Y per yeast protein extraction reagent kit

For enzymatic assays, 30 mL of bioreactor culture was harvested by centrifugation (5 min, 7745 ×g) after 17 h of cultivation in the presence of IPTG. CFE were made according to the Y-PER Yeast Protein Extraction Reagent kit instructions (Thermo Scientific). Protein concentrations were determined by using the Total Protein Kit, Micro Lowry, Onishi and Barr Modification (Sigma–Aldrich).
The activity of CadA and Irg1 was measured with cis- and trans-aconitate according to Vuoristo et al. (2015) (link). Besides, the Irg1 activity was measured by using a method adapted from Michelucci et al. (2013) (link) with the following modifications: CFE’s were incubated with 200 mM of cis-aconitate in 25 mM HEPES buffer (pH 7.1) supplied with proteinase inhibitor (cOmplete Protease Inhibitor Cocktail Tablets, Roche) for 50 min at 30°C.
For whole cell assays, 20 mL of bioreactor culture was harvested by centrifugation (5 min, 7745 × g) after 17 h of cultivation in the presence of IPTG. Cells were washed with M9 salts in 100 mM MOPS (pH 7.1) and resuspended in 10 ml of the same medium. Twenty-millimeter of substrate (cis- or trans-aconitate) was added to 2 mL of the cell suspension and incubated at 30°C and 100 rpm. 1 M HCl was added to terminate the reaction, after which the supernatants were analyzed for itaconate formation by HPLC.

For SDS-PAGE, E. coli BW25113 (DE3) Δpta–ΔldhA containing either pKV-GA, pKV-GAC^opt, pKV-GAC^har, pKV-GAI^opt, or pKV-GAI^har was cultivated for 16 h at 37°C and 200 rpm in 2 mL MM. Subsequently, the overnight cultures were transferred into 20 mL of fresh MM and incubated at 25°C and 200 rpm. When the cultures reached an OD₆₀₀ of approximately 0.4, they were induced with 0.5 mM IPTG and incubated overnight at 25°C and 200 rpm.
Sixteen-milliliter of the cultures were used to make cell free extracts (CFE) according to the Y-PER Yeast Protein Extraction Reagent kit instructions (Thermo Scientific). Four-milliliter of the cultures were harvested by centrifugation (2 min, 14000 rpm), resuspended in a small volume of water, and loaded on pre-casted Criterion XT SDS-PAGE gels (Bio-Rad) together with the CFE’s according to manufacturer’s instructions (Criterion^TM Cell, Bio-Rad). The proteins were stained by Bio-Safe Coomassie staining (Bio-Rad). Protein sizes were determined by using Precision Plus Protein^TM All Blue Standards ladder (Bio-Rad).