Plant diseases pose a significant threat to global crop production. Most disease resistance genes used in crop breeding programs encode nucleotide-binding leucine-rich repeat receptors (NLRs) that are limited in pathogen specificity and durability. In this study, we leveraged synthetic biology to develop an inducible broad-spectrum resistance in tomatoes. Constitutive expression of autoactive NLRs in plants leads to robust resistance against multiple pathogens but significantly stunts growth. We expressed autoactive NLRs under the control of pathogen-inducible (PI) promoters to mitigate the fitness costs. Taking advantage of extensive, new genomic and transcriptomic resources, we identified PI promoters that responded to multiple pathogens but not abiotic stress. We further validated functionality of predicted elements through a promoter luciferase assay. We generated significant resistance in transgenic tomatoes but we also encountered unwanted expression induction of the native promoter regions in flowers which led to lethal fruit development. Thus, we pursued promoter engineering for fine-tuning the induction. We identified cis-regulatory regions responsible for pathogen-inducibility through promoter bashing experiments and recombined the native promoter with the inducible part and the core promoter. Furthermore, we rationally created synthetic promoters showing a gradient of expression levels, which will allow for selection for transgenic tomatoes with the best performance. We found that the spacing between functional sequences, repeat number of inducible sequences, and core promoters all influence the outcome of engineering. Our study outlines a framework for developing broad-spectrum synthetic immune constructs with reduced fitness cost and provides examples of pathogen-inducible promoter engineering.
植物病害对全球作物生产构成重大威胁。作物育种计划中使用的大多数抗病基因编码的核苷酸结合富亮氨酸重复受体（NLRs）在病原体特异性和持久性方面受到限制。在这项研究中，我们利用合成生物学技术开发了番茄的诱导性广谱抗性。植物中自身活性 NLRs 的显性表达可产生对多种病原体的强大抵抗力，但会显著抑制生长。我们在病原体诱导型（PI）启动子的控制下表达了自活性 NLRs，以减轻健康成本。利用广泛的新基因组和转录组资源，我们确定了对多种病原体而非生物胁迫有反应的 PI 启动子。我们通过启动子荧光素酶检测进一步验证了预测元件的功能。我们在转基因西红柿中产生了明显的抗性，但我们也遇到了原生启动子区域在花朵中不必要的表达诱导，导致果实发育致死。因此，我们对启动子工程进行了微调。我们通过启动子撞击实验确定了病原体诱导性的顺式调控区，并将原生启动子与诱导部分和核心启动子重组。此外，我们还合理地创建了合成启动子，使其表达水平呈现梯度，从而筛选出性能最佳的转基因番茄。我们发现，功能序列之间的间距、可诱导序列的重复数量以及核心启动子都会影响工程设计的结果。我们的研究勾勒出了一个开发广谱合成免疫构建体的框架，降低了适应性成本，并提供了病原体诱导启动子工程的实例。
{"title":"Engineering pathogen-inducible promoters for conferring disease resistance in tomato","authors":"Wei Wei, Doogie Kim, Naio Koehler, Ashley Bendl, Myeong-Je Cho, Ksenia Krasileva","doi":"10.1101/2024.08.30.610566","DOIUrl":"https://doi.org/10.1101/2024.08.30.610566","url":null,"abstract":"Plant diseases pose a significant threat to global crop production. Most disease resistance genes used in crop breeding programs encode nucleotide-binding leucine-rich repeat receptors (NLRs) that are limited in pathogen specificity and durability. In this study, we leveraged synthetic biology to develop an inducible broad-spectrum resistance in tomatoes. Constitutive expression of autoactive NLRs in plants leads to robust resistance against multiple pathogens but significantly stunts growth. We expressed autoactive NLRs under the control of pathogen-inducible (PI) promoters to mitigate the fitness costs. Taking advantage of extensive, new genomic and transcriptomic resources, we identified PI promoters that responded to multiple pathogens but not abiotic stress. We further validated functionality of predicted elements through a promoter luciferase assay. We generated significant resistance in transgenic tomatoes but we also encountered unwanted expression induction of the native promoter regions in flowers which led to lethal fruit development. Thus, we pursued promoter engineering for fine-tuning the induction. We identified cis-regulatory regions responsible for pathogen-inducibility through promoter bashing experiments and recombined the native promoter with the inducible part and the core promoter. Furthermore, we rationally created synthetic promoters showing a gradient of expression levels, which will allow for selection for transgenic tomatoes with the best performance. We found that the spacing between functional sequences, repeat number of inducible sequences, and core promoters all influence the outcome of engineering. Our study outlines a framework for developing broad-spectrum synthetic immune constructs with reduced fitness cost and provides examples of pathogen-inducible promoter engineering.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142226314","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 : 2024-08-31DOI: 10.1101/2024.08.31.610601
