1

Supplementary Figures

Intr

acel

lula

r Ly

sine

(g

Lys/

g D

CW

)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

WL0(M9)

WL1(M9)

WL2(M9)

WL2(CM9)

WL3(CM9)

Supplementary Figure S1. Intracellular lysine concentrations of the constructed variants.

The intracellular lysine concentration of each strain was measured as described in Supplementary

Methods. WL0, WL1, and WL2 strains were cultured in M9 media. For the comparison between

WL2 and WL3 strains, they were cultured in complemented M9 (CM9) media (supplemented

with L-methionine and L-threonine to complement the auxotrophic phenotype of the WL3 strain).

The experiments were replicated twice.

2

Flu

ores

cenc

e (a

.u)

0

5

10

15

20

25

30

WLN0 WLN1

Supplementary Figure S2. The 5’ leader region of lysC in E. coli as a lysine riboswitch. The

fluorescence intensity of two strains with different intracellular concentrations of lysine was

measured when the 5’ leader region of lysC in E. coli was located at the region upstream of sgfp

(see Supplementary Methods for details). The y-axis is fluorescence in arbitrary units. The

experiments were replicated twice.

3

Sp

ecifi

cG

row

thR

ate

(h-1

)

0.00

0.05

0.10

0.15

0.20

WLR0 WLR1

Sp

ecifi

cG

row

thR

ate

(h-1

)

0.00

0.05

0.10

0.15

0.20

0.25

WLN0 WLN1

Sp

ecifi

cG

row

thR

ate

(h-1

)

0.00

0.05

0.10

0.15

0.20

0.25

WLM0 WLM1

Sp

ecifi

cG

row

thR

ate

(h-1

)

0.00

0.05

0.10

0.15

0.20

WLM0 WLM1

a b

c d

NiCl2

NiCl2

NiCl2

Tet

Supplementary Figure S3. Implementation of the Lysine Riboselector in WL0 and WL1

strains. (a) The relatively high-lysine producer grew faster under selection pressure (9 μM NiCl2

in M9) following introduction of the Lysine Riboselector. (b) The cellular growth rates of both

strains were similar without the Lysine Riboselector under selection pressure (9 μM NiCl2 in

M9). Strains harboring disabled Lysine Riboselector (LysRiboMut; Lysine binding was

destroyed and thus, tetA is constitutively expressed) (c) similarly grew in tetracycline (120

μg/mL in M9) (d) but did not grow in nickel (9 μM NiCl2 in M9). The y-axis represents the

specific growth rate (h-1), and the experiments were replicated twice.

4

a b

c d

Tet

NiCl2

NiCl2

Tet

Sp

ecifi

cG

row

thR

ate

(h-1

)

0.00

0.05

0.10

0.15

0.20

WLR1 WLR2

Sp

ecifi

cG

row

thR

ate

(h-1

)

0.00

0.05

0.10

0.15

0.20

WLN1 WLN2

Sp

ecifi

cG

row

thR

ate

(h-1

)

0.00

0.05

0.10

0.15

0.20

WLM1 WLM2

Sp

ecifi

cG

row

thR

ate

(h-1

)

0.00

0.05

0.10

0.15

0.20

WLM1 WLM2

Supplementary Figure S4. Implementation of the Lysine Riboselector in WL1 and WL2

strains. (a) The relatively high-lysine producer grew slower in tetracycline (120 μg/mL in M9)

following introduction of the Lysine Riboselector, indicating that the difference in growth rate in

NiCl2 was due to a change in tetA expression. (b) Without the Lysine Riboselector, both strains

grew well under the same selection pressure (21 μM NiCl2 in M9). Strains harboring disabled

Lysine Riboselector (c) similarly grew in tetracycline (120 μg/mL in M9) (d) but did not grow in

nickel (21 μM NiCl2 in M9). The y-axis represents the specific growth rate (h-1), and the

experiments were replicated twice.

