Supplemental Information 1: Expression temperatures and screening conditions used for
respective enzymes
Enzyme
Assay Buffer
SeKARI
Expression
Substrate
Temperature
20 °C
(S)-2-acetolactate
AtGR1
25 °C
Sodium glyoxylate
100 mM HEPES, pH
7.8
ScADH6
20 °C
Trans-cinnamaldehyde 50 mM sodium
phosphate, pH 7.0
LlAdhA
20 °C
Isobutyraldehyde
50 mM sodium
phosphate, pH 7.0
DmAdhA
20 °C
Isobutyraldehyde
50 mM sodium
phosphate, pH 7.0
KpDhaT
20 °C
Isobutyraldehyde
50 mM sodium
phosphate, pH 7.0
EcFucO
20 °C
Furfural
50 mM sodium
phosphate, pH 7.0
TeXR
20 °C
D-xylose
50 mM potassium
phosphate, pH 7.0
LsNOX
20 °C
Oxygen gas
50 mM sodium
phosphate, pH 7.0
50 mM sodium
phosphate, pH 7.0
Supplemental Information 2: Brief discussion of the enzymes studied in this work.







Saccharomyces cerevisiae cinnamyl alcohol dehydrogenase (ScADH6) is used by
yeast for the detoxification of aromatic aldehydes (Larroy et al. 2002).
Arabidopsis thaliana glyoxylate reductase (AtGR1) is involved in the glyoxylateglycolate shuttle for the regulation of photosynthesis (G. J. Hoover et al. 2007).
Lactococcus lactis and Drosophila melanogaster alcohol dehydrogenases
(LlAdhA and DmADH) are promiscuous alcohol dehydrogenases previously
investigated for use in microbial isobutanol production (Liu et al. 2012).
Klebsiella pneumoniae propanediol dehydrogenase (KpDhaT) is one of the main
enzymes for the metabolic pathway in K. pneumoniae that enables the species to
metabolize glycerol as a sole source of carbon and energy by reduction of 3hydroxypropanal to propane-1,3-diol (Marcal et al. 2009).
Escherichia coli lactaldehyde reductase (EcFucO) catalyzes the inter-conversion
between L-lactaldehyde and L-1,2-propanediol during the anaerobic dissimilation
of fucose (Cocks et al. 1974) and aerobic growth on L-1,2-propanediol (Chen et
al. 1989; Wang et al. 2011). It also reduces furfural, the dehydration product of
xylose, an important fermentation inhibitor in sugar syrups derived from woody
biomass to the less toxic furfuryl alcohol (Wang et al. 2011).
Xylose reductase is the first enzyme of xylose metabolism in fungi; the enzyme
from Talaromyces emersonii (TeXR) is a highly active and thermostable member
of this family (Fernandes et al. 2009).
NADH oxidase from Lactobacillus sanfranciscensis (LsNOX) is an important
enzyme in maintaining redox balance (Jansch et al. 2011) and has been used for
NAD(P)+ regeneration (Petschacher et al. 2014).
relative activity normalized to
optimum [%]
Supplemental Information 3: pH-activity profiles and thermostability of indicated
enzymes. T50 is defined as the temperature at which 50% of the initial activity is retained
after 10 min incubation.
DmADH WT
100
80
50 mM Citrate buffer
60
50 mM Sodium phosphate
buffer
40
50 mM TRIS-HCl buffer
20
50 mM Carbonatebicarbonate buffer
0
2
3
4
5
6
7
8
9
10
11
relative activity normalized to
optimum [%]
pH
DmADH V108I
100
80
50 mM Citrate buffer
60
50 mM Sodium phosphate
buffer
40
50 mM TRIS-HCl buffer
20
50 mM Carbonatebicarbonate buffer
0
2
3
4
5
6
7
pH
Enzyme
DmADH WT
T50 (°C)
46.6 ± 0.6
DmADH V108I
48.8 ± 0.8
EcFucO WT
44.7 ± 0.7
EcFucO M185A
45.3 ± 4.0
EcFucO M185C
40.8 ± 2.9
8
9
10
11
Supplemental Information 4: Kinetic parameters of characterized enzymes. Numbers in
parentheses refer to α, the Hill constant.
Mutation
kcat
(min-1)
NADH
NADPH
NADH
KM
(μM)
NADPH
Substrate
AtGR1
Wild Type
7.3 ± 0.9
27 ± 3.9
76 ± 24
20 ± 6
690 ± 160
AtGR1
C68E
2.7 ± 0.2
13 ± 0.8
66 ± 29
10 ± 3
60 ± 11
AtGR1
C68R
13 ± 3.1
7.3 ± 1.8
53 ± 11
24 ± 9
120 ± 46
ScADH6
Wild Type
1300 ± 370
16000 ± 2300
130 ± 51
140 ± 15
170 ± 26
ScADH6
T255K
3200 ± 87
9000 ± 1100
240 ± 13
37 ± 21
56 ± 4
DmADH
Wild Type
7.8 ± 1.6
-
57 ± 6
-
130 ± 33
DmADH
V108I
9.2 ± 3.4
-
-
100 ± 24
EcFucO
Wild Type
1.8 ± 0.2
-
-
1400 ± 130
EcFucO
M185A
6.2 ± 2.0
-
-
390 ± 87
EcFucO
M185C
6.5 ± 1.8
-
-
910 ± 140
LsNOX
Wild Type
1200 ± 94
890 ± 170
42 ± 6
70 ± 8
(2.9 ± 0.8)
39 ± 4
(2.7 ± 0.6)
55 ± 2
(3.0 ± 0.3)
85 ± 12
73 ± 60
-
LsNOX
I122V
4000 ± 240
1400 ± 280
20 ± 9.3
110 ± 64
-
LsNOX
I155L
2700 ± 520
3500 ± 360
100 ± 80
32 ± 8.4
-
LsNOX
I243M
I122VI155L
I22VI243M
I155LI243M
I122VI155LI243M
2800 ± 370
3100 ± 120
150 ± 175
56 ± 48
-
2700 ± 490
5000 ± 540
70 ± 12
120 ± 30
-
5500 ± 290
4500 ± 49
59 ± 22
31 ± 21
-
2500 ± 290
2400 ± 93
83 ± 7.6
120 ± 24
-
7900 ± 520
11000 ± 360
93 ± 96
140 ± 300
-
LsNOX
LsNOX
LsNOX
LsNOX
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