JPH02255082A - Transformant having tryptophanase productivity and production of l-tryptophan - Google Patents

Abstract

PURPOSE:To obtain the subject transformant of a bacterium belonging to Providencia genus producing an enzyme capable of converting pyruvic acid or L-serine to L-tryptophan in a high efficiency by carrying out transformation using a DNA containing a gene controlling tryptophanase production. CONSTITUTION:A gene controlling tryptophanase production is subjected to self-cloning using P.rettgeri plasmid to form a complex plasmid having the gene controlling tryptophanase production. Using the resultant complex plasmid, a bacterium belonging to Providencia genus is transformed.

Description

Detailed Description of the Invention <Industrial Application Field> The present invention provides a transformant having a recombinant DNA containing a gene governing the production of tritifphanase, and a transformant for producing tritiftophanase using the transformant. The present invention relates to a method for producing L-tryptophan by reaction.

<Prior art> L-)Lyptophan can be produced by direct fermentation using strains of the genus Brevibacterium, Corynebacterium, Bacillus, and Escherichia; ) is known to be produced by enzymatic methods using bacterial strains such as As a method for producing L-tryptophan using an enzymatic method, Providencia rettgeri (formerly Proteus rettgeri, hereafter P,
triftphanase (E
A method using C4,1,99,1) (Reference 1) is known as an excellent method.

<Problem to be solved by the invention> Applying genetic manipulation to produce this tryptophanase is expected to improve the productivity of tryptophanase, and further improve the productivity of L-tryptophan. .

In view of this situation, the present inventors have made ingenuity, and P.
Using the 1 lasmid of E. rettgeri, self-cloning the gene governing tritifphanase production to create a composite plasmid having the gene governing tritifphanase production, and transforming Providencia bacteria with this composite plasmid, Using this transformant, we succeeded in producing tritifphanase and in improving L-tryptophan productivity through the tritiftophanase reaction, thus completing the present invention.

Means for Solving the Problems> That is, the present invention provides a recombinant DN containing a gene governing the production of tryptophanase of P, ret(leri).
A transformant having A, and a culture, bacterial cells, or a treated product of the transformant are allowed to act on pyruvate or L-serine to produce and accumulate L-tryptophan,
The present invention provides a method for producing tri-11-phane, which is characterized by collecting L-tryptophan from the reaction solution.

The present invention will be explained in detail below.

The plasmids used as vectors are p, rettg
Use one derived from eri. For example p, rettge
rATCC25932, ATCC,9919, ATCC
Can it be extracted from 29944?p, rettgerA
Plasmid pYU300 extracted from TCC25932
P. reHgeri A obtained by transforming P. reHgeri ATCC21118 by
TCC21118 (PYU300) (Microtechnical Laboratory No. 84
It is more convenient to extract and use plasmid pYU300 from (No. 75).

As a donor of the gene responsible for triftphanase production,
Bacteria of the genus Propitensia can be used, for example P
, Rettgeri ATCC29944 can be used, but is not limited to this strain. Chromosomal DNA can be extracted according to a conventional method such as that described in Reference 2, for example. In this case, the lytic process is 30-6
Self-cloning of the gene governing 6L-threonine fermentation production can be carried out at 5° C. for 20 to 60 minutes to increase the yield of NA, but is not limited to this method. Shotgun (Sho)
This is done using the tgun) method.

As a host, a tryptophanase-deficient mutant strain of P. rettger i can be used, but E. coli (
The host may be a tryptophanase-deficient mutant strain of E. colt, which is further transformed with P. rettger i.

Fragmentation of chromosomal DNA and cleavage of plasmids for vectorization can be carried out by conventional enzymatic reactions using restriction enzymes such as old ndll. The DNA fragment and the vector can be ligated by a conventional ligation reaction using an enzyme such as T4 DNA ligase, and the host can be made competent and transformed by a conventional ligation reaction as described in Reference 4. method can be applied. Screening of transformants was performed using markers for rescue of host tritifphanase deficiency (Ilarker
It is more efficient to use the vector's drug resistance as an indicator, or to apply selective pressure for drug resistance of the vector. Furthermore, if the restricted ends of the vector are treated with alkaline phosphatase as described in Reference 5, self-cyclization of the vector can be prevented.

A recombinant plasmid is extracted from the obtained transformant, for example, by the method described in Reference 6, and this recombinant plasmid is used to extract p, rettg, which has tritrophanase productivity.
Transform the eri strain. Transformants are selected using the drug resistance or high tryptophan productivity of the plasmid as an indicator. The production of tryptophan can be carried out according to a known method (Reference 1).

Furthermore, ordinary methods can be applied to separate and purify L-tryptophan produced and accumulated in the reaction solution.

By using the transformant microorganism of the present invention, not only the accumulated concentration of L-tryptophan can be increased, but also the culture time can be shortened, compared to the case where the conventionally known P. rettgeri) liptophanase-producing bacteria are used.

