Pseudomonas putida

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Lars M Blank - One of the best experts on this subject based on the ideXlab platform.

  • tn7 based device for calibrated heterologous gene expression in Pseudomonas putida
    ACS Synthetic Biology, 2015
    Co-Authors: Sebastian Zobel, Victor De Lorenzo, Ilaria Benedetti, Lara Eisenbach, Nick Wierckx, Lars M Blank
    Abstract:

    The soil bacterium Pseudomonas putida is increasingly attracting considerable interest as a platform for advanced metabolic engineering through synthetic biology approaches. However, genomic context, gene copy number, and transcription/translation interplay often introduce considerable uncertainty to the design of reliable genetic constructs. In this work, we have established a standardized heterologous expression device in which the promoter strength is the only variable; the remaining parameters of the flow have stable default values. To this end, we tailored a mini-Tn7 delivery transposon vector that inserts the constructs in a single genomic locus of P. putida’s chromosome. This was then merged with a promoter insertion site, an unvarying translational coupler, and a downstream location for placing the gene(s) of interest under fixed assembly rules. This arrangement was exploited to benchmark a collection of synthetic promoters with low transcriptional noise in this bacterial host. Growth experiments ...

  • Tn7-Based Device for Calibrated Heterologous Gene Expression in Pseudomonas putida.
    ACS synthetic biology, 2015
    Co-Authors: Sebastian Zobel, Victor De Lorenzo, Ilaria Benedetti, Lara Eisenbach, Nick Wierckx, Lars M Blank
    Abstract:

    The soil bacterium Pseudomonas putida is increasingly attracting considerable interest as a platform for advanced metabolic engineering through synthetic biology approaches. However, genomic contex...

  • Metabolic engineering of Pseudomonas putida KT2440 to produce anthranilate from glucose
    Frontiers Media S.A., 2015
    Co-Authors: Jannis Ekuepper, Jasmin Edickler, Michael Ebiggel, Swantje Ebehnken, Gernot Ejaeger, Nick Ewierckx, Lars M Blank
    Abstract:

    The Pseudomonas putida KT2440 strain was engineered in order to produce anthranilate (oAB, ortho-aminobenzoate), a precursor of the aromatic amino acid tryptophan, from glucose as sole carbon source. To enable the production of the metabolic intermediate oAB, the trpDC operon encoding an anthranilate phosphoribosyltransferase (TrpD) and an indole-3-glycerol phosphate synthase (TrpC), were deleted. In addition, the chorismate mutase (pheA) responsible for the conversion of chorismate over prephenate to phenylpyruvate was deleted in the background of the deletion of trpDC to circumvent a potential drain of precursor. To further increase the oAB production, a feedback insensitive version of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase encoded by the aroGD146N gene and an anthranilate synthase (trpES40FG) were overexpressed separately and simultaneously in the deletion mutants. With optimized production conditions in a tryptophan-limited fed-batch process a maximum of 1.54 ±0.3 g L-1 (11.23 mM) oAB was obtained with the best performing engineered P. putida KT2440 strain (P. putida ∆trpDC pSEVA234_aroGD146N_trpES40FG)

  • selected Pseudomonas putida strains able to grow in the presence of high butanol concentrations
    Applied and Environmental Microbiology, 2009
    Co-Authors: Jana Ruhl, Andreas Schmid, Lars M Blank
    Abstract:

    Adapted Pseudomonas putida strains grew in the presence of up to 6% (vol/vol) butanol, the highest reported butanol concentration tolerated by a microbe. P. putida might be an alternative host for biobutanol production, overcoming the primary limitation of currently used strains—insufficient product titers due to low butanol tolerance.

Victor De Lorenzo - One of the best experts on this subject based on the ideXlab platform.

  • Pseudomonas putida in the quest of programmable chemistry
    'Elsevier BV', 2021
    Co-Authors: Martínez-garcía Esteban, Victor De Lorenzo
    Abstract:

    Traditional microbial biotechnology is in the midst of a profound transformation brought about not only by many conceptual and technical breakthroughs (e.g. systems and synthetic biology, the CRISPR revolution) but also by the major change of socioeconomic context generically known as the 4th Industrial Revolution. Owing to its naturally evolved properties of stress endurance, metabolic versatility, and physiological robustness the soil bacterium Pseudomonas putida has recently received a considerable attention as the basis for developing whole-cell catalysts. The review below sketches the ongoing journey of this bacterium from being a soil-dweller, root-colonizer microbe all the way to become a programmable catalyst for executing complex biotransformations at very different scales—having in the background the contemporary developments in non-biological programmable chemistry.The work in Authors’ Laboratory was funded by the HELIOS Project of the Spanish Ministry of Science BIO 2015-66960-C3-2-R (MINECO/FEDER); the ARISYS (ERC-2012-ADG-322797), EmPowerputida (EU-H2020-BIOTEC-2014-2015-6335536), MADONNA (H2020-FET-OPEN-RIA-2017-1-766975), BioRoboost (H2020-NMBP-BIO-CSA-2018), and SYNBIO4FLAV (H2020-NMBP/0500) Contracts of the European Union and the S2017/BMD-3691 InGEMICS-CM funded by the Comunidad de Madrid (European Structural and Investment Funds)

