Pub Date : 2024-09-18DOI: 10.1101/2024.09.17.611575
xiaoshu ma, lei yang, hua ye
Saccharomyces cerevisiae is widely used in DNA assembly due to their efficient homologous recombination [1], but DNA assembly through yeast recombination in vivo usually requires the vector to have the ability to replicate in yeast. The CRISPR-Cas9 system can efficiently edit DNA [2,3], and the system can also be used for DNA editing of plasmids. In this paper, a yeast universal element is selected, which can be inserted into the vector, so that the vector can replicate in yeast cells, and then the intermediate plasmid containing yeast universal element can be obtained by recombination in yeast. At the same time, a pCas-SmR plasmid was designed in this paper. After Donor DNA is added, the CRISPR-Cas9 system can accurately and efficiently knock out the yeast universal element in the intermediate plasmid, remove the pCas-SmR plasmid through sucrose screening, and finally obtain a pure plasmid. Saccharomyces cerevisiae cells are widely used in DNA assembly due to their efficient homologous recombination [1], but DNA assembly through yeast recombination in vivo usually requires the vector to have the ability to replicate in yeast. The CRISPR-Cas9 system can efficiently edit DNA [2,3], and the system can also be used for DNA editing of plasmids. In this paper, a yeast universal element is selected, which can be inserted into the vector, so that the vector has the ability to replicate in yeast cells, and then the intermediate plasmid containing yeast universal element can be obtained by recombination in yeast. At the same time, a pCas-SmR plasmid was designed in this paper. After Donor DNA is added, the CRISPR-Cas9 system can accurately and efficiently knock out the yeast universal element in the intermediate plasmid, remove the pCas-SmR plasmid through sucrose screening, and finally obtain a pure knocked out plasmid.
酵母菌因其高效的同源重组而被广泛用于 DNA 组装[1]，但在体内通过酵母重组进行 DNA 组装通常需要载体具有在酵母中复制的能力。CRISPR-Cas9 系统可以高效地编辑 DNA [2,3]，该系统也可用于质粒的 DNA 编辑。本文选择了一种酵母通用元件，将其插入载体中，使载体可以在酵母细胞中复制，然后通过酵母重组获得含有酵母通用元件的中间质粒。同时，本文还设计了一种 pCas-SmR 质粒。加入Donor DNA后，CRISPR-Cas9系统可以准确有效地敲除中间质粒中的酵母通用元件，并通过蔗糖筛选去除pCas-SmR质粒，最终获得纯质粒。酵母细胞因其高效的同源重组而被广泛应用于DNA组装[1]，但在体内通过酵母重组进行DNA组装通常需要载体具有在酵母体内复制的能力。CRISPR-Cas9 系统可以高效地编辑 DNA [2,3]，该系统也可用于质粒的 DNA 编辑。本文选择了一种酵母通用元件，将其插入载体中，使载体具有在酵母细胞中复制的能力，然后通过酵母重组获得含有酵母通用元件的中间质粒。同时，本文还设计了一种 pCas-SmR 质粒。加入Donor DNA后，CRISPR-Cas9系统可以准确高效地敲除中间质粒中的酵母通用元件，并通过蔗糖筛选去除pCas-SmR质粒，最终获得纯合的敲除质粒。
{"title":"Cas9AEY (Cas9-facilitated Homologous Recombination Assembly of non-specific Escherichia coli yeast vector) method of constructing large-sized DNA.","authors":"xiaoshu ma, lei yang, hua ye","doi":"10.1101/2024.09.17.611575","DOIUrl":"https://doi.org/10.1101/2024.09.17.611575","url":null,"abstract":"Saccharomyces cerevisiae is widely used in DNA assembly due to their efficient homologous recombination [1], but DNA assembly through yeast recombination in vivo usually requires the vector to have the ability to replicate in yeast. The CRISPR-Cas9 system can efficiently edit DNA [2,3], and the system can also be used for DNA editing of plasmids. In this paper, a yeast universal element is selected, which can be inserted into the vector, so that the vector can replicate in yeast cells, and then the intermediate plasmid containing yeast universal element can be obtained by recombination in yeast. At the same time, a pCas-SmR plasmid was designed in this paper. After Donor DNA is added, the CRISPR-Cas9 system can accurately and efficiently knock out the yeast universal element in the intermediate plasmid, remove the pCas-SmR plasmid through sucrose screening, and finally obtain a pure plasmid. Saccharomyces cerevisiae cells are widely used in DNA assembly due to their efficient homologous recombination [1], but DNA assembly through yeast recombination in vivo usually requires the vector to have the ability to replicate in yeast. The CRISPR-Cas9 system can efficiently edit DNA [2,3], and the system can also be used for DNA editing of plasmids. In this paper, a yeast universal element is selected, which can be inserted into the vector, so that the vector has the ability to replicate in yeast cells, and then the intermediate plasmid containing yeast universal element can be obtained by recombination in yeast. At the same time, a pCas-SmR plasmid was designed in this paper. After Donor DNA is added, the CRISPR-Cas9 system can accurately and efficiently knock out the yeast universal element in the intermediate plasmid, remove the pCas-SmR plasmid through sucrose screening, and finally obtain a pure knocked out plasmid.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"52 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250791","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 : 2024-09-18DOI: 10.1101/2024.09.18.613743
