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Supplementary Methods, Results, Tables and Figures
Systems metabolic engineering of Escherichia coli for Lthreonine production
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Kwang Ho Lee1,3, Jin Hwan Park1, Tae Yong Kim1, Hyun Uk Kim1, Sang Yup Lee1,2,*
1
Metabolic and Biomolecular Engineering National Research Laboratory, Department
of Chemical and Biomolecular Engineering (BK21 program), BioProcess Engineering
Research Center, Center for Ultramicrochemical Process Systems, Center for Systems
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and Synthetic Biotechnology, and Institute for the BioCentury, Korea Advanced
Institute of Science and Technology (KAIST), Daejeon 305-701, Korea
2
Department of BioSystems and Bioinformatics Research Center, KAIST, Daejeon
305-701, Korea
3
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R&D Center for Bioproducts, CJ Corp., Seoul 157-724, Korea
* Correspondence author. Metabolic and Biomolecular Engineering National Research
Laboratory, Department of Chemical and Biomolecular Engineering, Korea Advanced
Institute of Science and Technology (KAIST), Daejeon 305-701, Korea. Tel.: +82 42
869 3930; Fax: +82 42 869 8800; E-mail: [email protected]
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Supplementary Methods
Strains and plasmids
For the construction of pKKThrABC plasmid, structural genes, including mutant thrA
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(thrAC1034T), thrB, and thrC, were amplified by PCR with oligonucleotide primers
Thr_Xma and Thr_Hin using the genomic DNA of E. coli TH01 (W3110 △lacI
thrAC1034T) as a template. The PCR product was digested with XmaI and HindIII, and
ligated with XmaI-HindIII-digested DNA fragment of pKK223-3 vector. The resulting
plasmid, pKKThrABC, contains the thrAC1034T, thrB, and thrC genes under the control
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of strong tac promoter. Then, the NdeI-SalI-digested 1.6-kb DNA fragment of pBR322
was ligated with the 7.8-kb NdeI-SalI-digested DNA fragment of the pKKThrABC to
make a medium copy number plasmid pBRThrABC. To make pBRThrABCR, rhtC gene
was amplified with primers FrhtCEcBa and RrhtCSpMl, digested with BamHI and SphI,
and ligated with the BamHI-SphI-digested 9.2-kb fragment of pBRThrABC. To make
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pBRThrABCR2, rhtA gene was amplified with primers FrhtAMlu and RrhtASalPst,
digested with MluI and SalI, and ligated with the MluI-SalI-digested pBRThrABCR.
Finally, for the amplification of the rhtB gene, PCR was performed with primers
FrhtBPst and RrhtBEagAat, digested with PstI and EagI, and ligated with the PstIEagI-digested 10.8-kb fragment of the pBRThrABCR2 to make pBRThrABCR3
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(Supplementary Figure 1G).
To amplify aceBA genes, PCR was performed with primers EaceBA1 and EaceBA2
using the genomic DNA of E. coli W3110 as a template. The PCR product was digested
with AatII and PstI, and was cloned into the same site of pACYC177, resulting in
pACYCaceBA. The ppc gene was amplified with primers Eppc1 and Eppc2, digested
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with EcoRI and PstI, and ligated with the EcoRI-PstI-digested pUC19 to generate
pUCppc. For pACYCppc, the pACYC177 was digested with AatII, gap-filled with
Klenow fragments, and digested again with PstI to liberate a 3.3-kb DNA fragment.
This DNA fragment was ligated with FspI-PstI-digested 3.1-kb fragment of pUCppc.
Plasmid pMloxC containing a selectable antibiotic marker flanked by mutant loxP sites
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(Albert et al, 1995) was constructed. PCR was performed with pACYC184 as a
template using primers ECmlox1 and ECmlox2 that contain lox66 sequence and lox71
sequence, respectively. The resulting PCR product was digested with HindIII and SmaI
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sequentially, and was ligated with the HindIII-EcoRV-digested pUG6 (Guldener et al,
1996).
Genome engineering: Promoter replacement and site-directed mutagenesis
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The primers used for chromosomal manipulation are listed in Supplementary Table I.
Replacement of native promoter in threonine operon with the strong tac promoter,
removal of feedback inhibitions of aspartokinae I and III, and reduction of threonine
dehydratase activity were performed using sacB homologous recombination system
(Schweizer et al, 1992). The levansucrase, encoded by the Bacillus subtilis sacB gene,
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which is inducible in the presence of sucrose, causes lethal cell lysis by an uncontrolled
accumulation of levan in the periplasm of gram-negative bacteria. Hence, cells
harboring the sacB gene by first homologous recombination have to release this gene by
second homologous recombination to survive in the presence of sucrose.
Substitution of native promoter in threonine operon with the strong tac promoter was
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performed as follows (Supplementary Figure 1A and F). A 0.7-kb PCR product
amplified with primers thrAT1 and thrAT2 was digested with PvuII and SphI, followed
by cloning into the same sites of pKK223-3 to make pKKthrL. Another 0.7-kb PCR
product amplified with primers thrAT3 and thrAT4 was digested with EcoRI and PstI,
and then it was cloned into the EcoRI-PstI-digested pKK223-3 to make pKKthrLR. The
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PvuII-PstI digested 1.8-kb DNA fragment containing the tac promoter flanked by 0.7kb homologous DNA fragments was cloned into pSacHR06 (Park et al, 2007) to make
pSacAT01. This construct was digested with NheI and a 5.2-kb DNA fragment was selfligated to remove the 0.9-kb fragment containing the pMB1 origin of replication. The
final construct was electroporated into E. coli, and the clones having the native
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promoter changed with the tac promoter by double homologous recombination were
identified by positive selection based on the conditional lethal effect of the sacB gene in
E. coli. Replacement of endogenous promoter of L-threonine operon by the tac
promoter in the chromosomal DNA was confirmed by DNA sequence analysis.
