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Figures
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405
Figure 1. Metabolic engineering of caffeic acid producing E. coli. The blue X indicates disruption of
406
the ptsH, ptsI, trpE and pheA genes. Red or orange shows genes involved in caffeic acid synthesis
407
overexpressed by plasmid or genomic integration. G6P, glucose 6-phosphate; PEP,
408
phosphoenolpyruvate; E4P, erythrose 4-phosphate; DAHP, 3-deoxy-d-arabinoheptulosonic acid 7-
409
phosphate; PP pathway, pentose–phosphate pathway; SHK pathway, shikimate pathway; RgTAL,
410
tyrosine ammonia-lyase from Rhodotorula glutinis; SeC3H, p-coumaric acid 3-hydroxylase from
411
Saccharothrix espanaensis; PaHpaBC, hydroxylase complexes 4HPA3H from Pseudomonas
412
aeruginosa; tyrAfbr, feedback-resistant chorismate mutase/prephenate dehydrogenase; galP, d-
413
galactose transporter; glk, glucokinase; ptsH, phosphocarrier protein HPr; ptsI, phosphoenolpyruvate-
414
protein phosphotransferase; pykF, pyruvate kinaseⅠ; pykA, pyruvate kinaseⅡ; tktA, transketolase;
415
aroGfbr, feedback-resistant 3-deoxy-7-phosphoheptulonate synthase; trpE, anthranilate synthase
416
component I; pheA, chorismate mutase/prephenate dehydratase.
417
16
0.5
Caffeic acid [g/L]
0.4
0.3
0.2
0.1
pZE12-RS pTrcHisB-RS
PHLA-RS
pSAK-RS
418
419
Figure 2. Caffeic acid production after 48 h of cultivation in M9Y medium containing 0.5 g/L L-
420
tyrosine and 20 g/L glucose using the strains harboring plasmid pZE12-RS, pTrcHisB-RS, pHLA-RS,
421
or pSAK-RS. The data shown are as the means and standard deviations of three independent
422
experiments.
2.0
p-Coumaric acid
Caffeic acid
Caffeic acid [g/L]
1.5
1.0
0.5
CA0/RS
CA1
CA2
CA3
423
424
Figure 3. Caffeic acid production after 48 h of cultivation in M9Y medium containing 20 g/L glucose
425
using strains CA1, CA2, and CA3. The data shown are as the means and standard deviations of three
426
independent experiments
427
17
428
429
Figure 4. Caffeic acid production after 48 h of cultivation in M9Y medium containing 20 g/L glucose
430
using the strains CA4-CA8. The data shown are as the means and standard deviations of three
431
independent experiments.
4.0
p-Coumaric acid
Caffeic acid
3.5
Caffeic acid [g/L]
3.0
2.5
2.0
1.5
1.0
0.5
CA8
CA9
CA10
CA11
432
433
Figure 5. Caffeic acid production after 48 h of cultivation in M9Y medium containing 20 g/L glucose
434
using the strains CA8-CA11. The data shown are as the means and standard deviations of three
435
independent experiments
436
18
7.0
6.0
OD600 [-] / Glucose [g/L]
40
5.0
30
4.0
3.0
20
2.0
10
1.0
p-Coumaric acid / Caffeic acid [g/L]
50
24
437
48
Time [h]
72
96
438
Figure 6. CA8 culture profiles in a jar fermenter. Blue squares, green triangles, light-brown and dark-
439
brown symbols indicate cell growth, glucose concentration, p-coumaric acid concentration and caffeic
440
acid concentration, respectively. The data shown are as the means and standard deviations of three
441
independent experiments.
442
19
443
Table 1. Strains and plasmids used in this study.
