JPS61239896A - Production of l-tryptophan - Google Patents

Abstract

PURPOSE:To produce L-tryptophan in high yield efficiently, by cultivating a specific bacterium belonging to the genus Escherichia coli in a medium containing indoleacrylic acid. CONSTITUTION:A bacterium belonging to the genus Escherichia coli which is transformed with a plasmid containing a DNA fragment having a gene to conduct biosynthesis of tryptophan synthetase and a DNA fragment having a gene to rule a multiplication control distribution system derived from F factor plasmid is inoculated into a medium containing indoleacrylic acid having 25mug/ml final addition amount concentration or its salt and 10-600 times as much M of glucose as it by molar amount and cultivated at 5-9pH at 20-50 deg.C for 24-120hr to form tryptophan in the medium. In is treated with an ion exchange resin, separated and recovered.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-tryptophan, and more particularly, to a method for producing tryptophan synthase.
L-trypto7ane is produced using a microorganism belonging to the genus Escherichia that has been transformed with a plasmid containing a DNA fragment containing a gene that controls synthesis and a DNA fragment containing a gene that controls the growth control distribution system derived from the F factor plasmid. It relates to a method for producing efficiently with high yield.

Conventionally, L-t') butophane-producing microorganisms were used to produce L-
)! In the fermentation production of J-buto-7-an, a method is known in which anthinyl acid or indole is added to the culture medium to increase the productivity of L-tryp-7-an (Japanese Patent Publication No. 12384/1984). reference). Furthermore, Z-)!
A method using anthranilic acid-resistant microorganisms has also been proposed as an improved method to improve the productivity of J.
Japanese Patent Publication No. 53-33152 and Japanese Patent Publication No. 59-130
However, it has been pointed out that the production of L-tryptophan by conventional fermentation methods has a limited productivity and is problematic in practice.

In addition, a method has been proposed to improve the productivity of L-1) butophane using genetic recombination technology (Japanese Patent Application Laid-open No.
9-140891), but as is generally accepted, plasmids created by genetic recombination technology are genetically unstable, and the plasmids gradually fall off from the transformed microorganism during cultivation of the microorganism. This happens often. In particular, Z-) as a host microorganism! In the case of a microorganism deficient in the tritfan repressor gene suitable for the production of Jbut7an or when using a medium supplemented with indole acrylic acid, the recombinant plasmid shedding phenomenon is severe, and the stability of the recombinant plasmid within the host microorganism may be affected. Unless a method for recombination is established, L-t! There are problems with the production method of J Gutfan for practical use (T, IMANAKA
,7. General Mtcrobtol, 11
8.255-261 (1980)]. Although it has been proposed to add drugs such as antibiotics to the culture medium as a method for stabilizing plasmids, it cannot be said to be an effective method from an economical standpoint.

As a result of intensive research into a method for producing L-tryptophan that does not have the drawbacks described above, the present inventors have discovered that D.
When culturing a microorganism belonging to the genus Escherichia transformed with a plasmid containing an NA fragment and a DNA fragment containing a gene controlling the growth control distribution system derived from the F factor plasmid, in a medium containing indoleacrylic acid or its salts, The present inventors have discovered that L-)ligtophan can be efficiently produced in high yields with almost no plasmid shedding phenomenon, and have completed the present invention.

Therefore, according to the present invention, a DNA fragment containing a gene governing the biosynthesis of tryptophan synthase and a DNA fragment containing a gene governing the growth control distribution system derived from the F factor plasmid are combined.
A microorganism belonging to the genus Escherichia transformed with a plasmid having a fragment of
Provided is a method for producing L-tryptophan, which comprises recovering ammonium.