Rong Zheng, Abhay Prasad, Deeksha Satyabola, Yang Xu, Hao Yan
Constraining proximity-based drugs, such as proteolysis-targeting chimeras (PROTACs), into its bioactive conformation can significantly impact their selectivity and potency. However, traditional methods for achieving this often involve complex and time-consuming synthetic procedures. Here, we introduced an alternative approach by demonstrating DNA-templated spatially controlled PROTACs (DTACs), which leverage the programmability of nucleic-acid based self-assembly for efficient synthesis, providing precise control over inhibitors spacing and orientation. The resulting constructs revealed distance-and orientation-dependent selectivity and degradation potency for the CyclinD1-CDK4/6 protein complex in cancer cells. Notably, an optimal construct DTAC-V1 demonstrated the unprecedented synchronous degradation of entire CyclinD1-CDK4/6 complex. This resulted in the effective cell cycle arrest in G1 phase, and further therapeutic studies showed its potent anti-tumor effects compared to inhibitors alone. These findings present a novel framework for PROTACs design, offering critical insights that may inform the development of other proximity-induced therapeutic modalities.
{"title":"DNA-templated spatially controlled proteolysis targeting chimeras for CyclinD1-CDK4/6 complex protein degradation","authors":"Rong Zheng, Abhay Prasad, Deeksha Satyabola, Yang Xu, Hao Yan","doi":"10.1101/2024.09.18.613743","DOIUrl":"https://doi.org/10.1101/2024.09.18.613743","url":null,"abstract":"Constraining proximity-based drugs, such as proteolysis-targeting chimeras (PROTACs), into its bioactive conformation can significantly impact their selectivity and potency. However, traditional methods for achieving this often involve complex and time-consuming synthetic procedures. Here, we introduced an alternative approach by demonstrating DNA-templated spatially controlled PROTACs (DTACs), which leverage the programmability of nucleic-acid based self-assembly for efficient synthesis, providing precise control over inhibitors spacing and orientation. The resulting constructs revealed distance-and orientation-dependent selectivity and degradation potency for the CyclinD1-CDK4/6 protein complex in cancer cells. Notably, an optimal construct DTAC-V1 demonstrated the unprecedented synchronous degradation of entire CyclinD1-CDK4/6 complex. This resulted in the effective cell cycle arrest in G1 phase, and further therapeutic studies showed its potent anti-tumor effects compared to inhibitors alone. These findings present a novel framework for PROTACs design, offering critical insights that may inform the development of other proximity-induced therapeutic modalities.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250790","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 : 2024-09-17DOI: 10.1101/2024.09.17.613521
Andrew P Hren, Joshua P Abraham, Melissa P. Tumen-Velasquez, Michael Melesse Vergara, Adam M Guss, William G Alexander, Brian F Pfleger, Jerome M Fox, Carrie A Eckert
Cyanobacteria are promising microbial platforms for a myriad of biotechnological applications, from sustainable biomaterials to photosynthetic chemical production, but still lack the breadth of genetic tools available for more commonly engineered microbes such as Escherichia coli. This study develops genetic tools to enhance the transformation efficiency and heterologous gene expression in Picosynechococcus sp. PCC 7002, a fast-growing, halotolerant, and naturally competent strain. Integration of fluorescent reporter cassettes across the genome revealed an integration site that yields a fourfold improvement in gene expression relative to previously reported sites. Protocol optimization and engineered DNA methylation in E. coli increased transformation efficiency by over tenfold. This work provides an experimental framework for efficient genome editing and metabolic engineering in the model cyanobacterium PCC 7002.
