To prepare the pre-culture, the engineered E. coli was cultured in 100 mL of TB medium at 30 °C and 180 rpm. The batch fermentation was performed by inoculating 1% (v/v) of pre-culture into 400 mL TB medium supplemented with 10 g/L glycerol and overlaid with 80 mL n-dodecane in a 1 L fermenter. The cultures were incubated at 30 °C and air-aerated with a flow rate of 1 volume of air per volume of medium per min (vvm), the agitation speed remained fixed at 200 rpm in all experiments. Glycerol was used as a carbon source at a concentration of 10 g/L. The pH was balanced to pH 7.0 by the addition of 1 M HCl and 25% NH4OH solutions. n-Dodecane was overlaid up to 20% (v/v) of culture volume to continuously extract (−)-α-bisabolol from the culture broth during fermentation. The fed-batch fermentation began as a batch operation and was switched to the fed-batch mode at 24 h, when OD600 reached 3.0. At the beginning of fed-batch fermentation, TB medium was supplemented with 10 g/L glycerol, 0.1% (v/v) trace element solution (27 g/L FeCl3·6H2O, 2 g/L ZnCl2·4H2O, 2 g/L CoCl2·6H2O, 2 g/L Na2MoO4·2H2O, 1 g/L CaCl2·2H2O, 1.3 g/L CuCl2·6H2O, and 0.5 g/L H3BO3) and 1% (v/v) vitamin solution (Sigma Aldrich, Cat. No. M6895). Glycerol was then fed at a rate of 0.01 L/h and 10% (v/v) canola oil (instead of 20% (v/v) n-dodecane) was used to overlay 30 L TB medium in a 50 L fermenter.