CN1316015C - New-mutant carbamyl-phosphate synthesized enzyme and method for producing compound derivated from carbamyl-phosphate - Google Patents

Abstract

L-arginine, citrulline and pyrimidine derivatives including orotic acid, uridine, uridine-5'-phosphate (UMP), cytidine and cytidine-5'-phosphate (CMP) are prepared utilizing new-mutant carbamyl-phosphate synthesized enzyme, as a bacteria belonging to escherichia, wherein an amino acid sequence corresponding to 947-951 position of mutant carbamyl-phosphate synthesized enzyme is replaced by any one amino acid sequence selected from SEQ ID NO: 1-9 and the feedback inhibition effect of the uridine-5'-phosphate in the bacteria is desensitized.

Description

New-mutant carbamyl-phosphate synthesized enzyme and production are by the method for carbamyl phosphate derived compounds

Technical field

The present invention relates to the microbiology industry, be specifically related to produce method by the carbamyl phosphate derived compounds.More particularly, the present invention relates to utilize the arginine that participates in coli strain and the new anti-feedback enzyme of pyrimidine biosynthetic pathway to produce, as arginine, citrulline with comprise the pyrimidine derivatives of vitamin B13, uridine, uridine 5 '-a phosphoric acid (UMP), cytidine and cytidine 5 '-a phosphoric acid (CMP) by the carbamyl phosphate derived compounds.

Background technology

The catalysis of colibacillary carbamyl phosphate synthetase (CPSase) energy is by the building-up process of the complexity of supercarbonate, glutamine and two Mg-ATP molecule generation carbamyl phosphates (CP), discharge L-glutamic acid, phosphoric acid and two Mg-ADP[Meister A. simultaneously, Advan.Enzymol.Mol.Biol., 62 volumes, the 315-374 page or leaf, 1989].The synthetic of CP is the intermediate steps of two kinds of biosynthetic pathways, i.e. pyrimidine nucleotide and arginic biosynthetic pathway intermediate steps.In first approach, CP and aspartate carbamoyltransferase (ATCase) coupling causes forming vitamin B13 by two steps.Vitamin B13 is the biosynthetic important metabolic intermediate of pyrimidine derivatives, and described derivative comprises pyrimidine, as uridylic; Pyrimidine nucleoside is as orotidine, uridine and cytidine; And pyrimidine nucleotide, as orotidine 5 '-a phosphoric acid (OMP), UMP and CMP.Confirmed already, and in the fermenting process of various bacteria, in substratum, existed vitamin B13 obviously to help pyrimidine derivatives, be i.e. the production of uridylic and accumulation (US patent 3214344).In second approach, CP constitutes the 6th step (being begun by L-glutamic acid) of arginine biosynthetic pathway by 2,5-diaminovaleric acid carbamyl transferase (OTCase) and 2,5-diaminovaleric acid coupling.CPSase is activated by ornithine and IMP (precursor of purine nucleotides), and is suppressed by UMP.Carbamyl phosphate synthetase is made up of two subunits.For coryneform bacteria (EP1026247A1) with belong to Escherichia and the bacterium of bacillus, confirmed already that described subunit was by carA and carB genes encoding.The accumulative total inhibition of transcribing the end product that is subjected to two approach of carAB operon [Charlier D. etc., molecular biology magazine, 226 volumes, 367-386 page or leaf, 1992; Wang H. etc., molecular biology magazine, 277 volumes, 805-824 page or leaf, 1998; Glansdorff N. etc., pyrimidine approach, 6 volumes, 53-62 page or leaf, 1998].The heterodimer that natural intestinal bacteria CPSase is made up of the big subunit of the small subunit of a 41270Da and a 117710Da, described two subunits are respectively by carA and carB genes encoding.The hydrolysis of small subunit catalysis glutamine, and responsible NH ₃To the transfer of big subunit, the reality of carrying out CP on big subunit is synthetic.Big subunit comprises that substrate is the binding site of carbonic acid ammonia salt, ammonia, and two independently are used in conjunction with the site of Mg-ATP and constitute 18kDa C-terminal district [Rubio V. etc., biological chemistry, 30 volumes, 1068-1075 page or leaf, 1991 in adjusted and controlled territory; Cervera J. etc., biological chemistry, 35 volumes, 7247-7255 page or leaf, 1996].In addition, the someone thinks that big subunit has the activity of the building-up reactions of catalysis carbamyl phosphate (Stephen D.Rubino etc., journal of biological chemistry, 206,4382-4386,1987) alone.

