Filter and export

Publications

What is a Publication?
6 Publications visible to you, out of a total of 6
The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: highlights from a multi-level omics approach

Abstract (Expand)

BACKGROUND: Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made … biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures. RESULTS: We found that 26, 62, and 81% of the cell dry weight consist of PHA under conditions of carbon, dual, and nitrogen limitation, respectively. Under nitrogen limitation a specific PHA production rate of 0.43 (g.(g.h)-1) was obtained. The residual biomass was not constant for dual- and strict nitrogen-limiting growth, showing a different feature in comparison to other P. putida strains. Dual limitation resulted in patterns of gene expression, protein level, and metabolite concentrations that substantially differ from those observed under exclusive carbon or nitrogen limitation. The most pronounced differences were found in the energy metabolism, fatty acid metabolism, as well as stress proteins and enzymes belonging to the transport system. CONCLUSION: This is the first study where the interrelationship between nutrient limitations and PHA synthesis has been investigated under well-controlled conditions using a system level approach. The knowledge generated will be of great assistance for the development of bioprocesses and further metabolic engineering work in this versatile organism to both enhance and diversify the industrial production of PHAs.

Authors: , I. F. Escapa, C. Jager, J. Puchalka, , , ,

Date Published: 20th Mar 2012

Publication Type: Not specified

Functional genomics of the initial phase of cold adaptation of Pseudomonas putida KT2440

Abstract (Expand)

The cold stress response of Pseudomonas putida KT2440 was investigated by genomewide deep cDNA sequencing and gel-free MS-based protein profiling. Transcriptome and proteome profiles were assessed at … 30 degrees C and 2 h after a downshift from 30 to 10 degrees C. Pseudomonas putida adapted to lower ambient temperature by the activation of ribosome-associated functional modules that facilitate translational efficiency. The outer membrane profile was reorganized, anabolic pathways and core as well as energy metabolism were repressed and the alginate regulon and sugar catabolism were activated. At the investigated early time point of cold adaptation, the transcriptome was reprogrammed in almost all functional categories, but the protein profile had still not adapted to the change of living conditions in the cold.

Authors: , F. Schmidt, , C. F. Davenport, M. Gesell Salazar, U. Volker,

Date Published: 1st Mar 2011

Publication Type: Not specified

Pseudomonas putida KT2440 genome update by cDNA sequencing and microarray transcriptomics

Abstract (Expand)

Pseudomonas putida KT2440 is a completely sequenced biosafety strain that has retained its capability to survive and function in the environment. The global mRNA expression profiles of the KT2440 strain … grown at 10 degrees C and 30 degrees C were determined by deep cDNA sequencing to refine the genome annotation. Transcriptome sequencing identified 36 yet unknown small non-coding RNAs, 143 novel ORFs in 106 intergenic regions, 42 unclassified genes and eight highly expressed leaderless mRNA transcripts. The genome coordinates of eight genes and the organization of 57 operons were corrected. No overrepresented sequence motifs were detected in the 5'-untranslated regions. The 50 most highly expressed genes made up 60% of the total mRNA pool. Comparison of cDNA sequencing, Affymetrix and Progenika microarray data from the same mRNA preparation revealed a higher sensitivity and specificity of cDNA sequencing, a relatively poor correlation between the normalized cDNA reads and microarray signal intensities, and a systematic signal-dependent bias of microarrays in the detection of differentially regulated genes. The study demonstrates the power of next-generation cDNA sequencing for the quantitation of mRNA transcripts and the refinement of bacterial genome annotation.

Authors: , , P. Hagendorf, R. Geffers, U. Schock, T. Pohl, C. F. Davenport,

Date Published: 28th Feb 2011

Publication Type: Not specified

Identification and characterization of the PhhR regulon inPseudomonas putida

Abstract (Expand)

Pseudomonas putida is a soil microorganism that utilizes aromatic amino acids present in root exudates as a nitrogen source. We have previously shown that the PhhR transcriptional regulator induces … phhAB genes encoding a phenylalanine hydroxylase. In this study we show, using microarray assays and promoter fusions, that PhhR is a global regulator responsible for the activation of genes essential for phenylalanine degradation, phenylalanine homeostasis and other genes of unknown function. Recently, it has been shown that phenylalanine catabolism occurs through more than one pathway. One of these possible pathways involves the metabolism of phenylalanine via tyrosine, p-hydroxyphenylpyruvate, and homogentisate. We identified two genes within this pathway that encode an acyl-CoA transferase involved in the metabolism of acetoacetate. All genes in this pathway were induced in response to phenylalanine in a PhhR-proficient background. The second potential degradative pathway involves the degradation of phenylalanine to produce phenylpyruvate, which seems to be degraded via phenylacetyl-CoA. A number of mutants in the paa genes encoding phenylacetyl-CoA degradation enzymes fail to grow on phenylpyruvate or phenylacetate, further supporting the existence of this second pathway. We found that the PhhR regulon also includes genes involved in the biosynthesis of aromatic amino acids that are repressed in the presence of phenylalanine, suggesting the possibility of feedback at the transcriptional level. In addition, we found that PhhR modulates the level of expression of the broad-substrate-specificity MexEF/OprN efflux pump. Expression from this pump is under the control of mexT gene product because phenylalanine-dependent transcription from the mexE promoter does not occur in a mexT mutant background. These results place PhhR as an important regulator in the control of bacterial responses to aromatic amino acids.

