Pub Date : 2024-06-27DOI: 10.1101/2024.06.27.601038
Erik Kubaczka, Maximilian Gehri, Jérémie J. M. Marlhens, Tobias Schwarz, Maik Molderings, Nicolai Engelmann, Christian Hochberger, Heinz Koeppl
Energy and its dissipation are fundamental to all living systems, including cells. Insufficient abundance of energy carriers -as caused by the additional burden of artificial genetic circuits- shifts a cell's priority to survival, also harming the functionality of the genetic circuit. Moreover, recent works have shown the importance of energy expenditure in information transmission. Despite living organisms being non-equilibrium systems, non-equilibrium models capable of accounting for energy dissipation and non-equilibrium response curves are not yet employed in genetic design automation (GDA) software. To this end, we introduce Energy Aware Technology Mapping, the automated design of genetic logic circuits with respect to energy efficiency and functionality. The basis for this is an energy aware non-equilibrium steady state (NESS) model of gene expression, capturing characteristics like energy dissipation -which we link to the entropy production rate- and transcriptional bursting, relevant to eukaryotes as well as prokaryotes. Our evaluation shows that a genetic logic circuit's functional performance and energy efficiency are disjoint optimization goals. For our benchmark, energy efficiency improves by 37.2% on average when comparing to functionally optimized variants. We discover a linear increase in energy expenditure and overall protein expression with the circuit size, where Energy Aware Technology Mapping allows for designing genetic logic circuits with the energy efficiency of circuits that are one to two gates smaller. Structural variants improve this further, while results show the Pareto dominance among structures of a single Boolean function. By incorporating energy demand into the design, Energy Aware Technology Mapping enables energy efficiency by design. This extends current GDA tools and complements approaches coping with burden in vivo.
{"title":"Energy Aware Technology Mapping of Genetic Logic Circuits","authors":"Erik Kubaczka, Maximilian Gehri, Jérémie J. M. Marlhens, Tobias Schwarz, Maik Molderings, Nicolai Engelmann, Christian Hochberger, Heinz Koeppl","doi":"10.1101/2024.06.27.601038","DOIUrl":"https://doi.org/10.1101/2024.06.27.601038","url":null,"abstract":"Energy and its dissipation are fundamental to all living systems, including cells. Insufficient abundance of energy carriers -as caused by the additional burden of artificial genetic circuits- shifts a cell's priority to survival, also harming the functionality of the genetic circuit. Moreover, recent works have shown the importance of energy expenditure in information transmission. Despite living organisms being non-equilibrium systems, non-equilibrium models capable of accounting for energy dissipation and non-equilibrium response curves are not yet employed in genetic design automation (GDA) software. To this end, we introduce Energy Aware Technology Mapping, the automated design of genetic logic circuits with respect to energy efficiency and functionality. The basis for this is an energy aware non-equilibrium steady state (NESS) model of gene expression, capturing characteristics like energy dissipation -which we link to the entropy production rate- and transcriptional bursting, relevant to eukaryotes as well as prokaryotes. Our evaluation shows that a genetic logic circuit's functional performance and energy efficiency are disjoint optimization goals. For our benchmark, energy efficiency improves by 37.2% on average when comparing to functionally optimized variants. We discover a linear increase in energy expenditure and overall protein expression with the circuit size, where Energy Aware Technology Mapping allows for designing genetic logic circuits with the energy efficiency of circuits that are one to two gates smaller. Structural variants improve this further, while results show the Pareto dominance among structures of a single Boolean function. By incorporating energy demand into the design, Energy Aware Technology Mapping enables energy efficiency by design. This extends current GDA tools and complements approaches coping with burden <em>in vivo</em>.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"341 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141503904","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}
Pub Date : 2024-06-27DOI: 10.1101/2024.06.26.600856
Diego M. Roldan, Vanesa Amarelle
Finding novel promoter sequences is a cornerstone of synthetic biology. To contribute to the expanding catalog of biological parts, we employed a promoter-trap approach to identify novel sequences within an Antarctic microbial community that act as broad host-range promoters functional in diverse Pseudomonadota. Using Pseudomonas putida KT2440 as host, we generated a library comprising approximately 2,000 clones resulting in the identification of thirteen functional promoter sequences, thereby expanding the genetic toolkit available for this chassis. Some of the discovered promoter sequences prove to be broad host-range as they drove gene expression not only in P. putida KT2440 but also in Escherichia coli DH5α, Cupriavidus taiwanensis R1T, Paraburkholderia phymatum STM 815T, Ensifer meliloti 1021, and an indigenous Antarctic bacterium, Pseudomonas sp. UYIF39. Our findings enrich the existing catalog of biological parts, offering a repertoire of broad host-range promoter sequences that exhibit functionality across diverse members of the phylum Pseudomonadota, proving Antarctic microbial community as a valuable resource for prospecting new biological parts for synthetic biology.
