SI APPENDIX
Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into
fuels and chemical commodities.
M. Kalim Akhtar a, b
, Nicholas J. Turner b, 1
, and Patrik R. Jones a, 1
a Department of Biochemistry and Food Chemistry, University of Turku, Tykistökatu 6A, 6krs, 20520 Turku,
Finland
b School of Chemistry, Manchester Institute of Biotechnology, University of Manchester,131 Princess Street,
Manchester, M1 7DN
1 To whom correspondence should be addressed: Patrik R. Jones, [email protected] & Nicholas J. Turner,
Methods
Strains and plasmids
The genes encoding for M. marinum CAR and B. Subtilis Sfp were synthesized with codon-
optimization for E. coli (Genscript, USA), while tesA, slr1192 and ahr were amplified from
E. coli BL21(DE3) and Synechocystis sp. PCC 6803 genomes using the following PCR
program: 95 °C for 2 min; 40 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for 1 min;
and 72 °C for 20 min. Genetic inserts along with T7-based commercial vectors (Novagen)
were cut with the appropriate restriction enzymes (New England Biolabs) and ligated using
T4 DNA ligase (Fermentas) (Tables S3-S5). Multiple genes were assembled in artificial
operons as described previously (1). Plasmids were used to transform E. coli BL21 (DE3) to
generate the strains listed in Table S6. Table S7 summarizes the gene-products used to
engineer the recombinant pathways.
Protein expression and purification
Overnight LB-grown pre-cultures of BL21(DE3) were used to inoculate 20 ml cultures of
Overnight Express™ Instant TB Medium (Novagen) at 2% (v/v). Cultures were incubated
overnight (18-24 h, 30 oC, 250 rpm). Cells were pelleted and resuspended in a lysis buffer
containing lysozyme (2 mg/ml) and 2% (v/v) hexane, and incubated for 30 min at room
temperature with gentle inversion. The insoluble debris was centrifuged (17,000g, 5 min) and
the supernatant was applied to a microfuge spin column pre-filled with 200 l His-Select®
Nickel Affinity Gel (Sigma Aldrich). The column was washed five times with 0.5 ml 0.1 M
Tris-HCl (pH 7.5, 21 oC) and the recombinant his-tagged protein eluted with 100 l 0.4 M
imidazole, prepared in 50 mM Tris-HCl buffer (pH 7.5, 21 oC). Protein recovery was
estimated using the Bio-Rad Protein Assay (Biorad).
Enzyme characterization
All enzyme reactions were performed in triplicates and monitored at 340 nm for up to 15 min
in 96-well microplates (Tecan M200 Affinity). Control reactions without addition of the
purified enzyme were also included to take into account any background oxidation of
NADPH. For the CAR assay, a 100 μl reaction volume typically contained the following
components: CARhis (0-10 g/ml), 1 mM NADPH, 1 mM ATP, 10 mM MgCl2 and 0.5 mM
fatty acids. For Km and Vmax determinations, the various (co)substrates: fatty acids, NADPH
and ATP were prepared at 11 different concentrations with the concentration range specified
in the figure legends. Based on the initial reaction rates, the apparent Km and Vmax values
were determined using the enzyme kinetics module of SigmaPlot (Systat Software, San Jose,
CA). For the AHR assay, reactions typically contained the following components: YjgBhis (1
or 10 g/ml), 1 mM NADPH and 0.5 mM C4-C12 aldehydes or C4-C12 alcohols. For
qualitative confirmation of aldehyde (in the case of CARhis) and alcohol synthesis (in the case
of YjgBhis), reaction mixtures (500 l) were mixed with an organic solvent (75 l) and the
organic phase analyzed by GC-MS, as described below.
In vivo production of fatty alcohols and alkanes
Strains were cultivated either in Overnight Express™ Instant TB Medium (Novagen) or in a
defined minimal medium containing: M9 salts (Sigma Aldrich); BME vitamins (Sigma
Aldrich); 100 mM potassium phosphate buffer (pH 7.5, 21 oC); 2 mM MgSO4; 0.1 mM
CaCl2; micronutrient mix consisting of 10 nM FeSO4, 3 μM (NH4)6Mo7O24, 0.4 mM boric
acid, 30 μM CoCl2, 15 μM CuSO4, 80 μM MnCl2 and 10 μM ZnSO4; 2% (w/v) glucose;
appropriate antibiotics, 50 g/ml ampicillin and/or 50 g/ml spectinomycin; and 50 M
IPTG. Cultures (2-5ml) were incubated for up to 48 h with shaking at 180 - 200 rpm in 50 ml
sterile Falcon tubes. For total fatty alcohol and alkane quantification, 100 l cell culture was
vigorously mixed with 200 l acetone, microfuged (17,000g, 5 min) and the resulting
supernatant analyzed by GC-MS as described below. Glucose levels were quantified at
340nm, based on the reduction of NAD+ catalyzed by glucose-6-phosphate dehydrogenase.
In vitro conversion of fatty acids to fatty alcohols and alkanes
In vitro alkane synthesis was carried out as described previously (2) with the addition of
CARhis (100 g/ml), 1 mM NADPH, 1 mM ATP and 0.5 mM fatty acid (C4-C16); C14-C16
fatty acid substrates were added as suspensions due to poor solubility. For in vitro fatty
alcohol formation, ADChis was replaced with Ahrhis (10 g/ml) and NADH, N-
phenylmethazonium methosulphate (PMS) & ferrous ammonium sulphate were omitted. All
tubes were incubated in a heat block at 30 oC for up to 4 h without shaking. For analyte
extraction, reaction mixtures were terminated either by the addition of acetone or chloroform.
The organic phase/supernatant was analyzed by GC-MS as described below.
In vitro conversion of fatty acids to fatty alcohols and alkanes
For in vitro alkane synthesis, reactions were performed in 500 l reaction volumes: 50 mM,
Tris-HCl (pH 7.5, 21oC), CARhis (100 g/ml), ADChis (200 g/ml), 1 mM NADPH, 1 mM
ATP, 10 mM MgCl2, 20 M PMS, 1 mM NADH, 20 M ferrous ammonium sulphate and
0.5 mM fatty acid substrates ranging from C4 (butyric acid) to C16 (hexadecanoic acid). The
protocol was modified according to ADC assays described previously (2). For in vitro fatty
alcohol formation, ADChis was replaced with YjgBhis (10 g/ml) and the following
components were omitted: NADH, PMS & ferrous ammonium sulphate. Due to their
hydrophobicity, the C14 and C16 fatty acid substrates were added to the assays as suspensions.
