JPS58103398A - Microbial gene, plasmid and microbial manufacture of aromatic amino acid - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to imitation 8:@manufacture of aromatic azure. More specifically, the present invention provides genetic modification of microorganisms to increase their ability to efficiently produce aromatic atonosomes.
l (ganatio modification)
Regarding K.

The aromatic iso1llfs, tyrosine, tributophane and phenylalanine are essential nutrients for humans and most animals. These compounds have become valuable commercial products for use as intermediates in gourds and other useful products used in nutritional formulations and nutritional supplements.

Currently, the production of aromatic amino acids is coupled on a large scale by fermentation methods using tyrosine, tryptophan or phenylalanine biogenic IR strains of bacteria. Bacterial biosynthesis of all eight amino acids involves a common metabolic pathway starting with the precursors erythro-phosphate (B-4-P) and phosphoenorbilpate (PNP). These precursors combine to form the intermediate 8-deoxy-D-appinohegullosone hk-7-phosphate (DA) IF). The metabolic pathway continues from DAHP through a number of intermediates, including colic acid, which is a common intermediate among the aromatic compounds of the eight Cypresses. Chorismic acid & is also subsequently converted to anthranilic acid, an intermediate of tryptophan, and to pre-7enoic acid, intermediate between phenylalanine and tyrosine.

The first step in the metabolic pathway (the reaction of E-4-P and PEP to produce DAHP) is mediated by am finci, collectively known as DAHPV ncetases. The biosynthesis of each of these isozymes is controlled by feedback inhibition by IJIg of the products of 8JI4, tyrosine, tryptophan, and phenylalanine. Other enzymatic reactions in the metabolic pathway are also controlled by feedback inhibition by end products or intermediates.

Feedback inhibition is a natural means by which organisms prevent excessive production of certain complements, but in industrial settings, feedback inhibition limits the efficiency of fermentation and the total amount of product that can be recovered.

Feedback-resistant strains of the Rose-tribe Aono #jusei were isolated from wild burials and were often obtained by means of mutation and selection.

Ha14, W, A, and 8m1th, O, H,, J
own or BaBa-0terLOIO10
1, No, l, 202-208 (19? 0)
Although overall fermentation efficiency has been improved using a variety of strains, it has been previously impossible to combine feedback resistance mutations with other desirable culture characteristics in a single microorganism. .

Genetic engineering techniques offer the possibility of introducing genes specifying feedback resistance into the genome of aromatic amino-dwelling adult cells. Large Mall (E.

The coll) genes are known as aroF, aroG and aroH. He1d and 8ugly x
th (5upra, ) describes an 8°0011 mutant strain (MAR1$) derived from mW1486 with aroG for DAHP synthetase that is resistant to feedback inhibition by phenylalanine.

A sample of this strain is from the Amarxoan Type Culture Collection W y (Amarxoan Typ@cuxt
ur@eoll-・otion, Rookvtl
ATCC819 to ls, MarylaM)
It is stored as 98.

According to the present invention, 2-deoxy-2-keto-low arabinohegutulosone-7-phosphate (DAH) is resistant to feedback inhibition by aromatic amino acids.
P) A single microbial gene specifying synthetase biosynthesis is provided. In other aspects, the invention encompasses a plus strain into which such feedback resistance genes are cloned, and a microorganism transformed by such a grass tip.

Feedback resistance aroF' for cloning
, a mutant strain is selected that produces DAHP synthetase in the presence of relatively high concentrations of aromatic amino acids as the source of the aro(j or tortoise roH gene).

Five keyaki trees are known in tanks (for example.

H@l(1, W, A,, @t al,, -aup
ra, @sho) or by well-known artificial mutation and selection procedures. E, 0
B. ooli strains are a convenient source of such genes because the genetics of B. ell have been extensively studied and are relatively well understood. However, DAHP
- Any microorganism that utilizes the shikimin passageway is a residual source of feedback resistance [vI! Can be used.

Genetically mutated organisms containing a cloned feedback-resistant DAHP synthetase gene involve several steps.

As a first step, the gene is isolated from the feedback-resistant mutant and placed in a cloning vector (ol
onlng motor).

