DESCRIPTION
Invention Title
A MICROORGANISM OF GENUS CORYNEBACTERIUM HAVING AN ABILITY
TO PRODUCE L-ARGININE AND A METHOD FOR PRODUCING L-ARGININE
5 USING THE SAME
Technical Field
The present invention relates to a microorganism of the genus Corynebacterium
having an ability to produce L-arginine and a method of producing L-arginine using
10 the same.
Background Art
L-arginine is an amino acid widely used in amino acid supplements, pharmaceutical
drugs, foods, etc., and there has been demand for the development of efficient L-
15 arginine production in the related industries.
The method of producing L-arginine by a conventional biological fermentation
method is a method to produce L-arginine directly from carbon and nitrogen sources,
and various methods including a method using an induced modified strain from a
microorganism of the genus Brevibacterium or Corynebacterium, a method using a
20 bacterial cell line developed to have enhanced amino acid-producing ability by cell
fusion, etc., have been reported. Recently, a method of using a genetic recombinant
strain, wherein a gene which inhibits the expression of arginine-biosynthesizing
operon argR was inactivated (U.S. Patent No. 7,160,705), and a method of using the
overexpression of argF in the arginine operon (Korean Patent No. 10-0854234), etc.,
25 were reported. In particular, the deletion in argR, which controls the arginine
operon, has been considered as an important factor in arginine production.
According to the facts known so far, in a microorganism of Corynebacterium,
argCJBDFR gene, which is involved in arginine biosynthesis, is constituted in the
form of an operon and is subjected to feedback-inhibition by intracellular arginine
30 (Vehary Sakanyan, et al., Microbiology, 142:9-108, 1996), thus imposing a limitation on
2
its high-yield L-arginine production.
Disclosure
Technical Problem
5 Accordingly, the present inventors, while endeavoring to increase the production
yield of L-arginine, discovered that L-arginine can be produced in higher yield
compared to the parental L-arginine-producing strain, by enhancing the activities of
the arginine operon and ornithine carbamoyltransferase, without any deletion in
arginine repressor (argR), which has conventionally been known as an important
10 factor, thereby completing the present invention.
Technical Solution
An object of the present invention is to provide a microorganism of the genus
Corynebacteriumhaving an ability to produce L-arginine.
15 Another object of the present invention is to provide a method of producing Larginine
using the microorganism of the genus Corynebacterium.
Advantageous Effects
L-arginine can be produced in high yield using an L-arginine-producing
20 microorganism of the genus Corynebacterium with enhanced activities of an arginine
operon and ornithine carbamoyltransferase (ArgF or ArgF2) according to the present
invention. Additionally, the L-arginine produced in high yield can be effectively
used in human pharmaceutical and pharmacological industries.
25 Best Mode
In an aspect to achieve the above-identified objects, the present invention provides a
microorganism of the genus Corynebacterium capable of producing L-arginine with
enhanced activities of an arginine operon and ornithine carbamoyltransferase.
In the present invention, the arginine operon is an operon consisting of enzymes
30 involved in the mechanism of L-arginine biosynthesis, and in particular, arginine
3
operon is consisted of enzymes constituting the cyclic steps of L-arginine
biosynthesis. Specifically, the arginine operon consists of N-acetylglutamyl
phosphate reductase (ArgC), glutamate N-acetyltransferase (ArgJ), Nacetylglutamate
kinase (ArgB), acetylornithine aminotransferase (ArgD), ornithine
5 carbamoyltransferase (ArgF), and arginine repressor (ArgR), and these enzymes are
involved in the continuous enzyme reactions of L-arginine biosynthesis.
These enzymes that constitute the arginine operon are involved in the final Larginine
biosynthesis using L-glutamate as a precursor. The glutamate Nacetyltransferase
(ArgJ) synthesizes N-acetylglutamate using L-glutamate as a
10 precursor, and it may be one encoded by argJ gene. In particular, the acetyl group
is obtained by decomposing N-acetylornithine into L-ornithine. It has been known
that glutamate N-acetyltransferase is involved in a recycling reaction for L-arginine
biosynthesis in microorganisms belonging to the genus Corynebacterium.
