Figure 3: Construction and characterization of P.
taiwanensis VLB120 pSEVA_CL_1 (A, B, C, F) and pSEVA_CL_2 (A, D, E,
F). (A) Graphical representation of expression units in the plasmids
pSEVA_CL_1 and pSEVA_CL_2. (B) and (D) Time courses for the
production of cyclohexanol, ε-CL, and 6HA and for whole-cell activities
for the total product (sum of ε-CL and 6HA) formation in resting cell
bioconversions. Cells were cultivated as described in the legend of
Figure 2 and induced for 4 h. Resting cell bioconversions were performed
with a biomass concentration of
0.5 gCDW L-1 in 10 mL KPi-g buffer and
started by adding 10 µL of pure cyclohexane (180 µM dissolved in the
aqueous phase, 9.2 mM in total concerning the aqueous phase volume). (C)
and (E) SDS-PAGE analyses of P. taiwanensis VLB120 containing
pSEVA_CL_1 and pSEVA_CL_2, respectively, showing bands for Cyp (47.4
kDa), CDH (26.5 kDa), and CHMO (58.8 kDa) after different times of
induction and compared with cells containing an empty vector. (F)
Resting cell bioconversions to study cascade inhibition by cyclohexanol.
Varying cyclohexanol concentrations were applied in 1 mL KPi-g buffer
with a cell concentration of
0.25 gCDW L-1. The graphs depict
cyclohexanone and ε-CL concentrations as well as the whole-cell activity
(ε-CL formation) for an assay time of 10 min. Graphs represent average
values and standard deviations of two independent biological replicates.