Next, cells are transformed with the Sunset-Jung vector to amplify the gene, and transformants can be selected! - selected based on the ability of the cell to produce I) AHP quincetase activity that is not inhibited by the carcinogen or by the presence of aromatic alkyl salts or analogues thereof.

After amplifying the gene.

Cut it from the cloning vector (・xoL-
s@) and can be inserted into a dynamic display vector, which is then used to transform the aromatic amino acid-producing host cell 1@.

Isolation of feedback-resistant DAHP synthetase genes from feedback-resistant mutants is achieved using well-known procedures. Generally, cells are disrupted, for example, by mechanical homogenization and/or enzymatic digestion, and solid cell debris is removed by centrifugation.

Proteins can be phenol precipitated from the cytoplasm and removed by centrifugation, and the DNA can then be recovered by ethanol precipitation.

The total cellular DNA is then digested with restriction endonucleases to generate a mixture of restriction fragments of various sizes. Partial restriction digestion using such restriction endonucleases is one problem in that a complete AHD synthetase (i.e., feedback-resistant aroF, aroG
or aroH) are preferred because they have increased formic acid +1] ability. Any restriction endonuclease with filamentous sequences often present in microbial DNA can be used for this digestion. 1ooRI is a preferred restriction endonuclease because its linear sequence is common in microbial DNA and the EooRI site is a convenient site for cloning vectors often used to amplify genes. .

The restriction fragments resulting from the digestion reaction are advantageously sized to isolate larger fragments that are likely to contain the gene in question in full length. Conventional sizing techniques can be used, such as electrophoresis and density gradient centrifugation, with density gradient centrifugation being a particularly preferred technique. The DAHI' synthase gene is believed to have a size of about 1.1 lkk (the complete gene contains fragments, but it is possible to obtain larger fragments, e.g., about Bkb or more, preferably about 10 mb or more). By selecting the fragment [,
Improved. On the other hand, large pieces.

When inserted into the R-Royung vector, it becomes insensitive! can produce large recombinant molecules. Therefore, it is advantageous to use a control lI4 slice sound that is smaller than about 5 skb.

Restriction fragments containing preferably one feedback resistance gene can be obtained together with a phenotypic marker. For example, Loolz's MAR18 (5upra
An 81 kb fragment isolated from the complete NA of A., ) was found to contain the galactose operon (G11) in close proximity to the feedback resistance aroO gene.

Cells containing the ga,l operon can be easily marked by their ability to produce a red pigment on indicator plates, such as V. onkay's medium containing galactose. . Therefore s g'
The l operon can be used as an a-salt marker to select transformants that are likely to contain the aroo gene.

As described below, it was found that performing the cloning step in eight steps resulted in a higher frequency of transformants containing the cloned gene of interest. The sized restriction fragments are advantageously first inserted into a single copy plasmid. The grasmids exhibit tightly controlled replication and generally only 1 molecule of srI! per cell! ,
exists outside the body. It is believed that the use of copying plasmids increases the opportunity to effectively clone larger fragments containing a large number of known and unknown genes. A particularly preferred cloning vector is the single copy plasmid pMF8. This plasmid was developed by Maalson and Klzna, M.
jG, 152.175-182 (197?) K
Listed E, 0O11 strain BK26g (001dS
pr1ngHarbor No., 50). A sample of this strain is from the American Type Culture Collection (＾+oer1can Type@Cu
1ture Co11eotzon, Hoo-kvl
lla, Maryland) i'c ATCC81
It is stored as 994.

A recombinant plasmid potentially containing a restriction fragment carrying the feedback-resistant DAHP syntheter (gene @
The restriction fragments are incubated with restriction-digested grasmids in the presence of DNA ligase for one round of annealing and one round of ligation. Conjugation of restriction fragments to linearized plasmids can be accomplished by any of a number of known procedures. For example, a preferred procedure consists of cleaving grasmids using the same restriction endonucleases used to produce the restriction fragments. Alternatively, complementary homopolymer tails (eg, poly d[C] and poly d[T]) can be ligated to the five-sided ends of the restriction fragment and plasmid, respectively. Jackson,・tal,, Proo, Na
tl, Aoad, 801. U8A, 69. $
904-2909 (197!). Other techniques, such as 51-endonuclease treatment, subsequent blunt end ligation, or short segments of DNA (oligonucleotide linkers) can be used if desired.

