KR100628394B1 - - preparation of alanine auxotroph by deletion of alanine aminotransferase gene in Corynebacterium and production method of L-lysine using the same - Google Patents

Abstract

The present invention produces alanine liquor by destroying the alanine aminotrasnferase gene of Corynebacterium, and cultures it by direct fermentation to accumulate L-lysine in fermentation broth, and recover L-lysine therefrom. The present invention relates to a method for producing L-lysine.

Corynebacterium, Alanine, Aminotransferase, L-Lysine, L-Amino Acid

Description

Preparation of alanine auxotroph by deletion of alanine aminotransferase gene in Corynebacterium and production method of L-lysine using the same }

1 is Corynebacterium of alanine amino transferase raised in the present invention: a recombinant vector pCR-Δaat containing (alanine aminotransferase aat) the aat gene fragment of SEQ ID NO: 2, produced for the purpose of destroying the gene.

The present invention is to produce alanine liquor by destroying the alanine aminotransferase ( aat ) gene of Corynebacterium, and cultured by direct fermentation method to accumulate L- lysine in the fermentation broth and then from there L- lysine The present invention relates to a method for producing L-lysine, characterized by recovering lysine.

Corynebacterium strain (Corynebacterium), in particular Corynebacterium glutamicum (Corynebacterium glutamicum) is a Gram-positive microorganism which is widely used in production of L- amino acids. L-amino acids, especially L-lysine, are used in animal feed, human medicine and pharmaceutical industry and are produced by fermentation of Corynebacterium strains. Thus, since the production of L-amino acid using the Corynebacterium strain is important, many attempts have been made to improve the production method thereof.

Among them, there have been many studies to amplify each L-amino acid biosynthesis-related gene using recombinant DNA technology to study the effect on L-amino acid production and to improve L-amino acid producing Corynebacterium strains. , "Glutamic Acid Bacteria" in Biology of industrial Microorganisms, Demain and Solomon (Eds), Benjamin Chummings, London, UK, 1985, 115-142; Hiliger, BioTec 2, 40-44 (1991); Eggeling, Amino Acids 6, 261-272 (1994); Jetten and Sinskey, Critical Reviews in Biotechnology 15, 73-103 (1995); And Sahm et al, Annuals of the New York Academy of Science 782, 25-39 (1996). In addition, there have been studies to improve L-amino acid producing Corynebacterium strains by destroying or underexpressing specific genes. For example, Korean Patent Publication Nos. 2001-51915 and 2001-62279 to Degussa-Wheels Actiengegelshaft disclose attenuated expression of sucC and sucD genes and zwa2 genes derived from Corynebacterium glutamicum. A method of increasing the productivity of L-amino acids from coryneform bacteria is described.

In addition, genes derived from other bacteria may be introduced. In Japanese Patent Laid-Open No. 7-121228, a method of introducing a gene encoding citric acid synthase derived from Escherichia coli is described.

In the case of L-amino acid-producing Corynebacterium strains, pyruvate produced through glycolysis from various carbon sources is composed of various amino acids (valine, isoleucine, leucine, and leucine). ), Lysine, alanine, and the like. For this reason, if one wants to increase the production of a particular amino acid, the strain is modified so that only the desired amino acid is ultimately produced by inhibiting the production of other amino acids derived from pyruvate.

Currently, all of the metabolic pathways for producing amino acids such as valine, isoleucine, leucine, and lysine from pyruvate in corynebacterium have been identified, and studies on improving various amino acid producing strains using them are being actively conducted. In the case of alanine, however, no enzyme has yet been reported in the pathway for producing alanine from pyruvate.