{"title":"Identification of novel broad host-range promoter sequences functional in diverse Pseudomonadota by a promoter-trap approach","authors":"Diego M. Roldan, Vanesa Amarelle","doi":"10.1101/2024.06.26.600856","DOIUrl":"https://doi.org/10.1101/2024.06.26.600856","url":null,"abstract":"Finding novel promoter sequences is a cornerstone of synthetic biology. To contribute to the expanding catalog of biological parts, we employed a promoter-trap approach to identify novel sequences within an Antarctic microbial community that act as broad host-range promoters functional in diverse Pseudomonadota. Using Pseudomonas putida KT2440 as host, we generated a library comprising approximately 2,000 clones resulting in the identification of thirteen functional promoter sequences, thereby expanding the genetic toolkit available for this chassis. Some of the discovered promoter sequences prove to be broad host-range as they drove gene expression not only in P. putida KT2440 but also in Escherichia coli DH5α, Cupriavidus taiwanensis R1T, Paraburkholderia phymatum STM 815T, Ensifer meliloti 1021, and an indigenous Antarctic bacterium, Pseudomonas sp. UYIF39. Our findings enrich the existing catalog of biological parts, offering a repertoire of broad host-range promoter sequences that exhibit functionality across diverse members of the phylum Pseudomonadota, proving Antarctic microbial community as a valuable resource for prospecting new biological parts for synthetic biology.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141503905","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}
Pub Date : 2024-06-27DOI: 10.1101/2024.06.26.600737
Guadalupe Alvarez Gonzalez, Micaela Chacon, Thomas Butterfield, Neil Dixon
Transcription factor-based biosensors are genetic tools that aim to predictability link the presence of a specific input stimuli to a tailored gene expression output. The performance characteristics of a biosensor fundamentally determines its potential applications. However, current methods to engineer and optimise tailored biosensor responses are highly nonintuitive, and struggle to investigate multidimensional sequence/design space efficiently. In this study we employ a design of experiments (DoE) approach to build a framework for efficiently engineering activator-based biosensors with tailored performances, and we apply the framework for the development of biosensors for the polyethylene terephthalate (PET) plastic degradation monomer terephthalate (TPA). We simultaneously engineer the core promoter and operator regions of the responsive promoter, and by employing a dual refactoring approach, we were able to explore an enhanced biosensor design space and assign their causative performance effects. The approach employed here serves as a foundational framework for engineering transcriptional biosensors and enabled development of tailored biosensors with enhanced dynamic range and diverse signal output, sensitivity, and steepness. We further demonstrate its applicability on the development of tailored biosensors for primary screening of PET hydrolases and enzyme condition screening, demonstrating the potential of statistical modelling in optimizing biosensors for tailored industrial and environmental applications.
