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The glucose-tolerant strain of Synechocyst … The glucose-tolerant strain of Synechocystis sp. PCC 6803, isolated by Williams (Williams, 1988), was grown in modified BG-11 medium, consisting of BG-110 liquid medium (Rippka, 1988) supplemented with 5 mM NH4Cl (buffered with 20 mM HEPES–KOH, pH 7.8). The GT-I strain, among GT substrains, was used in the current study (Kanesaki et al., 2012). Liquid cultures were bubbled with 1% (v/v) CO2 in air and incubated at 30°C under continuous white light (~50–70 μmol photons m−2 s−1). For the mutant strains, 10, 0.3, and 10 μg/mL of kanamycin, gentamycin and chloramphenicol, respectively, were added for preculturing. Modified BG-11 medium (containing 10 mM NH4Cl in liquid medium) was solidified with agar (1.5% w/v) for plate cultures, and similarly incubated in air at 30°C under continuous white light (~50–70 μmol photons m−2 s−1). Cell densities were measured at A730 using a Hitachi U-3310 spectrophotometer (Hitachi High-Tech., Tokyo, Japan).
For succinate production, cells grown in 70 mL modified BG-11 medium (started from A730 = 0.4) for 3 days were concentrated into 10 mL HEPES buffer (20 mM HEPES–KOH, pH 7.8) or modified BG-11 medium to A730 = 20 in a GC vial. The vial was sealed using butyl rubber, and N2 gas was introduced using syringes for 1 h to produce anaerobic conditions. After removing the syringes, the vial was wrapped with aluminum foil and shaken at 30°C. Cell cultures were then centrifuged at 5800 × g for 2 min, the supernatant was filtrated, and 1 mL supernatant was freeze-dried for 1 day. The dried sample was used for high-performance liquid chromatography analysis. erformance liquid chromatography analysis.
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