ACS Synthetic Biology最新文献

{"title":"Dual-Plasmid Mini-Tn5 System to Stably Integrate Multicopy of Target Genes in <i>Escherichia coli</i>.","authors":"Menghui Liu, Wei Ge, Guomei Zhong, Yuqing Yang, Luying Xun, Yongzhen Xia","doi":"10.1021/acssynbio.4c00140","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00140","url":null,"abstract":"<p><p>The efficiency of valuable metabolite production by engineered microorganisms underscores the importance of stable and controllable gene expression. While plasmid-based methods offer flexibility, integrating genes into host chromosomes can establish stability without selection pressure. However, achieving site-directed multicopy integration presents challenges, including site selection and stability. We introduced a stable multicopy integration method by using a novel dual-plasmid mini-Tn5 system to insert genes into <i>Escherichia coli</i>'s genome. The gene of interest was combined with a removable antibiotic resistance gene. After the selection of bacteria with inserted genes, the antibiotic resistance gene was removed. Optimizations yielded an integration efficiency of approximately 5.5 × 10^-3 per recipient cell in a single round. Six rounds of integration resulted in 19 and 5 copies of the <i>egfp</i> gene in the RecA⁺ strain MG1655 and the RecA^- strain XL1-Blue MRF', respectively. Additionally, we integrated a polyhydroxybutyrate (PHB) synthesis gene cluster into <i>E. coli</i> MG1655, yielding an 8-copy integration strain producing more PHB than strains with the cluster on a high-copy plasmid. The method was efficient in generating gene insertions in various <i>E. coli</i> strains, and the inserted genes were stable after extended culture. This stable, high-copy integration tool offers potential for diverse applications in synthetic biology.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142453376","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}

{"title":"Reconfiguring the <i>Escherichia coli</i> Electron Transport Chain to Enhance <i>trans</i>-2-Decenoic Acid Production.","authors":"Ben Liu, HaoYang Wang, ChunLi Su, SiFan ShangGuan, YiSang Zhang, ShiHao Nie, Ruiming Wang, Piwu Li, Junqing Wang, Jing Su","doi":"10.1021/acssynbio.4c00451","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00451","url":null,"abstract":"<p><p><i>trans</i>-2-Decenoic acid is a pivotal α,β-medium-chain unsaturated fatty acid that serves as an essential intermediary in the synthesis of 10-hydroxy-2-decenoic acid and various pharmaceutical compounds. Biosynthesis yield of <i>trans</i>-2-decenoic acid by decanoic acid has significantly improved in recent years; however, the oxidative stress of <i>Escherichia coli</i> at high fatty acid concentrations restricts the conversion rate. Here, we introduced a combination of rational design and metabolic rewiring of the <i>E. coli</i> electron transport chain (ETC) to improve <i>trans</i>-2-decenoic acid production. Overexpressing ubiquinone (UbQ) biosynthesis genes enhanced the expression of ETC complex III: UbQ to reduce reactive oxygen species (ROS) accumulation. Furthermore, applying rotenone to inhibit ETC complex I improved the electron transfer efficiency of complex II. The integration of Vitamin B₅ and B₂ into the fermentation process increased the activities of fatty acyl-CoA synthetase (^<i>Ma</i><i>MACS</i>) and fatty acyl-CoA dehydrogenase (^<i>Pp</i><i>fadE</i>). Finally, the constructed <i>E. coli</i> BL21(DE3)(Δ<i>fadBJR</i>/pCDFDuet-1-^<i>Pp</i><i>fadE</i>-^<i>Ma</i><i>MACS</i>/pRSFDuet-1-sumo-^<i>Ct</i><i>ydiI</i>-<i>ubiI</i>) strain exhibited a 51.50% decrease in ROS and a 93.33% enhancement in <i>trans</i>-2-decenoic acid yield, reaching 1.45 g/L after 66 h, which is the highest yield reported for flask fermentation. This study reports the feasibility of rewiring the ETC regulation and energy metabolism to improve α,β-UCA biosynthesis efficiency.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142453380","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}