Substitution of the endogenous promoter of the ppc gene by the trc promoter was
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performed by PCR-mediated -Red recombination (Yuan et al, 2006) (Supplementary
Figure 1B). The PCR fragment required for the promoter replacement was amplified in
three steps. A 1047-bp DNA fragment of fused lox71-chloramphenicol marker-lox66
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was generated by the first PCR reaction with primers FPppc1 and RPppc1 which
contain lox71 and lox66, respectively, using pACYC184 as a template. To introduce the
trc promoter, the second PCR reaction was performed with primers FPppc2 and RPppc2
using the 1047-bp DNA fragment as a template. The primer RPppc2 contains the trc
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promoter sequence. To introduce homologous regions into the final PCR product, the
third PCR reaction was carried out with primers FPppc3 and RPppc3 using the second
PCR product as a template. The final PCR product was electroporated into E. coli cells
carrying -Red recombinase expression plasmid pKD46, and cells in which double
homologous recombination occurred were selected on the agar plate containing
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chloramphenicol and screened by direct colony PCR. Replacement of the promoter of
the ppc gene with the trc promoter was confirmed by DNA sequence analysis.
Substitution of a native promoter of the acs gene by the trc promoter was performed
in the same manner as described for the ppc gene. A 1195-bp fused lox71chloramphenicol marker-lox66 DNA fragment was obtained by the first PCR reaction
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with primers FPacs1 and RPacs1 using pMloxC as a template. To introduce the trc
promoter, the second PCR reaction was performed with primers FPacs2 and RPacs2
using the 1195-bp DNA fragment as a template. The primer RPacs2 contains the trc
promoter sequence. To introduce homologous regions into the final PCR product, the
third PCR reaction was carried out with primers FPacs3 and RPacs3 using the second
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PCR product as a template. The final PCR product was electroporated into E. coli cells
carrying -Red recombinase expression plasmid pKD46, and cells in which double
homologous recombination occurred were selected on the agar plate containing
chloramphenicol, and screened by direct colony PCR. Replacement of the promoter of
the acs gene with the trc promoter was confirmed by DNA sequence analysis.
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The feedback-resistant aspartokinase I was constructed by replacing the 345th serine
with phenylalanine (Supplementary Figure 1C). Oligonucleotide primers thrA1 and
thrA2, containing a BamHI site and a mutated nucleotide (22nd GA), respectively,
were used to amplify a 664-bp fragment. Another 657-bp fragment was amplified using
primers thrA3 and thrA4, containing a mutated nucleotide (22nd CT) and a SalI site,
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respectively. Two DNA fragments were purified and mixed, and the complete 1279-bp
fragment was amplified by overlapping PCR using the primers thrA1 and thrA4. The
BamHI-SalI-digested 1279-bp PCR fragment was ligated into the BamHI-SalI-digested
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pSacHR06 to make pSacthrA. The resultant construct was digested with NheI, and selfligated to remove the pMB1 origin of replication. The final construct was then
electroporated into E. coli cells harboring pKD46, and the target clone was subsequently
identified by positive selection based on the conditional lethal effect of the sacB gene in
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E. coli. Substitution of a single nucleotide was confirmed by DNA sequence analysis.
The feedback-resistant aspartokinase III was generated by replacing threonine at
position 352 by isoleucine (Supplementary Figure 1D). Oligonucleotide primers lysC1
and lysC2, containing a BamHI site and a mutated nucleotide (23rd GA), respectively,
were used to amplify a 719-bp fragment. Another 808-bp fragment was amplified using
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primers lysC3 and lysC4, containing a mutated nucleotide (22nd CT) and a SalI site,
respectively. Two DNA fragments were purified and mixed, and the complete 1404-bp
fragment was amplified by overlapping PCR using the primers lysC1 and lysC4. The
BamHI-SalI-digested 1404-bp PCR fragment was ligated into the BamHI-SalI-digested
pSacHR06 to make pSaclysC. The resultant construct was digested with NheI, and self-
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ligated to remove the pMB1 origin of replication. The final construct was then
electroporated into E. coli cells carrying pKD46, and the target clone was subsequently
identified by positive selection based on the conditional lethal effect of the sacB gene in
E. coli. Substitution of a single nucleotide was confirmed by DNA sequence analysis.
Threonine dehydratase was mutated by replacing the 97th serine with phenylalanine
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(Supplementary Figure 1E). Oligonucleotide primers ilvA1 and ilvA2, containing a
BamHI site and a mutated nucleotide (20th GA), respectively, were used to amplify a
648-bp fragment. Another 676-bp fragment was amplified using primers ilvA3 and
ilvA4, containing a mutated nucleotide (19th CT) and a PstI site, respectively. Two
DNA fragments were purified and mixed, and the complete 1325-bp fragment was
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amplified by overlapping PCR using primers ilvA1 and ilvA4. The BamHI-PstIdigested 1325-bp PCR fragment was ligated into the BamHI-PstI-digested pSacHR06 to
make pSacilvA. The resultant construct was digested with NheI, and self-ligated to
remove the pMB1 origin of replication. The final construct was then electroporated into
E. coli cells carrying pKD46, and the target clone was subsequently identified by
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positive selection based on the conditional lethal effect of the sacB gene in E. coli.
Substitution of a single nucleotide was confirmed by DNA sequence analysis.
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Knocking-out chromosomal genes
Deletion of the lysA, metA, tdh, ppc, and iclR genes was performed using one-step
inactivation method (Datsenko and Wanner, 2000). The schematic procedure of gene
inactivation is shown in Supplementary Figure 1F. The -Red recombinase expression
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plasmid pKD46 was used to disrupt the genes in the chromosome of E. coli W3110 with
appropriate antibiotic markers. Recombinant E. coli W3110 harboring the pKD46 was
cultivated at 30C, and the expression of λ recombinase was induced by adding Larabinose (10 mM). Then electrocompetent cells were prepared by standard protocol.
PCR was performed using plasmid pKD3 or pKD4, which contains the antibiotic
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resistance gene flanked by FRT sequence (FLP recognition target) as templates and
primers listed in Supplementary Table I. The PCR products were transformed into the
electrocompetent E. coli W3110 harboring the pKD46. Colonies were selected on LB
agar plates containing kanamycin (40 μg/ml) or chloramphenicol (34 μg/ml). Successful
gene replacement with the antibiotic marker was confirmed by direct colony PCR. The
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antibiotic marker was eliminated by using a helper plasmid pCP20 encoding the FLP
recombinase. Plasmid pCP20 contains ampicillin and chloramphenicol resistant
markers, and shows temperature-sensitive replication6. The CmR or KmR knockout
mutants were transformed with the pCP20, and ampicillin-resistant transformants were
selected on LB agar plates at 30°C. Several colonies were cultivated without antibiotic
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marker in LB liquid media at 43°C, and then examined for the loss of all antibiotic
resistances. Elimination of the antibiotic markers was verified by PCR.
