Robin Weihmann, Sonja Kubicki, Nora Lisa Bitzenhofer, Andreas Domröse, Isabel Bator, Lisa-Marie Kirschen, Franziska Kofler, Aileen Funk, Till Tiso, Lars M Blank, Karl-Erich Jaeger, Thomas Drepper, Stephan Thies, Anita Loeschcke
{"title":"模块化pYT载体系列用于染色体基因整合和表达,以在P.putida中产生咔唑和糖脂。","authors":"Robin Weihmann, Sonja Kubicki, Nora Lisa Bitzenhofer, Andreas Domröse, Isabel Bator, Lisa-Marie Kirschen, Franziska Kofler, Aileen Funk, Till Tiso, Lars M Blank, Karl-Erich Jaeger, Thomas Drepper, Stephan Thies, Anita Loeschcke","doi":"10.1093/femsmc/xtac030","DOIUrl":null,"url":null,"abstract":"<p><p>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 <i>Pseudomonas putida</i> KT2440. To this end, we designed an integrative sequence, allowing customisation regarding the modes of integration (random, at <i>att</i>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 <i>rrn</i> operons. Integration in the <i>att</i>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 <i>P. putida</i> for the first time, we compared different integration and expression modes, revealing integration at <i>att</i>Tn7 and expression with NagR/P<i><sub>nagAa</sub></i> to be most suitable. In summary, the new toolbox can be utilised for the rapid generation of various types of <i>P. putida</i> expression and production strains.</p>","PeriodicalId":73024,"journal":{"name":"FEMS microbes","volume":"4 ","pages":"xtac030"},"PeriodicalIF":0.0000,"publicationDate":"2022-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/9c/90/xtac030.PMC10117823.pdf","citationCount":"1","resultStr":"{\"title\":\"The modular pYT vector series employed for chromosomal gene integration and expression to produce carbazoles and glycolipids in <i>P. putida</i>.\",\"authors\":\"Robin Weihmann, Sonja Kubicki, Nora Lisa Bitzenhofer, Andreas Domröse, Isabel Bator, Lisa-Marie Kirschen, Franziska Kofler, Aileen Funk, Till Tiso, Lars M Blank, Karl-Erich Jaeger, Thomas Drepper, Stephan Thies, Anita Loeschcke\",\"doi\":\"10.1093/femsmc/xtac030\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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 <i>Pseudomonas putida</i> KT2440. To this end, we designed an integrative sequence, allowing customisation regarding the modes of integration (random, at <i>att</i>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 <i>rrn</i> operons. Integration in the <i>att</i>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 <i>P. putida</i> for the first time, we compared different integration and expression modes, revealing integration at <i>att</i>Tn7 and expression with NagR/P<i><sub>nagAa</sub></i> to be most suitable. In summary, the new toolbox can be utilised for the rapid generation of various types of <i>P. putida</i> expression and production strains.</p>\",\"PeriodicalId\":73024,\"journal\":{\"name\":\"FEMS microbes\",\"volume\":\"4 \",\"pages\":\"xtac030\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2022-12-19\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/9c/90/xtac030.PMC10117823.pdf\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"FEMS microbes\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1093/femsmc/xtac030\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2023/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"FEMS microbes","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1093/femsmc/xtac030","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2023/1/1 0:00:00","PubModel":"eCollection","JCR":"","JCRName":"","Score":null,"Total":0}
The modular pYT vector series employed for chromosomal gene integration and expression to produce carbazoles and glycolipids in P. putida.
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 Pseudomonas putida KT2440. To this end, we designed an integrative sequence, allowing customisation regarding the modes of integration (random, at attTn7, 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 rrn operons. Integration in the attTn7 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 P. putida for the first time, we compared different integration and expression modes, revealing integration at attTn7 and expression with NagR/PnagAa to be most suitable. In summary, the new toolbox can be utilised for the rapid generation of various types of P. putida expression and production strains.