WO2008007914A1 - A nucleotide sequence of a mutant argf with increased activity and a method for producing l-arginine using a transformed cell containing the same - Google Patents

Abstract

Disclosed herein is a nucleic acid molecule of a mutant argF gene, which is involved in arginine biosynthesis. The protein product of the mutant argF gene, in which a partial nucleotide or amino acid sequence is substituted, has increased ornithine carbamoyltransferase activity. Also, disclosed are a recombinant vector containing the nucleic acid molecule and a cell transformed with the recombinant vector. Further, disclosed is a method of producing high levels of L-arginine by culturing the transformant. The recombinant microorganism provides an increased yield of L-arginine.

Description

Description

A NUCLEOTIDE SEQUENCE OF A MUTANT ARGF WITH INCREASED ACTIVITY AND A METHOD FOR PRODUCING L-ARGININE USING A TRANSFORMED CELL CONTAINING

THE SAME Technical Field

[1] The present invention relates to a nucleotide sequence of a mutant argF gene, which is involved in arginine biosynthesis. More particularly, the present invention relates to a nucleotide sequence of a mutant argF gene, which has a partial nucleotide sequence varied and thus results in an amino acid sequence varied from the wild type sequence, so as to increase ornithine carbamoyltransferase activity, a recombinant vector carrying the nucleotide sequence, and a cell transformed with the recombinant vector. Also, the present invention is concerned with a method of producing high levels of L-arginine by culturing the transformant.

[2]

Background Art

[3] L-arginine, a semi-essential amino acid that is naturally produced in the body, has been widely used in medicaments, food, animal feedstuffs, and other products. L- arginine is useful as a drug for improving hepatic function and brain function and treating male sterility and as an ingredient of multiple amino acid supplements. Also, L-arginine has been used as a food additive in fish cakes and health beverages, and has recently gained interest as a salt substitute for hypertension patients.

[4] Conventional methods for L-arginine production by biological fermentation are based on producing L-arginine directly from carbon and nitrogen sources. For example, L-arginine can be produced using a mutant strain derived from a glutamate- producing microorganism belonging to the genus Brevibacterium or Corynebacterium (Japanese Pat. Laid-Open Publication Nos. Sho57-163487, ShoβO-83593 and Sho62-265988), or using an amino acid-producing microorganism having improved growth properties by cell fusion (Japanese Pat. Laid-Open Publication No. Sho59-158185). Recently, another method using a recombinant strain, in which the argR gene, which mediates the repression of arginine biosynthetic operons, is inactivated, was developed (U.S. Pat. Application No. US2002/0045223A1).

[5] In microorganisms, the biosynthesis of L-arginine proceeds in eight enzymatic steps starting from L-glutamate through two different pathways, the linear pathway or the cyclic pathway. In the linear pathway, L-arginine is synthesized from L-glutamate via N-acetylglutamate, N-acetylornithine, ornithine, citralline and argininosuccinate. These intermediates are synthesized through consecutive reactions catalyzed by the following enzymes, N-acetylglutamate synthase, N-acetylglutamate kinase, N- acetylglutamylphosphate reductase, acetylornithine aminotransferase, acetylornithine deacetylase, ornithine carbamoyltransferase, argininosuccinate synthase and argini- nosuccinase. These enzymes are encoded by argA, argB, argC, argD, argE, argF, argG and argH genes, respectively.

[6] The present inventors, in order to develop a microbial strain capable of producing

L-arginine in higher yields, intended to amplify the argF gene encoding ornithine carbamoyltransferase, which catalyzes the conversion of ornithine into citrulline in the arginine biosynthetic pathway, and then to achieve the improved productivity of L- arginine using a mutant argF gene, which allows enhancement of the gene-encoded enzyme activity compared to the wild type, thereby leading to the present invention.

