The linear correlation between growth, production of lysine and dihydroxyacetone (DHA) and consumption of glucose indicates metabolic steady-state during the cultivation

<p><b>Copyright information:</b></p><p>Taken from "Metabolic responses to pyruvate kinase deletion in lysine producing "</p><p>http://www.microbialcellfactories.com/content/7/1/8</p><p>Microbial Cell Factories 2008;7():8-8.</p><p>Published online 13 Mar 2008</p><p>PMCID:PMC2322953.</p><p></p

The labelling patterns of the amino acids were determined from protein hydrolysates harvested at different cell dryx mass (CDM) concentrations during the cultivation

The amino acids shown here exemplarily stem form different parts of the metabolic network and comprise alanine (solid square), phenylalanine (open square), valine (closed circle), glycine (open circle), glutamate (closed triangle), threonine (open triangle) and serine (closed diamond). M(non labelled), M(single labelled) and M(double labelled) denote the relative fractions of the corresponding mass isotopomers.<p><b>Copyright information:</b></p><p>Taken from "Metabolic responses to pyruvate kinase deletion in lysine producing "</p><p>http://www.microbialcellfactories.com/content/7/1/8</p><p>Microbial Cell Factories 2008;7():8-8.</p><p>Published online 13 Mar 2008</p><p>PMCID:PMC2322953.</p><p></p

The actual flux values are represented by the thickness of the corresponding arrows

<p><b>Copyright information:</b></p><p>Taken from "Metabolic responses to pyruvate kinase deletion in lysine producing "</p><p>http://www.microbialcellfactories.com/content/7/1/8</p><p>Microbial Cell Factories 2008;7():8-8.</p><p>Published online 13 Mar 2008</p><p>PMCID:PMC2322953.</p><p></p

MOESM1 of Metabolic engineering of Corynebacterium glutamicum for the production of cis, cis-muconic acid from lignin

Additional file 1: Figure S1. Confirmation of the deletion of the catB gene in Corynebacterium glutamicum ATCC 13032, using colony PCR. To this end, C. glutamicum ATCC 13032, had been transformed with the integrative plasmid pClik int sacB ΔcatB, followed by recombination and selection. The primers ΔcatB TS1 FW and ΔcatB TS2 RV were used for the PCR (Table 1). The positive clone, indicated by the white arrow, revealed the small fragment, size expected for the deletion. It was designated C. glutamicum LIMA-1. M, 1 kb DNA ladder; 1, blank; 2, positive clone; WT, wild type. Figure S2. Growth and cis–cis-muconic acid (MA) production from small aromatics, using Corynebacterium glutamicum MA-1. The yield for MA on benzoic acid (20 mM), catechol (10 mM), and phenol (5 mM) was obtained from measurement of substrates and product at the beginning and after 24 h of incubation (A). The tolerance to catechol was obtained from cell growth measurement (B). All data represent mean values and standard deviations from three biological replicates. Figure S3. Tolerance of Corynebacterium glutamicum MA-1 against benzoic acid (A), catechol (B), and phenol (C). In addition, glucose was added as growth substrate. The final cell concentration was measured after 24 h of cultivation. The data represent mean values and standard deviations from three biological replicates. Figure S4. Kinetics and stoichiometry of cis–cis-muconic acid (MA) production, using the first generation producer Corynebacterium glutamicum MA-1. The aromatics benzoic acid (A), catechol (B) and phenol (C) were used for production. In addition, glucose was added as growth substrate. The data represent mean values and standard deviations from three biological replicates. Figure S5. Characteristics of catechol-1,2-dioxygenase (CatA) in Corynebacterium glutamicum, grown on different aromatics. The data comprise the specific enzyme activity of the first generation producer C. glutamicum MA-1 (A), the kinetics of the enzyme with a fit of the experimental data from benzoate-grown cells to a Michaelis–Menten type kinetics (B), and the specific enzyme activity of the second generation producer C. glutamicum MA-2 (C). The data represent mean values and standard deviations from three biological replicates. Figure S6. Confirmation of overexpression of the catA gene under control of the tuf promoter in Corynebacterium glutamicum MA-2, using colony PCR. To this end, C. glutamicum MA-1 had been transformed with the integrative plasmid pClik int sacB PtufcatA, followed by recombination and selection. The primers Pef-tu-catA TS1 FW and Pef-tu-catA TS2 RV were used for the PCR (Table 1). The positive clone, indicated by the white arrow, revealed the increased fragment size, expected for the promoter exchange. M, 1 kb DNA ladder; 1, positive clone; WT, wild type. Figure S7. Kinetics and stoichiometry of cis–cis-muconic acid (MA) production, using the second generation producer Corynebacterium glutamicum MA-2 on catechol (A) and the first generation producer C. glutamicum MA-1 on catechol and benzoic acid (B). Glucose was added as growth substrate. The data represent mean values and standard deviations from three biological replicates. Figure S8. Fed-batch production of cis–cis-muconic acid (MA) from catechol by metabolically engineered Corynebacterium glutamicum MA-2. Substrate consumption, growth and MA formation (A). Volumetric productivity (B). Yields for MA from catechol, and for MA from catechol plus glucose (C). Pulse-wise feeding of catechol (D). Glucose was added continuously to maintain the glucose level in the range between about 5 to 15 g L−1 (A). The vertical lines represent individual catechol feed pulses (D). The feed frequency was variably adjusted, depending on the signal of dissolved oxygen, which precisely indicated the time point of catechol depletion. As example, feeding was halted once during the initial phase, corresponding to transient catechol accumulation and was accelerated later in response to the faster conversion. The data represent mean values from two replicates

Robustness and Plasticity of Metabolic Pathway Flux among Uropathogenic Isolates of <i>Pseudomonas aeruginosa</i>

