Supplementary Materials
Title: Enhanced Production of Branched-Chain Fatty Acids by Replacing β-Ketoacyl-(acylcarrier-protein) Synthase III (FabH)
Authors: Wen Jiang1§, Yanfang Jiang1§, Gayle J. Bentley2, Di Liu1, Yi Xiao1, Fuzhong Zhang1,2*
1
Department of Energy, Environmental and Chemical Engineering, 2Division of Biological &
Biomedical Sciences, Washington University in St. Louis, Saint Louis, MO 63130, USA
*
Correspondence:
Dr. Fuzhong Zhang
Department of Energy, Environmental & Chemical Engineering, Washington University
1 Brookings Drive, St. Louis, MO, 63130, USA
fzhang@seas.wustl.edu
Tel: 314-935-7671
§
These authors contributed equally to the work
Supplementary Materials and Methods
Plasmid construction
Genes encoding S. aureus FabH (SaFabH) was codon-optimized for E. coli expression and
synthesized by Integrated DNA Technologies (Coralville, IA, USA), whereas other genes were
amplified by PCR using primers and templates listed in Supplementary Table 2.
To create plasmids pSa-PecfabH-SafabH, pSa-PecfabH-BsfabH2, and pSa-PecfabH-BsfabH1, four
fabH genes were separately cloned 3’ of the E. coli fabH promoter and assembled into the
backbone of a BioBrick plasmid (Lee et al. 2011) pBbS7a-RFP using Golden-Gate DNA
assembly method (Engler et al. 2008). To create plasmids pE8c-tesA-bkd and pB5c-tesA-bkd, an
E. coli cytosolic thioesterase gene tesA (‘tesA; leader sequence deleted) (Steen et al. 2010) and a
branched-chain α-keto acid dehydrogenase operon bkd from B. subtilis were amplified and
inserted into either pBbB5c-RFP or pBbE8c-RFP (Lee et al. 2011) using restriction sites
EcoRI/BamHI, BamHI/XhoI, and EcoRI/XhoI, respectively. Plasmid pB8c-tesA-bkd was created
by transferring the pBBR1 replication origin from pBbB5c-RFP to plasmid pE8c-tesA-bkd using
SacI and AvrII. Plasmids pE1k-BsfabH2, pB8k-BsfabH2, pB5c-bkd, pE8c-bkd, and pA5a-tesA
were constructed by cloning target genes into the backbone of different BioBrick plasmids. To
create plasmid pE2k-alsS-ilvCD and pE2k-leuABCD-alsS-ilvCD, the alsS-ilvCD fragment was
PCR amplified from pSA69 (Atsumi et al. 2008), the leuABCD operon was PCR amplified from
E. coli, and the corresponding fragments were then ligated with the backbone of pBbE2k-RFP
using Gibson isothermal assembling method (Gibson et al. 2009).
Composition of M9 MOPS Minimal Medium
Minimal medium (M9 medium supplemented with 75 mM MOPS at pH 7.4, 2 mM MgSO4, 1
mg/L thiamine, 50 μg/mL lipoic acid, 10 μM FeSO4, 0.1 mM CaCl2 and micronutrients including
3 μM (NH4)6Mo7O24, 0.4 mM boric acid, 30 μM CoCl2, 15 μM CuSO4, 80 μM MnCl2, and 10
μM ZnSO4) containing 2% glucose as carbon source was used for cell growth and fatty acid
production. For strains derived from CL111, 0.5% yeast extract was added for cell growth.