[7] In E. coli, the expression of the argF gene is controlled by the repressor protein argR and intracellular arginine. In high levels of arginine, the argR protein negatively regulates the expression of the argF gene (Dongbin Lim, Werner K. Mass, 1987, PNAS 84: 6697-6701). In some microorganisms, the activity of the argF protein has been reported to be suppressed by the presence of high concentrations of arginine (Raymon Cunin, Victor Stalon, 1986, Microbiological Reviews 50: 314-352). Also, the inactivation of the argR gene increases the expression of the argF gene and enhances the production efficiency of arginine (U.S. Pat. Application No. US2002/0045223A1). According to the information reveled to date, in C. glutamicum, argCJBDFR genes are organized in an operon, of which the properties are very similar to those of argF enzyme in other microorganisms (Jae-Yeon Chun, Myeong-Sok Lee, 1999, Molecules and Cells 9:333-337). Thus, when the expression of the argF gene is increased, the arginine-mediated suppression of ArgF activity can be overcome even though arginine is accumulated in high levels intracellularly or extracelluarly, thereby strengthening arginine biosynthetic flux. This may lead to enhanced arginine production.

[8] In this regard, the present inventors have constructed an expression vector that comprises the argF gene obtained from a mutant strain that produces arginine in high levels using a genetic engineering technique, and have introduced the expression vector into an arginine-producing strain, thereby developing a strain capable of producing high concentrations of L-arginine. When the strain was substantially used in fermentation for arginine production, the yield of L-arginine remarkably increased.

[9]

Disclosure of Invention Technical Problem [10] It is therefore an object of the present invention to provide a recombinant microorganism for producing L-arginine, the microorganism being capable of producing the amino acid L-arginine.

[11] In particular, the present invention aims to provide a recombinant microorganism producing the amino acid L-arginine in higher yields and a preparation method thereof comprising preparing a modified argF gene or a DNA fragment thereof, constructing a vector capable of expressing the mutant argF gene in C. glutamicum, and introducing the vector into an L-arginine-producing strain.

[12] It is another object of the present invention to provide a method of producing the amino acid L-arginine, comprising culturing the recombinant microorganism capable of producing the amino acid L-arginine and isolating the amino acid L-arginine from the culture fluid.

[13]

Technical Solution

[14] The present inventors have constructed an expression vector that comprises the argF gene obtained from a mutant strain that produces arginine at high levels using a genetic engineering technique, and have introduced the expression vector into an arginine- producing strain, thereby developing a strain capable of producing high concentrations of L-arginine. When the strain was substantially used in fermentation for arginine production, the yield of L-arginine remarkably increased.

Advantageous Effects

[15] The L-arginine-producing microorganism overexpressing the argF gene according to the present invention increases the yield of L-arginine.

[16]

Brief Description of the Drawings

[17] FIG. 1 shows a process for constructing a recombinant plasmid pECCGl 17-argF according to the present invention.

[18]

Best Mode for Carrying Out the Invention

[19] The present invention is characterized by targeting the argF gene, encoding ornithine carbamoyltransferase, in order to improve the productivity of L-arginine. In detail, the present invention involves obtaining the argF gene from an L- arginine-producing microorganism, inserting the argF gene into a vector in order to construct an argF expression vector and introducing the expression vector into an L- arginine-producing strain using a genetic engineering technique, thereby increasing the productivity of L-arginine even in the presence of high concentrations of arginine during fermentation. [20] This concept of the present invention is applicable to all microorganisms, including prokaryotes and eukaryotes, that are able to produce L-arginine. Representative microorganisms may include E. coli, coryneform bacteria, and Bacillus bacteria, which have been conventionally used to produce L-arginine. Preferred is a strain of Corynebacterium glutamicum, which is resistant to an L-arginine analogue and is capable of producing L-arginine.

[21] The expression of the argF gene, located on a chromosome of a microorganism producing L-arginine, may be achieved using a common genetic engineering technique. The nucleotide sequence of the gene may be analyzed using a fluorescent DNA sequencing method.

