshua T. Atkinson, Benjamin M. Bonis, Jeffrey A. Gralnick, Mohamed Y. El-Naggar and James Q. Boedicker*,
Optogenetics is a powerful tool for spatiotemporal control of gene expression. Several light-inducible gene regulators have been developed to function in bacteria, and these regulatory circuits have been ported to new host strains. Here, we developed and adapted a red-light-inducible transcription factor for Shewanella oneidensis. This regulatory circuit is based on the iLight optogenetic system, which controls gene expression using red light. A thermodynamic model and promoter engineering were used to adapt this system to achieve differential gene expression in light and dark conditions within a S. oneidensis host strain. We further improved the iLight optogenetic system by adding a repressor to invert the genetic circuit and activate gene expression under red light illumination. The inverted iLight genetic circuit was used to control extracellular electron transfer within S. oneidensis. The ability to use both red- and blue-light-induced optogenetic circuits simultaneously was also demonstrated. Our work expands the synthetic biology capabilities in S. oneidensis, which could facilitate future advances in applications with electrogenic bacteria.
光遗传学是一种对基因表达进行时空控制的强大工具。目前已开发出多种光诱导基因调控因子在细菌中发挥作用,这些调控回路已被移植到新的宿主菌株中。在这里,我们开发了一种红光诱导转录因子,并将其应用于Shewanella oneidensis。该调控电路基于 iLight 光遗传系统,该系统利用红光控制基因表达。我们利用热力学模型和启动子工程改造了这一系统,使其在S. oneidensis宿主菌株中实现了光照和黑暗条件下的不同基因表达。我们进一步改进了 iLight 光遗传系统,添加了一个抑制因子,使基因回路反转,在红光照射下激活基因表达。倒置的 iLight 基因回路被用于控制 S. oneidensis 的胞外电子传递。同时使用红光和蓝光诱导的光遗传回路的能力也得到了证实。我们的工作拓展了 S. oneidensis 的合成生物学能力,这将促进未来电生细菌应用的进步。
{"title":"Red-Light-Induced Genetic System for Control of Extracellular Electron Transfer","authors":"Fengjie Zhao, Christina M. Niman, Ghazaleh Ostovar, Marko S. Chavez, Joshua T. Atkinson, Benjamin M. Bonis, Jeffrey A. Gralnick, Mohamed Y. El-Naggar and James Q. Boedicker*, ","doi":"10.1021/acssynbio.3c00684","DOIUrl":"10.1021/acssynbio.3c00684","url":null,"abstract":"<p >Optogenetics is a powerful tool for spatiotemporal control of gene expression. Several light-inducible gene regulators have been developed to function in bacteria, and these regulatory circuits have been ported to new host strains. Here, we developed and adapted a red-light-inducible transcription factor for <i>Shewanella oneidensis</i>. This regulatory circuit is based on the iLight optogenetic system, which controls gene expression using red light. A thermodynamic model and promoter engineering were used to adapt this system to achieve differential gene expression in light and dark conditions within a <i>S. oneidensis</i> host strain. We further improved the iLight optogenetic system by adding a repressor to invert the genetic circuit and activate gene expression under red light illumination. The inverted iLight genetic circuit was used to control extracellular electron transfer within <i>S. oneidensis</i>. The ability to use both red- and blue-light-induced optogenetic circuits simultaneously was also demonstrated. Our work expands the synthetic biology capabilities in <i>S. oneidensis</i>, which could facilitate future advances in applications with electrogenic bacteria.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140829854","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}
Enzymatic cascades have become a green and sustainable approach for the synthesis of valuable chemicals and pharmaceuticals. Using sequential enzymes to construct a multienzyme complex is an effective way to enhance the overall performance of biosynthetic routes. Here we report the design of an efficient in vitro hybrid biocatalytic system by assembling three enzymes that can convert styrene to (S)-1-phenyl-1,2-ethanediol. Specifically, we prepared the three enzymes in different ways, which were cell surface-displayed, purified, and cell-free expressed. To assemble them, we fused two orthogonal peptide–protein pairs (i.e., SpyTag/SpyCatcher and SnoopTag/SnoopCatcher) to the three enzymes, allowing their spatial organization by covalent assembly. By doing this, we constructed a multienzyme complex, which could enhance the production of (S)-1-phenyl-1,2-ethanediol by 3 times compared to the free-floating enzyme system without assembly. After optimization of the reaction system, the final product yield reached 234.6 μM with a substrate conversion rate of 46.9% (based on 0.5 mM styrene). Taken together, our strategy integrates the merits of advanced biochemical engineering techniques, including cellular surface display, spatial enzyme organization, and cell-free expression, which offers a new solution for chemical biosynthesis by enzymatic cascade biotransformation. We, therefore, anticipate that our approach will hold great potential for designing and constructing highly efficient systems to synthesize chemicals of agricultural, industrial, and pharmaceutical significance.