5

a b

c d

Tet

NiCl2

NiCl2

Tet

Sp

ecifi

cG

row

thR

ate

(h-1

)

0.00

0.05

0.10

0.15

0.20

0.25

WLR2 WLR3

Sp

ecifi

cG

row

thR

ate

(h-1

)

0.00

0.05

0.10

0.15

0.20

0.25

WLN2 WLN3

Sp

ecifi

cG

row

thR

ate

(h-1

)

0.00

0.05

0.10

0.15

0.20

0.25

WLM2 WLM3

Sp

ecifi

cG

row

thR

ate

(h-1

)

0.00

0.05

0.10

0.15

0.20

0.25

WLM2 WLM3

Supplementary Figure S5. Implementation of the Lysine Riboselector in WL2 and WL3

strains. (a) The relatively high-lysine producer grew slower in tetracycline (120 μg/mL in

complemented M9) following introduction of the Lysine Riboselector, indicating that the

difference in growth rate in NiCl2 was due to a change in tetA expression. (b) Without the Lysine

Riboselector, both strains grew well under the same selection pressure (240 μM NiCl2 in

complemented M9). Strains harboring disabled Lysine Riboselector (c) similarly grew in

tetracycline (120 μg/mL in complemented M9) (d) but did not grow in nickel (240 μM NiCl2 in

complemented M9). The y-axis represents the specific growth rate (h-1), and the experiments

were replicated twice.

6

Spe

cific

gro

wth

rat

e (h

-1)

0.00

0.05

0.10

0.15WLR3WLN3

60 μMNiCl2

90 μMNiCl2

120 μMNiCl2

Supplementary Figure S6. Setting the selection pressure for enrichment. The cellular growth

rates of the parental strain, with and without lysine riboselector (WLR3 and WLN3), were

measured in different concentrations of NiCl2. The y-axis represents the specific growth rate (h-1),

and the experiments were replicated twice.

7

Time (h)

0 4 8 12 16 20 24

OD

600

0.1

1G

luco

se(g

/L)

0

1

2

3

4

Time (h)

0 4 8 12 16 20 24

Lysine

(g/L)

0.0

0.2

0.4

0.6

0.8WLRE2 WLRE3

Supplementary Figure S7. Lysine production by the enriched strains using Lysine

Riboselector. Culture data for the enriched strains WLRE2 (left panel) and WLRE3 (right panel)

are shown. The left y-offset and right y-axis represent glucose (circles) and lysine (triangles)

concentration (g/L), respectively. The left y-axis represents optical density (rectangles) at 600 nm

in log scale. The x-axis represents the culture time (h). The experiments were replicated twice.

8

Rel

ativ

e ppc

expr

essi

on le

vel

0

1

4

6

8

10

WLR

E1

WL

RE

2

WL

RE

3

WR

3

WLC

P1

WL

CP

2

WLC

P3

Supplementary Figure S8. Comparison of ppc expression levels between enriched (plasmid-

based and chromosome-based) and parental strains. Relative changes in ppc mRNA

expression were monitored by RT-QPCR (Reverse Transcription Quantitative PCR) and

calculated using the comparative CT method (2-ΔΔCt) with gapA as a reference (See

Supplementary Methods for details). The y-axis represents the relative expression level. The

experiments were replicated twice.

9

Time (h)

0 4 8 12 16 20 24

OD

600

0.1

1

Glu

cose

(g/L

)

0

1

2

3

4

Time (h)

0 4 8 12 16 20

Time (h)

0 4 8 12 16 20 24 28

Lysine

(g/L)

0.0

0.2

0.4

0.6

0.8WLCP1 WLCP2 WLCP3

Supplementary Figure S9. Lysine production by the strains whose chromosomal ppc

promoters are switched with the enriched promoters (WLCP1, WLCP2, and WLCP3).

Culture data for the strains WLCP1 (left panel), WLCP2 (middle panel), and WLCP3 (right

panel) are shown. The left y-offset and right y-axis represent glucose (circles) and lysine

(triangles) concentration (g/L), respectively. The left y-axis represents optical density

(rectangles) at 600 nm in log scale. The x-axis represents the culture time (h). The experiments

were replicated twice.