  • ribonucleases control distinct traits of Pseudomonas putida lifestyle
    Environmental Microbiology, 2021
    Co-Authors: Patricia Apura, Victor De Lorenzo, Cecilia M Arraiano, Esteban Martinezgarcia, Sandra C Viegas
    Abstract:

    The role of archetypal ribonucleases (RNases) in the physiology and stress endurance of the soil bacterium and metabolic engineering platform Pseudomonas putida KT2440 has been inspected. To this end, variants of this strain lacking each of the most important RNases were constructed. Each mutant lacked either one exoribonuclease (PNPase, RNase R) or one endoribonuclease (RNase E, RNase III, RNase G). The global physiological and metabolic costs of the absence of each of these enzymes were then analysed in terms of growth, motility and morphology. The effects of different oxidative chemicals that mimic the stresses endured by this microorganism in its natural habitats were studied as well. The results highlighted that each ribonuclease is specifically related with different traits of the environmental lifestyle that distinctively characterizes this microorganism. Interestingly, the physiological responses of P. putida to the absence of each enzyme diverged significantly from those known previously in Escherichia coli. This exposed not only species-specific regulatory functions for otherwise known RNase activities but also expanded the panoply of post-transcriptional adaptation devices that P. putida can make use of for facing hostile environments.

  • the ssr protein t1e_1405 from Pseudomonas putida dot t1e enables oligonucleotide based recombineering in platform strain p putida em42
    Biotechnology Journal, 2016
    Co-Authors: Tomas Aparicio, Victor De Lorenzo, Sheila Ingemann Jensen, Alex Toftgaard Nielsen, Esteban Martinezgarcia
    Abstract:

    Some strains of the soil bacterium Pseudomonas putida have become in recent years platforms of choice for hosting biotransformations of industrial interest. Despite availability of many genetic tools for this microorganism, genomic editing of the cell factory P. putida EM42 (a derivative of reference strain KT2440) is still a time-consuming endeavor. In this work we have investigated the in vivo activity of the Ssr protein encoded by the open reading frame T1E_1405 from Pseudomonas putida DOT-T1E, a plausible functional homologue of the β protein of the Red recombination system of λ phage of Escherichia coli. A test based on the phenotypes of pyrF mutants of P. putida (the yeast's URA3 ortholog) was developed for quantifying the ability of Ssr to promote invasion of the genomic DNA replication fork by synthetic oligonucleotides. The efficiency of the process was measured by monitoring the inheritance of the changes entered into pyrF by oligonucleotides bearing mutated sequences. Ssr fostered short and long genomic deletions/insertions at considerable frequencies as well as single-base swaps not affected by mismatch repair. These results not only demonstrate the feasibility of recombineering in P. putida, but they also enable a suite of multiplexed genomic manipulations in this biotechnologically important bacterium.

  • tn7 based device for calibrated heterologous gene expression in Pseudomonas putida
    ACS Synthetic Biology, 2015
    Co-Authors: Sebastian Zobel, Victor De Lorenzo, Ilaria Benedetti, Lara Eisenbach, Nick Wierckx, Lars M Blank
    Abstract:

    The soil bacterium Pseudomonas putida is increasingly attracting considerable interest as a platform for advanced metabolic engineering through synthetic biology approaches. However, genomic context, gene copy number, and transcription/translation interplay often introduce considerable uncertainty to the design of reliable genetic constructs. In this work, we have established a standardized heterologous expression device in which the promoter strength is the only variable; the remaining parameters of the flow have stable default values. To this end, we tailored a mini-Tn7 delivery transposon vector that inserts the constructs in a single genomic locus of P. putida’s chromosome. This was then merged with a promoter insertion site, an unvarying translational coupler, and a downstream location for placing the gene(s) of interest under fixed assembly rules. This arrangement was exploited to benchmark a collection of synthetic promoters with low transcriptional noise in this bacterial host. Growth experiments ...

  • Tn7-Based Device for Calibrated Heterologous Gene Expression in Pseudomonas putida.
    ACS synthetic biology, 2015
    Co-Authors: Sebastian Zobel, Victor De Lorenzo, Ilaria Benedetti, Lara Eisenbach, Nick Wierckx, Lars M Blank
    Abstract:

    The soil bacterium Pseudomonas putida is increasingly attracting considerable interest as a platform for advanced metabolic engineering through synthetic biology approaches. However, genomic contex...

Bernard R Glick - One of the best experts on this subject based on the ideXlab platform.

Norbert Strater - One of the best experts on this subject based on the ideXlab platform.

