[Description]
[Invention Title]
GLUCONATE REPRESSOR VARIANT, MICROORGANISM CONTAINING THE
SAME PRODUCING L-LYSINE, AND METHOD FOR PRODUCING L-LYSINE
[Technical Field]
The present invention relates to a novel gluconate repressor variant, a microorganism
containing the same, and a method for producing L-lysine using the same.
[Background Art]
For the mass production of useful products such as amino acids, regulation of
glucose uptake and pentose phosphorylation pathway in the Corynebacterium strain is very
important (Handbook of Corynebacterium glutamicum. 2005. 215-240).
Gluconate repressor (GntR) is an important regulatory protein that regulates carbon
flow through glucose uptake and pentose phosphorylation pathways. It is known that two
gluconate repressors (GntR1 and GntR2) are found in the Corynebacterium glutamicum
strain.
GntR1 and GntR2 strongly inhibit the expression of genes encoding gluconate
permease (gntP), gluconate kinase (gntK), and 6-phosphogluconate dehydrogenase (gnd)
which are related to gluconate metabolism, and serve a role of weakly inhibiting the
expression of tkt-tal-zwf-opcA-devB operon, which is a major gene of the pentose
phosphorylation pathway. Meanwhile, the expression of genes encoding glucose-specific
transporter enzyme II (ptsG) and sucrose-specific transporter enzyme II (ptsS) of phosphotransferase
system (PTS), which is a major sugar-infusion metabolic pathway of the
Corynebacterium strain, is promoted to be significantly involved in sugar infusion (Julia
Frunzke, Verena Engels, Sonja Hasenbein, Cornelia Gatgens and Michael Bott, Molecular
Microbiology (2008) 67(2), 305-322).
[Disclosure]
[Technical Problem]
The present inventors have tried to find a gene that has an effective effect on L-lysine
productivity and developed a novel variant gluconate repressor 1 (GntR1), and it was
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confirmed that the microorganism containing the same producing L-lysine, compared to the
microorganism comprising wild-type gluconate repressor 1, enhanced sugar consumption rate
and L-lysine productivity, and thereby the present inventors completed the invention.
[Technical Solution]
An object of the present invention is to provide a novel variant of the gluconate
repressor protein.
Another object of the present invention is to provide a polynucleotide encoding the
variant and an expression vector comprising the polynucleotide.
Still another object of the present invention is to provide a microorganism producing
L-lysine, which expresses a variant of the gluconate repressor protein.
Yet another object of the present invention is to provide a method for producing Llysine
comprising: (a) culturing the microorganism producing L-lysine in a medium; and (b)
recovering L-lysine from the cultured microorganism or the medium in step (a).
[Advantageous Effects]
The microorganism containing a gluconate repressor variant producing L-lysine
according to the present invention exhibits an improved sugar consumption rate as compared
with the microorganism not containing the variant and can lead to effective production of Llysine.
In particular, L-lysine is an essential amino acid of animal feed and is required to be
industrially mass produced, and when L-lysine is produced with high efficiency according to
the present invention, it can also reduce the cost of producing feed.
[Best Mode]
An embodiment of the present invention provides a novel variant of the gluconate
repressor protein.
As used herein, the term “gluconate repressor protein” refers to a regulatory protein
involved in gluconate metabolism or sugar uptake. The gluconate repressor protein is not
particularly limited, but may be a microorganism belonging to the genus Corynebacterium,
specifically, a gluconate repressor protein derived from Corynebacterium glutamicum.
More specifically, the gluconate repressor protein may be GntR1 protein derived from
Corynebacterium glutamicum, but is not limited thereto. The gluconate repressor protein
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may be, but is not limited to, GntR1 comprising the amino acid sequence of SEQ ID NO: 4.
For example, the gluconate repressor protein may be an amino acid sequence comprising an
amino acid sequence of SEQ ID NO: 4 and having a homology of at least 80%, specifically at
least 90%, more specifically at least 95%, and even more specifically at least 99%. In
addition, the gluconate repressor protein comprising the amino acid sequence of SED ID NO:
4 may be encoded by the gntR1 gene comprising the nucleotide sequence of SEQ ID NO: 3,
but is not limited thereto. For example, it may be the nucleotide sequence of SEQ ID NO: 3
or may be a nucleotide sequence having a homology of at least 80%, specifically at least 90%,
more specifically at least 95%, or even more specifically at least 99%.