The tdcC gene was deleted in essentially the same manner, except that pMloxC
plasmid was used as a template DNA for PCR. For the elimination of antibiotic
resistance gene, a helper plasmid pJW168 (Palmeros et al, 2000) encoding the cre
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recombinase was used.
Flask cultivation
Flask cultures were carried out using 250-ml baffled Erlenmeyer flasks containing 30
ml of TPM1 medium at 31°C in a rotary shaker at 250 rpm for 48 hours. The TPM1
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medium contains per liter: glucose, 50 g; yeast extract, 2 g; MgSO4.7H2O, 2 g; KH2PO4,
4 g; (NH4)2SO4, 14 g; betaine, 1 g; L-methionine, 0.149 g; L-lysine, 0.164 g; trace metal
solution, 5 ml and CaCO3, 30 g. The trace metal solution contains per liter: FeSO4.7H2O,
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10 g; CaCl2, 1.35 g; ZnSO4.7H2O, 2.25 g; MnSO4.4H2O, 0.5 g; CuSO4.5H2O, 1 g;
(NH4)6Mo7O24.4H2O, 0.106 g; Na2B4O7.10H2O, 0.23 g; 35% HCl, 10 ml. The final pH
was adjusted to 7.2 by adding 4N KOH. Chloramphenicol (35 μg/ml), kanamycin (40
μg/ml) and ampicillin (50 μg/ml) were added to the medium when necessary.
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Supplementary Results
Transcriptome analysis of Thr producing strain
In order to identify notable changes in transcript level during Thr production,
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transcriptome profiling was carried out. Transcriptome profiles of TH07 (pBRThrABC)
were compared with those of control WL3110 (pBR322) strain. Samples for
transcriptome profiling were taken at the OD600 of 5.13 (the former) and 5.21 (the latter)
(Supplementary Figure 2). They were both at exponential growth phase and the specific
growth rates were 0.328 /h for the former and 0.357 /h for the latter. At the time of
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sampling, the TH07C (pBRThrABC) strain produced 0.71 g/l of Thr. Genes that were
found to be significantly upregulated or downregulated are listed in Supplementary
Table V and VI, respectively, and categorized in Supplementary Table VII. The
expression levels of genes involved in glycolysis, TCA cycle and pentose phosphate
pathway were not significantly changed (Supplementary Figure 3). Interestingly, the
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expression levels of the genes involved in glyoxylate cycle were considerably
upregulated, while those of hdeA, hdeB, gadA, and gadB, which are known to be
induced by acetate (Arnold et al, 2001), were found to be downregulated in TH07C
(pBRThrABC). This result can be explained because the acetic acid concentration in
TH07C (pBRThrABC) is much lower than that in WL3110 (pBR322) at early stationary
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phase (0.52 g/l vs. 1.61 g/l). The expression level of the rhtC gene, which encodes a Thr
exporter, increased to 2.99-fold, while that of the ppc gene decreased to 0.43-fold.
As expected, the thrABC genes, which were amplified both chromosomally
(attenuation removal) and extrachromosomally (plasmid-based amplification), were
significantly upregulated by 43.60 (thrA), 39.05 (thrB) and 23.55-folds (thrC),
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respectively. The expression levels of the genes involved in L-isoleucine biosynthesis
were significantly increased. This seems to be caused by the decreased activity of
threonine dehydratase in TH07C (pBRThrABC).
Effects of amplifying the aceBA genes
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We examined by flux response analysis whether further amplification of the aceBA
genes would increase Thr production. As shown in Figure 2B, the response of Thr
production rate to increasing ICL flux showed similar profile with that observed earlier
-8-
for increasing the PPC flux. Thus, we concluded that knocking out the iclR gene was
good enough for increasing the Thr production. To prove whether this is true, we
constructed pACYCaceBA to further amplify the aceBA genes. The specific activity of
ICL and the Thr concentration obtained with TH09C (pBRThrABC, pACYCaceBA)
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were 337.2% and 90.6%, respectively, of those obtained with TH09C (pBRThrABC,
pACYC177) (Supplementary Table IV and Supplementary Figure 4A), suggesting that
further amplification of the aceBA genes had a negative effect on Thr production as
predicted by flux response analysis.
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Reduction of acetic acid formation during the fed-batch culture
As can be seen from Figure 2C, the acetic acid production can be reduced by (i)
reducing glycolytic flux, (ii) increasing pentose phosphate pathway flux, (iii) increasing
the anaplerotic flux (ppc or aceBAK), (iv) decreasing the flux through the acetate
pathway (pta-ack, poxB), and (v) increasing the flux of acetate utilization by acetyl-CoA
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synthetase (acs). The first option is not desirable as it will also decrease cell growth and
Thr production. The second option involves amplification of multiple enzymes, which is
not desirable either considering the metabolic burden of overexpression. The third
option of increasing the anaplerotic fluxes was already implemented by altering both
PPC expression level and the regulation of the glyoxylate bypass. The fourth option of
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knocking out the genes in the acetate pathway (pta-ack or poxB) was not chosen
because it can retard growth and/or increase pyruvate excretion. The last strategy of
amplifying the acs gene was selected to reduce acetic acid production.
Acetyl-CoA synthetase encoded by the acs gene catalyzes an irreversible reaction
that converts acetate to acetyl-CoA. The native acs promoter in the chromosome was
25
replaced with the trc promoter to increase the expression of the acs gene. This method
of promoter replacement was employed to avoid metabolic burden to be caused by
additional plasmid-based overexpression and to allow constitutive expression of the acs
gene in the lacI mutant host strain used in this study. The increased expression of the
acs gene was validated by real time RT-PCR analysis (see below).
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Additional fed-batch cultures of TH28C (pBRThrABCR3) strain
During the fed-batch culture of TH28C (pBRThrABCR3) strain shown in Figure 3B in
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the text, samples were taken 33 times to accurately analyze the fermentation
performance. However, due to the frequent sampling, there was noticeable culture
volume change which may affect the real fermentation time profile. Thus, fed-batch
culture of TH28C (pBRThrABCR3) strain was repeated under the same condition, but
5
with minimal number of samplings. Samples were taken out only three times as shown
in Supplementary Figure 6A online. The final Thr concentration reached in 52.4 h was
76.5 g/l Thr, with the volumetric Thr productivity of 1.46 g/l/h (Supplementary Figure
6A and Supplementary Table III). The concentration of acetic acid accumulated was
2.41 g/l. Furthermore, the pH-stat fed-batch culture of TH28C (pBRThrABCR3) strain
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with automatic feeding also allowed production of Thr to 75.1 g/l with the productivity
of 1.436 g/l/h. (Supplementary Figure 6B). These results suggest that the performance
of rationally engineered TH28C (pBRThrABCR3) strain is comparable to the randomly
mutagenized strain employed in industry.