10

Enrichment cycles

1st 2nd 3rd 4th

Per

cent

age

(%)

0

10

20

30

40

WLNE1WLNE2WLNE3WLNE4WLNE5WLNE6WLNE7WLNE8WLNE9WLNE10

Supplementary Figure S10. A population analysis of the enriched strains without Lysine

Riboselector (RiboNULL). Next-generation sequencing was used to examine the promoter

region of each variant for population analysis. The y-axis represents the percentage of each

variant in the total population. The x-axis represents the enrichment cycle, where each

enrichment cycle corresponds to three serial cultures.

11

Time (h)

0 4 8 12 16 20 24

OD

600

0.1

1

Glu

cose

(g/L

)

0

1

2

3

4

Time (h)

0 4 8 12 16 20 24

Time (h)

0 4 8 12 16 20 24

Lysine

(g/L)

0.0

0.2

0.4

0.6

0.8WLNE1 WLNE2 WLNE3

Supplementary Figure S11. Lysine production by the enriched strains without Lysine

Riboselector (RiboNULL). Culture data for the enriched strains without Lysine Riboselector

WLNE1 (left panel), WLNE2 (middle panel), and WLNE3 (right panel) are shown. The left

y-offset and right y-axis represent glucose (circles) and lysine (triangles) concentration (g/L),

respectively. The left y-axis represents optical density (rectangles) at 600 nm in log scale. The x-

axis represents the culture time (h). The experiments were replicated twice.

12

Supplementary Tables

Supplementary Table S1. Sequences of the enriched ppc promoters using LysRibo.

Strains Promoter Sequences (5’-3’)a WLRE1 GAACAAGCTAGCTCAGTCCTAGGCGGCAAGCTAGC WLRE2 CCCCCAGCTAGCTCAGTCCTAGGTAGCGGGCTAGC WLRE3 CAAATTGCTAGCTCAGTCCTAGGAAAAGGGCTAGC

a Bold letters indicate randomized regions.

13

Supplementary Table S2. Sequences of the selected ppc promoters when enriched using RiboNULL.

Strains Promoter Sequences (5’-3’)a WLNE1 GCACCTGCTAGCTCAGTCCTAGGCACAGCGCTAGC WLNE2 AAAGAGGCTAGCTCAGTCCTAGGGGTCCAGCTAGC WLNE3 CTATCTGCTAGCTCAGTCCTAGGCATGGTGCTAGC WLNE4 ACGTAAGCTAGCTCAGTCCTAGGTAATCGGCTAGC WLNE5 TCTAACGCTAGCTCAGTCCTAGGATAACGGCTAGC WLNE6 GCGGTAGCTAGCTCAGTCCTAGGTTTGTCGCTAGC WLNE7 CATACAGCTAGCTCAGTCCTAGGACTGACGCTAGC WLNE8 TGAATCGCTAGCTCAGTCCTAGGAGCTCGGCTAGC WLNE9 ACGAAAGCTAGCTCAGTCCTAGGTGCAGCGCTAGC WLNE10 AGGTAAGCTAGCTCAGTCCTAGGAATAGCGCTAGC

a Bold letters indicate randomized regions.

14

Supplementary Table S3. Strains and plasmids used in this study.

Name Relevant characteristics Source Strains

Mach1-T1R F- φ80(lacZ)ΔM15 ΔlacX74 hsdR(r

K

-m

K

+)

ΔrecA1398 endA1 tonA

Invitrogen

DH5α F- φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (r

k-, m

k+) phoA supE44 λ- thi-1

gyrA96 relA1

Invitrogen

DH10B-T1R φ80lacZ ΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ- rpsL nupG tonA

Invitrogen

W3110 (WL1) F- λ- rph-1 IN(rrnD, rrnE)1 ATCC 27325 WL0 W3110 ΔlysC This study WL2 WL0 BBa_J23100_lysCfbr This study WL3 WL2 PdapA::BBa_J23100, PdapB::BBa_J23100,