[22] In one aspect, the gene expression vector of the present invention is constructed according to the following procedure. The genomic DNA is isolated from a microbial strain capable of producing L-arginine. A polymerase chain reaction (PCR) is performed using the isolated genomic DNA as a template in accordance with the standard protocols in order to amplify the ORF of the argF gene, the argC promoter region and the rrnB terminator region. The nucleotide sequence of the argF gene thus obtained is determined using a common DNA sequencing method. The amplified promoter, argF gene and terminator are sequentially cloned into a suitable plasmid or vector. The resulting recombinant vector or plasmid is transformed into a host cell, such as E. coli. The transformant is cultivated to be propagated, and the recombinant vector, carrying, sequentially, the promoter, argF gene and terminator, is extracted from the culture. The extracted recombinant vector is introduced into a microbial strain capable of producing L-arginine using a common technique, such as electroporation. A strain harboring the recombinant vector is then selected using its antibiotic resistance.

[23] It will be understood by those skilled in the art that the nucleotide sequence analysis and DNA fragment preparation of the present invention can be performed using standard DNA sequencing and cloning methods. PCR is preferably carried out using oligonucleotide primers targeting the argC promoter, argF gene and rrnB terminator according to the present invention. The PCR method is well known in the art (see, PCR Protocols: A Guide to Method and Application, Ed. M. Innis et al., Academic Press (1990)). The PCR is performed in a PCR mixture containing genomic DNA, an appropriate polymerase, primers and buffer conveniently using a PTC-200 Peltier Thermal Cycler (MJ Research, USA). Positive PCR results are determined, for example, by detecting a suitable size of a DNA fragment through electrophoresis on an agarose gel.

[24] In a preferred aspect, the mutant strain capable of producing L-arginine has a resistance to an L-arginine analogue. The L-arginine analogue includes canavanine and arginine hydroxamate. [25] In a particularly preferred aspect, the present inventors constructed a recombinant plasmid pECCGl 17-argF, and introduced the recombinant plasmid into C. glutamicum CJR0500, having resistance to an L-arginine analogue using electroporation, thereby developing a novel strain that overexpresses the mutant argF gene and thus produces higher concentrations of L-arginine compared to the parent strain. The present applicant deposited the novel mutant strain of Corynebacterium, CJR0586, at the Korean Culture Center of Microorganisms (KCCM) on March 15, 2006 so as to make it available to those skilled in the art, and the deposit was assigned accession number KCCM- 10742P.

[26] The novel mutant strain CJR0586 of the present invention is derived from the parent strain CJR0500, which is induced from a glutamine-producing strain KFCC- 10680 (Korean Pat. Publication No. 91-7818). The parent strain CJR0500 is a mutant strain, which is induced by treating Corynebacterium glutamicum KFCC- 10680 with N-methyl-N-nitro-N-nitrosoguanidine (NTG), as a general method for mutagenesis, smearing said treated C. glutamicum KFCC- 10680 onto a minimal medium (1.0% glucose, 0.4% ammonium sulfate, 0.04% magnesium sulfate, 0.1% potassium phosphate monobasic, 0.1% urea, 0.0001% thiamine, 200 μg/L biotin, 2% agar, pH 7.0) supplemented with canavanine and arginine hydroxamate, and selecting a clone resistant to 500 mg/L of each of canavanine and arginine hydroxamate.

[27] In the production of L-arginine, citrulline, an intermediate of the metabolic pathway of arginine, is of importance in nitrogen metabolism along with the urea cycle. The CJR0586 strain exhibited increased expression of the argF gene since the strain was prepared by introducing the recombinant expression vector, constructed by inserting into a vector the ornithine carbamoyltransferase gene (argF), amplified by PCR using the genomic DNA from the L-arginine-producing strain C. glutamicum CJR0500, into the parent strain CJR0500. The elevated expression of the argF gene activated the arginine biosynthetic pathway and thus increased the yield of arginine.

[28] All processes for the mass production of L-arginine, comprising the fermentation of the recombinant strain capable of producing L-arginine in high efficiency through the elevated expression of the argF gene and the isolation of L-arginine from the fermentation fluid, are well known in the art.

[29]

Mode for the Invention

[30] A better understanding of the present invention may be obtained through the following examples, which are set forth to illustrate, but are not to be construed as the limit of the present invention.