{"title":"High-efficiency transformation and gene expression in Picosynechococcus sp. PCC 7002","authors":"Andrew P Hren, Joshua P Abraham, Melissa P. Tumen-Velasquez, Michael Melesse Vergara, Adam M Guss, William G Alexander, Brian F Pfleger, Jerome M Fox, Carrie A Eckert","doi":"10.1101/2024.09.17.613521","DOIUrl":"https://doi.org/10.1101/2024.09.17.613521","url":null,"abstract":"Cyanobacteria are promising microbial platforms for a myriad of biotechnological applications, from sustainable biomaterials to photosynthetic chemical production, but still lack the breadth of genetic tools available for more commonly engineered microbes such as <em>Escherichia coli</em>. This study develops genetic tools to enhance the transformation efficiency and heterologous gene expression in <em>Picosynechococcus</em> sp. PCC 7002, a fast-growing, halotolerant, and naturally competent strain. Integration of fluorescent reporter cassettes across the genome revealed an integration site that yields a fourfold improvement in gene expression relative to previously reported sites. Protocol optimization and engineered DNA methylation in <em>E. coli</em> increased transformation efficiency by over tenfold. This work provides an experimental framework for efficient genome editing and metabolic engineering in the model cyanobacterium PCC 7002.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"49 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250799","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 : 2024-09-17DOI: 10.1101/2024.09.17.613466
Anupama K Puppala, Andrew C Nielsen, Maureen R Regan, Georgina E Mancinelli, Renee F DePooter, Stephen Arnovitz, Caspian Harding, Michaele McGregor, Nikolas G Balanis, Ryan Clarke, Brad J Merrill
Although genomes encode instructions for mammalian cell differentiation with rich syntactic relationships, existing methods for genetically programming cells have modest capabilities for stepwise regulation of genes. Here, we developed a sequential genetic system that enables transcriptional activation of endogenous genes in a preprogrammed, stepwise manner. The system relies on the removal of an RNA polymerase III termination signal to induce both the transcriptional activation and the DNA endonuclease activities of a Cas9-VPR protein to effect stepwise progression through cascades of gene activation events. The efficiency of the cascading system enables a new dimension for cellular programming by allowing the manipulation of the sequential order of gene activation for directing the differentiation of human stem cells.
尽管基因组编码的哺乳动物细胞分化指令具有丰富的句法关系，但现有的细胞基因编程方法在逐步调控基因方面能力有限。在这里，我们开发了一种顺序遗传系统，能以预编程的方式逐步激活内源基因的转录。该系统依靠去除 RNA 聚合酶 III 终止信号来诱导 Cas9-VPR 蛋白的转录激活和 DNA 内切酶活性，从而通过级联基因激活事件实现逐步推进。级联系统的高效性为细胞编程提供了一个新的维度，它允许操纵基因激活的顺序来指导人类干细胞的分化。
{"title":"A modular system for programming multistep activation of endogenous genes in stem cells","authors":"Anupama K Puppala, Andrew C Nielsen, Maureen R Regan, Georgina E Mancinelli, Renee F DePooter, Stephen Arnovitz, Caspian Harding, Michaele McGregor, Nikolas G Balanis, Ryan Clarke, Brad J Merrill","doi":"10.1101/2024.09.17.613466","DOIUrl":"https://doi.org/10.1101/2024.09.17.613466","url":null,"abstract":"Although genomes encode instructions for mammalian cell differentiation with rich syntactic relationships, existing methods for genetically programming cells have modest capabilities for stepwise regulation of genes. Here, we developed a sequential genetic system that enables transcriptional activation of endogenous genes in a preprogrammed, stepwise manner. The system relies on the removal of an RNA polymerase III termination signal to induce both the transcriptional activation and the DNA endonuclease activities of a Cas9-VPR protein to effect stepwise progression through cascades of gene activation events. The efficiency of the cascading system enables a new dimension for cellular programming by allowing the manipulation of the sequential order of gene activation for directing the differentiation of human stem cells.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"53 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250794","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 : 2024-09-17DOI: 10.1101/2024.09.17.613456
Carolina Jerez-Longres, Wilfried Weber