{"title":"An In Vitro Hybrid Biocatalytic System Enabled by a Combination of Surface-Displayed, Purified, and Cell-Free Expressed Enzymes","authors":"Ying Liu, Shuhui Huang, Wan-Qiu Liu, Fang Ba, Yifan Liu, Shengjie Ling and Jian Li*, ","doi":"10.1021/acssynbio.4c00201","DOIUrl":"10.1021/acssynbio.4c00201","url":null,"abstract":"<p >Enzymatic cascades have become a green and sustainable approach for the synthesis of valuable chemicals and pharmaceuticals. Using sequential enzymes to construct a multienzyme complex is an effective way to enhance the overall performance of biosynthetic routes. Here we report the design of an efficient <i>in vitro</i> hybrid biocatalytic system by assembling three enzymes that can convert styrene to (<i>S</i>)-1-phenyl-1,2-ethanediol. Specifically, we prepared the three enzymes in different ways, which were cell surface-displayed, purified, and cell-free expressed. To assemble them, we fused two orthogonal peptide–protein pairs (i.e., SpyTag/SpyCatcher and SnoopTag/SnoopCatcher) to the three enzymes, allowing their spatial organization by covalent assembly. By doing this, we constructed a multienzyme complex, which could enhance the production of (<i>S</i>)-1-phenyl-1,2-ethanediol by 3 times compared to the free-floating enzyme system without assembly. After optimization of the reaction system, the final product yield reached 234.6 μM with a substrate conversion rate of 46.9% (based on 0.5 mM styrene). Taken together, our strategy integrates the merits of advanced biochemical engineering techniques, including cellular surface display, spatial enzyme organization, and cell-free expression, which offers a new solution for chemical biosynthesis by enzymatic cascade biotransformation. We, therefore, anticipate that our approach will hold great potential for designing and constructing highly efficient systems to synthesize chemicals of agricultural, industrial, and pharmaceutical significance.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140830043","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 : 2024-04-29DOI: 10.1021/acssynbio.3c00761
Zachary P. Harmer, Jaron C. Thompson, David L. Cole, Ophelia S. Venturelli, Victor M. Zavala and Megan N. McClean*,
The ability to control cellular processes using optogenetics is inducer-limited, with most optogenetic systems responding to blue light. To address this limitation, we leverage an integrated framework combining Lustro, a powerful high-throughput optogenetics platform, and machine learning tools to enable multiplexed control over blue light-sensitive optogenetic systems. Specifically, we identify light induction conditions for sequential activation as well as preferential activation and switching between pairs of light-sensitive split transcription factors in the budding yeast, Saccharomyces cerevisiae. We use the high-throughput data generated from Lustro to build a Bayesian optimization framework that incorporates data-driven learning, uncertainty quantification, and experimental design to enable the prediction of system behavior and the identification of optimal conditions for multiplexed control. This work lays the foundation for designing more advanced synthetic biological circuits incorporating optogenetics, where multiple circuit components can be controlled using designer light induction programs, with broad implications for biotechnology and bioengineering.
{"title":"Dynamic Multiplexed Control and Modeling of Optogenetic Systems Using the High-Throughput Optogenetic Platform, Lustro","authors":"Zachary P. Harmer, Jaron C. Thompson, David L. Cole, Ophelia S. Venturelli, Victor M. Zavala and Megan N. McClean*, ","doi":"10.1021/acssynbio.3c00761","DOIUrl":"10.1021/acssynbio.3c00761","url":null,"abstract":"<p >The ability to control cellular processes using optogenetics is inducer-limited, with most optogenetic systems responding to blue light. To address this limitation, we leverage an integrated framework combining Lustro, a powerful high-throughput optogenetics platform, and machine learning tools to enable multiplexed control over blue light-sensitive optogenetic systems. Specifically, we identify light induction conditions for sequential activation as well as preferential activation and switching between pairs of light-sensitive split transcription factors in the budding yeast, <i>Saccharomyces cerevisiae</i>. We use the high-throughput data generated from Lustro to build a Bayesian optimization framework that incorporates data-driven learning, uncertainty quantification, and experimental design to enable the prediction of system behavior and the identification of optimal conditions for multiplexed control. This work lays the foundation for designing more advanced synthetic biological circuits incorporating optogenetics, where multiple circuit components can be controlled using designer light induction programs, with broad implications for biotechnology and bioengineering.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.3c00761","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140829994","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}