Two strains of E. coli BL21(DE3) containing plasmids pETBCS-ACAT(Eco) and pETBCS-HMGS(Sce) were grown in a 1-liter fermenter at 37°C (250 rpm) at constant pH 6.95 in rich medium [glucose (18 g liter−1), yeast extract (16 g liter−1), tryptone (10 g liter−1), NaCl (5 g liter−1), potassium phosphate dibasic (K2HPO4; 7 g liter−1), and potassium phosphate monobasic (KH2PO4; 3 g liter−1)] containing carbenicillin (100 μg ml−1; IBI Scientific, Peosta, IA). After induction with 0.1 mM isopropyl-β-d-thiogalactopyranoside at OD600 (optical density at 600 nm) of 0.6, growth continued for 4 hours at 30°C until harvest by centrifugation. See (38) for plasmid sequences and expression characterization. E. coli cells were lysed, and crude lysates were generated using methods previously described (38).

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