JPS58224684A - L-threonine-producing bacteria, preparation of l-threonine using the same, and cultivation of said bacteria - Google Patents

Abstract

PURPOSE:To prepare L-threonine, in high efficiency, by culturing L-threonine- producing bacteria belonging to Brevibacterium genus and obtained by cell fusion. CONSTITUTION:An L-threonine-producing bacterial strain requiring L-isoleucine and L-lysine, resistant to alpha-amino-beta-hydroxyvaleric acid and S-(2-aminoethyl) cysteine, belonging to Brevibacterium genus, and obtained by the cell fusion of an L-lysine-requiring strain and S-(2-aminoethyl)cysteine-resistant strain both belonging to Brevibacterium genus, e.g. Brevibacterium lactofermentum AJ 11788 (FERM-P No.6411), is inoculated in a nutrient medium and cultured under aerobic condition at 5-8pH and 29-37 deg.C for 1-4 days. L-threonine is separated from the culture liquid medium.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an L-threonine producing strain and a method for producing l-threonine using the same.

L-threonine-producing strains, especially V. The four threonine-producing strains belonging to the genus V.
Artificial mutant strain resistant to hydroxy-valeric acid (hereinafter referred to as rAHV J) (Japanese Patent Publication No. 45-26708) AH
Artificial mutant strain having Vtn resistance and requiring L-1 gin or L-inleucine (Japanese Patent Publication No. 48-44876),
An artificial mutant strain that is resistant to AHV and 5-(2-7minoethyl)-7 stain (rAEcJ) and co-produces L-1 gin (US Pat. No. 3,732,144), which is resistant to AHV and ABC. Artificial mutant strain that has L-leunone auxotrophy and co-produces monolysine (JP-A-5O-31093)
etc. are known.

The present inventors conducted research to obtain a strain with higher L-inleucine production ability, and found that it has resistance to AHV and AECr of the genus L-inleucine and L-inleucine. We have succeeded in breeding a strain that has L-lysine auxotrophy, and found that this strain produces L-threonia in high yield.

A strain with AHV and AECt stiffness and an auxotrophy for mono-inleucine and L-'J lysine may be difficult to obtain if the experimental results of the present inventors are taken into consideration. Therefore, it was not possible to induce a strain resistant to AHV and ABC and auxotrophic for inuroinone and L-lysine by artificial mutation. Scolding L
- Obtaining a strain that has both L-lysine auxotrophy and ABC resistance for the first time by cell fusion of a lysine auxotrophic strain and an ABC-resistant strain? This was successful.

One of the parent strains (parent strain a) used for cell fusion to induce the threonine-producing bacteria of the present invention has a lysine auxotrophy of the genus Brevibacterium, and the following parent strain b h'-
A HII l111J property and L-inleucine requirement, or if neither of these properties is present.
A mutant strain that has properties that the parent strain of Kokeso does not have and does not have resistance to 5-(2-aminoethyl)-cysteine (specifically, L-jJ lysine auxotroph A)
)] V rigid strain, L-1 di/auxotrophic, AHV resistant and L-isoroinone auxotrophic strain, L-lysine and L-isoleucine auxotrophic strain), and the other parent strain (parent strain b) is Brevibacterium If the above parent strain a does not have the properties of AHV rigidity and/or L-isoloy7 requirement, then the moss has the properties that the parent strain a does not have. and mutant strains that do not have L-lysine auxotrophy (specifically, AEC resistant strains, ABC rigid and AHV resistant strains, EC resistant, AHV resistant and L-isoleucine auxotrophic strains, EC resistant and L-lysine auxotrophic strains, Isoloi 7 auxotrophic strain). AHV resistance and L-inleucine auxotrophy In this case, one of the parent strains must have this property, but even if both parent strains have AHV resistance and/or L-inleucine auxotrophy, good.

The original strain of these mutant strains is preferably a so-called L-glutamic acid producing bacterium belonging to the genus Brevibacterium.

Specific examples include the following.