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- 10 -
Supplementary Table I - Bacterial strains and plasmids used in this study
Descriptiona
Strain/plasmid
Reference/Source
Strains
CGSCb
E. coli W3110
Coli Genetic Stock Center strain (CGSC) No. 4474
E. coli TOP10
F_ mcrA _(mrr-hsdRMS-mcrBC) (_80lacZ_M15 _lacX74 deoR recA1
araD139 _(ara-leu)7697galU galK rpsL endA1 nupG
WL3110
W3110 lacI
TH01
W3110 lacI, thrAC1034T
TH02
Invitrogenc
Park et al, 2007
This study
W3110 lacI, thrA
C1034T
C1055T
TH03
W3110 lacI, thrA
C1034T
C1055T
, Pthr::Ptac
This study
TH04
W3110 lacI, thrA
C1034T
C1055T
, Pthr::Ptac, lysA
This study
TH05
W3110 lacI, thrAC1034T, lysCC1055T, Pthr::Ptac, lysA, metA
This study
TH06
W3110 lacI, thrAC1034T, lysCC1055T, Pthr::Ptac, lysA, metA, ilvAC290T
This study
TH07
TH09C
TH11C
TH19C
TH20C
TH27C
TH28C
This study
, lysC
, lysC
, lysC
W3110 lacI, thrAC1034T, lysCC1055T, Pthr::Ptac,
tdh
W3110 lacI, thrAC1034T, lysCC1055T, Pthr::Ptac,
tdh, iclR::CmR
W3110 lacI, thrAC1034T, lysCC1055T, Pthr::Ptac,
tdh, ppc::CmR
W3110 lacI, thrAC1034T, lysCC1055T, Pthr::Ptac,
tdh, Pppc::Ptrc
W3110 lacI, thrAC1034T, lysCC1055T, Pthr::Ptac,
tdh, iclR::CmR, Pppc::Ptrc
W3110 lacI, thrAC1034T, lysCC1055T, Pthr::Ptac,
tdh, iclR, Pppc::Ptrc, tdcC::CmR
W3110 lacI, thrAC1034T, lysCC1055T, Pthr::Ptac,
tdh, iclR, Pppc::Ptrc, tdcC, Pacs::CmR-Ptrc
lysA, metA, ilvAC290T,
lysA, metA, ilvA
C290T
lysA, metA, ilvA
C290T
lysA, metA, ilvA
C290T
lysA, metA, ilvA
C290T
lysA, metA, ilvA
C290T
,
,
,
,
,
lysA, metA, ilvAC290T,
This study
This study
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This study
This study
This study
This study
Plasmids
pKD46
pKD3
pCP20
pJW168
pUG6
ApR,  Red recombinase expression plasmid,
expression, temperature sensitive replication, 6.3-kb
R
ara-inducible
R
Cm , Ap , oriR plasmid containing an FRT-aph-FRT cassette, 2.8-kb
ApR, CmR, repA(Ts), pSC101 based vector expressing the yeast Flp
recombinase
ApR, repA(Ts), pSC101 based vector expressing cre-recombinase, 5.5kb
ApR, loxP-kanMX-loxP, 4.0-kb
Datsenko et al, 2000
Datsenko et al, 2000
Datsenko et al, 2000
Palmero et al, 2000
Guldener et al, 1996
R
R
pMloxC
Ap , HindIII-SmaI-digested 1.1-kb PCR product of lox66-Cm -lox71
ligated with HindIII-EcoRV-digested 2.4-kb fragment of the pUG6, 3.5kb
pACYC177
KmR, ApR; p15A ori, 3.9-kb
New England Biolabsd
pACYC184
TcR, CmR, p15A ori, 4.2-kb
New England Biolabs
pUC19
This study
R
New England Biolabs
R
Ap , pMB1 ori, 2.7-kb
pKK223-3
Ap , tac promoter, pMB1 ori, 4.6-kb
Pharmacia Bioteche
pBR322
ApR, TcR, rop, pMB1 ori, 4.4-kb
Pharmacia Biotech
pSacHR06
KmR, homologous recombination vector, 4.3-kb
pUCppc
R
Ap , ppc cloned in EcoRI and PstI site of pUC19, 5.7-kb
R
Park et al, 2007
This study
pACYCppc
Km , ppc cloned in AatII and PstI site of pACYC177, 6.3-kb
This study
pACYCaceBA
KmR, aceBA cloned in AatII and PstI site of pACYC177, 6.7-kb
This study
- 11 -
Supplementary Table I - continued
Descriptiona
Strain or plasmid
pKKthrL
ApR, thrL fragment cloned in SphI and PvuII site of pKK223-3, 3.8-kb
R
This study
pKKthrLR
Ap , thrR fragment cloned in EcoRI and PstI site of pKKthrL, 4.5-kb
This study
pSacAT01
KmR, thrL-Ptac-thrR fragment cloned in EcoRV and PstI site of
pSacHR06, 6.1-kb
This study
pSacthrA
KmR, mutant thrA cloned in BamHI and SalI site of pSacHR06, 5.6-kb
This study
pSaclysC
pSacilvA
pKKthrABC
pBRThrABC
pBRThrABCR
pBRThrABCR2
pBRThrABCR3
R
This study
R
This study
Km , mutant lysC cloned in BamHI and SalI site of pSacHR06, 5.8-kb
Km , mutant ilvA cloned in BamHI and PstI site of pSacHR06, 5.6-kb
R
Ap , mutant threonine operon cloned in XmaI and HindIII site, 9.4-kb
ApR, NdeI-SalI-digested 7.8-kb fragment of pKKThrABC ligated with
NdeI-SalI -digested 1.6-kb fragment of the pBR322, 9.4-kb
ApR, BamHI-SphI-digested 1.5-kb PCR product of rhtC ligated with
BamHI-SphI-digested 9.2-kb fragment of the pBRThrABC, 10.7-kb
ApR, MluI-SalI-digested 1.1-kb PCR product of rhtA ligated with MluISalI- digested 10.0-kb fragment of the pBRThrABCR, 11.1-kb
ApR, PstI-EagI-digested 0.9-kb PCR product of rhtB ligated with PstIEagI-digested 10.8-kb fragment of the pBRThrABCR2, 11.7-kb
a
Abbreviations: Ap, ampicillin; Cm, chloramphenicol; Km, kanamycin; Tc, tetracycline; R, resistance.