Living natural materials have remarkable sensing abilities that translate external cues into functional changes of the material. The reconstruction of such sensing materials in bottom-up synthetic biology provides the opportunity to develop synthetic materials with life-like sensing and adaptation ability. Key to such functions are material modules that translate specific input signals into a biomolecular response. Here, we engineer a synthetic organelle based on liquid-liquid phase separation that translates a metabolic signal into the regulation of gene transcription. To this aim, we engineer the pyruvate-dependent repressor PdhR to undergo liquid-liquid phase separation in vitro by fusion to intrinsically disordered regions. We demonstrate that the resulting coacervates bind DNA harbouring PdhR-responsive operator sites in a pyruvate dose-dependent and reversible manner. We observed that the activity of transcription units on the DNA was strongly attenuated following recruitment to the coacervates. However, the addition of pyruvate resulted in a reversible and dose-dependent reconstitution of transcriptional activity. The coacervate-based synthetic organelles linking metabolic cues to transcriptional signals represent a materials approach to confer stimulus-responsiveness to minimal bottom-up synthetic biological systems and open opportunities in materials for sensor applications.
{"title":"Metabolite-responsive Control of Transcription by Phase Separation-based Synthetic Organelles","authors":"Carolina Jerez-Longres, Wilfried Weber","doi":"10.1101/2024.09.17.613456","DOIUrl":"https://doi.org/10.1101/2024.09.17.613456","url":null,"abstract":"Living natural materials have remarkable sensing abilities that translate external cues into functional changes of the material. The reconstruction of such sensing materials in bottom-up synthetic biology provides the opportunity to develop synthetic materials with life-like sensing and adaptation ability. Key to such functions are material modules that translate specific input signals into a biomolecular response. Here, we engineer a synthetic organelle based on liquid-liquid phase separation that translates a metabolic signal into the regulation of gene transcription. To this aim, we engineer the pyruvate-dependent repressor PdhR to undergo liquid-liquid phase separation in vitro by fusion to intrinsically disordered regions. We demonstrate that the resulting coacervates bind DNA harbouring PdhR-responsive operator sites in a pyruvate dose-dependent and reversible manner. We observed that the activity of transcription units on the DNA was strongly attenuated following recruitment to the coacervates. However, the addition of pyruvate resulted in a reversible and dose-dependent reconstitution of transcriptional activity. The coacervate-based synthetic organelles linking metabolic cues to transcriptional signals represent a materials approach to confer stimulus-responsiveness to minimal bottom-up synthetic biological systems and open opportunities in materials for sensor applications.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250793","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 : 2024-09-17DOI: 10.1101/2024.09.17.613417
Nishu Kanwa, Shunshi Kohyama, Leonard Fröhlich, Amogh Desai, Petra Schwille
Cell membranes in bacteria are laterally polarized to produce specific environments for membrane proteins, e.g., proteins involved in cell division which accumulate at mid-cell or the cell poles. An interesting result of such membrane-lipid interplay is the reorganization of lipid domains together with membrane-bound proteins at the onset of cell division, suggesting a functional significance of membrane compartments in the cell cycle. Here, by adopting the key bacterial division proteins MinCDE and FtsZ as an archetypal spatial patterning system, we present a simple vesicle-based in vitro model to explore the mutual dependence of protein pattern formation and membrane heterogeneity. Like many other peripheral membrane proteins, MinDE exhibit preferential binding and macro-scale pattern formation at Ld domains, which leads to altered oscillation mode selection in phase-separated membrane compartments (GUVs). Moreover, incorporating bacterial division proteins within phase-separated GUVs leads to blebbing-like membrane deformations followed by the reorganization of Lo domains aligning at the neck region of the bleb, which agrees well with the domain rearrangement in bacterial membranes immediately preceding the radial constriction process. Overall, the presented in vitro model system showcases a basic framework to better comprehend the cellular division mechanism in consideration of complex cellular lipid environments.