Tomasz Czerniak, James Peter Saenz
RNA plays crucial roles in cellular organization and metabolism, and modulating its activity is essential for maintaining cellular functions. RNA activity, involving both catalytic (ribozymes) and translation processes, is controlled via myriad mechanisms involving different binding partners such as proteins and smaller polar solutes. We previously reported that lipid membranes can directly interact with the artificial R3C ribozyme, changing its activity; however, the effect of lipids on naturally occurring ribozymes remains unknown. Here, we report that both catalytic activity and RNA integrity can be controlled by the presence of different lipid membranes. Lipid gel membranes decreased the activity of hepatitis delta virus (HDV) and increased the hammerhead (HH) ribozyme reaction yield. The presence of lipid liquid membrane lattices triggered RNA degradation, with greater degradation occurring in the single-stranded regions of RNA. The interplay between RNA activity and stability in the presence of different lipid membranes introduces multiple possibilities, where different combinations of ribozyme and lipid membrane composition could produce different effects on activity. Taken together, these observations support the hypothesis that the activity of both natural and artificial RNAs can be modulated by lipid membranes, which, in turn, contribute to the development of novel riboswitch-like molecules and lipid membrane-based RNA-biosensors.
{"title":"Effects of lipid membranes on RNA catalytic activity and stability","authors":"Tomasz Czerniak, James Peter Saenz","doi":"10.1101/2024.08.31.610601","DOIUrl":"https://doi.org/10.1101/2024.08.31.610601","url":null,"abstract":"RNA plays crucial roles in cellular organization and metabolism, and modulating its activity is essential for maintaining cellular functions. RNA activity, involving both catalytic (ribozymes) and translation processes, is controlled via myriad mechanisms involving different binding partners such as proteins and smaller polar solutes. We previously reported that lipid membranes can directly interact with the artificial R3C ribozyme, changing its activity; however, the effect of lipids on naturally occurring ribozymes remains unknown. Here, we report that both catalytic activity and RNA integrity can be controlled by the presence of different lipid membranes. Lipid gel membranes decreased the activity of hepatitis delta virus (HDV) and increased the hammerhead (HH) ribozyme reaction yield. The presence of lipid liquid membrane lattices triggered RNA degradation, with greater degradation occurring in the single-stranded regions of RNA. The interplay between RNA activity and stability in the presence of different lipid membranes introduces multiple possibilities, where different combinations of ribozyme and lipid membrane composition could produce different effects on activity. Taken together, these observations support the hypothesis that the activity of both natural and artificial RNAs can be modulated by lipid membranes, which, in turn, contribute to the development of novel riboswitch-like molecules and lipid membrane-based RNA-biosensors.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"30 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206735","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 : 2024-08-30DOI: 10.1101/2024.08.29.610422
Simeon D. Castle, Adrian Woolfson, Gregory Linshiz, Blake T. Riley, Ifor D.W. Samuel, Philipp Holliger, Lauren Oldfield, Andrew Hessel, Thomas E. Gorochowski
DNA polymerases are complex molecular machines able to replicate genetic material using a template-driven process. While the copying function of these enzymes is well established, their ability to perform untemplated DNA synthesis is less well characterized. Here, we explore the ability of DNA polymerases to synthesize DNA fragments in the absence of template. We use long-read nanopore sequencing and real-time PCR to observe the synthesis of pools of DNA products derived from a diverse set of natural and engineered DNA polymerases across varying temperatures and buffer compositions. We detail the features of the DNA fragments generated, enrichment of select sequence motifs, and demonstrate that the sequence composition of the synthesized DNA may be altered by modifying environmental conditions. This work provides an extensive data set to better discern the process of untemplated DNA polymerase activity and may support its potential repurposing as a technology for the guided synthesis of DNA sequences on the kilobase-scale and beyond.
