Pseudomonas putida rDNA is a favored site for the expression of biosynthetic genes

Domröse, Andreas; Hage-Hülsmann, Jennifer; Thies, Stephan; Weihmann, Robin; Kruse, Luzie; Otto, Maike; Wierckx, Nick; Jaeger, Karl‐Erich; Drepper, Thomas; Loeschcke, Anita

doi:10.1038/s41598-019-43405-1

Cited by 20 publications

(37 citation statements)

References 69 publications

(102 reference statements)

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“…These experiments demonstrate that variation due to integration locus is not unique to fluorescent protein expression, but also affects functional proteins such as a transporter. In contrast to the previous integration locus analysis ( Domröse et al., 2019 ) where high expression was desirable and integration loci were chosen accordingly, utility of mucK was highest when expression was moderate. Notably, growth on glucose with the insertion of mKate at PP_2224 and PP_1642 was substantially decreased, where growth on glucose in all three mucK strains was sustained at wild type levels.…”

Section: Resultsmentioning

confidence: 85%

“…A previous study ( Domröse et al., 2019 ) used Tn5 transposition to randomly integrate a 21 kb prodigiosin gene cluster across the P. putida KT2440 chromosome. In that example, it was found that the mutants with the highest expression were biased to one specific location, one of seven rrn operons.…”

Section: Resultsmentioning

confidence: 99%

“…The characterization of chromosomal integration loci for this purpose has been explored to various degrees in several organisms such as Escherichia coli ( Sousa et al. 1997 ; Bassalo et al., 2016 ), Bacillus subtilis ( Sauer et al., 2016 ) , Saccharomyces cerevisiae ( Flagfeldt et al., 2009 ; Yamane et al., 1998 ), Lactococcus lactis ( Thompson and Gasson, 2001 ), P. putida KT2440 ( Domröse et al., 2019 ), and Drosophila melanogaster ( Markstein et al., 2008 ). In this study, we have rationally selected seven insertion loci on the P. putida KT2440 chromosome to test for the expression of a fluorescent reporter gene and the effect of the insertion of the reporter on the growth of the host.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of chromosomal insertion loci in the Pseudomonas putida KT2440 genome for predictable biosystems design

Chaves

Wilton

Gao

et al. 2020

Metabolic Engineering Communications

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The development of Pseudomonas strains for industrial production of fuels and chemicals will require the integration of heterologous genes and pathways into the chromosome. Finding the most appropriate integration site to maximize strain performance is an essential part of the strain design process. We characterized seven chromosomal loci in Pseudomonas putida KT2440 for integration of a fluorescent protein expression construct. Insertion in five of the loci did not affect growth rate, but fluorescence varied by up to 27-fold. Three sites displaying a diversity of phenotypes with the fluorescent reporter were also chosen for the integration of a gene encoding a muconate importer. Depending on the integration locus, expression of the importer varied by approximately 3-fold and produced significant phenotypic differences. This work demonstrates the impact of the integration location on host viability, gene expression, and overall strain performance.

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Section: Resultsmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evaluation of chromosomal insertion loci in the Pseudomonas putida KT2440 genome for predictable biosystems design

Chaves

Wilton

Gao

et al. 2020

Metabolic Engineering Communications

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“…Theses promoter systems without the need of induction are especially interesting for industrial processes where protein expression independent of the addition of an inducer is desired to reduce the metabolic burden during exponential growth phase (Meyers et al 2019 ). Furthermore, rrn operons have demonstrated a strong preference for biosynthetic gene cluster integration in P. putida , due to stable integration and strong expression by the native promoters (Domröse et al 2019 ). Finally, it was recently demonstrated that fine-tuning of metabolic pathways via targeted proteolysis enables a new control layer of engineered pathways (Calles et al 2019 ; Volke et al 2020b ) .…”