The "DNA fragment containing the gene governing the biosynthesis of tryptophan synthase" (hereinafter sometimes abbreviated as "T fragment"), which constitutes the plasmid used for the transformation of microorganisms in the present invention, refers to indole and L -or DL
- from serine to L-) refers to a DNA fragment containing the gene responsible for the biosynthesis of ligtophan; therefore, the T fragment includes the tryptophan operon, a large NA fragment containing the tryptophan operon, or tryptophan j
A DNA fragment in which a promoter and operator are linked to Trp A and Trp B, which are the genes responsible for the biosynthesis of tryptophan synthase in the operon, or T
rp A and Trp B, Trp C, Trp D and T
A DNA fragment that combines at least one type of rpE with a promoter and an operator (e.g., TrpA, Trp
B, a DNA fragment in which a promoter and an operator are linked to Trp C and Trp E), etc. These T fragments include those derived from prokaryotic microorganisms, 7age, etc., and those derived from Escherichia coli are preferably used. Among them, the tryptophan operon itself and the gene of phage φ8opt are digested with the restriction enzyme BamH.
A DNA fragment containing the tryptophan operon with a molecular weight of approximately 22.6 kb obtained by cleavage with I is advantageously used.

As described above, the plasmid used in the present invention is characterized in that the T fragment described above is combined with a DNA fragment containing a gene controlling the growth control distribution system derived from the F factor plasmid. The F factor plasmid has a molecular weight of 94.5 kb (62 x 10' dol
ton) (D) A known plasmid that normally exists in intestinal bacteria such as reeds and Escherichia coli with a copy number of 91 to 2 copies per cell chromosome. (This mechanism for keeping the copy number at a low level and increasing it at exactly the same pace as the host's proliferation is called 5trin (Hnt-like proliferation control).) F
Such a stringent in the factor plasmid
It has already been determined that the control function for growth is carried out by a fragment called mini-F, which has a molecular weight of 9.1 kb and can reproduce autonomously. It is also known that it can be cut out by

However, the "DNA fragment containing the gene controlling the proliferation control distribution system derived from the F factor plasmid" (hereinafter abbreviated as F fragment) is a mechanism for accurately transmitting the F factor plasmid to daughter cells. It refers to a gene fragment with a molecular weight of about 9jkb, and a typical example of a more F fragment is the mltt-F fragment having a molecular weight of about 9jkb.

In addition to the T fragment and F fragment described above, the plasmid used in the present invention contains other gene fragments, such as gene fragments that control drug resistance in various bacteria, such as kanamycin resistance, such as genes that control kanamycin resistance. It may also contain fragments and the like.

Typical examples of such plasmids include the tryptophan operon fragment and m1nt-F derived from the F factor plasmid.
It contains a fragment, has a molecular weight of about 31.7 kb, and exhibits the following sensitivity to the following restriction enzymes, namely, (α) has five recognition sites for Ec, o R1, and (6) Bam. Plasmid pMTY-1 is characterized in that it has two recognition sites for BI (sb) and three recognition sites for (c) H4sd (2).

This plasmid pMTY-1 is about 31.7 mm as described above.
It has a molecular weight of &&, which was determined by agarose gel electrophoresis.

In addition, the sensitivity of plasmid pMTY-1 to various restriction enzymes is shown in (α) to (C) above. It is.

1 (α) ECa R1: 9.1.7.
0, &4.6.1, 51(6) Bam HI:
22.6.9.1(C)1isdm: 1aO11(L
8.2.9 Plasmid pM with characteristics as described above
TY-1 can be manufactured, for example, as follows.

First, the preparation of the tributophane operon fragment is carried out by e.g.
Escherichia coli that has a triglophane operon in its chromosomal gene, for example, EachariahtαcoHK-12 (I
FO55Q1, ATCC10798, ATCCe255
62), etc., for example, phage φ80 (A
TCC11456α-B1) etc. were infected, and lysogenization and induction phenomena were used to prepare a large amount of phage that incorporated the tryptophan operon into the phage DNA [R
, M., Dasnay, C., YaxofskyH7, B.
aetertol, 118.505 (1974)], then the conventional method (A?, F, Frltsch.

Sambrook, “Mo1ecular alon.
t%g1 (1982) 7.164-165, ColdS
This can be carried out by extracting the phage DNA according to the method described in the p-in Arbor Laboratory] and excising the DNA fragment containing the trypto-7 operon using restriction enzymes such as Bα normal H1, Eco R1, etc.