As used herein, the term “homology” refers to the percentage of identity between two
polynucleotides or polypeptide moieties. The homology between sequences from one
moiety to another moiety can be determined by known techniques. For example, homology
can be determined by aligning sequence information and directly aligning the sequence
information between two polynucleotide molecules or two polypeptide molecules using a
computer program that is already available. The computer program may be BLAST (NCBI),
CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc.), etc. Further, homology
between polynucleotides can be determined by hybridizing polynucleotides under conditions
that form a stable double strand between homologous regions, then resolving the singlestrand-
specific nucleases to determine the size of the resolved fragment.
The variant of the gluconate repressor protein may be a polypeptide in which
arginine (R), which is the 102nd amino acid in the amino acid sequence of SEQ ID NO: 4, is
substituted with an amino acid other than arginine. Specifically, the variant of the gluconate
repressor protein may be a polypeptide comprising an amino acid sequence of SEQ ID NO: 1,
wherein the amino acid at position 102 is substituted with cysteine (C).
Such a variant of the gluconate repressor protein includes not only a polypeptide
comprising an amino acid sequence of SEQ ID NO: 1, but also a variant having a homology
of 80% or more, specifically 90% or more, more specifically 95% or more, even more
specifically 99% or more, and substantially, it is obvious that an amino acid sequence having
biological activity identical to or corresponding to a protein having an amino acid sequence
of SEQ ID NO: 1, even in cases where some of the sequences have deletion, modification,
substitution, or addition of amino acid sequences, is included in the scope of the present
invention.
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Another embodiment of the present invention is a polynucleotide encoding a variant
of the gluconate repressor protein and an expression vector comprising the polynucleotide.
The gluconate repressor protein and a variant thereof are as described above.
Specifically, a polynucleotide encoding a variant of the gluconate repressor protein
may be a polynucleotide encoding a protein comprising an amino acid sequence of SEQ ID
NO: 1, or may be a polynucleotide having a homology of at least 80%, specifically at least
90%, more specifically 95%, even more specifically at least 99%. The polynucleotide
encoding a variant of the gluconate repressor protein may have, for example, the nucleotide
sequence of SEQ ID NO: 2, but is not limited thereto.
As used herein, the term “polynucleotide” refers to a polymer of nucleotides in which
the nucleotide unit is linked in a long chain by covalent bonds. Generally, it refers to DNA
or RNA strands longer than a certain length. The polynucleotide may have a variety of
nucleotide sequences that encode the same amino acid sequence due to genetic code
degeneracy of the genetic code. In addition, codon-optimized sequences may be employed
to optimize expression depending on the type of host cells.
As used herein, the term “vector” refers to any nucleic acid molecule for cloning
and/or transfer of a polynucleotide to a host cell. A vector may be a replicon that can bring
about the replication of a joined fragment of another DNA fragment. A “replication unit”
refers to any genetic unit (e.g., plasmid, phage, cosmid, chromosome, and virus). In the
present invention, the vector is not particularly limited as long as it is replicable in a host, and
any vector known in the art can be used. The vector used to construct the recombinant
vector may be a plasmid, a cosmid, a virus, and a bacteriophage in a natural or recombinant
state. For example, pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII, λgt10,
λgt11, Charon4A, Charon21A, etc. can be used as the phage vector or cosmid vector, and as
the plasmid vector, pDZ vector, pBR system, pUC system, pBluescriptII system, pGEM
system, pTZ system, pCL system, pET system, etc. can be used. The available vectors are
not particularly limited, and known expression vectors can be used. Specifically, pDZ
(Korean Registered Patent No. 10-0924065; incorporated herein by reference in its entirety)
may be used, but is not limited thereto.
As used herein, the term “transformation” is carried out by introducing a
polynucleotide encoding a variant of the gluconate repressor protein into a host cell so that
the polynucleotide can be expressed in the host cell, and the polynucleotide which is
transformed, as long as it can be expressed in the host cell, includes, without limitation, those
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which are inserted in a chromosome or located outside the chromosome.
The polynucleotide may be introduced into the host cell in the form of an expression
cassette, which is a construct containing all elements necessary for expression by itself. The
expression cassette can generally include a promoter operably linked to the gene, a
transcription termination signal, a ribosome binding site, and a translation termination signal.
The expression cassette may be in the form of an expression vector capable of self-replication.
Further, the polynucleotide may be introduced into the host cell itself or in the form of an
expression cassette and operatively linked to the sequence necessary for expression in the
host cell, but is not limited thereto.
