KR100854234B1 - 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

The present invention relates to the arginine biosynthesis related gene argF sequence, specifically argF Tin ornithine The nucleotide sequence and amino acid sequences of the gene part is substituted carbamoyl trans peora enzyme activity is directed towards the improved argF gene sequence and a recombinant vector and a transformant with the recombinant vector cell containing said sequence, and wherein said transformed The present invention relates to a method for producing L-arginine at high concentration by culturing a conversion strain. The recombinant microorganisms of the invention provide increased yield of L-arginine.

L-arginine, amino acid, argF, ornithine carbamoyltransferase, recombinant vector

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}

1 is a schematic of the recombinant plasmid pECCG117- argF according to the present invention.

L-arginine is a semi-essential natural amino acid widely used in medicines, foods, and other animal feeds. It is used in medicine for liver function, brain function, male infertility, and comprehensive amino acid preparations. Fish jelly additives, health beverage additives, and salt substitutes for high blood pressure patients have recently been in the spotlight.

The known method for producing L-arginine by a biological fermentation method is a method for producing L-arginine directly from a carbon source and a nitrogen source. The method of using (open Japanese digestion 57-163487, 60-83593, 62-265988), the method of using the amino acid production strain improved growth by cell fusion (Japanese published digestion 59-158185). Recently, a method using a recombinant strain in which the gene argR that inhibits the expression of arginine biosynthetic operon has been inactivated (US Patent US2002 / 0045223A1) and the like.

Biosynthesis of L-arginine in microorganisms takes place in eight different enzymatic steps from L-glutamate, via two different pathways, linear and cyclic. In a linear step, L-arginine is derived from N-acetylglutamate, N-acetylornithine, ornithine, citrulline, and argininosuccinic acid in L-glutamate. Synthesized via argininosuccinate. These intermediates include N-acetylglutamate synthase, N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, and acetylornithine aminotransfer. It is synthesized by a continuous chemical reaction of enzymes, acetylornithine aminotransferase, N-acetylornisynthase, and argininosuccinase. This enzyme, respectively argA, argB, argC, argD, argE, argF, argG And Encrypted by argH .

The present inventors have developed an enzyme ornithine carbamoyltransferase, which catalyzes the conversion of ornithine, which is an intermediate step in arginine biosynthesis, to citurulin in order to develop a strain that can further increase the yield of L-arginine. ArgF to code In order to amplify the gene, the present invention was completed by aiming to improve the productivity of L-arginine by targeting the argF gene with improved titer than the wild type.

It has been shown in E. coli that the expression of the argF gene is regulated by argR inhibitory proteins and intracellular arginine, and at high alginine concentrations, the argR protein is known to inhibit the expression of the argF gene (Dongbin Lim, Werner K). Mass, 1987, PNAS 84: 6697-6701) Some microorganisms have been reported to inhibit ArgF protein titers by high concentrations of arginine. (Raymon Cunin, Victor Stalon, 1986, Microbiological Reviews 50: 314-352) There is also a patent that increases the expression of argF gene when argR gene is inactivated and increases the efficiency of arginine production (US Pat. No. US2002 / 0045223A1). According to the information revealed so far, in the case of C. glutamicum , the argCJBDFR gene is composed of operon and is very similar to the characteristics of ArgF enzyme of other microorganisms. (Jae-Yeon Chun, Myeong-Sok Lee, 1999, Molecules and Cells 9: 333-337) Therefore, when increasing the expression of the argF gene, may be accumulated to a high concentration with Al in intracellular ArgF activity can be overcome inhibition by arginine can enhance the biosynthesis of flux (flux) with Al It is possible to increase productivity by knowing.

Accordingly, the present inventors have developed an argF gene expression vector from a mutant strain producing alginine at high concentration, and introduced it into an alginine producing strain to develop a high concentration of L-arginine producing strain and substantially the strain. Was used in fermentation to obtain L-arginine in significantly increased yield.

It is an object of the present invention to provide a recombinant microorganism for producing L-arginine capable of producing the amino acid L-arginine.