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

Example 1 (1) Extraction and separation of plasmid P, rettaeri ATCC21118 (pYU
300) (Feikoken Bibori No. 8475) was added to LB medium (tryptone 1%, yeast extract 0.5%, sodium chloride 1%).
, pH 7.5) Cells cultured aerobically for 117 hours at 37°C in ljt were added to 3ml of 0.1ml containing 15ml lysozyme hydrochloride.
5MNaCj201 MF, DTA-50m14Tri
s-HCj! (pH 8) and incubated at 37°C for 15 minutes, followed by cryoabduction and then 25 ml of 0, I
HTris-HCj! (pH9) -1%5DS-0
,1M NaCj! was added and incubated at 60°C for 20 minutes to dissolve. 30. O0OrpF, 30
Add 10μ/ml RNaseA to the supernatant obtained by centrifugation for 1 minute.
After 1 hour of incubation at 37°C, crude DNA was extracted by phenol extraction and ethanol precipitation.
I got it.

This was subjected to biogel column chromatography and cesium chloride-ethidium bromide density gradient equilibrium centrifugation, and 55 μg of plasmid DNA (pYU300
).

The length of plasmid pYU300 was 4.2 Kb, and the drug resistance was Tc and CmR. Further, the restriction map of pYU300 is as shown in FIG.

(2) Extraction of chromosomal DNA p. rettaeriA cultured in LB medium
After the cells of TCC29944 were lysed by the method described in Section 1, they were lysed using the method of Saito-Miura (Reference 2).
Chromosomal DNA of (2) was obtained.

(3) Digestion and fractionation of DNA with restriction enzymes The DNA obtained in step (2) of 0.25 μg/μ (0.25 μg/μ) was digested with 0.12 units/μj! of the restriction enzyme old ndl[I
The decomposition product (125 μg of DNA) that was digested for 2 hours was
Centrifuge in 2 ml of 10-40% sucrose density gradient 0.5
Inl fractions were collected. Centrifugation was performed using a Hitachi RPS40T rotor at 20° C. for 1124 hours (25.0 O Orl).

Measure the length of each DNA segment by electrophoresis, and
Both parts of b were ligated to a plasmid.

Restriction enzymes manufactured by Takara Shuzo ■ were used, and the activity units were as defined in the attached manual.

(4) Preparation of vector DNA pBR322 manufactured by Takara Shuzo
[After complete decomposition, alkaline forsterase treatment was performed according to Reference 5.

(5) Preparation of host Logarithmic-phase E. coli M M 294 cultured in 5n+l LB medium for 37°C for 13 hours was transfected with T according to Reference 8.
After mutagenesis using 1100 u/ml NTQ in Mff buffer, a tritifphanase-deficient mutant of this strain was isolated using an indole productivity test as an indicator.

(6) DNA ligation and transformation 10 μg of DNA fragment obtained in (3) and PB obtained in (4)
10 μg of R322 plasmid vector DNA was mixed with 05 units of 74 DNA ligase based on the method described in Reference 5.
6mM Mgcj in 00μ! 2 6mM β-mercaptoethanol-0,5mMATP-6mHTris-HC
j! After incubating overnight at 14°C in pH 7.6, 10 μl of this reaction solution was incubated overnight at 14°C. E,co prepared in (5)
Bacterial solution 2 in which the li host was made competent by the method described in Reference 4
After mixing with 00μβ and incubating at 4°C for 45 minutes, then at 42°C for 90 seconds, and at 4°C for 1 minute, LB medium was added to make 1f111, and the mixture was cultured with shaking at 37°C for 1 hour to perform transformation.

(7) Screening The transformant mixture prepared in the previous section (6) was
Minimal medium (02% glucose,
0.2%KH,2PO4,0,7% 2HPO4,0
, 01% MgSO4.7H20, 0.05% Sodium citrate, L-tryptophan Ig/J! Sprinkle on top and heat to 37℃
The transformants were selectively cultured by static culture. Colonies generated on the third day of culture were transplanted again to a minimal medium containing chloramphenicol to reconfirm their proliferation ability, and then
The plasmid was extracted and the digested product of old ndl was subjected to electrophoresis according to the method of . inserter) DN in plasmid
Observe a complex plasmid of PBR322 with A length of 3°6 Kb and name it pUsO4. pUsO4 is shown in Figure 2.

(8) Cloning of tritifphanase gene into pYtJ300 From E. coli containing pUsO4, p
UsO4 was extracted and completely digested with pYU300 using restriction enzyme layer ndl [using method (4)], and then DNA was ligated with method (6) to make competent E, col.
i host was transformed.

(9) The transformant mixture prepared in screening (8) was added to 7,5
Using a minimal medium containing μg/ml chloramphenicol and using L-Trp as the sole nitrogen source shown in (7),
Screening was performed in the same manner as in 7).