qRT-PCR
Strains DH1(ΔfadE), BC13, and BC13A were cultivated as described in section 2.3 and induced
with 1 mM of IPTG and the supplementation of 1 g/L 4-methyl-2-oxopentanoic acid. Total
RNAs were extracted from exponentially growing cells in duplicate using TRIzol Max Bacterial
RNA Isolation Kit (life technologies). Contaminating DNA was removed with RNase-free
DNase I (Thermo Scientific), and cDNAs were synthesized using a Revert Aid First Strand
cDNA Synthesis Kit (Thermo Scientific) with random hexamer primers following the
manufacturer’s protocol. The synthesized cDNAs were normalized to 0.2 μg/μL, and 1 μL was
amplified using the Power SYBR Green PCR Master Mix (Applied Biosystems) and primers
specific to the genes of interest (Supplementary Table 3) in a 20 μL reaction system. The reaction
for each gene in each sample was performed in triplicates. qRT-PCR assays were carried out on
an ABI7500 fast machine with the thermal cycling conditions recommended by the
manufacturer. For data analysis, expression levels of the house keeping gene dnaK were used as
a control for normalization between samples. Fold changes of genes of interest were calculated
as 2-ΔΔCT.
Quantification of free fatty acids
For the quantification of free fatty acids, 1 mL of cell culture was acidified with 100 μL of
concentrated HCl (12N). Free fatty acids were extracted twice with 0.5 mL ethyl acetate, which
was spiked with 20 μg/mL of C19:0 fatty acid as an internal standard. The extracted fatty acids
were methylated to fatty acid methyl esters (FAMEs) by adding 10 μL concentrated HCl, 90 μL
methanol, and 120 μL of TMS-diazomethane, and incubated at room temperature for 15 min.
FAMEs were quantified using a GC-MS (Hewlett-Packard model 7890A, Agilent Technologies)
equipped with a 30 m DB5-MS column (J&W Scientific) and a mass spectrometer (5975C,
Agilent Technologies) or a FID (Agilent Technologies) detector. For each sample, the column
was equilibrated at 80 °C for 1 min, followed by a ramp to 280 °C at 30°C/min, and was then
held at 280 °C for 3 min. Individual BCFA peaks were identified by comparing their retention
time to those of standard BCFA methyl esters (Bacterial Acid Methyl Ester Mix, Sigma Aldrich)
and by comparing their mass spectra to the Probability Based Matching (PBM) Mass
Spectrometry Library. Concentrations of each fatty acid were determined by comparing the area
of each FAME peak to a standard curve generated by standard FAME mixtures (GLC-20, GLC30, and Bacterial Acid Methyl Ester Mix, Sigma Aldrich) eluted using the same method. BCFA
titer for each strain was measured in biological triplicate (starting from three different colonies)
and average values are reported.
Supplementary Table 1 Oligonucleotides used in this research
Name Sequence
SA1
TCAGCAGGTCTCAGTACATGAATGTAGGTATTAAAGGCTTCG
SA2
TCTGGTCTCAATCCTTATTTA CCCCACTTAATGGTCATC
BSA1 TCAGCAGGTCTCAGTACATGAAAGCTGGAATACTTGGTGTT