A type of enzyme involved in amino acid biosynthesis and degradation, aminotransferase is an enzyme that mediates the transfer of amino groups between amino acids and oxo acids. Such aminotransferases in Corynebacterium have various substrate specificities and show redundancy with respect to various substrates, and until recently, few characteristics have been reported. According to a recently reported article ( Journal of Biotechnology 104 (2003) 229-240), the aminotransferase genes of Corynebacterium are searched and classified by an analysis based on the Hidden-Markov model. However, the function of each aminotransferase is not clearly defined. In particular, no alanine biosynthesis-related aminotransferases have been reported so far, but the Japanese Agrochemical Society in 2002 clinicians cloned alanine aminotransferases ( aat ) from Corynebacterium lactofermentum 13869 strains. Genetic deletion experiments have revealed that the sugar gene is the only aminotransferase involved in alanine biosynthesis of Corynebacterium (published number 3-5Bp14).

Accordingly, the present inventors confirmed that the gene of Corynebacterium glutamicum ATCC 13032 strain corresponding to alanine aminotransferase was a gene registered under NCBI accession number AY238317, and the present gene was deleted by intracellular pyruvation. Increasing the concentration of baits attempted to increase the productivity of L-lysine.

The present invention aims to increase L-lysine productivity by increasing the intracellular concentration of pyruvate, which is a major substrate of L-lysine biosynthesis, and destroys the alanine aminotransferase gene to produce a requirement for alanine and its production of L-lysine. The purpose of the present invention is to provide a method for producing L-lysine using the present invention.

The object of the present invention was achieved by confirming that L-lysine production is further increased when the alanine aminotransferase ( aat ) gene involved in alanine biosynthesis in Corynebacterium is destroyed.

The present invention is to prepare alanine liquor by destroying the alanine aminotransferase ( aat ) gene of Corynebacterium, and cultured by direct fermentation to accumulate L- lysine in the fermentation broth, from which L-lysine is obtained. The present invention relates to a method for producing L-lysine, characterized in that it is collected.

The present invention is described in detail below.

First, the present invention provides a recombinant vector containing an aat gene fragment produced for the purpose of destroying the alanine aminotransferase ( aat ) gene of Corynebacterium.

In the above, the recombinant vector is characterized in that it comprises an alanine aminotransferase ( aat ) gene fragment of Corynebacterium of SEQ ID NO: 2.

The present invention also provides a transformed cell in which the alanine aminotransferase gene is destroyed using the recombinant vector. As host cells usable in the present invention, microorganisms belonging to Gram-positive bacteria can be used, and in particular, it is preferable to use microorganisms belonging to the genus Corynebacterium .

Furthermore, the present invention provides a method for producing L-lysine by culturing the transformed microorganism provided above under conditions suitable for producing L-lysine and recovering L-lysine.

Meanwhile, the Corynebacterium lysine producing strain, Corynebacterium glutamicum (CF3224-0412), in which the gene aat was provided by the present invention, was deposited and deposited with the Korea Microorganism Conservation Center on December 10, 2004. No. KCCM-10636 has been assigned.

As used herein, the term 'vector' refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expression of the DNA in a suitable host. Such regulatory sequences include promoters for carrying out transcription, any operator sequence for regulating such transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control termination of transcription and translation. The vector may be a plasmid, phage particles, or simply a potential genomic insert. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Because plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention.

Corynebacteria strains used in the present invention, Corynebacterium glutamicum ATCC13032 in addition to other strains of the genus Corynebacterium, particularly suitable strains of Corynebacterium glutamicum species known wild type strain, Coryne Bacterium thermoaminogenes FERMBP-1539, Brevibacterium flavum ATCC14067, Brevibacterium lactofermentum ATCC13869, and L-amino acid producing mutants or strains prepared therefrom For example, Corynebacterium glutamicum KFCC10881, Corynebacterium glutamicum KFCC11001, and the like.

Corynebacteria strains used in accordance with the present invention can be cultured continuously or batchwise in a batch process or in a fed batch or repeated fed batch process. These known culture methods are described in Chmiel, (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991); and Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)). .