The produced N-acetylglutamate is synthesized into N-acetylglutamyl phosphate by
15 N-acetylglutamate kinase (ArgB), ADP is produced by consuming ATP as a
coenzyme, and may be one encoded by argB gene. Since it is known to be subjected
to feedback inhibition by the final product, L-arginine, modifications releasing the
feedback inhibition by L-arginine have been known, and there were reports that Larginine
productivity can be improved utilizing the same (Chinese Patent No.
20 102021154, and Amino Acids. 2012 Jul;43(1): 255-66. doi: 10.1007/s00726-011-1069-x.
Epub 2011 Sep 8).
N-acetylglutamyl phosphate reductase (ArgC) is also called acetylglutamate
semialdehyde dehydrogenase in E. coli or yeasts, and may be encoded by argC gene.
N-acetylglutamyl phosphate is converted into N-acetylglutamate 5-semialdehyde by
25 this enzyme. NADPH is used as a coenzyme to supply energy. The produced Nacetylglutamate
5-semialdehyde is converted into N-acetylornithine using Lglutamate
as an amino acid donor, and this reaction is mediated by acetylornithine
aminotransferase (ArgD). Acetylornithine aminotransferase may be encoded by
argD gene. The converted N-acetylornithine delivers its acetyl group to L-
30 glutamate by the recycling reaction of glutamate N-acetyltransferase (ArgJ), and
4
reacts as L-ornithine.
Ornithine carbamoyltransferase (ArgF) is generally called ornithine carbamoylase,
and may be encoded by argF or argF2 genes. L-ornithine binds to carbamoyl
phosphate to form L-citrulline, and a phosphate is produced as a side reaction
5 product. The produced L-citrulline is finally synthesized into L-arginine by the
enzyme reactions of argininosuccinic acid synthase (ArgG) and argininosuccinic acid
lyase (ArgH), which are present separated from the arginine operon, mentioned
above. L-arginine is synthesized by a total of 8 biosynthetic steps, and in the present
invention, the enhancement of L-arginine productivity was induced by
10 strengthening the activity of the arginine operon (argCJBDFR).
The enzymes that constitute the arginine operon may be included within the scope
of the present invention as long as they have the activities described above, and
specifically, the enzymes may be proteins derived from a microorganism of the
genus Corynebacterium. More specifically, glutamate N-acetyltransferase (ArgJ) may
15 include the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence
which has a homology to the sequence of at least 70%, specifically 80%, and more
specifically 90% or higher. N-acetylglutamate kinase (ArgB) may include the amino
acid sequence of SEQ ID NO: 21, or an amino acid sequence which has a homology
of at least 70% to the sequence, specifically 80%, and more specifically 90% or higher.
20 Additionally, in the case of the corresponding enzyme, modifications known in the
art may be introduced in order to release feedback inhibition by arginine. Nacetylglutamyl
phosphate reductase (ArgC) may include the amino acid sequence of
SEQ ID NO: 23, or an amino acid sequence which has a homology of at least 70%to
the sequence, specifically 80%, and more specifically 90% or higher.
25 Acetylornithine aminotransferase (ArgD) may include the amino acid sequence of
SEQ ID NO: 25, or an amino acid sequence which has a homology of at least 70% to
the sequence, specifically 80%, and more specifically 90% or higher. Ornithine
carbamoyltransferase (ArgF) may include an amino acid sequence of SEQ ID NO: 1
or SEQ ID NO: 3, or may include an amino acid sequence which has a homology of
30 at least 70% to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
5
Specifically, ornithine carbamoyltransferase (ArgF) may include an amino acid
sequence which has a homology of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
or 99% or higher to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In
addition, it is obvious that amino acid sequences including a deletion, modification,
5 substitution or addition in one or more amino acid residues fall within the scope of
the present invention, as long as they have the homology with the above proteins
and have substantially the same or corresponding biological activity to the above
proteins.
