Bacterial strains, media, and culture conditions

Strains used in this study are listed in Table 1. E. coli DH5α was used for cloning experiments and (−)-α-bisabolol production. BL21(DE3) and MG1655 strains were used to compare effects of different host strains on the production and toxicity of (−)-α-bisabolol. E. coli strains were grown in Luria Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl) for cloning experiments and (−)-α-bisabolol production. To produce (−)-α-bisabolol in E. coli, we used various media at 30 °C and 200 rpm; terrific broth (TB) medium (12 g/L enzymatic casein digest, 24 g/L yeast extract, 9.4 g/L K₂HPO₄, and 2.2 g/L KH₂PO₄), 2xYT medium (16 g/L tryptone, 10 g/L yeast extract, and 5 g/L NaCl), and M9 minimal medium (6.78 g/L Na₂HPO₄, 3 g/L KH₂PO₄, 0.5 g/L NaCl, 1 g/L NH₄Cl, 0.241 g/L MgSO₄, 0.0111 g/L CaCl₂, and 0.1 g/L thiamine). M9 minimal medium was supplemented with 4 g/L of glucose. Ampicillin (100 μg/mL), chloramphenicol (34 μg/mL), or isopropyl β-d-1-thiogalactopyranoside (IPTG) as required.

List of strains, plasmids, and primers used in this study

Overlap region for Gibson Assembly is underlined

^aPrimer sequences are indicated in the 5′–3′ direction

n-Dodecane was used as an overlay to prevent the loss of volatiles. Furthermore, n-dodecane was used to solubilize and extract (−)-α-bisabolol, which is toxic to E. coli cell growth in high concentrations, from the culture media during cultivation. MVA was prepared from mevalonolactone (Sigma Aldrich) as previously described [16]. Cell growth was monitored by measuring the optical density at a wavelength of 600 nm (OD₆₀₀) with a spectrophotometer (Ultrospec 8000, GE Healthcare, Uppsala, Sweden). The inhibitory effect of (−)-α-bisabolol on the growth of E. coli strains was investigated in LB liquid medium supplemented with various concentrations of (−)-α-bisabolol. Diluted samples from cultures during the stationary phase were plated on LB solid medium and the colony-forming units (CFUs) were determined after overnight incubation at 30 °C.