Another embodiment of the present invention is a microorganism producing L-lysine
which expresses a variant of the gluconate repressor protein.
The gluconate repressor protein and a variant thereof have been described above.
As used herein, the term “L-lysine” is an essential amino acid that is not synthesized
in the body as a basic α-amino acid and refers to a kind of L-amino acid having the chemical
formula NH2(CH2)4CH(NH2)COOH. L-Lysine is biosynthesized from oxaloacetic acid
through the lysine biosynthetic pathway, and NADPH-dependent reductase catalyzes the
intermediate process for lysine biosynthesis. In the course of L-lysine biosynthesis of one
molecule, three molecules of NADPH are directly consumed by the sugar enzymes, and one
molecule of NADPH is indirectly used.
The microorganism may include variants of the gluconate repressor protein by
mutation or may be transformed with a vector comprising a polynucleotide encoding a
variant of the gluconate repressor protein.
The microorganism includes a protein having mutant activity of the gluconate
repressor protein or a prokaryotic microorganism and eukaryotic microorganism if the
microorganism is transformed so that the glycosyltransferase protein is expressed. For
example, microorganism strains such as the genus Escherichia, the genus Erwinia, the genus
Serratia, the genus Providencia, the genus Enterobacteria, the genus Salmonella, the genus
Streptomyces, the genus Pseudomonas, the genus Brevibacterium, or the genus
Corynebacterium, etc. may be included. Specifically, they may be microorganisms
belonging to the genus Corynebacterium, and more specifically, Corynebacterium
glutamicum, but are not limited thereto.
When the mutant protein of the gluconate repressor protein of the present invention
is contained in the microorganism producing L-lysine, L-lysine production capacity can be
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increased by effectively consuming the saccharide as compared with the microorganism
containing the wild-type gluconate repressor protein.
The microorganism producing L-lysine may include any type of microorganism if it
has L-lysine producing ability, and includes all forms of natural strains and recombinant
strains.
The microorganism of the genus Corynebacterium capable of having L-lysine
producing ability may be a mutant strain resistant to the L-lysine analogue. L-Lysine
analogues inhibit the growth of Coryne microorganisms, but such inhibition is completely or
partially cleared when L-lysine coexists in the medium. Examples of the L-lysine analogues
include oxa L-lysine, L-lysine hydroxamate, S-(2-aminoethyl)-L-cysteine (AEC), γ-methyl
L-lysine, α-chlorocaprolactam, etc., but are not limited thereto. The variant having
resistance to such L-lysine analogues can be obtained by treating the microorganism of the
genus Corynebacterium with a conventional artificial mutagenesis treatment, but is not
limited thereto. Non-limiting examples of this include Corynebacterium glutamicum
KCCM11016P (Korean Registered Patent No. 10-0159812), Corynebacterium glutamicum
KFCC 11001, and Corynebacterium glutamicum KCCM11347P (Korean Registered Patent
No. 10-0073610), but are not limited thereto.
In addition, the microorganism of the genus Corynebacterium having L-lysine
producing ability may be modified such that the activity of the L-lysine biosynthesis-related
protein is enhanced as compared with non-modified strains. That is, the expression of one
or more types of L-lysine biosynthesis-related genes is enhanced. Such expression
enhancement may include, but is not limited to, gene amplification, replacement or alteration
of sequences such as promoters or initiation codons, introduction of modifications to improve
expression, etc. Non-limiting examples of this include Corynebacterium glutamicum CJ3P
(Binder et al. Genome Biology 2012, 13:R40), but are not limited thereto.
In addition, examples of the L-lysine biosynthesis-related gene include genes located
on the L-lysine biosynthetic pathway, and specifically, dihydrodipicolinic acid synthase gene
(dapA), aspartokinase gene (lysC), dihydrodipicolinic acid reductase gene (dapB),
diaminopimelate decarboxylase gene (lysA), diaminopimelate dehydrogenase gene (ddh),
phosphoenolpyruvate carboxylase gene (ppc), aspartate semialdehyde dehydrogenase gene
(asd), aspartase gene (aspA), and pyruvate carboxylase (pyc), but are not limited thereto.
Further, transketolase (tkt) present on the pentose phosphate pathway, etc. may be referenced,
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but the gene is not limited thereto.
Meanwhile, the microorganism of the genus Corynebacterium which can have the Llysine
producing ability by including modifications known in the art associated with L-lysine
production, but is not limited thereto.
Another aspect of the present invention is a method for producing L-lysine
comprising: (a) culturing a microorganism producing L-lysine in a medium; and (b)
recovering L-lysine from the cultured microorganism or culture medium in step (a).
The L-lysine and the microorganism producing it are as described above.
As used herein, the term “culture” refers to cultivation of the microorganism under
moderately artificially controlled environmental conditions. The culturing process of the
present invention can be carried out according to a suitable culture medium and culture
conditions known in the art. Conditions such as the specific culture temperature, incubation
time, and pH of the medium can be carried out according to the general knowledge of the
person skilled in the art or according to conventionally known methods. Specifically, these
known culture methods are described in [Chmiel; Bioprozesstechnik 1. Einfuhrung indie
Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991), and Storhas; Bioreaktoren und
periphere Einrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)]. Further, the
culture method includes a batch culture, a continuous culture, and a fed-batch culture.
Specifically, a batch process or a fed batch or a repeated fed batch process can be
continuously cultured, but the culturing process is not limited thereto.
The medium used for culture shall meet the requirements of specific strains in a
proper manner. Carbon sources which may be used for the medium include saccharides and
carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, cellulose, etc.;
oil and fat such as soybean oil, sunflower oil, castor oil, coconut oil, etc.; fatty acid such as
palmitic acid, stearic acid, linoleic acid, etc.; glycerol; alcohol such as ethanol; and organic
acid such as acetic acid, but are not limited thereto. These substances can be used
individually or as a mixture and are not limited thereto. Nitrogen sources which may be
used include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal, and
urea or inorganic compounds, for example, ammonium sulfate, ammonium chloride,
ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., but are not limited
thereto, and nitrogen sources can also be used individually or as a mixture. Available
phosphate sources include potassium dihydrogen phosphate, dipotassium hydrogen phosphate,
or corresponding sodium-containing salts, etc. but are not limited thereto. Further, the
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culture medium may contain metallic salts such as magnesium sulfate or iron sulfate, but the
salts are not limited thereto. Finally, in addition to the above materials, essential growth
substances such as amino acids and vitamins may be used. Further, appropriate precursors
may be used for the culture medium. Raw materials described above may be added batchwise
or continuously to an incubator in the incubation process, but are not limited thereto.
In addition, chemical compounds such as ammonium hydroxide, potassium
hydroxide, ammonia, phosphoric acid, and sulfuric acid may be appropriately added to the
incubator during culture to adjust the pH of the culture medium. During incubation,
production of bubbles can be inhibited by using antifoaming agents such as fatty acid
polyglycol. Further, in order to maintain the aerobic condition of the incubator or
incubation, oxygen or oxygen-containing gas can be added to the incubator, or in order to
maintain anaerobic conditions, nitrogen, hydrogen, or carbon dioxide gas may be injected or
gas may not be injected. The temperature of the incubation is typically between 20°C to
40°C, specifically 30°C to 35°C, but is not limited thereto. The incubation period can be
continued until the desired amount of useful material is achieved, and specifically may be 10
hours to 100 hours. L-Lysine may be excreted from the culture medium or included in
microorganisms.
In addition, methods for producing L-lysine of the present invention or methods for
recovering L-lysine from the culture medium are widely known in the art. For the methods
for recovering L-lysine, filtration, negative ion exchange chromatography, crystallization, and
HPLC, etc. may be used, but the methods are not limited to these examples.
[Best Mode]
Hereinbelow, the present invention will be described in more detail through the
following Examples. However, these Examples are intended to illustrate the present
invention in an exemplary manner, and the scope of the present invention is not limited to
these Examples.
Example 1: Preparation of recombinant Corynebacterium glutamicum genomic
DNA library
In the present Example, in order to discover the producing ability of L-lysine and
genes and variations that discover beneficial effects to productivity, after inducing artificial
mutations on Corynebacterium glutamicum ATCC13032 strains by N-methyl-N-nitro-NWO
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nitroguanidine (NTG), genomic DNA was extracted by extraction methods conventionally
known in the art. Restriction enzyme Sau3AI was applied to the prepared DNA obtain
partial fragments having a size of 1 kb to 3 kb. After connecting the fragments to
transformant shuttle vector pECCG122 to E. coli and Corynebacterium to restriction enzyme
BamHI end, they were transformed to E. coli DH5α and smeared to LB solid medium
including kanamycin (25 mg/L). After selecting colonies transformed into vectors inserted
with the fragments through PCR (using primers of SEQ ID NOs: 5 and 6), all of these were
mixed together to extract plasmids by a commonly known extraction method.
