ACS Synthetic Biology最新文献

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ShuffleAnalyzer: A Comprehensive Tool to Visualize DNA Shuffling. ShuffleAnalyzer:可视化 DNA 洗牌的综合工具
IF 3.7 2区 生物学
ACS Synthetic Biology Pub Date : 2024-07-11 DOI: 10.1021/acssynbio.4c00251
Franz Schweiggert, Gregor Habeck, Patrick Most, Martin Busch, Jörg Schweiggert
{"title":"ShuffleAnalyzer: A Comprehensive Tool to Visualize DNA Shuffling.","authors":"Franz Schweiggert, Gregor Habeck, Patrick Most, Martin Busch, Jörg Schweiggert","doi":"10.1021/acssynbio.4c00251","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00251","url":null,"abstract":"<p><p>DNA shuffling is a powerful technique for generating synthetic DNA via recombination of homologous parental sequences. Resulting chimeras are often incorporated into complex libraries for functionality screenings that identify novel variants with improved characteristics. To survey shuffling efficiency, subsequences of chimeras can be computationally assigned to their corresponding parental counterpart, yielding insight into frequency of recombination events, diversity of shuffling libraries and actual composition of final variants. Whereas tools for parental assignment exist, they do not provide direct visualization of the results, making the analysis time-consuming and cumbersome. Here we present ShuffleAnalyzer, a comprehensive, user-friendly, Python-based analysis tool that directly generates graphical outputs of parental assignments and is freely available under a BSD-3 license (https://github.com/joerg-swg/ShuffleAnalyzer/releases). Besides DNA shuffling, peptide insertions can be simultaneously analyzed and visualized, which makes ShuffleAnalyzer a highly valuable tool for integrated approaches often used in synthetic biology, such as AAV capsid engineering in gene therapy applications.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141588903","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Degron-Based bioPROTACs for Controlling Signaling in CAR T Cells. 用于控制 CAR T 细胞信号的 Degron-Based bioPROTACs。
IF 3.7 2区 生物学
ACS Synthetic Biology Pub Date : 2024-07-11 DOI: 10.1021/acssynbio.4c00109
Matthew S Kim, Hersh K Bhargava, Gavin E Shavey, Wendell A Lim, Hana El-Samad, Andrew H Ng
{"title":"Degron-Based bioPROTACs for Controlling Signaling in CAR T Cells.","authors":"Matthew S Kim, Hersh K Bhargava, Gavin E Shavey, Wendell A Lim, Hana El-Samad, Andrew H Ng","doi":"10.1021/acssynbio.4c00109","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00109","url":null,"abstract":"<p><p>Chimeric antigen receptor (CAR) T cells have made a tremendous impact in the clinic, but potent signaling through the CAR can be detrimental to treatment safety and efficacy. The use of protein degradation to control CAR signaling can address these issues in preclinical models. Existing strategies for regulating CAR stability rely on small molecules to induce systemic degradation. In contrast to small molecule regulation, genetic circuits offer a more precise method to control CAR signaling in an autonomous cell-by-cell fashion. Here, we describe a programmable protein degradation tool that adopts the framework of bioPROTACs, heterobifunctional proteins that are composed of a target recognition domain fused to a domain that recruits the endogenous ubiquitin proteasome system. We develop novel bioPROTACs that utilize a compact four-residue degron and demonstrate degradation of cytosolic and membrane protein targets using either a nanobody or synthetic leucine zipper as a protein binder. Our bioPROTACs exhibit potent degradation of CARs and can inhibit CAR signaling in primary human T cells. We demonstrate the utility of our bioPROTACs by constructing a genetic circuit to degrade the tyrosine kinase ZAP70 in response to recognition of a specific membrane-bound antigen. This circuit can disrupt CAR T cell signaling only in the presence of a specific cell population. These results suggest that bioPROTACs are powerful tools for expanding the CAR T cell engineering toolbox.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141588902","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Bioinformatic Prediction and High Throughput In Vivo Screening to Identify Cis-Regulatory Elements for the Development of Algal Synthetic Promoters. 通过生物信息学预测和高通量体内筛选确定用于开发藻类合成启动子的顺式调控元件。
