Metabolic engineering of strains of Ralstonia eutropha and Pseudomonas putida for biotechnological production of 2-methylcitric acid

Ewering, Christian; Heuser, Florian; Benölken, Jens Klaus; Brämer, Christian O; Steinbüchel, Alexander

doi:10.1016/j.ymben.2006.05.007

Cited by 44 publications

(30 citation statements)

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“…However, if any one of these predicted target genes was inactivated, the solution space was altered so that RehMBEL1391 is enabled to produce 2-methylcitric acid even at the optimal growth rate, which decreased to about 53% (see the enlarged panel in Figure 4) because metabolic fluxes were redirected to the product without being consumed by these three enzymes. These results are consistent with Ewering's study (2006) on the improvement of R. eutropha for the biotechnological production of 2-methylcitric acid [43]. They also disrupted the 2-methylisocitric acid dehydratase encoded by acnM in R. eutropha and confirmed the enhanced capability of the mutant for producing 2-methylcitric acid.…”

Section: Resultssupporting

confidence: 91%

“…As a proof-of-concept biotechnological application of R. eutropha as a production host, RehMBEL1391 was used to predict production of an important industrial product 2-methylcitric acid, which appears in propionic acid metabolism in many organisms across prokaryotes and eukaryotes, including R. eutropha [41-43], yeasts [44], and fungi [45]. 2-methylcitric acid and its derivatives have been considered for potential pharmaceutical applications, such as growth inhibitor agent in cancer treatment and antiperspirants [43].…”

Section: Resultsmentioning

confidence: 99%

“…2-methylcitric acid and its derivatives have been considered for potential pharmaceutical applications, such as growth inhibitor agent in cancer treatment and antiperspirants [43]. For the overproduction of 2-methylcitric acid in R. eutropha, in silico single gene knockout simulation was conducted using flux balance analysis (FBA) and MOMA in order to redesign the metabolic network and redirect metabolic fluxes towards production of 2-methylcitric acid [30].…”

Section: Resultsmentioning

confidence: 99%

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Genome-scale reconstruction and in silico analysis of the Ralstonia eutropha H16 for polyhydroxyalkanoate synthesis, lithoautotrophic growth, and 2-methyl citric acid production

2011

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BackgroundRalstonia eutropha H16, found in both soil and water, is a Gram-negative lithoautotrophic bacterium that can utillize CO2 and H2 as its sources of carbon and energy in the absence of organic substrates. R. eutropha H16 can reach high cell densities either under lithoautotrophic or heterotrophic conditions, which makes it suitable for a number of biotechnological applications. It is the best known and most promising producer of polyhydroxyalkanoates (PHAs) from various carbon substrates and is an environmentally important bacterium that can degrade aromatic compounds. In order to make R. eutropha H16 a more efficient and robust biofactory, system-wide metabolic engineering to improve its metabolic performance is essential. Thus, it is necessary to analyze its metabolic characteristics systematically and optimize the entire metabolic network at systems level.ResultsWe present the lithoautotrophic genome-scale metabolic model of R. eutropha H16 based on the annotated genome with biochemical and physiological information. The stoichiometic model, RehMBEL1391, is composed of 1391 reactions including 229 transport reactions and 1171 metabolites. Constraints-based flux analyses were performed to refine and validate the genome-scale metabolic model under environmental and genetic perturbations. First, the lithoautotrophic growth characteristics of R. eutropha H16 were investigated under varying feeding ratios of gas mixture. Second, the genome-scale metabolic model was used to design the strategies for the production of poly[R-(-)-3hydroxybutyrate] (PHB) under different pH values and carbon/nitrogen source uptake ratios. It was also used to analyze the metabolic characteristics of R. eutropha when the phosphofructokinase gene was expressed. Finally, in silico gene knockout simulations were performed to identify targets for metabolic engineering essential for the production of 2-methylcitric acid in R. eutropha H16.ConclusionThe genome-scale metabolic model, RehMBEL1391, successfully represented metabolic characteristics of R. eutropha H16 at systems level. The reconstructed genome-scale metabolic model can be employed as an useful tool for understanding its metabolic capabilities, predicting its physiological consequences in response to various environmental and genetic changes, and developing strategies for systems metabolic engineering to improve its metabolic performance.