5
Source
b
Coli Genetic Stock Center.
c
Invitrogen, Corp., Carlsbad, CA.
d
New England Biolabs, Inc., Beverly, Mass.
e
Pharmacia Biotech, Uppsala, Sweden.
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Supplementary Table II - Oligonucleotides used in this study
Sequence (5’3’)a
Name
thrA1
ACGCGGATCCATCGCCATTATGGCCGGCGTATTAGAAGC
thrA2
GATTGCGTAATCAGCACCACGAAAATACGGGCGCGTGACATCG
thrA3
CGATGTCACGCGCCCGTATTTTCGTGGTGCTGATTACGCAATC
thrA4
CACGCGTCGACCTGGAAGTGCAGTTAACAATGACCGGG
lysC1
CTGATGTCGACCCTGCTGTTTGTTGAGATCCTGCGC
lysC2
GGTTGAACCGGTGGTATCAAGGATAATGCCACGCTCACTTCTG
lysC3
CAGAAGTGAGCGTGGCATTAATCCTTGATACCACCGGTTCAACC
lysC4
CCAGCTAAATGACGCTTCAGGATCCGGTTTATAAG
ilvA1
GACGGGATCCGCAAAGCCTGTGCGCTGATCACCGACGG
ilvA2
CACGCCTAACCGCGCAGAAAAAAACGCGACGCCCTGCG
ilvA3
CGCAGGGCGTCGCGTTTTTTTCTGCGCGGTTAGGCGTG
ilvA4
CAGGTACTGCAGACCGGAAAGAATATGCGCCAGCCGTTCG
KOmetA1
GTGTGCCGGACGAGCTACCCGCCGTCAATTTCTTGCGTGAAGAAAACGTCTTTGTGATTGCAGCATTACACGTCTTG
KOmetA2
CGGGATGGCCCGTCACAAAGGCAATGCGCTTATCTTTACTGGCAAACAGACACTTAACGGCTGACATGGGA
KOlysA1
ATGCCACATTCACTGTTCAGCACCGATACCGATCTCACCGCCGAAAATCTGATTGCAGCATTACACGTCTTG
KOlysA2
GTTGATAAGGAACAGAAAGCCCACCGCCCGCAGAAATAGCCTGTAAATCCCACTTAACGGCTGACATGGGA
thrAT1
GCAGCCAGCTGTAGCGATCTGCGGATTGTCGATAGT
thrAT2
CAGGAGCATGCCAGAAGCTGCTATCAGACACTCTTT
thrAT3
CAGCAGAATTCATGCGAGTGTTGAAGTTCGGCGGTA
thrAT4
CAGAGCTGCAGTCCGTCCAAATCTCGCAACAATCGG
Thr_Xma
GTTGCCCGGGATGCGAGTGTTGAAGTTCGG
Thr_Hin
GCGTCAAGCTTCGGCGGTTGTTATTCTCCGC
KOtdh1
ATGAAAGCGTTATCCAAACTGAAAGCGGAAGAGGGCATCTGGATGACCGAGATTGCAGCATTACACGTCTTG
KOtdh2
ATCACTTTGGTCCAGTCGATAGACATATCAGACGGCGGAATACCCAGCATCACTTAACGGCTGACATGGGA
KOiclR1
TGAAAATGATTTCCACGATACAGAAAAAAGAGACTGTCATGGTCGCACCCGATTGCAGCATTACACGTCTTG
KOiclR2
ATAGAAATTGCGGCAAACGGTTCACGGTGCTCATCGAAAATACACGCTGCCACTTAACGGCTGACATGGGA
KOppc1
ATGAACGAACAATATTCCGCATTGCGTAGTAATGTCAGTATGCTCGGCAAGATTGCAGCATTACACGTCTTG
KOppc2
TTACCTAACGGCCACAGTGCTTTGTCTACCAGGCGTTGGTCATAGTATTCCACTTAACGGCTGACATGGGA
KOF1tdcC
GCGTAAATCAGATACCACATGGACGTTAGGCTTGTTTGGTACGGCAATCG TAGGTGACACTATAGAACGCG
KOF2tdcC
ATGAGTACTTCAGATAGCATTGTATCCAGCCAGACAAAACAATCGTCCTGGCGTAAATCAGATACCACAT
KOR1tdcC
CCAGTGTAATCGCGAACGTTGTTTTGGTACCGGTCATGGACGCAAAGTGGTAGTGGATCTGATGGGTACC
KOR2tdcC
GAAGAAAGATTTGAAGATAGCCACGAGTGCGATGATGGAAGCCGCATATTCCAGTGTAATCGCGAACGT
FPppc1
GTTTGCTGAAGCGATTTCGCTACCGTTCGTATAGCATACATTATACGAAGTTATTAACGACCCTGCCCTGAACC
FPppc2
CTTATTTAAAGCGTCGTGAATTTAATGACGTAAATTCCTGCTATTTATTCGTTTGCTGAAGCGATTTCGC
FPppc3
ACACCTTTGGTGTTACTTGGGGCGATTTTTTAACATTTCCATAAGTTACGCTTATTTAAAGCGTCGTGAA
RPppc1
GAGCCGGATGATTAATTGTCAACAGCTACCGTTCGTATAATGTATGCTATACGAAGTTATATGAGACGTTGATCGGCACG
RPppc2
ATTGTTCGTTCATGGTCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTC
RPppc3
CGCATCCTTGATGGTTTCTCCCAGCACTTTGCCGAGCATACTGACATTACTACGCAATGCGGAATATTGTTCGTTCATGGTCTGT
FPacs1
GCCCCTATGTGTAACAAATAACCACACTGTGAATGTTGTCTAGGTGACACTATAGAACGCG
FPacs2
TCACGACAGTAACCGCACCTACACTGTCATGACATTGCTCGCCCCTATGTGTAACAAATA
FPacs3
CGAATTGCGCCATTGTTGCAATGGCGGTTTTTATTGTTTTTCACGACAGTAACCGCACCT
RPacs1
TGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACAGCTAGTGGATCTGATGGGTACC
RPacs2
CGATGTTGGCAGGAATGGTGTGTTTGTGAATTTGGCTCATGGTCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCC
RPacs3
TTGTTGATACATCGCCTCGTACTGCTGAGGGTTTATCAGGCAACGGTCTGCGATGTTGGCAGGAATGGTG
Eppc1
CAGCGAATTCTCGGATGCGATACTTGCG
Eppc2
CGATCCTGCAGCGAATGTAACGACAATTCC
EaceBA1
CGACTGACGTCTGATAGTCGATCGTTAAGC
EaceBA2
CGATCCTGCAGAATCAATAATTCCAGGCC
ECmlox1
ATATAAGCTTTACCGTTCGTATAGCATACATTATACGAAGTTATCTGCCCTGAACCGACGACCG
ECmolx2
AATTCCCGGGTACCGTTCGTATAATGTATGCTATACGAAGTTATGCATCACCCGACGCACTTTGC
FrhtCEcBa
CTGAGAATTC GGATCCAGATGGCTGAACAGATGC
RrhtCSpMl
CCTACGCATGC ACGCGTCAAAGCAGATGAAGGCGC
FrhtAMlu
CTGAACGCGTGAACTGCGTAAGTATTACG
RrhtASalPst
CTGACGTCGAC CTGCAGACCATGCAGAAATGTAAAT
FrhtBPst
CGTAGCTGCAG TCCACACCAGTAAACTCTG
RrhtBEagAat
CATTTCGGCCG GACGTCAGTCGGATAAGGCGTTTAC
a
Restriction sites are underlined and sequences altered with respect to the template for site directed mutagenesis are in
boldface.