{"title":"Tuning the performance of a TphR-based terephthalate biosensor with a design of experiments approach","authors":"Guadalupe Alvarez Gonzalez, Micaela Chacon, Thomas Butterfield, Neil Dixon","doi":"10.1101/2024.06.26.600737","DOIUrl":"https://doi.org/10.1101/2024.06.26.600737","url":null,"abstract":"Transcription factor-based biosensors are genetic tools that aim to predictability link the presence of a specific input stimuli to a tailored gene expression output. The performance characteristics of a biosensor fundamentally determines its potential applications. However, current methods to engineer and optimise tailored biosensor responses are highly nonintuitive, and struggle to investigate multidimensional sequence/design space efficiently. In this study we employ a design of experiments (DoE) approach to build a framework for efficiently engineering activator-based biosensors with tailored performances, and we apply the framework for the development of biosensors for the polyethylene terephthalate (PET) plastic degradation monomer terephthalate (TPA). We simultaneously engineer the core promoter and operator regions of the responsive promoter, and by employing a dual refactoring approach, we were able to explore an enhanced biosensor design space and assign their causative performance effects. The approach employed here serves as a foundational framework for engineering transcriptional biosensors and enabled development of tailored biosensors with enhanced dynamic range and diverse signal output, sensitivity, and steepness. We further demonstrate its applicability on the development of tailored biosensors for primary screening of PET hydrolases and enzyme condition screening, demonstrating the potential of statistical modelling in optimizing biosensors for tailored industrial and environmental applications.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"18 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141524889","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}
The reconstitution of a cell nucleus in a lipid bilayer-enclosed synthetic cell makes great strides in bottom-up synthetic biology. In this study, we propose a method for assembling a nucleus in giant unilamellar vesicles (GUVs). To induce reconstitution of the nucleus, we utilise interphase egg extract of African clawed frogs Xenopus laevis, known as a biochemically controllable cell-free system capable of transforming an added sperm chromatin into a nucleus in vitro. We enhanced GUV-formation efficiency by the inverted emulsion method through incorporating prolonged waiting time and adding chloroform into lipid-dispersed oil, facilitating subsequent nuclear assembly reactions in the GUVs. Characterisation of nucleus-like structures formed in the GUVs revealed the presence of dense DNA and accumulated GFP-NLS in the structure, indicative of functional nuclear import. Immunostaining further validated the presence of nuclear pore complexes on the surfaces of these nucleus-like structures. Moreover, we observed a positive correlation between sizes of GUV and nucleus-like structure/nucleus. Our approach provides insights into nuclear assembly in lipid bilayer-enclosed cell-like confinement and becomes a platform for constructing artificial cellular systems that closely mimic eukaryotic cells.
{"title":"Nuclear assembly in giant unilamellar vesicles encapsulating Xenopus egg extract","authors":"Sho Takamori, Hisatoshi Mimura, Toshihisa Osaki, Tomo Kondo, Miyuki Shintomi, Keishi Shintomi, Miho Ohsugi, Shoji Takeuchi","doi":"10.1101/2024.06.25.600006","DOIUrl":"https://doi.org/10.1101/2024.06.25.600006","url":null,"abstract":"The reconstitution of a cell nucleus in a lipid bilayer-enclosed synthetic cell makes great strides in bottom-up synthetic biology. In this study, we propose a method for assembling a nucleus in giant unilamellar vesicles (GUVs). To induce reconstitution of the nucleus, we utilise interphase egg extract of African clawed frogs Xenopus laevis, known as a biochemically controllable cell-free system capable of transforming an added sperm chromatin into a nucleus in vitro. We enhanced GUV-formation efficiency by the inverted emulsion method through incorporating prolonged waiting time and adding chloroform into lipid-dispersed oil, facilitating subsequent nuclear assembly reactions in the GUVs. Characterisation of nucleus-like structures formed in the GUVs revealed the presence of dense DNA and accumulated GFP-NLS in the structure, indicative of functional nuclear import. Immunostaining further validated the presence of nuclear pore complexes on the surfaces of these nucleus-like structures. Moreover, we observed a positive correlation between sizes of GUV and nucleus-like structure/nucleus. Our approach provides insights into nuclear assembly in lipid bilayer-enclosed cell-like confinement and becomes a platform for constructing artificial cellular systems that closely mimic eukaryotic cells.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141524999","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}