All reactions were performed in a heat block at 30 oC for up to 4 h without shaking, and
terminated either by addition of acetone or chloroform. The supernatant or organic phase was
analyzed by GC-MS as described below. Given that conversion was dependent on two
NAPH-requiring reactions, fatty alcohol formation was also monitored by following the
oxidation of NADPH at 340 nm.
In vitro conversion of TAGs to aldehydes
Reactions were performed in a 1 ml volume containing the following: 50mM Tris-HCl (pH
7.5, 21oC), CARhis (300 g/ml), lipase (100 g/ml), 1 mM NADPH, 1 mM ATP, 10 mM
MgCl2 and 1 mM suspensions or emulsions of the commercially purified TAGs: C8 TAG
(glyceryl trioctanoate) and C12 TAG (glyceryl tridodecanoate). All tubes were incubated in a
heat block at 30 oC for up to 4 h. Reactions were terminated by vigorously mixing with an
equal of chloroform. The lower organic phase was analyzed directly by size-exclusion HPLC
as described below. For reactions containing C12 TAG, samples were concentrated by
evaporation of solvent in a Genevac (30 oC, 10 min) prior to HPLC analysis.
Lipase-mediated in vivo formation of fatty alcohols
A 1 ml preinduced cell culture (OD ~10) of the strain, PC-Ahrhis, encoding for the CARhis and
Ahrhis enzymes was centrifuged (7000g, 10 min). The cell pellet was resuspended in an equal
volume of 100 mM potassium phosphate buffer (pH 7.5, 21oC) together with three distinct
sources of TAGs: (i) harvested cells of the cell wall-less Chlamydomonas reinhardtii strain
cc406, (ii) palm oil (Afroase) and (iii) coconut oil (Biona Organic). Cells were supplemented
with 100mM glucose and incubated at 30 oC with 100 l samples taken at 30 min, 1 h, 2 h
and 5 h. Fatty alcohols were analyzed by GC-MS as described below.
Hydrocarbon analysis by GC-MS
Metabolite analysis was performed with an Agilent 7890A gas chromatograph equipped with
a 5975 mass spectrometry detector. All samples (1 l) were analyzed in splitless injection
mode with the inlet temperature set at 300 °C and passed through an Equity-1 fused silica
capillary column (Supelco) (30 m x 320 m x 1 m) at a flow rate of 1.9 ml/min, using
helium as the carrier gas. The oven was initially held at 45 °C for 2.5 min and ramped up to
300 °C at a rate of 20 °C/min. Data was acquired within the 25–350 m/z range. For analyte
identification, fragmentation patterns and retention times of the analytes were compared with
the NIST mass spectral library and commercially available standards of fatty alcohols, acids
and alkanes. A standard curve for quantification was prepared with commercial preparations
of fatty alcohols and alkanes.
Hydrocarbon analysis by HPLC-size exclusion chromatography
For separation and detection of the TAGs, fatty acids and aldehydes, size-exclusion HPLC
analysis was carried out using an Agilent 1200 Series HPLC module coupled to an Agilent
refractive index detector. For analyte separation, samples (10 µl) were injected and passed
through 3 size-exclusion columns (300 x 7.8 mm x 5 or 10 µm; Phenomenex) of different
pore sizes; 50, 100 and 500 Å; and connected in series, at a flow rate 0.8 ml/min. The
chromatography was performed in isocratic mode using super-purity grade chloroform
(Romil) as the mobile phase with column and refraction index detector temperatures set at 40
°C. The retention times of the analytes were confirmed using commercial standards.
Stoichiometric evaluation
The stoichiometric conversion efficiency (percent conversion, mole product per mole
glucose) was calculated by two independent methods, as described in detail below:
A maximum potential yield of C12 fatty alcohol per glucose is 0.33 and 0.288, respectively,
without biomass formation. All stoichiometric evaluations were made in minimal media.
Where an error is given in the text it represents the standard error (SEM, n=2-4).
The experimentally measured distribution of fatty alcohols in all TPC-Ahr strains was highly
similar regardless of cultivation conditions, as summarized below:
The molar demand for glucose that is required for each of the main fatty alcohols, identified
in the TPC-Ahr strain, was estimated based on the assumption that approximately 3 mole of
CO2 is released per mole of catabolized glucose through substrate oxidation. The molar
demand for glucose was related to the distribution of fatty alcohols that was repeatedly
observed; this is summarized in the Table below:
A B C D
Average Stoichiometric Carbon distribution Stoichiometric yield
distribution (%) glucose fatty alcohol fatty alcohol
required (mM) per 100 mM glucose (%) per 100 mM glucose (%)
C12:1ol 8.7 26.1 7.8 2.6
C12ol 41.3 124 37.1 12.4
C14:1ol 9.4 33 9.9 2.8
C14ol 23.9 83.5 25 7.1
C16:1ol 12.2 48.8 14.6 3.7
C16ol 2.2 8.9 2.7 0.7
C18:1ol 1.7 7.5 2.3 0.5
C18ol 0.5 2.4 0.7 0.2
Biomass -- -- 0 --
Sum 100 335.5 100 29.9
C12:1ol C12ol C14:1ol C14ol C16:1ol C16ol C18:1ol C18ol
Complex media TPC-Ahrhis 8 38 13 22 14 1 2 0
TPC-Ahrhis 9 37 13 22 14 2 3 0
Minimal media TPC2 8 41 8 26 12 3 2 0
TPC2 8 40 9 26 12 3 2 0
TPC-Ahr 8 47 7 24 10 2 1 0
TPC-Ahr 8 47 7 23 10 3 1 0
TPC-Ahrhis 10 49 8 21 10 2 1 0
TPC-Ahrhis 10 48 8 21 10 2 1 0
TPC-Ahrhis 10 36 11 25 12 2 1 2
TPC-Ahrhis 8 30 11 27 17 3 1 3
Average 8.7 41.3 9.4 23.9 12.2 2.2 1.7 0.5
Standard error 0.3 2.1 0.8 0.7 0.8 0.2 0.2 0.4
The analysis suggests that the maximum potential molar yield of fatty alcohols with the TPC-
Ahr strains is 30% (mole of fatty alcohols per mole of glucose), however, under these
conditions there would be no biomass formation. Since fatty alcohol production was studied
under exponential batch growth conditions, the same analysis was also repeated with the
assumption that 25% of all glucose is used for biomass-formation. The maximum yield of
fatty alcohols is then 22.4%.