The frequency with which restriction fragments are cloned can be improved by first treating the linearized plasmid DNA to remove the 51 phosphate group. Removal of these groups is
Prevents the recyclization of grasmids and the formation of two-ill and three-cell bodies. Phosphate groups are advantageously removed by treatment with DNA4 alkaline phosphatase, such as that obtained from calf intestine. Nrstrut
aat@, @t al,.

NAR4(lia), 4165-4174 (19
7? ).

After ligation of the restriction fragment into a cloning vector, one recombinant plasmid is used to transform host cells. Lo
olx cells are preferably used for this purpose, and other bacterial cells compatible with the cloning vector can also be used. Cells use calcium ions to
processing to render it competent (oo) for the reception of foreign DNA.
normal methods, including processing to
[Can be transformed by Cohen, 8.
N,, 6ta1. , PNA869, 211011
14 (19? 2) o The transformed cells are then cultured in half-culture in a nutrient medium designed to select transformants based on the phenotypic properties specified by the recombinant grasmid in question. For example, (al gene?!
- When selecting transformants containing a MAR1B fragment with -, a nutrient medium supplemented with galactose can be advantageously used.

Same 4m! Additionally, betel antibiotics or growth factors can be used depending on the composition of the cloning vector.

As long as the size of the original grasmid and restriction insert is known, further selections can be made based on the size of the plasmid in the transformed cells.

If primary cloning is accomplished using a single copy plasmid, as described above, the gene is preferably subcloned into the plasmid multiple times (ie, one that exhibits a relaxed mode of replication). In order to couple this subc.

Transformants carrying the four-fold feedback-resistant DAHP synthetase gene are cultured on the field to grow sufficient gill cells for subsequent handling. Plasmid DNA is recovered from these am, isolated, and excised by digesting the CDAHP synthetase gene with restriction endonucleases, preferably the same restriction endonucleases used to digest the cellular DNA. The Kit' is used to excise the DAHP synthetase gene. The gene is then isolated, e.g., by sucrose gradient centrifugation, and inserted into a multilayer glass assembly. This insertion is carried out by known techniques. The procedure is carried out substantially as previously described in connection with single copy plasmids. Such multicopy putosmids of bridges can be conveniently employed, and the well known plasmid designated pBR81a is preferred. pBR888
The plus construct has an EooRI restriction site, so the gooRI excised gene can be conveniently engineered into the pBR82B plasmid by a single ligation step.

The recombinant plasmid is again used to transform host cells and transformants containing the feedback-resistant DAHP synthetase gene are isolated based on the plasmid, visual marker on the gene container, and based on size. and select.

It is advantageous to use a step to reduce the size of the DNA fragment containing the feedback-resistant DAHP synthetase gene, before or after cloning it into a multi-transfer plus construct. For example, the aro(j gene was found to be contained within a fragment that could be isolated by restriction with the endonuclease H1ndll.
It was found that the Hlnd III tlJtM fragment obtained from the gene was further reduced in size to a fragment containing approximately 84 kbytes in vivo in g, ooll. This internal deletion was found to reduce the size of the Htn41 fragment by approximately 504, but it did not delete any part of the aroG gene. Further reductions in the size of cloned DNA fragments can be achieved by selective restriction endonuclease digestion. For example, 8*1
l-Hlnl In digestion reduces the size of the aroG fragment to M
It has been used to reduce ARlB to approximately 8 kb.

In addition to the DAHP synthetase gene, the cloned DNA fragment advantageously also contains one of the genes! Contains the nucleotide sequence that controls Jllll. These sequences were used to transform the gene by JIIL transformer (e.g.,
DAH) 'synthetase enzyme production). Alternatively, such control signals can be provided as part of the vector into which the gene is cloned, or can be spliced onto the gene prior to insertion into the vector.

It was found that g, colx cells obtained by this method produced significantly more DAHP synthetase in wax than in normal wax. Moreover, the DAHP synthetase so produced is active in the presence of relatively high degree aromatic amino acids. Feedback Resistance DA H)” Synthetase Gene When activated, this gene selectively binds to the host strain and other genetic mutants to produce highly efficient aromatic and noic acid producing strains. Due to the degeneracy of the genetic code, the nucleotide sequence order of the DAHP gene shown in the drawings can be substantially changed without changing the amino acid sequence order of the aggregated protein. Although the nucleotide and amino acid sequence order and seventy isolation and cloning procedures have been described, the present invention encompasses changes in the nucleotide sequence order permitted by the genetic code.