Pub Date : 2024-06-25DOI: 10.1101/2024.06.25.600629
Kasey S Love, Christopher P Johnstone, Emma L Peterman, Stephanie Gaglione, Kate E Galloway
To realize the potential of engineered cells in therapeutic applications, transgenes must be expressed within the window of therapeutic efficacy. Differences in copy number and other sources of extrinsic noise generate variance in transgene expression and limit the performance of synthetic gene circuits. In a therapeutic context, supraphysiological expression of transgenes can compromise engineered phenotypes and lead to toxicity. To ensure a narrow range of transgene expression, we design and characterize Compact microRNA-Mediated Attenuator of Noise and Dosage (ComMAND), a single-transcript, microRNA-based incoherent feedforward loop. We tune the ComMAND output profile, and we model the system to explore additional tuning strategies. By comparing ComMAND to two-gene implementations, we highlight the precise control afforded by the single-transcript architecture, particularly at relatively low copy numbers. We show that ComMAND tightly regulates transgene expression from lentiviruses and precisely controls expression in primary human T cells, primary rat neurons, primary mouse embryonic fibroblasts, and human induced pluripotent stem cells. Finally, ComMAND effectively sets levels of the clinically relevant transgenes FMRP1 and FXN within a narrow window. Together, ComMAND is a compact tool well-suited to precisely specify expression of therapeutic cargoes.
要实现工程细胞在治疗应用中的潜力，转基因必须在治疗效果窗口内表达。拷贝数的差异和其他外在噪声源会导致转基因表达的差异，并限制合成基因回路的性能。在治疗方面，转基因的超生理表达会损害工程表型并导致毒性。为了确保转基因的窄范围表达，我们设计并鉴定了基于单转录本、microRNA 的非相干前馈环路--Compact microRNA-Mediated Attenuator of Noise and Dosage (ComMAND)。我们调整了 ComMAND 的输出曲线，并建立了系统模型，以探索其他调整策略。通过将 ComMAND 与双基因实现进行比较，我们强调了单转录本架构所提供的精确控制，尤其是在拷贝数相对较低的情况下。我们的研究表明，ComMAND 能严格调控慢病毒的转基因表达，并能精确控制原代人类 T 细胞、原代大鼠神经元、原代小鼠胚胎成纤维细胞和人类诱导多能干细胞的表达。最后，ComMAND 能有效地将与临床相关的转基因 FMRP1 和 FXN 的水平设定在一个狭窄的窗口内。总之，ComMAND 是一种结构紧凑的工具，非常适合精确指定治疗载体的表达。
{"title":"Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells","authors":"Kasey S Love, Christopher P Johnstone, Emma L Peterman, Stephanie Gaglione, Kate E Galloway","doi":"10.1101/2024.06.25.600629","DOIUrl":"https://doi.org/10.1101/2024.06.25.600629","url":null,"abstract":"To realize the potential of engineered cells in therapeutic applications, transgenes must be expressed within the window of therapeutic efficacy. Differences in copy number and other sources of extrinsic noise generate variance in transgene expression and limit the performance of synthetic gene circuits. In a therapeutic context, supraphysiological expression of transgenes can compromise engineered phenotypes and lead to toxicity. To ensure a narrow range of transgene expression, we design and characterize Compact microRNA-Mediated Attenuator of Noise and Dosage (ComMAND), a single-transcript, microRNA-based incoherent feedforward loop. We tune the ComMAND output profile, and we model the system to explore additional tuning strategies. By comparing ComMAND to two-gene implementations, we highlight the precise control afforded by the single-transcript architecture, particularly at relatively low copy numbers. We show that ComMAND tightly regulates transgene expression from lentiviruses and precisely controls expression in primary human T cells, primary rat neurons, primary mouse embryonic fibroblasts, and human induced pluripotent stem cells. Finally, ComMAND effectively sets levels of the clinically relevant transgenes FMRP1 and FXN within a narrow window. Together, ComMAND is a compact tool well-suited to precisely specify expression of therapeutic cargoes.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"48 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141525001","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}
Pub Date : 2024-06-25DOI: 10.1101/2024.06.24.600531
Lin Wang, Li-shuang Zhang, Mei-ling Zhang, Ya-xin He, Yuan Yu, Ke Xu
Ethanol production from renewable cellulosic materials is a globally significant research area. However, the high temperatures and acetic acid generated during cellulose pretreatment can inhibit Saccharomyces cerevisiae growth, reducing ethanol yields. This study investigates the impact of glutaredoxin family genes (GRXs) over-expression on S. cerevisiae cell growth and fermentation performance under thermal and acetic acid stress. Engineered strains overexpressing GRX1, GRX2, and GRX5 demonstrated enhanced growth at 42 centigrade, while those overexpressing GRX1, GRX2, GRX6, and GRX7 showed improved growth at 1 g/L acetic acid. These results suggest that GRX over-expression can remediate S. cerevisiae, potentially accelerating advancements in green biomanufacturing.