An alternative approach to estimate the maximum potential yield of fatty alcohols is to carry
out the so-called 'flux balance analysis'. The maximum potential molar yield was calculated
for C12 alcohol with a stoichiometric model of iJR904 E. coli to which the TPC pathway was
added. Within the E. coli iJR904 stoichiometric model (3), the following three reactions were
added:
TES: C12-ACP + H2O = C12 fatty acid + ACP
CAR: C12 fatty acid + NADPH + H+ + ATP = C12 aldehyde + NADP
+ + AMP + PPi
AHR: C12 aldehyde + NADPH → C12 alcohol + NADP+
The iJR904 (3) and iAF1260 (4) models do not include any pathways for C14-ACP and C16-
ACP formation. The calculation of the potential conversion rate for all fatty alcohols is
therefore estimated according to C12 fatty alcohol synthesis even though the C14 and C16
alcohols are produced with and without mono-unsaturation.
After loading the iJR904_GlcMM model into COBRA Toolbox 1.3.3 (5), the three reactions
are added accordingly:
modelTes = addReaction(model,'TesA',{'ddcaACP[c]','h2o[c]','dodecanoate[c]','ACP[c]'},[-1
-1 1 1], false);
modelCAR =
addReaction(modelTes,'CAR',{'dodecanoate[c]','nadph[c]','h[c]','atp[c]','dodecanal[c]','nadp[c
]','amp[c]','ppi[c]'},[-1 -1 -1 -1 1 1 1 1], false);
modelAHR =
addReaction(modelCAR,'AHR',{'dodecanal[c]','nadph[c]','dodecanol[c]','nadp[c]'},[-1 -1 1 1],
false);
Then, an exchange reaction is also added for C12 alcohol:
modelTPC = addReaction(modelAHR,'dodecanolEX',{'dodecanol[c]'},[-1], true);
In order to obtain the maximum potential conversion rate, we changed the objective function
from biomass to 'dodecanolEX', which represents the rate of accumulation of C12 fatty
alcohol given that C12 fatty alcohol does not excrete from wild-type E. coli cultivated in
minimal media, and optimize, as follows:
modelTPCobjC12 = changeObjective(modelTPC, 'dodecanolEX');
solution = optimizeCbModel(modelTPCobjC12,'max',false,false)
A solution of f: 1.7266 was obtained using glpk solver.
Since the maximum glucose uptake rate is 6 mmol per gram dry weight per h, the maximum
potential molar conversion efficiency is:
1.7266/6 = 0.288 mol C12 fatty alcohol per mole glucose. Under such conditions, there would
be no biomass formation as the robustness analysis shows above.
If the above analysis is carried out also for the cyanobacterial pathway,
AAR: C12-ACP + NADPH → C12 aldehyde + NADP + ACP
with the following reactions added to COBRA:
modelSchirmer =
addReaction(model,'ACR',{'ddcaACP[c]','nadph[c]','dodecanal[c]','ACP[c]','nadp[c]'},[-1 -1 1
1 1], false);
modelAHR2 =
addReaction(modelSchirmer,'AHR',{'dodecanal[c]','nadph[c]','dodecanol[c]','nadp[c]'},[-1 -1
1 1], false);
modelTPC2 = addReaction(modelAHR2,'dodecanolEX',{'dodecanol[c]'},[-1], true);
robustnessAnalysis(modelTPC2, 'dodecanolEX');
we obtain an f-value of 1.7600, 1.9% greater potential yield than the CAR pathway, without
any biomass formation.
In order to provide insight into the relative distribution of fluxes in the optimized models, the
fluxes for key enzymes in central carbon metabolism and the major excretion products were
extracted from the optimized solution. The values are plotted on the next page, with enzyme
names as given for the iJR904 E. coli genome-scale model (3). The main difference between
the two pathways is the additional ATP requirement of the CAR-dependent pathway.
Notably, the simulation is unrealistic as there is no biomass-formation. Still, the below bar
graph suggests that the task of reducing NADP+ is shifted slightly away from the proton
gradient dependent transhydrogenase PntAB (THD2) towards the pentose phosphate pathway
(as shown by the ratio of flux through PGI (phosphoglucoseisomerase) vs. G6PDH2r
(glucose-6-phosphate dehydrogenase, Zwf)). As enhanced flux through the oxidative pentose
phosphate pathway results in increased loss of carbon, in the form of CO2, there is less fatty
alcohol produced.
All estimates of Gibbs free energy changes under standard conditions were obtained using the
eQuilibrator online platform (6).
1 2 3 4 5 6 7 8 9
Fig. S1 SDS-PAGE of purified preparations of CARhis, Ahrhis and ADChis. Proteins were
separated on 12% (w/v) acrylamide gel and stained with Coomassie Blue (7). Lane 1,
molecular-mass markers (in kDa); lane 2, crude fraction of BL21(DE3); lane 3, crude fraction
of PC; lane 4, crude fraction of Ahrhis; lane 5, crude fraction of ADChis; lane 6, his-tag eluate
fraction from BL21(DE3); lane 7, purified CARhis; lane 8, purified crude Ahrhis; and lane 9,
purified ADChis. Purified recombinant proteins are marked with an arrow and their theoretical
molecular weights indicated in brackets.
YjgBhis (36.5kDa)
ADChis (26.2kDa)
23
CARhis (127.8kDa) 175
80
58
46
30
Fig. S2 NADPH specificity of CARhis. Reactions were carried out in 50 mM Tris-HCl (pH
7.5, 21oC) containing purified CARhis (0.25 g/ml), 2 mM benzoic acid, 10 mM MgCl2 and
1mM ATP with either 1 mM NADPH or 1 mM NADH as the source of reductant.
Absorbance was monitored at 340 nm at 30 s intervals over a 10 min period.
0
1
2
3
4
5
6
7
0 2 4 6 8 10
Fo
rmati
on
of
NA
D(P
)+
(m
ol/
min
/mg
CA
Rh
is)
Time (min)
NADPH
NADH
A B
Benzoic acid (mM)
0 1 2 3 4 5
Ra
te (
µm
ol/m
in/m
g C
AR
his
)
0.0
0.5
1.0
1.5
2.0
2.5
NADPH (µM)
0 200 400 600 800 1000
Ra
te (
µm
ol/m
in/m
g C
AR
his
)
0.0
0.5
1.0
1.5
2.0
2.5
C
ATP (mM)
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Ra
te (
µm
ol/m
in/m
g C
AR
his
)
0.0
0.5
1.0
1.5
2.0
2.5
Fig. S3 Kinetic analysis of CARhis in the presence of benzoic acid. Reactions were carried out
in the presence of varying concentrations of (A) benzoic acid (0.2-0.5 mM), (B) NADPH
(250-1000 M) and (C) ATP (0.05-3 mM). The Km and Vmax were determined from non-
linear regression plots, using the enzyme kinetics module of Sigmaplot (Systat Software, San
Jose, CA). Results are means ± SEM of triplicate experiments.
