JPS6196990A - Production of l-triphtophane - Google Patents

Abstract

PURPOSE:A recombinant plasmid which has a promoter sequence so that another structural gene may not come into the uppersteam of a specific gene is introduced into a microorganism in Bacilus and the microorganism is cultured to produce L-tripatophane. CONSTITUTION:A plasmid which has a promotor sequence where no other structural gene is included in the trpB gene or in its upper stream is introduced into a microorganism in Bacillus and the microorganaism is cultured to produce L-triptophane. In another way, the cell bodies or their treated product of a microorganism in Bacillus into which the recombinant plasmide has been introduced is brought into contact with anthranylic acid or indole in an aqueous medium to produce L-triptophane.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application This invention relates to a method for producing L-tryptophan, and more specifically, to a method for producing L-tryptophan using a microorganism belonging to the genus Bacillus having a recombinant plasmid into which the trpB gene has been inserted.
Concerning a method for producing tryptophan.

Conventional technology When trying to produce L-tryptophan using carbohydrates such as glucose as a raw material as a method for producing L-)+7gutoquan using microorganisms, most wild strains produce L-)IJgutophan outside the bacterial body. Since the L-tritophane does not produce L-tritophane, a method has been adopted in which the wild strain is artificially mutated to give it the ability to produce L-tritophane.

As a conventionally known artificial mutant strain having the ability to produce L-trithophan, there is a mutant strain of the genus Bacillus that is resistant to 5-fluorotryptophan (Japanese Patent Application Laid-open No. 85289-1989).
), mutant strains of the genus Brevibacterium that are resistant to 5-methyltryptophan and m-fluorotrigdoane (Japanese Patent Application Laid-Open No. 50-42091) are known.

On the other hand, several attempts have recently been reported to utilize recombinant DNA technology, which is different from breeding by artificial mutation as described above, for breeding L-)lyptophan-producing bacteria. For example, Appl, Environ, Microbiol
.

38, (2), 181-190. (1979) described that a particular mutant strain of Escherichia coli containing a plasmid carrying the Escherichia coli trpE472 gene produced approximately 1.3 g/l of tricytophane. Also, [Japan Society of Fermentation Engineering, Showa 5]
Abstracts of the 5th Annual Conference, p. 170 (1980) J 9 A mutant strain of Escherichia coli containing a plasmid incorporating the Escherichia coli tritphan operon produced 360 rrug/l of L-tryptophan. is listed.

Further, JP-A-57-208994 describes the production of L-tryptophan using Bacillus microorganisms grown by recombinant DNA technology.

On the other hand, many methods for producing tryptophan using indole or anthranilic acid as raw materials are also known. (For example, JP 43-20711, JP 46-16955, JP 47-46348, JP 47-29584, JP 48-80785, JP 48-87085, JP 50-95484, etc.).

In addition, 9L-
A method for producing 1-lysotophan is also known (Japanese Patent Application Laid-Open No. 58-89194).

Problems to be Solved by the Invention The present invention provides a more efficient L
-) The purpose is to discover a method for producing lysotophan.

Means for Solving the Problems The present inventors have introduced a recombinant plasmid having a trpB gene and a promoter sequence arranged upstream of the trpB gene so that no other structural genes are interposed. It has been found that microorganisms of the genus Bacillus produced L-)1.1 butophane with high efficiency.

The trpB gene is tryptophan synthase B (L
-5erine hydrolyage (EC4,2
, 1, 20)+hereinafter referred to as r TS-B J). The above enzyme is an extremely important enzyme that catalyzes the final reaction of the L-tryptophan biosynthesis system that produces L-serine and indole (9L-) liptophan.

The Grasmid vector used in the present invention may be any vector as long as it replicates using a microorganism belonging to the genus Bacillus as a host.

For example, pT127 derived from Staphylococcus microorganisms
．． pc194. pc221. pC223, pUB
112 (Proc.

Natl, Acad, Sci, USA, 74:
1680-1682 (1977)), pUB
llo (J, Bactarol, 134:318-
329 (1978)), pTP4. pTP5
(Mlcroblol Letters, 5:55-
59 (1978)), pLs15. derived from Bacillus subtilis.
pLs28 (J, Bacteriol, 1
3, 699-701 (1977) + pLs13
(J, Bactsriol, +129:1487-
1494 (1977)), pPL.

pPL2 (J, Bacteriol, 12
4, 484 (1975)).