{"title":"Effect of Over-expression of GRXs on Thermo and Acetic Acid Stress Tolerance of Saccharomyces cerevisiae","authors":"Lin Wang, Li-shuang Zhang, Mei-ling Zhang, Ya-xin He, Yuan Yu, Ke Xu","doi":"10.1101/2024.06.24.600531","DOIUrl":"https://doi.org/10.1101/2024.06.24.600531","url":null,"abstract":"Ethanol production from renewable cellulosic materials is a globally significant research area. However, the high temperatures and acetic acid generated during cellulose pretreatment can inhibit Saccharomyces cerevisiae growth, reducing ethanol yields. This study investigates the impact of glutaredoxin family genes (GRXs) over-expression on S. cerevisiae cell growth and fermentation performance under thermal and acetic acid stress. Engineered strains overexpressing GRX1, GRX2, and GRX5 demonstrated enhanced growth at 42 centigrade, while those overexpressing GRX1, GRX2, GRX6, and GRX7 showed improved growth at 1 g/L acetic acid. These results suggest that GRX over-expression can remediate S. cerevisiae, potentially accelerating advancements in green biomanufacturing.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"43 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141524998","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}
Pub Date : 2024-06-25DOI: 10.1101/2024.06.24.600474
Joaquin Caro Astorga, Koray Malci, Tom Ellis, Paul James, Erika Debenedictis, Hia Ming, Mat Rogan
Building DNA constructs of increasing complexity is key to synthetic biology. Golden Gate methods led to the creation of cloning toolkits - collections of modular standardized DNA parts hosted on hierarchic plasmids, developed for yeast, plants, Gram-negative bacteria, and human cells. However, Gram-positive bacteria have been neglected. Bacillus subtilis is a Gram-positive model organism and a workhorse in the bioindustry. Here, we present the SubtiToolKit, a high-efficiency cloning toolkit for B. subtilis and Gram-positive bacteria. Its design permits DNA constructs for transcriptional units, operons, knock-in and knock-out applications. It contains libraries of promoters, RBSs, fluorescent proteins, protein tags, terminators, genome integration parts, a no-leakage genetic device to control the expression of toxic products during E. coli assembly, and a toolbox for industrially relevant strains of Geobacillus and Parageobacillus as an example of SubtiToolKit versatility for other Gram-positive bacteria and its future perspective as a reference toolkit.
