Weihmann Robin, Kubicki Sonja, Bitzenhofer Nora Lisa, Domröse Andreas, Bator Isabel, Kirschen Lisa-Marie, Kofler Franziska, Funk Aileen, Tiso Till, Blank Lars M, Jaeger Karl-Erich, Drepper Thomas, Thies Stephan, Loeschcke Anita
Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH, 52428 Jülich, Germany.
Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
FEMS Microbes. 2022 Dec 19;4:xtac030. doi: 10.1093/femsmc/xtac030. eCollection 2023.
The expression of biosynthetic genes in bacterial hosts can enable access to high-value compounds, for which appropriate molecular genetic tools are essential. Therefore, we developed a toolbox of modular vectors, which facilitate chromosomal gene integration and expression in KT2440. To this end, we designed an integrative sequence, allowing customisation regarding the modes of integration (random, at Tn7, or into the 16S rRNA gene), promoters, antibiotic resistance markers as well as fluorescent proteins and enzymes as transcription reporters. We thus established a toolbox of vectors carrying integrative sequences, designated as pYT series, of which we present 27 ready-to-use variants along with a set of strains equipped with unique 'landing pads' for directing a pYT interposon into one specific copy of the 16S rRNA gene. We used genes of the well-described violacein biosynthesis as reporter to showcase random Tn5-based chromosomal integration leading to constitutive expression and production of violacein and deoxyviolacein. Deoxyviolacein was likewise produced after gene integration into the 16S rRNA gene of operons. Integration in the Tn7 site was used to characterise the suitability of different inducible promoters and successive strain development for the metabolically challenging production of mono-rhamnolipids. Finally, to establish arcyriaflavin A production in for the first time, we compared different integration and expression modes, revealing integration at Tn7 and expression with NagR/P to be most suitable. In summary, the new toolbox can be utilised for the rapid generation of various types of expression and production strains.
细菌宿主中生物合成基因的表达能够获取高价值化合物，为此合适的分子遗传学工具必不可少。因此，我们开发了一套模块化载体工具箱，便于在KT2440中进行染色体基因整合和表达。为此，我们设计了一个整合序列，可在整合模式（随机、Tn7位点或16S rRNA基因）、启动子、抗生素抗性标记以及荧光蛋白和酶作为转录报告基因方面进行定制。我们由此建立了一套携带整合序列的载体工具箱，命名为pYT系列，我们展示了27个即用型变体以及一组配备独特“着陆垫”的菌株，用于将pYT插入子定向到16S rRNA基因的一个特定拷贝中。我们使用了已充分描述的紫罗碱生物合成基因作为报告基因，以展示基于随机Tn5的染色体整合，从而导致紫罗碱和脱氧紫罗碱的组成型表达和产生。将基因整合到操纵子的16S rRNA基因后，同样产生了脱氧紫罗碱。在Tn7位点的整合用于表征不同诱导型启动子的适用性以及后续菌株开发用于单鼠李糖脂代谢挑战性生产的情况。最后，为了首次在中建立刺孢黄素A的生产，我们比较了不同的整合和表达模式，发现Tn7位点整合和NagR/P表达最为合适。总之，这个新的工具箱可用于快速生成各种类型的表达和生产菌株。