The culture medium used must meet the requirements of the particular strain in an appropriate manner. Culture media for Corynebacteria strains can be found in "Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.C., USA, 1981). Sugar sources that can be used include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid Fatty acids such as linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials can be used individually or as a mixture. Nitrogen sources that can be used include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Nitrogen sources can also be used individually or as a mixture. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, the culture medium should contain metal salts such as magnesium sulfate or iron sulfate required for growth. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used. In addition, suitable precursors to the culture medium may be used. The above-mentioned raw materials may be added batchwise or continuously in a manner appropriate to the culture during the culturing process.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acid compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner to adjust the pH of the culture. In addition, antifoaming agents such as fatty acid polyglycol esters can be used to inhibit bubble generation. Inject oxygen or oxygen-containing gas (eg, air) into the culture to maintain aerobic conditions. The temperature of the culture is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. Incubation is continued until the maximum amount of L-amino acid desired is produced. For this purpose it is usually achieved in 10 to 160 hours.

L-amino acids can be analyzed by anion exchange chromatography and subsequent ninhydrin derivatization (Spackman et al. (Analytical Chemistry, 30, (1958), 1190).

Hereinafter, the configuration of the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1: Corynebacterium glutamicum ATCC 13032 derived aat  Cloning of genes and aat  Recombinant vector construction for gene destruction

Gene of the Corynebacterium glutamicum ATCC 13032 strain corresponding to alanine aminotransferase ( aat ) of Corynebacterium lactofermentum 13869 strain, published by the Japan Agricultural and Chemical Society in 2002 (NCBI accession No. AY238317, SEQ ID NO: 1 ), A pair of primers (SEQ ID NO: 3, SEQ ID NO: 4) described in Table 1 below were synthesized based on the registered nucleotide sequence, and the chromosomes of Corynebacterium glutamicum wild strain (ATCC 13032). Chain polymerization method (PCR method) using DNA as a template [Sambrook et al, Molecular Cloning, a Laboratory Manual (1989), Cold Spring Harbor Laboratories] (PCR conditions: Denaturing = 96 ° C., 30 seconds / Annealing = 48 ° C., 30) 1/314 base pair aat gene amplified by second / Polymerization = 72 ° C., 1 minute 10 seconds, 30 times), and then cloned into E. coli plasmid pCR2.1 (Invitrogen) using the TOPO Cloning Kit (Invitrogen) and pCR-aat plasmid Got.

Aat-F 5'- GTG ACT TCT AGA AAG CGC AAA ACC -3 ' Aat-r 5'- CTA CTG CTT GTA AGT GGA CAG GAA -3 '

Then Corey four bacteria to remove genomic activity of the aat gene on, by the fragment has two ends portions removed to amplify only the middle portion of the gene (Δaat) the insertion of the vector and Δaat by recombinant and aat gene on the genome aat gene To remove the activity. First, to prepare a Δaat fragment (SEQ ID NO: 2) by amplifying only the nucleotide sequence of 383..958 bp of SEQ ID NO: 1, a pair of primers (SEQ ID NO: 5, SEQ ID NO: 6) were prepared as shown in Table 2 below. Chain polymerization method (PCR method) (PCR conditions: Denaturing = 96 ° C., 30 seconds / Annealing = 48 ° C., 30 seconds / Polymerization = 72 ° C., 30 seconds, 30 times) with pCR-aat prepared as a template. After amplifying the Δaat fragment (SEQ ID NO: 2), it was cloned into E. coli plasmid pCR2.1 (Invitrogen) using a TOPO Cloning Kit (Invitrogen) to obtain pCR-Δaat (FIG. 1).

Δaat-F 5'- TTG ATG ATG TGT TCT TAG GCA ACG -3 ' Δaat-R 5'- GAA TAG CGT CGT GAG CTG GGA CAT -3 '

Example 2: L-Lysine Production in Corynebacterium aat  Destructive and Alanine Requirements

PCR-Δaat prepared in Example 1 was transformed into L-lysine producing strain Corynebacterium glutamicum KFCC 10749 by electric pulse method (Appl. Microbiol. Biotechnol. (1999) 52: 541-545 Transformation method), and transformed strains were obtained in a selection medium containing 25 mg / L of kanamycin. A strain in which the aat gene was destroyed by pCR-Δaat inserted into the genome by primary cross-over was obtained (KFCC10749 / Δaat).

