Cell culture and media
Seed cultures were started in 14 mL round-bottom falcon tubes, which were incubated at 37 °C/250 rpm overnight. Cell density was measured by Varioskan LUX multimode microplate reader (Thermo Fisher Scientific) at the wavelength of 600 nm. The overnight grown seed culture was used to inoculate (1.5%, v/v) 25 mL of K3 medium (Zhou et al., 2015) in 250 mL shake flasks. Subsequent cell culture processes were conducted at 30 °C/250 rpm. The K3 medium composition was as follows (working concentration): 10 g/L glucose, 10 g/L tryptone, 5 g/L yeast extract, 13.3 g/L KH2PO4, 4 g/L (NH4)2HPO4, 0.0084 g/L EDTA, 0.0025 g/L CoCl2, 0.015 g/L MnCl2, 0.0015 g/L CuCl2, 0.003 g/L H3BO3, 0.0025 g/L Na2MoO4, 0.008 g/L Zn (CH3COO)2, 0.06 g/L Fe(III) citrate and 1.3 g/L MgSO4. Antibiotics (100 µg/mL ampicillin and/or 50 µg/mL spectinomycin) were added according to Table 1 , and pH was adjusted to 7 using 400 g/L sodium hydroxide solution. Inducers were added when cell density (OD600) reached ~0.5 to trigger expression. 0.01 mM IPTG, 4 g/L L-arabinose and 0.03 mM cumic acid were added at this stage when gene expression was controlled by PT7, PBADand PcymO, respectively. Cells were collected 6 hours after induction.
The grown cells were centrifuged at 4,000 g for 8 min. Cell pellets were resuspended in 5 mL of fresh K3 medium to a final OD of 20. In the fresh K3 medium, 20 g/L L-arabinose, 10 g/L tryptone and 5 g/L yeast extract were used as carbon sources. 0.05 mM IPTG or 0.03 mM cumic acid was used in the second gene expression stage using PT7 or PcymO. One millilitre of re-suspended cells was transferred to a 14 mL round-bottom falcon tube. A total of 2 g/L isoprenol and prenol at a molar ratio of 3:1 was supplemented as substrate. A 15% v/v hexadecane layer was used under same conditions (as specified in Results and Discussion ). The culture was incubated at 30 °C/250 rpm for 24 h. For the biotransformation of nerol, perillyl alcohol and farnesol, 2 g/L of nerol, perillyl alcohol and farnesol were added instead of isoprenol and prenol.