<div><p><i>Pseudomonas aeruginosa</i> is a human pathogen that frequently causes urinary tract and catheter-associated urinary tract infections. Here, using ¹³C-metabolic flux analysis, we conducted quantitative analysis of metabolic fluxes in the model strain <i>P. aeruginosa</i> PAO1 and 17 clinical isolates. All <i>P. aeruginosa</i> strains catabolized glucose through the Entner-Doudoroff pathway with fully respiratory metabolism and no overflow. Together with other NADPH supplying reactions, this high-flux pathway provided by far more NADPH than needed for anabolism: a benefit for the pathogen to counteract oxidative stress imposed by the host. <i>P. aeruginosa</i> recruited the pentose phosphate pathway exclusively for biosynthesis. In contrast to glycolytic metabolism, which was conserved among all isolates, the flux through pyruvate metabolism, the tricarboxylic acid cycle, and the glyoxylate shunt was highly variable, likely caused by adaptive processes in individual strains during infection. This aspect of metabolism was niche-specific with respect to the corresponding flux because strains isolated from the urinary tract clustered separately from those originating from catheter-associated infections. Interestingly, most glucose-grown strains exhibited significant flux through the glyoxylate shunt. Projection into the theoretical flux space, which was computed using elementary flux-mode analysis, indicated that <i>P. aeruginosa</i> metabolism is optimized for efficient growth and exhibits significant potential for increasing NADPH supply to drive oxidative stress response.</p></div

In vivo carbon flux distributions in central metabolism of uropathogenic <i>P. aeruginosa</i> isolates during growth on glucose.

<p>Flux is given as average flux of all strains and is expressed as a molar percentage of the average glucose uptake rate of all strains (8.6 mmol g⁻¹ h⁻¹, calculated from the individual rates in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone-0088368-t001" target="_blank">Table 1</a>). Open arrows indicate flux toward biomass. For reversible reactions, the direction of net flux is indicated by a dashed arrow. The errors given for each flux reflect the corresponding 90% confidence intervals. The full flux data sets are presented in Supporting Information. Metabolic and isotopic steady states are ensured by constant stoichiometry, kinetics, and the constant ¹³C-labeling patterns of recruited metabolites during cultivation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone.0088368.s001" target="_blank">Figure S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone.0088368.s005" target="_blank">Figure S5</a>). Abbreviations are as follows: Entner-Doudoroff pathway (EDP), Embden-Meyerhof-Parnas pathway (EMPP), pentose phosphate pathway (PPP), glyoxylate (Gly) shunt, and tricarboxylic acid (TCA) cycle.</p

Kinetics and stoichiometry of glucose-grown <i>P. aeruginosa</i> PAO1 and uropathogenic <i>P. aeruginosa</i> isolates obtained from patients with catheter-associated urinary tract infection or urinary tract infections.

<p>The corresponding cultivation profiles for all strains are provided in Supporting Information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone.0088368.s003" target="_blank">Figure S3</a>). Values indicate means and standard deviations of three biological replicates. By-products in the supernatants were not detected; concentrations were below the detection limit (1 µM for amino acids and 10 µM for organic acids).</p

Metabolic flux in <i>P. aeruginosa</i> PAO1 and select gram-negative and gram-positive bacteria.

<p>Flux refers to the Entner-Doudoroff pathway (EDP), Embden-Meyerhof-Parnas pathway (EMPP), pentose phosphate pathway (PPP), and glyoxylate (Gly) shunt and reflects relative values normalized to the corresponding glucose uptake rate, defined as 100%.</p><p>* functional pathway not encoded.</p><p>** n.d. = not determined.</p

Metabolic adaption of pathogenic <i>P. aeruginosa</i>.

<p>The network representation integrates changes of flux determined for uropathogenic isolates and <i>P. aeruginosa</i> PAO1 (this work) and changes in transcription transcript level previously determined for successive isolates of <i>P. aeruginosa</i> from patients with chronic cystic fibrosis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone.0088368-Hoboth1" target="_blank">[11]</a>. The variation of metabolic flux among strains is indicated by color (black = conserved, green = changed). Genes with changed or unchanged transcript levels are shown in green or black, respectively. Abbreviations are as follows: Entner-Doudoroff pathway (EDP), Embden-Meyerhof-Parnas pathway (EMPP), pentose phosphate pathway (PPP), glyoxylate (Gly) shunt, and tricarboxylic acid (TCA) cycle.</p

Metabolic properties of uropathogenic <i>P. aeruginosa</i> isolates and <i>P. aeruginosa</i> PAO1.

<p>Integration of flux phenotypes into the theoretical flux space on basis of anabolism (considering the biomass yield coefficient Y_x/s, given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone-0088368-t001" target="_blank">Table 1</a>) and of NADPH metabolism (considering the NADPH balance as described below). In order to evaluate individual flux phenotypes, experimental values for biomass yield and apparent NADPH excess, respectively, (black squares) are integrated into the flux space created by elementary flux mode analysis (grey squares). Most strains localize close to the growth optimum. (A), Correlation of biomass formation and NADPH metabolism (B), contribution of isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase to supply of reducing power (C), correlation of biomass formation and flux through the oxidative pentose phosphate pathway (D). NADPH metabolism was inspected by balancing of the redox cofactor. For this purpose, the NADPH formation flux by concerted action of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase, and malic enzyme was balanced with the NADPH consumption flux for anabolism. For all strains, NADPH formation was higher than consumption. The resulting apparent excess flux of NADPH supply is presented here. The full flux data sets for all strains are presented in Supporting Information. The flux values are normalized to the corresponding glucose uptake rate for each strain (set to 100%).</p