BSA2 TCAGCAGGTCTCAATCCTTATCGGCCCCAGCGGA
BSB1 TCAGCAGGTCTCAGTACATGTCAAAAGCAAAAATTACAGCTATC
BSB2 TCAGCAGGTCTCAATCCTTACATCCCCCATTTAATAAGCA
BKD1 TTTTTGGATCCAGACAGACAGGAGTGAGTCAC
BKD2 TTTTTCTCGAGTTAGTAAACAGATGTCTTCTCGTC
TES1 TTTTTTGAATTCAAAAGATCTAAAGGAGGCCATCCTATGGC
TES2 TTTTTTCTCGAGAAAGGATCCTTATGAGTCATGATTTACTAAAGG
PE1
TCAGCAGGTCTCACGTCAGCGTTGGCTACAAAAGAGAC
PE2
TCAGCAGGTCTCAGTACGCTCAGTCACTTTTCGGTTA
PSA1 TCAGCAGGTCTCAGGATCCAAACTCGAGTAAGGATC
PSA2 TCAGCAGGTCTCAGACGTCAGGTGGCACTTTTC
E2K1 GGATCCAAACTCGAGTAAG
E2K2 ATGTATATCTCCTTCTTAAAAGATCTTTTG
AI1
tttaagaaggagatatacatATGTTGACAAAAGCAACAAAAG
AI2
actcgagtttggatccTTAACCCCCCAGTTTCGA
AI3
ttatgaattaaAGATCTTTTAAGAAGGAGATATACATATG
AI4
cgggttagAGATCTTTTAAGAAGGAGATATACATATG
LEU1 tttaagaaggagatatacatATGAGCCAGCAAGTCATTATTTTC
LEU2 cttaaaagatctTTAATTCATAAACGCAGGTTG
Supplementary Table 2 Primers used for the construction of plasmids in this study
Plasmid
Primer 1
pSa-PecfabHBsfabH1
PSA1
pSa-PecfabHBsfabH2
PSA1
pSa-PecfabHSafabH
PSA1
pB5c-tesAbkd/pE8ctesA-bkd
pE2k-alsSilvCD
Vector PCR
Primer 2
Template
PSA2
PSA2
PSA2
pE2kleuABCDalsS-ilvCD
E2K1
E2K1
E2K2
E2K2
pE2k-alsSilvCD
E2K1
E2K2
Primer 1
Primer 2
Insert PCR
Template
PE1
PE2
E. coli gDNA
BSA1
BSA2
B.
gDNA
PE1
PE2
E. coli gDNA
E.
coli
promoter
BSB1
BSB2
B.
gDNA
B. subtilis fabH2
PE1
PE2
E. coli gDNA
SA1
SA2
TES1
TES2
BKD1
BKD2
pBbE2kRFP
pBbE2kRFP
AI1
AI2
pSA69
B. subtilis alsS
E. coli ilvC ilvD
AI3
AI2
pE2k-alsSilvCD
B. subtilis alsS
E. coli ilvC ilvD
pBbE2kRFP
LEU1
LEU2
E. coli gDNA
E. coli leuABCD
pBbS7aRFP
pBbS7aRFP
pBbS7aRFP
subtilis
subtilis
Chemical
synthesis
E. coli gDNA
B.
subtilis
gDNA
Gene of Interest
E.
coli
fabH
promoter
B. subtilis fabH1
E.
coli
promoter
fabH
fabH
S. aureus fabH
E. coli tesA
B. subtilis bkd
Supplementary Table 3 Primers used for the qPCR in this study
Gene Sequence
fabD
Forward: GGCCAGCTGAAGAACTGAATA
Reverse: CATCATTGCCGGTGCTTTAC
fabG
Forward: GGCAAAGGTCTGATGTTGAATG
Reverse: CCGGCATTATTGACCAGGATA
dnaK Forward: AATCGAACTGTCTTCCGCTC
Reverse: TCTTCAACCAGGCTTTCCAG
Supplementary Table 4 Substrate specificity of four FabHs in this study
Substrates
EcFabH
BsFabH1
BsFabH2
EcFabH
SaFabH
Acetyl-CoA
494 ± 11
14 ± 0.5
113 ± 10
50.5 ± 4.5 1.0 ± 0.1
2-methylpropanoyl-CoA
<1
58 ± 2
149 ± 9
5.3 ± 0.8
31.4 ± 1.4
3-methylbutyryl-CoA
<1
16 ± 0.8
162 ± 5
3.8 ± 0.2
14.9 ± 0.5
2-methylbutyryl-CoA
<1
155 ± 10
79 ± 9
n.d.
n.d.
Reference
(Choi et al. 2000)
(Qiu et al. 2005)
(A)
160
BC01 strain
FFA Production (mg/L)
140
120
100
80
60
40
20
0
C12:0 C13:0 C14:0 C14:1 C14:0 C15:0 C15:0 C16:1 C16:0 C17:0 C18:1 C18:0
iso
iso
anteiso
iso
(B)
80
BC13A strain
FFA Production (mg/L)
70
60
50
40
30
20
10
0
C12:0 C13:0 C13:0 C14:1 C14:0 C15:0 C15:0 C16:1 C16:0 C17:0 C18:1 C18:0
iso anteiso
iso anteiso
iso
Supplementary Figure 1. Fatty acid production profiles of (A) strain BC01 (containing
EcFabH& BsFabH2) and (B) the top-performing strain, BC13A (containing SaFabH), with the
supplement of 1 g/L 4-methyl-2-oxopentanoic acid.