{"title":"Engineering Cyborg Pathogens through Intracellular Hydrogelation.","authors":"Shahid Khan, Pin-Ru Lin, Cheemeng Tan","doi":"10.1021/acssynbio.4c00420","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00420","url":null,"abstract":"<p><p>Synthetic biology primarily focuses on two kinds of cell chassis: living cells and nonliving systems. Living cells are autoreplicating systems that have active metabolism. Nonliving systems, including artificial cells and nanoparticles, are nonreplicating systems typically lacking active metabolism. In recent work, Cyborg bacteria that are nonreplicating-but-metabolically active have been engineered through intracellular hydrogelation. Intracellular hydrogelation is conducted by infusing gel monomers and photoactivators into cells, followed by the activation of polymerization of the gel monomers inside the cells. However, the previous work investigated only <i>Escherichia coli</i> cells. Extending the Cyborg-Cell method to pathogenic bacteria could enable the exploitation of their pathogenic properties in biomedical applications. Here, we focus on different strains of <i>Pseudomonas aeruginosa</i>, <i>Staphylococcus aureus</i>, and <i>Klebsiella pneumoniae</i>. To synthesize the Cyborg pathogens, we first reveal the impact of different hydrogel concentrations on the metabolism, replication, and intracellular gelation of Cyborg pathogens. Next, we demonstrate that the Cyborg pathogens are taken up by macrophages in a similar magnitude as wild-type pathogens through confocal microscopy and real-time PCR. Finally, we show that the macrophage that takes up the Cyborg pathogen exhibits a similar phenotypic response to the wild-type pathogen. Our work generalizes the intracellular hydrogelation approach from lab strains of <i>E. coli</i> to bacterial pathogens. The new Cyborg pathogens could be applied in biomedical applications ranging from drug delivery to immunotherapy.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142453377","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}

{"title":"Formaldehyde: An Essential Intermediate for C1 Metabolism and Bioconversion.","authors":"Mengshi Jia, Mengge Liu, Jiawen Li, Wankui Jiang, Fengxue Xin, Wenming Zhang, Yujia Jiang, Min Jiang","doi":"10.1021/acssynbio.4c00454","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00454","url":null,"abstract":"<p><p>Formaldehyde is an intermediate metabolite of methylotrophic microorganisms that can be obtained from formate and methanol through oxidation-reduction reactions. Formaldehyde is also a one-carbon (C1) compound with high uniquely reactive activity and versatility, which is more amenable to further biocatalysis. Biosynthesis of high-value-added chemicals using formaldehyde as an intermediate is theoretically feasible and promising. This review focuses on the design of the biosynthesis of high-value-added chemicals using formaldehyde as an essential intermediate. The upstream biosynthesis and downstream bioconversion pathways of formaldehyde as an intermediate metabolite are described in detail, aiming to highlight the important role of formaldehyde in the transition from inorganic to organic carbon and carbon chain elongation. In addition, challenges and future directions of formaldehyde as an intermediate for the chemicals are discussed, with the expectation of providing ideas for the utilization of C1.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142453378","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}

{"title":"Macroscopic Assembly of Materials with Engineered Bacterial Spores via Coiled-Coil Interaction.","authors":"Lucas Korbanka, Ju-An Kim, Seunghyun Sim","doi":"10.1021/acssynbio.4c00468","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00468","url":null,"abstract":"<p><p>Herein, we report macroscopic materials formed by the assembly of engineered bacterial spores. Spores were engineered by using a T7-driven expression system to display a high density of pH-responsive self-associating proteins on their surface. The engineered surface protein on the spore surface enabled pH-dependent binding at the protein level and enabled the assembly of granular materials. Mechanical properties remained largely constant with changing pH, but erosion stability was pH-dependent in a manner consistent with the pH-dependent interaction between the engineered surface proteins. Our finding utilizes synthetic biology for the design of macroscopic materials and illuminates the impact of coiled-coil interaction across various length scales.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142407417","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}

{"title":"Cell-Free Systems Biology: Characterizing Central Metabolism of <i>Clostridium thermocellum</i> with a Three-Enzyme Cascade Reaction.","authors":"S Bilal Jilani, Markus Alahuhta, Yannick J Bomble, Daniel G Olson","doi":"10.1021/acssynbio.4c00405","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00405","url":null,"abstract":"<p><p>Genetic approaches have been traditionally used to understand microbial metabolism, but this process can be slow in nonmodel organisms due to limited genetic tools. An alternative approach is to study metabolism directly in the cell lysate. This avoids the need for genetic tools and is routinely used to study individual enzymatic reactions but is not generally used to study systems-level properties of metabolism. Here we demonstrate a new approach that we call \"cell-free systems biology\", where we use well-characterized enzymes and multienzyme cascades to serve as sources or sinks of intermediate metabolites. This allows us to isolate subnetworks within metabolism and study their systems-level properties. To demonstrate this, we worked with a three-enzyme cascade reaction that converts pyruvate to 2,3-butanediol. Although it has been previously used in cell-free systems, its pH dependence was not well characterized, limiting its utility as a sink for pyruvate. We showed that improved proton accounting allowed better prediction of pH changes and that active pH control allowed 2,3-butanediol titers of up to 2.1 M (189 g/L) from acetoin and 1.6 M (144 g/L) from pyruvate. The improved proton accounting provided a crucial insight that preventing the escape of CO₂ from the system largely eliminated the need for active pH control, dramatically simplifying our experimental setup. We then used this cascade reaction to understand limits to product formation in <i>Clostridium thermocellum</i>, an organism with potential applications for cellulosic biofuel production. We showed that the fate of pyruvate is largely controlled by electron availability and that reactions upstream of pyruvate limit overall product formation.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142453364","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}

