ACS Synthetic Biology最新文献_第9页

Orthogonal Serine Integrases Enable Scalable Gene Storage Cascades in Bacterial Genome. 正交丝氨酸整合酶使细菌基因组中可扩展的基因存储级联成为可能

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2024-09-20 Epub Date: 2024-09-06 DOI: 10.1021/acssynbio.4c00505

Yufei Zhang, Fang Ba, Shuhui Huang, Wan-Qiu Liu, Jian Li

{"title":"Orthogonal Serine Integrases Enable Scalable Gene Storage Cascades in Bacterial Genome.","authors":"Yufei Zhang, Fang Ba, Shuhui Huang, Wan-Qiu Liu, Jian Li","doi":"10.1021/acssynbio.4c00505","DOIUrl":"10.1021/acssynbio.4c00505","url":null,"abstract":"Genome integration enables host organisms to stably carry heterologous DNA messages, introducing new genotypes and phenotypes for expanded applications. While several genome integration approaches have been reported, a scalable tool for DNA message storage within site-specific genome landing pads is still lacking. Here, we introduce an iterative genome integration method utilizing orthogonal serine integrases, enabling the stable storage of multiple heterologous genes in the chromosome of Escherichia coli MG1655. By leveraging serine integrases TP901-1, Bxb1, and PhiC31, along with engineered integration vectors, we demonstrate high-efficiency, marker-free integration of DNA fragments up to 13 kb in length. To further simplify the procedure, we then develop a streamlined integration method and showcase the system's versatility by constructing an engineered E. coli strain capable of storing and expressing multiple genes from diverse species. Additionally, we illustrate the potential utility of these engineered strains for synthetic biology applications, including in vivo and in vitro protein expression. Our work extends the application scope of serine integrases for scalable gene integration cascades, with implications for genome manipulation and gene storage applications in synthetic biology.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142138546","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

Adeno-Associated Virus 5 Protein Particles Produced by E. coli Cell-Free Protein Synthesis. 大肠杆菌细胞自由蛋白质合成产生的腺相关病毒 5 蛋白颗粒。

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2024-09-20 Epub Date: 2024-08-23 DOI: 10.1021/acssynbio.4c00403

Danielle Deuker, Ernest Asilonu, Daniel G Bracewell, Stefanie Frank

{"title":"Adeno-Associated Virus 5 Protein Particles Produced by E. coli Cell-Free Protein Synthesis.","authors":"Danielle Deuker, Ernest Asilonu, Daniel G Bracewell, Stefanie Frank","doi":"10.1021/acssynbio.4c00403","DOIUrl":"10.1021/acssynbio.4c00403","url":null,"abstract":"Recombinant adeno-associated viruses (rAAVs) have emerged as important tools for gene therapy and, more recently, vaccine development. Nonetheless, manufacturing can be costly and time-consuming, emphasizing the importance of alternative production platforms. We investigate the potential of E. coli-based cell-free protein synthesis (CFPS) to produce recombinant AAV5 virus-like particles (VLPs). AAV5 virus protein 3 (VP3) constructs, both with and without Strep-tag II, were expressed with CFPS. Lower reaction temperatures resulted in increased solubility, with the untagged variant containing nearly 90% more soluble VLP VP3 protein at 18 °C than at 37 °C. Affinity chromatography of N-terminally Strep(II)-tagged VP3 enabled successful isolation with minimal processing. DLS and TEM confirmed the presence of ∼20 nm particles. Furthermore, the N-terminally tagged AAV5 VP3 VLPs were biologically active, successfully internalizing into HeLa cells. This study describes an innovative approach to AAV VLP production using E. coli-based CFPS, demonstrating its potential for rapid and biologically active AAV VLP synthesis.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11421080/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142043776","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}

引用次数: 0

A Simple and Effective Strategy for the Development of Robust Promoter-Centric Gene Expression Tools. 开发以启动子为中心的强大基因表达工具的简单有效策略

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2024-09-20 Epub Date: 2024-08-09 DOI: 10.1021/acssynbio.4c00092