PlysA::BBa_J23100, lacZYA::BBa_J23100-ddh, ΔmetL, ΔthrA, ΔiclR

This study

WLN1 W3110 / RiboNULL This study WLN0 WL0 / RiboNULL This study WLN2 WL2 / RiboNULL This study WLN3 WL3 / RiboNULL This study WLN4 WLN3Δppc This study WLNE1 WLN4 / pCDF-WLNE1ppc This study WLNE2 WLN4 / pCDF-WLNE2ppc This study WLNE3 WLN4 / pCDF-WLNE3ppc This study WLM1 W3110 / LysRiboMut This study WLM0 WL0 / LysRiboMut This study WLM2 WL2 / LysRiboMut This study WLM3 WL3 / LysRiboMut This study WLM4 WLM3Δppc This study WLR1 W3110 / LysRibo This study WLR0 WL0 / LysRibo This study WLR2 WL2 / LysRibo This study WLR3 WL3 / LysRibo This study WLR4 WLR3Δppc This study WLRE1 WLR4 / pCDF-WLRE1ppc This study WLRE2 WLR4 / pCDF-WLRE2ppc This study WLRE3 WLR4 / pCDF-WLRE3ppc This study WLCP1 WL3 ppc::WLRE1ppc This study WLCP2 WL3 ppc::WLRE2ppc This study WLCP3 WL3 ppc::WLRE3ppc This study WT0 W3110 ΔaroG This study WTR1 W3110 / TrpRibo This study WTR0 WT0 / TrpRibo This study

15

Plasmids

pKD46 Red recombinase expression vector; AmpR pCP20 FLP expression vector; AmpR pACYCDuet Expression vector, CmR, p15A ori Novagen pCDFDuet Expression vector, SmR, cloDF13 ori Novagen pGEM T-Easy Cloning vector, AmpR Promega pMD20 Cloning vector, AmpR Takara pGFKF pGEM-FRT-KanR-FRT-BBa_J23100 This study pGFKF1 pGEM-FRT-KanR-FRT-NdeI-XhoI This study pGFKF2 pGEM-FRT-KanR-FRT-KpnI-SacI This study pGFKF1-ddh pGFKF1-NdeI-BBa_J23100-ddh-XhoI This study pGFKF2-lysC pGFKF2-KpnI-BBa_J23100-lysCfbr-SacI This study pMD20-FKF(f72)

pMD20-FRT(f72)-KanR-FRT(f27) This study

pMD20-FKF(f72)-WLRE1ppc

pMD20- FRT(f72)-KanR-FRT(f27)-KpnI-WLRE1ppc-SacI

This study

pMD20-FKF(f72)-WLRE2ppc

pMD20- FRT(f72)-KanR-FRT(f27)-KpnI-WLRE2ppc-SacI

This study

pMD20-FKF(f72)-WLRE3ppc

pMD20- FRT(f72)-KanR-FRT(f27)-KpnI-WLRE3ppc-SacI

This study

LysRibo pACYCDuet-KpnI-BBa_J23100-lysC UTR-tetA-SacI

This study

LysRiboMut pACYCDuet-KpnI-BBa_J23100-lysC UTR*(G31C)-tetA-SacI

This study

RiboNULL pACYCDuet-KpnI-BBa_J23100-lysC UTR-sgfp-SacI

This study

pCDF-ppc pCDFDuet-KpnI-ppc-SacI This study pCDF-ppc-Lib pCDFDuet- KpnI-N6-gctagctcagtcctagg-N6-

gctagc-UTR-ppc-SacI This study

TrpRibo_Lib pACYCDuet-J23100-TrpApt-UTR-N10-tetA-sgfp

This study

TrpRibo pACYCDuet-J23100-TrpApt-UTR-gagggtaaga-tetA-sgfp

This study

16

Supplementary Table S4. Primers used in this study.

Name Sequence (5’-3’)a,b M-pGFKF2-F ggaactgtcaacgGGTACCtctcgacatcatcac M-pGFKF2-R gtgatgatgtcgagaGGTACCcgttgacagttcc FRT(f72)-Kan-F

gcatgaccggcgcgatgcgaagttcctatactttctacagaataggaacttctcaagatcccctcacgctgccg

FRT(f72)-Kan-R

gctcagcggatctcatgcgcgaagttcctattctgtagaaagtataggaacttcagagcgcttttgaagctggggtgg

FKF(f72)-TA-F agcatgaccggcgcgatgc FKF(f72)-TA-R cctaggactgagctagccgtcaactcgaggctcagcggatctcatgcgc pMD20-FKF(f72)-AD-KpnI-F

