Metabolic network analysis of Streptomyces tenebrarius, a Streptomyces species with an active entner-doudoroff pathway

Abstract

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 by-product. 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-Meyerhof-Parnas (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.

FIG. 1.

Chemical structures of tobramycin, tobramycin carbamate, kanamycin B, and kanamycin B carbamate.

FIG. 2.

Final concentrations of antibiotics in S. tenebrarius cultivations on glucose and on an equimolar mixture of glucose and glycerol. Tobramycin shows the summed concentrations of tobramycin and tobramycin carbamate, and kanamycin shows the summed concentrations of kanamycin B and kanamycin B carbamate. Concentrations shown are means of three (glucose) or two (glucose-glycerol) cultivations.

FIG. 3.

Cultivation profile on minimal medium with [1-¹³C]glucose (A) and [1-¹³C]glucose-glycerol (B) as the carbon sources. The arrows indicate when samples for biomass labeling analysis were taken. Biomass dry weight □, glucose ▴, glycerol ♦, tobramycin and tobramycin carbamate •, kanamycin and kanamycin carbamate ▿, and apramycin ▪.

FIG. 4.

Labeling of pyruvate atoms in cultivation on glucose. The SFL of the pyruvate second and third carbon atoms was calculated as an average of SFLs of Ala116, Ala99, half-SFLs of Val127, and half-SFLs of Val144. The labeling of the first pyruvate carbon atom was found as an average between differences: Ala158 − PYR(2, 3) and Val186 − PYR(2, 3). The SFL of the pyruvate second carbon was calculated as the difference between Val143 and PYR(1). Eventually, the SFL of the third position was found as the difference between SFL of PYR(2, 3) and PYR(2).

FIG. 5.

Labeling of the TCA cycle metabolites oxaloacetate and α-ketoglutarate in cultivation on glucose. The labelings are normalized with respect to carbon atoms.

FIG. 6.

The metabolic fluxes as calculated in a computer simulation. The values are averages between fluxes obtained in two simulations. Numbers in boldface type are fluxes for the glucose cultivation, and numbers in italics are fluxes for the glucose-glycerol cultivation. All fluxes are given in mmol · g (dry weight)⁻¹ · h⁻¹. The numbers in circles show exchange coefficients for reversible reactions, calculated as MIN (v_forward, v_reverse)/100 + MIN (v_forward, v_reverse), where v_forward is the forward flux, and v_reverse is reverse flux. The function MIN (v_forward, v_reverse) gives the smaller flux (forward or reverse) as the result.

FIG. 7.

Specific reducing cofactor production in the cultivation on glucose and on glucose-glycerol.

FIG. 8.

Unrooted phylogenetic tree derived by neighbor-joining analysis of Edd amino acid sequences from different bacteria species with a proven Entner-Doudoroff pathway presence. The bootstrap values on the branches indicate the number of times a given branch appeared in 1,000 bootstrap replications. The sequences were obtained from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov). ClustalX 1.8 was used for making multiple sequence alignment and for construction of the tree. The tree was drawn with Njplot.