JP2001057896A - Production of l-lysine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing L-lysine by a fermentation process using a vitamin B12 non-demanding thermophilic bacterium belonging to the genus Bacillus. SOLUTION: A methanol-assimilating thermophilic bacterium belonging to the genus Bacillus, capable of producing L-lysine, not demanding B12, to be concrete, Bacillus methanolicus having L-lysine analog resistance, is cultured in a medium. L-Lysine is formed and accumulated in a culture product and L-lysine is collected from the culture product to give L-lysine.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a process for producing L- lysine BACKGROUND OF THE INVENTION, particularly, relates to a method for producing L- lysine using the vitamin B ₁₂ Non-auxotrophic methylotrophic thermophilic Bacillus bacteria. L-lysine is widely used as a feed additive or the like.

[0002]

2. Description of the Related Art At present, L-lysine is mainly produced by fermentation using L-lysine-producing bacteria belonging to the genus Brevibacterium, Corynebacterium, Bacillus, or Escherichia. During the fermentation, the temperature of the culture medium rises, so that it is necessary to cool the culture medium during the fermentation in order to inactivate enzymes necessary for the fermentation or to kill the producing bacteria.

On the other hand, enzymes and proteins produced by thermophilic bacteria are generally stable at high temperatures, and are increasingly used as diagnostic agents and industrial catalysts. It is also expected that thermophilic bacteria themselves will be used for fermentative production. If fermentative production of L-lysine at a high temperature using such a thermophilic bacterium becomes possible, cooling of the culture medium becomes unnecessary, and cooling cost during fermentation can be reduced. In addition, if fermentation can be performed at a high temperature, it is considered that the reaction rate can be improved.

[0004] Heterotrophic organisms that require an organic compound as a carbon source, such as microorganisms commonly used for fermentative production of amino acids and the like, use sugars metabolized by glycolysis as carbon sources. Often used as.
On the other hand, alcohols such as ethanol and methanol, cellulose, carbon dioxide, and the like are also being sought as carbon sources for fermentation. These carbon sources are currently selected mainly for economic reasons because of the high ratio of raw material costs to fermentation production. However, diversification of technology,
Alternatively, from the viewpoint of future technology development, fermentative production of amino acids using these carbon sources is considered to be an important technology.

[0005] As thermophilic bacteria, vitamin B ₁₂ request of Bacillus sp. An L-lysine-producing bacterium, which is a mutant strain of MGA3, has been reported (WO90 / 12105, Lee, G. et al.
H., et al., Biotechnology and Bioengineering, 49,
639-653 (1996)) is, in vitamin B ₁₂ Non-auxotrophic thermophilic Bacillus bacteria not report on L- lysine production.

[0006]

[0008] The present invention has been made from the viewpoint, to provide a method of producing L- lysine by fermentation using vitamin B ₁₂ Non-auxotrophic thermophilic Bacillus bacteria As an issue.

[0007]

Means for Solving the Problems The present inventors have solved the above problems.
As a result of intensive studies to solve the problem, vitamin B_{1 Two}
Unrequired methanol-utilizing thermophilic bacterium Bacillus methanoli
Mutation having resistance to L-lysine analog from a moss strain
That the mutant has L-lysine-producing ability
And completed the present invention. That is, the present invention
Is as follows.

(1) L-lysine producing ability, vitamin
Culturing the B ₁₂ unsolicited of methylotrophic thermophilic Bacillus bacteria in a culture medium, to produce and accumulate an L- lysine in culture,
Collecting L-lysine from the culture;
-A method for producing lysine. (2) The method according to (1), wherein the bacterium belongs to Bacillus methanolicus. (3) The bacterium is Bacillus methanolicus PB1 (NCIMB13
113) The method of (2) which is derived. (4) The method according to any one of (1) to (3), wherein the bacterium has L-lysine analog resistance.

In the present invention, L-lysine-producing ability refers to
The ability to accumulate L-lysine in the medium when the bacteria used in the present invention are cultured in the medium.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
Bacterium used to the preparation of L- lysine of the present invention has a L- lysine producing ability is vitamin B ₁₂ Non-auxotrophic methylotrophic thermophilic Bacillus bacteria. The vitamin B ₁₂ Non-auxotrophic methylotrophic thermophilic Bacillus bacteria include Bacillus methanolicus. In addition, as Bacillus methanolicus, specifically, Bacillus methanolicus PB
1 (NCIMB13113). Bacillus methanolicus strain PB1 (Arfman, N. et al, Int. J. Syst. Bacterio
l., 42 (3), 439 (1992)) is the NCIMB (National Collect
ions of Industrial and Marine Bacteria Ltd.
23 St. Machar Drive, Aberdeen AB2 1RY, Scotland, Un
ited Kingdom).

