The major concern at the metabolite level during secondary metabolism is sufficient supply of precursor metabolites and energy. Especially many secondary metabolites have a high demand of NADPH. This is usually solved at the cellular level by increasing pentose phosphate fluxes [37,38]. However, though these high demands on the molecule basis in principle also exist for Act and Red production (see formula in Figure 3 bottom), the relatively low
Inhibitors,research,lifescience,medical production of these secondary metabolites are in the present case unlikely to be limited by NADPH pool levels. 3. Experimental 3.1. Strain and General Cultivation Parameters Experiments were performed using S. coelicolor M145 and phoP deletion mutant INB201 derived thereof [19,20].
Spores prepared on SFM Inhibitors,research,lifescience,medical solid Selleckchem Integrase inhibitor medium were used as the inoculum in all cultivations. Spores were germinated for 5 h at 30 °C and 250 rpm in 250 mL baffled shake-flasks containing 50 mL 2× YT medium and 2 g of 3 mm glass beads. Cultivations were performed in 3-liter fermentors (Applikon) with an initial culture volume of 1.8 L. The optimized growth medium Inhibitors,research,lifescience,medical used for studying the effect of phosphate depletion during batch fermentation (SSBM-P) consisted of Na-glutamate, 55.2 g/L; Inhibitors,research,lifescience,medical D-glucose, 40 g/L; MgSO4, 2.0 mM; phosphate, 4.6 mM; supplemented minimal medium trace element solution SMM-TE , 8 mL/L and TMS1, 5.6 mL/L. TMS1 consisted of FeSO4 × 7 H2O, 5 g/L; CuSO4 × 5 H2O, 390 mg/L; ZnSO4 × 7 H2O, 440 mg/L; MnSO4 × H2O, 150 mg/L; Na2MoO4 × 2 H2O, 10 mg/L; CoCl2
× 6 H2O, 20 mg/L, and HCl, 50 ml/L. The optimized medium for studying the effect of L-glutamate depletion (SSBM-E) was identical to SSBM-P except for the concentrations of Na-glutamate and phosphate adjusted Inhibitors,research,lifescience,medical to be 15 g/L and 9.2 mM, respectively. Clerol FBA 622 fermentation defoamer (Diamond Shamrock Scandinavia) was added to the growth medium before inoculation. Dissolved oxygen levels were maintained at a minimum of 50% by automatic adjustment Megestrol Acetate of the stirrer speed (minimal agitation 325 rpm). The aeration rate was constant 0.5 L sterile air/L culture/min. Dissolved oxygen, agitation speed and CO2 concentration in off-gas were measured and logged on-line, while samples for the determination of cell dry weight, levels of key growth medium components and production of secondary metabolites were taken throughout the fermentation trials. For details on off-line analyses, it is referred to Nieselt et al. . 3.2.