Fig. S4 Effect of pH on CARhis activity. Reactions were carried out in 50 mM Tris-HCl
buffer (21oC) containing purified CARhis, 2 mM benzoic acid, 1 mM ATP and 1 mM
NADPH within the pH range of 7 to 8.5. The initial rate of CAR activity was determined
from the rate of NADPH oxidation at 340 nm. Results are means ± SEM of duplicate
experiments.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
7.0 7.5 8.0 8.5
Init
ial
rate
of
acti
vit
y
(m
ol
NA
DP
+/m
in/m
g C
AR
)
pH
Fig. S5 Thermostability of CARhis. In the absence of reducing agents, CARhis was incubated
without shaking at three different temperatures; 25oC, 30
oC and 37
oC; for up to 96 h in 50
mM Tris-HCl buffer (pH 7.5, 21oC). Samples were taken at specific time intervals and the
initial rate of CAR activity determined in the presence of 2 mM benzoic acid, 1 mM ATP, 10
mM MgCl2 and 1 mM NADPH. Results are means ± SEM. of duplicate experiments.
0
20
40
60
80
100
0 20 40 60 80 100
Sp
ecif
ic a
cti
vit
y (
%)
Time (h)
25°C
30°C
37°C
A C4 aldehyde (910, 971)
B C6 aldehyde (970, 971)
C C8 aldehyde (929, 953)
0
1
2
3
2.6 3.6 4.6 5.6 6.6
Ion
72 s
ign
al
inte
nsit
y
(x10
5)
Retention time (min)
Scan 38 (2.859 min): 1mM C4 Acid1 Xylene.D\ data.ms ButanalHead to Tail MF=910 RMF=970
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290
0
100
100
30
43
43
5057
57
72
72
81 87 93 100 110 118 128 136 145 157 167 177 191 205 267 281
0
1
2
3
6.1 6.6 7.1 7.6 8.1
Ion
82 s
ign
al
inte
nsit
y
(x10
6)
Retention time (min)
Scan 621 (7.035 min): 1mM C6 Acid1.D\ data.ms HexanalHead to Tail MF=965 RMF=966
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220
0
100
100
30
41
44
50
56
56
62
62
67
67
72
72 77
82
82 87
98
99
153 207
0
1
2
9.2 9.4 9.6 9.8
Ion
82 s
ign
al
inte
nsit
y
(x10
6)
Retention time (min)
Scan 960 (9.463 min): 1mM C8 Acid1.D\ data.ms OctanalHead to Tail MF=929 RMF=953
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290
0
100
100
30
41
41
57
57
68
69
84
84
100
100
110
110
121 133 193 207 281
D C10 aldehyde (968, 973)
E C12 aldehyde (888, 971)
F C14 aldehyde (978, 986)
0
1
2
3
4
9.5 9.7 9.9 10.1
Ion
82 s
ign
al
inte
nsit
y
(x10
6)
Retention time (min)
Scan 932 (9.763 min): 1mM C10 Acid1.D\ data.ms DecanalHead to Tail MF=873 RMF=881
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220
0
100
100
30
41
43
57
57
68
70
82
82
91
95
95
100
112
114
119
128
128
134
138
141 150
155
156 207
0
1
2
3
11 11.2 11.4 11.6
Ion
82 s
ign
al
inte
nsit
y
(x10
6)
Retention time (min)
Scan 1139 (11.245 min): 1mM C12 Acid1.D\ data.ms DodecanalHead to Tail MF=888 RMF=971
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290
0
100
100
30
41
43
57
57
6068
68
82
82
9096
96
109
109
123
123
140
140
148
151
156 164
166
177
180
193 207 267 281
0
1
2
3
4
11.8 12 12.2 12.4
Ion
82 s
ign
al
inte
nsit
y
(x10
6)
Retention time (min)
Scan 1442 (12.063 min): C14A.D\ data.ms TetradecanalHead to Tail MF=978 RMF=986
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280
0
100
100
29
29
41
43
57
57
67
68
82
82
96
96
109
109
123
124
137
137 146
152
154
168
168
176
179
184 192
194
203 211
211
219 249 265
G C16 aldehyde (858, 990)
H C18 aldehyde (728, 843)
I C18:1 aldehyde (942, 973)
0
1
2
3
4
13.8 14 14.2 14.4
Ion
82 s
ign
al
inte
nsit
y
(x10
6)
Retention time (min)
Scan 1650 (13.242 min): C16C.D\ data.ms HexadecanalHead to Tail MF=849 RMF=989
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280
0
100
100
29
29
41
43
57
5768
69
82
82
91
96
96
109
109
123
124
137
137
151
152
165
165
173
180
181 194
196
207
212
222
222
230 238 246 254 267
0
2
4
6
8
14.1 14.3 14.5 14.7
Ion
80 s
ign
al
inte
nsit
y
(x10
5)
Retention time (min)
Scan 1407 (14.266 min): 18 Enz.D\ data.ms OctadecanalHead to Tail MF=728 RMF=843
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360
0
100
100
28
29
43
43
57
57
67
68
82
82
96
96
109
109 123
133
137
147
152
161
165
177
178
191
194
207
207
221
222
234
235
249
250
267
268
281
300 313 325 338
0
1
2
3
14 14.2 14.4 14.6
Ion
80 s
ign
al
inte
ns
ity
(x10
6)
Retention time (min)
Scan 1394 (14.169 min): 18-1 VA Enz.D\ data.ms 9-Octadecenal, (Z)-Head to Tail MF=948 RMF=984
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340
0
100
100
29
41
41
55
55
69
69
81
83
95
98
111
111
135
135
149
149
163
163
175
177
187
194
207
209
220
222
233 248
248
265
266
281 295 325 339
Fig. S6 GC-MS confirmation of aldehyde synthesis by CARhis. Reactions were carried out in
the presence of CARhis (100-300 g/ml), 1 mM NADPH, 1 mM ATP, 10 mM MgCl2 and
0.5mM C4-C18 fatty acids (A-K). Reactions were incubated at 30 oC for up to 4 h or in the
case of the longer chain substrates (≥ C16) up to 12 h. Reactions were mixed with chloroform
and the organic phase analyzed by GC-MS (see Methods). For clarity, only the peak
representing the aldehyde is shown. Dashed lines represent chromatograms without the
addition of CARhis. Aldehyde formation was confirmed by comparing the mass spectrum of
the analyte (in red) against reference standards (in blue) from the NIST spectral database. The
match factors and reverse match factors quantitatively describe the spectral match between a
sample spectrum and the library spectrum, and are indicated in brackets. The reverse match
factor is obtained by excluding peaks that are present in the sample spectrum but not the
library spectrum. Both values are derived from a modified cosine of the angle between the
spectra, otherwise known as the normalized dot product. A value of 900 or greater signifies
an excellent match; 800–900, a good match; and 700–800, a fair match.