The following examples further elucidate the invention.

Example ■ Strain W1481S (MARlB) was grown to stationary phase by incubation for 18 hours at 87°C in L broth.
After extraction with Itphenol and chloroform, DNA was isolated by ethanol precipitation.

After precipitating with ethanol, DNA: wound three times,
T B@@Ksra turbid in liquid (Marmur, J
,.

1961, J. Mo1. Blot,, 8,11
8-818). 80μI of MARlB DNA,
8 with 40 units of EooRI restriction enzyme at 8F”C.
I spent time. This DNA was placed in a logarithmic sucrose gradient of At'15-8s96 and centrifuged at 87°C and 4°C for 18 hours.

Eight fractions containing arrowhead-sized DNA fragments were pooled, precipitated with DNAV ethanol, and the DNA
The aroG gene was analyzed using A(gμs)v.

Example ■ As described in Example ■ 1IllII! Shita. Control 1d, approximately 80 kb in size, purified on a sucrose gradient.
# The cord digested fragments are treated with plus hydro Ml' by the following procedure.
-8: The pMF'-4 positive electrode of p14μI was digested with 87 units of BooRI endonuclease for 1 hour. These mixtures were then heated to 65°C for 1
The endonuclease reaction was stopped by heating for 5 minutes and the plasmid DNA was precipitated by the addition of ethanol in an inpropanol/dry ice bath. The linearized plasmid DNA was then treated with alkaline phosphatase to remove the 51 phosphate group. Alkaline phosphatase was prepared as follows: Calf alkaline phosphatase solution (Boehrlnger Mannhsim, Ind.
xanapolzs, obtained from In+n-ana) at 16.000 rpm at 0°C.
Centrifuged for 5 minutes. The solution was then removed from the centrifuge, the supernatant was removed with a piece of paper, and the enzyme pellets were removed.
Resuspend in Y 100111j glycine buffer. This suspension was then diluted with 4.9 ml of glycine buffer.
, diluted and placed this preparation on ice for a minute.

Then linearized positive pellets) Y80μI T
P4Jl was suspended in i1l buffer and half KIOμj of bovine alkaline hostaty 1 preparation was added to this suspension. This solution was then incubated at 87°C for 8 hours.

The DNA from this S* was then ethanol precipitated, centrifuged, and stored at -80°C.

Eight pools of MARlB DNA were prepared in a sucrose gradient as described in Example IK.
) and MIC4 of 10 mmol Tris-6
The treated plus 2 DNA was then resuspended in 56 μl of Tris-MICl buffer (pH 1,4) and 14 MARlB D
Num was added to the suspension. To this mixture, add 1.0 μl of 1
molar of MpCI, 1.0 μl of dithiothreitol and 0.5 μl of 10 molar of AT.
P- [added. 6 μIY of this reaction mixture, 0.74 agarose (Agaroms) as a control for unbound material.
) Removed for gel electrophoresis. The remainder of this mixture was treated with T4-DNA ligase (Bsthesaa Re
5earoh Laboratorxes, Rookv
xll@.

One volume (1 vol) of a 1:10 dilution (obtained from MD) was added. This binding mixture was then incubated at 1°C for 8 hours. 5μjt of the reaction mixture was taken out again from the agarose gel electrophoresis, and the degree of binding vtiL was determined.
μlt added: 962 μ-10 mmol Tris-6ζ mmol MI
Clm (pH 7,4) 1 mol MICI of 10 μm! .

10 of 7 voices J < 1 of Rimol's dithiothreitol 6 voices I
0 mmol ATP 86 voices I:10 T4 ligase dilution.

This mixture was then cultured at 1g°C for 8 hours.

With this method m1ll! Using the recombinant plasmid,
The following 5K E, 0O1l cells were transformed with Y: the host cells were from a strain with a deletion Y:lr for the '(jal-aroG) domain. A sample of this strain was an American- Collection of tights culture (mu-r
xoan Type@Culture Co11aoti
on, Rookvzlle.