Plasmids were extracted from the strains obtained from the screening in the same manner as in (7), and the length of the insert DNA in the plasmid was 3.6 Kb, as confirmed by electrophoresis of the old ndl [digested product]. Recognize the complex plasmid and name it pUS301. In addition, pUs30
1 is shown in FIG.

(10) P of pUs301, rettgeri AT
E, col beam (1) containing pUs301 obtained by transformation into CC29944 (9)
pUs301 was extracted by the method of p, rettger; ATCC299 was made competent by the method of reference 4.
44 in the same manner as (6).

(11) Screening The transformant mixture prepared in the previous section (10) was added to LB containing 5 μg/ml of chloramphenicol.
The transformants were plated on an agar medium and statically cultured at 37°C to selectively culture the transformants.

After reconfirming the growth ability of the colonies generated after 1 day of culture in LB medium containing chloramphenicol, the plasmid was extracted, and the old ND target digest was electrophoresed to determine plJs30.
1 was confirmed.

(12)) Production of L-tryptophan by liptophanase reaction (culture) P. rettgeri ATCC299
44, P, rettoeri ATCC29944<
pUS301) in a test tube containing LB10o+I.
Do not incubate with shaking at 124°C.

II containing the above culture solution with 100 1 medium shown in Table 1
The cells were placed in an Erlenmeyer flask and cultured with rotational shaking at 30°C for 16 hours.

table L-) Liatfan Solpol W2O0 +1! Ribebuton S cornstarch liquor yeast extract Coacid acid g 0g 0g 60+r g g L-cystine Shi-Arginine L-methionine shi-proline H2PO4 Mg5Ol・FH2O 0,6z 0.6g o, 3f 0.31 g Reconstitute with ion-exchanged water.

pH 7.0<KOH is used) After culturing, solid matter other than bacterial cells is removed by low-speed centrifugation (100 rpm for 1 minute), followed by high-speed centrifugation (6000 rpm for 110 minutes).
Bacteria were collected.

The reaction solution shown in Table 2 was added to the above bacterial cells, and inosine 1
4 g was added and shaken at 30°C for 24 hours.

Table 2 Sodium pyruvate or L-serine ammonium acetate a2SO3 Indole pyridoxal phosphate (dissolved in 10 ml of methanol) 8.0 g 8.0 g o, 1 g 10 6.0 g Make up to 110 ml with ion-exchanged water.

pH 8.8 (adjusted with KOH) After reaction, adjust pH to 10-11 with NaOH, then adjust pH to 1-2
After stirring for a certain amount of time, increase the volume to a certain level and use Leuconostoc.
The amount of L-tryptophan produced was determined by a bioassay method using Mesenteroides ATCC8042.

The results are shown in Table 3.

As shown in Table 3, when using either sodium pyruvate or L-serine as a substrate, compared to the non-transformed strain, the
The amount of L-)liptophan produced was doubled.

<Effects of the Invention> According to the present invention, it is possible to obtain a transformant with improved productivity of tryptophanase, and the production of L-tryptophan can be significantly improved.

Reference 1, H. Nakazawa et at: FE
BS Lett 25 432, Care Hyuga: Proteins, Nucleic Acids, Oxygen 11.446-450 (1966) 3, V, Hershfield et at:
Proc, Natl, ^cad, Sci.

U, S, A, 71. 1) 3455-3459 (1
974) 4, Katsuya Shigesada: Cell Engineering 2.616-626
(15, edited by R, W, Davis et al.: ^dvance
d Bacter + al Genetics, A
Manual for GeneticEnqin
earing p738-739 (1980)6
, H.C., Birnboin and J.Do.
ly: Nucleic Ac1d Res
7 1513-1523 (1979) 7,
Edited by T., Maniatis et al.: Molecular C
loning, A Laboratory Manu
al p 86-96 (1982,) Cold
Spring Harbor Laboratory
y, New York 8, Tatsuo Ishikawa (ed.) “Experimental Methods of Microbial Genetics” I) 86-88 (1982) Kyoritsu Shuppan

[Brief explanation of drawings]

FIG. 1 shows the restriction map of plasmid pYU300, FIG. 2 shows the restriction map of plasmid pUsO4, and FIG. 3 shows the restriction map of plasmid pUs301.

Claims (3)

[Claims] (1) A transformant of a fungus belonging to the genus Providencia, which has a recombinant DNA containing a gene governing tritifphanase production. (2) The transformant according to claim (1), wherein the gene controlling tritifphanase production is derived from a strain of the genus Providencia. (3) L-tryptophan is produced and accumulated by allowing the culture, bacterial cells, or treated product of the transformant according to claim (1) to act on pyruvate or L-serine, and L-tryptophan is collected from the reaction solution. A method for producing L-tryptophan, characterized in that: