Supporting information
Figure S1 A): CO difference spectrum of P. taiwanensis VLB120 pCom10_chx growing in LB
medium. Cultures were induced with DCPK at OD450nm 0.5. Samples were taken after 4h, 6h and
8h after induction. B) SDS PAGE of samples taken from the culture after induction at 0 – 4h.
CYP 47.4kDa and the FAD-ferredoxin reductase (FAD-R) 42.7kDa are indicated. Marker:
SM0661 from Life Science, Germany.
A maximal intracellular CYP concentration of 80 nmol gCDW-1 was detected after 4h of induction
in the growing culture. The protein bands of the cytochrome P450 enzyme (47 kDa) and of the
FAD-ferredoxin reductase (42 kDa) were clearly visualized by SDS-PAGE(Figure S1B). No
band corresponding to a ferredoxin (12 kDa) could be detected, most probably due to its small
size. Functional expression of CYP_CHX genes in P. taiwanensis VLB120 pCom10_chx was
successful and enabled characterizing the biocatalytic performance of these enzymes.
Material and methods: All aqueous solutions were prepared with deionized water obtained
through a Seralpur Pro 90 CN (Seral, Ransbach-Baumbach, Germany). The biomass
concentration was monitored by measuring optical density at 450 nm (OD450), using a
photometer (Libra S11, Biochrom Ltd., Cambridge, UK). The correlation factor between OD450
and cell dry weight (CDW) concentration was deduced from Blank et al., 2008, where 1 OD450
corresponds to 0.166, 0.233 and 0.186 for E.coli, P.putida and P. taiwanensis, respectively.
Acidovorax sp. CHX100 was grown as described by Salamanca et al., 2015. Protein
concentration was measured by the method given by Bradford (Bradford 1976), using the quick
start Bradford dye (Bio-Rad, Munich, Germany), with a standard curve prepared using bovine
serum albumin.
Genome sequencing and annotation: Genomic DNA was isolated from Acidovorax CHX100
using a peqGOLD Bacterial DNA Kit (Peqlab, Erlangen, Germany). Genome sequencing and
automated annotation was carried out by CeBiTec and the genome was integrated into the
platform GenDB (Meyer et al. 2003).
Visualization of cell permeabilization: Propidium iodide dye PI (Invitrogen, OR) was used to
stain permeabilized cells. P. taiwanensis VLB120 pCom10_chx cells were induced and
harvested after 4h, as described above. Subsequently, cells at 0.5 gCDW L-1 were incubated in 15
mL Pyrex tubes and in the presence of 100 mM cyclohexane in 1 mL 50 mM Kpi buffer pH 7.4
containing 1% citrate. After 10 min, cells were centrifuged at 4°C, 4595 g, 10 minutes (Thermo
Electron Corporation, Langenselbold, Germany) and resuspended in 1 mL 50 mM Kpi buffer pH
7.4, supplemented with 1% citrate. Propidium iodide, 1 µL, was added and after 20 minutes of
incubation, the cells were washed in 50 mM Kpi buffer pH 7.4. Permeabilized cells were
inspected at 535 nm excitation and 617 nm emission using a Zeiss LSM5 Pascal confocal laser
scanning microscope (CLSM; Carl Zeiss, Jena Geramny) as described by Halan and co-workers
(Halan et al. 2011).
References:
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities
of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72(12):248-254.
Meyer F, Goesmann A, McHardy AC, Bartels D, Bekel T, Clausen J, Kalinowski J, Linke B,
Rupp O, Giegerich R and others. 2003. GenDB—an open source genome annotation
system for prokaryote genomes. Nucleic Acids Research 31(8):2187-2195.
Halan B, Schmid A, Buehler K. 2011. Real-Time Solvent Tolerance Analysis of Pseudomonas
sp. Strain VLB120ΔC Catalytic Biofilms. Applied and Environmental Microbiology
77(5):1563-1571.