IF 3.7 2区 生物学
ACS Synthetic Biology Pub Date : 2024-07-10 DOI: 10.1021/acssynbio.4c00199
Y Torres-Tiji, H Sethuram, A Gupta, J McCauley, J-V Dutra-Molino, R Pathania, L Saxton, K Kang, N J Hillson, S P Mayfield
{"title":"Bioinformatic Prediction and High Throughput In Vivo Screening to Identify Cis-Regulatory Elements for the Development of Algal Synthetic Promoters.","authors":"Y Torres-Tiji, H Sethuram, A Gupta, J McCauley, J-V Dutra-Molino, R Pathania, L Saxton, K Kang, N J Hillson, S P Mayfield","doi":"10.1021/acssynbio.4c00199","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00199","url":null,"abstract":"<p><p>Algae biotechnology holds immense promise for revolutionizing the bioeconomy through the sustainable and scalable production of various bioproducts. However, their development has been hindered by the lack of advanced genetic tools. This study introduces a synthetic biology approach to develop such tools, focusing on the construction and testing of synthetic promoters. By analyzing conserved DNA motifs within the promoter regions of highly expressed genes across six different algal species, we identified cis-regulatory elements (CREs) associated with high transcriptional activity. Combining the algorithms POWRS, STREME, and PhyloGibbs, we predicted 1511 CREs and inserted them into a minimal synthetic promoter sequence in 1, 2, or 3 copies, resulting in 4533 distinct synthetic promoters. These promoters were evaluated in vivo for their capacity to drive the expression of a transgene in a high-throughput manner through next-generation sequencing post antibiotic selection and fluorescence-activated cell sorting. To validate our approach, we sequenced hundreds of transgenic lines showing high levels of GFP expression. Further, we individually tested 14 identified promoters, revealing substantial increases in GFP expression─up to nine times higher than the baseline synthetic promoter, with five matching or even surpassing the performance of the native AR1 promoter. As a result of this study, we identified a catalog of CREs that can now be used to build superior synthetic algal promoters. More importantly, here we present a validated pipeline to generate building blocks for innovative synthetic genetic tools applicable to any algal species with a sequenced genome and transcriptome data set.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Cell-Free Translation Quantification via a Fluorescent Minihelix. 通过荧光小螺旋进行细胞自由翻译定量。
IF 3.7 2区 生物学
ACS Synthetic Biology Pub Date : 2024-07-09 DOI: 10.1021/acssynbio.4c00266
Jessica A Willi, Ashty S Karim, Michael C Jewett
{"title":"Cell-Free Translation Quantification via a Fluorescent Minihelix.","authors":"Jessica A Willi, Ashty S Karim, Michael C Jewett","doi":"10.1021/acssynbio.4c00266","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00266","url":null,"abstract":"<p><p>Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye. The fluorescent reporter helix is short enough to be encoded on a primer pair to tag any protein of interest via PCR. Both the tagged protein and the standalone reporter can be detected quantitatively in real time or at the end of cell-free expression reactions with standard 96/384-well plate readers, an RT-qPCR system, or gel electrophoresis without the need for staining. The fluorescent signal is stable and correlates linearly with the protein concentration, enabling product quantification. We modified the reporter to study cell-free expression dynamics and engineered ribosome activity. We anticipate that the fluorescent minihelix reporter will facilitate efforts in engineering <i>in vitro</i> transcription and translation systems.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141557366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening. 利用高通量声学筛选实现声学报告基因的定向进化。
IF 3.7 2区 生物学
ACS Synthetic Biology Pub Date : 2024-07-09 DOI: 10.1021/acssynbio.4c00283
Robert C Hurt, Zhiyang Jin, Mohamed Soufi, Katie K Wong, Daniel P Sawyer, Hao K Shen, Przemysław Dutka, Ramya Deshpande, Ruby Zhang, David R Mittelstein, Mikhail G Shapiro