In particular, the present invention provides amino acid L-arginine with improved yield by preparing a argF gene or a DNA fragment thereof mutated from a wild strain, and preparing a vector that can be expressed in C. glutamicum and introducing it into an L-arginine producing strain. It is an object of the present invention to provide a recombinant microorganism and a method for producing the same.

Another object of the present invention is to provide a method for producing amino acid L-arginine, which is characterized by culturing a strain capable of producing amino acid L-arginine and separating amino acid L-arginine from the culture.

Ornithine carbamoyltransferase Targeting the argF gene is characterized by improved productivity of L-arginine. More specifically, the present invention, by using a gene recombination technology to obtain the argF gene from the L-arginine producing microorganism and inserting into the vector to produce an argF expression vector, and the expression vector to the L- arginine production strain By introducing it, the productivity of L-arginine is increased even under high concentration of arginine fermentation conditions.

The idea of the present invention can be applied to all microorganisms capable of producing L-arginine including prokaryotic and eukaryotic, and representative microorganisms have been used to produce L-arginine in the prior arts such as E. coli, coryneform bacteria, Bacteria of the genus Bacillus. Preferably, they are microorganisms belonging to Corynebacterium glutamicum that are resistant to L-arginine analogs and are capable of producing L-arginine.

According to the present invention, a method for expressing an argF gene present in the chromosome of an L-arginine-producing microorganism may be performed by a conventional gene recombination technique, and the nucleotide sequence of the gene may be analyzed by a sequencing method using fluorescent material. Can be.

In one embodiment, the gene expression vector preparation process of the present invention includes the following process. Genomic DNA is isolated from a strain capable of producing L-arginine, and as a template, argF by polymerase chain reaction (PCR) using conventional techniques. To amplify the ORF, the promoter region, terminator region of the rrnB gene of argC. At this time, the nucleotide sequence of the obtained argF gene is analyzed by a conventional sequencing method. Also the promoter region obtained from the stomach, argF The gene, terminator site DNA is cloned in sequence into a suitable plasmid or other vector and the resulting recombinant vector is introduced into a host cell such as E. coli by transformation. After growing and transforming the transformant, the recombinant vector in which the promoter, argF and terminator DNA are inserted in this order is extracted. The extracted recombinant vector is introduced into a strain capable of producing L-arginine by a conventional technique such as an electroshock method, and then antibiotic resistance is used to isolate a strain comprising the recombinant vector.

Those skilled in the art will appreciate that the sequencing and construction of DNA fragments of the present invention can be accomplished by conventional sequencing and cloning methods. Preference is given to PCR amplification methods using oligonucleotide primers targeting the argC promoter, argF gene, and rrnB terminator according to the present invention. PCR amplification methods are well known in the art (PCR Protocols: A Guide to Method and Application, Ed. M. Innis et al., Academic Press (1990)). PCR includes genomic DNA, suitable polymerases, primers and buffers and is conveniently carried out in PTC-200 Peltier Thermal Cycler (MJ Researchh, USA). Positive PCR results are determined, for example, by detecting DNA fragments of appropriate size by agarose gel electrophoresis.

In a preferred embodiment, the present invention uses strains that are resistant to L-arginine analogs as variant strains capable of producing L-arginine. L-arginine analogs include canavatin and arginine hydroxamate.

In a particularly preferred embodiment, the inventors have constructed a recombinant plasmid pECCG117-argF and introduced the plasmid by electroshock into C. glutamicum CJR0500, which includes resistance to L-arginine analogs, to overexpress the mutant argF gene and to the parent strain. In comparison, a new strain was developed that produces a high concentration of L-arginine. Applicant has deposited the microbial Corynebacterium CJR0586 with Accession No. KCCM-10742P as of March 15, 2006 to the Korea Microorganism Conservation Center for general distribution to third parties.

The new strain CJR0586 of the present invention is derived from the parent strain CJR0500 and strain CJR0500 is derived from glutamine producing strain KFCC-10680 (Korean Patent Publication No. 91-7818). Parent strain CJR0500 was treated with N-methyl-N-nitro-N-nitrosoguanidine (NTG), a common mutation treatment method, in Corynebacterium glutamicum KFCC-10680, followed by minimal addition of cannabanine and arginine hydroxamate. Smear in medium (1.0% glucose, 0.4% ammonium sulfate, 0.04% magnesium sulfate, 0.1% potassium phosphate, 0.1% urea, 0.0001% thiamine, 200 ug / L biotin, 2% agar, pH7.0) for each It is a mutant strain derived by obtaining a strain having in common 500 mg / L resistance.