As used herein, the term “homology” refers to the degree of similarity between two
10 amino acid sequences or nucleotide sequences for comparison, and their homology
may be determined by comparing with the naked eye or using a bioinformatic
algorithm, which provides analysis results of a degree of homology by aligning
sequences for comparison. The homology between the two amino acid sequences
may be indicated in percentages. The useful automated algorithms may be used in
15 GAP, BESTFIT, FASTA, and TFASTA computer software modules of Wisconsin
Genetics Software Package (Genetics Computer Group, Madison, WI, USA). Other
useful algorithms and homology determinations on alignment are already
automated in software such as FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL
W.
20 In the present invention, the enhancement of the activity of the arginine operon may
refer to the enhancement of the activity in at least one enzyme among the enzymes
present in the arginine operon, however, it does not include the single enhancement
of argR gene alone. For example, the enhancement of the arginine operon activity
may refer to the enhancement of the activities of all enzymes present in the operon
25 through the enhancement of the promoter for one enzyme present in the arginine
operon, and specifically, may refer to the enhancement of the activity of the entire
operon by the enhancement of the promoter for the N-acetylglutamyl phosphate
reductase. Additionally, in the present invention, the increase in expression of a
gene encoding at least one enzyme among the enzymes constituting the arginine
30 operon may also be considered as the enhancement of the arginine operon activity.
6
As used herein, the term “enhancement” of activity refers to the provision of a
microorganism without a particular activity of a protein with the activity of the
protein, or increasing the intracellular activity in the microorganism possessing the
activity of the protein, etc., and refers to the increasing of the intracellular activity of
5 the protein compared to the intrinsic activity of the protein. As used herein, the
term intrinsic activity refers to the active state of the enzyme possessed in the natural
or pre-modified state by the microorganism belonging to the genus Corynebacterium.
For enhancing or increasing the activity of the enzyme, various methods known in
the art may be applicable. Examples of the method, although they are not limited
10 thereto, may include a method of increasing the copy number of nucleotide
sequences encoding enzymes by further inserting a polynucleotide including a
nucleotide sequence encoding the corresponding enzyme into a chromosome or
introducing the polynucleotide into a vector system, etc., a method of replacing
enzyme promoters with strong promoters, and specifically, may include a method of
15 introducing a modification on the promoters, and a method of modifying the
enzyme into one with strong activity by genetic modification.
Specific examples in the present invention may include a method of modifying the
enzyme promoter present in the arginine operon to a promoter which is strong
compared to the endogenous promoter, via modification or substitution of the
20 promoter. An improved promoter or heterogeneous promoter with a nucleotide
substitution modification may be connected instead of the promoter for the
endogenous enzyme, and examples of the heterogeneous promoters may include
pcj7 promoter(Korean Patent No. 10-0620092), lysCP1 promoter(Korean Patent No.
10-0930203), EF-Tu promoter, groEL promoter, aceA promoter, aceB promoter, etc.,
25 but are not limited thereto.
As used herein, the term “promoter” refers to a non-encoded nucleic acid sequence
upstream of an encoding region, which includes a polymerase-binding region and
has an activity of transcription initiation into mRNA of a gene downstream of the
promoter, i.e., the DNA region where the polymerase binds and initiates the
30 transcription of the gene, and is located on the 5’ region of the mRNA transcription
7
initiation region.
In the present invention, the enhancement of the ornithine carbamoyltransferase
activity may be performed using various methods well known in the art, and they
are the same as described above. Specifically, the enhancement may be achieved by
5 transformation of an expression vector including a polynucleotide encoding the
ornithine carbamoyltransferase into a bacterial strain, but is not limited thereto.
As used herein, the term “transformation” refers to an introduction of DNA into a
host, thereby making the inserted DNA replicable as an extrachromosomal factor or
by chromosomal integration. Specifically, the transformant of the present invention
10 may be inserted into a chromosome via homologous recombination between the
sequence of a nucleic acid molecule, which has the promoter activity within a vector
after the transformation of the vector including the above DNA into a host cell, and
the sequence in the promoter region of the endogenous target gene, or may be
retained in the form of a plasmid.
15 The method of vector transformation of the present invention may include any
method that can introduce a nucleic acid into a cell, and any appropriate standard
technology known in the art may be selected and performed according to each host
cell. For example, electroporation, calcium phosphate (CaPO4) precipitation,
calcium chloride (CaCl2) precipitation, microinjection, a polyethylene glycol (PEG)
20 method, a DEAE-dextran method, a cationic liposome method, a lithium
acetate/DMSOmethod, etc., may be used, but the method is not limited thereto.
As used herein, the term “a microorganism of the genus Corynebacterium
(Corynebacterium sp.)” may refer to all the strains belonging to the genus
Corynebacterium having the L-arginine-producing ability, e.g., Corynebacterium
25 glutamicum, Corynebacteriumammoniagenes, Corynebacteriumthermoaminogenes,
Brevibacterium flavum, Brevibacterium fermentum, etc., but is not limited thereto.
Specifically, Corynebacterium glutamicum may be used, but the microorganism is not
limited thereto.
In another aspect, the present invention provides a method for producing L-arginine
30 including culturing an L-arginine-producing microorganism of the genus
8
Corynebacterium in proper culture media.
In the present invention, the microorganism culture may be performed according to
methods widely known in the art, and the conditions of culture temperature, culture
hours, pH of culture medium, etc., may be appropriately adjusted. The known
5 culture methods are described in detail in references (Chmiel; Bioprozesstechnik 1.
Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991), and
Storhas; Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig /
Wiesbaden, 1994)). Additionally, culture methods may include batch culture,
continuous culture, and fed-batch culture, and specifically, cultures may be
10 performed continuously by a batch process or fed batch or repeated fed batch
process, but are not limited thereto.
The culture media to be used should appropriately meet the required conditions of a
particular strain. Culture media used for various microorganisms are already
known (e.g., “Manual of Methods for General Bacteriology” from American Society
15 for Bacteriology (Washington D.C., USA, 1981)). Carbon sources to be contained in
the media may include saccharides and carbohydrates (e.g., glucose, sucrose, lactose,
fructose, maltose, molasses, starch and cellulose), oils and fats (e.g., soybean oil,
sunflower seed oil, peanut oil, and coconut oil), fatty acids (e.g., palmitic acid, stearic
acid, and linoleic acid), alcohols (e.g., glycerol and ethanol), organic acids (e.g., acetic
20 acid), etc. These materials may be used individually or as a mixture, but are not
limited thereto. Nitrogen sources to be contained in the media may include
nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat gravy,
malt extract, corn steep liquor, soybean meal powder, and urea), and inorganic
compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate,
25 ammonium carbonate, and ammonium nitrate), and these materials may also be
used individually or as a mixture, but are not limited thereto. Phosphorous sources
to be contained in the media may include potassium dihydrogen phosphate or
dipotassium hydrogen phosphate or an equivalent sodium-containing salt thereof,
but are not limited thereto. Culture media may contain metal salts essential for
30 growth (e.g., magnesium sulfate or iron sulfate), and essential growth-promoting
9
materials such as amino acids and vitamins may be used, in addition to the materials
described above. Additionally, an appropriate precursor may be further added to
the culture media. The materials to be supplied described above may be added to
the media at once or appropriately during the culture.
5 The pH of culture media may be appropriately adjusted using a basic compound
(e.g., sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound
(e.g., phosphoric acid or sulfuric acid).