DNA 聚合酶是一种复杂的分子机器，能够利用模板驱动过程复制遗传物质。虽然这些酶的复制功能已得到公认，但它们进行无模板 DNA 合成的能力却没有得到很好的描述。在这里，我们探讨了 DNA 聚合酶在没有模板的情况下合成 DNA 片段的能力。我们使用长线程纳米孔测序和实时聚合酶链式反应（real-time PCR）技术，观察了不同温度和缓冲液成分下由多种天然和工程 DNA 聚合酶合成的 DNA 产物池。我们详细介绍了所生成 DNA 片段的特征、所选序列图案的富集情况，并证明合成 DNA 的序列组成可能会因环境条件的改变而改变。这项工作提供了一个广泛的数据集，以更好地辨别非模板 DNA 聚合酶的活动过程，并可能支持将其重新用作千碱基级甚至更高的 DNA 序列引导合成技术。
{"title":"Analysis and control of untemplated DNA polymerase activity for guided synthesis of kilobase-scale DNA sequences","authors":"Simeon D. Castle, Adrian Woolfson, Gregory Linshiz, Blake T. Riley, Ifor D.W. Samuel, Philipp Holliger, Lauren Oldfield, Andrew Hessel, Thomas E. Gorochowski","doi":"10.1101/2024.08.29.610422","DOIUrl":"https://doi.org/10.1101/2024.08.29.610422","url":null,"abstract":"DNA polymerases are complex molecular machines able to replicate genetic material using a template-driven process. While the copying function of these enzymes is well established, their ability to perform untemplated DNA synthesis is less well characterized. Here, we explore the ability of DNA polymerases to synthesize DNA fragments in the absence of template. We use long-read nanopore sequencing and real-time PCR to observe the synthesis of pools of DNA products derived from a diverse set of natural and engineered DNA polymerases across varying temperatures and buffer compositions. We detail the features of the DNA fragments generated, enrichment of select sequence motifs, and demonstrate that the sequence composition of the synthesized DNA may be altered by modifying environmental conditions. This work provides an extensive data set to better discern the process of untemplated DNA polymerase activity and may support its potential repurposing as a technology for the guided synthesis of DNA sequences on the kilobase-scale and beyond.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"59 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206733","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 : 2024-08-30DOI: 10.1101/2024.08.29.610230
Hao Wang, Zhengxin Dong, Jingyi Shi, Lei Chen, Tao Sun, Weiwen Zhang
The fusion expression of DNA replication-related proteins with nucleotide deaminase enzymes promotes random mutations in bacterial genomes, thereby increasing genetic diversity. However, most previous studies have focused on cytosine deaminase, which only produces C to T mutations, significantly limiting the variety of mutation types. In this study, we developed a fusion expression system in Escherichia coli by combining DnaG (RNA primase) with adenine deaminase TadA-8e (DnaG-TadA), which rapidly introduced A to G mutations into the E. coli genome, resulting in a 664-fold increase in mutation rate. Additionally, we engineered a dual-functional TadA variant, TadAD, and fused it with DnaG. This construct introduced both C to T and A to G mutations into the genome, with the mutation rate further increased by 370-fold upon co-expression with an uracil glycosylase inhibitor (DnaG-TadAD-UGI). We applied DnaG-TadA and DnaG-TadAD-UGI to adaptive laboratory evolution for Cd2+ and kanamycin resistance, achieving an 8.0 mM Cd2+ and 200 ug/mL kanamycin tolerance within just 17 days and 132 hours, respectively. Compared to standard evolution methods, the final tolerance levels increased by 320% and 266%, respectively. Our work offers a novel strategy for random mutagenesis in E. coli and potentially other species.
