Supplementary Information
Supplementary materials and methods
Construction of the bacterial strains
All chromosomal modifications were performed using the method of Datsenko and Wanner
(2000). In this study, the phage λ Xis/Int system, instead of FLP recombinase, was used to excise
the selective marker. The oligonucleotides are listed in Supplementary Table 1.
For the inactivation of the adhE, iclR, fadR, and aceBA genes, linear DNA fragments
carrying the chloramphenicol acetyltransferase (cat) gene flanked by λattR and λattL were
obtained by PCR using primer pairs P1, P2; P5, P6; P9, P10; and P20, P21 as well as the
pMW118-(λattL-Cm-λattR) plasmid (Katashkina et al. 2005) as a template. The PCR products
were integrated separately into the chromosome of the E. coli MG1655 strain harbouring the
pKD46 (Datsenko and Wanner 2000) helper plasmid. Further excision of the chloramphenicol
resistance marker from the chromosomes of the target strains was performed using the pMWtsInt/Xis plasmid, as described previously (Gulevich et al. 2009). The correspondence of the
desired and the experimentally obtained chromosome structures of the strains with the
individually inactivated genes adhE, iclR and fadR was confirmed by PCR analysis using the
locus-specific primers P3, P4; P7, P8; P11, P12; and P22, P23, respectively.
To provide constitutive expression of the aceEF-lpdA operon genes, the artificial genetic
element PL-SDaceF containing a lambda phage PL promoter and the Shine-Dalgarno sequence (SD
sequence) of the aceF gene was integrated into the E. coli MG1655 chromosome upstream of the
coding region of the aceE gene. The DNA fragment containing the BglII site, the PL promoter,
the aceF SD sequence, and the nucleotides complementary to the 5`-end of the open reading
frame of the aceE gene was obtained by PCR using primer pairs P13, P14 and P13, P15. The
DNA fragment containing the BglII restriction site, the CmR marker encoded by the cat gene and
sequences homologous to the DNA region upstream of the coding region of the aceE genes was
obtained by PCR using primers P16 and P17 as well as the plasmid pMW118-(λattL-Cm-λattR)
as a template. The amplified DNA fragments were digested with BglII restrictase followed by
ligation using T4 DNA ligase. The product obtained after ligation was amplified by PCR using
primers P15 and P16 followed by an integration of the PCR product into the chromosome. The
correspondence between the desired and the obtained structure of the new hybrid regulatory
element introduced upstream of the coding region of the aceE gene was confirmed by a sequence
analysis using primers P18 and P19.
The individual modifications were combined in the chromosomes of the target strains by
sequential P1-mediated transductions.
The transformation of strains with the pPYC plasmid was performed using the standard
technique.
Analytical techniques
The concentrations of organic acids and glucose in the culture media were measured by highperformance liquid chromatography using a Waters HPLC system (Waters). For organic acid
measurements, a reversed-phase column ReproSil-Pur C18-AQ (4 x 250 mm, 5 μm, Dr. Maisch)
was used with detection at 210 nm. An aqueous solution of phosphoric acid (100 mM) with
acetonitrile and methanol (each at 0.5% (v/v)) was used as a solvent, at a flow rate of 1.0 ml/min.
For the glucose measurements, a Waters HPLC system equipped with a refractive index Waters
2414 detector and a Spherisorb-NH2 column (4.6 x 250 mm, 5 μm, Waters) was used. The
mobile phase contained acetonitrile/ethyl acetate/water in a ratio of 76/4/20 (v/v/v) at a flow rate
of 1.0 ml/min. Samples were identified by comparing the retention times with those of the
corresponding standards.
The amounts of ethanol in the culture media were determined by gas chromatography
using a flame ionisation detector. The system consisted of a Shimadzu GC-17A chromatograph
equipped with an AOC-20i autosampler. An OmegaWax (Supelco) fused-silica column (30 m,
0.25 mm i.d., 0.25 μm film thickness) was used. Helium was used as the carrier gas at a constant
flow of 1.5 ml/min. The column oven was programmed from 40°C (hold 4 min) to 200°C (hold 1
min) at 30°C /min. The split injection mode was used (1:5 split ratio, injection volume 1 μl). The
temperature of the injector was 150°C. The detector was maintained at 250°C. Chromatographic
data acquisition and processing were performed with Chrom&Spec software.
Supplementary Table 1 Primer sequences
Primer name Sequence 5`→3`
P1
tatggctgttactaatgtcgctgaacttaacgcactcgctcaagttagtataaaaaagctgaac
P2
ttaagcggattttttcgcttttttctcagctttagctgaagcctgcttttttatactaagttgg
P3
cagtgagtgtgagcgcgag
P4
gaagccgttatagtgcctcag
P5
aatgaaaatgatttccacgatacagaaaaaagagaccgctcaagttagtataaaaaagctgaac
P6
tcagcgcattccaccgtacgccagcgtcacttcctttgaagcctgcttttttatactaagttgg
P7
cgaccaccacgcaacatgag
P8
gtcagcgcattccaccgtac
P9
tatggtcattaaggcgcaaagcccggcgggtttcgccgctcaagttagtataaaaaagctgaac
P10
ttatcgcccctgaatggctaaatcacccggcagatttgaagcctgcttttttatactaagttgg
P11
gctatcagcgtagttagccc
P12
cagcatcgagttgctggaac
P13
tgcgacagatctctcacctaccaaacaatgccc
P14
tattcttttacctcttaacggccaatgcttcgtttc
P15
gatcggatccacgtcatttgggaaacgttctgacattattcttttacctcttaacggccaatg
P16
aaaactcaacgttattagatagataaggaataaccccgctcaagttagtataaaaaagctgaac
P17
ctagtaagatcttgaagcctgcttttttatactaagttgg
P18
gcaactaaacgtagaacctgtc
P19
tgagcacgctcaacaccttc
P20
gatgactgaacaggcaacaacaaccgatgaactggccgctcaagttagtataaaaaagctgaac
P21
ttagaactgcgattcttcagtggagccggtcagcgctgaagcctgcttttttatactaagttgg
P22
gaaacgtacctcagcaggtg
P23
gccttatccagcctacgttcg
Supplementary References
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia
coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640-6645
Katashkina JI, Skorokhodova AY, Zimenkov DV, Gulevich AY, Minaeva NI, Doroshenko VG,
Biryukova IV, Mashko SV (2005) Tuning the expression level of a gene located on a bacterial
chromosome. Mol Biol (Mosk) 39:719-726
Gulevich AYu, Skorokhodova AYu, Ermishev VYu, Krylov AA, Minaeva NI, Polonskaya ZM,
Zimenkov DV, Biryukova IV, Mashko SV (2009) A new method for the construction of
translationally coupled operons in a bacterial chromosome. Mol Biol (Mosk) 43:505-514