Examples of promoter sequences known to function in microorganisms belonging to the genus Bacillus include those shown in Table 1.

1st Table 1 35 areas - 10 areas sealed (1)...TTGACA ・
・・TATAAT・・・Common array Veg' gone(2) =4TGAC,A-=4A
CAAT-penicillinase (3)...TTGC
AT... ...AATACT...(
B, 11chenlformis) α-amylase (
4) ...TTGTTAT...
...TAAAAT...CB, amylo -o
r =・TTATTA --TATAAT -1iqu
ifacien8) tms (5) ・” TTGAAA --TA
TATT -Erm (6) -TTCATA
--TdTAAT -Cam (7) -TTG
ATT --TCTAAT-5poVC(8)
...GGAGGTTTAAA--GTATTGTTT
-s po VG (9) -CGAGGATT
T--AATTGATA-(1) Ann, Rev
, Gen5t-, 13, 319 (1979) (2) M
o1. Gen, Genet, 186, 339 (198
2)(3) Gene, 15, 343 (1982)
(4) Gone + Dan, 43 (1981) (5) C
e1l, 25, 582 (1981) (6) J,
Bacteriol, 150.804 (1982)
(7) J. Bacteriol, 150,815
(1982) (8) Nucleic Ac1ds R
es, +dan, 5979 (1981) (9) Ce1l
, 25, 783 (1981) Structural genes other than the trpB gene include those whose molecular weight exceeds about 20,000, regardless of the type of protein encoded.

Between the trp B gene and the promoter sequence, there is no need for the other structural genes mentioned above, and of course, a control gene such as an operator sequence may be present.

As a method for constructing a recombinant grasmid having a promoter sequence arranged so as not to be interposed by a synthetic gene, conventional methods such as cleavage with a restriction endonuclease and ligation using a ligase can be used.

The microorganism of the genus Bacillus that is the recipient of the above recombinant plasmid may be a trpB gene-deficient strain, as long as it can accept the above recombinant plasmid. It is desirable that the production capacity is high.

To introduce a recombinant plasmid into NA recipient bacteria as described above, see e.g. J, Bactariol, +81
+741 (1961), Mol@c, Gen,
Conventional transformation methods can be used, such as those described in G@net, 168, 111 (1979).

Methods for producing L-tryptophan using the obtained L-)ligdoane-producing bacteria can be roughly divided into the following methods.

However, the purpose of explaining these separately is to improve understanding of the invention.
This is for the sake of simplicity; strictly speaking, it may not be clear which of the following cases can be classified.

The first method is to produce L-)ligdoane using carbohydrates as raw materials. That is, the medium is a conventional medium containing carbohydrates as a carbon source, a nitrogen source, inorganic ions, and, if necessary, organic micronutrients such as amino acids and vitamins. As the carbohydrate, glucose, sucrose, fructose, lactose, etc., and starch hydrolyzate containing these, whey, molasses, etc. are used. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, etc. can be used.

The culture is carried out under aerobic conditions, with appropriate adjustment of the medium and temperature, and substantially L-)! This is usually carried out for 2 to 5 days until production and accumulation of J-butophane is stopped. In this way, a significant amount of L-)ligdoane is produced and accumulated in the culture medium.

The second method is to add indole or anthranilic acid to the medium in the first method or to the medium during cultivation. This method is similar to the first method in that L-tryptophan is produced under conditions that allow L-tryptophan-producing bacteria to proliferate, but anthranilic acid or indole is also used as a raw material for L-tryptophan. This method differs from the first method in that it is used.

Anthranilic acid or indole may be added to the culture medium at the beginning of the culture. During the culture, especially after the L-tryptophan-producing bacteria have grown sufficiently, so as not to inhibit the growth of the L-tryptophan-producing bacteria. You may do so. The amount of anthranilic acid or indole added to the medium is L-)! It is desirable that the concentration in the medium does not exceed the concentration that inhibits the growth of butophane-producing bacteria, and for this purpose, small amounts of anthranilic acid or indole may be added to the medium continuously or in several portions. good. Furthermore, better results may be obtained by adding one or more of the below-mentioned glucose donor and alanine side chain donor to the medium.