DNA 复制相关蛋白与核苷酸脱氨酶的融合表达促进了细菌基因组的随机突变，从而增加了遗传多样性。然而，以往的研究大多集中在胞嘧啶脱氨酶上，它只能产生 C 到 T 的突变，大大限制了突变类型的多样性。在这项研究中，我们在大肠杆菌中开发了一种融合表达系统，将 DnaG（RNA 引物酶）与腺嘌呤脱氨酶 TadA-8e （DnaG-TadA）结合在一起，迅速将 A 到 G 的突变引入大肠杆菌基因组，使突变率提高了 664 倍。此外，我们还设计了一种双功能 TadA 变体 TadAD，并将其与 DnaG 融合。这种构建体在基因组中引入了 C 到 T 和 A 到 G 的突变，在与尿嘧啶糖基化酶抑制剂（DnaG-TadAD-UGI）共同表达时，突变率进一步增加了 370 倍。我们将 DnaG-TadA 和 DnaG-TadAD-UGI 应用于实验室对 Cd2+ 和卡那霉素抗性的适应性进化，分别在 17 天和 132 小时内实现了对 8.0 mM Cd2+ 和 200 ug/mL 卡那霉素的耐受性。与标准进化方法相比，最终耐受水平分别提高了 320% 和 266%。我们的工作为大肠杆菌及其他潜在物种的随机诱变提供了一种新策略。
{"title":"Genome-wide A to G and C to T Mutations Induced by Functional TadA Variants in Escherichia coli","authors":"Hao Wang, Zhengxin Dong, Jingyi Shi, Lei Chen, Tao Sun, Weiwen Zhang","doi":"10.1101/2024.08.29.610230","DOIUrl":"https://doi.org/10.1101/2024.08.29.610230","url":null,"abstract":"The fusion expression of DNA replication-related proteins with nucleotide deaminase enzymes promotes random mutations in bacterial genomes, thereby increasing genetic diversity. However, most previous studies have focused on cytosine deaminase, which only produces C to T mutations, significantly limiting the variety of mutation types. In this study, we developed a fusion expression system in Escherichia coli by combining DnaG (RNA primase) with adenine deaminase TadA-8e (DnaG-TadA), which rapidly introduced A to G mutations into the E. coli genome, resulting in a 664-fold increase in mutation rate. Additionally, we engineered a dual-functional TadA variant, TadAD, and fused it with DnaG. This construct introduced both C to T and A to G mutations into the genome, with the mutation rate further increased by 370-fold upon co-expression with an uracil glycosylase inhibitor (DnaG-TadAD-UGI). We applied DnaG-TadA and DnaG-TadAD-UGI to adaptive laboratory evolution for Cd2+ and kanamycin resistance, achieving an 8.0 mM Cd2+ and 200 ug/mL kanamycin tolerance within just 17 days and 132 hours, respectively. Compared to standard evolution methods, the final tolerance levels increased by 320% and 266%, respectively. Our work offers a novel strategy for random mutagenesis in E. coli and potentially other species.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206732","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}
Mitochondria play a key role in energy production and cellular metabolism, making them a promising target for metabolic engineering and disease treatment. However, despite the known influence of passenger proteins on localization efficiency, only a few protein-localization tags have been characterized for mitochondrial targeting. To address this limitation, we exploited Variational Autoencoder (VAE), an unsupervised deep learning framework, to design novel mitochondrial targeting sequences (MTSs). In silico analysis revealed that a high fraction of generated peptides are functional and possess features important for mitochondrial targeting. Additionally, we devised a sampling scheme to indirectly address biases arising from the differences in mitochondrial protein import machinery and characterized artificial MTSs in four eukaryotic organisms. These sequences displayed significant diversity, sharing less than 60% sequence identity with MTSs in the UniProt database. Moreover, we trained a separate VAE and employed latent space interpolation to design dual targeting sequences capable of targeting both mitochondria and chloroplasts, shedding light on their evolutionary origins. As a proof-of-concept, we demonstrate the application of these artificial MTSs in increasing titers of 3-hydroxypropionic acid through pathway compartmentalization and improving 5-aminolevulinate synthase delivery by 1.62-fold and 4.76-fold, respectively. Overall, our work not only demonstrates the potential of generative artificial intelligence in designing novel, functional mitochondrial targeting sequences but also highlights their utility in engineering mitochondria for both fundamental research and practical applications in biology.