Tongjian Yang, Yihua Chen, Xiaozhou Luo, Jay D Keasling, Keqiang Fan, Guohui Pan

{"title":"A Simple and Effective Strategy for the Development of Robust Promoter-Centric Gene Expression Tools.","authors":"Tongjian Yang, Yihua Chen, Xiaozhou Luo, Jay D Keasling, Keqiang Fan, Guohui Pan","doi":"10.1021/acssynbio.4c00092","DOIUrl":"10.1021/acssynbio.4c00092","url":null,"abstract":"Promoter-centric genetic tools play a crucial role in controlling gene expression for various applications, such as strain engineering and synthetic biology studies. Hence, a critical need persists for the development of robust gene expression tools. Streptomyces are well-known prolific producers of natural products and exceptional surrogate hosts for the production of high-value chemical compounds and enzymes. In this study, we reported a straightforward and effective strategy for the creation of potent gene expression tools. This was primarily achieved by introducing an additional -35-like motif upstream of the original -35 region of the promoter, coupled with the integration of a palindromic cis-element into the 5'-UTR region. This approach has generated a collection of robust constitutive and inducible gene expression tools tailored for Streptomyces. Of particular note, the fully activated oxytetracycline-inducible gene expression system containing an engineered kasOp* promoter (OK) exhibited nearly an order of magnitude greater activity compared to the well-established high-strength promoter kasOp* under the tested conditions, establishing itself as a powerful gene expression system for Streptomyces. This strategy is expected to be applicable in modifying various other promoters to acquire robust gene expression tools, as evidenced by the enhancement observed in the other two promoters, PL and P21 in this study. Moreover, the effectiveness of these tools has been demonstrated through the augmented production of transglutaminase and daptomycin. The gene expression tools established in this study, alongside those anticipated in forthcoming research, are positioned to markedly advance pathway engineering and synthetic biology investigations in Streptomyces and other microbial strains.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141904956","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

Systems-Level Modeling for CRISPR-Based Metabolic Engineering. 基于 CRISPR 的代谢工程的系统级建模。

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2024-09-20 Epub Date: 2024-08-09 DOI: 10.1021/acssynbio.4c00053

Ryan A L Cardiff, James M Carothers, Jesse G Zalatan, Herbert M Sauro

引用次数: 0

Metabolic Engineering of High L-Lysine-Producing Escherichia coli for de Novo Production of L-Lysine-Derived Compounds. 从新生产 L-赖氨酸衍生化合物的高产 L-赖氨酸大肠杆菌代谢工程。

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2024-09-20 Epub Date: 2024-08-19 DOI: 10.1021/acssynbio.4c00356

Yonghua Chen, Wenzhu Song, Guodong Wang, Yuanwei Wang, Shitong Dong, Yingshuai Wu, Ruiming Wang, Chunling Ma

{"title":"Metabolic Engineering of High L-Lysine-Producing Escherichia coli for de Novo Production of L-Lysine-Derived Compounds.","authors":"Yonghua Chen, Wenzhu Song, Guodong Wang, Yuanwei Wang, Shitong Dong, Yingshuai Wu, Ruiming Wang, Chunling Ma","doi":"10.1021/acssynbio.4c00356","DOIUrl":"10.1021/acssynbio.4c00356","url":null,"abstract":"5-Aminovalerate (5-AVA), 5-hydroxyvalerate (5-HV), and 1,5-pentanediol (1,5-PDO) are l-lysine derivatives with extensive applications in the production of materials such as polyesters, polyurethane, plasticizers, inks, and coatings. However, their large-scale production is limited by the lack of efficient synthetic pathways. Here, we aimed to construct multiple synthetic pathways by screening the key enzymes involved in the synthesis of these compounds in Escherichia coli. The engineered pathway utilizing RaiP demonstrated a superior catalytic efficiency. The LER strain that overexpressed only raiP successfully synthesized 9.70 g/L 5-HV and 8.31 g/L 5-AVA, whereas the strain LERGY that overexpressed raiP, gabT, and yahK accumulated 9.72 g/L 5-HV and 7.95 g/L 5-AVA from 20 g/L glucose. The introduction of exogenous transaminases and dehydrogenases enhanced cell growth and fermentation efficiency with respect to 5-HV synthesis, albeit without significantly impacting the yield. Strain LE05, incorporating only two exogenous enzymes, RaiP and CaR, produced 1.87 g/L 1,5-PDO, 3.85 g/L 5-HV, and 4.78 g/L 5-hydroxyglutaraldehyde from 20 g/L glucose after 6 days. The strain LE02G, fortified with transaminase, dehydrogenase, and NADPH regeneration system, accumulated 7.82 g/L 1,5-PDO, whereas the aldp-knock out LE02G2 synthesized 10.98 g/L 1,5-PDO from 50 g/L glucose in fed-batch fermentation after 6 days, yielding 0.22 g/g glucose (0.37 mol/mol). Introducing the NADPH regeneration pathway and deleting the NADPH-consuming pathways increased the 1,5-PDO yield and decreased the precursor concentration. The proposed pathways and engineering strategies presented in this study can prove instrumental in developing biological routes for the practical production of 5-AVA, 5-HV, and 1,5-PDO.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141998858","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