gggatccgattgcatggtaccggcgcgatgcg

FKF(f72)-AD-KpnI-R

cgcatcgcgccggtaccatgcaatcggatccc

D-lysC-F gactttggaagattgtagcgccagtcacagaaaaatgtgatggttttagtgcgatgcctcatccgcttctc

D-lysC-R gacaagaaaatcaatacggcccgaaatatagcttccaggccatacagtatgcaacgcagtagctggagtc

S-lysC-up-F acatatgtctgaaattgttgtctccaaatttgg M-lysC-up-R ccggtggtatcaaggattaatgccacgctcac M-lysC-down-F caccacgtcagaagtgagcgtggcattaatccttgataccaccggttcaacctccac S-lysC-down-R actcgagttactcaaacaaattactatgcagtttttgcac

P-lysC-F gactttggaagattgtagcgccagtcacagaaaaatgtgatggttttagtgcgttgctcctgacatggctc

P-lysC-R gacaagaaaatcaatacggcccgaaatatagcttccaggccatacagtatgcatccaacgcgttgggagctcc

ph-DNdeI-F gaatatcctccttagttcctattccgaagttcc ph-DNdeI-R ggatgaacgaaatagacagatcgc

P-dapA-F ggaaagcataaaaaaaacatgcatacaacaatcagaacggttctgtctgcatgggaattagccatggtccatatg

P-dapA-R atcgcgacaatacttcccgtgaacatgggccatcctctgtgcaaacaagtgctagcactgtacctaggactgagc

P-dapB-F gtaacctgtcacatgttattggcatgcagtcattcatcgactcatgccatgggaattagccatggtcc

P-dapB-R ggatgtttgcatcatgcatagctattctcttttgttaatttgcatagaccgctagcactgtacctaggactgagc

P-lysA-F gccattagcgctctctcgcaatccggtaatccatatcatttttgcatagagaatatcctccttagttcctattccgaag

P-lysA-R agattttcggcggtgagatcggtatcggtgctgaacagtgaatgtggcatatgtatatctccttcttaaagttaaacaa

S-ddh-F aCATATGaccaacatccgcgtagctatcgtgg S-ddh-R aCTCGAGctaaattagacgtcgcgtg P-ddh-F ttaaactgacgattcaactttataatctttgaaataatagtgcttatcccgtctatttcgttcatccgaatatc

17

ctcc

P-ddh-R gcggtatggcatgatagcgcccggaagagagtcaattcagggtggtgaatgagctccaaaaaacccctcaagac

D-met-F atgagtgtgattgcgcaggcaggggcgaaaggtcgtcagctgcataaattctagtgctggagcgaactgc

D-met-R aaaccataaacccgaaaacatgagtaccgggcattattaaatttctgaaaggagtactcgcggttgactg

D-thr-F gcgtacaggaaacacagaaaaaagcccgcacctgacagtgcgggctttttgttagcccgtctgtcccaa

D-thr-R tgattcatcatcaatttacgcaacgcagcaaaatcggcgggcagattatgcatactcgctcttgggtcgg

D-iclR-F ctgtggtaaaagcgaccaccacgcaacatgagatttgttcaacattaactcgtagcaccgagtcgtaccag

D-iclR-R gcattccaccgtacgccagcgtcacttccttcgccgctttaatcaccatccggtactggcctaacgcact

D-ppc-F ccagtgccgcaataatgtcggatgcgatacttgcgcatcttatccgaccgttagcccgtctgtcccaa D-ppc-R gcagacagaaatatattgaaaacgagggtgttagaacagaagtaTcatactcgctcttgggtcgg

D-aroG-F gatctcgtttttcgcgacaatctggcgtttttcttgctaattccaggatgagatgggaattagccatggtcc