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Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Genome-scale reconstruction and in silico analysis of the Ralstonia eutropha H16 for polyhydroxyalkanoate synthesis, lithoautotrophic growth, and 2-methyl citric acid production

2011

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“…Propionyl-CoA derived from levulinic acid, propionate or other carbon sources is then utilized via the 2-methylcitric acid cycle (2MCC) and not via the methyl-malonylCoA pathway [Brämer and Steinbüchel, 2001;Ewering et al, 2006a]. An engineered strain of R. eutropha , in which the 2-methyl-cis -aconitate hydratase (acnM) gene was disrupted by directed insertion of an additional copy of a 2-methylcitrate synthase gene (prpC) , could be utilized for production of high concentrations of 2-methylcitrate in the medium when cultivated with levulinic acid plus succinate as carbon sources [Ewering et al, 2006b]. Levulinic acid is in addition also a suitable carbon source to produce 4-hydroxyvalerate containing PHAs [Gorenflo et al, 2001].…”

Section: Important Features Of the Carbon Metabolism Of R Eutropha H16mentioning

confidence: 99%

<i>Ralstonia eutropha</i> Strain H16 as Model Organism for PHA Metabolism and for Biotechnological Production of Technically Interesting Biopolymers

Reinecke¹,

2008

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The Gram-negative, facultative chemolithoautotrophic bacterium Ralstonia eutropha has been intensively investigated for almost 50 years. Today it is the best studied ‘Knallgas’ bacterium and producer of poly(3-hydroxybutyric acid). This polyester provides the basis for renewable resource-based biodegradable plastic materials and has attracted much biotechnological interest. The polymer is accumulated in large amounts in the cell and can be used for various applications ranging from replacement of fossil resource-based bulk plastics to high-value special purpose polymers. To further enhance productivity and to allow tailormade poly(hydroxyalkanoic acids) (PHA) with different monomer compositions by metabolic engineering, the knowledge of metabolic pathways and of the biochemical properties of the enzymes involved is essential. Furthermore, proteins covering the PHA granule surface, which are referred to as phasins, and fusions of these phasins to other proteins are promising candidates for various protein technologies. The recently published genome sequence of strain H16 allows researchers to take a closer look at the genetic potential of this versatile bacterium. R. eutropha is, however, not limited to PHAs and to PHA-related polymers like poly(mercaptoalkanoic acids) as it can also be employed for production of a range of other interesting polymers including polyamides like cyanophycin.

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“…putida strains have been ranked among the most solvent-tolerant bacteria known (Nicolaou et al, 2010), suitable for enhancing their biotechnological production of compounds by metabolic engineering (Ewering et al, 2006). Numerous…”

Section: Introductionmentioning

confidence: 99%

Linking genes to microbial growth kinetics—An integrated biochemical systems engineering approach

Koutinas

Kiparissides

Silva‐Rocha

et al. 2011

Metabolic Engineering

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Metabolic engineering of strains of Ralstonia eutropha and Pseudomonas putida for biotechnological production of 2-methylcitric acid

Cited by 44 publications

References 33 publications

Genome-scale reconstruction and in silico analysis of the Ralstonia eutropha H16 for polyhydroxyalkanoate synthesis, lithoautotrophic growth, and 2-methyl citric acid production

Genome-scale reconstruction and in silico analysis of the Ralstonia eutropha H16 for polyhydroxyalkanoate synthesis, lithoautotrophic growth, and 2-methyl citric acid production

<i>Ralstonia eutropha</i> Strain H16 as Model Organism for PHA Metabolism and for Biotechnological Production of Technically Interesting Biopolymers

Linking genes to microbial growth kinetics—An integrated biochemical systems engineering approach

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