Supplementary Information
Metabolic Engineering of Corynebacterium glutamicum
for the Production of L-ornithine
Seo Yun Kim,1 Joungmin Lee,1 and Sang Yup Lee1,2
1
Metabolic and Biomolecular Engineering National Research Laboratory, Department of
Chemical and Biomolecular Engineering (BK21 plus program), Center for Systems and
Synthetic Biotechnology, BioProcess Engineering Research Center, and, Institute for the
BioCentury, KAIST, 291 Daehakro, Yuseong-gu, Daejeon 305-701, Republic of Korea;
telephone: +82-42-350-3930; fax: +82-42-350-8800; e-mail: leesy@kaist.ac.kr
2
Bioinformatics Research Center, KAIST, Daejeon, Republic of Korea
Correspondence to: S.Y. Lee
1
Supplementary Materials and Methods
Construction of the plasmids
All primers used in this study are listed in Supplementary Table I. Pfu-X DNA polymerase
(Solgent, Daejeon, Korea), restriction enzymes (New England Biolabs, Ipswich, MA), and T4
DNA ligase (Elpis Biotechnology, Daejeon, Korea) were used for the cloning. Plasmids were
purified using a DNA-Spin purification kit (iNtRON Biotechnology, Daejeon, Korea), and
genomic DNAs using an ExgeneTM Cell SV kit (GeneAll Biotechnology, Seoul, Korea). The
DNA sequences of the constructed plasmids were confirmed by Sanger sequencing.
For the construction of pSY927 and pSY223, the argCJBD genes including the
native promoters were amplified with their native promoters from genomic DNA of C.
glutamicum ATCC 13032 and ATCC 21831 with the primers WT_F/WT_R and
AT1_F/AT1_R, respectively. Each PCR product was assembled with the pEK0 that was
amplified by inverse PCR with K0_F/K0_R (Gibson et al., 2009).
For the construction of pSY01, partial fragments of the proB gene were amplified
from
the
genomic
DNA of
C.
glutamicum
ATCC
13032
with
the
primers
ProB_LA_F/ProB_LA_R and ProB_RA_F/ProB_RA_R (Supplementary Table I). The
resulting two distant homologous arms, each having a length of ca. 0.5 kb, were fused by
overlapping PCR. The finally assembled fragment was cloned through Gibson assembly with
the inverse PCR product of pK19mobsacB plasmid amplified with the primer pair pK19_F/R.
The pSY03 plasmid (for argR knockout) was constructed in the same way as pSY01 using
the primer pairs ArgR_LA_F/ ArgR_LA_R and ArgR_RA_F/ ArgR_RA_R. For the
construction of the pSY02 plasmid (for argF knockout), two homologous arms of the argF
gene were amplified from C. glutamicum ATCC 13032 using primers ArgF_LA_F/
ArgF_LA_R and ArgF_RA_F/ ArgF_RA_R. These two fragments are fused by overlapping
2
PCR, digested with BamHI/HindIII, and ligated into BamHI/HindIII double digested
pK19mobsacB. The pSY04 and pSY05 plasmids, for the start codon replacement of the pgi
and zwf genes, respectively, were constructed in the same manner as pSY01. For the
construction of pSY06, two homologous regions flanking the native promoter of the tkt
operon and the sod promoter were amplified from the genomic DNA of C. glutamicum ATCC
13032
with
the
primer
pairs
Tkt_LA_F/Tkt_LA_R,
Tkt_RA_F/Tkt_RA_R,
and
Sod_F/Sod_R, respectively. These fragments were assembled together into pK19mobsacB by
Gibson assembly, so that the sod promoter located between two homologous arms.
Transformation and chromosomal manipulation in C. glutamicum
The C. glutamicum cells was grown in 25 mL of the CR medium up to OD600 0.7 to 0.8 and
harvested by centrifuge. The pellet was washed twice using 25 mL of EPB1 buffer containing
20 mM of HEPES and 5% (w/v) of glycerol. Finally, cells were resuspended in 1 mL of
EPB2 buffer containing 5 mM of HEPES and 15% (w/v) of glycerol. The 0.1 ml of
competent cells and DNA were mixed and put into a 2-mm gapped cuvette. The cuvette was
subject to an exponential decay pulse of 1.8 kV, 200 Ω, and 25 μF. Immediately after the
pulse, 1 mL of the CR medium was added into the cuvette, and the cells were cultivated at
30°C for 2 hours for the recovery. Cells were spread onto a CR agar supplemented with Km.
Gene knockout or allelic replacement in C. glutamicum was conducted according to Schafer
et al. (1994). A knockout plasmid or a replacement plasmid was introduced into C.
glutamicum via electroporation, and single-crossover mutants were selected by kanamycin
resistance. Double-crossover mutants were selected by the loss of sucrose sensitivity on CR
medium supplemented with 15% (w/v) sucrose. Knockout mutants were first verified by the
loss of kanamycin resistance and finally by colony PCR. Those with codon or promoter
replacement were verified by sequencing of the PCR products from the mutants.
3
Supplementary Table I. Plasmids used in this study.