[31] [32] EXAMPLE 1: Recombinant plasmid construction

[33]

[34] The genomic DNA was isolated from the L-arginine-producing strain CJR0500 using a QIAGEN Genomic-tip system. PCR was carried out using the genomic DNA as a template in order to amplify the argC promoter region and the open reading frame (ORF) of the argF gene, thereby obtaining two DNA fragments (362 bp and 1059 bp, respectively). In order to fuse the argC promoter region to the argF gene, another PCR was carried out using the two amplified DNA fragments for the argC promoter and the argF gene as a template, thereby obtaining a fused fragment of 1421 bp. The argC promoter region was amplified using a pair of primers: 5'- cgcggatccggctacttcc- gaggaatcttc-3' (SEQ ID No. 3) and 5'-acaccatacacgttatgcatga-3' (SEQ ID No. 4), and PCR conditions included 20 cycles of denaturation at 94⁰C for 30 sec, annealing at 55⁰C for 30 sec and extension at 72⁰C for 30 sec. The resulting PCR reaction mixture was electrophoresed on a 1.0% agarose gel, and a band of 0.4 kb was excised from the gel.

[35] The argF gene was amplified using a pair of primers:

5'-tcatgcataacgtgtatggtgtatgacttcacaaccacaggttc-3' (SEQ ID No. 5) and 5'-acgcgatatccatgtcttacctcggctggt-3' (SEQ ID No. 6). PCR conditions included 20 cycles of denaturation at 94⁰C for 30 sec, annealing at 55⁰C for 30 sec and extension at 72⁰C for 1 min. The resulting PCR reaction mixture was electrophoresed on a 0.8% agarose gel, and a band of 1 kb was excised from the gel.

[36] The PCR for obtaining the fused fragment of the argC promoter and the argF gene was performed using a pair of primers: 5'-cgcggatccggctacttccgaggaatcttc-3' (SEQ ID No. 3) and 5'-acgcgatatccatgtcttacctcggctggt-3' (SEQ ID No. 6). PCR conditions included 20 cycles of denaturation at 94⁰C for 30 sec, annealing at 55⁰C for 30 sec and extension at 72⁰C for 1 min 30 sec. The resulting PCR reaction mixture was electrophoresed on a 0.8% agarose gel, and a band of 1.4 kb was excised from the gel.

[37] The rrnB terminator was amplified using pKK233 as a template DNA with a pair of primers: 5'-acgcgatatcctgttttggcggatgagaga-3' (SEQ ID No. 7) and 5'-cggggtacccaaaaaggccatccgtcag-3' (SEQ ID No. 8). PCR conditions included 30 cycles of denaturation at 94⁰C for 30 sec, annealing at 55⁰C for 30 sec and extension at 72⁰C for 30 sec. The resulting PCR reaction mixture was electrophoresed on a 1.0% agarose gel, and a band of 0.4 kb was excised from the gel.

[38] A plasmid pECCGl 17 was digested with BamHI and Kpnl, and a band of about 5.9 kb was excised from a 0.8% agarose gel. The fusion PCR product of the argC promoter and the argF gene was digested with BamHI and EcoRV, and a band of about 1.4 kb was excised from a 0.8% agarose gel. The PCR product for the rrnB terminator was digested with EcoRV and Kpnl, and a band of about 0.4 kb was excised from a 1% agarose gel. The three DNA fragments were ligated and inserted into the linearized pECCGl 17 plasmid, thereby obtaining a recombinant plasmid pECCGl 17-argF (about 7.7 kb) (FIG. 1).

[39] The recombinant plasmid pECCGl 17-argF was introduced into an L- arginine-producing strain (CJR0500) using electroporation, and was smeared onto a solid medium containing kanamycin. The emerged colonies were subjected to an arginine productivity test in a flask.

[40]

[41] EXAMPLE 2: Evaluation of the selected clones for arginine productivity in

Erlenmeyer flask

[42]

[43] Ten of the colonies selected on the solid medium containing the antibiotic ganamycin were cultured in an L- arginine seed medium, described in Note 1, below, and were then evaluated for L-arginine productivity using a medium for an L-arginine productivity assay, described in Note 2, below, in an Erlenmeyer flask. The mean values of the L-arginine productivity were calculated and compared between the ten clones.

