JPS62143682A - Coryne-type bacterium having recombinant dna and production of tyrosine and/or phenylalanine using same - Google Patents

Abstract

PURPOSE:To produce tyrosine, etc., in high efficiency, by culturing a coryne-type bacterial cell having a recombinant DNA in which a gene coding shikimic acid kinase is connected to a plasmid vector proliferative in a coryne-type bacterial cell. CONSTITUTION:A coryne-type bacterial cell containing a recombinant DNA in which a gene manifesting in a coryne-type bacterial cell and coding shikimic acid kinase is connected to a plasmid vector proliferative in coryne-type bacterial cell is prepared beforehand. Cultivation of the bacterial strain gives tyrosine and/or phenylalanine. It is preferable to insert a prephenic acid dehydratase gene, prephenic acid transaminase, etc., in addition to shikimic acid kinase gene in the case of producing a phenylalanine or tyrosine-producing strain.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application This invention relates to a coryneform bacterium having a recombinant DNA incorporating shikimate kinase and a method for producing tyrosine and/or phenylalanine using the coryneform bacterium.

Background Art Shikimate kinase (hereinafter referred to as rsKJ) is an enzyme that catalyzes the reaction of converting shikimate to shikimate 3-phosphate, and shikimate 3-phosphate is converted via chorismate. Converted to phenylalanidi, tyrosine or tritophane.

On the other hand, there are some known methods of breeding these aromatic amino acid producing bacteria using recombinant DNA methods (for example, JP-A-57-208994, JP-A-57-71397,
JP-A-58-89194, JP-A-58-134994, etc.), the gene encoding SK (hereinafter '711-SK
41.1).

Problems to be Solved by the Invention The object of the present invention is to obtain microorganisms with high productivity of tyrosine and/or phenylalanine, and thereby to find a highly efficient method for producing tyrosine and/or phenylalanine. .

Means for Solving the Problems In order to solve the above problems, the present inventors conducted research and found that the gene encoding SK, which is expressed in coryneform bacterial cells, can proliferate within coryneform bacterial cells. We have successfully isolated coryneform bacteria having recombinant DNA connected to a plasmid vector, and found that the resulting coryneform bacteria have high productivity of thytecine and/or phenylalanine.

Coryneform bacterium according to the present invention
aeterla)
Determinative bacteriology (Barg y
's Manualof D@terminative@
Bacterology) 8th edition 599. Among these coryneform bacteria, the following coryneform glutamate-producing bacteria are particularly preferred in the present invention.

Examples of wild strains of coryneform glutamate-producing bacteria include the following.

Brepinogacterium diparicatum AT
CC14020 Brevibacterium salacalolyticum ATCC14066 Brevibacterium inmariophyllum ATCC14068 Brevibacterium roserum ATCC1382
5 Brevibacterium furanogum
ATCC13826 Previpacterium thiodanitalis ATCC19240 Corynebacterium
Acetoacidophilum ATCC13870 Corynebacterium acetoglutamicum ATCC158
06 Corynebacterium carnae
ATCC15991 Corynebacterium glutamicum ATCC13032,13060 Corynebacterium Lilium ATCC159
90 Corynebacterium Melasecola
ATCC17965 A method for isolating a wild strain SK gene having glutamic acid productivity as described above is to first extract the chromosomal gene from a strain having the SK gene of a coryneform bacterium, for example, H,5alto and K, Miu.
ra Biochem.

Bloph)+8. Acta 72, 619, (
(1963) can be used. ), which is then cut with an appropriate restriction enzyme.

Next, the recombinant DNA was connected to a plasmid vector capable of propagating within coryneform bacteria cells, and the resulting recombinant DNA was used to transform an SK-deficient mutant strain of coryneform bacteria to obtain a strain that possessed SK-producing activity. We can isolate and separate 98 genes.

In order to cleave chromosomal genes, a wide variety of restriction enzymes can be used by adjusting the degree of cleavage by adjusting the cleavage reaction time and the like.

The Grasmid vector used in the present invention may be any vector as long as it can proliferate within coli vernal bacterial cells. Specific examples include the following.