Section: Recent Advances In Genetic Engineeringmentioning

confidence: 99%

Industrial biotechnology of Pseudomonas putida: advances and prospects

Weimer

Kohlstedt

Volke

et al. 2020

Appl Microbiol Biotechnol

137

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Pseudomonas putida is a Gram-negative, rod-shaped bacterium that can be encountered in diverse ecological habitats. This ubiquity is traced to its remarkably versatile metabolism, adapted to withstand physicochemical stress, and the capacity to thrive in harsh environments. Owing to these characteristics, there is a growing interest in this microbe for industrial use, and the corresponding research has made rapid progress in recent years. Hereby, strong drivers are the exploitation of cheap renewable feedstocks and waste streams to produce value-added chemicals and the steady progress in genetic strain engineering and systems biology understanding of this bacterium. Here, we summarize the recent advances and prospects in genetic engineering, systems and synthetic biology, and applications of P. putida as a cell factory. Key points • Pseudomonas putida advances to a global industrial cell factory. • Novel tools enable system-wide understanding and streamlined genomic engineering. • Applications of P. putida range from bioeconomy chemicals to biosynthetic drugs. Electronic supplementary material The online version of this article (10.1007/s00253-020-10811-9) contains supplementary material, which is available to authorized users.

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“…xylene, what plausibly favored implantation in regions of high transcriptional activity (37)(38)(39). Yet, when…”

Section: Paw140 Involved Random Chromosomal Insertion Of Tol Genes Anmentioning

confidence: 99%

The subcellular architecture of thexylgene expression flow of the TOL catabolic plasmid ofPseudomonas putidamt-2

Kim

Goñi-Moreno

2020

Preprint

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Despite intensive research on the biochemical and regulatory features of the archetypal catabolic TOL system borne by pWW0 of Pseudomonas putida mt-2, the physical arrangement and tridimensional logic of the xyl gene expression flow remains unknown. In this work, the spatial distribution of specific xyl mRNAs with respect to the host nucleoid, the TOL plasmid and the ribosomal pool has been investigated. In situ hybridization of target transcripts with fluorescent oligonucleotide probes revealed that xyl mRNAs cluster in discrete foci, adjacent but clearly separated from the TOL plasmid and the cell nucleoid. Also, they co-localize with ribosome-rich domains of the intracellular milieu. This arrangement was kept even when the xyl genes were artificially relocated at different chromosomal locations. The same happened when genes were expressed through a heterologous T7 polymerase-based system, which originated mRNA foci outside the DNA. In contrast, rifampicin treatment, known to ease crowding, blurred the confinement of xyl transcripts. This suggested that xyl mRNAs intrinsically run away from their initiation sites to ribosome-rich points for translation—rather than being translated coupled to transcription. Moreover, the results suggest that the distinct subcellular motion of xyl mRNAs results both from innate properties of the sequence at stake and the physical forces that keep the ribosomal pool away from the nucleoid in P. putida. This scenario is discussed on the background of current knowledge on the 3D organization of the gene expression flow in other bacteria and the environmental lifestyle of this soil microorganism.IMPORTANCEThe transfer of information between DNA, RNA and proteins in a bacterium is often compared to the decoding of a piece of software in a computer. However, the tridimensional layout and the relational logic of the cognate biological hardware i.e. the nucleoid, the RNA polymerase and the ribosomes, are habitually taken for granted. In this work we inspected the localization and fate of the transcripts that stem from the archetypal biodegradative plasmid pWW0 of soil bacterium Pseudomonas putida KT2440 through the non-homogenous milieu of the bacterial cytoplasm. The results expose that—similarly to computers also—the material components that enable the expression flow are well separated physically and they decipher the sequences through a distinct tridimensional arrangement with no indication of transcription/translation coupling. We argue that the resulting subcellular architecture enters an extra regulatory layer that obeys a species-specific positional code that accompanies the environmental lifestyle of this bacterium.

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Pseudomonas putida rDNA is a favored site for the expression of biosynthetic genes

Cited by 20 publications

References 69 publications

Evaluation of chromosomal insertion loci in the Pseudomonas putida KT2440 genome for predictable biosystems design

Evaluation of chromosomal insertion loci in the Pseudomonas putida KT2440 genome for predictable biosystems design

Industrial biotechnology of Pseudomonas putida: advances and prospects

The subcellular architecture of thexylgene expression flow of the TOL catabolic plasmid ofPseudomonas putidamt-2

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