{"title":"Fine-Regulating the Carbon Flux of l-Isoleucine Producing Corynebacterium glutamicum WM001 for Efficient l-Threonine Production","authors":"Guihong Zhao,&nbsp;Dezhi Zhang,&nbsp;Benzheng Zhou,&nbsp;Zihan Li,&nbsp;Geer Liu,&nbsp;Hedan Li,&nbsp;Xiaoqing Hu and Xiaoyuan Wang*,&nbsp;","doi":"10.1021/acssynbio.4c0051810.1021/acssynbio.4c00518","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00518https://doi.org/10.1021/acssynbio.4c00518","url":null,"abstract":"<p ><span>l</span>-Threonine, an essential amino acid, is widely used in various industries, with an annually growing demand. However, the present <i>Corynebacterium glutamicum</i> strains are difficult to achieve industrialization of <span>l</span>-threonine due to low yield and purity. In this study, we engineered an <span>l</span>-isoleucine-producing <i>C. glutamicum</i> WM001 to efficiently produce <span>l</span>-threonine by finely regulating the carbon flux. First, the threonine dehydratase in WM001 was mutated to lower the level of <span>l</span>-isoleucine production, then the homoserine dehydrogenase and aspartate kinase were mutated to release the feedback inhibition of <span>l</span>-threonine, and the resulting strain TWZ006 produced 14.2 g/L <span>l</span>-threonine. Subsequently, aspartate ammonia-lyase and aspartate transaminase were overexpressed to accumulate the precursor <span>l</span>-aspartate. Next, phosphoenolpyruvate carboxylase, pyruvate carboxylase and pyruvate kinase were overexpressed, and phosphoenolpyruvate carboxykinase, oxaloacetate decarboxylase were inactivated to fine-regulate the carbon flux among oxaloacetate, pyruvate and phosphoenolpyruvate. The resulting strain TWZ017 produced 21.5 g/L <span>l</span>-threonine. Finally, dihydrodipicolinate synthase was mutated with strong allosteric inhibition from <span>l</span>-lysine to significantly decrease byproducts accumulation, <span>l</span>-threonine export was optimized, and the final engineered strain TWZ024/pXTuf-<i>thrE</i> produced 78.3 g/L of <span>l</span>-threonine with the yield of 0.33 g/g glucose and the productivity of 0.82 g/L/h in a 7 L bioreactor. To the best of our knowledge, this represents the highest <span>l</span>-threonine production in <i>C. glutamicum</i>, providing possibilities for industrial-scale production.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142450525","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}

{"title":"Characterizing and Engineering Rhamnose-Inducible Regulatory Systems for Dynamic Control of Metabolic Pathways in Streptomyces","authors":"Qian Yang,&nbsp;Mengao Luan,&nbsp;Meiyan Wang,&nbsp;Yuxin Zhang,&nbsp;Guoqiang Liu and Guoqing Niu*,&nbsp;","doi":"10.1021/acssynbio.4c0062610.1021/acssynbio.4c00626","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00626https://doi.org/10.1021/acssynbio.4c00626","url":null,"abstract":"<p >Fine-tuning gene expression is of great interest for synthetic biotechnological applications. This is particularly true for the genus <i>Streptomyces</i>, which is well-known as a prolific producer of diverse natural products. Currently, there is an increasing demand to develop effective gene induction systems. In this study, bioinformatic analysis revealed a putative rhamnose catabolic pathway in multiple <i>Streptomyces</i> species, and the removal of the pathway in the model organism <i>Streptomyces coelicolor</i> impaired its growth on minimal media with rhamnose as the sole carbon source. To unravel the regulatory mechanism of RhaR, a LacI family transcriptional regulator of the catabolic pathway, electrophoretic mobility shift assays (EMSAs) were performed to identify potential target promoters. Multiple sequence alignments retrieved a consensus sequence of the RhaR operator (<i>rhaO</i>). A synthetic biology-based strategy was then deployed to build rhamnose-inducible regulatory systems, referred to as <i>rhaRS1</i> and <i>rhaRS2</i>, by assembling the repressor/operator pair RhaR/<i>rhaO</i> with the well-defined constitutive <i>kasO*</i> promoter. Both <i>rhaRS1</i> and <i>rhaRS2</i> exhibited a high level of induced reporter activity, with no leaky expression. <i>rhaRS2</i> has been proven successful for the programmable production of actinorhodin and violacein in <i>Streptomyces</i>. Our study expanded the toolkit of inducible regulatory systems that will be broadly applicable to many other <i>Streptomyces</i> species.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142450868","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}