J C18:2 aldehyde (799, 878)
K C18:3 aldehyde (831, 901)
0
1
2
3
4
14 14.2 14.4 14.6
Ion
80 s
ign
al
inte
nsit
y
(x10
6)
Retention time (min)
Scan 1386 (14.111 min): 18-2A Enz.D\ data.ms 9,12-OctadecadienalHead to Tail MF=799 RMF=878
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350
0
50
100
50
100
29
29
41
41
55
55
67
67
81
81
95
95
109
109
123
123
133
137
147
147
161 177 191
207
221 233 246 264
264
281295 325 339
0
2
4
6
8
10
14 14.2 14.4 14.6
Ion
80 s
ign
al
inte
nsit
y
(x10
6)
Retention time (min)
Scan 1391 (14.147 min): 18-3 2ul Enz.D\ data.ms 9,12,15-OctadecatrienalHead to Tail MF=831 RMF=901
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350
0
50
100
50
100
29
29
41
41
55
55
67
67
79
79
93
95
108
108
121
121
135
135
149
149
161 175 191
193 206
207216 233 247 262
262
281295 311 325 339
A B
C4 fatty acid (mM)
0 5 10 15 20
Ra
te (
µm
ol/m
in/m
g C
AR
his
)
0.0
0.2
0.4
0.6
0.8
C6 fatty acid (mM)
0 1 2 3 4 5
Ra
te (
µm
ol/m
in/m
g C
AR
his
)
0.0
0.2
0.4
0.6
0.8
1.0
C D
C8 fatty acid (mM)
0.0 0.5 1.0 1.5 2.0 2.5
Ra
te (
µm
ol/m
in/m
g C
AR
his
)
0.0
0.2
0.4
0.6
0.8
C10 fatty acid (µM)
0 200 400 600 800
Ra
te (
µm
ol/m
in/m
g C
AR
his
)
0.0
0.1
0.2
0.3
0.4
0.5
E
C12 fatty acid (µM)
0 20 40 60 80 100
Ra
te (
µm
ol/m
in/m
g C
AR
his
)
0.00
0.05
0.10
0.15
0.20
0.25
Fig. S7. Kinetic analysis of CARhis based on C4 to C12 saturated fatty acid substrates.
Reactions were carried out in 50mM Tris-HCl buffer containing CAR (0.5 to 4 g/ml), 1 mM
NADPH and fatty acid substrates ranging from 0 to 20 mM. For the C4 substrate, the
buffering capacity was increased to 100mM. Reactions were monitored at 340nm at 30 s
intervals. The apparent Km and Vmax values were determined from non-linear regression plots
using the enzyme kinetics module of SigmaPlot (Systat Software, San Jose, CA). The
saturated fatty acids evaluated were: (A) C4 fatty acid (0.5-20 mM), (B) C6 fatty acid (0.2-5
mM), (C) C8 fatty acid (0.1-2.5 mM), (D) C10 fatty acid (10-750 M) and (E) C12 fatty acid
(5-100 M). Results are means ± SEM of triplicate experiments.
A
1 2 3 4 5 6 7 8
B
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
AH
R a
cti
vit
y w
ith
C12 a
ldeh
yd
e
(m
ol
NA
DP
+/m
in/m
g c
rud
e p
rote
in)
Strain
CARhis (127.8kDa)
slr1192(his)/Ahr(his) (36.5kDa)
175
80
58
46
30
Fig. S8. Activity and expression levels of AHR in crude soluble fractions. (A) The crude
fractions were analyzed by SDS-PAGE for expression levels of AHR. Lane 1, molecular-
mass markers (in kDa); lane 2, crude fraction of TesA; lane 3, crude fraction of TPC; lane 4,
crude fraction of TPC-slr1192; lane 5, crude fraction of TPC-slr1192his; lane 6, crude fraction
of TPC-Ahr; lane 7, crude fraction of TPC-Ahrhis; and lane 8, molecular-mass markers (in
kDa). Proteins bands representing the CAR and AHR enzymes are indicated by red arrows.
(B) AHR activity was determined in crude fractions of all fatty alcohol-producing strains
using C12 aldehyde as the substrate. The TesA strain was included as a negative control.
Results are means ± SEM of duplicate experiments.
slr1192 1 --MIKAYAALEANGKLQPFEYDPGALGANEVEIEVQYCGVCHSDLSMINNEWGISNY
Ahr 1 MSMIKSYAAKEAGGELEVYEYDPGELRPQDVEVQVDYCGICHSDLSMIDNEWGFSQY
slr1192 56 PLVPGHEVVGTVAAMGEGVN--HVEVGDLVGLGWHSGYCMTCHSCLSGYHNLCATAE
Ahr 58 PLVAGHEVIGRVVALGSAAQDKGLQVGQRVGIGWTARSCGHCDACISGNQINCEQGA
slr1192 112 STIVGHYGGFGDRVRAKGVSVVKLPKGIDLASAGPLFCGGITVFSPMVELSLKPTAK
Ahr 116 VPTIMNRGGFAEKLRADWQWVIPLPENIDIESAGPLLCGGITVFKPLLMHHITATSR
slr1192 169 VAVIGIGGLGHLAVQFLRAWGCEVTAFTSSARKQTEVLELGAHHILDSTNPEAIASA
Ahr 173 VGVIGIGGLGHIAIKLLHAMGCEVTAFSSNPAKEQEVLAMGADKVVNSRDPQALKAL
slr1192 226 EGKFDYIISTVNLKLDWNLYISTLAPQGHFHFVGVVLEPLDLNLFPLLMGQRSVSAS
Ahr 230 AGQFDLIINTVNVSLDWQPYFEALTYGGNFHTVGAVLTPLSVPAFTLIAGDRSVSGS
slr1192 282 PVGSPATIATMLDFAVRHDIKPVVEQFSFDQINEAIAHLESGKAHYRVVLSHSKN
Ahr 286 ATGTPYELRKLMRFAARSKVAPTTELFPMSKINDAIQHVRDGKARYRVVLKADF-
Fig. S9. Amino acid sequence alignment of E. coli Ahr and Synechocystis sp. PCC 6803
slr1192. Sequences were aligned using ClustalW 1.8 (http://www.ebi.ac.uk/Tools/msa/) (8)
and formatted using Boxshade 3.21 (http://www.ch.embnet.org/software/BOX_form.html).
Dark-shaded boxes depict identical amino acids, while lighter-shaded boxes signify amino
acid residues with similar properties. Conserved amino acid residues for zinc binding are
highlighted in red.
Fig. S10. NADPH specificity of Ahrhis. Reactions were carried out in 50 mM Tris-HCl (pH
7.5, 21oC) buffer containing Ahrhis (1 g /ml), 0.5 mM NADPH or NADH and 0.5 mM C6
aldehyde. Absorbance was monitored at 340 nm at 15 s intervals over a 15 min period.