Strains and Plasmids
Strains
Genotype
Reference
NovaBlue
endA1 hsdR17(rK12-mK12+) supE44 thi-1 gyrA96 relA1
Novagen
lac recA1/F [proAB+ laclqZΔM15 Tn10(TetR)]
ATCC31882
L-Phenylalanine-overproducing
strain
ATCC
(aroF aroG tyrR pheA tyrA trpE)
CFT1
ATCC31882ΔptsHI::PA1lacO1-glk-galP
Noda et al
CA0
CFT1ΔpheA
This study
CA0/RS
CFT1ΔtrpEΔpheA/pZE12-RS
This study
CA1
CFT1ΔtrpEΔpheA/pZE12-RS+pSAK-Ptrc-tyrAfbr
This study
CA2
CFT1ΔtrpE::Ptrc-tyrAfbrΔpheA/pZE12-RS
This study
CA3
CFT1ΔtrpE::PA1lacO1-tyrAfbrΔpheA/pZE12-RS
This study
CA4
CFT1ΔtrpE::PA1lacO1-tyrAfbrΔpheA/pZE12-RS+pSAK-BC
This study
CA5
CFT1ΔtrpE::PA1lacO1-tyrAfbrΔpheA::PA1lacO1-
This study
PaHpaB/pZE12-RS
CA6
CFT1ΔtrpE::PA1lacO1-tyrAfbrΔpheA/pZE12-RS+pSAK-B
This study
CA7
CFT1ΔtrpE::PA1lacO1-tyrAfbrΔpheA::PA1lacO1-
This study
PaHpaC/pZE12-RS
CA8
CFT1ΔtrpE::PA1lacO1-tyrAfbrΔpheA/pZE12-RS+pSAK-C
This study
CA9
CFT1ΔtrpE::PA1lacO1-tyrAfbrΔpheA::PA1lacO1-
This study
aroGfbr/pZE12-RS+pSAK-C
CA10
CFT1ΔtrpE::PA1lacO1-tyrAfbrΔpheA::PA1lacO1-tktA/pZE12-
This study
RS+pSAK-C
CA11
CFT1ΔtrpE::PA1lacO1-tyrAfbrΔpheAΔpykAF/pZE12-
This study
RS+pSAK-C
Plasmids
Characteristic
Reference
20
pZE12-MCS
PLlacO1, colE ori, and Ampr
Expressys
pTrcHisB
Ptrc, pBR322 ori, and Ampr
Life
Technologies
pHLA
PHCE, colE1 ori, and Ampr
Tanaka et al.,
2011
pSAK
PA1lacO1, SC101 ori, and Cmr
Noda et al.,
2017
pZE12-RS
pZE12 containing RgTAL and SeC3H
This study
pTrcHisB-RS
pTrcHisB containing RgTAL and SeC3H
This study
pHLA-RS
pHLA containing RgTAL and SeC3H
This study
pSAK-RS
pSAK containing RgTAL and SeC3H
This study
pSAK-Ptrc-tyrAfbr
pSAK-Ptrc containing tyrAfbr
Fujiwara et al.,
2020
pSAK-BC
pSAK containing PaHpaBC
This study
pSAK-B
pSAK containing PaHpaB
This study
pSAK-C
pSAK containing PaHpaC
This study
pSAK-aroGfbr
pSAK containing aroGfbr
This study
pSAK-tktA
pSAK containing tktA
This study
pTargetF
Constitutive expression of sgRNA
Addgene
pCas
Constitutive expression of cas9 and inducible expression
Addgene
of λ RED and sgRNA
pTΔtrpE
Constitutive expression of sgRNA with donor editing
Fujiwara et al.,
template DNA for trpE disruption
2020
Constitutive expression of sgRNA with donor editing
Fujiwara et al.,
template DNA for Ptrc-tyrAfbr into trpE gene loci
2020
pTΔtrpE::PA1lacO1-
Constitutive expression of sgRNA with donor editing
Fujiwara et al.,
tyrAfbr
template DNA for PA1lacO1-tyrAfbr into trpE gene loci
2020
pTΔtrpE::Ptrc-tyrAfbr
21
pTΔpykA
pTΔpykF
pTΔpheA
Constitutive expression of sgRNA with donor editing
Noda et al.,
template DNA for pykA disruption
2016
Constitutive expression of sgRNA with donor editing
Noda et al.,
template DNA for pykF disruption
2016
Constitutive expression of sgRNA with donor editing
This study
template DNA for pheA disruption
pTΔpheA::PA1lacO1-
Constitutive expression of sgRNA with donor editing
PaHpaB
template DNA for PA1lacO1-PaHpaB into pheA gene loci
pTΔpheA::PA1lacO1-
Constitutive expression of sgRNA with donor editing
PaHpaC
template DNA for PA1lacO1-PaHpaC into pheA gene loci
pTΔpheA::PA1lacO1-
Constitutive expression of sgRNA with donor editing
aroGfbr
template DNA for PA1lacO1-aroGfbr into pheA gene loci
pTΔpheA::PA1lacO1-
Constitutive expression of sgRNA with donor editing
tktA
template DNA for PA1lacO1-tktA into pheA gene loci
444
445
Graphical abstract
Glucose
Escherichia coli
G6P
PEP
E4P
SHK
Pathw ay
Chorismic acid
L-Tyrosine
RgTAL
TCA
cycle
446
p-Coumaric acid
PaHpaB
PaHpaC
SeC3H
Caffeic acid
Caffeic acid
22
This study
This study
This study
This study
...
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