细菌的细胞膜是横向极化的，为膜蛋白创造了特定的环境，例如，参与细胞分裂的蛋白质聚集在细胞中部或细胞两极。这种膜-脂相互作用的一个有趣结果是，在细胞分裂开始时，脂质结构域与膜结合蛋白一起重组，这表明膜区在细胞周期中具有功能意义。在这里，我们以关键的细菌分裂蛋白 MinCDE 和 FtsZ 为原型空间模式系统，提出了一个简单的基于囊泡的体外模型，以探索蛋白模式形成与膜异质性的相互依存关系。与许多其他外周膜蛋白一样，MinDE在Ld结构域表现出优先结合和大尺度模式形成，这导致相分离膜区（GUVs）中振荡模式选择的改变。此外，在相分离的 GUVs 中加入细菌分裂蛋白会导致类似裂片的膜变形，随后在裂片颈部区域对齐的 Lo 结构域会重组，这与紧接着径向收缩过程之前细菌膜中的结构域重组非常吻合。总之，所介绍的体外模型系统展示了一个基本框架，以便在考虑复杂的细胞脂质环境时更好地理解细胞分裂机制。
{"title":"Mutual dependence between membrane phase separation and bacterial division protein dynamics in synthetic cell models","authors":"Nishu Kanwa, Shunshi Kohyama, Leonard Fröhlich, Amogh Desai, Petra Schwille","doi":"10.1101/2024.09.17.613417","DOIUrl":"https://doi.org/10.1101/2024.09.17.613417","url":null,"abstract":"Cell membranes in bacteria are laterally polarized to produce specific environments for membrane proteins, e.g., proteins involved in cell division which accumulate at mid-cell or the cell poles. An interesting result of such membrane-lipid interplay is the reorganization of lipid domains together with membrane-bound proteins at the onset of cell division, suggesting a functional significance of membrane compartments in the cell cycle. Here, by adopting the key bacterial division proteins MinCDE and FtsZ as an archetypal spatial patterning system, we present a simple vesicle-based in vitro model to explore the mutual dependence of protein pattern formation and membrane heterogeneity. Like many other peripheral membrane proteins, MinDE exhibit preferential binding and macro-scale pattern formation at Ld domains, which leads to altered oscillation mode selection in phase-separated membrane compartments (GUVs). Moreover, incorporating bacterial division proteins within phase-separated GUVs leads to blebbing-like membrane deformations followed by the reorganization of Lo domains aligning at the neck region of the bleb, which agrees well with the domain rearrangement in bacterial membranes immediately preceding the radial constriction process. Overall, the presented in vitro model system showcases a basic framework to better comprehend the cellular division mechanism in consideration of complex cellular lipid environments.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"40 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250795","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 global crisis of polyethylene terephthalate (PET) waste demands innovative solutions for sustainable management. Current approaches are often inefficient, energy-intensive, and result in incomplete depolymerization. Here, we introduce SPEED (Scaffold-enabled PET Enzyme Ensemble-augmented Degradation), a transformative biocatalytic platform engineered for the superior degradation across diverse PET substrates. Through the strategic combination of complementary PET hydrolases on a tailored protein nano-scaffold and extensive optimization, SPEED achieves near-complete depolymerization of PET into its constituent monomers, exceeding existing biocatalytic systems' efficiency by up to two orders of magnitude. The platform's versatility and industrial relevance are further demonstrated through successful integration with metal-organic frameworks (MOFs) for enhanced stability and reusability, enabling PET upcycling into valuable products, and its compatibility with a yeast-based live cell system for surface display. SPEED's high efficiency, adaptability, and cost-effectiveness position it as a powerful technology to accelerate sustainable plastic waste management and drive a circular PET economy.