SEQ ID NO: TCAGGGTGTAGCGGTTCGGTTTAT
SEQ ID NO: CCGCGCGTAATACGACTCACTATA
Example 2: Introduction of artificial variant library and L-lysine producing
ability enhancement
By having KCCM11016P strain as a parent strain (the microorganism was disclosed
as KFCC10881 and re-deposited into an international depository institution under the
Budapest Treaty and assigned an accession number of KCCM11016P, Korean Patent
Registration Number 10-0159812), the prepared vector was transformed and smeared to a
composite plate medium below.

Glucose 20 g, (NH4)2SO4 50 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4
5 g, K2HPO4 10 g, MgSO4 7H2O 0.5 g, biotin 100 μg, thiamine chloride salt 1,000 μg,
calcium-pantothenic acid 2,000 μg, nicotinamide 2,000 μg, agar 20 g, and kanamycin 25 mg
(1 L of distilled water).
About 10,000 colonies were seeded to each of 300 μL of selection media and
cultured for about 16 hours at 1,000 rpm at 32°C in a 96 deep well plate. In order to analyze
the production amount of L-lysine produced during culture, the ninhydrin method was used (J.
Biol. Chem. 1948. 176:367-388). After the culture was completed, 10 μL of culture
supernatant and 190 μL of ninhydrin reaction solution (63% glycerol, 27% ninhydrin solution)
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were reacted at 65°C for 30 minutes, absorbance was measured by a spectrophotometer at a
wavelength of 570 nm, and compared to absorbance of control group
(KCCM11016P/pECCG122), 248 species of variant strain exhibiting similar or increased
absorbance were selected. During culture under the condition, since the control group strain
had residual sugar of 1 g/L to 2 g/L, sugar consumption rate per control group was fast, and
strains with increased lysine producing ability could be selected.

Glucose 10 g, ammonium sulfate 5.5 g, MgSO4·7H2O 1.2 g, KH2PO4 0.8 g,
K2HPO4 16.4 g, biotin 100 μg, thiamine HCl 1,000 μg, calcium-pantothenic acid 2,000 μg,
and nicotinamide 2,000 μg (1 L of distilled water standard).
In addition, based on strains of 248 selected species, the method was repeatedly
performed. As a result, variant strains of the above 29 species which have an enhanced
producing ability of L-lysine by more than 10% compared to KCCM11016P/pECCG122
strain were selected.
Example 3: L-lysine producing ability of selected artificial variant strains and
analysis of sugar consumption rate
L-Lysine producing ability of the strains of the 29 selected species of Example 2 and
sugar consumption rate were cultured according to the method below and analyzed.
Each strain was seeded into a 250 mL corner-baffle flask and shake-cultured at 30°C
for 20 hours at 200 rpm. Afterwards, 1 mL of a species culture solution was seeded into a
250 mL corner-baffle flask containing 24 mL of a production medium. Using HPLC, the
concentration of L-lysine was analyzed, and in order to measure sugar consumption rate of
the strains and productivity thereof, the concentration of remaining sugar concentration
among the culture solution per time was measured.

Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g,
MgSO4·7H2O 0.5 g, biotin 100 μg, thiamine HCl 1,000 μg, calcium-pantothenic acid 2,000
μg, nicotinamide 2,000 μg (1 L of distilled water standard).
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Glucose 100 g, (NH4)2SO4 40 g, soybean protein 2.5 g, corn steep solids 5 g, urea 3
g, KH2PO4 1 g, MgSO4·7H2O 0.5 g, biotin 100 μg, thiamine chloride salt 1,000 μg, calcium
pantothenic acid 2,000 μg, nicotinamide 3,000 μg, and CaCO3 30 g (1 L of distilled water
standard).
Among variant strains of 29 species, the concentration of L-lysine compared to the
control group was above an equivalent level, and three types of the strains with increased
sugar consumption rate and productivity were selected for repeatedly performing the culture
and analysis, and the analyzed results are shown in Table 1.