On the other hand, the preparation of the m1nt F fragment can be carried out using microorganisms harboring the F factor plasmid, such as Escherichia coli (E, ea
rs) f-12 strain (ATCC13153, ATCCg
23589, ATCC g23590) etc. in a manner known per se.

Gwttrry, D.L., LeBLanc, S.
.

Falkow HJ, Eact, 116.1064 (
It can be prepared by extracting the F factor plasmid by the method described in the literature such as (1973) and cutting out the mtystF fragment having a molecular weight of about 91 kb using the restriction enzyme EcoRI.

The mini F fragment thus prepared was treated with the same restriction enzyme used to excise the DNA fragment containing the tryptophan operon, combined with the gene fraction containing the tryptophan operon prepared above, and treated with ligase. , for example, by binding with T47age-derived glue gauze, etc., the desired plasmid pM
TY-1 is obtained.

A specific method for preparing plasmid pMTY-1 is described in detail in Example 1 of JP-A-60-2189.

The plasmid pMTY-1 prepared in this way is
Although the number of copies is small, it has the excellent property that it is accurately passed on to daughter cells during host cell division and is rarely shed.

According to the present invention, a microorganism belonging to the genus Escherichia is transformed with the plasmid thus produced. As the microorganism (host) belonging to the genus Escherichia to be transformed, for example, Escherichia coli (Esaht
richa con) K-12 series microorganisms,
Among these, preferred are strains that do not have L-tributophane degrading activity (ie, tryptophanase-deficient mutant strains).

The above-mentioned plasmid can be introduced into the host microorganism using methods known per se, such as Ji, Maysdel.

A, Htga HJ, Mo1. Biol, 53.159
(1970) and the like.

The transformed microorganism is usually cultured aerobically in a medium containing indole-acrylic acid or its salt. Cultivation can be carried out by a method known per se; for example, a natural or synthetic medium containing carbon sources, nitrogen sources, inorganic salts, etc. similar to those used for the cultivation of ordinary microorganisms can be used. can.

As a carbon source, various carbohydrates such as glucose, glycerol, fructose, sucrose, and molasses can be used, and as a nitrogen source, for example, tryptone, yeast extract, corn staple liquor, and casein hydrate can be used. Natural organic nitrogen sources such as decomposition products can be used. Many natural organic nitrogen sources can be used as carbon sources as well as nitrogen sources. Examples of inorganic salts that can be used include potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, calcium chloride, manganese chloride, and the like. Furthermore, other nutrients necessary for the growth of microorganisms can be added to the medium as necessary.

According to the method of the present invention, indoleacrylic acid or a salt thereof is added to a medium containing the carbon source, nitrogen source, inorganic salt, etc. as described above. Indole acrylic acid or its salt may be added at the beginning of the culture, or may be added during the culture. If added midway through, it is desirable to add at the latest the end of the logarithmic growth phase of the microorganism. The addition may be made at once, or may be made in multiple portions or continuously.

The amount of indole acrylic acid or its salt added is not strictly limited, but in general, it can be added to the medium to a final concentration of at least 25 μm17d, preferably 80 to 400 μy/-. It is advantageous to add to a final concentration in the range, more preferably in the range 100 to 200 Ag/-.

In addition, as a salt of indole acrylic acid, for example, indole ac 1 such as sodium indole acrylate
Included are alkali metal salts of noric acids.

Here, the "final concentration" refers to the total concentration of indoleacrylic acid or its salt added to the medium by the end of culture.

Furthermore, indole or anthranilic acid or a salt thereof (the same salts as those mentioned above for the salt of indole acrylic acid can be used as the salt) can be appropriately added to the medium as a precursor of L-tributophane. The addition of indole or anthranilic acid or its salts is
As with indole acrylic acid or its salt, it may be carried out at the beginning of the culture, or during the culture. If it is carried out during the culture, it is desirable to add it at the latest at the end of the logarithmic growth phase of the microorganism, and the addition may be done at once, divided into multiple times, or continuously.

