Production of cis, cis-muconate from benzoate at high rate and yield by a mutant of P. putida KT2440
Ontology highlight
ABSTRACT:
ORGANISM(S): Pseudomonas putida KT2440 Pseudomonas putida
PROVIDER: GSE25329 | GEO | 2013/05/15
SECONDARY ACCESSION(S): PRJNA134853
REPOSITORIES: GEO
Similar Datasets
Project description:Genome-wide scanning of gene expression by microarray techniques was successfully performed on RNA extracted from a sterilized soil inoculated with Pseudomonas putida KT2440/pSL1, which contains a chloroaromatic degrading plasmid, in the presence or absence of 3-chlorobenzoic acid (3CB). The genes showing significant changes in their expression in both triplicate microarray analyses using amplified RNA and single microarray analysis using unamplified RNA were investigated. Pathway analysis revealed that the benzoate degradation pathway underwent the most significant changes following treatment with 3CB. Analysis based on categorization of differentially expressed genes against 3CB revealed new findings about the cellular responses of the bacteria to 3CB, including upregulation of the genes specifically involved in transport of 3CB, and induction of a K+/H+ antiporter complex, an universal stress protein, two cytochrome P450 proteins and an efflux transporter. Downregulated expression of some genes involved in carbon metabolism and the genes belong to a prophage in the presence of 3CB was observed. This study demonstrated the applicability of the method of soil RNA extraction for microarray analysis through a proof-of-concept experiment using a sterilized soil inoculated with Pseudomonas putida KT2440/pSL1. A study using total RNA extracted from soil cultures of Pseudomonas putida KT2440/pSL1. Each chip measures the expression level of 5,341 genes from the Pseudomonas putida KT2440 genome with two sets of six 60-mer probes per gene.
2011-03-29 | E-GEOD-28215 | biostudies-arrayexpress
Project description:The metabolically versatile Pseudomonas putida strain KT2440 is the first Gram-negative soil bacterium certified as a biosafety strain and is being used for applications in agriculture, biotechnology and bioremediation. P. putida has to cope in its niche with numerous abiotic stresses. The stress response to 4°C, pH 4.5, 0.8 M urea or 45 mM sodium benzoate, respectively, was analyzed by the global mRNA expression profile and screening for stress-intolerant Tn5 transposon mutants. In total we identified 49 gene regions to be differentially expressed and 32 genes in 22 operons to be indispensable for growth during exposure to one or the other abiotic stresses. We propose that stress is sensed by the outer membrane proteins OmlA and FepA and the inner membrane constituents PtsP, PhoPQ and CbrAB. The metabolic response is regulated by the cyo operon, the RelA/SpoT modulon, PcnB and VacB that control mRNA stability and BipA that exerts transcript-specific translational control. The adaptation of the membrane barrier, the uptake of phosphate, the maintenance of intracellular pH and redox status and the translational control of metabolism are the indispensable key mechanisms of the P. putida stress response. Keywords: functional genomics
2006-07-27 | GSE4048 | GEO
Project description:The entire set of flagellar structural components and flagellar-specific transcriptional regulators, as well as much of the core chemotaxis machinery, is encoded into a >70 kbp cluster in Pseudomonas putida KT2440 genome. We have performed RNA-seq of the wild-type strain in order to identify operon boundaries and promoters location in this cluster.
2021-05-05 | GSE173832 | GEO
Project description:Pseudomonas putida KT2440 has received increasing attention as an important biocatalyst for the conversion of diverse carbon sources to multiple products, including the olefinic diacid, cis,cis-muconic acid (muconate). P. putida has been previously engineered to produce muconate from glucose; however, periplasmic oxidation of glucose causes substantial 2-ketogluconate accumulation, reducing product yield and selectivity. Deletion of the glucose dehydrogenase gene (gcd) prevents 2-ketogluconate accumulation, but dramatically slows growth and muconate production. In this work, we employed adaptive laboratory evolution to improve muconate production in strains incapable of producing 2-ketogluconate. Growth-based selection improved growth, but reduced muconate titer. A new muconate-responsive biosensor was therefore developed to enable muconate-based screening using fluorescence activated cell sorting. Sorted clones demonstrated both improved growth and muconate production. Mutations identified by whole genome resequencing of these isolates indicated that glucose metabolism may be dysregulated in strains lacking gcd. Using this information, we used targeted engineering to recapitulate improvements achieved by evolution. Deletion of the transcriptional repressor gene hexR improved strain growth and increased the muconate production rate, and the impact of this deletion was investigated using transcriptomics. The genes gntZ and gacS were also disrupted in several evolved clones, and deletion of these genes further improved strain growth and muconate production. Together, these targets provide a suite of modifications that improve glucose conversion to muconate by P. putida in the context of gcd deletion. Prior to this work, our engineered strain lacking gcd generated 7.0 g/L muconate at a productivity of 0.07 g/L/h and a 38% yield (mol/mol) in a fed-batch bioreactor. Here, the resulting strain with the deletion of hexR, gntZ, and gacS achieved 22.0 g/L at 0.21 g/L/h and a 35.6% yield (mol/mol) from glucose in similar conditions. These strategies enabled enhanced muconic acid production and may also improve production of other target molecules from glucose in P. putida.