{"title":"Design of diverse, functional mitochondrial targeting sequences across eukaryotic organisms using variational autoencoder","authors":"Aashutosh Girish Boob, Shih-I Tan, Airah Zaidi, Nilmani Singh, Xueyi Xue, Shuaizhen Zhou, Teresa A Martin, Li-Qing Chen, Huimin Zhao","doi":"10.1101/2024.08.28.610205","DOIUrl":"https://doi.org/10.1101/2024.08.28.610205","url":null,"abstract":"Mitochondria play a key role in energy production and cellular metabolism, making them a promising target for metabolic engineering and disease treatment. However, despite the known influence of passenger proteins on localization efficiency, only a few protein-localization tags have been characterized for mitochondrial targeting. To address this limitation, we exploited Variational Autoencoder (VAE), an unsupervised deep learning framework, to design novel mitochondrial targeting sequences (MTSs). In silico analysis revealed that a high fraction of generated peptides are functional and possess features important for mitochondrial targeting. Additionally, we devised a sampling scheme to indirectly address biases arising from the differences in mitochondrial protein import machinery and characterized artificial MTSs in four eukaryotic organisms. These sequences displayed significant diversity, sharing less than 60% sequence identity with MTSs in the UniProt database. Moreover, we trained a separate VAE and employed latent space interpolation to design dual targeting sequences capable of targeting both mitochondria and chloroplasts, shedding light on their evolutionary origins. As a proof-of-concept, we demonstrate the application of these artificial MTSs in increasing titers of 3-hydroxypropionic acid through pathway compartmentalization and improving 5-aminolevulinate synthase delivery by 1.62-fold and 4.76-fold, respectively. Overall, our work not only demonstrates the potential of generative artificial intelligence in designing novel, functional mitochondrial targeting sequences but also highlights their utility in engineering mitochondria for both fundamental research and practical applications in biology.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"53 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206736","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 : 2024-08-29DOI: 10.1101/2024.08.29.610322
Farren Isaacs, Colin Hemez, Kyle Mohler, Felix Radford, Jack Moen, Jesse Rinehart
Genomically recoded organisms hold promise for many biotechnological applications, but they may exhibit substantial fitness defects relative to their non-recoded counterparts. We used targeted metabolic screens, genetic analysis, and proteomics to identify the origins of fitness impairment in a model recoded organism, Escherichia coli C321.∆A. We found that defects in isoleucine biosynthesis and release factor activity, caused by mutations extant in all K-12 lineage strains, elicited profound fitness impairments in C321.∆A, suggesting that genome recoding exacerbates suboptimal traits present in precursor strains. By correcting these and other C321.∆A-specific mutations, we engineered C321.∆A strains with doubling time reductions of 17% and 42% in rich and minimal medium, respectively, compared to ancestral C321. Strains with improved growth kinetics also demonstrated enhanced ribosomal non-standard amino acid incorporation capabilities. Proteomic analysis indicated that C321.∆A lacks the ability to regulate essential amino acid and nucleotide biosynthesis pathways, and that targeted mutation reversion restored regulatory capabilities. Our work outlines a strategy for the rapid and precise phenotypic optimization of genomically recoded organisms and other engineered microbes.
