KR20050065712A - Corynebaterium sp. transformed with a heterogenous fructokinase gene and method for producing l-lysine using the same - Google Patents

Abstract

The present invention provides a Corynebacterium sp. Strain transformed by a foreign fructokinase gene and having L-lysine production ability, and a method for producing L-lysine using the same.

Description

Corynebacterium spp. Transformed with fructokinase gene and method for producing L-lysine using the same {Corynebaterium sp. transformed with a heterogenous fructokinase gene and method for producing L-lysine using the same}

The present invention relates to a Corynebacterium sp. Strain having a high L-lysine production capacity and a method for producing L-lysine using the same.

A strain of the genus Corynebacterium , in particular Corynebacterium glutamicum , is a Gram-positive microorganism that is widely used for L-amino acid production. L-amino acids, in particular L-lysine, are used in the animal feed, human medicine and pharmaceutical industries 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, many 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)). There have also been studies to improve L-amino acid producing Corynebacterium strains by disrupting or underexpressing specific genes. For example, Korean Patent Publication Nos. 2001-51915 and 2001-62279 to Degussa-Wheels Actiengegelshaft disclose attenuated expression of the sucC and sucD genes and zwa2 genes derived from Corynebacterium glutamicum. A method for increasing the productivity of L-amino acids from four bacteria is described. In addition, genes derived from other external bacteria may be introduced. Japanese Patent Laid-Open No. 7-121228 describes a method of introducing a gene encoding a citric acid synthase derived from Escherichia coli .

On the other hand, the strain of the genus Corynebacterium can use both sucrose (sucrose), glucose (glucose), fructose (fructose) as a carbon source. Among them, sucrose is phosphorylated by the phosphotransferase system (PTS) and introduced into cells, and phosphorylated sucrose is directly degraded into 6-phosphorylated glucose and fructose by an invertase. 6-Phosphorylated Glucose immediately enters glycolysis, and fructose leaves the cell and is then phosphorylated by the phosphotransferase system to enter glycolysis. These fructose release and resorption processes require additional energy as a whole. This process is believed to be due to the absence of fructokinase expression in Corynebacterium (H. Dominguez et al., Applied and Environmental Microbiology, 62,3878-3880 (1996)). On the other hand, in bacteria expressing such fructokinase, fructose degraded by invertase is directly phosphorylated by fructokinase and enters glycolysis.

Therefore, conventionally known Corynebacterium strains do not express fructokinase, so there is a problem that additional energy must be consumed in order to use fructose. In addition, there is no known example of using a gene encoding a fructokinase for breeding of Corynebacterium strains.

Accordingly, the present inventors isolate the gene encoding the above-described fructokinase from foreign individuals and transform it into a Corynebacterium strain, thereby searching for a Corynebacterium strain having improved L-lysine production ability. It was completed.

Accordingly, it is an object of the present invention to provide a strain of Corynebacterium sp. (L. lysine) which has been transformed by a foreign fructokinase gene.

It is also an object of the present invention to provide a method for producing L-lysine using the Corynebacterium sp. Strain of the present invention.

The present invention provides a strain of Corynebacterium sp. Transformed by a foreign fructokinase gene and having L-lysine production ability.

In the present invention, the foreign fructokinase gene is included as long as it is a polynucleotide encoding a polypeptide having an activity of converting fructose to fructose 6-phosphate. Examples of such foreign fructokinase genes include fructokinase genes derived from animals, plants, and bacteria. Preferably, it is a fruckinase gene derived from bacteria, more preferably a fructokinase derived from Gram-positive bacteria. Such Gram-positive bacteria include, for example, Clostridium genus and Bacillus genus bacteria. In the present invention, most preferably, the foreign fructokinase gene is a fructokinase gene derived from Clostridium acetylbutylicum or Bacillus subtilus . Specifically, it may have a nucleotide sequence of GenBank Accession No. NP_347064.