5
- 13 -
Supplementary Table III - Comparison of fermentation results obtained with metabolically engineered E. coli strains
Conc. of fermentation products (g/l)
L-threonine
acetic acid
succinic
acid
pyruvic acid
Biomass
yield
(g DCW/g
of glucose)
5.83
1.48  0.01
0.70  0.02
0.02  0.01
0.13  0.01
0.563
0.143
0.137
5.88
1.46  0.02
0.64  0.04
0.01  0.01
0.12  0.01
0.565
0.140
0.124
6.04
1.21  0.01
0.34  0.04
0.10  0.02
0.04  0.01
0.586
0.118
0.113
5.82
1.93  0.02
0.36  0.03
0.04  0.00
0.02  0.01
0.560
0.186
0.149
5.77
1.92  0.01
0.36  0.02
0.04  0.01
0.04  0.01
0.560
0.186
0.140
5.80
1.74  0.03
0.05  0.01
0.15  0.02
0.02  0.00
0.558
0.169
0.095
1.79
0.19  0.01
1.91  0.06
n.d.
2.21  0.12
0.263
0.028
0.010
TH19C (pBRThrABC)
5.88
1.89  0.01
0.51  0.04
0.02  0.01
0.05 0.01
0.571
0.183
0.194
TH20C (pBRThrABC)
5.78
2.24  0.01
0.24  0.01
0.03  0.00
0.03  0.00
0.556
0.213
0.220
TH20C (pBRThrABC)b
14.20
6.39  0.07
0.62  0.05
0.473
0.213
0.335
TH27C (pBRThrABC)
14.03
7.39  0.07
0.77  0.10
0.468
0.246
0.456
TH27C (pBRThrABCR)
11.32
11.10  0.22
0.80  0.08
0.377
0.370
0.766
TH27C (pBRThrABCR2)
11.20
11.59  0.28
0.59  0.02
0.373
0.386
0.768
TH27C (pBRThrABCR3)
10.99
11.81  0.16
0.45  0.05
0.366
0.393
0.777
31.93
77.1  1.5
7.85  0.25
0.114
0.275
1.374
35.60
82.4  1.1
2.35  0.13
0.127
0.294
1.648
33.35
76.5  1.0
2.41  0.17
0.119
0.273
1.460
32.97
75.1  1.5
2.16  0.10
0.118
0.268
1.436
Strain
TH07 (pBRThrABC)a
TH07
(pBRThrABC, pACYC177)
TH07
(pBRThrABC, pACYCppc)
TH09C (pBRThrABC)
TH09C
(pBRThrABC, pACYC177)
TH09C
(pBRThrABC, pACYCaceBA)
TH11C
(pBRThrABC, pACYC177)
TH27C (pBRThrABCR3)c
fed-batch
TH28C (pBRThrABCR3)
fed-batch
TH28C (pBRThrABCR3)
fed-batch (minimum sampling)
TH28C (pBRThrABCR3)
fed-batch (pH-stat)
Biomass
(g
DCW/l)
d
L-threonine
yield
(g/g of
glucose)
Volumetric
L-threonine
productivity
(g/l/h)
Batch cultures of recombinant strains were carried out in TPM2 medium containing 10 g/l glucosea or 30 g/l glucoseb as described in materials and methods. Standard deviations were
calculated from the results of three independent experiments.
- 14 -
c
Fed-batch cultures were carried out in TPM2 medium containing 20 g/l glucose at 31°C. The pH was controlled at 6.5 by automatic feeding of 25% (v/v) NH4OH. The feeding solutions were
introduced into the fermentor as described in Materials and methods.
d
Not detected.
- 15 -
Supplementary Table IV - The specific activities of phosphoenolpyruvate
carboxylase and isocitrate lyasea
Phosphoenolpyruvate
carboxylase (PPC)
Isocitrate lyase
(ICL)
TH07 (pBRThrABC, pACYC177)
32.0  1.8
2.2  0.1
TH07 (pBRThrABC, pACYCppc)
298.4  14.9
1.8  0.2
TH09C (pBRThrABC, pACYC177)
23.1  1.1
16.4  0.7
TH09C (pBRThrABC, pACYCaceBA)
17.4  1.1
55.3  3.8
n.d.b
5.3  0.2
TH19C (pBRThrABC)
119.1  5.1
2.3  0.2
TH20C (pBRThrABC)
82.5  3.2
14.1  0.4
Strain
TH11C (pBRThrABC, pACYC177)
a
All enzyme assays were performed as described in the materials and method. The unit of the enzyme activity is
nmol/min/ mg (protein).
b
Not detected.