[44]

[45] Note 1. L-arginine seed medium: 5% glucose, 1% bactopeptone, 0.25% sodium chloride, 1% yeast extract, 3 μg/L biotin, 0.4% urea, pH 7.0

[46] Note 2. Medium for L-arginine productivity assay: 4.0% glucose, 3% ammonium sulfate, 0.3% urea, 0.1% potassium phosphate monobasic, 0.1% potassium phosphate dibasic, 0.025% magnesium sulfate heptahydrate, 2.0% CSL (corn steep liquor), 200 μg/L biotin, pH 7.2

[47]

[48] The cells were cultured in the seed medium in an incubator at 3O⁰C for 16 hrs. 1 ml of the seed culture was inoculated in 24 ml of the medium for L-arginine productivity assay, and culturing was carried out at 3O⁰C for 72 hrs with agitation at 250 rpm. The results for L-arginine productivity are given in Table 1, below. The parent strain CJR0500 exhibited L-arginine productivity of 2.8 g/L. The recombinant strain CJR0586, overexpressing the argF gene, displayed a higher arginine productivity of 3.1 g/L, which was increased by about 10% compared to the parent strain.

[49] Table 1

[50]

[51] EXAMPLE 3: Determination of the nucleotide sequence of the mutant argF gene

[52]

[53] In order to identify the mutated regions in the argF gene, the nucleotide sequence of the recombinant vector pECCGl 16-argF was analyzed. A PCR was carried out using 0.1 μg of pECCGl 16-argF as a template with 2 mM of a pair of primers of SEQ ID Nos. 3 and 6 and 1 μl of a BigDyeTM Terminator Cycle Sequencing v2.0 Ready Reaction (PE Biosystems). PCR conditions included 30 cycles of denaturation at 95⁰C for 30 sec, annealing at 55⁰C for 30 sec and extension at 72⁰C for 1 min 30 sec, followed by quenching at 4⁰C. A DNA fragment of 1.4 kb was then isolated. This DNA fragment was subjected to sequencing analysis using the primer of SEQ ID No. 3, and sequencing was performed on an ABI PRISM 3100 Genetic AnalyzerTM (Applied Biosystems).

[54] Sequencing analysis identified eight base substitutions in argF, the structural gene for ArgF protein. The base substitutions resulted in a substitution of threonine (T) for a serine (S) residue at position 88 in the ArgF protein. The determined nucleotide sequence of the mutant argF gene and the amino acid sequence having the aforementioned substitution are shown in SEQ ID Nos. 1 and 2, respectively.

[55]

Industrial Applicability

[56] As described hereinbefore, the L-arginine-producing microorganism overexpressing the argF gene according to the present invention increases the yield of L-arginine.

[57]

Claims

Claims

[1] A nucleic acid molecule encoding a polypeptide having the amino acid sequence represented by SEQ ID NO.l. [2] The nucleic acid molecule as set forth in claim 1, which has the nucleotide sequence represented by SEQ ID NO.

2.

[3] A recombinant vector comprising the nucleic acid molecule of claim 1 or 2.

[4] A host cell which comprises the nucleic acid molecule of claim 1 or 2 and is capable of producing L-arginine.

[5] The host cell as set forth in claim 4, which is a microorganism belonging to the genus Corynebacterium.

[6] The host cell as set forth in claim 5, which is C. glutamicum KCCM-10742P.

[7] A method of producing L-arginine, comprising culturing the host cell of claim 5 or 6. [8] A method of producing L-arginine, comprising:

(1) isolating an argF gene, which encodes ornithine carbamoyltransferase, from a strain capable of producing L-arginine;

(2) constructing an expression vector comprising the isolated argF gene;

(3) transforming the constructed argF expression vector into an L- arginine-producing strain; and

(4) culturing the strain transformed with the argF expression vector.