Supplementary Data
Supplementary Fig. 1 GC/MS spectrum of the silylated reaction product obtained from the
conversion of a-linolenic acid to reaction product by permeabilized S. nitritireducens cells. The
silylated reaction product was a 10-hydroxy-12,15(Z,Z)-octadecadienoic acid.
10-Hydroxy-12,15(Z,Z)
-octadecadienoic acid (g l-1)
12
10
8
6
4
2
ro
N
on
f
BC
Fr
ze
n
c
oz ells
en
ce
l
Sp ls
an
2
Sp 0
an
Tw 80
ee
n
Tw 20
ee
n
Tw 40
ee
n
Tr
80
ito
n
X10
0
C
TA
B
0
Permeabilizing agents
Supplementary Fig. 2 Effect of detergents on the permeabilization of S. nitritireducens for the
production of 10-hydroxy-12,15(Z,Z)-octadecaidenoic acid from α-linolenic acid. 10% (v/v)
(filled bar), 20% (v/v) (empty bar), 0.05% (v/v) (grey bar), or 0.1% (v/v) (diagonal line–
grey bar) detergent. Nonfrozen cells indicate non-permeabilized cells. The reactions were
performed in 50 mM citrate/phosphate buffer (pH 7.0), 50 g permeabilized cells l−1, 22.5 g αlinolenic acid l−1, and 0.02% (v/v) Tween 80 at 35°C for 2 h in anaerobic atmosphere. Data
represent the means of three separate experiments and error bars represent the standard
deviation.
a
10-Hydroxy-12,15(Z,Z)
-octadecadienoic acid (g l-1)
12
10
8
6
4
2
0
nc
ze
fro
Un
ell
s
nc
e
oz
Fr
ell
s
l
C
Na
TA
ED
Cl 2
Mg
DM
SA
Permeabilizing agents
b
10-Hydroxy-12,15(Z,Z)
-octadecadienoic acid (g l-1)
12
10
8
6
4
2
0
0.0
0.5
1.0
1.5
2.0
2.5
NaCl concentration (M)
Supplementary Fig. 3 Effect of salt or chemical treatment on the permeabilization of S.
nitritireducens for the production of 10-hydroxy-12,15(Z,Z)-octadecaidenoic acid from αlinolenic acid. a Effect of salt or chemical type. 0.1 M (filled bar) or 0.5 M (empty bar) salt
and 2 mM (grey bar) or 5 mM (diagonal line–grey bar) chemical. Nonfrozen cells indicate
non-permeabilized cells. b Effect of NaCl concentration for cell permeabilization. The
reactions were performed in 50 mM citrate/phosphate (pH 7.0) buffer containing 50 g
permeabilized cells l−1, 22.5 g α-linolenic acid l−1, and 0.02% (v/v) Tween 80 at 35°C for 2 h
in anaerobic atmosphere. Data represent the means of three separate experiments and error
bars represent the standard deviations.
10-Hydroxy-12,15(Z,Z)
-octadecadienoic acid (g l-1)
16
12
8
4
0
Co
nt
ro
l
o
(f r
ze
n)
Na
Cl
h
et
an
ol
M
C
Na
l+
M
h
et
an
ol
Supplementary Fig. 4 Effects of NaCl and/or methanol treatments on the permeabilization of
S. nitritireducensfor the conversion of α-linolenic acid to 10-hydroxy-12,15(Z,Z)octadecadienoic acid. The concentrations of NaCl and methanol were 0.5 M and 1.25% (v/v),
respectively.The reactions were performed in 50 mM citrate/phosphate buffer (pH 7.0)
containing 50 g permeabilized cells l−1, 22.5 g α-linolenic acid l−1, and 0.02% (v/v) Tween 80
at 35°C for 2 h in anaerobic atmosphere. Data represent the means of three separate
experiments and error bars represent the standard deviation
a
120
Relative activity (%)
100
80
60
40
20
0
20
25
30
35
40
45
50
8.0
8.5
Temperature (oC)
b
120
Relative activity (%)
100
80
60
40
20
0
5.5
6.0
6.5
7.0
7.5
pH
Supplementary Fig. 5 Effects of temperature and pH on the conversion of α-linolenic acid to
10-hydroxy-12,15(Z,Z)-octadecadienoic acid by S. nitritireducens. a Effect of temperature. The
reactions were performed in 50 mM citrate/phosphate buffer (pH 7.0) containing 0.7 g cells
l−1,0.14 g α-linolenic acid l−1 in 0.02% (v/v) Tween 80 for 15 min in anaerobic atmosphere. At
the relative 100%, 22.8 mg 10-hydroxy-12,15(Z,Z)-octadecadienoic l−1 was produced. b Effect
of pH.The reactions were performed in 50 mM citrate/phosphate (pH 6.0−8.0) buffer
containing 0.7 g cells l−1, 0.14 g α-linolenic acid l−1, and 0.02% (v/v) Tween 80 at 35°C for 10
min in anaerobic atmosphere. At 100% relative activity, 24.5 mg 10-hydroxy-12,15(Z,Z)octadecadienoic l−1 was produced. Data represent the means of three separate experiments and
error bars represent standard deviation.
16
1 0 - H y d r o x y - 1 2 ,1 5 ( Z ,Z )
-1
- o c t a d e c a d ie n o ic a c id ( g l )
14
12
10
8
6
4
2
0
C
on
tro
l
Tw
ee
n
20
Tw
ee
n
40
Tw
ee
n
80
S
pa
n
20
S
pa
n
80
T
o
r it
n
X
-1
00
D e te rg e n ts
Supplementary Fig. 6 Effect of detergent on the conversion of α-linolenic acid to 10-hydroxy12,15(Z,Z)-octadecadienoic acid production by permeabilized cells of S. nitritireducens. The
reactions were performed in 50 mM citrate/phosphate buffer (pH 7.0) containing 50 g
permeabilized cells l−1, 22.5 g α-linolenic acid l−1, and 0.025% (v/v) (filled bar), or 0.05% (v/v)
(open bar) detergent at 35°C for 2 h in anaerobic atmosphere. Data represent the means of three
separate experiments, and error bars represent the standard deviations.