D-aroG-R cggttgcaaaccagggtaaagcgaagtaaacgtcattcgtttaaaatgaggtgtaggctggagctgcttc

C-lysC-F ctgccaggcagcggtctgc C-lysC-R cgggaattcgtttgcgagcagaac C-lysC-R2 cagctacgctggtgccgccaaatttggag C-dapA-F caacgcagtgatcaccagataatgttg C-dapA-R ttaccggaatgcgcccatcagc C-dapB-F ttaccggaatgcgcccatcagc C-dapB-R cagcgtaccttccggacggg C-lysA-F ggtgtgccgcctcagtcagg C-lysA-R tccactttcacgccctgctc C-ddh-F agtatcagcggcaattacctgatggactgg C-ddh-R cggtggcggcttcgttcatgac C-met-F cgctggcggaatcattaggg C-met-R cgaaaaatgaccaatgatggtg C-thr-F ccaatataggcatagcgcacag C-thr-R gcccgttatgggtcgatatccg C-iclR-F ctgcgcacgcagttgttcc C-iclR-R gtgtgtgaagtgtatgac C-ppc-F ccggtgagcgaacgctggcc C-ppc-R attccttaaggatatctgaagg C-aroG-F ccaggttatgaaacgcagcagagaatcttg C-aroG-R gttcgacgagaatttcaaaccgctgaaacg Pro-LUTR-F attgacggctagctcagtcctaggtacagtgctagcgtactacctgcgctagcgca

18

LUTR-R aactacctcgtgtcaggggatccat

L-sgfp-F ctcttcccttgtgccaaggctgaaaatggatcccctgacacgaggtagttatggctagcaagggcgaggagctgt

sgfp-R acaaaaaacccctcaagacccgtttagaggc KpnI-Pro-F aGGTACCttgacggctagctcagtcctaggtacagtg

L-tetA-F ctcttcccttgtgccaaggctgaaaatggatcccctgacacgaggtagttatgaaatctaacaatgcgctc

tetA-R aGAGCTCgtggccaggacccaacgctgcccga LysRiboMut-G31C-F

gcgcaggccagaagacgcgcgttgcccaag

LysRiboMut-G31C-R

cttgggcaacgcgcgtcttctggcctgcgc

M-pCDL-F ctcgagtctGGTACCgaaaccgctgctgcg M-pCDL-R cgcagcagcggtttcGGTACCagactcgag

S-ppc-F aGGTACCacaggttcagagttctacagtccgacatgagcaaaggtttcagtaggaggaaagaacaatgaacgaacaatattccgcattgcg

S-ppc-R aGAGCTCgagggtgttagaacagaagtatttc

FL-ppc-F gcagacagaaatatattgaaaacgagggtgttagaacagaagtatgagggtgttagaacagaagtatttcag

FL-ppc-R gtcggatgcgatacttgcgcatcttatccgaccgttagcccgtctgtcccggctcagcggatctcatgcgc

ph-UTR-ppc-F gaattcgtatgccgtcttctgcttgtcgaggaggatcccaatgaacgaacaatattccgcattgcg ph-UTR-ppc-R tcggactgtagaactctgaacctgtgg

ph-J23N-F gctagcNNNNNNcctaggactgagctagcNNNNNNcatgtcggactgtagaactctgaacc

ph-J23N-R ttcgtatgccgtcttctgcttgtcg

ph-T-Library-F atatttgacggctagctcagtcctaggtacagtgctagcctggacgacggggacgccactggactaggtaagccaggaccgtacgtcgggagccgtcagaataNNNNNNNNNNaaggagcatctatgaaatctaacaatgcgct

ph-T-Library-R ttaaggtaccgcgcaacgcaattaatgtaagtta T-tetA-F aGGTACCaaggagcatctatgaaatctaacaatgcgctcatcgtca

T-tetA-R gctcccaccgccactcccaccgccggacccaccgcccgacccaccgccggtcgaggtggcccggct

T-sgfp-F ggcggtgggtcgggcggtgggtccggcggtgggagtggcggtgggagcgctagcaagggcgaggagct

T-sgfp-R aGAGCTCtcacttgtacagctcgtccatgcc

a Capital letters indicate restriction sites.

b Underlined letters indicate homologous sequences for recombination and overlap PCR.

c Underlined and capital letters indicate randomized sequences for library construction.

d Primer names beginning with a “C” indicate primers used to check Red recombination.