Foaming may be adjusted using an antifoaming agent such as fatty acid polyglycol
ester. An aerobic condition may be maintained by introducing oxygen or an
10 oxygen-containing gas mixture, for example, air, into a culture medium. Culture
temperature may be from 20°C to 45°C, and specifically, from 25°C to 40°C.
Culturing may be continued until a maximum amount of the desired L-amino acid
production is obtained, and specifically from 10 hours to 160 hours. L-arginine
may be released into the culture medium or may remain contained in the cell.
15 Meanwhile, the method of producing L-arginine of the present invention including
culturing the microorganism described above may further include a step of
recovering L-arginine during the culture. That is, the method of producing Larginine
of the present invention may include culturing a microorganism of the
genus Corynebacterium in culture media and recovering the L-arginine from the
20 microorganism and the culture media. The step of recovering arginine may mean
to separate arginine from cells or culture media using a method of recovering
arginine widely known in the art. Methods of recovering L-arginine may include
centrifugation, filtration, extraction, spray, drying, evaporation, precipitation,
crystallization, electrophoresis, fractional dissolution, chromatography (e.g., ion
25 exchange, affinity, hydrophobicity, size exclusion, and high performance liquid
chromatography), etc., but are not limited thereto.
Mode for Invention
Hereinafter, the present invention will be described in more detail with reference to
30 the following Examples. However, these Examples are for illustrative purposes
10
only, and the invention is not intended to be limited by these Examples.
Example 1: Construction of a vector with an enhanced arginine operon
In order to enhance the arginine operon on the chromosome of a microorganism, a
5 vector where the self-promoter for N-acetylglutamyl phosphate reductase (ArgC)
was deleted and substituted with a different promoter was constructed. As the
substitution promoter, lysCP1 (SEQ ID NO: 18 disclosed in Korean Patent No. 10-
0930203), which has a strong expression-inducing activity, was used.
10 First, DNA fragments were amplified via primary polymerase chain reaction (PCR)
using the chromosomal DNA of a wild type strain of Corynebacterium glutamicum
(Accession No: ATCC13869) as a template, along with a primer pair of SEQ ID NO:
13 (SF_pargC_PR_pDC infusion primer; 5'-
CGAGCTCGGTACCCGGGCAAAGAATACGGCTTCCTTGGC-3') and SEQ ID NO:
15 14 (SR_pargC_PR_XbaI-XhoI-BamHI infusion/restriction enzyme primer; 5'-
CTGGATCCTCGAGTCTAGAGACGGGTTAGACATGCAAAA-3') and a primer
pair of SEQ ID NO: 15 (SF_pargC_PR_SpeI-ScaI-BamHI infusion/restriction enzyme
primer; 5'-GACTCGAGGATCCAGTACTAGTATGATAATCAAGGTTGCAAT-3')
and SEQ ID NO: 16 (SR_pargC_PR_pDC infusion primer; 5'-
20 TGCAGGTCGACTCTAGGGTAACGCCTTCTTTCAAAG-3'). The specific
conditions for PCR reaction were as follows: the PCR reaction was performed by
denaturing at 95°C for 10 minutes, annealing at 55°C for 30 seconds, and elongation
at 72°C for 1 minute using a PCR device (Bio-rad C1000 thermal cycler) and Pfu
polymerase (Macrogen), and repeated for 28 cycles.
25
The thus-obtained primary PCR fragments were purified using fragment DNA
purification kit (GeneAll), and then three DNA fragments were connected by mixing
them with a pD vector, which was already prepared by digesting with XmaI-XbaI
restriction enzymes. The connected DNA fragments were subjected to a reaction at
30 50°C for 10 minutes using the In-fusion Cloning Kit (Clontech), and thereby a pD-
11
RargC_PR vector was constructed.
