Optimized submerged batch fermentation strategy for systems scale studies of metabolic switching in Streptomyces coelicolor A3(2)

Abstract

Background: Systems biology approaches to study metabolic switching in Streptomyces coelicolor A3(2) depend on cultivation conditions ensuring high reproducibility and distinct phases of culture growth and secondary metabolite production. In addition, biomass concentrations must be sufficiently high to allow for extensive time-series sampling before occurrence of a given nutrient depletion for transition triggering. The present study describes for the first time the development of a dedicated optimized submerged batch fermentation strategy as the basis for highly time-resolved systems biology studies of metabolic switching in S. coelicolor A3(2).

Results: By a step-wise approach, cultivation conditions and two fully defined cultivation media were developed and evaluated using strain M145 of S. coelicolor A3(2), providing a high degree of cultivation reproducibility and enabling reliable studies of the effect of phosphate depletion and L-glutamate depletion on the metabolic transition to antibiotic production phase. Interestingly, both of the two carbon sources provided, D-glucose and L-glutamate, were found to be necessary in order to maintain high growth rates and prevent secondary metabolite production before nutrient depletion. Comparative analysis of batch cultivations with (i) both L-glutamate and D-glucose in excess, (ii) L-glutamate depletion and D-glucose in excess, (iii) L-glutamate as the sole source of carbon and (iv) D-glucose as the sole source of carbon, reveal a complex interplay of the two carbon sources in the bacterium's central carbon metabolism.

Conclusions: The present study presents for the first time a dedicated cultivation strategy fulfilling the requirements for systems biology studies of metabolic switching in S. coelicolor A3(2). Key results from labelling and cultivation experiments on either or both of the two carbon sources provided indicate that in the presence of D-glucose, L-glutamate was the preferred carbon source, while D-glucose alone appeared incapable of maintaining culture growth, likely due to a metabolic bottleneck at the oxidation of pyruvate to acetyl-CoA.

Figure 1

Listing of cultivation conditions applied in (upper part), and presentation and comparative characterization of specific productivities determined (lower part) for fermentations of the present study; single fermentations runs, except for where indicated a-c (three biological replicas). SMM-TE, trace elements solution of medium SMM [26]; PEG, polyethylene glycol 6000; TES, N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; TMS1, trace mineral solution 1; const. agit. or min. DO, cultivation performed at given constant agitation from the start/culture started at min. agitation (~325 rpm) or agitation was adjusted automatically to maintain at least the given percentage of dissolved oxygen (DO); 900/1100/1300 (ferm. #16a-c) indicates manual change of constant agitation from 900 to 1100 to 1300 rpm after 21 and 26 h, respectively, in each of three biological replicas to prevent a DO of lower than approx. 50%, returned to 900 rpm after 45 h. Ferm. #18a-c and #19a-c were experiments with massive volume reduction due to high resolution time-series sampling. Ferm. #21, L-glutamate amount exchanged with ammonium sulfate of the respective molar amount of nitrogen; ferm. #24/25, use of custom-made 350 mL fermentation vessels with 200 mL working volume; ferm. #25, use of 20 g/L fully ¹³ C-labelled D-glucose (*). All fermentations were performed at 30°C and pH 7.0, automatically adjusted with 2 M HCl and 2 M NaOH. Initial specific productivity q_Pinit of secondary metabolites determined by measuring the pigment levels in culture samples using the RED/TBP assays and plotting the data as a function of time, linear regression of the initial production data points after production start (usually within the first 15 h after sec. metabolites have first been detected); max CDW, maximum biomass concentration (cell dry weight). X symbols indicate the maximum biomass concentration, red and blue bars the initial specific productivity of RED and TBP, respectively. Bold font indicates changes in cultivation conditions in the order of presentation from ferm. #1 to ferm. #25.

Figure 2

On-line and off-line measurements as a function of time of strain M145 grown on (A) phosphate and (B) L-glutamate depletion medium. Upper panels: on-line measurements of three individual biological replicas, lower panels: average and standard deviations of off-line analyses obtained for three biological replicas. A, ferm. #18a-c; B, ferm. #19a-c. Results in (A) include data identical to those published by us before [19].

Figure 3

The split carbon metabolism of M145 grown on D-glucose and/or L-glutamate. (A) Summary of on-line and off-line analyses of fermentations performed in the 200 mL scale on ¹³ C₆ D-glucose and non-labelled L-glutamate (ferm. #25, red), and on non-labelled substrates only (ferm. #24, blue), reference fermentation 1.8 L standard volume (ferm. #23, grey). (B) Schematic representation of parts of the central carbon metabolism including the two carbon sources used, and results from metabolite profiling and summed fractional labelling (SFL) determination (grey boxes) of selected metabolites (green/blue); Only the most significant SFLs are displayed, obtained by analysis of GC-MS data based on samples from ferm. #24 and #25 28 h (growth phase, left) and 44 h (transition phase, right) after inoculation. Relative % standard deviations are given in brackets below the SFLs. (C) M145 culture growth and antibiotic production in the absence of D-glucose (ferm. #22, red), or with L-glutamate (ferm. #20, grey) or ammonium (ferm. #21, blue) as the sources of nitrogen. For the latter, no significant growth was detected, not giving rise to any significant production phenotype.