KR100645769B1 - - Method for the preparation of L-lysine using Corynebacterium having disrupted aspA gene - Google Patents

Abstract

A method for preparation of L-lysine by using Corynebacterium having disrupted aspA gene is provided to improve preparation yield and reduce preparation costs by disrupting the aspA gene encoding aspartase which catalyzes reversible conversion from aspartate into fumarate. The method for preparation of L-lysine comprises the steps of: disrupting the aspA gene of a Corynebacterium strain by transforming the Corynebacterium strain capable of producing L-lysine with a recombinant vector containing a portion of the aspA gene, wherein the recombinant vector is pCR-aspA; culturing the transformed Corynebacterium strain; and recovering L-lysine from the cultured medium. And the transformed Corynebacterium strain is Corynebacterium glutamicum KFCC 10881-CJP5111(KCCM-10706P).

Description

Method for the preparation of L-lysine using Corynebacterium having disrupted aspA gene}

1 is a cleavage map of pCR-aspA, a recombinant vector of the present invention.

The present invention is to cultivate the coryneform bacteria having L-lysine production capacity of the aspase encoding aspartase, an enzyme catalyzing the reversible conversion reaction between fumarate and oxaloacetate in a medium, L- in fermentation broth. A method for producing L-lysine, characterized by accumulating lysine and recovering L-lysine therefrom.

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 the animal feed, human pharmaceutical and cosmetic industries and are produced by fermentation of Corynebacterium strains. As described above, 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, a number of studies have been conducted to amplify each L-amino acid biosynthesis-related gene using recombinant DNA technology to study the effects on L-amino acid production and to improve L-amino acid producing Corynebacterium strains [Kinoshita, "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. Japanese Patent Laid-Open No. 7-121228 describes a method of introducing a gene encoding an Escherichia coli-derived citric acid synthase.

In the case of L-amino acid-producing Corynebacterium strains, pyruvate generated through glycolysis from various carbon sources is converted to aspartate via oxaloacetate, and aspartate isoleucine. (isoleucine), leucine, threonine, methionine and lysine, and are converted to amino acids such as pantothenate via betaalanine. It becomes a precursor to produce. Since aspartate acts as an important precursor of various aspartate-derived amino acids including lysine, increasing the amount of intracellular aspartate has been used as an important strategy for improving the lysine producing strain.

 To date, all metabolic pathways and major genes that produce isoleucine, leucine, threonine, methionine and lysine from aspartate in Corynebacterium have been identified, and studies on improving various amino acid producing strains using them are actively underway. No research has been done on the effects of lysine production on the manipulation of pathways with aspartate.

To biosynthesize lysine, threonine, methionine and isoleucine, aspartate must be converted to aspartylphosphate by the asparto kinase enzyme, and this pathway is competitive with other pathways using aspartate as a common precursor. In a relationship. These competitive pathways are roughly mediated by algininosuccinate synthetase (EC 6.3.4.5), pathways mediated by adenilosuccinate synthase (EC 6.3.4.4), aspartate carbamoyl conversion. Pathway mediated by enzyme (EC 2.1.3.2), pathway mediated by asparagine synthetase (EC 6.3.5.4), betaalanine synthesis pathway mediated by aspartate 1-decarboxylase (EC 4.1.1.11) And as mediated by aspartase (EC 4.3.1.1).

To examine the effect of the above metabolic pathways on the production of lysine, and to apply to the improvement of the lysine producer strain was produced each strain, the present invention is the effect of the aspA gene.