2022-03-26 | GSE198795 | GEO
Project description:Pseudomonas putida mt-2 metabolizes m-xylene and other aromatic compounds through the enzymes encoded by the xyl operons of the TOL plasmid pWW0 along with other chromosomally-encoded activities. Tiling arrays of densely overlapping oligonucleotides were designed to cover every gene involved in this process, allowing dissection of operon structures and exposing the interplay of plasmid and chromosomal functions. All xyl sequences were transcribed in response to aromatic substrates and the 3'-termini of both upper and lower mRNA operons extended beyond the ir coding regions, i.e., the 3â-end of the lower operon mRNA penetrated into the convergent xylS regulatory gene. Furthermore, xylR mRNA for the master m-xylene responsive regulator of the system was decreased by aromatic substrates, while the cognate upper operon mRNA was evenly stable throughout its full length. RNA-seq confirmed these data at a single-nucleotide level and refined the formerly misannotated xylL sequence. The chromosomal ortho route for degradation of benzoate (the ben, cat clusters and some pca genes) was activated by this aromatic, but not by the TOL substrates, toluene or m-xylene. We advocate this scenario as a testbed of natural 16 retroactivity between a pre-existing metabolic network and a new biochemical pathway implanted through gene transfer. Genome Analyzer IIx system (1x75bp) was used for sequencing total RNA extracted from P. putida (mt-2) containing the plasmid pWW0, in two different physiological states (treated with m-xylene and in C-runout condition).
2016-07-07 | E-GEOD-71852 | biostudies-arrayexpress
Project description:To know whether the microarray technique could be used to detect bacterial gene expression in soil, large quantity of RNA was extracted from soil cultures of Pseudomonas putida KT2440 containing a chloroaromatic degrading plasmid at the presence or absence of the growth substrate, 3-chlorobenzoate (3CB). The quality and quantity of the extracted RNA were proper for a typical microarray analysis. Gene expression patterns of soil cultures were analyzed by DNA microarray using the extracted RNA. Among 5346 genes on the array, 5% and 4.5% of genes showed up- or down-regulation. Analysis done at the DAVID Bioinformatics Resources server suggested that the benzoate degradation via hydroxylation pathway had the most significant changes after treatment with 3CB. Expression of the 3CB degradation genes located in the genome was confirmed by real-time RT-PCR. In addition, real time RT-PCR analysis revealed that the fluorescent signals from plasmid genes on the microarray were saturated so that the induction ratio of the genes located in the plasmid was underestimated in microarray analysis. To our best knowledge, this report represents the first trial to use microarray technique to detect genome-wide bacterial gene expression in soil. A study using total RNA extracted from soil cultures of Pseudomonas putida KT2440/pSL1. Each chip measures the expression level of 5,341 genes from Pseudomonas putida KT2440 genome and 5 genes from an introduced plasmid pSL1 with fourteen 60-mer probes per gene which have five-fold technical redundancy.
2010-04-01 | E-GEOD-19516 | biostudies-arrayexpress
Project description:Nogales2008 - Genome-scale metabolic network
of Pseudomonas putida (iJN746)
This model is described in the article:
A genome-scale metabolic
reconstruction of Pseudomonas putida KT2440: iJN746 as a cell
factory.
Nogales J, Palsson BØ, Thiele
I.
BMC Syst Biol 2008; 2: 79
Abstract:
BACKGROUND: Pseudomonas putida is the best studied pollutant
degradative bacteria and is harnessed by industrial
biotechnology to synthesize fine chemicals. Since the
publication of P. putida KT2440's genome, some in silico
analyses of its metabolic and biotechnology capacities have
been published. However, global understanding of the
capabilities of P. putida KT2440 requires the construction of a
metabolic model that enables the integration of classical
experimental data along with genomic and high-throughput data.