{"title":"Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening.","authors":"Robert C Hurt, Zhiyang Jin, Mohamed Soufi, Katie K Wong, Daniel P Sawyer, Hao K Shen, Przemysław Dutka, Ramya Deshpande, Ruby Zhang, David R Mittelstein, Mikhail G Shapiro","doi":"10.1021/acssynbio.4c00283","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00283","url":null,"abstract":"<p><p>A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 μm. Within the past decade, reporter genes for US have been introduced and engineered to link cellular functions to US signals <i>via</i> heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs). Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semiautomated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologues of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141561924","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Metabolic Profile of the Genome-Reduced Bacillus subtilis Strain IIG-Bs-27-39: An Attractive Chassis for Recombinant Protein Production. 基因组还原枯草芽孢杆菌菌株 IIG-Bs-27-39 的代谢概况:重组蛋白质生产的诱人底盘
IF 3.7 2区 生物学
ACS Synthetic Biology Pub Date : 2024-07-09 DOI: 10.1021/acssynbio.4c00254
Rocío Aguilar Suárez, Michael Kohlstedt, Ayşegül Öktem, Jolanda Neef, Yuzheng Wu, Kaiya Ikeda, Ken-Ichi Yoshida, Josef Altenbuchner, Christoph Wittmann, Jan Maarten van Dijl
{"title":"Metabolic Profile of the Genome-Reduced <i>Bacillus subtilis</i> Strain IIG-Bs-27-39: An Attractive Chassis for Recombinant Protein Production.","authors":"Rocío Aguilar Suárez, Michael Kohlstedt, Ayşegül Öktem, Jolanda Neef, Yuzheng Wu, Kaiya Ikeda, Ken-Ichi Yoshida, Josef Altenbuchner, Christoph Wittmann, Jan Maarten van Dijl","doi":"10.1021/acssynbio.4c00254","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00254","url":null,"abstract":"<p><p>The Gram-positive bacterium <i>Bacillus subtilis</i> is extensively used in the industry for the secretory production of proteins with commercial value. To further improve its performance, this microbe has been the subject of extensive genome engineering efforts, especially the removal of large genomic regions that are dispensable or even counterproductive. Here, we present the genome-reduced <i>B. subtilis</i> strain IIG-Bs-27-39, which was obtained through systematic deletion of mobile genetic elements, as well as genes for extracellular proteases, sporulation, flagella formation, and antibiotic production. Different from previously characterized genome-reduced <i>B. subtilis</i> strains, the IIG-Bs-27-39 strain was still able to grow on minimal media. We used this feature to benchmark strain IIG-Bs-27-39 against its parental strain 168 with respect to heterologous protein production and metabolic parameters during bioreactor cultivation. The IIG-Bs-27-39 strain presented superior secretion of difficult-to-produce staphylococcal antigens, as well as higher specific growth rates and biomass yields. At the metabolic level, changes in byproduct formation and internal amino acid pools were observed, whereas energetic parameters such as the ATP yield, ATP/ADP levels, and adenylate energy charge were comparable between the two strains. Intriguingly, we observed a significant increase in the total cellular NADPH level during all tested conditions and increases in the NAD<sup>+</sup> and NADP(H) pools during protein production. This indicates that the IIG-Bs-27-39 strain has more energy available for anabolic processes and protein production, thereby providing a link between strain physiology and production performance. On this basis, we conclude that the genome-reduced strain IIG-Bs-27-39 represents an attractive chassis for future biotechnological applications.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141561925","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
AutoBioTech─A Versatile Biofoundry for Automated Strain Engineering. AutoBioTech--用于自动菌株工程的多功能生物设施。
IF 3.7 2区 生物学
ACS Synthetic Biology Pub Date : 2024-07-08 DOI: 10.1021/acssynbio.4c00298
Tobias Michael Rosch, Julia Tenhaef, Tim Stoltmann, Till Redeker, Dominic Kösters, Niels Hollmann, Karin Krumbach, Wolfgang Wiechert, Michael Bott, Susana Matamouros, Jan Marienhagen, Stephan Noack