19

Supplementary Methods

Construction of bacterial strains and plasmids

Phusion polymerase and restriction endonuclease were purchased from New England

Biolabs (Beverly, MA, USA). pGFKF was constructed by subcloning the amplified FRT-KanR-

FRT-BBa_J23100 into the pGEM T-Easy vector. pGFKF1 was constructed by polymerize chain

reaction (PCR)-based blunt-end ligation with ph-DNdeI-F and ph-DNdeI-R primers using

pGFKF as a template. pGFKF2 was constructed by adding a KpnI restriction site through PCR-

based site-directed mutagenesis with M-pGFKF2-F and M-pGFKF2-R primers using pGKF1 as

a template. pMD20-FKF(f72) was constructed by subcloning FRT(f72)-KanR-FRT(f72) fragment

amplified by FRT(f72)-Kan-F and FRT(f72)-Kan-R into the pMD20 vector and adding a KpnI

restriction site through PCR-based site-directed mutagenesis with FKF(f72)-AD-KpnI-F and

FKF(f72)-AD-KpnI-R primers.

All chromosomal work was done using the Red recombination system with pKD46 and

pCP20, as described in previous studies44,45. The WL0 strain was constructed by deleting the

chromosomal lysC gene of E. coli W3110 with an FRT-KanR-FRT fragment amplified by D-

lysC-F and D-lysC-R primers. The WL2 strain was constructed by constitutively overexpressing

the feedback-resistant lysC (lysCfbr) in the WL0 strain. lysCfbr, constructed by overlap PCR using

primers S-lysC-up-F, M-lysC-up-R, M-lysC-down-F, and S-lysC-down-R to introduce a point

mutation at nucleotide 1055 of the lysC gene24, was amplified with S-lysC-up-F and S-lysC-

down-R primers and inserted into the KpnI and SacI restriction sites of pGFKF2. Thereafter,

BBa_J23100-lysCfbr-FRT-KanR-FRT, amplified with P-lysC-F and P-lysC-R primers, was

introduced into the position of lysC in the WL0 strain by recombination. The WL3 strain was

constructed by overexpressing native and heterologous pathway enzymes for lysine production

20

and deleting genes for competing pathways. This was accomplished by amplifying the ddh gene

from Corynebacterium glutamicum with S-ddh-F and S-ddh-R primers and subcloning it into the

NdeI and XhoI restriction sites of pGFKF2. Then, the BBa_J23100-ddh-FRT-KanR-FRT

fragment, amplified with P-ddh-F and P-ddh-R primers, was introduced into the position of the

lac operon of the WL2 strain by recombination. The promoter regions of dapA, dapB, and lysA

of this latter strain were replaced with the BBa_J23100 promoter using P-dapA-F and P-dapA-R,

P-dapB-F and P-dapB-R, and P-lysA-F and P-lysA-R primers. Lastly, metL, thrA, and iclR were

deleted using fragments amplified with D-met-F and D-met-R, D-thr-F and D-thr-R, and D-iclR-

F and D-iclR-R primers, respectively. The WL4 strain was constructed by deleting ppc from the

WL3 strain using a fragment amplified with D-ppc-F and D-ppc-R primers. The WT0 strain was

constructed by deleting the chromosomal aroG of E. coli W3110 with an FRT-KanR-FRT

fragment amplified with D-aroG-F and D-aroG-R primers. Each strain was confirmed by PCR

and sequencing using primers listed in Supplementary Table S4.

LysRiboMut was constructed by site-directed mutagenesis of LysRibo with

LysRiboMut-G31C-F and LysRiboMut-G31C-R primers. The region for the point mutation to

disable lysine binding to the aptamer was selected based on the previous study21. The mutation

that disabled the function of B. subtilis lysine riboswitch (G39C for B. subtilis lysine riboswitch)

was introduced in the corresponding conserved region of E. coli lysine riboswitch (G31C).

RiboNULL was constructed by assembling the BBa_J23100-lysC_UTR-sgfp fragment using

overlap PCR of two fragments: one amplified with Pro-LUTR-F and LUTR-R primers using

genomic DNA of E. coli W3110 as a template, and the other amplified with L-sgfp-F and sgfp-R

using the sgfp gene as a template. The overlap-PCR product, amplified by KpnI-Pro-F and sgfp-

R, was inserted into KpnI and SacI restriction sites of pACYCDuet. LysRibo was constructed as

21

described above for BBa_J23100-lysC_UTR-tetA except using L-tetA-F and tetA-R to amplify

tetA from pBR322 (Fermentas, Glen Burnie, MD, USA).