(1) pAM 330 JP-A-58-6769
See 9 (2) pAJ 1844 % 1977-1
Cleavage of the 92900 reference plasmid vector DNA is carried out by cleaving the DNA using a restriction enzyme that cuts at a single site or by partially cleaving the DNA using a restriction enzyme that cuts at multiple sites.

The vector DNA is cleaved by the restriction enzyme used to cleave the chromosomal gene, or oligonucleotides having complementary base sequences are connected to both ends of the chromosomal DNA cleavage fragment and the cleaved vector DNA. Seshime Seven, then Plus 5 Doctor and Chromosome D
It is subjected to a lidar reaction with an NA fragment.

In order to introduce the thus obtained recombinant DNA of the chromosomal DNA and the vector Grasmid into a recipient bacterium belonging to coryneform bacteria, it is necessary to introduce the recombinant DNA of the chromosomal DNA and the vector grasmid into a recipient bacterium belonging to coryneform bacteria, such as the one reported for -12 in Escherichia coli (Mandel +M, and
Hlgar A, +J0Mo1. +Bio1.53
1159 (1970)) to increase DNA permeability by treating recipient cells with calcium chloride, or as reported for No9tilus subtilis (Dune et al.
an *C, H,, Wilgon l G, A, an
d Young e F, E, * Gsne +1
, 153 (1977)) by introducing cells into a proliferation stage (so-called competent cells) in which they become capable of taking up DNA. Alternatively, as is known for Nonocilus subtilis, actinomycetes and yeasts (Chang r S, and Choan,
S, N,.

Mo1ec, Gen, , Genet, + 168.1
11 (1979): Bibb +M, J., Ward
l J, M, and Hopwood + O, A
, r Nature *274 + 398 (19
78) :Hlnnen +A, +H1eks +
J.B.

and Fink r G, R,, Proa, Nat
l,Aaad,Sai,USA+751929
(1978) ), the DNA recipient bacteria were transformed into Grasmid D
It is also possible to introduce the 7':7 smid into the DNA recipient bacteria as protoplasts or spheroplasts that readily take up NA.

In the protoplast method, it is possible to obtain a sufficiently high frequency using the above-mentioned method used for Tylus subtilis. Of course, it can be used. Equivalent results can also be obtained by adding oxymethyl cellulose, dextran, Ficoll, Tsururonic F68 (Serpa), etc. to the substitute of polyethylene glycol or polyvinyl alcohol to promote DNA infiltration. .

As a tyrosine and/or phenylalanine producing bacterium, S
A strain obtained by transforming a K-deficient strain as a host can be used, but if the following hosts are used, a strain with higher productivity of tyrosine and/or phenylalanine may be obtained.

In the case of tyrosine, Corynebacterium requires phenylalanine and 3-
Mutant strain H0Hagino + K, Na that is resistant to aminotyrosine, p-aminophenylalanine, p-fluorophenylalanine, and tyrosine hydroxamate
kayama: Agric, Blol, C
hem, + 37 +2013 (1973), Mutant strain of Brevibacterium showing resistance to m-fluorophenylalanine Sugimoto + Nakag
Awa r Tsuchida + 5hiio
, + Agric.

11io1, Chem, 37, 2327 (
1973) etc.

In the case of phenylalanine, a mutant strain of Brevibacterium resistant to m-fluorophenylalanine (8, Sugimoto + M.

Nakagawa IT, Tsuchida *I
, 8hi lo -t Agric, Biol.

Chem, 37.2327 (1973)),
A mutant strain of Brevibacterium that requires tyrosine and methionine and is resistant to 5-methyltryptophan and p-fluorophenylalanine.
), a mutant strain that requires tyrosine and methionine from the genus Brevibacterium and is resistant to and sensitive to high concentrations of 5-methyltryptophan and p-fluorophenylalanine (Japanese Patent Application Laid-open No. 59-34893), which requires tyrosine from the genus Corynebacterium. , p-fluorophenylalanine,
A mutant strain resistant to p-7 minophenylalanine (H
, Hagino, Nakayama, + Agric
, Bio1. Chsm, , 38, 157 (19
74)) etc.