[0011] The bacterium of the present invention, the above-mentioned vitamin B ₁₂ Non-auxotrophic methylotrophic thermophilic Bacillus bacteria (hereinafter, simply referred to as "thermophilic Bacillus bacterium") to L
-Obtained by imparting lysine-producing ability. L-
A thermophilic Bacillus bacterium having lysine-producing ability can be obtained by isolating an L-lysine analog-resistant mutant from a wild-type thermophilic Bacillus bacterium. Examples of L-lysine analogs include S-2-aminoethylcysteine (AEC), γ-methyllysine (ML), α-amino-β-
Hydroxyvaleric acid (AHV) and trans-4,5-dehydrolysine (DHL). Hereinafter, a method for obtaining a thermophilic Bacillus bacterium having L-lysine analog resistance will be described.

The thermophilic Bacillus bacterium having L-lysine analog resistance is obtained by culturing the thermophilic Bacillus bacterium in a minimal medium containing a concentration of the L-lysine analog that inhibits the growth of the bacterium, and selecting a strain that grows. Can be obtained by Here, growth inhibition refers to growth delay or growth inhibition. The selection may be performed once or plural times. The amount of the L-lysine analog to be added to the medium is not particularly limited as long as the growth of the parent strain to which L-lysine analog resistance is to be imparted is inhibited, but is 0.01 mg / ml or more, preferably 1 mg / ml to 50 mg / ml. Prior to selection, a thermophilic Bacillus bacterium may be subjected to a mutation treatment. Mutation treatment is performed by ultraviolet irradiation or N-methyl-N ′
It may be carried out by treatment with a mutagen commonly used for artificial mutation such as -nitro-N-nitrosoguanidine (NTG) or nitrous acid. Selection of an L-lysine analog-resistant strain may be performed for one type of L-lysine analog, or may be performed for a plurality of types of L-lysine analog. The selection may be performed once for one kind of L-lysine analog, or may be performed plural times.

The thermophilic Bacillus bacterium having L-lysine analog resistance obtained as described above can grow even in the presence of an L-lysine analog at a concentration at which the parent strain cannot grow.

The thermophilic Bacillus bacterium also has L-lysine caused by mutations other than the L-lysine analog resistance mutation, as is known from L-lysine-producing bacteria of the genus Brevibacterium or Corynebacterium. Production capacity can be provided. Mutants having such a mutation include L-leucine, L-homoserine, L-proline, L-serine, L-arginine, L-alanine, and L-leucine.
Mutants requiring amino acids such as valine (US Patent No. 3708)
Nos. 395 and 3825472), DL-α-amino-ε-caprolactam, α-amino-lauryl lactam, aspartic acid-analogs, sulfa drugs, quinoids, L-lysine-producing mutants resistant to N-lauroylleucine, L-Lysine-producing mutants showing resistance to oxaloacetate decarboxylase (decarboxylase) or respiratory enzyme inhibitors (Japanese Patent Application Laid-Open Nos. 50-53588, 50-31093, 52-102498)
No., JP-A-53-9394, JP-A-53-86089, JP-A-55-97
No. 83, JP-A-55-9759, JP-A-56-32995, JP-A-56-329
39778, JP-B-53-43591, JP-B-53-1833), L-lysine-producing mutants requiring inositol or acetic acid (JP-A-55-9784, JP-A-56-8692), fluoropyruvin L-lysine-producing mutants that are sensitive to acids (Japanese Patent Application Laid-Open Nos. 55-9783 and 53-86090), mutants resistant to ethylene glycol (US Patent No. 4411997), and the like are known. I have.

Furthermore, L-lysine-producing ability can be imparted or enhanced by enhancing the L-lysine biosynthetic enzyme gene in thermophilic Bacillus bacteria. Examples of the L-lysine biosynthetic enzyme gene include an aspartokinase gene, a dihydrodipicolinate reductase gene, a dihydrodipicolinate synthase gene, a diaminopimelate decarboxylase gene, a diaminopimelate dehydrogenase gene, and a phosphoenolpyruvate carboxylase gene.

