Fig. 4: Dynamic control of glycolysis with CRISPRi.

a CRISPRi was used to knock down pfkB. The sgRNA targeting the promoter region of pfkB or without targeting sequence was expressed from a constitutive promoter. dcas9 was expressed from the aTc-inducible P_tet. Strain 5, 6, and 7 (Table 1) were grown in M9P media with 40 g L⁻¹ glucose at 37 °C to OD₆₀₀ ~ 1, after which 1 mM IPTG and 100 ng mL⁻¹ aTc were added and cells were grown at 30 ˚C for 24 h. b High cell density D-psicose production. Strain 7 cultures was grown in M9P media with 40 g L⁻¹ glucose at 37 °C until an OD₆₀₀ of ~1, after which they were induced with 1 mM IPTG and 100 ng mL⁻¹ aTc and grown for another 30 min. Cultures were then spun down and resuspended in M9P media with 40 g L⁻¹ glucose, 1 mM IPTG, and 100 ng mL⁻¹ aTC to an OD₆₀₀ of ~10 and grown at 30 ˚C for 8 h. Error bars indicate s.d. (n = 3 biological replicates). c HPLC chromatogram of the high cell density experiment with Strain 7 cultured for 24 h. The elution of D-glucose occurs at ~3.36 min, and the elution of D-psicose occurs at ~5.45 min.