Although the amount of indole or anthranilic acid or its salt added is not strictly limited, for example, when using glucose as a carbon source, the amount of indole or anthranilic acid or its salt added is 0.
It is advantageous to add in an amount ranging from 0.5 to 1 mole, preferably from 0.1 to 0.5 mole.

Furthermore, in the method of the present invention, it is desirable to use glucose as at least a part of the carbon source, and the amount used is generally about
10 to 600 times the mole, preferably 50 to 400 times the mole,
More preferably, a range of 100 to 300 times the mole is appropriate.

The culture time varies depending on the type of microorganism, other culture conditions, etc., but when culturing is carried out by adding indole acrylic acid or its salt at the beginning of culture, it is usually about 24 to about 120 hours.
In addition, when indole acrylic acid or a salt thereof is added during the culture, the culture time can be generally about 1 to about 48 hours.

Furthermore, the culture temperature can generally be in the range of about 20 to about 50°C, and the pH of the culture medium is generally adjusted to the range of 5 to 9, preferably in the range of about 6 to about 8.

Furthermore, the culture is preferably carried out under aerobic conditions such as shaking or aeration.

Recovery of L-1) butophane from the culture obtained by culturing under the above-mentioned culture conditions can be carried out by methods known per se. For example, using ion exchange resin, activated carbon, etc. The adsorption-desorption process can be carried out by using the adsorption-desorption process.

According to the method of the present invention, a DNA fragment containing a gene governing the biosynthesis of tributophane synthase and F factor 7''
DN containing the gene governing the growth control distribution system derived from the 2 smid
A plasmid having the A fragment, for example plasmid pMT
When L-tryptophan is produced by culturing a microorganism belonging to the genus Escherichia transformed with Y-1, the production rate of tryptophan can be significantly increased, which is extremely advantageous for the Formula 1 industry.

Hereinafter, the method of the present invention will be explained in more detail with reference to Examples.

Transformation (a) Preparation of host bacteria Escherichia coli (E, cog) strain f-12 (rp
A tryptophan auxotrophic strain was prepared from Os5o1) by treating NTG with trosoguanidine (E, A, Adtl-berg et al.,
Bzeochetn, Eiophys.

Rea, Comm, 18.788 (1965)
reference〕.

That is, the above E. coli K-12 strain was cultured in L medium until the mid-logarithmic growth phase, the bacteria were collected by centrifugation, and the strain was suspended in Tris-maleic acid buffer (pH 6,0). 100μI/- to 200μg/ml NTG (N
-Methyl-N'-nitro N-nitrosoguanidine) and treat for 15-30 minutes. After washing the treated product with the same buffer, L medium (Bαeta tryptone 101
1 yeast extract 5IS NaC15JF, glucose II
I, water (11; pH 7, 2) and cultured with shaking at 37°C, and the culture was used in a conventional manner to isolate a tryptophan auxotrophic strain using a combination of penicillin concentration method and replica method.

(g) Transformation The E. coli strain obtained in (a) above was inoculated with the 112 strain (tryptophan auxotrophic mutant strain) into 10 tnt of the same L medium as above, and cultured with shaking at 37°C. After growing to the late stage, the bacteria were collected by centrifugation, and then cooled on ice at 0°C with 0.1M MQCI.

and O-1Mcacl, to prepare DNA receptive bacteria (competent bacteria). Plasmid pMTY-1 obtained according to the method described in Example 1 of JP-A-60-2189 was added to this, reacted for 40 minutes under water cooling at 0°C, and then heated at 40°C for 3 minutes. After that, the mixture was reacted again for 2 hours under water cooling at 0°C.

Next, the treated bacterial cells were inoculated into L medium, and after shaking culture at 37°C for 1 hour and 30 minutes, the bacteria were collected by centrifugation, and after washing the bacterial cells, HPO4711, HPO4711,
KE, Po, 29, MQSO, 7H.