A Synthetic Biology Approach to Transgene Expression in Insects. 昆虫转基因表达的合成生物学方法。

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2024-09-20 Epub Date: 2024-08-28 DOI: 10.1021/acssynbio.4c00250

Philip T Leftwich, Jessica C Purcell, Michelle A E Anderson, Rennos Fragkoudis, Sanjay Basu, Gareth Lycett, Luke Alphey

引用次数: 0

Genetic Code Expansion in Shewanella oneidensis MR-1 Allows Site-Specific Incorporation of Bioorthogonal Functional Groups into a c-Type Cytochrome. Shewanella oneidensis MR-1 中的遗传密码扩展允许在特定位点将生物正交功能基团结合到 c 型细胞色素中。

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2024-09-20 Epub Date: 2024-08-19 DOI: 10.1021/acssynbio.4c00248

Colin W J Lockwood, Benjamin W Nash, Simone E Newton-Payne, Jessica H van Wonderen, Keir P S Whiting, Abigail Connolly, Alexander L Sutton-Cook, Archie Crook, Advait R Aithal, Marcus J Edwards, Thomas A Clarke, Amit Sachdeva, Julea N Butt

{"title":"Genetic Code Expansion in Shewanella oneidensis MR-1 Allows Site-Specific Incorporation of Bioorthogonal Functional Groups into a c-Type Cytochrome.","authors":"Colin W J Lockwood, Benjamin W Nash, Simone E Newton-Payne, Jessica H van Wonderen, Keir P S Whiting, Abigail Connolly, Alexander L Sutton-Cook, Archie Crook, Advait R Aithal, Marcus J Edwards, Thomas A Clarke, Amit Sachdeva, Julea N Butt","doi":"10.1021/acssynbio.4c00248","DOIUrl":"10.1021/acssynbio.4c00248","url":null,"abstract":"Genetic code expansion has enabled cellular synthesis of proteins containing unique chemical functional groups to allow the understanding and modulation of biological systems and engineer new biotechnology. Here, we report the development of efficient methods for site-specific incorporation of structurally diverse noncanonical amino acids (ncAAs) into proteins expressed in the electroactive bacterium Shewanella oneidensis MR-1. We demonstrate that the biosynthetic machinery for ncAA incorporation is compatible and orthogonal to the endogenous pathways of S. oneidensis MR-1 for protein synthesis, maturation of c-type cytochromes, and protein secretion. This allowed the efficient synthesis of a c-type cytochrome, MtrC, containing site-specifically incorporated ncAA in S. oneidensis MR-1 cells. We demonstrate that site-specific replacement of surface residues in MtrC with ncAAs does not influence its three-dimensional structure and redox properties. We also demonstrate that site-specifically incorporated bioorthogonal functional groups could be used for efficient site-selective labeling of MtrC with fluorophores. These synthetic biology developments pave the way to expand the chemical repertoire of designer proteins expressed in S. oneidensis MR-1.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11421213/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141998857","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}

引用次数: 0

Optimized CRISPR Interference System for Investigating Pseudomonas alloputida Genes Involved in Rhizosphere Microbiome Assembly. 优化 CRISPR 干扰系统，用于研究参与根瘤菌微生物组组装的全生育期假单胞菌基因。

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2024-09-20 Epub Date: 2024-08-20 DOI: 10.1021/acssynbio.4c00312

Marissa N Roghair Stroud, Dua X Vang, Larry J Halverson

{"title":"Optimized CRISPR Interference System for Investigating Pseudomonas alloputida Genes Involved in Rhizosphere Microbiome Assembly.","authors":"Marissa N Roghair Stroud, Dua X Vang, Larry J Halverson","doi":"10.1021/acssynbio.4c00312","DOIUrl":"10.1021/acssynbio.4c00312","url":null,"abstract":"Pseudomonas alloputida KT2440 (formerly P. putida) has become both a well-known chassis organism for synthetic biology and a model organism for rhizosphere colonization. Here, we describe a CRISPR interference (CRISPRi) system in KT2440 for exploring microbe-microbe interactions in the rhizosphere and for use in industrial systems. Our CRISPRi system features three different promoter systems (XylS/Pm, LacI/Plac, and AraC/PBAD) and a dCas9 codon-optimized for Pseudomonads, all located on a mini-Tn7-based transposon that inserts into a neutral site in the genome. It also includes a suite of pSEVA-derived sgRNA expression vectors, where the expression is driven by synthetic promoters varying in strength. We compare the three promoter systems in terms of how well they can precisely modulate gene expression, and we discuss the impact of environmental factors, such as media choice, on the success of CRISPRi. We demonstrate that CRISPRi is functional in bacteria colonizing the rhizosphere, with repression of essential genes leading to a 10-100-fold reduction in P. alloputida cells per root. Finally, we show that CRISPRi can be used to modulate microbe-microbe interactions. When the gene pvdH is repressed and P. alloputida is unable to produce pyoverdine, it loses its ability to inhibit other microbes in vitro. Moreover, our design is amendable for future CRISPRi-seq studies and in multispecies microbial communities, with the different promoter systems providing a means to control the level of gene expression in many different environments.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11421427/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142007823","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}