If the following genes are inserted into the 8th gene, the productivity of tyrosine and/or phenylalanine will often be higher. 3-dehydetetraquinate synthase gene, shikimate dehydrogenase gene, 3-deoxy-
D-arabino-heptulonic acid-7-phosphate (DAHP)
) synthase gene, 3-dehydroquinate dehydratase gene, 5-enolbilbylshikimate-3-phosphate synthase, and chorizmate synthase gene.

In addition, when attempting to obtain phenylalanine or tyrosine 1-producing bacteria, the zolephenate dehydratase gene, prephenate transaminase, pretyrosine dehydrogenase gene, tyrosy y7 minotransferase gene, etc. must be inserted into the SK gene. is desirable.

The method of culturing the thus obtained coryneform bacteria capable of producing tyrosine and/or phenylalanine to produce and accumulate tyrosine and/or phenylalanine is a method for producing tyrosine and/or phenylalanine by conventional coryneform bacteria. There are no major differences from the methods used for this purpose.

That is, the culture medium is a usual one containing a carbon source, a nitrogen source, inorganic ions, and, if necessary, organic micronutrients such as amino acids and vitamins. As the carbon source, glucose, cinicrose, lactose, etc., and starch hydrolyzate containing these, whey, molasses, etc. are used. As the nitrogen source, ammonia gas, aqueous ammonia, anthonium salt, etc. can be used.

Cultivation is carried out under aerobic conditions, with appropriate adjustment of medium temperature and temperature, until production and accumulation of tyrosine and/or phenylalanine substantially ceases.

Example (1) Preparation of chromosome-DNA containing SK gene Brevibacterium lactofermentum AJ 11957
(FERM-P6673) in 11 CMG medium (peptone 11PA1., yeast extract 1? scraps, glucose 0.5
? and NaC20.5f/di, pH 7.2
The microorganisms were inoculated into a culture medium (adjusted to 100°C), cultured with shaking at 30°C for about 3 hours, and the cells in the logarithmic growth phase were collected.

After the bacterial cells were lysed with lysozyme/SDS, the chromosomal DNA was extracted and purified using a conventional phenol treatment method, and finally 3.5 μm of DNA was obtained.

(2) Preparation of vector DNA 1) AJ1844 (molecular weight 5.4 megadaltons) was used as a vector, and its DNA was prepared as follows.

First, Brevibacterium lactofermentum AJ 12037 carrying pAJ1844 as a plasmid
was grafted onto 100 d of CMG medium and cultured at 30°C until the late logarithmic growth phase, then lysed by lysozyme SDS treatment and ultracentrifuged at 30,0 OOX, p for 30 minutes to obtain a supernatant. . After the phenol treatment, 2 volumes of ethanol were added to precipitate and collect the DNA. Add this to a small amount of TEN buffer (20mM), Liss-HCl, 20mM NaC2, 1r
After dissolving in rM EDTA (pI(8,0)), it was separated by agarose gel electrophoresis, and then excised to form pAJ1.
844 f Lasmid DNA approximately 15μ? I got it.

(3) Insertion of chromosomal DNA fragment into vector Chromosomal DNA l0μ/ obtained in (1) and plasmid DNA obtained in (2)
A 5μ? and were each cleaved with restriction endonuclease Pst I by keeping each at 37°C for 1 hour.

After heating to 65°C for 10 minutes, both reaction solutions were mixed and AT
In the presence of P and dithiothreitol, the DNA was maintained at 10°C for 24 hours using DNA suga-se derived from T4 phage.
The chains were connected. The reaction solution was then heated at 65° C. for 5 minutes, and 2 volumes of ethanol was added to the reaction solution to collect a precipitate of ligated DNA.

(4) Cloning of SK gene Brevibacterium lactofermentum AJ12157 with deletion of SK gene (Brevibacterium lactofermentum AJ12036 was used as the parent strain and mutated with N-methyl-N-nitro-N-nitrosoguanidine) As a result, a mutant strain requiring three amino acids, diphenylalanine, tritophane, and tyrosine, was selected for growth.) was used as a recipient strain.