In the production of L-arginine, Cituruline is an intermediate in the metabolic pathway of arginine and is important for nitrogen metabolism along with the Urea cycle. CJR0586 strain is a parent strain of a recombinant expression vector produced by inserting ornithine carbamoyltransferase gene ( argF ) obtained from a chromosome of C. glutamicum CJR0500, a production strain of L-arginine, into a vector. By introducing into CJR0500, the expression of the argF gene was increased. As a result, the expression of the argF gene was increased, thereby activating the arginine biosynthetic pathway, thereby increasing the yield of arginine production.

All processes for separating the desired L-arginine from the fermentation broth by fermenting a recombinant strain capable of increasing the expression of the argF gene and efficiently producing L-arginine for mass production of L-arginine according to the present invention Is well known in the art.

The invention will be more specifically illustrated by the following examples. However, these examples are merely embodiments of the present invention and do not limit the scope of the present invention.

Example 1: Recombinant Plasmid Construction

Genomic DNA was extracted from the L-arginine producing strain CJR0500 strain using the QIAGEN Genomic-tip system, and polymerase chain reaction (PCR) was extracted from the genomic DNA template. using the DNA fragment comprising the ORF (Open Reading Frame) of the argC promoter region and the argF gene, respectively, it was amplified 362bp, 1059bp. In order to obtain a fusion fragment between the argC promoter and the argF gene, the argC promoter amplification fragment and the argF gene amplification fragment were mixed and subjected to polymerase chain reaction as a template, thereby amplifying a fragment of 1421 bp. The primers used to amplify the promoter region are argC 5'- cgcggatccggctacttccgaggaatcttc-3 '(SEQ ID NO: 3) and 5'-acaccatacacgttatgcatga-3' (SEQ ID NO: 4), with a modified (denaturation) step is 30 seconds at 94 ℃, annealing (annealing ) Step was performed at 55 ° C. for 30 seconds, and extension step was performed at 72 ° C. for 30 seconds, and 20 cycles were performed. The PCR product was electrophoresed on 1.0% agarose gel and eluted with a band of 0.4 Kb.

Primers used for argF gene amplification were 5'-tcatgcataacgtgtatggtgtatgacttcacaaccacaggttc-3 '(SEQ ID NO: 5) and 5'-acgcgatatccatgtcttacctcggctggt-3' (SEQ ID NO: 6) and the denaturation step was annealed at 94 ° C for 30 seconds. The step was performed at 55 ° C. for 30 seconds, the extension step was performed at 72 ° C. for 1 minute, and 20 cycles were performed. The PCR product was electrophoresed on a 0.8% agarose gel and eluted with a band of 1 Kb size.

The primers used for the argC promoter and the argF gene fusion fragment amplified are 5'- cgcggatccggctacttccgaggaatcttc-3 '(SEQ ID NO: 3) and 5'-acgcgatatccatgtcttacctcggctggt-3' (SEQ ID NO: 6), with a modified (denaturation) step is from 94 ℃ 30 chogan The annealing step was performed at 55 ° C. for 30 seconds, and the extension step was performed at 72 ° C. for 1 minute and 30 seconds, and 20 cycles were performed. The PCR product was electrophoresed in 0.8% agarose gel and eluted with a 1.4 Kb size band.

Primers used for amplification of the rrnB terminator site were 5'-acgcgatatcctgttttggcggatgagaga-3 '(SEQ ID NO: 7) and 5'-cggggtacccaaaaaggccatccgtcag-3' (SEQ ID NO: 8), and the denaturation step was annealed for 30 seconds at 94 ° C. ) Step was carried out for 30 seconds at 55 ℃, the extension (extension) was carried out for 30 seconds at 72 ℃, 30 cycles were carried out and used as the template DNA pKK233. The PCR product was electrophoresed on 1.0% agarose gel and eluted with a band of 0.4 Kb.