Brevibacterium deivaricatam ATC
C14 (120 Brevibacterium flavum
ATCC13826 Previbacterium lactofermentum ATCC13869 Brevibacterium roserum ATCC13B25
Brevibacterium thiogenitalis ATC
The CI 9240 parent strain was used as a auxotrophic mutant strain A.E. C4F resistance - ■■--- All of the mutant strains were used as markers for auxotrophy, etc., to distinguish them from the resulting fusion strain. Further suitable properties may be added.

The method of merging the parent strains is, for example, Agric, B, which has an example of fusion with Brevi 4cterium IAV.
iol, Chem, 43. 1007 (+97
Conventional methods such as those described in 9) can be used. For example, after exposing the parent strain to penicillin G for a while,
The parent cells are made into protoplasts by removing the cell wall with lysozyme in a hypertonic solution, and then the protoplasts of both parents are brought into contact with a trowel in a hypertonic solution containing polyethylene glycol, polyvinyl alcohol, etc. to fuse. After fusion, the cell wall may be regenerated by a conventional method. A completely conventional method can be used to select a strain having resistance to AHV and ΔHCt and a requirement for L-lysine and L-isoroinone from strains whose cell walls have been regenerated.

The method of culturing the obtained mono-threonine-producing bacteria to produce L-threonine differs from the usual method of culturing the mono-threonine-producing bacteria in that it contains nutrients, vitamins, amino acids, etc. as necessary. Ordinary products containing organic micronutrients can be used. As the carbon source, sugars such as glucose, nicococoose, and fructose, starch decomposition products containing these, invert sugar, molasses, fruit juice, etc. can be used.

Nitrogen sources include ammonia gas, ammonia water, ammonium salts, etc., and inorganic ions such as phosphate ions, potassium ions, sodium ions, iron ions, and magnenoium ions are added to the medium as appropriate.

Cultivation is preferably carried out while limiting the pH to a range of 5 to 8, and the most favorable results are obtained if the culture temperature is carried out in a range of 29 tl'' to 37 c.Culture is usually carried out under aerobic conditions for 1 day or more. It will be held for 4 days.

In this way, a significant amount of L-threonine is produced and accumulated in the culture solution. A conventional method may be used to collect L-threone from the culture solution.

Example 1 Brevibacterium lactofermentum AJ +
1786 ('FERM-P et al.') (A HV
resistance, ABC resistance, isoloinone auxotrophy, leuinone auxotrophy) and Brevibacterium lactofermentum AJ11787 (FERM-P gθ10 ) (lysine auxotrophy) with peptone 1y/de, yeast extract 197d
pH 7 with potassium hydroxide, containing sulfur chloride 0.5 f/de and euculose o, 5y/de.
The cells were cultured for 24 hours at 31.5 tr using a complete nutrient medium (hereinafter referred to as "CM medium") adjusted to 2.

31. The obtained bacterial cells were separately inoculated into large test tubes containing CM medium with 100 ml of water. ．． Cultured with shaking at 5 r to reach mid-logarithmic growth phase (I 08 cells/me).
(31] old 1 /I Ome ) was added to the medium. 9
Cultivation was continued for 0 minutes, and bacterial cells were collected from the culture solution, which was then diluted with a hypertonic diluted solution (hereinafter referred to as rHP solution-1, pH 7).
．． 25 M euculose, 0.25 M cono)phosphoric acid 2
Sodium, 100mM 00mM Calsulam Chloride M
The plate was washed with a diluted solution containing 1 potassium phosphate, 100 mM dipotassium phosphate, and 5 mM EDTA, adjusted to pH 7-10.5.