The protoplast transformation method was used as the transformation method. First, the strain was cultured in a 5-〇MG liquid medium until the early logarithmic growth phase, and penicillin G
After adding 0.6 units/yul, the culture was further incubated with shaking for 1.5 hours, and the bacterial cells were collected by centrifugation.
5 M sucrose, 20mM maleic acid, 20mM
Magnesium chloride, 3.5% penassai broth (Dir
SMMP medium (pH 6,5) 0.5-
Washed with. Next, the cells were suspended in SMMP medium containing 101n9/1rLl of lysozyme to form protoplasts at 30°C for 20 hours. After centrifugation at 4.6000× for 10 min, the protoplasts were washed with SMVP and 0.5× SM.
Resuspended in MP. The protoplasts obtained in this way and the DNA prepared in (3), 10μ? 5mM E
After mixing in the presence of DTA and adding polyethylene glycol to a final concentration of 3096, the mixture was left at room temperature for 2 minutes to allow DNA to be incorporated into protoplasts.

After washing the protoplasts with 1rrLl of 8MMP medium, resuspending them in 1d of SMMP medium, and for expression,
The cells were incubated at ℃ for 2 hours. This culture solution was spread on a protogellast regeneration medium at pH 7.0. Prodob 2-Stroke regeneration medium is 1 part distilled water and 12 parts Citris(hydroxymethyl)aminomethane. , KCl O,5t , glucose 10
? , MgCl2・6H208,1? , C&C12-2H
202,2p-, peptone 4iI-1 powdered yeast extract 4
No, Casamino acid (Direo) IP, K2HPO40
,2,P, Sodium succinate 135y-1 agar 8f and chloramphenicol 3μl"/11Ll t Contains
r.

After culturing at 30°C for 2 weeks, approximately 20,000 chloramphenicol-resistant colonies appeared, which were then grown in minimal medium (2% glucose, 1% ammonium chloride, 0.3
Thiurea, 0.1e16 potassium dihydrogen phosphate, 0.04
% magnesium sulfate heptahydrate, 2 ppm iron ion, 2
ppm manganese ion, 200μ? /l thiamine hydrochloride 1,50μif-/l biotin, chloramphenicol 10μ? Three strains were obtained which were resistant to chloramphenicol and had lost the requirement for phenylalanine, tricytophane, and tyrosine.

(5) Plasmid analysis of transformed strains For these strains, prepare a lysate using the method described in (2), and perform plasmid D by agarose gel electrophoresis.
When NA was detected, all strains showed pAJ1 of the vector.
Clearly large cilasmids were also detected in 844.

Restriction enzyme P using three strains of Grasmid for recombination
When cut with stl, all plasmids contain a 2.9kb D
An NA insertion fragment was observed. Therefore, the SK gene is 2.9
It appears to be present on the Pst I DNA fragment of kb. 2.9 at the P@t I breakpoint of vector pAJ1844
PSC the recombinant plasmid into which the kb DNA fragment has been inserted.
I named it 5. In addition, the strain containing PSC5 was transferred to AJlmo (
vERMP-8546).

(6) Re-transformation In order to confirm the presence of the SK gene on the recombinant plasmid containing the 2 and 9 kilobase DNA fragments detected in (5), this Grasmid DNA PSC5 was used to confirm that Brevibacterium. Lactofermentum AJ1215
7 was transformed again.

When we picked up 10 of each of the citachloramphenicol-resistant colonies and tested for triple auxotrophy for phenylalanine, tricytophane, and tyrosine, all of these auxotrophies disappeared. It has become clear that the SK gene exists in

(7) SK activity of the transformed strain The test strain was tested using the tyrosine and/or fluorinated nilua 2 shown in Table 2.
Bacterial cells cultured for 40 hours in a chlorine solution were treated with ultrasonic waves to prepare a lysate, which was then heated at 32,000X? , 20
The supernatant was obtained by centrifugation for a minute.

This supernatant was used as a crude fermentation liquid to prepare GAITON.
E.