L-)! The culturing of the Jbut7an-producing bacterium is carried out by a method that is substantially the same as the first method except for the points mentioned above.

The third method and the second method are that anthranilic acid and indole are used as raw materials for L-tryptophan.
Both methods are very similar, but differ from the first method in that L-tricytophan is produced under conditions that do not require the growth of L-tryptophan-producing bacteria or that coryneform bacteria do not grow. There is. That is, the third method is L-
This method differs from the first and second methods in that the growth of tryptophan-producing bacteria and the production of L-tricytophan are carried out substantially separately.

As a medium for growing L-tryptophan-producing bacteria, a conventional medium containing the carbon source, nitrogen source, inorganic ions, and, if necessary, organic micronutrients such as amino acids and vitamins can be used. Adding a small amount of anthranilic acid or indole to the medium often yields bacterial cells with high pL-) liptophan production activity. The culturing method is preferably carried out under the aerobic conditions described above.

After culturing, L-1j butophane-producing bacterial cells are once isolated. The culture solution may be used as the bacterial cells, but cells separated from the culture solution by filtration or centrifugation, etc., or separated bacterial cells washed with water, acetone, a surfactant, etc. can also be used. . Furthermore, it is also possible to use the above-mentioned bacterial cells that have been immobilized, crushed or ground, or from which protein fractions have been appropriately separated, and further, processed bacterial cells such as isolated TS-B protein. .

These bacterial cells or treated bacterial cells are added together with anthranilic acid or indole to an aqueous medium for producing L-1'lyptophan. The amount of bacterial cells or treated bacterial cells to be used is preferably 0.5 to 59/dll (in terms of dry matter) in terms of the amount of bacterial cells. When using the culture solution as it is as the bacterial cells, a culture solution or a culture solution diluted with water etc. may be used, and when using once isolated bacterial cells or a processed product of the bacterial cells, a buffer such as phosphate buffer may be used. It may also be simply water.

More favorable results can often be obtained by adding sodium sulfite, ethylenediaminetetraacetic acid, pyridoxal phosphoric acid, an organic solvent such as acetone or ethanol, a surfactant, etc. to the suspension. Furthermore, when inosine is added to the aqueous medium, a poorly soluble tryptophan-inosine complex is formed and tryptophan is removed from the reaction solution, so that the reaction favors the production of L-tryptophan.

The preferred concentration of anthranilic acid or indole in the aqueous medium is 0.1 to 597 dll.

When indole is o, i ~ 4.5 VdJJ.

When anthranilic acid is used as a raw material, a licose donor selected from redace, 5-phosphoridis, 1-phosphoridis or 5-phospholizo-spirophosphoric acid is added to the aqueous medium. The amount added is
All have good O11-51/dl, and when anthranilic acid is added as a substrate at a high concentration, it is desirable to increase the concentration of these additives accordingly.

When using indole as a raw material, an alanine side chain donor or pyruvate and ammonium ions are added to the aqueous medium. The alanine side chain donor is represented by the following general formula.

X-CH2-CH(■2)C02H (X is a hydroxyl group, 710rn, alkylmercapto group, mercapto group, alkoxy group, benzyloxy group, or thiobenzyl group.) Addition of alanine side chain donor and pyruvic acid The amount is 0.1M to IM each. In the case of anthranilic acid, the L-tryptophan production reaction is preferably performed by shaking an aqueous medium at 25 to 45°C for 10 to 48 hours. In the case of indole, it is preferable to leave the aqueous medium at 25 to 45° C. for 5 to 48 hours or to react with gentle shaking.

L-) produced in the culture solution or aqueous medium obtained by the first, second or third method. J-butophane can be separated and collected using conventional methods.

Example j - Method for constructing a plasmid having the trpB structural gene and promoter upstream thereof, the trpB structural gene is Bacillus subtilis AJ 1
1712 as the recipient strain and the chromosome of AJ11711 as the donor DNA.
pAT5 was obtained by cloning into 10 EcoRI sites (Fig. 1). A fragment containing trpB (1600b
p). In Bacillus subtilis, trp
The B structural gene is located in the second half of the tryptophan operon.Bloehem, Biophys, Rea,
Commun, +35+838 (1969),
It does not include the promoter immediately before it.