构建日益复杂的 DNA 结构是合成生物学的关键。金门方法催生了克隆工具包--寄存在分级质粒上的模块化标准化DNA部件集合，这些工具包是为酵母、植物、革兰氏阴性细菌和人类细胞开发的。然而，革兰氏阳性细菌却被忽视了。枯草芽孢杆菌是一种革兰氏阳性模式生物，也是生物产业的主力军。在这里，我们介绍一种用于枯草杆菌和革兰氏阳性菌的高效克隆工具包 SubtiToolKit。它的设计允许为转录单元、操作子、基因敲入和敲出应用构建 DNA。它包含启动子库、RBS、荧光蛋白、蛋白标签、终止子、基因组整合部件、用于控制大肠杆菌组装过程中有毒产物表达的无泄漏基因装置，以及用于工业相关革兰氏阳性菌株和副革兰氏阳性菌株的工具箱，作为 SubtiToolKit 可用于其他革兰氏阳性菌的多功能性及其作为参考工具箱的未来前景的范例。
{"title":"SubtiToolKit - a bioengineering kit for Bacillus subtilis and Gram-positive bacteria.","authors":"Joaquin Caro Astorga, Koray Malci, Tom Ellis, Paul James, Erika Debenedictis, Hia Ming, Mat Rogan","doi":"10.1101/2024.06.24.600474","DOIUrl":"https://doi.org/10.1101/2024.06.24.600474","url":null,"abstract":"Building DNA constructs of increasing complexity is key to synthetic biology. Golden Gate methods led to the creation of cloning toolkits - collections of modular standardized DNA parts hosted on hierarchic plasmids, developed for yeast, plants, Gram-negative bacteria, and human cells. However, Gram-positive bacteria have been neglected. Bacillus subtilis is a Gram-positive model organism and a workhorse in the bioindustry. Here, we present the SubtiToolKit, a high-efficiency cloning toolkit for B. subtilis and Gram-positive bacteria. Its design permits DNA constructs for transcriptional units, operons, knock-in and knock-out applications. It contains libraries of promoters, RBSs, fluorescent proteins, protein tags, terminators, genome integration parts, a no-leakage genetic device to control the expression of toxic products during E. coli assembly, and a toolbox for industrially relevant strains of Geobacillus and Parageobacillus as an example of SubtiToolKit versatility for other Gram-positive bacteria and its future perspective as a reference toolkit.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"28 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141525000","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}
Pub Date : 2024-06-24DOI: 10.1101/2024.06.23.600301
Fankang Meng, William M Shaw, Yue Kei Keith Kam, Tom Ellis
Coordination of behaviour in multicellular systems is one the main ways that nature increases the complexity of biological function in organisms and communities. While Saccharomyces cerevisiae yeast is used extensively in research and biotechnology, it is a unicellular organism capable of only limited multicellular states. Here we expand the possibilities for engineering multicellular behaviours in yeast by developing modular toolkits for two key mechanisms seen in multicellularity, contact-dependent signalling and specific cell-to-cell adhesion. MARS (Mating-peptide Anchored Response System) is a toolkit based on surface-displayed fungal mating peptides and G protein-coupled receptor (GPCR) signalling which can mimic juxtacrine signalling between yeasts. SATURN (Saccharomyces Adhesion Toolkit for multicellUlar patteRNing) surface displays adhesion-proteins pairs on yeasts and facilitates the creation of cell aggregation patterns. Together they can be used to create multicellular logic circuits, equivalent to developmental programs that lead to cell differentiation based on the local population. Using MARS and SATURN, we further developed JUPITER (JUxtacrine sensor for Protein-protein InTERaction), a genetic sensor for assaying protein-protein interactions in culture, demonstrating this as a tool to select for high affinity binders among a population of mutated nanobodies. Collectively, MARS, SATURN, and JUPITER present valuable tools that facilitate the engineering of complex multicellularity with yeast and expand the scope of its biotechnological applications.