Sequence(5’->3’)
Primer
pK19_F
GGGCGGTTTTATGGACAGCAAGCGA
pK19_R
GGCGAGCGGTATCAGCTCACTCAAA
AACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCAACG
ProB_LA_F
ATGGTAACGCTATGAA
ProB_LA_R
ATACCGGGCAAAAGAACGTCCCC
ProB_RA_F
GACGTTCTTTTGCCCGGTATTTGTATGCCGCAGATACTGCAGGA
GCTGGCAATTCCGGTTCGCTTGCTGTCCATAAAACCGCCCCGGC
ProB_RA_R
AAATCCGGGAACTCCG
ArgF_LA_F
ACCGAAGCTTGTTGTTCCCGATGTGGTGAC
ArgF_LA_R
ATCATCAGCCAGAAAATGGC
CGCCATTTTCTGGCTGATGATCAGCAGGCCTTAAGGGTAAGAAG
ArgF_RA_F
G
ArgF_RA_R
TCAAGGATCCTTACCTCGGCTGGTTGGCCA
AACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCTTGC
ArgR_LA_F
CACCGCGGGCATGGAT
ArgR_LA_R
AATGAGAGCTTGGCGTGCAG
ArgR_RA_F
CTGCACGCCAAGCTCTCATTCTCAGCGGGCGCACCACTTA
GCTGGCAATTCCGGTTCGCTTGCTGTCCATAAAACCGCCCCTCA
ArgR_RA_R
ACGAGGTGCTTGACGA
GGGCGGTTTTATGGACAGCAAGCGAGCGTTGCCAACGCTCAGC
Zwf_LA_F
G
Zwf_LA_R
GTTTGTGCTCATGATGGTA
4
Zwf_RA_F
CTACCATCATGAGCACAAAC
GGCGAGCGGTATCAGCTCACTCAAATGATCACGCGGCGCCATGC
Zwf_RA_R
T
Tkt_LA_F
ATGAGGATCCAGAGTGTTTACTCAAGACATTTTCTAAGA
Tkt_LA_R
CATTCGCAGGGTAACGGCCATCTTAAGTCTGGGAGCTGTGACG
Sod_F
AGCTGCCAATTATTCCGGGCTTG
Sod_R
TTTCCGCACCGAGCATATACATCTT
Tkt_RA_F
GATGTATATGCTCGGTGCGGAAATTGACCACCTTGACCTGTC
Tkt_RA_R
GAATTCCGCCCTCAGCAGCGG
K0_F
TGGGGTGCCTAATGAGTGAGCTAAC
K0_R
CTACGGTGTGCAATCTATTCACGAT
GTATCTGGGGGTGGCATCGTGAATAGATTGCACACCGTAGAATT
WT_F
CATGCTTTTACCCACTTGCAGTT
ACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCATTATG
WT_R
CGATTGTCTCGGCAATAG
GTATCTGGGGGTGGCATCGTGAATAGATTGCACACCGTAGAATT
AT1_F
CATGCTTTTACCCACTTGCA
ACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCATTATG
AT1_R
CGATTGTCTCGGCAATAGC
5
Table II. Production of L-ornithine by C. glutamicum strains in the flask culturea,b.
Strain
L-ornithine (mg/L)
Cell growth (OD600)
Lactic acid (g/L)
Acetic acid (g/L)
Wild-type
35.33 ± 0.01
35.33 ± 0.09
45.61 ± 0.12
4.67 ± 0.02
YW02
37.80 ± 0.12
37.80 ± 0.14
25.61 ± 0.12
3.61 ± 0.15
YW03
230.29 ± 0.07
33.02 ± 0.16
27.86 ± 0.08
3.32 ± 0.07
YW03 (pSY927)
1970.12 ± 0.25
23.31 ± 0.09
19.23 ± 0.03
1.32 ± 0.12
YW03 (pSY223)
7190.27 ± 0.21
31.07 ± 0.05
16.76 ± 0.07
1.60 ± 0.04
YW04
242.29 ± 0.21
29.29 ± 0.09
26.86 ± 0.18
4.38 ± 0.16
YW04 (pSY223)
6549.65 ± 0.13
22.20 ± 0.02
27.39 ± 0.11
1.91 ± 0.01
YW05
261.29 ± 0.37
24.57 ± 0.11
29.86 ± 0.27
3.72 ± 0.13
YW06
417.21 ± 0.35
19.33 ± 0.17
29.51 ± 0.14
3.29 ± 0.08
YW06 (pSY223)
8549.65 ± 0.10
23.29 ± 0.08
28.12 ± 0.09
1.86 ± 0.01
a
The values were obtained by triplicate experiments; (mean) ± (SD)
b
Glucose (initially 50 g/L) was completely consumed in every strain.
6
Supplementary Table III. Mutations identified in the argCJBD genes of the ATCC 21831
strain compared to those in ATCC 13032 straina.
Gene
Type
Mutation (nucleotide)
Mutation
(amino
acid)
argCa
Nonsynonymous
Deletion (1..30)b, C32T Deletion(1..10)a,
T12I
argJ
Nonsynonymous
C553T, T849C, T864G, G331D
G992A
argB
Nonsynonymous
T144C,
A160G, M64Vc
C354T, T576C, A594G,
C774T, G882A
argD
Synonymous
T94C, G1152A
None
a
According to Park et al. (2014)
b
This difference might be originated from the different annotation algorithms used for the
ATCC 13032 and 21832 strains; the sequence actually exists in the ATCC 21831 strain.
c
This mutation is identical to M31V reported by Ikeda et al. (2009) and Park et al. (2014).
7
References
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