The above-mentioned enzyme gene can be obtained, for example, from a gene library of a thermophilic Bacillus bacterium as a gene for restoring the auxotrophy of a mutant strain of a microorganism deficient in the enzyme. Thus was vitamin B was obtained ₁₂ request of Bacillus sp is thermophilic Bacillus. Disclosed are a gene encoding aspartokinase II from the MGA3 strain (US Pat. No. 5,243,039) and a gene encoding diaminopimelate decarboxylase (US Pat. No. 5,426,052). In the present invention, it is possible to use these vitamin B ₁₂ requirement for gene from thermophilic Bacillus, even these genes obtained from vitamin B ₁₂ non requirement thermophilic Bacillus in the same manner as the gene, It can be used as well.

Vectors for introducing the L-lysine biosynthesis gene into thermophilic Bacillus bacteria include pUB110, pHY3
00PLK, pHV1248, pE194, pC194, pBC16, pSA0501, pSA2
100, pAM77, pT181, pBD6, pBD8 and pBD64, pHV14, and their derivative plasmids. Also,
An endogenous plasmid of about 20.5 kbp or a derivative thereof, which is maintained by the Bacillus methanolicus PB1 strain, can also be used to introduce an L-lysine biosynthesis gene into a thermophilic Bacillus bacterium (Japanese Patent Application No. Hei 11 (1999)). -105812).

Transformation of a thermophilic Bacillus bacterium is carried out by introducing host cells into protoplasts or spheroplasts and introducing recombinant DNA into DNA recipients (Chang,
S. and Choen, SN, Molec. Gen. Genet., 168, 111
(1979); Bibb, MJ, Ward, JM and Hopwood, O.
A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J.
B. and Fink, GR, Proc. Natl. Acad. Sci. USA, 7
5, 1929 (1978)).

The vitamin B ₁₂ non-requiring methanol-assimilating thermophilic Bacillus bacterium obtained as described above is cultured in a medium, L-lysine is produced and accumulated in the culture, and L-lysine is produced from the culture. By collecting lysine, L-lysine can be produced.

The medium used for the cultivation includes a carbon source, a nitrogen source,
This is a normal medium containing inorganic ions and other organic components as required.

Examples of the carbon source include sugars such as glucose, maltose and ribose, methanol, mannitol,
Alcohols such as sorbitol can be used. In the present invention, methanol can be particularly preferably used.

As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like can be used.

As inorganic ions and their sources, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions and the like are added. As organic trace nutrients, biotin, folic acid, be suitable amount necessary required substances or yeast extract, and the like, such as vitamin B ₁ desirable.

The culturing is carried out under conditions suitable for the growth of the microorganism to be used. Usually, it is good to carry out under aerobic conditions for 16 to 72 hours, and the culture temperature is controlled at 20 ° C to 45 ° C, and the pH is controlled at 5 to 8.5 during the culture. For pH adjustment, an inorganic or organic acidic or alkaline substance, furthermore, ammonia gas or the like can be used. When a thermophilic bacterium is used as a host, the cultivation can be performed at a temperature of 42 ° C to 60 ° C.

The collection of L-lysine from the culture can be usually performed by a combination of an ion exchange resin method, a precipitation method and other known methods.

[0026]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples.

[0027]

[Example 1] L- from Bacillus methanolicus strain PB1
Acquisition of lysine analog resistant strain Vitamin B₁₂Unrequired methanol-utilizing thermophilic bacterium Bacill
S. methanolicus PB1 (NCIMB13113) was added to 5 ml of A medium (K_Two
HPO_Four3.8g / L, NaH_TwoPO_Four・ 2H_TwoO 3.1 g / L, (NH_Four)_TwoSO_Four 3.6g /
L, MgSO_Four・ 7H_TwoO 0.5g / L, FeSO_Four・ 7H_TwoO 2mg / L, CuSO_Four・ 5H
_TwoO 40μg / L, H_ThreeBO _Three30μg / L, MnSO_Four・ 4H_TwoO 200μg / L, Zn
SO_Four・ 7H_TwoO 200μg / L, Na_TwoMoO_Four・ 2H_TwoO 40μg / L, CaCl_Two・
2H_TwoO 5.3μg / L, CoCl_Two・ 6H_TwoO 40μg / L, thiamine hydrochloride
 100μg / L, calcium pantothenate 100μg / L, ribo
Flavin 100μg / L, biotin 100μg / L, nicotinic acid
 100 μg / L, pyridoxine hydrochloride 100 μg / L, amino ammonium
20 μg / L benzoic acid, 20 μg / L lipoic acid, 20 μg / L folic acid,
Inoculate a test tube containing 2% methanol (V / V) at 50 ℃
And cultured overnight with shaking.

The above culture solution was inoculated at 1% into a Sakaguchi flask containing 50 ml of A medium, grown at 50 ° C. until the logarithmic growth phase, and then collected.