In the present invention, the Corynebacterium sp. Used in the transformation of the foreign fructokinase gene belongs to Corynebacterium, which includes any one having L-lysine production ability. Examples of the Corynebacterium sp. Include Corynebacterium glutamicum (eg ATCC 13032), Corynebacterium thermoaminogenes (eg FERM BP- 1539), strains selected from the group consisting of Brevibacterium flavum (e.g. ATCC 14067), and Brevibacterium fermentum (e.g. ATCC 13869) have. In addition, variants having L-lysine producing ability derived from the strain may be included, for example, Corynebacterium glutamicum KFCC 10881 and KFCC 110011. However, it is not limited to the said example.

Corynebacterium sp. Strain of the present invention is preferably transformed with a fructokinase gene derived from Clostridium acetylbutylicum , and having L-lysine production ability Corynebacterium glutamicum CJ3930 (Accession No .: KCCM-10531).

As used herein, the term “transformation” means introducing a gene into a host cell so that it can be expressed in the host cell. As long as the transformed gene is in a state capable of being expressed in the host cell, it is included whether it is inserted into the chromosome of the host cell or located outside the chromosome. The gene also includes DNA and RNA as polynucleotides capable of encoding a polypeptide. The gene may be introduced in any form as long as it can be introduced into and expressed in a host cell. For example, the gene may be introduced into a host cell in the form of an expression cassette, which is a polynucleotide construct that contains all the elements necessary for expression of the gene. The expression cassette typically contains a promoter, transcription termination signal, ribosomal binding site and translation termination signal operably linked to the gene. The expression cassette may be in the form of an expression vector capable of self replication. In addition, the gene may be introduced into the host cell in itself or in the form of a polynucleotide structure, and may be operably linked to a sequence required for expression of the host cell.

The present invention also comprises the steps of culturing a Corynebacterium sp. Strain transformed by the foreign fructokinase gene of the present invention and having L-lysine production capacity; And it provides a method for producing L- lysine, comprising the step of separating the L- lysine from the culture.

In the L-lysine production method of the present invention, the culture of the Corynebacterium sp. Strain of the present invention can be cultured by various culture methods known in the art in a suitable medium. Examples of the culturing method include batch, continuous and fed-batch cultivation. The fed-batch culture includes, but is not limited to, injection batches and repeated injection batch cultures. Such various culture methods are described, for example, in Chmiel, Bipprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik, Gustav Fischer Verlag, Stuttgart, 1991; And Storhas, Bioreaktoren und periphere Einrichtungen, Vieweg Verlag, Braunschweig / Wiesbaden, 1994.

As a medium that can be used for the culture, a suitable medium known in the art may be selected depending on the culture method and the strain. Medium for Corynebacterium strains is disclosed, for example, in "Manual of Methods for General Bacteriology" by the American Society for Bacteriology, Washington, D.C., USA, 1981. The medium contains various carbon sources, nitrogen sources and trace element components. Examples of carbon sources that can be used include glucose, sucrose, lactose, fructose, maltose, starch, carbohydrates such as cellulose, soybean oil, sunflower oil, castor oil, fats such as coconut oil, palmitic acid, stearic acid, linoleic acid Fatty acids such as, alcohols such as glycelol and ethanol, and organic acids such as acetic acid. These carbon sources may be used alone or in combination. Examples of nitrogen sources that can be used include inorganic nitrogen sources such as peptone, yeast extract, gravy, malt extract, corn steep liquor, and organic nitrogen sources such as soybean wheat, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate Included. These nitrogen sources may be used alone or in combination. The medium may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodium-containing salts as a person. It may also include metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, appropriate precursors, and the like can be included. These media or precursors may be added batchwise or continuously to the culture.

During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, during the culture, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble generation. In addition, to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas (eg, air) is injected into the culture. The temperature of the culture is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. The incubation period may continue until the desired amount of L-lysine is obtained, preferably 10 to 160 hours.