全球聚对苯二甲酸乙二酯（PET）废弃物危机需要创新的可持续管理解决方案。目前的方法往往效率低下、能耗高，而且导致解聚不完全。在此，我们介绍 SPEED（Scaffold-enabled PET Enzyme Ensemble-augmented Degradation），这是一个变革性的生物催化平台，专为降解各种 PET 底物而设计。通过将互补的 PET水解酶战略性地结合到定制的蛋白质纳米支架上并进行广泛的优化，SPEED 实现了将 PET 近乎完全地解聚成其组成单体，其效率比现有的生物催化系统高出两个数量级。通过与金属有机框架（MOFs）的成功整合，该平台的多功能性和工业相关性得到了进一步证明，MOFs 可增强稳定性和可再利用性，使 PET 可以循环利用，转化为有价值的产品，而且该平台与基于酵母的活细胞系统兼容，可进行表面展示。SPEED 的高效率、适应性和成本效益使其成为加速可持续塑料废物管理和推动 PET 循环经济的强大技术。
{"title":"Modular Nano-Scaffold Biocatalysis for Superior PET Depolymerization and Valorization","authors":"Yujia Zhang, Chongsen Li, Ehsan Hashemi, Enting Xu, Xuemei Yang, Yanbing Lin, Hui Gao, Zhuobin Liang","doi":"10.1101/2024.09.16.613172","DOIUrl":"https://doi.org/10.1101/2024.09.16.613172","url":null,"abstract":"The global crisis of polyethylene terephthalate (PET) waste demands innovative solutions for sustainable management. Current approaches are often inefficient, energy-intensive, and result in incomplete depolymerization. Here, we introduce SPEED (Scaffold-enabled PET Enzyme Ensemble-augmented Degradation), a transformative biocatalytic platform engineered for the superior degradation across diverse PET substrates. Through the strategic combination of complementary PET hydrolases on a tailored protein nano-scaffold and extensive optimization, SPEED achieves near-complete depolymerization of PET into its constituent monomers, exceeding existing biocatalytic systems' efficiency by up to two orders of magnitude. The platform's versatility and industrial relevance are further demonstrated through successful integration with metal-organic frameworks (MOFs) for enhanced stability and reusability, enabling PET upcycling into valuable products, and its compatibility with a yeast-based live cell system for surface display. SPEED's high efficiency, adaptability, and cost-effectiveness position it as a powerful technology to accelerate sustainable plastic waste management and drive a circular PET economy.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"152 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250796","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}
In plant mitochondria and chloroplasts, cytidine-to-uridine RNA editing plays a crucial role in regulating gene expression. While natural PLS-type PPR proteins are specialized in this process, synthetic PPR proteins offer significant potential for targeted RNA editing. In this study, we engineered chimeric editing factors by fusing synthetic P-type PPR guides with the DYW cytidine deaminase domain of a moss mitochondrial editing factor, PPR56. These designer PPR editors (dPPRe) elicited efficient and precise de novo RNA editing in Escherichia coli, and in Nicotiana benthamiana chloroplasts and mitochondria. Chloroplast transcriptome-wide analysis of the most efficient dPPRe revealed minimal off-target effects, with only three non-target C sites edited due to sequence similarity with the intended target. This study introduces a novel and precise method for RNA base editing in plant organelles, paving the way for new approaches in gene regulation applicable to plants and potentially other organisms.
