bioRxiv - Synthetic Biology最新文献_第9页

Shikimate pathway-Dependent Catabolism: enabling near-to-maximum production yield of aromatics 依赖莽草酸途径的分解代谢：使芳香烃的产量接近最高水平

bioRxiv - Synthetic Biology Pub Date : 2024-07-07 DOI: 10.1101/2024.07.06.602327

Lyon Bruinsma, Christos Batianis, Sara Moreno Paz, Kesi Kurnia, Job Dirkmaat, Alexandra Muller, Jose Juncosa Nunez, Ruud Weusthuis, Vitor A.P. Martins dos Santos

{"title":"Shikimate pathway-Dependent Catabolism: enabling near-to-maximum production yield of aromatics","authors":"Lyon Bruinsma, Christos Batianis, Sara Moreno Paz, Kesi Kurnia, Job Dirkmaat, Alexandra Muller, Jose Juncosa Nunez, Ruud Weusthuis, Vitor A.P. Martins dos Santos","doi":"10.1101/2024.07.06.602327","DOIUrl":"https://doi.org/10.1101/2024.07.06.602327","url":null,"abstract":"Catabolism is a complex network of tightly regulated metabolic reactions that provides energy and carbon to fuel anabolism in all living organisms. Rewiring catabolism is essential for harnessing industrial biotechnology but remains a substantial metabolic engineering challenge due to its high genetic stability and tight regulation acquired through evolution. In this study, by combining metabolic modeling, rational engineering, and adaptive laboratory evolution, we fundamentally redesigned bacterial catabolism. We created a new-to-nature shikimate pathway-dependent catabolism (SDC) in Pseudomonas putida by reprogramming the shikimate pathway as the primary catabolic route. SDC supports growth by supplying the glycerol catabolic end-product pyruvate, enabling superior production of shikimate pathway-derived molecules. Through SDC, aromatics production reached over 89% of the pathway's maximum theoretical yield, setting a new benchmark for their microbial synthesis. Our study successfully repurposed an anabolic pathway for catabolism, exemplifying the high metabolic plasticity of microbes and providing a bacterial chassis for the efficient production of high-added value compounds.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"87 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141569785","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Tuning Ultrasensitivity in Genetic Logic Gates using Antisense RNA Feedback 利用反义 RNA 反馈调节遗传逻辑门的超灵敏度

bioRxiv - Synthetic Biology Pub Date : 2024-07-04 DOI: 10.1101/2024.07.03.601968

Nicolai Engelmann, Maik Molderings, Heinz Koeppl

引用次数: 0

Resource competition in CRISPRa genetic circuits CRISPRa 基因回路中的资源竞争

bioRxiv - Synthetic Biology Pub Date : 2024-07-04 DOI: 10.1101/2024.07.03.601429

Krishna Manoj, Domitilla Del Vecchio

{"title":"Resource competition in CRISPRa genetic circuits","authors":"Krishna Manoj, Domitilla Del Vecchio","doi":"10.1101/2024.07.03.601429","DOIUrl":"https://doi.org/10.1101/2024.07.03.601429","url":null,"abstract":"CRISPR-mediated gene activation (CRISPRa) allows concurrent regulation of transcription of many gene targets and has found widespread applications in genome-wide screening, bioproduction, and therapeutics. In principle, multiple gene targets can be regulated independently by choosing orthogonal scaffold RNA (scRNA); in practice, this is not the case due to competition for dCas9 and activator protein. While prior studies have extensively characterized competition effects in CRISPRi circuits such an investigation in CRISPRa circuits is still lacking. In this paper, we investigate the on-target and off-target effects of competition for the two resources during CRISPRa. We observe a biphasic response in the on-target response of a CRISPRa module, wherein increased expression of scRNA leads to target repression instead of activation. The addition of a competitor further leads to the repression of any other CRISPRa system, showing the lack of modularity of concurrent CRISPRa systems. These effects are significant even when both resources are expressed at their maximum tolerable limit. We finally show that competition can also be used to design the response of a CRISPRa module by reducing leakiness while keeping the same maximal activation. The results from this study can significantly affect the interpretation of data obtained when using multiple CRISPRi/a modules and can be used as a predictive tool to determine how the operation of any such module in a concurrent configuration is different from the one expected in isolation.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"47 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141550747","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Developing, characterizing and modeling CRISPR-based point-of-use pathogen diagnostics 开发、鉴定和模拟基于 CRISPR 的使用点病原体诊断方法