M, W, GORDON (1958, J, Biol
, Chsm 230:1043-1050), a slightly modified method, i.e., 50 mM Ver.
onal buffer (p'49.0), 1mM shikimic acid, 4
mM ATP, 5 mM MgCl2.10 m
The reaction was carried out for 30 minutes at 37°C in reaction solution 1- consisting of M NaF, and after the reaction was completed, 1M Tris-HCl buffer (pH 7,
8) Add 0.2 μg of water and treat at IOOO°C for 2 minutes to inactivate the enzyme. After cooling 3. O, d reaction solution (Shikimic acid 2
~10μ? (reaction solution appropriately diluted to contain 0.5
Add 1 nl of 1-periodic acid and leave at room temperature for 3 hours.
Add 0.5 WLl of IN sodium hydroxide, immediately add 0.3 wL of 0.1 M glycine,
Shishikimic acid was quantified by measuring the absorbance. SK activity was determined by subtracting the amount of shikimic acid after the reaction was completed from the amount of shikimic acid added to the reaction solution. Table 1 shows the measurement results.

Table 1 SK activity PX-10,54 (8) Tyrosine and/or phenylalanine production ability of each transformed strain Using the above PS05, m-fluorophenylalanine resistant strain Brevibacterium tyrosine and/or phenylalanine producing Lactofermentum MF-35
8 and its phenylalanine-requiring, tyrosine-producing FT-1 and tyrosine-requiring, phenylalanine-producing PX-1 were transformed by the method described in (4), and transformed using chloramphenicol resistance as an indicator. I chose convertible stock.

The thus obtained AJ12261 (FERM P-8
547).

AJ12262 (FERM P-8548), A
J12263 (FERM P-8549) was cultured,
When the ability to produce tyrosine and/or phenylalanine was investigated, the results shown in Table 3 were obtained.

For culture, 20 WL/l of the production medium shown in Table 2 was added to 50 Qm.
A! CaC was placed in a Yonosaka flask and sterilized separately.
1 O3? Inoculate the test bacterial strain into the mixture, and add 31.5
It was carried out under shaking for 72-92 hours at <RTIgt;C.

Phenylalanine or tyrosine may be collected from the culture solution according to a known method, and after separating and removing bacterial cells from the culture solution, it is collected by a method such as concentration crystallization or a method using an ion exchange resin.

Glucose 130? Ammonium sulfate 10 KH2PO41tt MgSO4・7H201g Fumaric acid 12 Acetic acid
3- Soy protein decomposition solution 50 〃Fe50
4/7H2010my Yashi MJtS04/4H201o Biotia, 501t? Thiamine hydrochloride * 20oO〃DL-Methionine 400 Ki*T-N@degree 4.0shibao To obtain MF-358, FT-1, PX-1, deposited AJ12261, AJ12262 and A
J12263 makes it possible to remove complex plasmids in host cells without damaging them. That is, plasmids can be naturally lost from the host, and
It can also be removed by "Remove" operation (Bact, R
e.v.

36, p361-405 (1972)). Examples of other removal operations are as follows. AJ12261,
AJ12262 and AJ12263 were inoculated into a CMG liquid medium, and after culturing at 37°C overnight (high temperature treatment), the culture solution was appropriately diluted, applied to a CMG agar medium containing or not containing chloramphenicol, and incubated at 30°C. Culture for 1 to 3 days.

Strains isolated as chloramphenicol-sensitive strains are MF-358, FT-1, and PX-1.

[Brief explanation of drawings]

FIG. 1 shows a restriction enzyme cleavage map of plasmid P8C5.

Claims (2)

[Claims] (1) A coryneform bacterium having a recombinant DNA in which a gene encoding shikimate kinase expressed in a coryneform bacterial cell is connected to a plasmid vector capable of propagating within the coryneform bacterial cell. (2) culturing a coryneform bacterium having a recombinant DNA in which a gene encoding shikimate kinase expressed in a coryneform bacterium cell is connected to a plasmid vector capable of propagating within a coryneform bacterium cell; A method for producing tyrosine and/or phenylalanine.