0
100
200
300
400
500
600
0 5 10 15
Fo
rmati
on
of
NA
D(P
)+
(m
ol/m
g A
hr h
is)
Time (min)
NADPH
NADH
Fig. S11. Substrate specificity of Ahrhis. Reactions were carried out in 50mM Tris-HCl buffer
containing Ahrhis (1 g or 10 g /ml) , 0.5 mM NAD(P)H or NAD(P)+ with either (A) 0.5mM
aldehydes (C4-C12) or (B) 0.5 mM alcohols (C4-C12). Initial rates for both reactions were
determined from the rate of NAD(P)H oxidation or NAD(P)+ reduction. Results are means ±
SEM of duplicate experiments.
A
B
0
50
100
150
Init
ial
rate
of
reacti
on
(m
ol
NA
D(P
)+/m
in/m
g A
hr h
is)
Substrate
NADPH
NADH
0.00
0.05
0.10
0.15
Init
ial
rate
of
reacti
on
(m
ol
NA
D(P
)H/m
in/m
g A
hr h
is)
Substrate
NADP+
NAD+
A C4 alcohol (899, 916)
B C6 alcohol (918, 923)
C C8 alcohol (940, 969)
0
1
2
3
4
3.1 4.1 5.1 6.1
Ion
56 s
ign
al
inte
nsit
y
(x10
6)
Retention time (min)
Scan 68 (3.472 min): C4A Diethyl ether.D\ data.ms 1-ButanolHead to Tail MF=899 RMF=916
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220
0
100
100
27
31
31
33
41
41
45
45
50
50
56
56
59
69
74
74
79 84 91 207
0
2
4
6
8
10
5.6 6.6 7.6 8.6
Ion
56 s
ign
al
inte
nsit
y
(x10
6)
Retention time (min)
Scan 546 (6.182 min): C6B CHCl3.D\ data.ms 1-HexanolHead to Tail MF=918 RMF=923
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280
0
100
100
31
31
41
43
55
56
69
69
7384
84
91 101
102
117 126 135 143 161 207 267
0
1
2
3
4
5
8 8.5 9 9.5 10
Ion
56 s
ign
al
inte
nsit
y
(x10
6)
Retention time (min)
Scan 900 (8.190 min): C8B CHCl3.D\ data.ms 1-OctanolHead to Tail MF=940 RMF=969
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280
0
100
100
28
29
41
41
56
56
69
69
83
84
97
97
105
111
113 121 129
129
141 149 161 207 235 254 268
Fig. S12. GC-MS verification of alcohol synthesis by Ahrhis. Reactions were carried out in
50mM Tris-HCl buffer containing Ahrhis (10-30 g/ml), 1 mM NADPH and 0.5mM C4-C12
aldehydes (A-E). Reactions were incubated at 30 oC for up to 2 h and terminated by addition
of chloroform. The organic phase analyzed by GC-MS (see Methods). For clarity, only the
peak representing the alcohol is shown. Dashed lines represent chromatograms without the
addition of the enzyme. Alcohol formation was confirmed by comparing the mass spectrum
of the analyte (in red) against reference standards (in blue) from the NIST spectral database.
The match factors and reverse match factors are indicated in brackets. Refer to Fig. S6 for
explanation of (reverse) match factors.
D C10 alcohol (971, 976)
E C12 alcohol (954, 961)
0
2
4
6
8
9.6 9.8 10 10.2 10.4
Ion
82 s
ign
al
inte
nsit
y
(x10
6)
Retention time (min)
Scan 1182 (9.788 min): C10B CHCl3.D\ data.ms 1-DecanolHead to Tail MF=971 RMF=976
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220
0
100
100
29
29
41
43
45
55
55
59
59
70
70
77
83
83
91
91
97
97
103112
112
119
123
126
129
133
138
140 147 161 175 207
0
2
4
6
8
10
11 11.2 11.4 11.6
Ion
56 s
ign
al
inte
nsit
y
(x10
6)
Retention time (min)
Scan 1429 (11.189 min): C12B CHCl3.D\ data.ms 1-DodecanolHead to Tail MF=954 RMF=961
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220
0
100
100
29
29
41
43
55
55
69
69
73
83
83
91
91
97
97
111
111
125
125
133140
140
147 156 168
168
207
Fig. S13. GC-MS confirmation of enzyme-mediated formation of alcohols and alkanes.
Alcohol (A-F) and alkane formation (G-K) were verified by comparing the mass spectrum of
the analyte (in red) against the reference standards (in blue) from the NIST spectral database.
The match factors and reverse match factors are indicated in brackets. Refer to Fig. S6 for
explanation of (reverse) match factors.