The insertion of the substituting promoter was performed in such a manner that
lysCP1 promoter was amplified using the pDZ-lysCP1 (Korean Patent No. 10-
5 0930203) as a template along with a primer pair of SEQ ID NO: 5 (SF_PlysCP1_XhoIXbaI
infusion primer; 5'-CCGTCTCTAGACTCGAGCCATCTTTTGGGGTGCGG-3')
and SEQ ID NO: 6 (SR_PlysCP1_SpeI infusion primer; 5'-
TTGATTATCATACTAGTCTTTGTGCACCTTTCGAT-3'), and connected by mixing
them with a pD-PargC_PR vector, which was already prepared by digesting with
10 XhoI-SpeI restriction enzymes. The methods of PCR and In-fusion Cloning are the
same as described above, and finally a pD-PargC::lysCP1 vector was constructed
through the methods.
Example 2: Construction of a vector with enhanced ornithine
carbamoyltransferase
In order to enhance ornithine carbamoyltransferase, one of arginine biosynthesis
enzymes, a recombinant expression vector was constructed. The p117-cj7-GFP
(Korean Patent No. 10-0620092) was used as the template vector, and the nucleotide
sequence encoding GFP in the template vector was removed by treating with EcoRVXba
I restriction enzymes, and inserted with argF, derived from a wild type strain of
Corynebacterium glutamicum ATCC13869, and argF2 (Korean Patent No. 10-0830290).
The DNA fragments of the argF gene were amplified via PCR using the
chromosomal DNA of a wild type strain of Corynebacterium glutamicum (Accession
25 No: ATCC13869) as a template, along with a primer pair of SEQ ID NO: 7
(SF_argF_EcoRV infusion primer; 5'-
ACGAAAGGAAACACTCGATATCATGACTTCACAACCACAGGT-3') and SEQ ID
NO: 8 (SR_argF_XbaI infusion primer; 5'-
GCCAAAACAGCTCTAGATTACCTCGGCTGGTGGGCCA-3'). PCR reaction was
30 performed by denaturing at 95°C for 10 minutes, annealing at 55°C for 30 seconds,
12
and elongation at 72°C for 2 minutes using Pfu polymerase, and repeated for 28
cycles. The thus-obtained PCR fragments were purified and mixed with p117-cj7-
GFP, which was already treated with EcoRV-XbaI restriction enzymes, and
connected by the In-fusion Cloning method, and thereby a recombinant expression
5 vector, p117-Pcj7-argF,was constructed.
The argF2 gene was amplified via PCR using the chromosomal DNA of a wild type
strain of Corynebacterium glutamicum (Accession No: ATCC13032) as a template,
along with a primer pair of SEQ ID NO: 9 (SF_argF2_EcoRV infusion primer; 5'-
ACGAAAGGAAACACTCGATATCATGGCCAGAAAACATCTGCT-3') and SEQ ID
NO: 10 (SR_argF2_XbaI infusion primer; 5'-
GCCAAAACAGCTCTAGACTACGCATTGATCGACCGAG-3') and Pfu polymerase
(Macrogen), via PCR by denaturing at 95°C for 10 minutes, annealing at 55°C for 30
seconds, and elongation at 72°C for 2 minutes using Pfu polymerase, which was
repeated for 28 cycles. The thus-obtained PCR fragments were purified and mixed
with p117-cj7-GFP, which was already treated with EcoRV-XbaI restriction enzymes,
and connected by the In-fusion Cloning kit, and thereby a recombinant expression
vector, p117-Pcj7-argF2,was constructed.