The homology with E. coli aspA gene from Brevibacterium Plastic boom MJ233 by year Yoko Asai 1995 for gene aspA coding for Aspergillus Zapata enzyme which catalyzes a reversible conversion between aspartate and fumarate be referred to in the present invention Cloning and sequencing were performed (Yoko, A. et al. Gene 158, 87-90 (1995); Japanese Patent JP 1993030977-A / 1), and the entire gene translation and analysis process of Corynebacterium glutamicum (Kalinowsky, J. et al. Journal of Biotechnology 104 (1-3), 5-25 (2003)), and overexpressed the aspA gene isolated from Escherichia coli in a medium containing fumarate. There is a patent on the production of aspartate strains of amino acids from the strains of Leeum glutamicum (Sahm, H. et al. Patent No. 0318663 (1987)). Not reported to the bar.

In order to solve the above problems, the present inventors carried out extensive research on the production method of L- lysine in order to lower the production cost and increase the yield in the production of L- lysine by direct fermentation method using coryneform bacteria As a result, the present inventors have found that the destruction of the gene aspA in coryneform bacteria can improve the productivity of L-lysine-producing microorganisms and completed the present invention.

That is, an object of the present invention is to provide a recombinant vector and a transformant comprising the same for producing coryneform bacteria in which aspA gene is destroyed.

Another object of the present invention is to provide a method for producing L-lysine with improved productivity by using the transformant.

The above and other objects of the present invention can be achieved by the present invention described below.

The present invention to achieve the above object,

Degrading the activity of the aspA gene of Corynebacterium genus microorganism in the mutant microorganism for L- lysine production; It provides a method for producing a mutant microorganism for producing L- lysine, characterized in that consisting of.

The method of destroying the activity of the aspA gene of the Corynebacterium genus microorganism may be to transform a recombinant vector containing a part of the aspA gene to L- lysine-producing microorganisms.

The recombinant vector may be a vector pCR-aspA having a cleavage map of FIG. 1.

The present invention also provides a mutant microorganism for producing L-lysine produced by the above method.

The microorganism is Corynebacterium glutamicum ( Corynebacterium glutamicum ) KFCC10881-CJP5111 (Accession No .: KCCM-10706P).

In addition, the present invention comprises the steps of culturing the microorganisms under conditions suitable for producing L-lysine; And it provides a method for producing L- lysine comprising a; recovering L- lysine in the culture solution.

Hereinafter, the present invention will be described in detail.

The present invention produces a high yield lysine producer by destroying the aspartase (hereinafter aspA ) gene of Corynebacterium, and cultured by direct fermentation to accumulate L- lysine in the fermentation broth, from which L-lysine The present invention relates to a method for producing L-lysine, characterized in that it is collected.

Aspartase enzyme catalyzes the reversible reaction between aspartate, a precursor of aspartate-based amino acid biosynthesis, and fumarate, an intermediate in the citric acid cycle, and this reversible reaction depends on the intracellular equilibrium of aspartate and fumarate concentrations. Is determined. In lysine producers, it is expected to contain only relatively low concentrations of fumarate because the intracellular aspartate concentration is high while the metabolic flow into the citric acid cycle is relatively weak. The characteristics of these strains are expected to create an intracellular environment that favors the conversion of aspartate to fumarate rather than the reaction of aspartase enzyme to the biosynthesis of aspartate-derived amino acids.

The present inventors expected the lysine production yield, productivity and cost reduction effect by increasing the intracellular aspartate concentration by destroying the aspA gene in the current lysine producing strain based on this prediction.

The present invention, after obtaining the gene aspA encoding aspartase of Corynebacterium from the National Institutes of Health (NIH GenBank), and secured, using the above sequence information to produce Corynebacterium L-lysine The aspA gene of the strain was destroyed, and the resulting strain was found to possess improved L-lysine production capacity.

In one aspect, the present invention on the chromosome using the Aspergillus Zapata dehydratase gene aspA Corey four bacteria having the nucleotide sequence of SEQ ID NO: 1 aspA Includes recombinant cells that have destroyed genes. 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 .

In another aspect, the present invention provides a method of producing L-lysine by culturing the provided Corynebacterium strains under conditions suitable for producing L-lysine and recovering L-lysine.

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.

