A Process for Microbial Hydrocarbon Synthesis: Overproduction of
Fatty Acids in Escherichia coli and Catalytic Conversion to Alkanes
Supplementary Material
Rebecca M. Lennen1,2, Drew J. Braden1,2, Ryan A. West1,2, James A. Dumesic1,2,
Brian F. Pfleger*1,2
1
Department of Chemical and Biological Engineering, University of Wisconsin-Madison
2
U.S. Department of Energy Great Lakes Bioenergy Research Center
*Corresponding author:
Brian F. Pfleger
3629 Engineering Hall
1415 Engineering Drive
Madison, WI 53706-1691
phone: (608) 890-1940
fax: (608) 262-5434
pfleger@engr.wisc.edu
Running title: Fatty acid overproduction and conversion to alkanes
Sequence of synthesized BTE gene
The full sequence of synthesized DNA includes restriction sites at the 5' (XmaI) and 3' (HindIII)
ends (italicized), an artificial ribosome binding site (shown in blue), a spacer sequence (shown in
red), an artificial start codon (bolded), and two bases following the start codon and before the
XbaI site to generate an in-frame sequence (shown in green) is shown below. The codon
corresponding to histidine-204 is underlined. The stop codon is bolded.
1
51
101
151
201
251
301
351
401
451
501
551
601
651
701
751
801
851
901
CCCGGGAGGA
CTGCCTCAAC
TCGTACTTTC
CCATCCTGGC
AAATCTGTTG
TAAACGTGAC
GCTACCCTAC
TCCGGTAACA
GGGCGAAATC
GCACTCGTCG
CCTGCTTTCA
GCAAAAACTG
CGCGCTGGAA
GTTGCTTGGG
CATTTCCTCT
TTCTGCGCAG
GTCTGCGACC
GCGTACGGAG
TAATTCCGGC
GGATTATAAA
TGCTGGATGA
GCAATTCGTT
CGTCATGAAC
GTATCCTGGG
CTGATGTGGG
TTGGGGTGAC
ATGGTATGCG
CTGACGCGTT
CCTGTCTACC
TCGATAACGT
AACGACTCCA
CGACCTGGAT
TCTTCGAGAC
TTTACTCTGG
CCTGACCACC
ATCTGCTGCA
TGGCGTCCAA
GGAACCTCGT
ATGACTCTAG
TCACTTCGGT
CTTATGAAGT
CACATGCAGG
TGATGGTTTC
TAGTGCGTCG
ACTGTGGAAG
TCGCGATTTT
GCACCTCCCT
ATCCCGGACG
GGCAGTTAAA
CCGCGGACTA
GTTAATCAGC
TGTGCCGGAC
AGTACCGTCG
GTAAGCGGCG
ACTGGAAGGC
AGCTGACGGA
GTTTAAGCTT
AGTGGAAACC
CTGCACGGTC
GGGTCCAGAT
AAGCCACCCT
GGCACTACTC
CACCCACGTA
TCGAGTGTTG
CTGGTCCGTG
GAGCGTTCTG
AAGTGCGCGG
GACGACGAAA
CATCCAGGGC
ATGTGAACAA
AGCATTTTCG
CGAATGTACT
GTTCTAGCGA
GGCTCCGAAG
TTCTTTCCGC
GAAACCAAAA
TGGTGTTTCG
CGTTCTACCT
GAATCACGCG
TGGAAATGTC
GCAGTAGAGC
GATTGGCGCG
ACTGTAAAAC
ATGAACACCC
TGAGATCGGT
TCAAGAAACT
GGTCTGACTC
CCTGAAATAC
AAAGCCATCA
CGCGACTCCG
GGCAGGTCTG
TCCTGCGTGC
GGCATCTCCG
MG1655
MG1655
K27
MG1655 ΔfadD
A
glycerol +
sodium oleate
K27
MG1655 ΔfadD
MG1655 ΔfadD ΔaraBAD
sodium oleate
B
glycerol +
L-arabinose
L-arabinose
Figure S1. Functional verification of gene deletions constructed in this study. (A) E.
coli K-12 MG1655, K27 (a non-functional fadD mutant (Overath et al., 1969)), and
MG1655 ΔfadD streaked on M9 minimal medium agar containing 0.4% glycerol and
0.1% sodium oleate (left), or 0.1% sodium oleate (right). K27 and the ΔfadD strain
cannot grow on the oleate-only plate, indicating shut-down of β-oxidation. (B) K-12
MG1655 and MG1655 ΔfadD ΔaraBAD (RL08) streaked on M9 minimal medium
agar containing 0.4% glycerol and 0.4% L-arabinose (left), and 0.4% L-arabinose
(right). The ΔaraBAD strain cannot grow on the arabinose-only plate.
%
57
100.0
43
75.0
71
50.0
85
25.0
99
0.0
50.0
75.0
100.0
112
126
125.0
141
156
150.0
184
175.0
210 221
200.0
225.0
255
250.0
288
275.0
300.0
319
325.0
Figure S2. Mass spectrum of tridecane in a decarboxylated decane extraction of the
34 hour culture of RL08/pBAD33-ACC/pBAD35-BTE. The molecular ion (m/z 184)
is present.
Figure S3. Representative mass spectra of various fatty acid methyl esters (10:1, top;
12:1, second from top; 12:0-OH, second from bottom; 14:1, bottom) not present in the
standard mixture. Spectra are from a 23 hour extraction of a strain expressing BTE on
plasmid pBAD35-BTE. The molecular ions for 12:1 (m/z 212), 12:0-OH (m/z 229),
and 14:1 (m/z 240) are all present.
Table SI: Molar concentrations of fatty acid methyl esters in fatty acid overproducing and
negative control cultures prior to decane extraction, and alkanes following decane extraction and
catalytic conversion.
Species:
Fatty acid methyl ester concentration (µmol mL-1 culture volume)
12:0
12:1
14:0
14:1
16:0
16:1
pBAD33-ACC/
pBAD18-BTE
1.44  0.11
0.23  0.01
0.21  0.00
0.21  0.01
0.22  0.00
0.10  0.00
pBAD33/
pBAD18-BTEH204A
0.01  0.00
ND*
0.05  0.00
ND*
0.24  0.01
0.02  0.00
Species:
Alkane concentration (µmol mL-1 extract volume)
undecane
tridecane
pentadecane
pBAD33-ACC/
pBAD18-BTE
27.9  1.8
5.1  0.5
2.6  0.0
pBAD33/
pBAD18-BTEH204A
3.3  0.2
0.8  0.1
0.9  0.1
*ND = not detectable
ACC (+) BTE (+)
8:0
10:0
10:1
12:0
15:0
14:1
12:0-OH
12:1
(std)
16:0
14:0
16:1
17:0
intensity
(std)
18:1 18:0
ACC (-) BTE (+)
ACC (+) BTE (-)
ACC (-) BTE (-)
standard mixture
3
6
9
12
15
18
21
24
time (min)
Figure S4. Representative chromatograms (from 34 hour samples, undiluted hexane
extracts) of strains in this study, with quantified peaks indicated. Very large increases
in the levels of C12 and C14 species are clearly observed in BTE-expressing cultures.
Labelled peaks that are not present in the standard mixture (10:1, 12:1, 12:0-OH, and
14:1) were identified by comparing their mass spectra to the NIST library.