Identification of the Entner–Doudoroff pathway in an antibiotic‐producing actinomycete species

Gunnarsson, Nina; Mortensen, Uffe Hasbro; Sosio, Margherita; Nielsen, Jens

doi:10.1111/j.1365-2958.2004.04028.x

Cited by 33 publications

(29 citation statements)

References 43 publications

(55 reference statements)

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“…ATCC 39727 (10). The edd gene has not been found in the sequenced genomes of the two Streptomyces species (S. coelicolor and S. avermitilis) (10). ED pathway activity was shown in S. tenebrarius by labeling experiments, and the presence of the characteristic gene edd was confirmed by PCR and subsequent sequencing.…”

Section: Discussionmentioning

confidence: 82%

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Metabolic Network Analysis of Streptomyces tenebrarius , a Streptomyces Species with an Active Entner-Doudoroff Pathway

Borodina

Schöller²,

Eliasson

et al. 2005

Appl Environ Microbiol

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Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted byproduct. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin.13 C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-MeyerhofParnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.The ability of actinomycetes to make secondary metabolites with different useful properties (antibacterials, antitumor agents, immunosuppressants, etc.) is widely exploited in the pharmaceutical industry. Two thirds of the antibiotics produced by microorganisms are made by actinomycetes. In particular, the Streptomyces genus is remarkable in this aspect, representing about 80% of the actinomycete antibiotics (16).Antibiotics are formed from specific precursors that are drained from the central carbon metabolism, and overproduction of antibiotics therefore requires that the precursors are supplied in sufficient quantities. Improvement of antibiotic production has traditionally been based on random mutagenesis methods, and in the future these methods will play an important role. However, metabolic engineering enables the introduction of rational changes to the central carbon metabolism to increase fluxes of precursors and cofactors to antibiotics. Metabolic flux analysis is a valuable tool in guiding metabolic engineering strategies, as it enables rapid phenotypic characterization of different mutants; through analysis of different mutants, one may gain insight into the correlation between antibiotic production and the fluxes through specific branches of the metabolic network.In this study, correlations between the primary and the secondary metabolism in the antibiotic...

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Section: Discussionmentioning

confidence: 82%

“…To the best of our knowledge, the only actinomycetes found to possess an ED pathway are Mycobacterium smegmatis (1) and Nonomuraea spp. ATCC 39727 (10). The edd gene has not been found in the sequenced genomes of the two Streptomyces species (S. coelicolor and S. avermitilis) (10).…”

Section: Discussionmentioning

confidence: 99%

Metabolic Network Analysis of Streptomyces tenebrarius , a Streptomyces Species with an Active Entner-Doudoroff Pathway

Borodina

Schöller²,

Eliasson

et al. 2005

Appl Environ Microbiol

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Section: Based Onmentioning

confidence: 99%

“…In this case, the estimated fluxes are the best fit to the available data within the specified metabolic model. Beyond the quantification of flux through the wellknown biochemical pathways, flux methods have recently demonstrated their value for the identification of novel (17) or unexpected (22,34,39,40) metabolic pathways.For obvious reasons, such flux methods were applied primarily to model microbes with industrial relevance, such as Escherichia coli (15,24,41), Bacillus subtilis (9, 40), Corynebacterium glutamicum (30,48), and Saccharomyces cerevisiae (3,19). While the accumulated biochemical and metabolic data on these species are also the basis of much of our textbook knowledge, it is clear that these model species are not representative for all, and perhaps not even for most, microbes.…”

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confidence: 99%

Experimental Identification and Quantification of Glucose Metabolism in Seven Bacterial Species