{"title":"Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting","authors":"Yiming Wan,&nbsp;Christopher Helenek,&nbsp;Damiano Coraci and Gábor Balázsi*,&nbsp;","doi":"10.1021/acssynbio.4c0027110.1021/acssynbio.4c00271","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00271https://doi.org/10.1021/acssynbio.4c00271","url":null,"abstract":"<p >The invention of RNA-guided DNA cutting systems has revolutionized biotechnology. More recently, RNA-guided RNA cutting by Cas13d entered the scene as a highly promising alternative to RNA interference to engineer cellular transcriptomes for biotechnological and therapeutic purposes. Unfortunately, “collateral damage” by indiscriminate off-target cutting tampered enthusiasm for these systems. Yet, how collateral activity, or even RNA target reduction depends on Cas13d and guide RNA abundance has remained unclear due to the lack of expression-tuning studies to address this question. Here we use precise expression-tuning gene circuits to show that both nonspecific and specific, on-target RNA reduction depend on Cas13d and guide RNA levels, and that nonspecific RNA cutting from <i>trans</i> cleavage might contribute to on-target RNA reduction. Using RNA-level control techniques, we develop new <i>Multi-Level Optimized Negative-Autoregulated Cas13d and crRNA Hybrid</i> (MONARCH) gene circuits that achieve a high dynamic range with low basal on-target RNA reduction while minimizing collateral activity in human kidney cells and green monkey cells most frequently used in human virology. MONARCH should bring RNA-guided RNA cutting systems to the forefront, as easily applicable, programmable tools for transcriptome engineering in biotechnological and medical applications.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.4c00271","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142450374","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Energy Aware Technology Mapping of Genetic Logic Circuits","authors":"Erik Kubaczka,&nbsp;Maximilian Gehri,&nbsp;Jérémie J. M. Marlhens,&nbsp;Tobias Schwarz,&nbsp;Maik Molderings,&nbsp;Nicolai Engelmann,&nbsp;Hernan G. Garcia,&nbsp;Christian Hochberger and Heinz Koeppl*,&nbsp;","doi":"10.1021/acssynbio.4c0039510.1021/acssynbio.4c00395","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00395https://doi.org/10.1021/acssynbio.4c00395","url":null,"abstract":"<p >Energy and its dissipation are fundamental to all living systems, including cells. Insufficient abundance of energy carriers─as caused by the additional burden of artificial genetic circuits─shifts a cell’s priority to survival, also impairing the functionality of the genetic circuit. Moreover, recent works have shown the importance of energy expenditure in information transmission. Despite living organisms being non-equilibrium systems, non-equilibrium models capable of accounting for energy dissipation and non-equilibrium response curves are not yet employed in genetic design automation (GDA) software. To this end, we introduce Energy Aware Technology Mapping, the automated design of genetic logic circuits with respect to energy efficiency and functionality. The basis for this is an energy aware non-equilibrium steady state model of gene expression, capturing characteristics like energy dissipation─which we link to the entropy production rate─and transcriptional bursting, relevant to eukaryotes as well as prokaryotes. Our evaluation shows that a genetic logic circuit’s functional performance and energy efficiency are disjoint optimization goals. For our benchmark, energy efficiency improves by 37.2% on average when comparing to functionally optimized variants. We discover a linear increase in energy expenditure and overall protein expression with the circuit size, where Energy Aware Technology Mapping allows for designing genetic logic circuits with the energetic costs of circuits that are one to two gates smaller. Structural variants improve this further, while results show the Pareto dominance among structures of a single Boolean function. By incorporating energy demand into the design, Energy Aware Technology Mapping enables energy efficiency by design. This extends current GDA tools and complements approaches coping with burden <i>in vivo</i>.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.4c00395","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142450377","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}