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.