{"title":"AutoBioTech─A Versatile Biofoundry for Automated Strain Engineering.","authors":"Tobias Michael Rosch, Julia Tenhaef, Tim Stoltmann, Till Redeker, Dominic Kösters, Niels Hollmann, Karin Krumbach, Wolfgang Wiechert, Michael Bott, Susana Matamouros, Jan Marienhagen, Stephan Noack","doi":"10.1021/acssynbio.4c00298","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00298","url":null,"abstract":"<p><p>The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering of microbes will not be sufficient to meet the ever-increasing demand for novel producer strains. Here we describe the AutoBioTech platform, a fully automated laboratory system with 14 devices to perform operations for strain construction without human interaction. Using modular workflows, this platform enables automated transformations of <i>Escherichia coli</i> with plasmids assembled via modular cloning. A CRISPR/Cas9 toolbox compatible with existing modular cloning frameworks allows automated and flexible genome editing of <i>E. coli</i>. In addition, novel workflows have been established for the fully automated transformation of the Gram-positive model organism <i>Corynebacterium glutamicum</i> by conjugation and electroporation, with the latter proving to be the more robust technique. Overall, the AutoBioTech platform excels at versatility due to the modularity of workflows and seamless transitions between modules. This will accelerate strain engineering of Gram-negative and Gram-positive bacteria.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141553549","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Reliable Genomic Integration Sites in Pseudomonas putida Identified by Two-Dimensional Transcriptome Analysis. 通过二维转录组分析确定假单胞菌的可靠基因组整合位点
IF 3.7 2区 生物学
ACS Synthetic Biology Pub Date : 2024-07-05 DOI: 10.1021/acssynbio.3c00747
Sebastian Köbbing, Thorsten Lechtenberg, Benedikt Wynands, Lars M Blank, Nick Wierckx
{"title":"Reliable Genomic Integration Sites in <i>Pseudomonas putida</i> Identified by Two-Dimensional Transcriptome Analysis.","authors":"Sebastian Köbbing, Thorsten Lechtenberg, Benedikt Wynands, Lars M Blank, Nick Wierckx","doi":"10.1021/acssynbio.3c00747","DOIUrl":"https://doi.org/10.1021/acssynbio.3c00747","url":null,"abstract":"<p><p>Genomic integration is commonly used to engineer stable production hosts. However, so far, for many microbial workhorses, only a few integration sites have been characterized, thereby restraining advanced strain engineering that requires multiple insertions. Here, we report on the identification of novel genomic integration sites, so-called landing pads, for <i>Pseudomonas putida</i> KT2440. We identified genomic regions with constant expression patterns under diverse experimental conditions by using RNA-Seq data. Homologous recombination constructs were designed to insert heterologous genes into intergenic sites in these regions, allowing condition-independent gene expression. Ten potential landing pads were characterized using four different <i>msfGFP</i> expression cassettes. An insulated probe sensor was used to study locus-dependent effects on recombinant gene expression, excluding genomic read-through of flanking promoters under changing cultivation conditions. While the reproducibility of expression in the landing pads was very high, the msfGFP signals varied strongly between the different landing pads, confirming a strong influence of the genomic context. To showcase that the identified landing pads are also suitable candidates for heterologous gene expression in other Pseudomonads, four equivalent landing pads were identified and characterized in <i>Pseudomonas taiwanensis</i> VLB120. This study shows that genomic \"hot\" and \"cold\" spots exist, causing strong promoter-independent variations in gene expression. This highlights that the genomic context is an additional parameter to consider when designing integrable genomic cassettes for tailored heterologous expression. The set of characterized genomic landing pads presented here further increases the genetic toolbox for deep metabolic engineering in Pseudomonads.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141537141","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Investigating and Engineering an 1,2-Propanediol-Responsive Transcription Factor-Based Biosensor. 基于转录因子的 1,2-丙二醇反应生物传感器的研究与工程设计
IF 3.7 2区 生物学
ACS Synthetic Biology Pub Date : 2024-07-05 DOI: 10.1021/acssynbio.4c00237