20 Additionally, a recombinant expression vector, which can simultaneously express
both argF and argF2 genes, was constructed. The thus-constructed expression
vector, p117-Pcj7-argF, was treated with NotI and then p117-Pcj7-argF2 was inserted
thereinto. Specifically, PCR reaction was performed using the recombinant plasmid,
p117-Pcj7-argF2, as a template, along with SEQ ID NO: 11 (SF_Pcj7_argF2_NotI
25 infusion primer; 5'-CCTTTTTGCGGCGGCCGCAGAAACATCCCAGCGCTACT-3')
and SEQ ID NO: 12 (SR_argF2_NotI infusion primer; 5'-
CACCGCGGTGGCGGCCGCCGCAAAAAGGCCATCCGTCA-3') primer and Pfu
polymerase, by denaturing at 95°C for 10 minutes, annealing at 55°C for 30 seconds,
and elongation at 72°C for 2.5 minutes, and was repeated for 28 cycles. The thus-
30 obtained PCR fragments were purified and mixed with p117-Pcj7-argF, which was
13
already treated with NotI restriction enzyme, and connected by the In-fusion
Cloning kit, and finally a recombinant expression vector, p117-Pcj7-argF/Pcj7-argF2,
was constructed.
5 Example 3: Construction of a strain having a recombinant vector inserted therein
3-1. Insertion of a vector with an enhanced arginine operon
In order to substitute a self-promoter of an arginine operon on the chromosome of
Corynebacterium, pD-PargC::lysCP1, the recombinant vector constructed in Example
1, was transformed into an existing arginine-producing Corynebacterium strain, and
10 thereby a Corynebacterium strain inserted with a recombinant vector was constructed.
Specifically, lysCP1 promoter sequence was inserted into the chromosome by
transforming pD-PargC::lysCP1, the recombinant vector constructed in Example 1,
into the existing arginine-producing strains of KCCM10741P (Korean Patent No. 10-
07916590) and ATCC21831, thereby substituting the self-promoter sequence
15 possessed by the parental strain with the promoter sequence of the vector via
homologous recombination.
In performing the transformation, the recombinant vector was first inserted into
KCCM10741P and ATCC21831 via an electric pulse method (Appl Microbiol
20 Biotechnol. 1999 Oct; 52(4): 541-5), and the strains with the insertions on their
chromosome by the recombination of homologous sequences were selected in media
containing 25 mg/L kanamycin. The selected primary strains were subjected to
cross-over, and thereby those strains, where the promoters were substituted with
lysCP1 promoter and the vector was removed, were selected. The presence of
25 promoter substitution in the final transformed strains was confirmed by PCR using a
primer pair of SEQ ID NO: 5 and SEQ ID NO: 6, and the strains were named as
KCCM10741P_ΔPargC::lysCP1 and ATCC21831_ΔPargC::lysCP1.
3-2. Insertion of a vector with enhanced ornithine carbamoyltransf erase
30 The recombinant expression vectors, p117-Pcj7-argF, p117-Pcj7-argF2, and p117-Pcj7-
14
argF/Pcj7-argF2 constructed in Example 2, was inserted into the strain
KCCM10741P_ΔPargC::lysCP1 and ATCC21831_ΔPargC::lysCP1 by electric pulse
method, selected in media containing 25 mg/L kanamycin, and the strains further
expressing argF, argF2, and argF/argF2 were finally constructed. The strains were
5 named as KCCM10741P_ΔPargC::lysCP1_Pcj7-argF,
KCCM10741P_ΔPargC::lysCP1_Pcj7-argF2, KCCM10741P_ΔPargC::lysCP1_Pcj7-
argF/Pcj7-argF2, ATCC21831_ΔPargC::lysCP1_Pcj7-argF,
ATCC21831_ΔPargC::lysCP1_Pcj7-argF2, and ATCC21831_ΔPargC::lysCP1_Pcj7-
argF/Pcj7-argF2, and among them, KCCM10741P_ΔPargC::lysCP1_Pcj7-argF2 was
10 renamed as CA06-2044, and deposited at Korean Culture Center of Microorganisms
(KCCM) under the Budapest Treaty on December 9, 2013 under the accession
number KCCM11498P.