[Table 1]
Strain number Residue sugar (g/L) L-Lysine
(g/L)
Average
18 hours 28 hours
KCCM11016P
/pECCG122
1 50.9 38.2 43.1 43.4
2 51.9 39.8 42.5
3 51.2 37.6 44.5
KCCM11016P/3 1 47.9 34.2 43.3 43.4
2 48.6 33.8 43.8
3 46.9 31.9 43.0
KCCM11016P/94 1 47 33.7 44.5 44.3
2 46.7 33.6 44.3
3 44.8 32.8 44.1
KCCM11016P/1160 1 42.2 26.8 44.2 43.9
2 43.5 25.4 44.1
3 42.9 24.3 43.5
It was confirmed that the three strains produced L-lysine above the equivalent level
of the control group, and the rate of consumption per unit hour was enhanced by improving
the fermentation productivity. Among the selected variation strains, strain
KCCM11016P/1160 with the most meaningfully enhanced productivity was ultimately
selected, and plasmids were extracted to search for nucleotide sequence mutations at the
fundamental genetic level that caused sugar consumption rate and productivity increase.
Afterwards, nucleotide sequences were analyzed using SEQ ID NOs: 5 and 6. Plasmids
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derived from KCCM11016P/1160 contained from about 450 bp region upstream of
NCgl2440 gene ORF initiation codon to the vicinity of about 350 bp downstream of the
termination codon. Further, through nucleotide sequence analysis of the gene based on the
National Institutes of Health (NIH) GenBank, the gene was confirmed to be gntR1, which is
one of gluconate repressors, and gntR1 variation which was introduced to
KCCM11016P/1160 strain was substituted from C to T at the 304th nucleotide sequence, and
it was confirmed that the 102nd amino acid was mutated from arginine to cysteine.
That is, it was confirmed that arginine, which is the 102nd amino acid of gntR1,
having an amino acid sequence of SEQ ID NO: 4, was substituted with cysteine.
Example 4: Vector preparation for introducing gntR1 variation into the
chromosome of L-lysine producing strain
In order to confirm the application effect of gntR1 variation which was confirmed in
Example 3, a vector which can introduce the same into chromosome was prepared.
Based on reported nucleotide sequences, a primer (SEQ ID NO: 7), in which EcoRI
restriction enzyme region was inserted to the 5’ end and SalI restriction enzyme region was
inserted to the 3’ end, was synthesized. Using such primer pairs, PCR was performed by
having a plasmid of KCCM11016P/1160 as a mold to amplify gntR1 gene fragments. The
PCR condition was repeating 30 times a cycle of denaturing at 94°C for 5 minutes,
denaturing at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and polymerizing at
72°C for 40 seconds, and a polymerization reaction was performed at 72°C for 7 minutes.
SEQ ID NO: 7: AAGAATTCAGCAGATCGAAGAAGAAGC
SEQ ID NO: 8: AAGTCGACGGGACTAAAAGCTGGCG
After treating gene fragments which were amplified with PCR with restriction
enzymes EcoRI and SalI, each DNA fragment was obtained, and after connecting the same to
pDZ vector (Korean Patent Registration Number 10-0924065) having restriction enzymes
EcoRI and SalI ends, it was transformed to E. coli DH5α and blotted out on an LB solid
medium containing kanamycin (25 mg/L). After selecting a colony which was transformed
with a vector in which the target gene was inserted through PCR, a plasmid was obtained
using a commonly known plasmid extraction method, and according to the variation which
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was inserted in gntR1 of the plasmid, it was named pDZ-gntR1 (R102C).
Example 5: Preparation of strain in which gntR1 variation is introduced into
KCCM11016P, an L-lysine production strain, and comparison of productivity
A novel variant introduction vector prepared in Example 4 was transformed into
Corynebacterium glutamicum KCCM11016P, which is an L-lysine production strain,
according to two-step homologous chromosome recombination. Afterwards, the strain in
which gntR1 variation on chromosome was introduced was selected according to
chromosome sequence analysis, and the strain in which the gntR1 variation was introduced
was named as KCCM11016P::gntR1(R102C).
By culturing with same methods as in Example 3, the concentration of L-lysine and
residual sugar concentration were measured therefrom. In addition, in order to confirm
culture effects from raw sugar, raw sugar production media instead of glucose production
media were used to analyze using the same methods [Table 2].

Raw sugar 100 g, (NH4)2SO4 40 g, soybean protein 2.5 g, corn steep solids 5 g,
enzyme 3 g, KH2PO4 1 g, MgSO4·7 H2O 0.5 g, biotin 100 μg, thiamine chloride acid salt
1,000 μg, calcium-pantothenic acid 2,000 μg, nicotinamide 3,000 μg, CaCO3 30 g (1 L level
of distilled water).