bioRxiv - Synthetic Biology Pub Date : 2024-07-03 DOI: 10.1101/2024.07.03.601853

Jaeyoung K. Jung, Kathleen S. Dreyer, Kate E. Dray, Joseph J. Muldoon, Jithin George, Sasha Shirman, Maria D. Cabezas, Anne E. D'Aquino, Matthew S. Verosloff, Kosuke Seki, Grant A. Rybnicky, Khalid K. Alam, Neda Bagheri, Michael C. Jewett, Joshua Nathaniel Leonard, Niall M. Mangan, Julius B Lucks

{"title":"Developing, characterizing and modeling CRISPR-based point-of-use pathogen diagnostics","authors":"Jaeyoung K. Jung, Kathleen S. Dreyer, Kate E. Dray, Joseph J. Muldoon, Jithin George, Sasha Shirman, Maria D. Cabezas, Anne E. D'Aquino, Matthew S. Verosloff, Kosuke Seki, Grant A. Rybnicky, Khalid K. Alam, Neda Bagheri, Michael C. Jewett, Joshua Nathaniel Leonard, Niall M. Mangan, Julius B Lucks","doi":"10.1101/2024.07.03.601853","DOIUrl":"https://doi.org/10.1101/2024.07.03.601853","url":null,"abstract":"Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20 - 200 aM sensitivity without any specialized equipment. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"26 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141550748","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Biochemical implementation of acceleration sensing and PIDA control 加速感应和 PIDA 控制的生化实现

bioRxiv - Synthetic Biology Pub Date : 2024-07-02 DOI: 10.1101/2024.07.02.601775

Emmanouil Alexis, Sebastian Espinel-Rios, Ioannis G. Kevrekidis, Jose L. Avalos

引用次数: 0

Simulating 500 million years of evolution with a language model 用语言模型模拟 5 亿年的进化过程

bioRxiv - Synthetic Biology Pub Date : 2024-07-02 DOI: 10.1101/2024.07.01.600583

Tomas Hayes, Roshan Rao, Halil Akin, Nicholas J Sofroniew, Deniz Oktay, Zeming Lin, Robert Verkuil, Vincent Q Tran, Jonathan Deaton, Marius Wiggert, Rohil Badkundri, Irhum Shafkat, Jun Gong, Alexander Derry, Raúl Santiago Molina, Neil Thomas, Yousuf A Khan, Chetan Mishra, Carolyn Kim, Liam J Bartie, Matthew Nemeth, Patrick D Hsu, Tom Sercu, Salvatore Candido, Alexander Rives

{"title":"Simulating 500 million years of evolution with a language model","authors":"Tomas Hayes, Roshan Rao, Halil Akin, Nicholas J Sofroniew, Deniz Oktay, Zeming Lin, Robert Verkuil, Vincent Q Tran, Jonathan Deaton, Marius Wiggert, Rohil Badkundri, Irhum Shafkat, Jun Gong, Alexander Derry, Raúl Santiago Molina, Neil Thomas, Yousuf A Khan, Chetan Mishra, Carolyn Kim, Liam J Bartie, Matthew Nemeth, Patrick D Hsu, Tom Sercu, Salvatore Candido, Alexander Rives","doi":"10.1101/2024.07.01.600583","DOIUrl":"https://doi.org/10.1101/2024.07.01.600583","url":null,"abstract":"More than three billion years of evolution have produced an image of biology encoded into the space of natural proteins. Here we show that language models trained on tokens generated by evolution can act as evolutionary simulators to generate functional proteins that are far away from known proteins. We present ESM3, a frontier multimodal generative language model that reasons over the sequence, structure, and function of proteins. ESM3 can follow complex prompts combining its modalities and is highly responsive to biological alignment. We have prompted ESM3 to generate fluorescent proteins with a chain of thought. Among the generations that we synthesized, we found a bright fluorescent protein at far distance (58% identity) from known fluorescent proteins. Similarly distant natural fluorescent proteins are separated by over five hundred million years of evolution.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"34 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141524887","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Enzyme-Responsive DNA Condensates 酶反应 DNA 凝聚物