多细胞系统中的行为协调是大自然增加生物体和群落中生物功能复杂性的主要方式之一。虽然酵母菌被广泛用于研究和生物技术领域，但它是一种单细胞生物，只能实现有限的多细胞状态。在这里，我们开发了模块化工具包，用于多细胞性中的两个关键机制，即依赖接触的信号传导和特定的细胞间粘附，从而拓展了酵母多细胞行为工程的可能性。MARS（交配肽锚定反应系统）是一个基于表面显示的真菌交配肽和G蛋白偶联受体（GPCR）信号的工具包，它可以模拟酵母菌之间的共生信号。SATURN（酵母菌多细胞粘附工具包）表面可显示酵母菌上的粘附蛋白对，并有助于创建细胞聚集模式。它们可共同用于创建多细胞逻辑电路，相当于根据本地群体进行细胞分化的发育程序。利用 MARS 和 SATURN，我们进一步开发了 JUPITER（用于蛋白质-蛋白质相互作用的 JUxtacrine 传感器），这是一种用于检测培养物中蛋白质-蛋白质相互作用的基因传感器。总之，MARS、SATURN 和 JUPITER 提供了宝贵的工具，有助于利用酵母进行复杂的多细胞工程，并扩大了生物技术的应用范围。
{"title":"Engineered yeast multicellularity via synthetic cell-cell adhesion and direct-contact signalling","authors":"Fankang Meng, William M Shaw, Yue Kei Keith Kam, Tom Ellis","doi":"10.1101/2024.06.23.600301","DOIUrl":"https://doi.org/10.1101/2024.06.23.600301","url":null,"abstract":"Coordination of behaviour in multicellular systems is one the main ways that nature increases the complexity of biological function in organisms and communities. While Saccharomyces cerevisiae yeast is used extensively in research and biotechnology, it is a unicellular organism capable of only limited multicellular states. Here we expand the possibilities for engineering multicellular behaviours in yeast by developing modular toolkits for two key mechanisms seen in multicellularity, contact-dependent signalling and specific cell-to-cell adhesion. MARS (Mating-peptide Anchored Response System) is a toolkit based on surface-displayed fungal mating peptides and G protein-coupled receptor (GPCR) signalling which can mimic juxtacrine signalling between yeasts. SATURN (Saccharomyces Adhesion Toolkit for multicellUlar patteRNing) surface displays adhesion-proteins pairs on yeasts and facilitates the creation of cell aggregation patterns. Together they can be used to create multicellular logic circuits, equivalent to developmental programs that lead to cell differentiation based on the local population. Using MARS and SATURN, we further developed JUPITER (JUxtacrine sensor for Protein-protein InTERaction), a genetic sensor for assaying protein-protein interactions in culture, demonstrating this as a tool to select for high affinity binders among a population of mutated nanobodies. Collectively, MARS, SATURN, and JUPITER present valuable tools that facilitate the engineering of complex multicellularity with yeast and expand the scope of its biotechnological applications.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"2015 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141503906","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}
Pub Date : 2024-06-22DOI: 10.1101/2024.06.22.600171
Stijn T. de Vries, Tania S. Koebel, Ahmet Sanal, Daniel Schindler
Golden Gate cloning has become one of the most important DNA assembly strategies. The construction of standardized and reusable part libraries, their assembly into transcription units, and the subsequent assembly of multigene constructs is highly reliable and sustainable. Researchers can quickly construct derivatives of their assemblies or entire pathways, and importantly, the standardization of Golden Gate assemblies is compatible with laboratory automation. Most Golden Gate strategies rely on four nucleotide overhangs generated by commonly used Type IIS enzymes. However, reduction to three nucleotide overhangs allows the use of codons as fusion sites and reduces potential scar sequences. This is particularly important when studying biological functions, as additional nucleotides may alter the structure or stability of the transcribed RNA. To address this issue we use SapI, a Type IIS enzyme generating three nucleotide overhangs, for transcription unit assembly, allowing for codon-based fusion in coding sequences. We created a corresponding plasmid toolbox for basic part generation and transcription unit assembly, a workflow we term In-Cloning. In-Cloning is downstream compatible with the Modular Cloning standard developed by Sylvestre Marillonnet's group for standardized assembly of multigene constructs. However, the multigene construct plasmids may not be compatible for use with the model organism of choice. Therefore, we have developed a workflow called Out-Cloning to rapidly generate Golden Gate acceptor plasmids. Out-Cloning uses standardized plasmid parts that are assembled into Golden Gate acceptor plasmids using flexible linkers. This allows the systematic construction of acceptor plasmids needed to transfer assembled DNA into the organism of interest.