【Table 2】
Strain number Residual sugar (g/L) L-Lysine
(g/L)
Average
18 hours 28 hours
Sugar KCCM11016P 1 47.1 32.5 43.8 43.5
2 46.2 33.8 42.6
3 45 32.0 44.1
KCCM11016P::GntR1(R
102C)
1 34.5 21.4 43.5 43.9
2 39.5 21.2 44.2
3 37 20.6 43.9
Raw
sugar
KCCM11016P 1 42.9 28.6 47.3 47.0
2 42.0 29.1 46.
3 41.0 28.4 47.6
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KCCM11016P::GntR1(R
102C)
1 34.1 17.2 47 47.4
2 35.2 16.3 47.7
3 33.7 15.6 47.4
As a result, it was confirmed that sugar consumption rate was increased without
affecting L-lysine production ability KCCM11016P::gntR1(R102C) more than KCCM11016,
which is a comparison group in glucose and original sugar media. Further, as a result of
confirming time points when all of glucose and original sugar were consumed, comparison
groups were completely consumed after 72 hours and 68 hours, and L-lysine was produced at
43.5 g/L and 47.0 g/L, respectively, and gntR1 variation introduction strains were produced at
43.9 g/L and 47.4 g/L after 60 hours and 58 hours. That is, in a case of strains in which
gntR1 variation is introduced, it was confirmed that production ability of L-lysine according
to time was increased by 21% and 18% with glucose and raw sugar, respectively [Table 3].
[Table 3]
Strain number Production
ability (g/h)
%
Glucose KCCM11016P 0.60 100
KCCM11016P::gntR1(R102C) 0.73 121
Raw sugar KCCM11016P 0.69 100
KCCM11016P::gntR1(R102C) 0.82 118
As can be seen from the above results, the introduction of gntR1 variation has Llysine
producing ability at the same level and is effective in glucose and raw sugar
consumption rates. That is, when gntR1 variation was introduced of the present invention, it
suggests excellence of effectiveness in L-lysine production ability. As such, the present
inventors named KCCM11016P::gntR1(R102C) as Corynebacterium glutamicum “CA01-
2293”, which has enhanced glucose consumption rate and productivity, and as of December 5,
2014, it was given an accession number of KCCM11628P, which is deposited at the Korea
Culture Center of Microorganisms (KCCM), an international depository institution.
Example 6: Preparation of strains in which gntR1 variation is introduced in Llysine
production strain and comparison of productivity
In order to determine effects of other strains belonging to Corynebacterium
WO 2016/171392 A1 PCT/KR2016/002480
16
glutamicum, a strain in which gntR1 variation is introduced into Corynebacterium
glutamicum KCCM11347P (the microorganism is disclosed as KFCC10750 and redeposited
in an international depository institution under the Budapest Treaty and assigned
KCCM11347P. Korean Patent Registration Number 10-0073610) was prepared and named
KCCM11347P::gntR1(R102C). Using the same method as in Example 5, the prepared
strains were cultured, and lysine production ability and residual sugar were compared [Table
4].
【Table 4】
Strain number Residual sugar (g/L) L-Lysine
(g/L)
Average
18 hours 28 hours
Glucose KCCM11347P 1 43.8 27.6 38.5 38.6
2 42.6 28.8 38.7
3 43.2 27.2 38.6
KCCM11347P::gntR1(R1
02C)
1 39.2 18.2 38.6 38.7
2 37.6 18.0 38.6
3 37.0 17.5 38.9
Residue
sugar
KCCM11347P 1 39.9 24.9 41.2 41.3
2 39.3 25.3 41.4
3 38.8 24.7 41.3
KCCM11347P::gntR1(R1
02C)
1 35.7 14.4 41.3 41.4
2 34.2 13.7 41.3
3 33.3 13.1 41.6
As a result, same as in Example 5, it was confirmed that all glucose consumption
rates were increased in glucose and raw sugar compared to the comparison group. Further,
as a result of determining time points at which glucose and raw sugar were all consumed, the
comparison group all consumed sugar at 72 hours and 69 hours, and L-lysine was produced at
38.6 g/L and 41.3 g/L, and gntR1 variation produced L-lysine at 61 hours and 58 hours at
38.7 g/L and 41.4 g/L. That is, in a case of strains wherein gntR1 variation is introduced,
the productivity of L-lysine according to time was confirmed to be increased by 18% and 19%
in glucose and raw sugar, respectively [Table 5].