bioRxiv - Synthetic Biology Pub Date : 2024-07-02 DOI: 10.1101/2024.07.02.601714

Juliette Bucci, Layla Malouf, Diana A Tanase, Nada Farag, Jacob R Lamb, Serena Gentile, Erica Del Grosso, Clemens F Kaminski, Lorenzo Di Michele, Francesco Ricci

{"title":"Enzyme-Responsive DNA Condensates","authors":"Juliette Bucci, Layla Malouf, Diana A Tanase, Nada Farag, Jacob R Lamb, Serena Gentile, Erica Del Grosso, Clemens F Kaminski, Lorenzo Di Michele, Francesco Ricci","doi":"10.1101/2024.07.02.601714","DOIUrl":"https://doi.org/10.1101/2024.07.02.601714","url":null,"abstract":"Membrane-less compartments and organelles are widely acknowledged for their role in regulating cellular processes, and there is an urgent need to harness their full potential as both structural and functional elements of synthetic cells. Despite rapid progress, synthetically recapitulating the nonequilibrium, spatially distributed responses of natural membrane-less organelles remain elusive. Here we demonstrate that the activity of nucleic-acid cleaving enzymes can be localised within DNA-based membrane-less compartments by sequestering the respective DNA or RNA substrates. Reaction-diffusion processes lead to complex nonequilibrium patterns, dependent on enzyme concentration. By arresting similar dynamic patterns, we spatially organise different substrates in concentric sub-compartments, which can be then selectively addressed by different enzymes, demonstrating spatial distribution of enzymatic activity. Besides advancing our ability to engineer advanced biomimetic functions in synthetic membrane-less organelles, our results may facilitate the deployment of DNA-based condensates as microbioreactors or platforms for the detection and quantitation of enzymes and nucleic acids.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"36 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141524884","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Self-growing protocell models in aqueous two-phase system induced by internal DNA replication reaction 内部 DNA 复制反应诱导水基两相体系中的自生长原细胞模型

bioRxiv - Synthetic Biology Pub Date : 2024-07-02 DOI: 10.1101/2024.07.01.599542

Yoshihiro Minagawa, Moe Yabuta, Masayuki Su'estugu, Hiroyuki Noji

{"title":"Self-growing protocell models in aqueous two-phase system induced by internal DNA replication reaction","authors":"Yoshihiro Minagawa, Moe Yabuta, Masayuki Su'estugu, Hiroyuki Noji","doi":"10.1101/2024.07.01.599542","DOIUrl":"https://doi.org/10.1101/2024.07.01.599542","url":null,"abstract":"The bottom-up reconstitution of self-growing artificial cells is a critical milestone toward realizing autonomy and evolvability. However, building artificial cells that exhibit self-growth coupled with internal replication of gene-encoding DNA has not been achieved yet. Here, we report self-growing artificial cell models based on dextran-rich droplets in an aqueous two-phase system of poly(ethylene glycol) (PEG) and dextran (DEX). Motivated by the finding that DNA induces the generation of DEX-rich droplets, we integrated DNA amplification system with DEX-rich droplets, which exhibited active self-growth. We implemented the protocells with cell-free transcription-translation (TXTL) systems coupled with DNA amplification/replication, which also showed active self-growth. We also observed self-growth activity of protocells carrying a single copy of DNA. Considering the simplicities in terms of the chemical composition and the mechanism, these results underscore the potential of DEX droplets as a foundational platform for engineering protocells, giving implications for the emergence of protocells under prebiotic conditions.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"57 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141524886","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Development of modular geminivirus-based vectors for high cargo expression and gene targeting in plants 开发基于模块化 geminivirus 的载体，用于植物的高负载表达和基因打靶

bioRxiv - Synthetic Biology Pub Date : 2024-06-29 DOI: 10.1101/2024.06.29.601216

Matthew Neubauer, Katie Vollen, Jose T Ascencio-Ibanez, Linda Hanley-Bowdoin, Anna N Stepanova, Jose M Alonso