A C6 alcohol (974, 984) B C8 alcohol (913, 908)
C C10 alcohol (976, 977) D C12 alcohol (968, 973)
E C14 alcohol (979, 984) F C16 alcohol (948, 980)
G C7 alkane (800, 755) H C9 alkane (866, 896)
I C11 alkane (848, 893) J C13 alkane (952, 973)
K C15 alkane (837, 935)
Scan 654 (6.795 min): C6-3B.D\ data.ms 1-HexanolHead to Tail MF=974 RMF=984
20 40 60 80 100 120 140 160 180 200 220 240 260 280
0
100
100
29
3143
43
46
56
56
69
69
77
77
84
84
91 101
101
119 133 147 161 179 191 207 267
Scan 991 (8.705 min): C8-2B.D\ data.ms 1-OctanolHead to Tail MF=908 RMF=913
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220
0
100
100
28
29
41
41
56
56
68
69
84
84
91 97
97
105
111
113 121 129
129
142 161 179 193 207
Scan 1095 (10.295 min): C10A Enz Alcohol.D\ data.ms 1-DecanolHead to Tail MF=976 RMF=977
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220
0
100
100
29
29
41
43
55
55
70
70
83
83
97
97
112
112123
125 135
138
147 207
Scan 1524 (11.727 min): C12-2B.D\ data.ms 1-TetradecanolHead to Tail MF=930 RMF=938
20 40 60 80 100 120 140 160 180 200 220 240 260 280
0
100
10029
31
41
43
55
55
69
69
83
83
90
97
97
111
111
125
125
140
140
150
150
160
168
177
182
193
196
207 267
Scan 1745 (12.980 min): C14-1B.D\ data.ms 1-TetradecanolHead to Tail MF=979 RMF=984
20 40 60 80 100 120 140 160 180 200 220 240 260 280
0
100
100
29
29
41
43
55
55
69
69
83
83
97
97
111
111
125
125
140
140
151
152
168
168
179 191
196
205 221 241 267
Scan 1944 (14.109 min): C16-1ConcB.D\ data.ms 1-HexadecanolHead to Tail MF=948 RMF=988
20 40 60 80 100 120 140 160 180 200 220 240 260 280
0
100
100
29
43
43
55
55
69
69
83
83
97
97
111
111
125
125
139
139
149
152
159
166
169 179 196
196
207 221
224
237 249 265
Scan 59 (4.221 min): CAR plus ADC C8.D\ data.ms HeptaneHead to Tail MF=755 RMF=800
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220
0
100
100
28
29
43
43
46
47
57
57
61
62
71
71
77
83
83
100
100
117 135 159 207
Scan 485 (6.637 min): CAR plus ADC C10.D\ data.ms NonaneHead to Tail MF=866 RMF=896
20 40 60 80 100 120 140 160 180 200 220 240 260 280
0
100
100
28
29
43
43
47
57
57
63
71
71
85
85
99
99 112
117 128
128
207 267
Scan 813 (8.496 min): C12AAA FA.D\ data.ms UndecaneHead to Tail MF=860 RMF=930
20 40 60 80 100 120 140 160 180 200 220 240 260 280
0
100
100
28
29
43
43
47
57
57
71
71
75
85
85
88 98
98
112
112
122
126
133 156
156
167 179 191 205 223 249 267
Scan 1088 (10.055 min): C14AAA FA.D\ data.ms TridecaneHead to Tail MF=952 RMF=973
20 40 60 80 100 120 140 160 180 200 220 240 260 280
0
100
100
28
29
43
43
47
57
57
71
71
85
85
89 98
99
112
112 125
127 140
140
151
154
161 184
184
207 267
Scan 1327 (11.410 min): C16AAA FA.D\ data.ms PentadecaneHead to Tail MF=837 RMF=935
20 40 60 80 100 120 140 160 180 200 220 240 260 280
0
100
100
28
29
43
43
57
57
71
71
85
85
99
99
112
113 126
127 137
140
147
154
157 167
168
178
182
188 207
212
221 235 249 265
A
B
C
0
1
2
3
4
24 26 28 30
RID
sig
na
l in
ten
sit
y
(x
10
3)
Retention time (min)
Lipase & CAR 0 min
Lipase & CAR 30 min
Lipase & CAR 1 h
Lipase & CAR 2 h
Lipase & CAR 4 h
0
1
2
3
4
24 26 28 30
RID
sig
nal
inte
nsit
y
(x10
3)
Retention time (min)
Lipase only 0 min
Lipase only 30 min
Lipase only 1 h
Lipase only 2 h
Lipase only 4 h
0
1
2
3
4
24 26 28 30
RID
sig
nal
inte
nit
y
(x10
3)
Retention time (min)
CAR only 0 min
CAR only 4h
C8 aldehyde
C8 triglyceride
C8 triglyceride
C8 fatty acid
C8 triglyceride
D
Fig. S14. Conversion of TAGs to fatty acids and aldehydes. Reactions were performed in 50
mM Tris-HCl (pH 7.5, 21oC) containing 2 mM NADPH, 2 mM ATP, 10 mM MgCl2 and 0.5
mM C8-TAG (glyceryl trioctanoate) in the presence of (A) lipase and CAR enzymes (B)
lipase only (0.1 mg/ml) and (C) CAR only (0.3 mg/ml). A further reaction using (D) C12-
TAG (glyceryl trilaurate) as the substrate was also carried out.
0
2
4
6
8
10
21 23 25 27
RID
sig
nal
inte
nsit
y
(x10
4)
Retention time (mins)
No enzymes
Lipase only
Lipase & CAR
C12 triglyceride
C12 aldehyde
C12 fatty acid
Fig. S15. In vivo synthesis of fatty alcohols using palm oil as substrate. Harvested cells from
a preinduced culture of PC-Ahrhis, were resuspended in 50 mM potassium phosphate buffered
medium, supplemented with glucose and lipase (1 mg/ml) and incubated at 30 oC with
shaking at 150 rpm for up to 5 h in the presence of palm oil. Asterisk denotes fatty acid.
0
1
2
3
4
5
6
13.3 13.8 14.3 14.8
Sig
nal
inte
nsit
y
(x10
7)
Retention time (mins)
0 min
30 min
2 h
5 h
C16
C18 & C18:1
*
*
Table S1: Key features of the fatty acyl-ACP/TAG to fatty alcohol-producing systems. Enzyme abbreviations according to the legend of Figure 1.
*Calculation of G values under standard conditions performed with eQuilibrator (4)
**Green box=detectable levels of fatty alcohols, grey box = undetected fatty alcohols
Fatty alcohol
producing system
Direct co-substrate requirement
Typical reaction(s) catalyzed* G
(kJ mol-1)
In vivo range of fatty alcohols** Yield of fatty alcohols
(mg L-1)
Reference
C8 C10 C12 C14 C16 C18
Lipase or TesA
+ Sfp
+ CAR
+ Ahr
ATP
2 NADPH
C8-TAG + 3 H2O → 3 C8 fatty acid + glycerol
C12-ACP + H2O → C12 fatty acid + ACP
C12 fatty acid + NADPH + H+ + ATP → NADP
+ + AMP + PPi + C12 aldehyde
C12 aldehyde + NADPH + H+ → C12 alcohol + NADP
+
Pathway summation of G
-32.1
-21.5
+4.4
-18.8
-46.5 or-35.9
360 This work
FatB or TesA
+ FadD
+ Acr1
ATP
2 NADPH
CoA
C12-ACP + H2O → C12 fatty acid + ACP
C12 fatty acid + CoA + ATP → C12-CoA + AMP + PPi
C12-CoA + NADPH + H+ →C12 aldehyde + NADP
+ + CoA
C12 aldehyde + NADPH + H+ → C12 alcohol + NADP
+
Pathway summation of G
-21.5
-10.5
+14.9
-18.8
-35.9
60 9
AAR
+ native aldehyde reductase activity
2 NADPH C12-ACP + NADPH + H+ → C12 aldehyde + NADP
+ + ACP
C12 aldehyde + NADPH + H+ → C12 alcohol + NADP
+
Pathway summation of G
+14.9
-18.8
-3.9 140 10
Table S2: Substrate specificity and turnovers of the metabolic enzymes required for fatty-acyl ACP to fatty alcohol conversion
Enzyme Accession number In vitro substrate specificity of metabolic enzymes* Apparent or observed
maximal Kcat (turnover/min) Reference
C6 C8 C10 C12 C14 C16 C18 C20 C22
TesA C6EKX0
5 (for C18:1) 11
CAR B2HN69 27 (for C18:1) This work
Ahr C6EC82 109 (for C12) This work
ADC Q7V6D4 0.1 (for C18:1) This work, 10
AAR Q54765 0.01 (for C18:1) 10
FadD C5W549 16 (for C18:1) 12
Acr1** P94129 0.003 (for C16)
13
ACR2*** A1U3L3 3 (for C18:1) (acyl coA to aldehyde)
550 (for C12) (aldehyde to alcohol) 14
*Green box=observed activity, blue box= undetected activity, grey box = untested substrates
**Partially purified enzyme.
***Purified ACR2 from Marinobacter aquaeolei VT8
Table S3. List of the primers and templates used for gene(s) amplification.