Yuxi Teng, Xinyu Gong, Jianli Zhang, Ziad Obideen, Yajun Yan
{"title":"Investigating and Engineering an 1,2-Propanediol-Responsive Transcription Factor-Based Biosensor.","authors":"Yuxi Teng, Xinyu Gong, Jianli Zhang, Ziad Obideen, Yajun Yan","doi":"10.1021/acssynbio.4c00237","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00237","url":null,"abstract":"<p><p>Transcription factor (TF)-based biosensors have arisen as powerful tools in the advancement of metabolic engineering. However, with the emergence of numerous bioproduction targets, the variety of applicable TF-based biosensors remains severely limited. In this study, we investigated and engineered an 1,2-propanediol (1,2-PD)-responsive transcription activator, PocR, from <i>Salmonella typhimurium</i> to enrich the current biosensor repertoire. Heterologous characterization of PocR in <i>E. coli</i> revealed a significantly limited operational range and dynamic range, primarily attributed to the leaky binding between PocR and its corresponding promoters in the absence of the 1,2-PD inducer. Promiscuity characterization uncovered the minor responsiveness of PocR toward glycerol and 1,2-butanediol (1,2-BD). Using AlphaFold-predicted structure and protein mutagenesis, we preliminarily explored the underlying mechanism of PocR. Based on the investigated mechanism, we engineered a PcoR-F46R/G105D variant with an altered inducer specificity to glycerol, as well as a PocR-ARE (Q107A/S192R/A203E) variant with nearly a 4-fold higher dynamic range (6.7-fold activation) and a 20-fold wider operational range (0-20 mM 1,2-PD). Finally, we successfully converted PocR to a repressor through promoter engineering. Integrating the activation and repression functions established a versatile 1,2-PD-induced bifunctional regulation system based on PocR-ARE. Our work showcases the exploration and exploitation of an underexplored type of transcriptional activator capable of recruiting RNA polymerase. It also expands the biosensor toolbox by providing a 1,2-PD-responsive bifunctional regulator and glycerol-responsive activator.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141537140","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Non-native Pathway Engineering with CRISPRi for Carbon Dioxide Assimilation and Valued 5-Aminolevulinic Acid Synthesis in Escherichia coli Nissle. 利用 CRISPRi 在大肠杆菌 Nissle 中进行二氧化碳同化和有价值的 5-氨基乙酰丙酸合成的非本地途径工程。
IF 3.7 2区 生物学
ACS Synthetic Biology Pub Date : 2024-07-02 DOI: 10.1021/acssynbio.4c00318
Sefli Sri Wahyu Effendi, I-Son Ng
{"title":"Non-native Pathway Engineering with CRISPRi for Carbon Dioxide Assimilation and Valued 5-Aminolevulinic Acid Synthesis in <i>Escherichia coli</i> Nissle.","authors":"Sefli Sri Wahyu Effendi, I-Son Ng","doi":"10.1021/acssynbio.4c00318","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00318","url":null,"abstract":"<p><p>Carbon dioxide emission and acidification during chemical biosynthesis are critical challenges toward microbial cell factories' sustainability and efficiency. Due to its acidophilic traits among workhorse lineages, the probiotic <i>Escherichia coli</i> Nissle (EcN) has emerged as a promising chemical bioproducer. However, EcN lacks a CO<sub>2</sub>-fixing system. Herein, EcN was equipped with a simultaneous CO<sub>2</sub> fixation system and subsequently utilized to produce low-emission 5-aminolevulinic acid (5-ALA). Two different artificial CO<sub>2</sub>-assimilating pathways were reconstructed: the novel ribose-1,5-bisphosphate (R15P) route and the conventional ribulose-5-phosphate (Ru5P) route. CRISPRi was employed to target the <i>pfk</i>AB and <i>zwf</i> genes in order to redirect the carbon flux. As expected, the CRISPRi design successfully strengthened the CO<sub>2</sub> fixation. The CO<sub>2</sub>-fixing route via R15P resulted in high biomass, while the engineered Ru5P route acquired the highest 5-ALA and suppressed the CO<sub>2</sub> release by 77%. CO<sub>2</sub> fixation during 5-ALA production in EcN was successfully synchronized through fine-tuning the non-native pathways with CRISPRi.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141489923","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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