The constraint-based reconstruction and analysis (COBRA)
approach has been successfully used to build and analyze in
silico genome-scale metabolic reconstructions. RESULTS: We
present a genome-scale reconstruction of P. putida KT2440's
metabolism, iJN746, which was constructed based on genomic,
biochemical, and physiological information. This
manually-curated reconstruction accounts for 746 genes, 950
reactions, and 911 metabolites. iJN746 captures
biotechnologically relevant pathways, including
polyhydroxyalkanoate synthesis and catabolic pathways of
aromatic compounds (e.g., toluene, benzoate, phenylacetate,
nicotinate), not described in other metabolic reconstructions
or biochemical databases. The predictive potential of iJN746
was validated using experimental data including growth
performance and gene deletion studies. Furthermore, in silico
growth on toluene was found to be oxygen-limited, suggesting
the existence of oxygen-efficient pathways not yet annotated in
P. putida's genome. Moreover, we evaluated the production
efficiency of polyhydroxyalkanoates from various carbon sources
and found fatty acids as the most prominent candidates, as
expected. CONCLUSION: Here we presented the first genome-scale
reconstruction of P. putida, a biotechnologically interesting
all-surrounder. Taken together, this work illustrates the
utility of iJN746 as i) a knowledge-base, ii) a discovery tool,
and iii) an engineering platform to explore P. putida's
potential in bioremediation and bioplastic production.
This model is hosted on
BioModels Database
and identified by:
MODEL1507180068.
To cite BioModels Database, please use:
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To the extent possible under law, all copyright and related or
neighbouring rights to this encoded model have been dedicated to
the public domain worldwide. Please refer to
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2015-07-30 | MODEL1507180068 | BioModels
Project description:Pseudomonas species thrive in different nutritional environments and can catabolize divergent carbon substrates. These capabilities have important implications for the role of these species in natural and engineered carbon processing. However, the metabolic phenotypes enabling Pseudomonas to utilize mixed substrates remain poorly understood. This work is part of a multi-omics approach involving stable isotope tracers, metabolomics, fluxomics, and proteomics in Pseudomonas putida KT2440 to investigate the constitutive metabolic network that achieves co-utilization of glucose and benzoate, respectively. The data used to estimate the changes in protein abundance are deposited here and were found to partially predicted the metabolic flux changes in cells grown on the glucose:benzoate mixture versus on glucose alone. Notably, flux magnitude and directionality were also maintained by metabolite levels and regulation of phosphorylation of key metabolic enzymes.
2021-09-08 | PXD013605 | Pride
Project description:Pseudomonas putida mt-2 metabolizes m-xylene and other aromatic compounds through the enzymes encoded by the xyl operons of the TOL plasmid pWW0 along with other chromosomally-encoded activities. Tiling arrays of densely overlapping oligonucleotides were designed to cover every gene involved in this process, allowing dissection of operon structures and exposing the interplay of plasmid and chromosomal functions. All xyl sequences were transcribed in response to aromatic substrates and the 3'-termini of both upper and lower mRNA operons extended beyond the ir coding regions, i.e., the 3’-end of the lower operon mRNA penetrated into the convergent xylS regulatory gene. Furthermore, xylR mRNA for the master m-xylene responsive regulator of the system was decreased by aromatic substrates, while the cognate upper operon mRNA was evenly stable throughout its full length. RNA-seq confirmed these data at a single-nucleotide level and refined the formerly misannotated xylL sequence. The chromosomal ortho route for degradation of benzoate (the ben, cat clusters and some pca genes) was activated by this aromatic, but not by the TOL substrates, toluene or m-xylene. We advocate this scenario as a testbed of natural 16 retroactivity between a pre-existing metabolic network and a new biochemical pathway implanted through gene transfer.
2016-07-07 | GSE71852 | GEO
Project description:Pseudomonas putida mt-2 metabolizes m-xylene and other aromatic compounds through the enzymes encoded by the xyl operons of the TOL plasmid pWW0 along with other chromosomally-encoded activities. Tiling arrays of densely overlapping oligonucleotides were designed to cover every gene involved in this process, allowing dissection of operon structures and exposing the interplay of plasmid and chromosomal functions. All xyl sequences were transcribed in response to aromatic substrates and the 3'-termini of both upper and lower mRNA operons extended beyond the ir coding regions, i.e., the 3'-end of the lower operon mRNA penetrated into the convergent xylS regulatory gene. Furthermore, xylR mRNA for the master m-xylene responsive regulator of the system was decreased by aromatic substrates, while the cognate upper operon mRNA was evenly stable throughout its full length. RNA-seq confirmed these data at a single-nucleotide level and refined the formerly misannotated xylL sequence. The chromosomal ortho route for degradation of benzoate (the ben, cat clusters and some pca genes) was activated by this aromatic, but not by the TOL substrates, toluene or m-xylene. We advocate this scenario as a testbed of natural 16 retroactivity between a pre-existing metabolic network and a new biochemical pathway implanted through gene transfer.
2016-07-07 | GSE71851 | GEO