{"title":"De novo RNA base editing in plant organelles with engineered synthetic P-type PPR editing factors","authors":"Sebastien Mathieu, Elena Lesch, Shahinez Garcia, Stefanie Graindorge, Mareike Schallenberg-Rudinger, Kamel Hammani","doi":"10.1101/2024.09.13.612905","DOIUrl":"https://doi.org/10.1101/2024.09.13.612905","url":null,"abstract":"In plant mitochondria and chloroplasts, cytidine-to-uridine RNA editing plays a crucial role in regulating gene expression. While natural PLS-type PPR proteins are specialized in this process, synthetic PPR proteins offer significant potential for targeted RNA editing. In this study, we engineered chimeric editing factors by fusing synthetic P-type PPR guides with the DYW cytidine deaminase domain of a moss mitochondrial editing factor, PPR56. These designer PPR editors (dPPRe) elicited efficient and precise <em>de novo</em> RNA editing in <em>Escherichia coli</em>, and in <em>Nicotiana benthamiana</em> chloroplasts and mitochondria. Chloroplast transcriptome-wide analysis of the most efficient dPPRe revealed minimal off-target effects, with only three non-target C sites edited due to sequence similarity with the intended target. This study introduces a novel and precise method for RNA base editing in plant organelles, paving the way for new approaches in gene regulation applicable to plants and potentially other organisms.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"40 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250798","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 : 2024-09-15DOI: 10.1101/2024.09.14.613006
Xiaoyou Zheng, Peifeng Xie, Andrew Chen Cai, Yuze Jiang, Sirui Huang, Xiaochong Ma, Honghao Su, Boxiang Wang
Mycosporine-like amino acids (MAAs) are potent natural UV-protectants, but their industrial production is hindered by efficiency and sustainability issues of large-scale extraction of their native hosts. Heterologous expression of MAA biosynthesis pathway genes in chassis organisms provides a promising alternative route, though the substrate promiscuity of the ATP-grasp ligase MysD complicates the biosynthesis of specific MAAs. In this study, we developed a Saccharomyces cerevisiae strain with enhanced capacity of producing mycosporine-glycine (MG), through genomic expression of biosynthesis pathway genes and knockout of competing pathway genes. This strain serves as an efficient MysD expression platform, which converts MG into shinorine and porphyra-334. Through structural modelling, site-directed mutagenesis and mutant characterization, we identified two residues on the omega-loop of MysD involved in determining product specificity. We further characterized the product specificity of 20 MysDs from diverse cyanobacterial lineages and confirmed the residue pattern-product specificity correlation. Our findings provide guidance for screening, selecting, and designing novel MysDs for industrial-scale MAA production through heterologous expression.
类霉菌素氨基酸（MAAs）是一种有效的天然紫外线防护剂，但其工业化生产却受到从其原生宿主中大规模提取的效率和可持续性问题的阻碍。在底盘生物中异源表达 MAA 生物合成途径基因提供了一条很有前景的替代途径，但 ATP-抓取连接酶 MysD 的底物杂合性使特定 MAA 的生物合成变得复杂。在这项研究中，我们通过生物合成途径基因的基因组表达和竞争途径基因的敲除，培育出了一株具有更强生产霉菌素-甘氨酸（MG）能力的酿酒酵母菌株。该菌株是一个高效的 MysD 表达平台，可将 MG 转化为鞘氨醇和卟啉-334。通过结构建模、定点突变和突变体表征，我们确定了 MysD ω-环上两个参与决定产物特异性的残基。我们进一步鉴定了来自不同蓝藻品系的 20 个 MysD 的产物特异性，并证实了残基模式与产物特异性之间的相关性。我们的发现为筛选、选择和设计新型 MysDs 提供了指导，以便通过异源表达实现工业规模的 MAA 生产。
{"title":"Decoding Specificity of Cyanobacterial MysDs in Mycosporine-Like Amino Acid Biosynthesis through Heterologous Expression in Saccharomyces cerevisiae","authors":"Xiaoyou Zheng, Peifeng Xie, Andrew Chen Cai, Yuze Jiang, Sirui Huang, Xiaochong Ma, Honghao Su, Boxiang Wang","doi":"10.1101/2024.09.14.613006","DOIUrl":"https://doi.org/10.1101/2024.09.14.613006","url":null,"abstract":"Mycosporine-like amino acids (MAAs) are potent natural UV-protectants, but their industrial production is hindered by efficiency and sustainability issues of large-scale extraction of their native hosts. Heterologous expression of MAA biosynthesis pathway genes in chassis organisms provides a promising alternative route, though the substrate promiscuity of the ATP-grasp ligase MysD complicates the biosynthesis of specific MAAs. In this study, we developed a <em>Saccharomyces cerevisiae</em> strain with enhanced capacity of producing mycosporine-glycine (MG), through genomic expression of biosynthesis pathway genes and knockout of competing pathway genes. This strain serves as an efficient MysD expression platform, which converts MG into shinorine and porphyra-334. Through structural modelling, site-directed mutagenesis and mutant characterization, we identified two residues on the omega-loop of MysD involved in determining product specificity. We further characterized the product specificity of 20 MysDs from diverse cyanobacterial lineages and confirmed the residue pattern-product specificity correlation. Our findings provide guidance for screening, selecting, and designing novel MysDs for industrial-scale MAA production through heterologous expression.