Table 1 Composition of minimal medium Sucrose 2. Oy/de ammonium sulfate 0.5 f/de prefecture
Elementary 0.3
f/deKH,PO40,1f/de MgSO4・7H200,04f/deFeSO4-
7H201, Om9/deMnSO4・4H20+, 0
+I+g/deNaCl
10 rng/diMSG
10 ■/deL-7 Lanin 40 1
ng/de Amino Nicotinic acid 1 5 m97de
Adeni/-101+1g/d Musaiamine Hydrochloride 200/If/l Hyotine I O0711/lpH
7,2 (KOH) The bacterial cells were centrifuged, and the resulting bacterial cells were 5000μy/m
e, The cells were resuspended in a minimal medium (Table 1) containing lysozyme, 0.41 M sucrose and 0.01 M magnesium sulfate, and allowed to stand for 31.5 tl:t. The formation of protoplasts was observed using an optical microscope, and after centrifugal washing of the cells with HP solution, one was diluted with HP solution to 0%.
The cells were inoculated onto a minimal medium containing 5M disodium cophosphate, and further layered with the same medium containing Vco, 8y/ae soft agar. The other is CM medium 1 diluted with phosphate buffer.
This was inoculated and further layered with the same medium containing soft agar. Cultured at 5C. The colonies that appear on the plate are those that grow under hypertonic conditions (vegetative cells and protoplasts) and those that grow under hypotonic conditions (vegetative cells only), and the experimental results are shown in Table 21.
I showed this.

Table 2 2 9,! Xl08
4.2XI084 5000 1.9XIO”
7.2XI082.1XIO829, 4XIO87
, lXl08 4 1000 3.2X10I' 8.1XI
081.0Xlo'21
2.1XI071.8XI06 Lysothyme treatment 2
In 1 hour, peninoline-treated cells became protoplasts □.

The treated bacterial cells were mixed with 33% polyethylene glycol (hereinafter referred to as r).
PEGJ) and fusion was performed at 36tT for 30 minutes. The PEG-treated solution containing the fused protoplasts was centrifuged and then suspended in HP solution. 3 o of this suspension
my/lie lysine, 3o, mg/deia 0in 7.
500011? /meA HV, 5000μv/rag
The cells were inoculated into a hypertonic minimal medium containing AEC and 0.5 M disodium phosphate, overlaid with a minimal medium containing 0.8% soft agar, and cultured at 31.5 t for 10 days. I did it. Colonies that appeared on the hypertonic medium were isolated.

Each isolated colony was examined for threonine production ability,
AJ11788 (FERM-Pl, (
1// ) was separated. Table 41 shows the results of examining the degree of growth of AJ'11788 and the parent strain using L-lysine or inleucine as an auxotroph. 562
This was done by measuring the absorbance at nm. This strain was a recombinant strain that had the phenotype of both parent strains, and its phenotype was completely stable.

Table 4 AJ11787 AJ11788 AJ117860m
1glde0 0 0L-Lys
30rn'j/de O,480,0B
0.09omg/deo.

L-Lys+I-lle each 30 mg/de
O,520,430,080mg/de 0 0
0 L Lys+L-11e +L-Leu each 30m97de O,
420, 450, 5601t? /ral O,54
, 0,460,09*I AHV 5000μm7me O,100
, 410,068, °"'shoulder °°"0゛4" °°゛.

AEC+Thr 5000pfAll O,0
70,370,07*IL Minimal medium containing 30my/me each of L-lysine and L-inuroinone HV
1 or ΔEC was added.

A medium having the following composition was steam sterilized in a 500 ml shaking flask at 110C for 10 minutes at 20 ml.

One loopful of this was inoculated with the strains shown in Table 5, which had been previously grown on CM medium (Slan I).
Shaking culture was carried out on an rt trowel for 72 hours. Each strain of L
- The amount of accumulated threonine is shown in Table 5f.

Medium composition Glucose + o ylde (
NH4),,So4' 4.5 f/d
(!KH,,PO40,+5 f/de MgSO4・7H,00,04f/deFeSO4・
7H201'm'j/, deMnSO4j 4H201
mg/de Thiamine hydrochloride +000 Itg/l
Biotia 100 1if/1
Soybean protein hydrochloric acid hydrolyzate 1.0 me
/de antifoaming agent 20 ■/de calcium carbonate (separate sterilization) 5 ri/depH7,
2 (neutralized with KOH) Table 5 Example 2 Brevibacterium flavum AJ3419 (FERM
- PI70B) (8 EC"+Ile') and Brevibacterium flavum BL-4 (Weitechsen-B.
]] (A'HVr, Lys), protoplast formation and fusion were performed in the same manner as in Example 1. Colony A grown on minimal medium with the composition shown in Table 1 containing 30 mg/deL-lysine 1,'.
J I Is I 2 (FERM-P 6 (J-
1≠) was isolated and the properties of this strain were investigated.