Marylanl) K ATCC81995 [
It is kept. A culture of this cell was prepared by inoculating with 100 liters of cell culture in 100 liters of 100 liters of cell culture, and incubated at 87°C in medium V for 5 hours and 5 hours, ss optical- f(590nm)K culture. Cells were harvested, washed in ice-cold 100 mmol MIC1R, and washed in 40 liters of ice-cold 100 R17 mole Ca.
5m5a in C4-50 Yrimole MttCIm and kept on ice for 90 minutes. Add 1 cold 100 mmol of CaC4,-60i to 1 aliquot of M I CIs.
The cells were resuspended in water and stored at 4°C for 30 hours. The cells were then heat shocked at 48"C for 8 min, 1 lljV of LB broth supplemented with 8" galactose was added, and this mixture
The cells were incubated at 8°C for 90 minutes with shaking. 6 JVs of on-site warming plants, 2 mediums of pine conch (h4*oconk@y) supplemented with glass dose and ampicillin.
Plated at 0 μI/nxl. Out of a total of 808 colonies, 15 were red. Some of the galactose-positive colonies were cultured in horn broth and analyzed for DAHP synthetase activity until a colony with AroG feedback resistance activity of 1 to 1 was found. DAHP synthetase activity was , MaoCa
ndlzss, R, J,, at aL, J, B
I.O.I.

Chew,, 25B, No, 12.42! S9”
4265 (1978).

Example ■ Humphr@ys from a feedback resistant aroG clone prepared as described in Example ■.

・tll, Dan 1888: 457 (19? 5) and (luerry, @z al,, J, Baot
, 116: 1064 (197B) K. The standard plasmid isolation procedures described in K., 197B) yielded 1%-unifemale coiled plus DNA. This Grassund's 1μI f H
digested with the lnl m restriction enzyme, and similarly its Hln
3 μ of pBR822 DNA cut at the dl[[ site
Combined with I. These DNAs were combined, cells were transformed, and cells containing the cloned feedback-resistant tortoise roG gene were selected essentially as described in Example 5. Of all the ruined colonies.

Only one was above 8 (ie, galactose positive). This colony was cultured in liquid broth and calibrated for feedback-resistant aroC1DAHP synthetase activity, indicating the presence of such activity! confirmed. Analysis of the plasmid content of these cells revealed that approximately ? , it was shown that a deletion of 1 kml had occurred.

The size of the plasmid insert was further reduced by sequential restriction enzyme digestion. First of all, putusumido V# element 8 au8
After partial digestion with A, approximately 8.7kk was deleted from the insert, and cells with feedback-resistant aroG-active tlI were obtained. Resulting plasmid y Hxn6 i
Further digestion with a mixture of 1.1 and 8.
．． A positive clone was obtained with a 6kb deletion, an insert of approximately g,'7kk, and the desired DAHP syntheter-4 activity. A sample of this strain was derived from the American e-type culture collection (Am・-rxoan Type@C
u1tur@Co1x@otxon, Rookvxl
ls.

Maryland) 4CATTCC819911. The nucleotide sequence order of the DA) IP Kunsetai gene in this product was determined and is shown in FIG. This plus word is illustrated in Figure 2. The DAHP synthetase gene (aroG) extends approximately from the BIITI site to the HxnoIf site, which is the direction of transcription.

[Brief explanation of the drawing]

FIG. 1 is the nucleotide sequence order of the DAHP synthetase gene of the present invention. FIG. 2 is a restriction map and an iI diagram of a plasmid containing the DAHP synthetase gene of the present invention. Special pestle applicant Genex also engraves the corporation drawing (r'i''1'' change L)FIG, 1-J=5 Suji 5;
j4 i i 1FIG, 2 9BR322 Written amendment of the most aggravated rationale procedure (1 尭) 1. Indication of 1. Showa S1 patent application No. 178160 Relationship with f'l Patent applicant (1, place name ( Name) Genetics=-Boreishi Seal 4
, Agent ji; +ili 2-11-12 Yoyomoto, Shibuya-ku, Tokyo 151 Honmura Hire β Minami 5 Correction 1fj
Showa date, month, day (Shipping) 6,
Increase in the number of inventions due to hli correction @ Engraving of the record (no change in content) Procedural amendment (A1) 1. Indication of the case 1988 Patent application No. 8260 2.
Title of the invention Microbial genes, plasmids,
Agent address: 11-1 Yoyomoto 2Y, Shibuya-ku, Tokyo 151
No. 2 Honmura Building 7Wr5, Date of amendment order January 25, 1980 (shipped) 6. Number of inventions increased by amendment 7, Subject of amendment 8, Contents of amendment (1) Representative of the patent applicant in the application Add the name as shown in the attached sheet. (2) Add the power of attorney and its translation as attached. (3) Submit engraving drawings (no changes in content) as shown in the attached sheet.

Claims (1)

[Claims] 1. Biosynthesis of 3-deoxy-2-keto D-arabinohebtulosonic acid-7-phosphate (DAHP) synthetase that is resistant to feedback inhibition by aromatic amino acids. Identify (substantially) two isolated complete microbial genes. 0 YTZ YTZ CCX CCX
G'J'Xteu Leu
Pro pro Va105 35 50 65 1f0 95 10 25 AAa Arq LY@
Leu Leucys
pro Val Gly
Phe5fu0 85 GGX ACX AT)i AAM
GTXGly Thr Ila
Lys Va100 15 30 45 60 6fu5 90 Gly Lye Glu P
ro ^8n Fu35 Fu50 65 Fu80 Fu95 110 25 40 AAM AAM CAM ATG
GAYLys Lys Gin
Met Asp55 8f0 85 GGX GGX GAM AAM
GCXGly Gly Glu
Lye Ala00 915 30 45 60 9F5 90 005 020 where the 51-8' strand begins with an amine end and each triblet cipher is shown, and in each triplet, one is a deoxyadenyl , T is thiidyl, 0 is deoxyguanyl, C is deoxycytosyl, X is A, T, C or G, Y is C, and Y is C In some cases, 2 is A or 0, H is person, T
or C, and Q is T or a person, and when Q is T, R is C, and S is A%T%
C or G and Q is a person, R is 0, and 8 is T or C, M is A or G, L is A or C, and when L is a person , N is A or U, and when L is C, N is A%T%C or U, and G17 is glycine. Aja is alanine, Van is valine, L@u is leucine, 1j. is inleukine, 8.r is serine, Thr is threonine, Ph. is phenylalanine, and Tyr is It's tyrosine. Trp is tryptophan, 07- is kusukoin, M@z is methionine, A-p is aspartic acid, (jju is glutaic acid, Lyhata is lysine, ARg is arginine and Hl- is histidine; Pro is proline; ujn is glutaine; and Atn is asparagine. :LYII
Ala xh* ttis
LIYII150 ATCCTG AAA GGT A
ATXle Lau Lys
Gly Asn65 GA'i' GAT CGCC'l'G
'! 'TGkBp A! IP
Ar9 Lau Leu80 GTT GTG ATT GGCC
CAVal Val: [le
Gly Pr. 95 TGCTCA A'! 'T CAT
GATCys Sar Xis
Hls Asp10 25 GAG TA'r GCCACT
CGCGlu Tyr, Ala Thr
Arg40 G'! "I' CTG GCG C
TG CGTVal Leu
Ala Leu Arg55 GAA GAG CteG AAA
GATGlu Glu Leu
Lys Asp”
2) O GAG CTG GAA ATCG
TAGlu Lau Glu
Ile Va185 ATG CGCGTCT λT TTTM
at Axq Val Ty
r Phe00 GAA AAG CCG CGT
ACCGlu Lys Pro
Arg Thr15 ACG GTG GGCTGG A
AAThr Val Gly
Trp Lys30 GGG T'rG A'l"J"
AA-CGA'J'Gly Leu
Ile Asn A8p45 CCG CAT ATG GAT
AATPro Hls Met
Asp Asn60 AGC'E"J'CCAG ATCAAC5er
Phe Gin I
le AsnAsp Gly
Leu Arg Ile90 GCCCGT AAA TTG C
'! 'GAla Arg Lye
Leu Leu05 CTT GAT ATT AACG
ACLeu A! IP engineering is
Aan Asp20 35 GCA GGT GAG TTT
CTCAla Gly Glu
Phe Leu50 GAT A'! 'G ATCACCCCCA
A! IpMat Xis Thr
Pr. 65 CAA TAT C'! 'CGC'l'
GACGin %r Leu
Ala Asp8O CTG A'! 'G AGCTGG
GGCLau Met Ser
Trp GlyAla X1e
Gly Ala Arg1
0 Val Hls Arg G
lu Leu40 55 TGT CCG G'l'CGGCTTC
Cys Pro Val G
ly Phe70 AAA AAte GGCACCGACLys
Jvsn Gly 'l'
hr Asp85 GGT ACG ATT AAA
GTGGly Thr Ile
Lys Va100 GCT ATCGAT GCCATT included 1
a IIs AJP
Ala Ile enter an Ala
Ala Gly Al
aPro Hls Cys
Ph1l L@u45 TTCGTA ACG AAA'
l'GGPhe Val Thr
Lyss Trp60 75 90 05 Fu20 35 50 Fro5 GAA GGG CTG AACA
AAGlu Gly Leu
Asn Lys Fu80 95 CAG GTG ATG ATCG
ATGin Val Mat