The promoter library for ppc was constructed by first inserting ppc, amplified with S-

ppc-F and S-ppc-R primers, into the KpnI and SacI restriction sites of a modified pCDFDuet

plasmid. Additionally, adapter sequences for next generation sequencing were added by PCR-

based blunt-end ligation using ph-UTR-ppc-F and ph-UTR-ppc-R primers, resulting in pCDF-

ppc, which is the template for ppc promoter library generation. The ppc promoter library was

generated by PCR-based blunt-end ligation using ph-J23N-F and ph-J23N-R primers, which

fully randomize the -35 and -10 boxes of the BBa_J23100 promoter (pCDF-ppc-Lib). The top 3

enriched ppc promoter sequences were integrated into the genomic region. Each enriched

plasmid was digested by KpnI and SacI restriction sites and ligated into pMD20-FKF(f72). Using

FL-ppc-F and FL-ppc-R primers, each ppc region containing enriched promoter sequences was

amplified and introduced into the position of the genome through Red recombination.

The Tryptophan Riboselector library plasmid (TrpRibo_Lib) was constructed by

combining the tetA fragment (amplified by T-tetA-F and T-tetA-R primers using pBR322 as a

template) and the sgfp fragment (amplified by T-sgfp-F and T-sgfp-R using the sgfp gene as a

template), amplifying the resulting fragment by overlap PCR using T-tetA-F and T-sgfp-R (tetA

and sgfp are fused by a GGGSx4 linker16), and ligating it into KpnI and SacI restriction sites of a

modified pACYCDuet vector. This plasmid was amplified by ph-T-Library-F and ph-T-Library-

R primers and blunt-end ligated to generate the Tryptophan Riboselector library (TrpRibo_Lib)..

Detection of intracellular lysine concentrations

In order to measure the intracellular concentrations of lysine, cells were cultured in

22

same media used for survival tests in mixed culture (WL0, WL1, and WL2 in M9 and WL2 and

WL3 in complemented M9). At mid-exponential phase, cells were harvested and prepared as

described in a previous study46. The detection of intracellular lysine was performed by Korea

Basic Science Institute (KBSI) using phenylisothiocyanate (PITC) derivatization method coupled

with reversed-phase LC column chromatography (Nova-Pak C18; Waters, Milford, MA, USA)

using an Agilent 1100 Series HPLC system (Agilent, Palo Alto, CA, USA) with UV detector at

254 nm.

Fluorescence affected by the leader region of E. coli lysC

The response of the leader region of lysC to intracellular lysine concentration was tested

by inoculating fresh seeds of WLN0 and WLN1 strains in 4 mL of M9 medium containing 34

μg/mL of chloramphenicol and all amino acids, except lysine. After 6 hours of incubation,

fluorescence intensity was measured. Specific SGFP activity was defined as fluorescence

normalized to the OD600 value of the culture after subtracting normalized fluorescence values of

negative controls. The density of E. coli was measured at a wavelength of 600 nm using a UV-

1700 spectrophotometer (Shimadzu, Kyoto, Japan). Fluorescence was detected using a

VICTOR3TM 1420 Multilabel Counter (PerkinElmer, Waltham, MA, USA). The detection was

carried out using a 486-nm excitation filter and a 535-nm emission filter with a 1-sec

measurement time.

23

Supplementary References

44 Lim, S. I., Min, B. E. & Jung, G. Y. Lagging strand-biased initiation of red recombination

by linear double-stranded DNAs. J. Mol. Biol. 384, 1098-1105 (2008).

45 Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in

Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U. S. A. 97, 6640-6645

(2000).

46 Tweeddale, H., Notley-McRobb, L. & Ferenci, T. Effect of slow growth on metabolism of

Escherichia coli, as revealed by global metabolite pool ("metabolome") analysis. J.

Bacteriol. 180, 5109-5116 (1998).