Désirée Körner, Niklas M. Schäfer, Antonio Lagares Jr., Lukas Birmes, Niels N. Oehlmann, Holly Addison, Sebastian Pöhl, Martin Thanbichler, Johannes G. Rebelein, Jörn Petersen and Anke Becker*,
{"title":"扩大阿尔法蛋白细菌宿主范围的模块化低拷贝数罗杆菌质粒载体","authors":"Désirée Körner, Niklas M. Schäfer, Antonio Lagares Jr., Lukas Birmes, Niels N. Oehlmann, Holly Addison, Sebastian Pöhl, Martin Thanbichler, Johannes G. Rebelein, Jörn Petersen and Anke Becker*, ","doi":"10.1021/acssynbio.4c00062","DOIUrl":null,"url":null,"abstract":"<p >Members of the alphaproteobacterial order Rhodobacterales are metabolically diverse and highly abundant in the ocean. They are becoming increasingly interesting for marine biotechnology, due to their ecological adaptability, wealth of versatile low-copy-number plasmids, and their ability to produce secondary metabolites. However, molecular tools for engineering strains of this bacterial lineage are limited. Here, we expand the genetic toolbox by establishing standardized, modular <i>repABC</i>-based plasmid vectors of four well-characterized compatibility groups from the Roseobacter group applicable in the Rhodobacterales, and likely in further alphaproteobacterial orders (Hyphomicrobiales, Rhodospirillales, Caulobacterales). We confirmed replication of these newly constructed pABC vectors in two members of Rhodobacterales, namely, <i>Dinoroseobacter shibae</i> DFL 12 and <i>Rhodobacter capsulatus</i> B10S, as well as in two members of the alphaproteobacterial order Hyphomicrobiales (synonym: Rhizobiales; <i>Ensifer meliloti</i> 2011 and “<i>Agrobacterium fabrum</i>” C58). Maintenance of the pABC vectors in the biotechnologically valuable orders Rhodobacterales and Hyphomicrobiales facilitates the shuttling of genetic constructs between alphaproteobacterial genera and orders. Additionally, plasmid replication was verified in one member of Rhodospirillales (<i>Rhodospirillum rubrum</i> S1) as well as in one member of Caulobacterales (<i>Caulobacter vibrioides</i> CB15N). The modular construction of pABC vectors and the usage of four compatible replication systems, which allows their coexistence in a host cell, are advantageous features for future implementations of newly designed synthetic pathways. The vector applicability was demonstrated by functional complementation of a nitrogenase mutant phenotype by two complementary pABC-based plasmids in <i>R. capsulatus</i>.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Modular Low-Copy-Number Plasmid Vectors for Rhodobacterales with Extended Host Range in Alphaproteobacteria\",\"authors\":\"Désirée Körner, Niklas M. Schäfer, Antonio Lagares Jr., Lukas Birmes, Niels N. Oehlmann, Holly Addison, Sebastian Pöhl, Martin Thanbichler, Johannes G. Rebelein, Jörn Petersen and Anke Becker*, \",\"doi\":\"10.1021/acssynbio.4c00062\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >Members of the alphaproteobacterial order Rhodobacterales are metabolically diverse and highly abundant in the ocean. 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Modular Low-Copy-Number Plasmid Vectors for Rhodobacterales with Extended Host Range in Alphaproteobacteria
Members of the alphaproteobacterial order Rhodobacterales are metabolically diverse and highly abundant in the ocean. They are becoming increasingly interesting for marine biotechnology, due to their ecological adaptability, wealth of versatile low-copy-number plasmids, and their ability to produce secondary metabolites. However, molecular tools for engineering strains of this bacterial lineage are limited. Here, we expand the genetic toolbox by establishing standardized, modular repABC-based plasmid vectors of four well-characterized compatibility groups from the Roseobacter group applicable in the Rhodobacterales, and likely in further alphaproteobacterial orders (Hyphomicrobiales, Rhodospirillales, Caulobacterales). We confirmed replication of these newly constructed pABC vectors in two members of Rhodobacterales, namely, Dinoroseobacter shibae DFL 12 and Rhodobacter capsulatus B10S, as well as in two members of the alphaproteobacterial order Hyphomicrobiales (synonym: Rhizobiales; Ensifer meliloti 2011 and “Agrobacterium fabrum” C58). Maintenance of the pABC vectors in the biotechnologically valuable orders Rhodobacterales and Hyphomicrobiales facilitates the shuttling of genetic constructs between alphaproteobacterial genera and orders. Additionally, plasmid replication was verified in one member of Rhodospirillales (Rhodospirillum rubrum S1) as well as in one member of Caulobacterales (Caulobacter vibrioides CB15N). The modular construction of pABC vectors and the usage of four compatible replication systems, which allows their coexistence in a host cell, are advantageous features for future implementations of newly designed synthetic pathways. The vector applicability was demonstrated by functional complementation of a nitrogenase mutant phenotype by two complementary pABC-based plasmids in R. capsulatus.
期刊介绍:
The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism.
Topics may include, but are not limited to:
Design and optimization of genetic systems
Genetic circuit design and their principles for their organization into programs
Computational methods to aid the design of genetic systems
Experimental methods to quantify genetic parts, circuits, and metabolic fluxes
Genetic parts libraries: their creation, analysis, and ontological representation
Protein engineering including computational design
Metabolic engineering and cellular manufacturing, including biomass conversion
Natural product access, engineering, and production
Creative and innovative applications of cellular programming
Medical applications, tissue engineering, and the programming of therapeutic cells
Minimal cell design and construction
Genomics and genome replacement strategies
Viral engineering
Automated and robotic assembly platforms for synthetic biology
DNA synthesis methodologies
Metagenomics and synthetic metagenomic analysis
Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction
Gene optimization
Methods for genome-scale measurements of transcription and metabolomics
Systems biology and methods to integrate multiple data sources
in vitro and cell-free synthetic biology and molecular programming
Nucleic acid engineering.