16
Table V - Genes that are significantly upregulated in L-threonine
producing straina
Gene name
thrA
thrB
thrC
ycbR
b0005
ilvB
hisG
srlE
fecI
ilvN
narG
ilvC
deoR
yfiA
ybjZ
rep
hisD
b1983
ilvG_1
b1758
yagU
ycjS
ptsA
ftn
ytfM
b1200
ytfH
ytfE
ilvD
nirB
b2433
b0298
nikD
yggU
narK
prfH
yajR
tnaB
yaaA
yjgQ
gatY
ansP
ynaF
rhtC
feoA
lacZ
iadA
marA
ompF
yphC
acrF
hycD
grxA
yaaJ
fliE
yifM_2
fepD
aer
aceB
aceA
tdcC
Functions
Relative expression ratio
aspartokinase I
homoserine kinase
threonine synthase
putative chaperone
hypothetical protein
acetolactate synthase I
ATP phosphoribosyltransferase
PTS system
probable RNA polymerase sigma factor
acetolactate synthase I
nitrate reductase 1
ketol-acid reductoisomerase
transcriptional repressor for deo operon
putative yhbH sigma 54 modulator
putative ATP-binding component of a transport system
rep helicase
L-histidinal:NAD+ oxidoreductase; L-histidinol:NAD+ oxidoreductase
hypothetical protein
acetolactate synthase II
putative cytochrome oxidase
hypothetical protein
putative dehydrogenase
PEP-protein phosphotransferase system enzyme I
cytoplasmic ferritin (an iron storage protein)
hypothetical protein
putative dihydroxyacetone kinase (EC 2.7.1.2)
hypothetical protein
hypothetical protein
dihydroxyacid dehydratase
nitrite reductase (NAD(P)H) subunit
hypothetical protein
putative factor
ATP-binding protein of nickel transport system
hypothetical protein
nitrite extrusion protein
probable peptide chain release factor
putative transport protein
low affinity tryptophan permease
hypothetical protein
hypothetical protein
tagatose-bisphosphate aldolase 1
L-asparagine permease
putative filament protein
hypothetical protein
ferrous iron transport protein A
beta-D-galactosidase
isoaspartyl dipeptidase
multiple antibiotic resistance; transcriptional activator of defense
systems
outer membrane protein 1a (Ia;b;F)
putative oxidoreductase
integral transmembrane protein; acridine resistance
membrane-spanning protein of hydrogenase 3 (part of FHL complex)
glutaredoxin1
redox
coenzyme
for
glutathione-dependent
ribonucleotide
reductase
inner
membrane
transport protein
flagellar biosynthesis; basal-body component
hypothetical protein
ferric enterobactin (enterochelin) transport
aerotaxis sensor receptor
malate synthase A
isocitrate lyase
anaerobically inducible L-threonine transporter
43.59
39.05
23.55
16.66
12.11
11.89
8.04
7.51
7.17
6.10
5.87
5.16
5.09
4.81
4.59
4.57
4.46
4.28
4.27
4.25
4.18
4.11
4.10
3.99
3.94
3.87
3.78
3.69
3.68
3.67
3.59
3.47
3.45
3.39
3.36
3.35
3.34
3.21
3.17
3.17
3.07
3.04
2.99
2.99
2.95
2.83
2.80
2.72
2.68
2.59
2.54
2.50
2.45
2.43
2.36
2.35
2.32
2.32
2.23
1.80
1.70
a
Transcriptome profiles of TH07 (pBRThrABC) were compared with those of control WL3110 (pBR322) strain.
17
Table VI - Genes that are significantly downregulated in L-threonine
producing straina
Gene name
gadA
gadB
hdeA
hdeB
betI
evgA
fhuF
sseA
lysA
dnaK
sodA
metA
xasA
yojH
metE
ftsL
dniR
rpsU
b1422
yadF
b1836
purM
hslV
accB
mopA
mopB
b2351
pyrB
rplU
crl
yhiW
cspA
carA
exbD
adk
tdh
yhgI
yrbA
ydfK
ytfK
infA
yigW_1
yciG
avtA
yajG
yhiF
htpG
yadQ
ygiB
yegQ
yadR
rpsB
ydcN
fnr
clpB
rplY
prfB
ogt
yejG
pyrD
miaA
rplL
yfhE
ppc
Functions
Relative expression ratio
glutamate decarboxylase isozyme
glutamate decarboxylase isozyme
hypothetical protein
hypothetical protein
probably transcriptional repressor of bet genes
putative positive transcription regulator (sensor EvgS)
hypothetical protein
putative thiosulfate sulfurtransferase
diaminopimelate decarboxylase
chaperone Hsp70; DNA biosynthesis; autoregulated heat shock proteins
superoxide dismutase
homoserine transsuccinylase
acid sensitivity protein
hypothetical protein
tetrahydropteroyltriglutamate methyltransferase
cell division protein; ingrowth of wall at septum
transcriptional regulator for nitrite reductase (cytochrome c552)
30S ribosomal subunit protein S21
putative transcriptional regulator LYSR-type
putative carbonic anhdrase (EC 4.2.1.1)
hypothetical protein
phosphoribosylaminoimidazole synthetase = AIR synthetase
heat shock protein hslVU
acetylCoA carboxylase
GroEL
GroES
putative glycan biosynthesis enzyme
aspartate carbamoyltransferase
50S ribosomal subunit protein L21
transcriptional regulator of cryptic csgA gene for curli surface fibers
putative ARAC-type regulatory protein
cold shock protein 7.4
carbamoyl-phosphate synthetase
uptake of enterochelin; tonB-dependent uptake of B colicins
adenylate kinase activity; pleiotropic effects on glycerol-3-phosphate
acyltransferase
activity
threonine
dehydrogenase
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
protein chain initiation factor IF-1
hypothetical protein
hypothetical protein
alanine-alpha-ketoisovalerate (or valine-pyruvate) transaminase
putative polymerase/proteinase
hypothetical protein
chaperone Hsp90
putative channel transporter
hypothetical protein
hypothetical protein
hypothetical protein
30S ribosomal subunit protein S2
hypothetical protein
transcriptional regulation of aerobic
heat shock protein
50S ribosomal subunit protein L25
peptide chain release factor RF-2
O-6-alkylguanine-DNA/cysteine-protein methyltransferase
hypothetical protein
dihydro-orotate dehydrogenase
delta(2)-isopentenylpyrophosphate tRNA-adenosine transferase
50S ribosomal subunit protein L7/L12
hypothetical protein
phosphoenolpyruvate carboxylase
0.07
0.07
0.10
0.11
0.11
0.12
0.13
0.13
0.15
0.17
0.18
0.18
0.18
0.18
0.21
0.21
0.22
0.22
0.22
0.24
0.25
0.26
0.26
0.26
0.26
0.26
0.26
0.27
0.27
0.27
0.27
0.28
0.28
0.28
0.29
0.29
0.29
0.29
0.30
0.30
0.30
0.30
0.31
0.31
0.31
0.31
0.31
0.32
0.33
0.33
0.33
0.34
0.34
0.34
0.35
0.35
0.35
0.36
0.36
0.36
0.36
0.36
0.36
0.43
a
Transcriptome profiles of TH07 (pBRThrABC) were compared with those of control WL3110 (pBR322) strain.
