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, patjon@utu.fi & Nicholas J. Turner,

nicholas.turner@manchester.ac.uk

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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