Selleckchem Lazertinib Strain Description Reference MG1655 wild type Coli Genetic Stock Center MG1655 ΔarcA ArcA knockout strain This study MG1655 ΔiclR Foretinib IclR knockout strain This study MG1655 ΔarcAΔiclR ArcA-IclR double knockout strain This study BL21 (DE3) wild type Coli Genetic Stock Center Media Luria Broth (LB) medium consisted of 10 g.L -1 tryptone peptone (Difco, Belgium), 5 g.L -1 yeast extract (Difco) and 10 g.L -1 sodium chloride. Shake flask medium (S) contained 2 g.L -1 NH4Cl, 5 g.L -1 (NH4)2SO4, 2.993 g.L -1 KH2PO4, 7.315 g.L -1 K2HPO4, 8.372 g. L -1 MOPS, 0.5 g. L -1 NaCl, 0.5 g.L -1 MgSO4 · 7 H2O, 16.5 g.L -1 glucose · H2O, 1 mL.L -1 trace element solution and 100

μL.L -1 molybdate solution. The medium was set to a pH of 7 with 1 M KH2PO4. The minimal medium during fermentations (M1) in a benchtop bioreactor contained 6.75 g.L -1 NH4Cl, 1.25 g.L -1 (NH4)2SO4, 1.15 g.L -1 KH2PO4, 0.5 g.L -1 NaCl, 0.5 g.L -1 MgSO4

· 7 H2O, 16.5 g.L -1 glucose · H2O, 1 mL.L -1 trace element solution and 100 μL.L -1 molybdate solution. In 13C-flux analysis experiments, minimal medium for minireactors (M2) was used. This medium contained 1 g.L PERK modulator inhibitor -1 NH4Cl, 1 g.L -1 (NH4)2SO4, 3 g.L -1 KH2PO4, 7.315 g.L -1 Na2HPO4, 0.5 g.L -1 NaCl, 0.5 g.L -1 MgSO4 · 7 H2O, 3 g.L -1 glucose, 1 mL.L -1 trace element solution, 100 μL.L -1 molybdate solution. The glucose used in this M2 medium was added as a mixture of 20% U-13C glucose (99% purity) and 80%

naturally labeled glucose or as a mixture of 50% 1-13C glucose (99% purity) and 50% naturally labeled glucose depending on the flux ratios that needed to be identified. Trace element solution consisted of 3.6 g.L -1 FeCl2 · 4 H2O, 5 g.L -1 CaCl2 · 2 H2O, 1.3 g.L -1 MnCl2 · 2 H2O, 0.38 g.L -1 CuCl2 · 2 H2O, 0.5 g.L -1 CoCl2 · 6 H2O, 0.94 g.L -1 ZnCl2, 0.0311 g.L -1 H3BO4, 0.4 g.L -1 Na2EDTA · 2 H2O, 42 g.L -1SeO2 and 1.01 g.L -1 thiamine · HCl. The molybdate solution contained 0.967 g.L -1 Na2MoO4 · 2 H2O. If not specifically mentioned, all chemicals were second purchased at Sigma, Belgium. Cultivation conditions To determine substrate uptake and product secretion rates, enzyme activities, and glycogen and trehalose contents, cells were cultivated in 2L benchtop bioreactors, since higher volume vessels improve accuracy of the measurements. However, in order to map the metabolic fluxes in the cell, expensive 13C-labeled substrates are necessary and therefore alternative miniscale reactors were chosen as the method of cultivation. Earlier studies have shown that similar growth conditions were achieved in the benchtop and miniscale reactor setups [69, 70]. For experiments in bioreactors, a preculture in a test tube filled with 5 mL LB medium was inoculated with a single colony from a LB-plate and incubated during 8 hours at 37°C on an orbital shaker at 200 rpm.