2005

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The structurally conserved and ubiquitous pathways of central carbon metabolism provide building blocks and cofactors for the biosynthesis of cellular macromolecules. The relative uses of pathways and reactions, however, vary widely among species and depend upon conditions, and some are not used at all. Here we identify the network topology of glucose metabolism and its in vivo operation by quantification of intracellular carbon fluxes from 13 C tracer experiments. Specifically, we investigated Agrobacterium tumefaciens, two pseudomonads, Sinorhizobium meliloti, Rhodobacter sphaeroides, Zymomonas mobilis, and Paracoccus versutus, which grow on glucose as the sole carbon source, represent fundamentally different metabolic lifestyles (aerobic, anaerobic, photoheterotrophic, and chemoheterotrophic), and are phylogenetically distinct (firmicutes, ␥-proteobacteria, and ␣-proteobacteria). Compared to those of the model bacteria Escherichia coli and Bacillus subtilis, metabolisms of the investigated species differed significantly in several respects: (i) the Entner-Doudoroff pathway was the almost exclusive catabolic route; (ii) the pentose phosphate pathway exhibited exclusively biosynthetic functions, in many cases also requiring flux through the nonoxidative branch; (iii) all aerobes exhibited fully respiratory metabolism without significant overflow metabolism; and (iv) all aerobes used the pyruvate bypass of the malate dehydrogenase reaction to a significant extent. Exclusively, Pseudomonas fluorescens converted most glucose extracellularly to gluconate and 2-ketogluconate. Overall, the results suggest that metabolic data from model species with extensive industrial and laboratory history are not representative of microbial metabolism, at least not quantitatively. Based on13 C tracer experiments, metabolic-flux analysis emerged as a key methodology to identify the network topology of active reactions and to quantify the in vivo distribution of molecular fluxes throughout metabolism (38, 47). In contrast to global protein, mRNA, or metabolite concentration analyses that assess network composition, flux methods directly assess the operation of metabolic networks by quantifying in vivo reaction velocities. The general principle is based on mass spectrometry (MS) or nuclear magnetic resonance detection of 13 C patterns in products of metabolism. Often, protein-bound amino acids that preserve the carbon backbone of eight metabolic key intermediates are used. The detected 13 C isotope patterns then reflect the activity of intracellular pathways and reactions, whose fluxes can be quantified from the isotope data by using mathematical models with various levels of complexity. In the simplest approach, algebraic equations are used to determine strictly local ratios of converging fluxes analytically by so-called metabolic-flux ratio (METAFoR) analysis (3,16,41,44). Absolute intracellular fluxes in millimoles per gram of biomass per hour may be estimated indirectly by combining such 13 C data with quantitative physiological ...

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“…In particular, when considering evolution of metabolic functions, more direct information on intracellular flux rerouting would be necessary to reveal the molecular mechanisms that cause a given improved phenotype. Such in vivo reaction rates are accessible through methods of 13 Cbased metabolic flux analysis (12), which have been used successfully to identify functional flux states in various microbes (13)(14)(15)(16)(17). Potentially, flux data can fill the gap between the intrinsically noisy and indirect transcriptome, proteome, or metabolome data and the actual phenotype (18,19).…”

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confidence: 99%

Latent Pathway Activation and Increased Pathway Capacity Enable Escherichia coli Adaptation to Loss of Key Metabolic Enzymes

Fong

Nanchen

Palsson

et al. 2006

Journal of Biological Chemistry

172

205

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The ability of biological systems to adapt to genetic and environmental perturbations is a fundamental but poorly understood process at the molecular level. By quantifying metabolic fluxes and global mRNA abundance, we investigated the genetic and metabolic mechanisms that underlie adaptive evolution of four metabolic gene deletion mutants of Escherichia coli (⌬pgi, ⌬ppc, ⌬pta, and ⌬tpi) in parallel evolution experiments of each mutant. The initial response to the gene deletions was flux rerouting through local bypass reactions or normally latent pathways. The principal effect of evolution was improved capacity of already active pathways, whereas new flux distributions were not observed. Combinatorial changes in capacity and pathway activation, however, led to different intracellular flux states that enabled evolution in three of the four parallel cases tested. The molecular bases of the evolved phenotypes were then elucidated by global mRNA transcript analyses. Activation of latent pathways and flux changes in the tricarboxylic acid cycle were found to correlate well with molecular changes at the transcriptional level. Flux alterations in other central metabolic pathways, in contrast, were apparently not connected to changes in the transcriptional network. These results give new insight into the dynamics of the evolutionary process by demonstrating the flexibility of the metabolic network of E. coli to compensate for genetic perturbations and the utility of combining multiple high throughput data sets to differentiate between causal and noncausal mechanistic changes.

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Identification of the Entner–Doudoroff pathway in an antibiotic‐producing actinomycete species

Cited by 33 publications

References 43 publications

Metabolic Network Analysis of Streptomyces tenebrarius , a Streptomyces Species with an Active Entner-Doudoroff Pathway

Metabolic Network Analysis of Streptomyces tenebrarius , a Streptomyces Species with an Active Entner-Doudoroff Pathway

Experimental Identification and Quantification of Glucose Metabolism in Seven Bacterial Species

Latent Pathway Activation and Increased Pathway Capacity Enable Escherichia coli Adaptation to Loss of Key Metabolic Enzymes

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