Supplementary Table 1: Strains used in this study
Strain
BW25113
AG977
AG978
JME1
JME2
JME3
JME4
JME6
JME7
JME15
JME16
JME17
JME50
Genotype
F-, Δ(araD-araB)567, lacZ4787(del)::rrnB-3, λ-,
rph-1, Δ(rhaD-rhaB)568, hsdR514
BW25113 ΔompT ΔpflB
BW25113 ompT::pcaHGBDC pflB::pcaIJFK
Evolved variant of AG978
Evolved variant of AG978
Evolved variant of AG978
Evolved variant of AG978
Evolved variant of AG978
AG978 elfC::praI
AG978 flu(Δ6)
AG978 flu(Δ6) pcaH(R4)
AG978 pcaH(R4)
JME17 elfC::praI
Reference
Datsenko and Wanner, 2000
This study
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Supplementary Table 2: Plasmids used in this study
Plasmid
pCas
pTarget
pKD46
pKD46-I-SceI
pET30a
pET30a-dest
pET30a-LPpcaIJFK
pET30a-LPpcaHGBDC
pET30a-LPcontrol
pJM157
pJM160
pJM168
pJM172
pJM179
pJM180
pJM182
Description
Expresses Cas9 and λ-RED
Expresses sgRNA
PBAD λ-RED, AmpR
PBAD λ-RED, I-SceI, AmpR
LacI+, KanR
MCS, KanR
pcaIJFK integration, KanR
Reference
Jiang et al., 2015
Jiang et al., 2015
Datsenko and Wanner, 2000
This study
EMD Millipore, Billerica, MA
This study
This study
pcaHGBDC integration, KanR
This study
Gene deletion, KanR
This study
Expresses elfC sgRNA
Contains praI expression construct with elfC
homology
Expresses gfcAB sgRNA
Contains pobA expression construct with
gfcAB homology
Expresses flu sgRNA
Expresses pcaH sgRNA #1
Expresses pcaH sgRNA #2
This study
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Supplementary Table 3: Primers used in this study
Name
dest-FWD
Sequence
TAGGGATAACAGGGTAATCAGTCGTGTCATCTG
ATTACCTGGCGGAAATTAAACTAAGAGAGAGC
TCTAAATCATGAAAAATTTATTTGCTTTGTG
dest-REV CGAATTAGCCTGCCAGCCCTGTTTTTATTAGTGC
ATTTTGCGCGAGGTCAAAATAAATTTTTCATGA
TTT
I-SceI-LP- TCCGATTAGGGATAACAGGGTAATGTGTAGGCT
T5-FWD
GGAGCTGCTTCTACGGCCCCAAGGTCCAAACGG
TGAAAATCATGAAAAATTTATTTGCTTTGTG
LP-IJFK- ACAAGGCCATGGCTGATCATATGAATATCCTCC
REV
TTAGTTGGCTTCAGGGATGAGGCGCCATCCTGG
GCGGTTCTGATAACGA
LPACAAGGCCATGGCTGATCATATGAATATCCTCC
HGBDC- TTAGTTGGCTTCAGGGATGAGGCGCCATCTTTG
REV
CCTGGCGGCAGTAG
LPACAAGGCCATGGCTGATCATATGAATATCCTCC
controlTTAGTTGGCTTCAGGGATGAGGCGCCATCAAAT
REV
AAATTTTTCATGATTT
ompT-LP- ATATAAAAAATACATATTCAATCATTAAAACGA
up
TTGAATGGAGAACTTTTCATATGAATATCCTCC
TTAGTTGGCTTCAG
ompT-LP- AGTTATTCCCCGGGGCGATTTTCACCTCGGGGA
down
AATTTTAGTTGGCGTTCGTGTAGGCTGGAGCTG
CTTC
pflB-LPTTTTACTGTACGATTTCAGTCAAATCTAATTACA
up
TAGATTGAGTGAAGGTGTGTAGGCTGGAGCTGC
TTC
pflB-LPCGAAGTACGCAGTAAATAAAAAATCCACTTAA
down
GAAGGTAGGTGTTACATGCATATGAATATCCTC
CTTAG
ompTTAAACAAAATATAAACAGTGGAGC
FWD
ompTCTATAATTGATGGTTTTCTATGTG
REV
pflB-FWD TTTTACTGTACGATTTCAGTCAAAT
pflB-REV CGAAGTACGCAGTAAATAAAAAATC
pTarget
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG
FWD
pTarget
ACTAGTATTATACCTAGGACTGAG
REV
elfC N20F TTCCTTATGCCACTTCTTAT
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG
GCTAGTC
Purpose
pET30a-dest
pET30a-dest
pET30a-LP-pcaIJFK,
pET30a-LP-control
pET30a-LP-pcaIJFK
pET30a-LP-pcaHGBDC
pET30a-LP-control
I-SceI, landing pad, and
CmR cassette
integration at ompT
I-SceI, landing pad, and
CmR cassette
integration at ompT
I-SceI, landing pad, and
CmR cassette
integration at pflB
I-SceI, landing pad, and
CmR cassette
integration at pflB
PCR screen at ompT
PCR screen at ompT
PCR screen at pflB
PCR screen at pflB
Inverse PCR of pTarget
Inverse PCR of pTarget
Construction of elfC
sgRNA
elfC N20R ATAAGAAGTGGCATAAGGAA
ACTAGTATTATACCTAGGACTGAGCTAGCTGTC
AAGGATC
pcaH
TTTTACCTCCTCTCTAGAAG
N20F1
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG
GCTAGTC
pcaH
CTTCTAGAGAGGAGGTAAAA
N20R1
ACTAGTATTATACCTAGGACTGAGCTAGCTGTC
AAGGATC
pcaH
AGAGGAGGTAAAACATATGC
N20F2
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG
GCTAGTC
pcaH
GCATATGTTTTACCTCCTCT
N20R2
ACTAGTATTATACCTAGGACTGAGCTAGCTGTC
AAGGATC
flu N20F
TACGAACTTCCCTGTGCGGG
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG
GCTAGTC
flu N20R
CCCGCACAGGGAAGTTCGTA
ACTAGTATTATACCTAGGACTGAGCTAGCTGTC
AAGGATC
Construction of elfC
sgRNA
Construction of pcaH
sgRNA
Construction of pcaH
sgRNA
Construction of pcaH
sgRNA
Construction of pcaH
sgRNA
Construction of flu
sgRNA
Construction of flu
sgRNA
Supplementary Table 4: Mutations observed in resequenced isolates. All nucleotide references
are relative to AG978 (BW25113 ompT::pcaHGBDC pflB::pcaIJFK)
Strain
JME1
Nucleotide Mutation
Deletion, 266,713-365,993
Duplication, 570,874-689,163
Duplication, 2,065,704-2,105,550
JME2
G2,076,174T
C2,853,953T
Deletion, 2,971,431-2,971,442
Duplication, 3,624,082-3,767,566
Deletion, 4,409,630-4,409,639
Deletion, 266,713-365,993
Duplication, 570,874-689,163
JME3
Deletion, 1,198,056-1,213,105
T1,311,771G
Deletion, 2,967,196-2,967,197
Deletion, 4,409,630-4,409,639
Duplication, 570,874-689,163
JME4
Duplication, 3,624,082-3,767,566
Duplication, 3,940,660-4,034,652
T3,991,518A
C4,174,713A
T580,465G
JME6
Deletion, 1,198,056-1,213,105
C2,072,530T
Deletion, 2,072,288-2,072,443
Duplication, 3,624,082-3,767,566
IS5 insertion
Duplication, 2,065,704-2,105,550
Deletion, 2,072,681-2,072,941
Duplication, 3,624,082-3,767,566
Deletion, 3,816,442
Notes
Contains lacI, mhpABC
Contains pcaHBGDC
IS5 flanks each side of duplication
Contains flu
IS5 flanks each side of duplication
Ag43 G684C, in both copies of flu
Intergenic
In-frame deletion of 12 bp in ptsP
Spans rhsB to rhsA, frequently duplicated
10 bp frameshift mutation in rnr
Contains lacI, mhpABC
Contains pcaHBGDC
IS5 flanks each side of duplication
Prophage excision
TopA M533R
2 bp frameshift mutation in ptsP
10 bp frameshift mutation in rnr
Contains pcaHBGDC
IS5 flanks each side of duplication
Spans rhsB to rhsA, frequently duplicated
CyaA W1072R
SecE A29E
pcaH RBS
See Supplementary Figure 1
Prophage excision
Silent mutation in Ag43
In-frame 156 bp deletion in flu
rhsB to rhsA
Between promoter and RBS of pcaH
See Supplementary Figure 1
flu at 2,071,645-2,074,769
IS5 flanks each side of duplication
In-frame 261 bp deletion in both copies of
flu
rhsB to rhsA
Intergenic
Supplementary Figure 1: Construct design. The eight-gene pathway for ortho PCA degradation,
along with the PCA transporter pcaK was designed as two operons, each expressed from a T5-lac
promoter. These operons were integrated into the indicated chromosomal loci in AG978. The
mutations to the ompT::pcaHGBDC operon found in JME4 and JME6 are indicated. A separate
expression construct for the 4-hydroxybenzoate 3-monooxygenase praI was inserted into the
elfC locus in strains JME7 and JME50.
Supplementary Figure 2: Mutations to flu decrease maximum optical density. A 261-bp deletion
in flu was introduced into AG978 and JME17, producing strains JME15 and JME16,
respectively. Strains were grown in minimal medium containing 1 g/L PCA as the sole source of
carbon and energy. In each case, the mutation reduces the maximum optical density of the
culture without improving the growth rate.
Supplementary Figure 3: A two-fold increase in RBS strength is the dominant strategy for PcaH
overexpression. (A) As measured by qPCR, the population averaged pcaH copy number
increases in replicate population B. The RBS mutation is first detected by targeted Sanger
sequencing at generation 500. Error bars show one standard deviation, calculated from three
biological replicates. The curve is a guide for the eye. (B) The RBS mutation, reconstructed in
JME17, is predicted to increase RBS strength roughly two-fold compared to the parent, AG978.
Engineered mutants with further increases in predicted RBS strength do not lead to a
corresponding increase in growth rate. Error bars show one standard deviation, calculated from
three biological replicates.