In order to confirm that the gene aat is actually an aminotransferase with specificity for alanine, and to determine the alanine requirement of the destroyed KFCC10749 / Δaat, a minimal medium containing no alanine [ Biotechnology Letters vol. 11 (1) 11- 16 (1989)] observed the growth of the cells. The results are shown in Table 3 below.

Alanine Requirements of KFCC10749 / Δaat Strains KFCC10749 KFCC10749 / Δaat Minimum medium + Alanine (100mg / L) +++++ +++++ Minimum badge +++++ +

As a result of Table 3, it was confirmed that the gene aat used in the present invention was actually an enzyme involved in alanine biosynthesis. Meanwhile, the KFCC10749 / Δaat identified as an alanine-required strain was named Corynebacterium glutamicum (CF3224-0412), and was deposited on the Korea Microorganism Conservation Center on Dec. 10, 2004. Accession No. KCCM-10636 Was granted.

Example 3: aat  Lysine Production in Destructive L-lysine Producing Strains

Corynebacterium glutamicum KFCC10749 / Δaat strain (KCCM-10636), an L-lysine producing strain having alanine requirement as in Example 2, was cultured in the following manner for L-lysine production.

A 250 ml corner-baffle flask containing 25 ml of the following species was inoculated with Corynebacterium glutamicum parent strain KFCC10749 and the strain KFCC10749 / Δaat of the present invention, respectively, and shaken (200 rmp) at 30 ° C. for 20 hours. . 250 ml corner baffle flasks containing 24 ml of the following medium were inoculated with 1 ml of seed culture and shaken (200 rpm) for 120 hours at 30 ° C. After the incubation, L-lysine production was measured by an amino acid analyzer. The results for alanine produced by L-lysine and by-products in the cultures for Corynebacterium glutamicum KFCC10749 and KFCC10749 / Δaat are shown in Table 4 below.

Strain Lysine Alanine KFCC10749 25.1 g / L 479mg / L KFCC10749 / Δaat 31.0 g / L 159 mg / L

Species Medium (pH 7.0)

: 20g raw sugar, 10g peptone, 5g yeast extract, 1.5g urea, KH ₂ PO ₄ 4g, K ₂ HPO ₄ 8g, MgSO ₄ · 7H ₂ O 0.5g, biotin 100㎍, thiamine HCl 1000㎍, calcium-pantothenic acid 2000㎍ , Nicotine amide 2000㎍ (based on 1 liter of process water)

Production Medium (pH 7.0)

: Glucose 100g, (NH ₄ ) 2SO ₄ 40g, Soy Protein 2.5g, Corn Steep Solids 5g, Urea 3g, KH ₂ PO ₄ 1g, MgSO ₄ · 7H ₂ O 0.5g, Biotin 100µg, Thiamine Hydrochloride 1000µg, Calcium-Pantothenic Acid 2000µg, Nicotinamide 3000µg, CaCO ₃ 30g (Based on 1 liter of process water)

As shown in Table 4, the gene aat disrupted strain (KFCC10749 / Δaat (KCCM-10636)) was significantly increased in lysine production compared to the parent strain KFCC10749, and production of alanine was reduced by about 67%. there was.

As described above, the present invention newly provides a transformed microorganism having an alanine requirement and further improving the production capacity of L-lysine by destroying the alanine aminotransferase aat gene of Corynebacterium. Incubating the converting microorganisms under conditions suitable for producing L-lysine and recovering L-lysine has the effect of providing an excellent method for producing L-lysine which can produce much more L-lysine than before.

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Claims (6)

Alanine aminotransferase of Corynebacterium comprising a fragment of the alanine aminotransferase ( aat ) gene derived from Corynebacterium according to SEQ ID NO: 2, and having a cleavage map of FIG. Recombinant vector for gene destruction. delete Corynebacterium glutamicum CF3224-0412 KCCM 10636, wherein the Corynebacterium alanine aminotransferase gene was transformed with the recombinant vector according to claim 1. delete delete A method for producing L-lysine, comprising recovering L-lysine after culturing the transformed microorganism of claim 3 under L-lysine production conditions.

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