Isolation of L-lysine from the culture can be isolated by conventional methods known in the art. In such a separation method, methods such as centrifugation, filtration, ion exchange chromatography, and crystallization may be used. For example, the supernatant obtained by removing the biomass by centrifugation of the culture at low speed can be separated by ion exchange chromatography.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

Example 1: Clostridium-derived fructokinase gene search and cloning

The base sequence of the fructokinase gene derived from Clostridium has already been clearly identified and disclosed. The gene information (registration number NP_347064) of Clostridium acetobutylicum of Clostridium acetobutylicum was obtained from NIH GenBank. Based on the reported nucleotide sequence, a pair of primers (SEQ ID NOs: 1 and 2) were synthesized, and the gene was amplified by PCR using chromosomal DNA of Clostridium acetobutylicum ATCC 824 as a template. PCR conditions were denatured 94 ℃, 20 seconds; Annealing 52 ° C., 20 seconds; Polymerization 72 degreeC, 1 minute 10 second was repeated 30 times. As a result, a 1200 bp fructokinase gene amplification product was obtained, and then cloned into E. coli plasmid pCR2.1 using a TOPO TA Cloning Kit (Invitrogen) to obtain a pCR-CFK plasmid.

Example 2 Preparation of Fructokinase Gene Expression Vectors

The vector pCR-CFK containing the fructokinase gene obtained in Example 1 was cleaved with restriction enzymes Nde I and Kpn I, respectively, to isolate only the gene fragment encoding the fructokinase, and then the pCJ (corynebacteria expression vector) PCJ-CFK was prepared by ligation to the nebacterium glutamicum cryptic plasmid pCG1 using a ligation enzyme (see FIG. 1). Next, the obtained pCJ-CFK was transformed into a strain of Corynebacterium glutamicum ATCC13032 using an electropulse method, and applied to LB agar medium containing 100 μg / ml of spectinomycin, followed by selection of transformants. , Obtained. PCJ-CFK was isolated and confirmed from each transgenic strain.

Example 3: Determination of fructokinase activity

The expression of fructokinase and activity in the cells from the fructokinase expression vector was measured by a method described in the literature (Andreas Pikis et al., Microbiology , 148, 843-852 (2002)). Corynebacterium glutamicum ATCC13032 (pCJ-CFK), a transforming strain containing pCJ-CFK, was cultured in LB medium for about one day, and then centrifuged to obtain only cells. The obtained cells were suspended in a suitable buffer, and then the cells were disrupted by ultrasonic disruption, followed by high-speed centrifugation to obtain a supernatant. The amount of NADPH produced by reacting the supernatant obtained by the above method with the reaction solution containing pectose, phosphoglucose isomerase, glucose-6-phosphate dehydrogenase, ATP and NADP ⁺ is absorbed at wavelength 340 nm. Table 2 shows the results of calculating the activity of fructokinase indirectly. As shown in Table 2, Corynebacterium glutamicum ATCC13032 (pCJ-CFK) exhibited about two times more activity than the Corynebacterium glutamicum ATCC13032 strain without the fructokinase expression vector. It was confirmed that the lactokinase gene was expressed.

Table 2.

Test strain ATCC13032 ATCC13032 (pCJ-CFK) Activity ^a 5.14 12.13

^a nmol (produced fructose-6-phosphate) min ^-1 mg (protein) ^-1

Example 4 Introduction of Fructokinase Gene Expression Vector and Lysine Production into Corynebacterial Strains with L-lysine Production Capacity

The transformed strain Corynebacterium glutanicum CJ3930 was obtained by transforming L-lysine producing strain Corynebacterium glutanicum KFCC10881 with the fructokinase expression vector pCJ-CFK by electric pulse method. The obtained transformed strains were first checked for the presence of a vector, and then cultured 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 a loop for inoculation with Corynebacterium glutamicum KFCC10881 and CJ3930 and shake-cultured at 220 ° C for 20 hours at 30 ° C. A 250 ml corner-baffle flask containing 25 ml of the following production medium was inoculated with 1 ml of seed culture and shaken at 220 rpm for 96 hours at 32 ° C. After the incubation was completed, the yield of L-lysine was measured by HPLC. As a result, L-lysine content in the culture for Corynebacterium glutamicum KFCC10881, and CJ3930 was as shown in Table 3 below.