{"title":"In- & Out-Cloning: Plasmid toolboxes for scarless transcription unit and modular Golden Gate acceptor plasmid assembly","authors":"Stijn T. de Vries, Tania S. Koebel, Ahmet Sanal, Daniel Schindler","doi":"10.1101/2024.06.22.600171","DOIUrl":"https://doi.org/10.1101/2024.06.22.600171","url":null,"abstract":"Golden Gate cloning has become one of the most important DNA assembly strategies. The construction of standardized and reusable part libraries, their assembly into transcription units, and the subsequent assembly of multigene constructs is highly reliable and sustainable. Researchers can quickly construct derivatives of their assemblies or entire pathways, and importantly, the standardization of Golden Gate assemblies is compatible with laboratory automation. Most Golden Gate strategies rely on four nucleotide overhangs generated by commonly used Type IIS enzymes. However, reduction to three nucleotide overhangs allows the use of codons as fusion sites and reduces potential scar sequences. This is particularly important when studying biological functions, as additional nucleotides may alter the structure or stability of the transcribed RNA. To address this issue we use SapI, a Type IIS enzyme generating three nucleotide overhangs, for transcription unit assembly, allowing for codon-based fusion in coding sequences. We created a corresponding plasmid toolbox for basic part generation and transcription unit assembly, a workflow we term In-Cloning. In-Cloning is downstream compatible with the Modular Cloning standard developed by Sylvestre Marillonnet's group for standardized assembly of multigene constructs. However, the multigene construct plasmids may not be compatible for use with the model organism of choice. Therefore, we have developed a workflow called Out-Cloning to rapidly generate Golden Gate acceptor plasmids. Out-Cloning uses standardized plasmid parts that are assembled into Golden Gate acceptor plasmids using flexible linkers. This allows the systematic construction of acceptor plasmids needed to transfer assembled DNA into the organism of interest.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141503907","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}
Pub Date : 2024-06-21DOI: 10.1101/2024.06.20.599979
Juli Wang, Guanqun Gavin Chen
Producing high-value plant-derived unusual fatty acids in microorganisms via synthetic biology is attractive, but increasing their contents through rational step-by-step gene stacking is challenging. Using Saccharomyces cerevisiae and pomegranate-derived punicic acid (PuA) as representatives, an efficient, result-driven gene shuffling strategy was developed to facilitate the production of value-added plant lipids. By targeting yeast Ty retrotransposon regions, candidate genes related to PuA production were directly shuffled within the yeast genome to create recombinant libraries. Subsequent screening and bioprocess optimization led to a recombinant yeast strain with 26.7% of total fatty acids as PuA through neosynthesis. Further analyses revealed that the strain hosts multiple genes, contains over 22% PuA in the storage lipid triacylglycerol, and has substantial changes in its lipidome. Overall, this work provided an efficient strategy for improving PuA content in yeast, which could be adopted to engineer microorganisms for the production of other high-value plant-derived fatty acids and bioproducts.
{"title":"Optimization of plant-derived punicic acid synthesis in Saccharomyces cerevisiae by Ty retrotransposon-targeted random gene shuffling","authors":"Juli Wang, Guanqun Gavin Chen","doi":"10.1101/2024.06.20.599979","DOIUrl":"https://doi.org/10.1101/2024.06.20.599979","url":null,"abstract":"Producing high-value plant-derived unusual fatty acids in microorganisms via synthetic biology is attractive, but increasing their contents through rational step-by-step gene stacking is challenging. Using Saccharomyces cerevisiae and pomegranate-derived punicic acid (PuA) as representatives, an efficient, result-driven gene shuffling strategy was developed to facilitate the production of value-added plant lipids. By targeting yeast Ty retrotransposon regions, candidate genes related to PuA production were directly shuffled within the yeast genome to create recombinant libraries. Subsequent screening and bioprocess optimization led to a recombinant yeast strain with 26.7% of total fatty acids as PuA through neosynthesis. Further analyses revealed that the strain hosts multiple genes, contains over 22% PuA in the storage lipid triacylglycerol, and has substantial changes in its lipidome. Overall, this work provided an efficient strategy for improving PuA content in yeast, which could be adopted to engineer microorganisms for the production of other high-value plant-derived fatty acids and bioproducts.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"36 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141525002","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}
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