Gene(s) PCR template Primers employeda
tesA E. coli BL21 (DE3) FP: 5’- aaccatggcggacacgttattgattctgggtgatagcc -3’
RP: 5’- aaaagcttttatgagtcatgatttactaaaggctgcaactgcttcg -3’
slr1192 Synechocytis sp. PCC 6803 FP: 5’- attaatcatatgattaaagcctacgctgccctggaag -3’
RP: 5’- aacctaggtatggctgagcactacccgataatgggc -3’
ahr E. coli BL21 (DE3) FP: 5’- attaatccatggtctagataattaatggatccaggaggaaacatatgtcgatgataaaaagctatgccgcaaaag -3’
RP: 5’- attaatcctaggaagcttctcgagtcaaaaatcggctttcaacaccacgcgg -3’
ahrhis E. coli BL21 (DE3) FP: 5’- attaatccatgggccaccaccaccaccaccacggcagccatatgtcgatgataaaaagctatgccgcaaaagaag -3’
RP: 5’- attaatcctaggaagcttctcgagtcaaaaatcggctttcaacaccacgcgg -3’
adchis pET28-ADChis FP: 5’- attaatccatggcacaccaccaccaccaccaccaa -3’
RP: 5’- attaatcctaggttacaccatccgtgccgcagcc -3’
sfp + carhis pET-TPC FP: 5’- attaatccatgggcaagatctacggcatatacatggaccgcc -3’
RP: 5’- aataatcctagggaattcttacagcaggcccag -3’
a Restriction enzyme sites are underlined and italicized
Abbreviations used: FP, forward primer; RP, reverse primer
Table S4. Summary of the vector backbone and gene(s) inserts used for plasmid construction.
Ligated fragmentsa
Plasmid Vector backbone Gene(s) insert
pET-TPC pET-BPC (NcoI/HindIII) tesA (NcoI/HindIII)
pET-TP pET-TPC (EcoRI) n/a
pET-PC pET-TP (NcoI/EcoRI) sfp + carhis (NcoI/EcoRI)
pET-TesA pET-TP (BamHI) n/a
pCDF-slr1192 pCDF-Ahr (NdeI/AvrII) slr1192 (NdeI/AvrII)
pCDF-slr1192his pCDF- Ahrhis (NdeI/AvrII) slr1192 (NdeI/AvrII)
pCDF-Ahr pCDF-Duet1 (NcoI/AvrII) ahr (NcoI/AvrII)
pCDF- Ahrhis pCDF-Duet1 (NcoI/AvrII) ahrhis (NcoI/AvrII)
pCDF- ADChis pCDF-Duet1 (NcoI/AvrII) adchis (NcoI/AvrII)
a Restriction enzyme cuts are specified in brackets.
Table S5. List of the plasmids used in the study including their properties.
Plasmid Recombinant
proteins encoded
Source
pETDuet-1 None Novagen
pCDFDuet-1 None Novagen
pET28-ADChis ADChis 2
pCDF-ADChis ADChis This study
pET-TesA TesA This study
pET-TP TesA & Sfp This study
pET-PC Sfp & Carhis This study
pET-TPC TesA, Sfp & Carhis This study
pCDF-slr1192 slr1192 This study
pCDF-slr1192his slr1192his This study
pCDF-Ahr Ahr This study
pCDF-Ahrhis Ahrhis This study
Table S6. List of E. coli strains engineered in the study.
Strain name Host strain Plasmids/genotype Recombinant proteins induced Source or
reference
BL21(DE3) ------------- F–ompT hsdSB(rB–mB–) gal dcm (DE3) ------------- Novagen
Control BL21(DE3) pETDuet-1, pCDFDuet-1 None This study
slr1192 BL21(DE3) pCDF-slr1192 slr1192 This study
slr1192his BL21(DE3) pCDF-slr1192his slr1192his This study
Ahr BL21(DE3) pCDF-Ahr Ahr This study
Ahrhis BL21(DE3) pCDF- Ahrhis Ahrhis This study
TesA BL21(DE3) pET-TesA, pCDF-Empty TesA This study
ADChis BL21(DE3) pET28-ADChis ADChis This study
TP BL21(DE3) pET-TP, pCDF-Empty TesA & Sfp This study
PC BL21(DE3) pET-PC, pCDF-Empty Sfp & CARhis This study
PC-Ahrhis BL21(DE3) pET-PC, pCDF- Ahrhis Sfp, CARhis & Ahrhis This study
TPC BL21(DE3) pET-TPC, pCDF-Empty TesA, Sfp & CARhis This study
TPC-slr1192 BL21(DE3) pET-TPC, pCDF-slr1192 TesA, Sfp, CARhis & slr1192 This study
TPC-slr1192his BL21(DE3) pET-TPC, pCDF-slr1192his TesA, Sfp, CARhis & slr1192his This study
TPC-Ahr BL21(DE3) pET-TPC, pCDF-Ahr TesA, Sfp, CARhis & Ahr This study
TPC-Ahrhis BL21(DE3) pET-TPC, pCDF-Ahrhis TesA, Sfp, CARhis & Ahrhis This study
TPC-ADChis BL21(DE3) pET-TPC, pCDF-ADChis TesA, Sfp, CARhis & ADChis This study
Table S7. List of the enzymes used to engineer the metabolic pathways
Enzyme Organism source Enzyme encoded for Enzyme modifications Enzyme accession number Reference
TesA E. coli Acyl-ACP/CoA thioesterase Signal sequence removed C6EKX0 15
Sfp B. subtilis Phosphopantetheinyl transferase None P39135 16
CARhis M. marinum Carboxylic acid reductase N-terminal 6x his tag B2HN69 This study
slr1192 Synechocystis sp PCC 6803 Aldehyde reductase None P74721 17
slr1192his Synechocystis sp PCC 6803 Aldehyde reductase N-terminal 6x his tag As above 17
ADChis P. marinus Aldehyde decarbonylase N-terminal 6x his tag NP_895059.1 2
Ahr E. coli Aldehyde reductase None C6EC82 This study
Ahrhis E. coli Aldehyde reductase N-terminal 6x his tag As above This study
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