Example 4: Evaluation of constructed strains
15 In order to examine the effect of enhancement of the arginine operon and ornithine
carbamoyltransferase on arginine-producing ability using Corynebacterium
glutamicum KCCM10741P_ΔPargC::lysCP1, KCCM10741P_ΔPargC::lysCP1_Pcj7-argF,
KCCM10741P_ΔPargC::lysCP1_Pcj7-argF2, KCCM10741P_ΔPargC::lysCP1_Pcj7-
argF/ Pcj7-argF2, ATCC21831_ΔPargC::ly sCP1, ATCC21831_ΔPargC::ly sCP1_Pcj7-
20 argF, ATCC21831_ΔPargC::lysCP1_Pcj7-argF2, and
ATCC21831_ΔPargC::lysCP1_Pcj7-argF/Pcj7-argF2, which are arginine-producing
strains constructed in Example 3, they were cultured as shown below. In particular,
Corynebacterium glutamicum KCCM10741P and ATCC21831, which are the parental
strains, were used as control, and a platinum loop of the strains was respectively
25 inoculated onto a 250 mL corner-baffled flask containing 25 mL (6% glucose, 3%
ammonium sulfate, 0.1% potassium phosphate, 0.2% magnesium sulfate
heptahydrate, 1.5% corn steep liquor (CSL), 1% NaCl, 0.5% yeast extract, and 100
μg/L biotin, pH 7.2) of a production medium, and incubated at 30°C at 200 rpm for
48 hours. Upon completion of culturing, the amount of L-arginine production was
30 measured by HPLC, and the results are shown in Table 1 below.
15
[Table 1]
Confirmation of arginine-producing abilities by parent strain and recombinant
strains
Strain
KCCM10741P
KCCM10741P _ΔPargC::lysCP1
KCCM10741P
_ΔPargC::lysCP1_Pcj7-argF
KCCM10741P
_ΔPargC::ly sCP1_Pcj7-argF2
KCCM10741P
_ΔPargC::lysCP1_Pcj7-
argF/Pcj7-argF2
ATCC21831
ATCC21831_ΔPargC::ly sCP1
ATCC21831_ΔPargC::lysCP1_Pc
j7-argF
ATCC21831_ΔPargC::lysCP1_Pc
j7-argF2
ATCC21831_ΔPargC::lysCP1_Pc
j7-argF/Pcj7-argF2
OD
91
72
69
70
69
102
86
86
88
85
Conc. of arginine
(g/L)
3.0
2.2
4.3
4.1
4.5
4.2
3.2
5.5
5.3
5.6
Conc. of ornithine
(g/L)
0.2
1.9
0.2
0.5
0.2
0.3
2.9
0.3
0.6
0.3
5
As shown in Table 1 above, the strains, where the genes encoding arginine operon
and ornithine carbamoyltransferase were simultaneously enhanced, showed a
maximum of 50% increase in arginine-producing ability compared to that of control.
Additionally, the increases in arginine concentration and ornithine concentration,
10 shown in the enhancement of the arginine operon alone
(KCCM10741P_ΔPargC::lysCP1 and ATCC21831_ΔPargC::lysCP1), were solved by
introducing argF, argF2 or argF and argF2, and eventually showing the result of
increase in arginine concentration.
15 From the foregoing, one of ordinary skill 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
16
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
5 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.
17
[CLAIMS]
[Claim 1]
A microorganism of the genus Corynebacterium having an ability to produce Larginine
with enhanced activities of an arginine operon and ornithine
carbamoyltransferase.
[Claim 2]
The microorganism of claim 1, wherein the ornithine carbamoyltransferase is an
amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3.
[Claim 3]
The microorganism of claim 1, wherein the microorganism of the genus
Corynebacterium is Corynebacteriumglutamicum.
[Claim 4]
A method of producing L-arginine, comprising:
culturing a microorganism of the genus Corynebacterium of any one of claims 1 to 3 in
a culture media; and
recovering the L-arginine from the microorganism or the media.