The bacterial cells obtained as above are washed with a potassium phosphate buffer (pH 7.4), and then washed with 50 mg / L of N-methyl-N'-nitro-N-nitrosoguanidine (NTG ), And suspended at 50 ° C. for 30 minutes. The cells treated with NTG were added to a potassium phosphate buffer solution.
After washing twice (pH 7.4), the cells were suspended in medium A and cultured with shaking overnight. Collect a portion of these cultures and add 1 mg / ml
S-2-aminoethylcysteine (AEC) was applied to an A agar medium (A medium containing 1.5% agar) and cultured at 50 ° C. for 2 to 5 days. The emerged colonies were streaked at 50 ° C. on an A agar medium containing AEC at the same concentration as above. Thereafter, the formed colony was inoculated into a test tube containing 5 ml of A medium, and cultured with shaking for 2 to 5 days. These cultures were analyzed by thin-layer chromatography (TLC) using a developing solution having a composition of n-butanol: acetic acid: water: diethylamine = 5: 5: 2: 1 (volume ratio). -Some lysine spots were detectable. Two strains where large L-lysine spots could be confirmed were selected and named # 1691 and # 1821, respectively.

# 1691 and # 1821 are AJ13602 and A, respectively.
J13603, and on June 4, 1999, Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry (Postal Code: 305-8566)
Deposited at 1-3-1-3 Higashi, Tsukuba, Ibaraki, Japan)
Accession number FERM P-17413 and FERM
P-17414 was granted and transferred to an international deposit under the Budapest Treaty on June 2, 2000.
Accession numbers for BP-7182 and FERM BP-7183 are assigned.

[0031]

Example 2 Production of L-Lysine by L-Lysine Analog-Resistant Strain The above two strains (# 1691 and # 1821) obtained in Example 1 were cultured on A agar medium at 50 ° C. for 2 days. One platinum loop of these cells was inoculated into a test tube containing 5 ml of A medium, and cultured with shaking at 50 ° C. for 2 days.

Next, 2 ml of each culture was added to a Sakaguchi flask containing 20 ml of the A medium, and shaking culture was similarly performed at 50 ° C. for 2 to 3 days. After completion of the culture, the cells were removed by centrifugation, and the concentration of L-lysine contained in the culture supernatant was quantified using an amino acid analyzer. The results are shown in Table 1.

[0033]

[Table 1] Table 1 {Lactate production of L-lysine (g / L)} ─────── PB1 0 # 1691 0.36 # 1821 0.64 ────────────────────

Further, the culture was carried out in the same manner using a 1 L jar fermenter. The medium used was the medium A as a basic medium, and the culture broth was sequentially collected during the culture, and the methanol concentration was analyzed by gas chromatography.When the methanol concentration became 0.5 to 1.0% (V / V), Methanol was added as appropriate.

The pH of the medium was maintained at pH 6.7 by adding ammonia, and foaming was controlled by appropriately adding a silicone-based antifoaming agent (GD-113). After completion of the culture, the concentration of L-lysine in the medium was quantified in the same manner as described above. Table 2 shows the culture time and the concentration of L-lysine HCl accumulated in the medium. In the table, "-" indicates that the test was not performed.

[0036]

[Table 2]

[0037]

According to the present invention can produce L- lysine using the vitamin B ₁₂ Non-auxotrophic thermophilic Bacillus bacteria. According to the present invention, without the addition of vitamin B ₁₂ in a medium, it can be produced L- lysine using a carbon source such as methanol.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Yoshio Kawahara 1-1, Suzukicho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Ajinomoto Co., Ltd. Fermentation Research Institute (72) Shinichi Sugimoto 1, Suzukicho, Kawasaki-ku, Kawasaki-shi, Kanagawa -1 F-term in Ajinomoto Co., Inc. Fermentation Research Laboratory (reference) 4B064 AE25 CA02 CD06 DA01 DA10 DA11

Claims (4)

[Claims] 1. A L- lysine has an ability to produce vitamin B ₁₂
A non-requiring methanol-assimilating thermophilic Bacillus bacterium is cultured in a medium, L-lysine is produced and accumulated in the culture, and L-lysine is collected from the culture. Manufacturing method. 2. The method according to claim 1, wherein the bacterium is a bacterium belonging to Bacillus methanolicus. 3. The bacterium is Bacillus methanolicus PB1.
The method according to claim 2, which is derived from (NCIMB13113). 4. The method according to claim 1, wherein the bacterium has L-lysine analog resistance.