Ila Asp810 Ser Ser Lys Gin
Phe40 AAA AAG CAG ATG G
ATLys Lye Gin Me
t AspVal Cyss Al
a Asp Va170 85 15 30 45 AGCC'J'CGAG AGCGGGGSer
Leu Glu Ser Gl
y60 GAG CCG C'l'G GC
CTACGlu Pro Lau
Ala Tyr9fu5 GGT AAG AGCATCACCGl
y Lys Ser Xle
Thr90 GA'l”GCCTGCATCGGCA!IP
Ala Cys Ile
Gly005 TGG GAA GAT ACCG
ATTrp Glu Asp Th
r Asp020 GCT CTG TTA CG,T
CAAAla Leu Lau
Arg Gin035 CTG GCG AAT GCA
GTALeu Ala A
Consisting of an Ala Val.
The microbial gene according to claim 1. The gene according to claim 1, wherein the gene is a bacterially sourced RidePack resistance aroG gene. a Gene is a feedback resistance source of micro#I source a r
The gene according to claim 1, which is an o k' gene. The gene according to claim 1qA, wherein the a gene is a native feedback resistance aroH gene. The τ gene is derived from E. coli (&ah@riohia 001
1) The gene according to claim 4, which is derived from 1). a Claims 1.9, 8, 5.6 or 7
A plasid consisting of genes capable of replicating in bacterial cells. a Bacterial Grasmid pH & -8. Claim 1a Grasmid of claim 1fi#E8qA, consisting of Grasmid pBR8Bg. IL Claim 1, which makes every effort to maintain and reproduce the plasid! I. Section 8. Microorganisms transformed with sensitive grass buds. □ l2 The grasmid can be recognized by the microorganism and contains a signal that controls the expression of a feedback-resistant DAHP synthetase gene, whereby the microorganism is able to recognize the presence of a normal, inhibitory amount of aromatic azunoacids. 1.1. The axis sigma microorganism of claim 1.1, having the ability to produce DAHP synthetase, which is active in . 1a The microorganism according to claim 11, wherein the plasmid is the plasmid according to claim 9. Table 1 The microorganism described in Claim 11, wherein the plasmid is Grasmid as described in Claim 1θ0. 1K First, insert the DNA segment into a single copy plasmid and change it to C#i14 to generate a plasmid.
Transforming microbial cells with the first recombinant grasmid,
isolating plasmid DNA from the microbial cell, excising the DNA segment or portion from the plasmid DNA, and then inserting the excised segment into a multicopy plasmid. A method for cloning a DNA segment comprising forming a second recombinant plus strain and transforming a microbial cell with said second recombinant plus strain. 16. The method of claim 15, wherein the DNA segment to be cloned is characterized by the feedback-resistant DAHI' synthetase gene. l? The method of claim 16, wherein the feedback-resistant DAHP synthetase gene is a feedback-resistant aro(j); the fist-copying push cell is pMF-8; ;, oolz)
The method according to claim 17, wherein: The above-mentioned Tatesha plus card is pBR8B8,
18. The method according to claim 17, wherein the microbial cell is a coliform (N, ooll).