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"65 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250797","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 : 2024-09-15DOI: 10.1101/2024.09.15.613133
Mousumi Akter, Hossein Moghimianavval, Gary D Luker, Allen P Liu
Synthetic cells offer a versatile platform for addressing biomedical and environmental challenges, due to their modular design and capability to mimic cellular processes such as biosensing, intercellular communication, and metabolism. Constructing synthetic cells capable of stimuli-responsive secretion is vital for applications in targeted drug delivery and biosensor development. Previous attempts at engineering secretion for synthetic cells have been confined to non-specific cargo release via membrane pores, limiting the spatiotemporal precision and specificity necessary for selective secretion. Here, we designed and constructed a protein-based platform termed TEV Protease-mediated Releasable Actin-binding protein (TRAP) for selective, rapid, and triggerable secretion in synthetic cells. TRAP is designed to bind tightly to reconstituted actin networks and is proteolytically released from bound actin, followed by secretion via cell-penetrating peptide membrane translocation. We demonstrated TRAP's efficacy in facilitating light-activated secretion of both fluorescent and luminescent proteins. By equipping synthetic cells with a controlled secretion mechanism, TRAP paves the way for the development of stimuli-responsive biomaterials, versatile synthetic cell-based biosensing systems, and therapeutic applications through the integration of synthetic cells with living cells for targeted delivery of protein therapeutics.
合成细胞具有模块化设计和模拟生物传感、细胞间通信和新陈代谢等细胞过程的能力，为应对生物医学和环境挑战提供了一个多功能平台。构建具有刺激响应分泌能力的合成细胞对于靶向给药和生物传感器开发中的应用至关重要。以前对合成细胞分泌工程的尝试仅限于通过膜孔释放非特异性货物，从而限制了选择性分泌所需的时空精确性和特异性。在这里，我们设计并构建了一个基于蛋白质的平台，称为 TEV 蛋白酶介导的可释放肌动蛋白结合蛋白（TRAP），用于在合成细胞中进行选择性、快速和可触发的分泌。TRAP 可与重组肌动蛋白网络紧密结合，并通过蛋白水解从结合的肌动蛋白中释放出来，然后通过细胞穿透肽膜转运进行分泌。我们证明了 TRAP 在促进荧光蛋白和发光蛋白的光激活分泌方面的功效。通过为合成细胞配备可控分泌机制，TRAP 为开发刺激响应型生物材料、基于合成细胞的多功能生物传感系统，以及通过将合成细胞与活细胞整合以靶向输送蛋白质治疗药物的治疗应用铺平了道路。
{"title":"Light-triggered protease-mediated release of actin-bound cargo from synthetic cells","authors":"Mousumi Akter, Hossein Moghimianavval, Gary D Luker, Allen P Liu","doi":"10.1101/2024.09.15.613133","DOIUrl":"https://doi.org/10.1101/2024.09.15.613133","url":null,"abstract":"Synthetic cells offer a versatile platform for addressing biomedical and environmental challenges, due to their modular design and capability to mimic cellular processes such as biosensing, intercellular communication, and metabolism. Constructing synthetic cells capable of stimuli-responsive secretion is vital for applications in targeted drug delivery and biosensor development. Previous attempts at engineering secretion for synthetic cells have been confined to non-specific cargo release via membrane pores, limiting the spatiotemporal precision and specificity necessary for selective secretion. Here, we designed and constructed a protein-based platform termed TEV Protease-mediated Releasable Actin-binding protein (TRAP) for selective, rapid, and triggerable secretion in synthetic cells. TRAP is designed to bind tightly to reconstituted actin networks and is proteolytically released from bound actin, followed by secretion via cell-penetrating peptide membrane translocation. We demonstrated TRAP's efficacy in facilitating light-activated secretion of both fluorescent and luminescent proteins. By equipping synthetic cells with a controlled secretion mechanism, TRAP paves the way for the development of stimuli-responsive biomaterials, versatile synthetic cell-based biosensing systems, and therapeutic applications through the integration of synthetic cells with living cells for targeted delivery of protein therapeutics.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250832","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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