{"title":"Genomically recoded Escherichia coli with optimized functional phenotypes","authors":"Farren Isaacs, Colin Hemez, Kyle Mohler, Felix Radford, Jack Moen, Jesse Rinehart","doi":"10.1101/2024.08.29.610322","DOIUrl":"https://doi.org/10.1101/2024.08.29.610322","url":null,"abstract":"Genomically recoded organisms hold promise for many biotechnological applications, but they may exhibit substantial fitness defects relative to their non-recoded counterparts. We used targeted metabolic screens, genetic analysis, and proteomics to identify the origins of fitness impairment in a model recoded organism, Escherichia coli C321.∆A. We found that defects in isoleucine biosynthesis and release factor activity, caused by mutations extant in all K-12 lineage strains, elicited profound fitness impairments in C321.∆A, suggesting that genome recoding exacerbates suboptimal traits present in precursor strains. By correcting these and other C321.∆A-specific mutations, we engineered C321.∆A strains with doubling time reductions of 17% and 42% in rich and minimal medium, respectively, compared to ancestral C321. Strains with improved growth kinetics also demonstrated enhanced ribosomal non-standard amino acid incorporation capabilities. Proteomic analysis indicated that C321.∆A lacks the ability to regulate essential amino acid and nucleotide biosynthesis pathways, and that targeted mutation reversion restored regulatory capabilities. Our work outlines a strategy for the rapid and precise phenotypic optimization of genomically recoded organisms and other engineered microbes.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"24 12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206737","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 : 2024-08-29DOI: 10.1101/2024.08.29.610350
Md Saifur Rahman
Molecular farming, which utilizes plants as biofactories for recombinant protein production, offers an innovative and cost-effective alternative to traditional expression systems. Despite its advantages, plant-based production faces challenges such as low transgene expression and protein instability. Recent studies have highlighted the potential of Nicotiana benthamiana axillary stem leaves to enhance protein yield. This study explored the development of N. benthamiana lines expressing TENGU without signal peptide (T-SP), a Phytoplasma effector known to induce plant dwarfism and increase shoot growth. TENGU and other effectors, such as SAP05 and SAP11, were introduced to create phenotypic variations that favor recombinant protein production. This study aimed to optimize these transgenic lines for increased biomass and protein yields by leveraging vertical farming conditions for scalable production. The results demonstrated significant improvements in leaf number, biomass, and five times more soluble protein content in T-SP lines compared to control lines, suggesting a promising approach for efficient molecular farming.
{"title":"Optimizing Plant Biofactories: Enhancing Recombinant Protein Production in Nicotiana benthamiana through Phytoplasma Effectors","authors":"Md Saifur Rahman","doi":"10.1101/2024.08.29.610350","DOIUrl":"https://doi.org/10.1101/2024.08.29.610350","url":null,"abstract":"Molecular farming, which utilizes plants as biofactories for recombinant protein production, offers an innovative and cost-effective alternative to traditional expression systems. Despite its advantages, plant-based production faces challenges such as low transgene expression and protein instability. Recent studies have highlighted the potential of Nicotiana benthamiana axillary stem leaves to enhance protein yield. This study explored the development of N. benthamiana lines expressing TENGU without signal peptide (T-SP), a Phytoplasma effector known to induce plant dwarfism and increase shoot growth. TENGU and other effectors, such as SAP05 and SAP11, were introduced to create phenotypic variations that favor recombinant protein production. This study aimed to optimize these transgenic lines for increased biomass and protein yields by leveraging vertical farming conditions for scalable production. The results demonstrated significant improvements in leaf number, biomass, and five times more soluble protein content in T-SP lines compared to control lines, suggesting a promising approach for efficient molecular farming.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206738","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 : 2024-08-28DOI: 10.1101/2024.08.27.609958
Giorgia Isinelli, Christopher Wintersinger, Pascal Lill, Olivia Young, Jie Deng, William Shih, Yang Zeng
DNA origami has enabled the development of responsive drug-delivery vehicles with precision features that were previously not attainable in bionanotechnology. To reduce the costs of creating therapeutics-scale amounts of DNA origami that need to bear costly modifications with high occupancy, we reused the excess staple oligonucleotides that are leftover from the folding process to fold additional origami. We determined that a DNA origami can be successfully folded with up to 80% cost savings by cyclic recovery and reuse of excess staple strands. We found evidence that higher quality staple strands are preferentially incorporated into origami, consistent with past reports, and therefore are preferentially depleted from the free-strand pool. The folding of DNA origami with staple strands that were reused up to eleven times was indistinguishable by our panel of assays versus a control folded with new strands, so long as the reused oligonucleotides were replenished each cycle with a small excess of fresh strands. We also observed a high degree of incorporation of guests on the DNA origami. By recovering, reusing, and replenishing excess staple oligonucleotides, it is possible to significantly lessen production costs to create well-formed origami, which is useful to allow more therapeutic designs to be tested.