The plasmid pECCG117 was treated with restriction enzymes BamHI and Kpn, followed by eluting a band of about 5.9 Kb on a 0.8% agarose gel, and the PCR product fused with the argC promoter and argF gene was treated with restriction enzymes BamHI and EcoRV 0.8%. A band of about 1.4 Kb was eluted from the agarose gel, and the rrnB terminator PCR result was treated with restriction enzymes EcoRV and KpnI, and then a band of about 0.4 Kb was eluted from the 1% agarose gel. These three DNA fragments were linked to obtain the recombinant plasmid pECCG117-argF (about 7.7 Kb) (FIG. 1).

The recombinant plasmid pECCG117-argF was introduced into the L-arginine producing strain (CJR0500) by electroporation and plated on a solid medium containing kanamycin, and then flask titration experiments were performed on colonies selected.

Example 2: Arginine Production Titer Comparison Laboratory in Erlenmeyer Flasks for Selected Strains

10 colonies for each strain selected by screening on solid medium containing antibiotic kanamycin were selected and cultured in L-arginine species medium shown in Week 1, followed by the Erlenmeyer flask using titer medium shown in Week 2. L-arginine productivity was compared as the mean value.

Note 1.L-arginine species: glucose 5%, bactopeptone 1%, sodium chloride 0.25%, yeast extract 1%, biotin 3ug / L, urea 0.4%, pH7.0

Note 2.L-arginine titer: 4.0% glucose, 3% ammonium sulfate, 0.3% urea, 0.1% potassium monophosphate, 0.1% potassium diphosphate, 0.025% magnesium sulfate hydrochloride, CSL (corn immersion liquid) 2.0%, Biotin 200ug / L, pH7.2

After incubation for 16 hours in a seed medium in a 30 ℃ incubator, the culture solution was inoculated in 1 ml each of 24 ml titer medium and incubated at 30 rpm for 250 hours at 250 rpm. The analysis results are shown in Table 1 below, and the parent strain CJR0500 showed 2.8 g / L of arginine production, whereas the recombinant strain overexpressing the argF gene, CJR0586, showed improved results of 3.1 g / L. Based on this result, the concentration was observed to be improved by about 10% compared to the parent strain.

Flask titer test results of recombinant strains Strain CJR0500 CJR0586 L-arginine 2.8 3.1

Example 3: argF DNA sequence identification experiment

In this example, to identify the mutation site, the nucleotide sequence of pECCG116- argF was analyzed. pECCG116- argF 0.1 ug was used as a template, PCR was performed using 2 mM primers of SEQ ID NO: 3 and SEQ ID NO: 6 and 1 μl of BigDye ^™ Terminator Cycle Sequencing v2.0 Ready Reaction (PE Biosystems) (modification: 95 ° C., 30 sec. Annealing: 55 ° C. 30 sec, polymerization: 72 ° C. 1 minute 30 sec, repeated 30 times, and the reaction was completed at 4 ° C.), and a 1.4 kb DNA fragment was isolated. Using this DNA fragment and the primer of SEQ ID NO: 3, ABI PRISM 3100 Genetic Analyzer ^™ The base sequence was determined by (Applied Biosystems).

As a result of analyzing the nucleotide sequence, it was confirmed that the nucleotide sequence of ArgF structural gene, argF , was substituted for eight sites, and thus the 88th amino acid serine (S) of ArgF protein was substituted with threonine (T). The base sequence and the substituted amino acid sequence of the obtained mutated argF gene are shown in SEQ ID NOs: 1 and 2.

L-arginine producing microorganisms overexpressing the argF gene according to the present invention increase the yield of L-arginine.

Attach sequence list electronic file  

Claims (8)

A polynucleotide encoding a polypeptide having an amino acid sequence of SEQ ID NO. According to claim 1, wherein the polynucleotide having a nucleotide sequence of SEQ ID NO: 2. A recombinant vector comprising the polynucleotide of claim 1. A host cell comprising the polynucleotide according to claim 1 or capable of producing L-arginine. The host cell of claim 4 which is a microorganism belonging to the genus Corynebacterium . 6. The host cell of claim 5, which is C. glutamicum KCCM-10742P. Method for producing L-arginine comprising culturing the host cell according to claim 4. delete

Images