AJI+812 and the parent strain were examined for growth using di/or L-inleucine as a required substance, and one AHV
Table 6V shows the results of investigating dust resistance by adding AEC chemicals.
I showed this. The determination of the degree of growth was the same as that described in Example 1t. This strain was a recombinant strain that had the phenotype of both parent strains, and its phenotype was completely stable.

Table 6 01ny/de 0 0 00mg/
de OO0 0mg1de O(l 001n/me
'0.45 0.42 0.39*1 *I 30m each of L-Rigi/ and L-Inroinone
Minimal medium containing g/de: A HV or ABC
was added.

Add 20 ml of culture medium with the following composition, shake 500 ml, and add 7 pots. Steam sterilize with IOr for 10 minutes.
Table 7 grown in CM medium (slant) in advance
A loopful of each of the following 9 strains was inoculated and cultured with shaking at 3 + and 5 r for 72 hours. The results are shown in Table 7.

Medium composition Lucose +o y/rte
(NH< )2 SO< 4.5
f/diKH2P0.
0.15t/deMgS04-7H200,04f
/deFeSO,・7H,01mg/de MnSO4'4H20+ mg/dethiamine・
Hydrochloride 1000 1t? /1 biochi:/
+00 lif/Soybean Protein Hydrochloric Acid Hydrolyzed Solution 1.0 me/de Antifoaming Agent
20 mg/rye carbonic acid power runium (separate sterilization)
5y/1tepH7,2 (neutralized with KOH) Table 7 AJ 3419 0A
, l I 1 8 1 2 1
7.6B L -49,8 Patent Applicant Ajinomoto Co., Ltd. Procedural Amendment 8 (Method) % Formula % 1, Display of the case 1982 Special W [Application No. 36901 2, Name of Mitsuma 1, - Thread A Ninno 1 production fii'i using it 1--
Threonine production method and A breeding method 3. Person making the amendment 1'1 Relationship Patent applicant 41 5-8-6 Kyobashi-chome, Chuo-ku, Tokyo Contents of the amendment Name of the invention in the book 11ll [ L-threonine-producing strain (method for producing 1-threonine using the same and breeding method thereof); Correction zuru.

Claims (1)

[Claims] 1. α-amino-β-hi I' of the genus Brevibacterium
A 1-threoni/producer strain having resistance to R-xyvaleric acid and S-(2-aminoethyl)-Schiff, and auxotrophy for L-isoleucine and L-lysine. 2 Brevibacterium species that are resistant to α-amino-β-hydroxyvaleric acid and 5-(2-aminoethyl)-cy,tin, and have L-inloidino and di-lysine requirements [-threonine production 1. A method for producing 1-threonine, which comprises culturing a bacterial strain and collecting 1-threonine produced and accumulated in the culture solution. 3. Has a sulfur-requiring property of the genus Brevibacterium,
If the following parent strain does not have the properties of α-amino-β-hydroxy-valeric acid resistance and -isoleucine requirement, or both, it has the properties that the parent strain does not have, and 5- (2-aminoethyl)-cysteine-resistant parent strain a, and the parent strain Suga, which has Brevibacterium resistance to 5-(2-aminoethyl)-cysteine, α-amino-B. - If the parent strain a does not have either or both of hydroquine valeric acid and L-isoleucine requirements, the parent strain a has the characteristics that the parent strain does not have, and the parent strain does not have the L-1+ ren requirement. L, L-170 and L which are resistant to α-amino-β-hydroxyvaleric acid and 5-(2-aminoethyl)-instein/
-Has lysine requirement