Authors: M. Carmen Herrera, , José J. Rodríguez-Herva, Ana M. Fernández-Escamilla,

Date Published: 2010

Publication Type: Not specified

Functional analysis of new transporters involved in stress tolerance inPseudomonas putidaDOT-T1E

Abstract (Expand)

Pseudomonas putida DOT-T1E is a highly solvent-tolerant strain. Although the main mechanism that confers solvent tolerance to the strain is the TtgGHI efflux pump, a number of other proteins are also … involved in the response to toluene. Previous proteomic and transcriptomic analysis carried out in our lab with P. putida DOT-T1E, and the solvent-sensitive strain, P. putida KT2440, revealed several transporters that were induced in the presence of toluene. We prepared five mutants of the corresponding genes in P. putida DOT-T1E and analysed their phenotypes with respect to solvent tolerance, stress endurance and growth with different carbon, nitrogen and sulfur sources. The data clearly demonstrated that two transporters (Ttg2ABC and TtgK) are involved in multidrug resistance and toluene tolerance, whereas another (homologous to PP0219 of P. putida KT2440) is a sulfate/sulfite transporter. No clear function could be assigned to the other two transporters. Of the transporters shown to be involved in toluene tolerance, one (ttg2ABC) belongs to the ATP-Binding Cassette (ABC) family, and is involved in multidrug resistance in P. putida DOT-T1E, while the other belongs to the Major Facilitator Superfamily and exhibits homology to a putative transporter of the Bcr/CflA family that has not previously been reported to be involved in toluene tolerance.

Authors: Vanina García, Patricia Godoy, Craig Daniels, Ana Hurtado, , Ana Segura

Date Published: 1st Nov 2009

Publication Type: Not specified

Genome-scale reconstruction and analysis of the Pseudomonas putida KT2440 metabolic network facilitates applications in biotechnology

Abstract (Expand)

A cornerstone of biotechnology is the use of microorganisms for the efficient production of chemicals and the elimination of harmful waste. Pseudomonas putida is an archetype of such microbes due to … its metabolic versatility, stress resistance, amenability to genetic modifications, and vast potential for environmental and industrial applications. To address both the elucidation of the metabolic wiring in P. putida and its uses in biocatalysis, in particular for the production of non-growth-related biochemicals, we developed and present here a genome-scale constraint-based model of the metabolism of P. putida KT2440. Network reconstruction and flux balance analysis (FBA) enabled definition of the structure of the metabolic network, identification of knowledge gaps, and pin-pointing of essential metabolic functions, facilitating thereby the refinement of gene annotations. FBA and flux variability analysis were used to analyze the properties, potential, and limits of the model. These analyses allowed identification, under various conditions, of key features of metabolism such as growth yield, resource distribution, network robustness, and gene essentiality. The model was validated with data from continuous cell cultures, high-throughput phenotyping data, (13)C-measurement of internal flux distributions, and specifically generated knock-out mutants. Auxotrophy was correctly predicted in 75% of the cases. These systematic analyses revealed that the metabolic network structure is the main factor determining the accuracy of predictions, whereas biomass composition has negligible influence. Finally, we drew on the model to devise metabolic engineering strategies to improve production of polyhydroxyalkanoates, a class of biotechnologically useful compounds whose synthesis is not coupled to cell survival. The solidly validated model yields valuable insights into genotype-phenotype relationships and provides a sound framework to explore this versatile bacterium and to capitalize on its vast biotechnological potential.

Authors: Jacek Puchałka, Matthew A Oberhardt, Miguel Godinho, Agata Bielecka, Daniela Regenhardt, , Jason A Papin,

Date Published: 27th Mar 2008

Publication Type: Not specified

Powered by
 (v.1.16.0)
About FAIRDOMHub | Funding and Programmes | Credits | Terms & Conditions | Imprint | Cookie preferences
Copyright © 2008 - 2024 The University of Manchester and HITS gGmbH