18
Table VII - Functional classification of differentially expressed genesa
Differentially expressed gene(s)
Functional group
Upregulated
Downregulated
Amino acid biosynthesis and metabolism
thrA, thrB, thrC, ilvB, ilvN, ilvC, hisG,
hisD, ilvD, asnB, ilvM
lysA, metA, metE, tdh, avtA,
serA, serC
Central intermediary metabolism
aceB, aceA
gadA, gadB, sseA
Energy metabolism
narG, nirB, nirD
ppc, dniR
Transport and binding proteins
fecI, ptsA, ftn, hikD, narK, tnaB
exbD
Cell structure
ompF, filE
crl
Nucleotide biosynthesis and metabolism
deoR
carA, pyrB, purM
Translation, posttranslational modification
prfH, iadA
rplU, hslV, rpsU
cell process
marA, acrF
htpG, cspA, mopB, sodA, betI
a
Transcriptome profiles of TH07 (pBRThrABC) were compared with those of control WL3110 (pBR322) strain.
19
Supplementary Figure 1 - Construction of plasmids (A) pSacAT01, (C)
pSacthrA, (D) pSaclysC, and (E) pSacilvA used for site-directed
mutagenesis. (B) The procedure for the replacement of the ppc gene
promoter in the chromosome by PCR-based homologous recombination
mediated by -Red recombinase. (F) Schematics of chromosomal
substitution and gene inactivation. It should be noted that the genes are
not shown in their actual location in the chromosome. (G) Construction of
plasmid pBRThrABCR3
20
Supplementary Figure 2 - Time profiles of cell growth and Thr production
during the batch cultures of Thr producing TH07C (pBRThrABC) strain
30
6
25
5
20
4
15
3
10
2
5
1
0
0
2
4
6
8
10
12
14
L-threonine (g/l)
Biomass (OD600)
and the control WL3110 (pBR322) strain
0
16
Time (h)
Symbols are: solid circles, the growth of WL3110 (pBR322); solid triangles, the growth of
TH27C (pBRThrABC); solid squares, L-threonine concentration during the culture of TH27C
(pBRThrABC). Arrows indicate the sampling points for transcriptome profiling.
21
Supplementary Figure 3 - Results of comparative transcriptome profiling
The numbers are the ratios of the expression levels in TH07C (pBRThrABC) versus WL3110
(pBR322). The shaded and boxed numbers indicate significantly up- and down-regulated genes,
respectively, in Thr producing strain, TH07C (pBRThrABC). Abbreviations are: GLC, glucose;
G6P, glucose-6-phosphate; 6PGL, gluconolactone-6-phosphate; 6PGC, 6-phosphogluconate;
RL5P, ribulose-5-phosphate; X5P, xylulose-5-phosphate; R5P, ribose-5-phosphate; S7P,
sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; FBP,
fructose-1,
6-bisphosphate;
GAP,
glyceraldehyde-3-phosphate;
DHAP,
dihydroxyacetonephosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; CIT, citrate; ICIT,
isocitrate; AKG, α-ketoglutarate; SUCOA, succinyl-CoA; SUC, succinate; FUM, fumarate; MAL,
22
malate; OAA, oxaloacetate; GLY, L-glycine; ASP, L-aspartate; AS4P, L-aspartyl-4-phosphate;
ASSA, aspartate semialdehyde; HS, homoserine; HSP, homoserine phosphate; THR, Lthreonine; LYS, L-lysine; MET, L-methionine; ILE, L-isoleucine; LEU, L-leucine.
23
Supplementary Figure 4 - Batch fermentation profiles of engineered E. coli
strains. (A) TH07C (pBRThrABC, pACYC177), (B) TH07C (pBRThrABC,
pACYCppc), (C) TH09C (pBRThrABC, pACYC177), (D) TH09C (pBRThrABC,
pACYCaceBA),
(E)
TH11C
(pBRThrABC,
(pBRThrABC), and (G) TH20C (pBRThrABC)
24
pACYC177),
(F)
TH19C
Cells were grown in TPM2 medium containing 10 g/l glucose as described in Materials and
methods. Symbols are: ○, cell growth (OD600); ■, L-threonine (g/l); ▲, glucose (g/l); □,
acetic acid (g/l); ◇, pyruvic acid (g/l); △, succinic acid (g/l)
25
Supplementary Figure 5 - Batch fermentation profiles of engineered E. coli
strains. (A) TH20C (pBRThrABC), (B) TH27C (pBRThrABC), (C) TH27C
(pBRThrR), (D) TH27C (pBRThrABCR2), and (E) TH27C (pBRThrABCR3)
Cells were grown in TPM2 medium containing 30 g/l glucose as described in Materials and
methods. Symbols are: ○, cell growth (OD600); ■, L-threonine (g/l); ▲, glucose (g/l); □,
acetic acid (g/l)
26
Supplementary
Figure
6
-
Fed-batch
culture
of
E.
coli
TH28C
(pBRThrABCR3). (A) time profiles of the fed-batch culture with minimum
samplings (B) time profiles of the pH-stat fed-batch culture
(A) Cells were cultured in TPM2 medium containing 20 g/l glucose at 31°C. The pH was
controlled at 6.5 by automatic feeding of 25% (v/v) NH4OH. When the glucose concentration in
the culture broth fell down to 1 g/l, 80 ml of feeding solution, which is equivalent to 40 g glucose,
1 g KH2PO4, 0.24 g L-methionine, and 0.35 g L-lysine, was added. Only three samplings were
taken to simulate the industrial fermentation process. (B) When the pH becomes higher than
6.59, 6 ml of feeding solution composed of 565 g/l glucose, 12.9 g/l KH2PO4, 3.59 g/l Lmethionine, and 5.27 g/l L-lysine was automatically added. Symbols are: ○, cell growth
(OD600); ■, L-threonine (g/l); ▲, glucose (g/l); □, acetic acid (g/l); ◇, lactic acid (g/l).
27
Supplementary References
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Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia
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