DNA 折纸使我们能够开发出具有精确特征的响应性给药载体，而这在以前的仿生技术中是无法实现的。为了降低制作治疗级 DNA 折纸的成本，我们重新利用折叠过程中剩余的主寡核苷酸来折叠更多的 DNA 折纸。我们确定，通过循环回收和重复使用多余的主链，可以成功折叠出 DNA 折纸，并节省高达 80% 的成本。我们发现有证据表明，质量较高的主链会优先融入折纸中，这与过去的报告一致，因此会优先从自由链池中耗尽。用重复使用多达 11 次的主链折叠 DNA 折纸，与用新链折叠的对照组相比，在我们的检测小组中没有区别，只要在每个周期用少量过量的新鲜链补充重复使用的寡核苷酸即可。我们还观察到客体在 DNA 折纸上的高度结合。通过回收、重复使用和补充多余的主链寡核苷酸，可以大大降低制作成型良好的折纸的生产成本，这有助于测试更多的治疗设计。
{"title":"Reusing excess staple oligonucleotides for economical production of DNA origami","authors":"Giorgia Isinelli, Christopher Wintersinger, Pascal Lill, Olivia Young, Jie Deng, William Shih, Yang Zeng","doi":"10.1101/2024.08.27.609958","DOIUrl":"https://doi.org/10.1101/2024.08.27.609958","url":null,"abstract":"DNA origami has enabled the development of responsive drug-delivery vehicles with precision features that were previously not attainable in bionanotechnology. To reduce the costs of creating therapeutics-scale amounts of DNA origami that need to bear costly modifications with high occupancy, we reused the excess staple oligonucleotides that are leftover from the folding process to fold additional origami. We determined that a DNA origami can be successfully folded with up to 80% cost savings by cyclic recovery and reuse of excess staple strands. We found evidence that higher quality staple strands are preferentially incorporated into origami, consistent with past reports, and therefore are preferentially depleted from the free-strand pool. The folding of DNA origami with staple strands that were reused up to eleven times was indistinguishable by our panel of assays versus a control folded with new strands, so long as the reused oligonucleotides were replenished each cycle with a small excess of fresh strands. We also observed a high degree of incorporation of guests on the DNA origami. By recovering, reusing, and replenishing excess staple oligonucleotides, it is possible to significantly lessen production costs to create well-formed origami, which is useful to allow more therapeutic designs to be tested.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"37 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206780","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 : 2024-08-28DOI: 10.1101/2024.08.28.610020
Or Willinger, Naor Granik, Sarah Goldberg, Roee Amit
To gain access to cells, viruses employ host proteins as receptors. In soluble form, these receptors are used as decoys to inhibit infection. We fused candidate soluble receptors to an RNA-binding protein, and using synthetic long non-coding RNA (slncRNA) cassettes that can undergo phase-separation we scaffolded the receptor fusions to generate antiviral decoy particles. Using confocal microscopy, we screened antiviral protein candidates by observing changes in phase-separation morphology when incubated with viral-mimicking components. We demonstrated that ACE2 decoy particles bind strongly to the coronavirus RBD, facilitating FRET, while sufficiently sialylated decoy particles form agglutinated structures with RNA peripheries in the presence of a sialolectin. Infection assays show ACE2 decoy particles fully inhibit the Delta and Omicron BA.1 coronavirus variants, and LAMP1 and GYPA decoy particles significantly reduce influenza infection in-cellulo. This work establishes a foundation for broad-spectrum antiviral decoy particles, composed of multiple receptors targeting various viruses.