A DNA fragment having promoter activity was inserted upstream of the trp B gene obtained in this way to obtain pAT2. Fragment B having promoter activity (2400 bp
), the part with the promoter sequence is C (approximately 400
bp) and the base sequence of this portion is shown in FIG.

Is it a pig? Example of TS-B activity of novel tryptophan-producing bacteria AJ12174 was transformed using the obtained cilasmids pAT5 and pAT2, and AJ12175 (F
ERM-P 7'? a2) and AJ12176 (F
EaM-p7? θ3) was obtained. These strains were cultured in an L-tryptophan production medium, and TS-B activity was measured. The results are shown in Table 2.

The medium was tryptophan production medium Ck:+-,z89/dl, NH4C in a 500fI11 flask.
l1f! /di, KCl0.2VU, KH2PO4
0.1%, MgSO4.7H20o, o4. F/#
, casamino acid 0.4VdA', FeSO4・41(
201m97'dl, MgSO4・4%0 1mfU
, L-Aru'l'nin+L-1:'Ishin each 20m'
7dl.

and CaCO34Vdl, adjusted to pH 7.0, were dispensed into 20-liter portions, and after inoculating with bacterial cells, they were incubated at 30°C for 24 hours.
Culture was performed for hours.

Furthermore, 5μ of kanamycin and Al were added to the medium of AJ12175 and AJ12176. The TS-B activity measurement method is S, O, ) (och et al. (Biochem, B
iophys, Rag, Common, +35.8
38 (1969)).

Table 2 Bacterial strain TS-B activity relative value AJ12174
1.5 1AJ12175 2.
2 1.4AJ 12176 15.6
10.4 (*nmole/min/mgprote
in) Butophane production (1) The strain obtained in Example 2 was cultured at 30°C for 96 hours in the same L-tryptophan production medium as in Example 2. L-)
The amount of lysotophan produced was measured.

At the same time, culturing anthinyl acid or indole48
Feed little by little between hours and 96 hours until the total amount is 0.
．． Feed 5%. The results are shown in Table 3 Table 3: Acid Indole Additive-free strain Added Added AJ12174 120 130 1
10.

AJ12175 180 210 1
50AJ 12176 490 850
45'0 (2) Each strain shown in Table 3 was inoculated with 1 platinum solution into 60 ml of a medium having the following composition and cultured at 31° C. for 20 hours.

Casamino i5 in 11 water, 2g yeast extract, 20g cornstarch liquor, KH2PO40,5I! .

MgSO4-7H200,5g, FeSO4・4H2
0107Q, and MuSO414H2010m9 (pH 7,0). Furthermore, AJ12175 and AJ12176
At that time, 5μ of kanamycin was added to the koji and cultured. Culture solution 1
Centrifuge the cells and collect the bacterial cells.
2gAlt, L-serine 0.2fl/di.

Na 2 SOs 0.1 g/'di, EDTA
O-3gAi, pyridoxal phosphate 0.01g/cM
, and inosine s were suspended in 100 ml of a reaction solution having a composition of 4.9/g (pH 8.5) and reacted at 30° C. for 20 hours. As a result, 0.189 ALvL -) liptophan was produced.

How to obtain AJ12174 AJ12174 consists of 10 g of polybutone, 1 yeast extract
AJ12175, which has been deposited, was planted in a test tube containing 41 nt of a pH 7.0 medium containing 0 g and NaCl2 i in 11 parts of water, and cultured with shaking at 30°C for 12 hours, and then further incubated at 41°C for 12 hours. It can be obtained by culturing with shaking and selecting strains that have lost the kanamycin resistance derived from the plasmid.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram of recombinant Grasmid pAT5 (comparative example) and recombinant plasmid pAT2. A is a DNA region containing trp B, B is a DNA region having promoter activity, and C is a region having a promoter sequence.

Claims (2)

[Claims] (1) It is characterized by culturing a microorganism of the genus Bacillus into which a recombinant plasmid having a trpB gene and a promoter sequence arranged upstream of the trpB gene so that no other structural genes are interposed is introduced. L-
Method for producing tryptophan. (2) Cells of microorganisms belonging to the genus Bacillus into which a recombinant plasmid having the trpB gene and a promoter sequence arranged so that no other structural genes are interposed upstream of the trpB gene are introduced, or a processed product thereof. A method for producing L-tryptophan, which comprises contacting it with anthranilic acid or indole in an aqueous medium.