  • cosubstrate induced dynamics of d 3 hydroxybutyrate dehydrogenase from Pseudomonas putida
    FEBS Journal, 2007
    Co-Authors: Karthik S Paithankar, Claudia Feller, Bartholomeus E Kuettner, Antje Keim, Marlis Grunow, Norbert Strater
    Abstract:

    D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida belongs to the family of short-chain dehydrogenases/reductases. We have determined X-ray structures of the D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida, which was recombinantly expressed in Escherichia coli, in three different crystal forms to resolutions between 1.9 and 2.1 A. The so-called substrate-binding loop (residues 187–210) was partially disordered in several subunits, in both the presence and absence of NAD+. However, in two subunits, this loop was completely defined in an open conformation in the apoenzyme and in a closed conformation in the complex structure with NAD+. Structural comparisons indicated that the loop moves as a rigid body by about 46°. However, the two small α-helices (αFG1 and αFG2) of the loop also re-orientated slightly during the conformational change. Probably, the interactions of Val185, Thr187 and Leu189 with the cosubstrate induced the conformational change. A model of the binding mode of the substrate D-3-hydroxybutyrate indicated that the loop in the closed conformation, as a result of NAD+ binding, is positioned competent for catalysis. Gln193 is the only residue of the substrate-binding loop that interacts directly with the substrate. A translation, libration and screw (TLS) analysis of the rigid body movement of the loop in the crystal showed significant librational displacements, describing the coordinated movement of the substrate-binding loop in the crystal. NAD+ binding increased the flexibility of the substrate-binding loop and shifted the equilibrium between the open and closed forms towards the closed form. The finding that all NAD+-bound subunits are present in the closed form and all NAD+-free subunits in the open form indicates that the loop closure is induced by cosubstrate binding alone. This mechanism may contribute to the sequential binding of cosubstrate followed by substrate.

  • cosubstrate induced dynamics of d 3 hydroxybutyrate dehydrogenase from Pseudomonas putida
    FEBS Journal, 2007
    Co-Authors: Karthik S Paithankar, Claudia Feller, Bartholomeus E Kuettner, Antje Keim, Marlis Grunow, Norbert Strater
    Abstract:

    D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida belongs to the family of short-chain dehydrogenases/reductases. We have determined X-ray structures of the D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida, which was recombinantly expressed in Escherichia coli, in three different crystal forms to resolutions between 1.9 and 2.1 A. The so-called substrate-binding loop (residues 187–210) was partially disordered in several subunits, in both the presence and absence of NAD+. However, in two subunits, this loop was completely defined in an open conformation in the apoenzyme and in a closed conformation in the complex structure with NAD+. Structural comparisons indicated that the loop moves as a rigid body by about 46°. However, the two small α-helices (αFG1 and αFG2) of the loop also re-orientated slightly during the conformational change. Probably, the interactions of Val185, Thr187 and Leu189 with the cosubstrate induced the conformational change. A model of the binding mode of the substrate D-3-hydroxybutyrate indicated that the loop in the closed conformation, as a result of NAD+ binding, is positioned competent for catalysis. Gln193 is the only residue of the substrate-binding loop that interacts directly with the substrate. A translation, libration and screw (TLS) analysis of the rigid body movement of the loop in the crystal showed significant librational displacements, describing the coordinated movement of the substrate-binding loop in the crystal. NAD+ binding increased the flexibility of the substrate-binding loop and shifted the equilibrium between the open and closed forms towards the closed form. The finding that all NAD+-bound subunits are present in the closed form and all NAD+-free subunits in the open form indicates that the loop closure is induced by cosubstrate binding alone. This mechanism may contribute to the sequential binding of cosubstrate followed by substrate.

Sebastian Zobel - One of the best experts on this subject based on the ideXlab platform.

  • tn7 based device for calibrated heterologous gene expression in Pseudomonas putida
    ACS Synthetic Biology, 2015
    Co-Authors: Sebastian Zobel, Victor De Lorenzo, Ilaria Benedetti, Lara Eisenbach, Nick Wierckx, Lars M Blank
    Abstract:

    The soil bacterium Pseudomonas putida is increasingly attracting considerable interest as a platform for advanced metabolic engineering through synthetic biology approaches. However, genomic context, gene copy number, and transcription/translation interplay often introduce considerable uncertainty to the design of reliable genetic constructs. In this work, we have established a standardized heterologous expression device in which the promoter strength is the only variable; the remaining parameters of the flow have stable default values. To this end, we tailored a mini-Tn7 delivery transposon vector that inserts the constructs in a single genomic locus of P. putida’s chromosome. This was then merged with a promoter insertion site, an unvarying translational coupler, and a downstream location for placing the gene(s) of interest under fixed assembly rules. This arrangement was exploited to benchmark a collection of synthetic promoters with low transcriptional noise in this bacterial host. Growth experiments ...

  • Tn7-Based Device for Calibrated Heterologous Gene Expression in Pseudomonas putida.
    ACS synthetic biology, 2015
    Co-Authors: Sebastian Zobel, Victor De Lorenzo, Ilaria Benedetti, Lara Eisenbach, Nick Wierckx, Lars M Blank
    Abstract:

    The soil bacterium Pseudomonas putida is increasingly attracting considerable interest as a platform for advanced metabolic engineering through synthetic biology approaches. However, genomic contex...

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