(A)
No Suppl.
BCFA Production (mg/L)
16
3-methyl-2-oxobutyric acid
14
12
10
8
6
4
2
0
C13:0
iso
C13:0
anteiso
C14:0
iso
C15:0
iso
C15:0
anteiso
C16:0
iso
C17:0
iso
C17:0
anteiso
(B)
No Suppl.
BCFA Production (mg/L)
40
4-methyl-2-oxopentanoic acid
35
30
25
20
15
10
5
0
C13:0
iso
C13:0
anteiso
C14:0
iso
C15:0
iso
C15:0
anteiso
C16:0
iso
C17:0
iso
C17:0
anteiso
(C)
No Suppl.
45
3-methyl-2-oxopentanoic acid
BCFA Production (mg/L)
40
35
30
25
20
15
10
5
0
C13:0
iso
C13:0
anteiso
C14:0
iso
C15:0
iso
C15:0
anteiso
C16:0
iso
C17:0
iso
C17:0
anteiso
Supplementary Figure 2. Effect of α-keto acid supplementation to BCFA productions. Strain
BC11A (containing BsFabH2) was cultivated as described in Methods and induced with 0.4%
arabinose. At the moment of induction, cell cultures were supplemented with 1 g/L of either 3methyl-2-oxobutyric acid (A, solid columns), 4-methyl-2-oxopentanoic acid (B, solid columns),
3-methyl-2-oxopentanoic acid (C, solid columns), or nothing (A-C, empty columns).
(A)
No suppl.
3-methyl-2-oxobutyric acid
BCFA Production (mg/L)
10
8
6
4
2
0
C13 iso
C13
C14 iso C15 iso
C15
C16 iso C17 iso
C17
anteiso
anteiso
anteiso
(B)
No suppl.
4-methyl-2-oxopentanoic acid
BCFA Production (mg/L)
20
16
12
8
4
0
C13 iso
C13
C14 iso C15 iso
C15
C16 iso C17 iso
C17
anteiso
anteiso
anteiso
(C)
No suppl.
3-methyl-2-oxopentanoic acid
BCFA Production (mg/L)
20
16
12
8
4
0
C13 iso
C13
C14 iso C15 iso
C15
C16 iso C17 iso
C17
anteiso
anteiso
anteiso
Supplementary Figure 3. Effect of α-keto acid supplementation to BCFA productions. Strain
BC12A (containing BsFabH1) was cultivated as described in Methods and induced with 0.4%
arabinose. At the moment of induction, cell cultures were supplemented with 1 g/L of either 3methyl-2-oxobutyric acid (A, solid columns), 4-methyl-2-oxopentanoic acid (B, solid columns),
3-methyl-2-oxopentanoic acid (C, solid columns), or nothing (A-C, empty columns).
(A)
Fatty Acid Production (mg/L)
37°C
30°C
25°C
100
80
60
40
20
0
(B)
Fatty Acid Production (mg/L)
without overlayer
90
75
60
45
30
15
0
with overlayer
(C)
37°C
30°C
25°C
37°C with dodecane layer
FFA Production (mg/L)
500
400
300
200
100
0
Total FFA
Unsaturated FFA
BCFA
Supplementary Figure 4. Optimization of cultivation conditions. Effect of cultivation
temperature (A) product trap (B) on BCFA production. Strain BC13A was induced with 0.1 mM
of IPTG and supplemented with 1 g/L of 4-methyl-2-oxopentanoic acid at the moment of
induction. (C) Total FFA, unsaturated FA, and BCFA titers of BC13A under different
conditions.
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