OcLlg, (NH,), So, 111, fh fucose 11 pure 17) was smeared on a plate medium prepared and incubated at 37°C.
After culturing the grown colonies on the same medium and purifying them again on the same medium, a transformed strain (loss of Trp auxotrophy, i.e., capable of biosynthesis using the tryptophan operon on the plasmid) was grown on the minimal medium. A viable strain) was obtained. This transformed strain was transformed into Escherichia coli (EscherichtaCol<3 with 12I'f2
As of July 6, 1981, it was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Higashi 1-1-3, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture, as 001, with the accession number: FEBN No. 7139 (FEBN).
P-7139).

Example 1 LL medium (tryptone 1011 yeast extract sy.

NαC131, glucose IF! , water 11. pH7.2
Dispense HC1d into a Daisei test tube (200 x 24φ) and add 1
After sterilization at 20℃ for 15 minutes,
Cori r-121'f2001 (FEBN P-715
9) was inoculated and cultured with shaking at 37°C for 1 g, which was used as a preculture. 2 m of the preculture thus obtained was treated with 100 ml of indole acrylic acid and 0.5 η of indole.
A culture medium having the following composition (100 μl dispensed into a 500 d Erlenmeyer flask) was inoculated and cultured with shaking at 37° C. for 48 hours.

After the cultivation was completed, the bacterial cells were removed by centrifugation, and L-1-1j gutophane in the supernatant was quantified by high-performance liquid chromatography (Shimadzu Lc-sA).
The formation of a cape/no was observed.

Composition of medium A ME, C141i KB, Po, 5gNα
t HP Oa・12H, 01℃Mxgso,・7H,
On, 5g CaC1,-2B, 0 15Wli
Casamino acids 5g yeast extract 2g glucose
30.9CaCO, (separate sterilization) 2ON water
117) H7,0 Example 2 Esieribi7-:y17f-12)'f2001(FE
RN P-7139) was precultured in the same manner as in Example 1. 2 d of the preculture thus obtained was mixed with 100 al/wl of indoleacrylic acid and 11 ml of anthranilic acid.
Medium A containing q/- (100 g
d) and cultured with shaking at 37°C for 48 hours.

After completion of the culture, L-tributophane in the supernatant obtained by centrifugation was quantitated, and the production of A111P/Rei was observed.

Claims (1)

[Scope of Claims] 1. A genus Escherichia transformed with a plasmid containing a DNA fragment containing a gene governing the biosynthesis of tryptophan synthase and a DNA fragment containing a gene governing the growth control distribution system derived from the F factor plasmid. 1. A method for producing soot L-tryptophan, which comprises culturing a microorganism belonging to the above group in a medium to which indole acrylic acid or a salt thereof is added, and recovering L-tryptophan from the culture. 2. The method according to claim 1, characterized in that indole or anthranilic acid or a salt thereof is further added to the medium. 3. Add at least 25 μg of indole acrylic acid or its salt to the culture medium by the end of the logarithmic growth phase of the microorganism at the latest.
2. A method according to claim 1, wherein the final concentration is 0.1/ml. 4. Claims 1 to 3, wherein the gene governing the biosynthesis of tryptophan synthase is a tryptophan operon.
The method described in any of the paragraphs. 5. The method according to any one of claims 1 to 4, wherein the DNA fragment containing the gene governing the biosynthesis of tryptophan synthase is derived from Escherichia coli. 6. The method according to any one of claims 1 to 5, wherein the DNA fragment containing the gene governing the growth control distribution system derived from the F factor plasmid is a mini-F fragment. 7. DN in which the plasmid contains the tryptophan operon
It contains the A fragment and the mini-F fragment derived from the F factor plasmid, has a molecular weight of approximately 31.7 kb, and contains the regulatory enzyme E.
7. The method according to any one of claims 1 to 6, wherein the plasmid pMTY-1 has recognition sites for _c_oRI, BamHI, and HindIII at 5, 2, and 3 sites, respectively. 8. Microorganisms belonging to the genus Escherichia are plasmid pMT
8. The method according to claim 7, which is an Escherichia coli K-12 microorganism transformed with Y-1. 9. Claim 1, wherein the microorganism belonging to the genus Escherichia is a strain that does not have L-tryptophan degrading activity.
The method according to any one of items 1 to 8.