{"title":"Development of modular geminivirus-based vectors for high cargo expression and gene targeting in plants","authors":"Matthew Neubauer, Katie Vollen, Jose T Ascencio-Ibanez, Linda Hanley-Bowdoin, Anna N Stepanova, Jose M Alonso","doi":"10.1101/2024.06.29.601216","DOIUrl":"https://doi.org/10.1101/2024.06.29.601216","url":null,"abstract":"Viral vectors can be useful tools for expressing recombinant proteins as well as delivering gene-editing machinery. Despite their utility, the development and subsequent optimization of these tools is often a difficult and tedious process. Thus, although considerable work has been done to create useful viral vectors for gene editing and protein expression, there is a lack of understanding of how best to design these vectors for specific applications. For instance, it is often unclear whether the inclusion of heterologous promoter sequences or different viral components will improve cargo expression or replicon accumulation. To address some of these hurdles, we designed a GoldenBraid (GB) compatible viral vector system based on the geminivirus Beet curly top virus (BCTV). This system allows for simple, modular cloning of a variety of reporter constructs. Making use of this modular cloning strategy, we compared a variety of alternative viral vector architectures. Interestingly, native BCTV promoters outperformed the constitutive 35S promoter, while the removal of the BCTV virion-sense genes promoted reporter expression. Intriguingly, these modifications had no effect on total replicon accumulation. These results show the utility of the new modular BCTV-based viral vectors for protein expression and gene targeting applications, as well as uncover design principles that may inform future geminivirus-based viral vector architectures. We anticipate that the availability of this new modular system will spark the broad adoption of replicon-based strategies in protein expression and gene editing experiments in plants.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"40 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141503854","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Creating a genetic toolbox for the carbon-fixing, nitrogen-fixing and dehalogenating bacterium Xanthobacter autotrophicus 创建固碳、固氮和脱卤黄杆菌基因工具箱

bioRxiv - Synthetic Biology Pub Date : 2024-06-27 DOI: 10.1101/2024.06.26.600795

Alexa F. Van Voorhis, Rebecca S. Sherbo

{"title":"Creating a genetic toolbox for the carbon-fixing, nitrogen-fixing and dehalogenating bacterium Xanthobacter autotrophicus","authors":"Alexa F. Van Voorhis, Rebecca S. Sherbo","doi":"10.1101/2024.06.26.600795","DOIUrl":"https://doi.org/10.1101/2024.06.26.600795","url":null,"abstract":"X. autotrophicus is a metabolically flexible microorganism with two key features: 1) The organism has adapted to grow on a wide variety of carbon sources including CO2, methanol, formate, propylene, haloalkanes and haloacids; and 2) X. autotrophicus was the first chemoautotroph identified that could also simultaneously fix N2, meaning the organism can utilize CO2, N2, and H2 for growth. This metabolic flexibility has enabled use of X. autotrophicus for gas fixation, the creation of fertilizers and foods from gases, and the dehalogenation of environmental contaminants. Despite the wide variety of applications that have already been demonstrated for this organism, there are few genetic tools available to explore and exploit its metabolism. Here, we report a genetic toolbox for use in X. autotrophicus. We first identified suitable origins of replication and quantified their copy number, and identified antibiotic resistance cassettes that could be used as selectable markers. We then tested several constitutive and inducible promoters and terminators and quantified their promoter strengths and termination efficiencies. Finally, we demonstrated that gene expression tools remain effective under both autotrophic and dehalogenative metabolic conditions to show that these tools can be used in the environments that make X. autotrophicus unique. Our extensive characterization of these tools in X. autotrophicus will enable genetic and metabolic engineering to optimize production of fertilizers and foods from gases, and enable bioremediation of halogenated environmental contaminants.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"209 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141524888","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0