Table 3.

Strain Lysine Concentration (g / l) Corynebacterium glutamicum KFCC10881 (Moju) 32.9 Corynebacterium glutamicum CJ3930 39.8

Composition of seed medium (pH 7.0):

50 g raw sugar, 10 g peptone, 10 g yeast extract, 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 (process Per liter of water).

Composition of Production Medium (pH 7.0):

Molasses or pretreated molasses (as reduced sugar) 50 g, raw sugar 50 g, yeast extract 4 g, (NH ₄ ) ₂ SO ₄ 40 g, urea 4 g, KH ₂ PO ₄ 1 g, NaCl 2.5 g, MgSO ₄ 7H ₂ O 1 g, FeSO ₄ 7H ₂ O 10 mg, MnSO ₄ 5H ₂ O 10 mg, Biotin 100 μg, Thiamine Hydrochloride 200 μg, CaCO ₃ 40 g, L-Leucine 0.4 g if necessary, L-Threonine 0.1 g if necessary, L-Methionine 0.1 g ( Per liter of process water).

Example 5 Mass Culture of Corynebacterium glutamicum CJ3930

Since the lysine concentration was increased in Example 4, the effect was tested in L-lysine production medium in a 30 L fermenter. The culture method was carried out by fed-batch fermentation method in which additional sugars and additional products were added during the cultivation. The medium components and culture conditions used were as follows, and the results are shown in Table 4 below.

Table 4.

Strain Average concentration (g / L) Incubation time (hr) Corynebacterium glutamicum KFCC10881 50.9 141 Corynebacterium glutamicum CJ3930 56.3 150

30L Fermenter Medium

Total sugar 335g / l (raw sugar, molasses), Corn steep liquor 100ml / l, (NH ₄ ) ₂ SO ₄ 35g / l, KH ₂ PO ₄ 1.75g / l, MgSO ₄ 7H ₂ O 0.7g / l, FeSO ₄ 7H ₂ O 13mg / l, biotin 500㎍ / l, thiamine hydrochloride 500㎍ / l, anti-foaming agents 3ml / l

Culture conditions: temperature 32 ℃, pH 6.9, stirring speed 550 rpm, air flow rate 0.5 vvm (air volume / medium / minute)

As can be seen in the above examples, it was confirmed that the Corynebacterium glutamicum CJ3930 strain of the present invention has excellent lysine production capacity. The Corynebacterium glutamicum CJ3930 strain of the present invention was deposited on November 28, 2003, at the Korea Microbial Preservation Center, an international depository institution under the Budapest Treaty (Admission Number: KCCM-10531).

Corynebacterium sp. Strain of the present invention can be used in a method for producing L-lysine in high yield.

According to the L-lysine production method of the present invention, it is possible to produce L-lysine in high yield.

Fig. 1 shows the expression vector pCJ-CFK of fructokinase derived from Clostridium.

<110> CJ Corporation <120> Corynebaterium sp. transformed with a heterogenous fructokinase gene and method for producing L-lysine using the same <130> P <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer: CFK_F <400> 1 catatgaata atgttttatg tatgggagaa 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer: CFK_R <400> 2 ataccattct agagggctta aagctaccgg 30

Claims (6)

Corynebacterium sp. Strain, which is transformed by a foreign fructokinase gene and has the ability to produce L-lysine. The strain of claim 1, wherein the foreign fructokinase gene has a nucleotide sequence of GenBank Accession No. NP_347064. According to claim 1, wherein the Corynebacterium sp. Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium thermoaminogenes ( Corynebacterium thermoaminogenes ), Bribibacterium flavomb ( Brevibacterium flavum ), and strains consisting of Brevibacterium fermentum . The strain according to claim 3, which is Corynebacterium glutamicum CJ3930 (Accession Number: KCCM-10531). Culturing the Corynebacterium sp. Strain according to any one of claims 1 to 4; And Separating the L-lysine from the culture method of producing L-lysine. 6. The method of claim 5, wherein the medium used for the culture comprises sucrose or fructose.