WO 2016/171392 A1 PCT/KR2016/002480
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【Table 5】
Strain number Productivity (g/h) %
Glucose KCCM11347P 0.54 100
KCCM11347P::gntR1(R102C) 0.63 118
Raw sugar KCCM11347P 0.60 100
KCCM11347P::gntR1(R102C) 0.71 119
Example 7: Preparation of strain in which gntR1 variation is introduced in Llysine
production strain CJ3P and comparison of productivity
In order to confirm effects in other strains belonging to Corynebacterium glutamicum,
a strain in which gntR1 variation is introduced in Corynebacterium glutamicum CJ3P (Binder
et al. Genome Biology 2012, 13: R40) was prepared, and it was named as
CJ3P::GntR1(R102C). CJ3P strain is a Corynebacterium glutamicum strain having L-lysine
producing ability in which 3 types of variations (pyc(Pro458Ser), hom(Val59Ala),
lysC(Thr311Ile)) were introduced in wild-type strains based on a disclosed technique. A
strain which is prepared using the same method as in Example 5 was prepared, and the
producing ability of lysine and concentration of residual sugar in a culture solution per time
was measured [Table 6].
【Table 6】
Strain number Residual sugar (g/L) L-Lysine
(g/L)
Average
12 시간 18 시간
Glucose CJ3P 1 37.2 23.6 8.1 8.2
2 37.9 23.0 8.2
3 37.4 21.1 8.2
CJ3P::gntR1(R102C) 1 35.0 17.7 8.4 8.4
2 35.5 17.5 8.3
3 34.2 16.7 8.4
Raw
sugar
CJ3P 1 34.3 21.5 8.7 8.8
2 34.1 20.9 8.8
3 32.7 19.2 8.8
CJ3P::gntR1(R102C) 1 30.8 16.1 9.0 9.0
2 31.8 15.9 8.9
3 31.3 15.2 9.0
As a result, similar to Examples 5 and 6, it was confirmed that sugar consumption
rate was increased in gntR1 variation insertion strain compared to the control group. Further,
WO 2016/171392 A1 PCT/KR2016/002480
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as a result of confirming the time point at which glucose and raw sugar were all consumed,
glucose and raw sugar were completely consumed at 48 hours and 42 hours in the control
group, and L-lysine was produced at 8.2 g/L and 8.8 g/L, respectively, and gntR1(R102C)
insertion strain produced lysine at 42 hours and 38 hours at 8.4 g/L and 9.0 g/L, respectively.
That is, in a case of a strain, gntR1 variation is introduced, it was confirmed that the
production rate of L-lysine according to time increased by 17% and 13%, respectively [Table
7].
【Table 7】
Strain Production rate
(g/h)
%
Glucose CJ3P 0.17 100
CJ3P::gntR1(R102C) 0.20 117
Raw sugar CJ3P 0.21 100
CJ3P::gntR1(R102C) 0.24 113
Accordingly, from the above results, when gntR1 variation was introduced in lysine
production strain in the Corynebacterium genus, it was confirmed that due to an increase in
sugar consumption rate, lysine production rate was effectively enhanced.
From the foregoing, a skilled person in the art to which the present invention pertains
will be able to understand that the present invention may be embodied in other specific forms
without modifying the technical concepts or essential characteristics of the present invention.
In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes
and should not be construed as limiting the scope of the present invention. On the contrary,
the present invention is intended to cover not only the exemplary embodiments but also
various alternatives, modifications, equivalents, and other embodiments that may be included
within the spirit and scope of the present invention as defined by the appended claims.
WO 2016/171392 A1 PCT/KR2016/002480

We Claim:
[Claim 1] A polypeptide having gluconate repressor activity comprising the amino acid
sequence of SEQ ID NO: 1.
[Claim 2] A polynucleotide encoding the polypeptide of claim 1.
[Claim 3]
The polynucleotide of claim 2, wherein the polynucleotide comprises the nucleotide
sequence of SEQ ID NO: 2.
[Claim 4] An expression vector comprising the polynucleotide of claim 2.
[Claim 5] A microorganism of the genus Corynebacterium producing L-lysine, expressing the
polypeptide of claim 1.
[Claim 6]
The microorganism of the genus Corynebacterium of claim 5, wherein the
microorganism of the genus Corynebacterium is Corynebacterium glutamicum.
[Claim 7] A method for producing L-lysine, comprising
(a) culturing the microorganism of the genus Corynebacterium of claim 5 or 6 in a
medium; and
(b) recovering L-lysine from the cultured microorganism or the medium in step (a).
Dated this 26th day of October, 2017