Universität zu Köln

Domroese, Andreas, Hage-Huelsmann, Jennifer, Thies, Stephan ORCID: 0000-0003-4240-9149, Weihmann, Robin ORCID: 0000-0002-9870-4086, Kruse, Luzie ORCID: 0000-0003-4224-9755, Otto, Maike ORCID: 0000-0001-6858-029X, Wierckx, Nick ORCID: 0000-0002-1590-1210, Jaeger, Karl-Erich, Drepper, Thomas and Loeschcke, Anita ORCID: 0000-0001-5184-8499 (2019). Pseudomonas putida rDNA is a favored site for the expression of biosynthetic genes. Sci Rep, 9. LONDON: NATURE PUBLISHING GROUP. ISSN 2045-2322

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Abstract

Since high-value bacterial secondary metabolites, including antibiotics, are often naturally produced in only low amounts, their efficient biosynthesis typically requires the transfer of entire metabolic pathways into suitable bacterial hosts like Pseudomonas putida. Stable maintenance and sufficient expression of heterologous pathway-encoding genes in host microbes, however, still remain key challenges. In this study, the 21 kb prodigiosin gene cluster from Serratia marcescens was used as a reporter to identify genomic sites in P. putida KT2440 especially suitable for maintenance and expression of pathway genes. After generation of a strain library by random Tn5 transposon-based chromosomal integration of the cluster, 50 strains exhibited strong prodigiosin production. Remarkably, chromosomal integration sites were exclusively identified in the seven rRNA-encoding rrn operons of P. putida. We could further demonstrate that prodigiosin production was mainly dependent on (i) the individual rrn operon where the gene cluster was inserted as well as (ii) the distance between the rrn promoter and the inserted prodigiosin biosynthetic genes. In addition, the recombinant strains showed high stability upon subculturing for many generations. Consequently, our findings demonstrate the general applicability of rDNA loci as chromosomal integration sites for gene cluster expression and recombinant pathway implementation in P. putida KT2440.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Domroese, Andreas UNSPECIFIED UNSPECIFIED UNSPECIFIED Hage-Huelsmann, Jennifer UNSPECIFIED UNSPECIFIED UNSPECIFIED Thies, Stephan UNSPECIFIED orcid.org/0000-0003-4240-9149 UNSPECIFIED Weihmann, Robin UNSPECIFIED orcid.org/0000-0002-9870-4086 UNSPECIFIED Kruse, Luzie UNSPECIFIED orcid.org/0000-0003-4224-9755 UNSPECIFIED Otto, Maike UNSPECIFIED orcid.org/0000-0001-6858-029X UNSPECIFIED Wierckx, Nick UNSPECIFIED orcid.org/0000-0002-1590-1210 UNSPECIFIED Jaeger, Karl-Erich UNSPECIFIED UNSPECIFIED UNSPECIFIED Drepper, Thomas UNSPECIFIED UNSPECIFIED UNSPECIFIED Loeschcke, Anita UNSPECIFIED orcid.org/0000-0001-5184-8499 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-148498
DOI:	10.1038/s41598-019-43405-1
Journal or Publication Title:	Sci Rep
Volume:	9
Date:	2019
Publisher:	NATURE PUBLISHING GROUP
Place of Publication:	LONDON
ISSN:	2045-2322
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language COMPLETE GENOME SEQUENCE; RIBOSOMAL-RNA OPERONS; ESCHERICHIA-COLI; MESSENGER-RNA; TRANSCRIPTION; GROWTH; DEGRADATION; BACTERIA; COPY; CONSTRUCTION Multiple languages Multidisciplinary Sciences Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/14849