Corynebacterium strains used in the present invention include Corynebacterium glutamicum ATCC13032 in addition to other strains of the genus Corynebacterium, in particular Corynebacterium glutamicum species known wild type strain, Corynebacterium C. thermoaminogenes FERM BP-1539, Brevibacterium flavum ) ATCC 14067, B. lactofermentum ATCC 13869, and L-amino acid producing mutants or strains prepared therefrom, such as Corynebacterium glutamicum KFCC10881, Corynebacte Leeum glutamicum KFCC11001.

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 the Manual of Methods for General Bacteriology by the American Society for Bacteriology (Washington DC, USA, 1981). Glucose, saccharose, Sugars and carbohydrates such as lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, fatty acids such as palmitic acid, stearic acid, linoleic acid, glycerol, ethanol Organic acids such as alcohols, acetic acid, etc. 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, Examples include ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Persons that can be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts, and the culture medium also contains metal salts such as magnesium sulfate or iron sulfate necessary for growth. Finally, in addition to these substances, essential growth substances such as amino acids and vitamins can be used, and appropriate precursors can be used in the culture medium. Or may be added continuously.

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 present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the examples.

[ Example  One]

Corynebacterium  origin aspA  Gene search and aspA Recombinant vector construction for gene destruction

Based on the aspA gene sequence of Brevibacterium plaboom reported by Yoko et al. In 1995 (Yoko, A. et al. Gene 158, 87-90 (1995); Japanese Patent JP 1993030977-A / 1) Corynebacterium The aspA gene sequence (SEQ ID NO: 1) was obtained from the National Institutes of Health gene bank (NIH GenBank) (NCgl1446). Based on the reported nucleotide sequence, a pair of primers (SEQ ID NO: 3, SEQ ID NO: 4) described in Table 1 below were synthesized, and the chain polymerization method using the chromosomal DNA of Corynebacterium glutamicum wild strain (ATCC13032) as a template (PCR method) [Sambrook et al, Molecular Cloning, a Laboratory Manual (1989), Cold Spring Harbor Laboratories] (PCR conditions: Denaturing = 96 ° C., 30 sec / Annealing = 52 ° C., 30 sec / Polymerization = 72 ° C., 30 Second, 30 times) amplified 500 base pair of aspA gene internal region fragment (SEQ ID NO: 2), and then cloned into the E. coli plasmid pCR-TOPO2.1 using the TOPO Cloning Kit (Invitrogen) pCR- aspA Plasmids were obtained.

Primer 1 5'-GGGCGAGTACCACATCCTGC-3 ' Primer 2 5'-TTGGACAGTTTCATGGCTGC-3 '

[ Example  2]

L-lysine production At the Corynebacterium aspA  Destruction

PCR- aspA prepared in Example 2 was transformed into L-lysine producing strain Corynebacterium glutamicum KFCC-10881 by an electric pulse method (Appl. Microbiol. Biotechnol. (1999) 52: 541-545 By transformation method). The pCR- aspA vector introduced into the cells is inserted by the homology between the aspA gene on the chromosome, which in turn destroys the aspA gene on the chromosome and also retains the kanamycin resistance antibiotic gene inherent in the pCR- aspA vector. Gain kanamycin resistance.

After the transformation experiment, the aspA gene disruption strain was selected from a selection medium containing 25 mg / L of kanamycin, and whether or not the gene was destroyed was determined by using the primers of SEQ ID NO: 3,4 or the sequence 3 and M13 reverse primer (Invitrogen). It was confirmed by DNA chain polymerization (PCR). PCR reaction conditions are as follows. (PCR conditions: Denaturing = 96 ° C., 30 seconds / Annealing = 52 ° C., 30 seconds / Polymerization = 72 ° C., 30 seconds, 30 times). As a result of confirming the product of the chain polymerization reaction by DNA electrophoresis, in the chain polymerization reaction using the sequence 3,4 primer combination, DNA of about 0.5 kb in the KFCC-10881 strain and the production strain KFCC-10881-CJP5111 used as a control as expected. The fragment was detected, and in the reaction using the combination of SEQ ID NO: 3 and the M13 reverse primer, no DNA fragment was detected in the KFCC-10881 strain and about 0.59 kb DNA fragment was detected in the KFCC-10881-CJP5111.