{"title":"Phase Separation-based Antiviral Decoy Particles as Basis for Programmable Broad-spectrum Therapeutics","authors":"Or Willinger, Naor Granik, Sarah Goldberg, Roee Amit","doi":"10.1101/2024.08.28.610020","DOIUrl":"https://doi.org/10.1101/2024.08.28.610020","url":null,"abstract":"To gain access to cells, viruses employ host proteins as receptors. In soluble form, these receptors are used as decoys to inhibit infection. We fused candidate soluble receptors to an RNA-binding protein, and using synthetic long non-coding RNA (slncRNA) cassettes that can undergo phase-separation we scaffolded the receptor fusions to generate antiviral decoy particles. Using confocal microscopy, we screened antiviral protein candidates by observing changes in phase-separation morphology when incubated with viral-mimicking components. We demonstrated that ACE2 decoy particles bind strongly to the coronavirus RBD, facilitating FRET, while sufficiently sialylated decoy particles form agglutinated structures with RNA peripheries in the presence of a sialolectin. Infection assays show ACE2 decoy particles fully inhibit the Delta and Omicron BA.1 coronavirus variants, and LAMP1 and GYPA decoy particles significantly reduce influenza infection in-cellulo. This work establishes a foundation for broad-spectrum antiviral decoy particles, composed of multiple receptors targeting various viruses.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206739","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}
Engineering multicellular consortia, where information processing is distributed across specialized cell types, offers a promising strategy for implementing sophisticated biocomputing systems. However, a major challenge remains in establishing orthogonal intercellular communication, or "wires," within synthetic bacterial consortia. In this study, we address this bottleneck by integrating phagemid-mediated intercellular communication with CRISPR-based gene regulation for multicellular computing in synthetic E. coli consortia. We achieve intercellular communication by regulating the transfer of single guide RNAs (sgRNAs) encoded on M13 phagemids from sender to receiver cells. Once inside the receiver cells, the transferred sgRNAs mediate gene regulation via CRISPR interference. Leveraging this approach, we successfully constructed one-, two-, and four-input logic gates. Our work expands the toolkit for intercellular communication and paves the way for complex information processing in synthetic microbial consortia, with diverse potential applications, including biocomputing, biosensing, and biomanufacturing.
{"title":"Engineering Intercellular Communication using M13 Phagemid and CRISPR-based Gene Regulation for Multicellular Computing in Escherichia coli","authors":"Hadiastri Kusumawardhani, Florian Zoppi, Roberto Avendaño, Yolanda Schaerli","doi":"10.1101/2024.08.28.610043","DOIUrl":"https://doi.org/10.1101/2024.08.28.610043","url":null,"abstract":"Engineering multicellular consortia, where information processing is distributed across specialized cell types, offers a promising strategy for implementing sophisticated biocomputing systems. However, a major challenge remains in establishing orthogonal intercellular communication, or \"wires,\" within synthetic bacterial consortia. In this study, we address this bottleneck by integrating phagemid-mediated intercellular communication with CRISPR-based gene regulation for multicellular computing in synthetic <em>E. coli</em> consortia. We achieve intercellular communication by regulating the transfer of single guide RNAs (sgRNAs) encoded on M13 phagemids from sender to receiver cells. Once inside the receiver cells, the transferred sgRNAs mediate gene regulation via CRISPR interference. Leveraging this approach, we successfully constructed one-, two-, and four-input logic gates. Our work expands the toolkit for intercellular communication and paves the way for complex information processing in synthetic microbial consortia, with diverse potential applications, including biocomputing, biosensing, and biomanufacturing.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206740","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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