引用次数: 0

Design of Thermoresponsive Genetic Controls with Minimal Heat-Shock Response. 设计热休克反应最小的抗热基因控制装置

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2024-09-20 Epub Date: 2024-08-16 DOI: 10.1021/acssynbio.4c00236

Haofeng Chen, Shan Jiang, Kaixuan Xu, Ziyu Ding, Jiangkai Wang, Mingfeng Cao, Jifeng Yuan

{"title":"Design of Thermoresponsive Genetic Controls with Minimal Heat-Shock Response.","authors":"Haofeng Chen, Shan Jiang, Kaixuan Xu, Ziyu Ding, Jiangkai Wang, Mingfeng Cao, Jifeng Yuan","doi":"10.1021/acssynbio.4c00236","DOIUrl":"10.1021/acssynbio.4c00236","url":null,"abstract":"As temperature serves as a versatile input signal, thermoresponsive genetic controls have gained significant interest for recombinant protein production and metabolic engineering applications. The conventional thermoresponsive systems normally require the continuous exposure of heat stimuli to trigger the prolonged expression of targeted genes, and the accompanied heat-shock response is detrimental to the bioproduction process. In this study, we present the design of thermoresponsive quorum-sensing (ThermoQS) circuits to make Escherichia coli record transient heat stimuli. By conversion of the heat input into the accumulation of quorum-sensing molecules such as acyl-homoserine lactone derived from Pseudomonas aeruginosa, sustained gene expressions were achieved by a minimal heat stimulus. Moreover, we also demonstrated that we reprogrammed the E. coli Lac operon to make it respond to heat stimuli with an impressive signal-to-noise ratio (S/N) of 15.3. Taken together, we envision that the ThermoQS systems reported in this study are expected to remarkably diminish both design and experimental expenditures for future metabolic engineering applications.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141992182","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

GOLDBAR: A Framework for Combinatorial Biological Design. GOLDBAR：组合生物设计框架。

IF 3.7 2区生物学

ACS Synthetic Biology Pub Date : 2024-09-20 Epub Date: 2024-08-20 DOI: 10.1021/acssynbio.4c00296

Nicholas Roehner, James Roberts, Andrei Lapets, Dany Gould, Vidya Akavoor, Lucy Qin, D Benjamin Gordon, Christopher Voigt, Douglas Densmore

{"title":"GOLDBAR: A Framework for Combinatorial Biological Design.","authors":"Nicholas Roehner, James Roberts, Andrei Lapets, Dany Gould, Vidya Akavoor, Lucy Qin, D Benjamin Gordon, Christopher Voigt, Douglas Densmore","doi":"10.1021/acssynbio.4c00296","DOIUrl":"10.1021/acssynbio.4c00296","url":null,"abstract":"With the rise of new DNA part libraries and technologies for assembling DNA, synthetic biologists are increasingly constructing and screening combinatorial libraries to optimize their biological designs. As combinatorial libraries are used to generate data on design performance, new rules for composing biological designs will emerge. Most formal frameworks for combinatorial design, however, do not yet support formal comparison of design composition, which is needed to facilitate automated analysis and machine learning in massive biological design spaces. To address this need, we introduce a combinatorial design framework called GOLDBAR. Compared with existing frameworks, GOLDBAR enables synthetic biologists to intersect and merge the rules for entire classes of biological designs to extract common design motifs and infer new ones. Here, we demonstrate the application of GOLDBAR to refine/validate design spaces for TetR-homologue transcriptional logic circuits, verify the assembly of a partial nif gene cluster, and infer novel gene clusters for the biosynthesis of rebeccamycin. We also discuss how GOLDBAR could be used to facilitate grammar-based machine learning in synthetic biology.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142002949","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