These results confirmed that the aspA gene on the chromosome of KFCC10881 was destroyed by the insertion of the pCR- aspA gene destruction vector. Thus, the Corynebacterium lysine producing strain (KFCC-10881-CJP5111), in which the gene aspA was destroyed, was deposited with the Korean spawn association of November 16, 2005, and was given accession number KCCM-10706P.

[ Example  3]

gene aspA  Destruction L-lysine In the production strain  Lysine production

L-lysine-producing strain Corynebacteria glutanicum KFCC-10881-CJP5111 strain prepared as in Example 3 was incubated in the following manner for the production of L-lysine.

A 250 ml corner-baffle flask containing 25 ml of the following species medium was inoculated with Corynebacterium glutamicum parent strains KFCC-10881 and KCCM-10706P (KFCC-10881-CJP5111) and incubated for 20 hours at 30 ° C. (200 rpm). A 250 ml corner-baffle flask containing 24 ml of the following production medium is 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 L-lysine in culture for Corynebacterium glutamicum KFCC-10881 and KCCM-10706P (KFCC-10881-CJP5111) are shown in Table 2 below.

Strain Lysine (g / l) KFCC-10881 45 KCCM-10706P (KFCC-10881-CJP5111) 47

Species  ( pH  7.0)

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

Production medium  ( pH  7.0)

Glucose 100 g, (NH ₄ ) ₂ SO ₄ 40 g, Soy Protein 2.5 g, Corn Steep Solids 5 g, Urea 3 g, KH ₂ PO ₄ 1 g, MgSO ₄ 7H ₂ O 0.5 g, Biotin 100 μg, Thiamine hydrochloride 1000 μg, calcium-pantothenic acid 2000 μg, nicotinamide 3000 μg, CaCO ₃ 30 g, (based on 1 liter of process water)

As shown in Table 2 above, the gene aspA disrupting strain In the case of KCCM-10706P (KFCC-10881-CJP5111), lysine production was increased by an average of 5% compared to the parent strain KFCC-10881.

As described above, the present invention provides an expression vector for destroying the gene aspA , which encodes aspartase , an enzyme catalyzing the reversible conversion reaction between fumarate and oxaloacetate, and an L-lysine producing strain including the same. It is a useful invention that has the effect of providing a method for producing L-lysine with improved productivity.

While the invention has been described in detail above with reference to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the scope and spirit of the invention, and such modifications and variations fall within the scope of the appended claims. It is also natural.

Attach sequence list electronic file

Claims (6)

Degrading the activity of the aspA gene of Corynebacterium genus microorganism in the mutant microorganism for L- lysine production; Method for producing a mutant microorganism for producing L- lysine, characterized in that consisting of. The method of claim 1, Method of destroying the activity of the aspA gene of the microorganism of the genus Corynebacterium , Method for producing a mutant microorganism for producing L- lysine, characterized in that for transforming the recombinant vector containing a portion of the aspA gene to the microorganism for producing L- lysine The method of claim 2, The recombinant vector is a vector pCR- aspA having a cleavage map of FIG. A mutant microorganism for producing L-lysine prepared by the method of any one of claims 1 to 3. The method of claim 4, wherein The microorganism is Corynebacterium glutamicum ( Corynebacterium glutamicum ) KFCC10881-CJP5111 (Accession No .: KCCM-10706P). Culturing the microorganism of claim 4 under conditions suitable for producing L-lysine; And Recovering L-lysine from the culture solution; Method for producing L-lysine, characterized in that consisting of

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