Crystalline texture [Thoden J. etc., biological chemistry, 36 volumes, 6305-6316 page or leaf, 1997 of the allosteric activated form of CPSase have been disclosed recently; Thoden J. etc., ActaCrystallogr.Sec.D., 55 volumes, 8-24 page or leaf, 1999].First three the independent structures territory that is called as A, B, C on the big subunit is closely similar in configuration aspects, but the 4th structural domain is different fully.D structural domain (937-1073 residue) is responsible for combination and is carried out the allosteric regulation and control by effector: IMP, UMP and ornithine.In addition, confirmed already that as if two residues, No. 948 Serines and No. 1042 Threonines for the allosteric regulation and control of CPSase play a crucial role [Delannay S. etc., molecular biology magazine, 286 volumes, 1217-1228 page or leaf, 1999].When No. 948 Serines were replaced by phenylalanine, it is insensitive to UMP and IMP that this kind of enzyme becomes, but still can be activated by ornithine, but the activated degree reduces.Enzyme with T1042I sudden change shows weakening greatly of ornithine activation.

Say that in principle anti-feedback (fbr) phenotype of enzyme is to occur as the result with the corresponding amino-acid residue of another kind of aminoacid replacement on one or several site on the aminoacid sequence, and this replacement can cause the activity of enzyme to reduce.For example, with the natural Met256 on each replacement intestinal bacteria serine acetyltransferase (SAT) (cysE gene) of 19 kinds of other amino-acid residues, in most of the cases can cause the fbr phenotype, but, mutant SAT albumen can not recover activity level [NakamoriS. etc., AEM, 64 volumes of natural SAT, the 1607-1611 page or leaf, 1998].Therefore, the defective of the mutant enzyme that obtains by aforesaid method is, compares with wild-type enzyme, and the activity of mutant enzyme reduces.

Summary of the invention

The present invention relates to be structured in anti-feedback, the highly active enzyme that plays a crucial role in intestinal bacteria pyrimidine and arginine or the citrulline biosynthesizing.

In the present invention, the novel method that has proposed to utilize the completely random of carB gene fragment to be combined to mass mutation type carB gene.Some amino-acid residue on the substituted amino acid sequence fragment (wherein, can locate the fbr sudden change) simultaneously can produce activity level is returned to mutein near native protein, and this is because the three-dimensional structure and the natural structure of this kind of enzyme more meet.Therefore, finished following described the present invention.

In other words, the invention provides:

(1) the big subunit of carbamyl phosphate synthetase, wherein, the aminoacid sequence that is equivalent to the 947-951 position of SEQ ID NO:20 is replaced by any aminoacid sequence among the SEQ ID NO:1-9, and the feedback inhibition of uridine 5 '-a phosphoric acid is desensitized;

(2) as the big subunit of the carbamyl phosphate synthetase of (1), wherein, carbamyl phosphate synthetase is the intestinal bacteria carbamyl phosphate synthetases.

(3) as the big subunit of the carbamyl phosphate synthetase of (1), wherein, the aminoacid sequence of the 947-951 position of SEQ ID NO:20 is replaced by any aminoacid sequence among the SEQ ID NO:1-9, and the feedback inhibition of uridine 5 '-a phosphoric acid is desensitized;

(4) as the big subunit of the carbamyl phosphate synthetase of (1), it is included in one or more locational one or several amino acid whose disappearance, replacement, insertion or interpolation except the 947-951 position, wherein, the feedback inhibition of uridine 5 '-a phosphoric acid is desensitized;

(5) carbamyl phosphate synthetase, it comprises the big subunit as each carbamyl phosphate synthetase in (1)-(4);

(6) DNA of each carbamyl phosphate synthetase in coding as (1)-(4), wherein, the feedback inhibition of uridine 5 '-a phosphoric acid is desensitized;

(7) DNA of the small subunit of big subunit of carbamyl phosphate synthetase that the feedback inhibition of each uridine 5 '-a phosphoric acid is desensitized in coding as (1)-(4) and intestinal bacteria carbamyl phosphate synthetase;

(8) belong to the bacterium of Escherichia, it has the DNA as (6) or (7);

(9) as the bacterium of (8), it has generation and is selected from L-arginine, the ability of the compound of citrulline and pyrimidine derivatives;

(10) as the bacterium of (9), wherein, described pyrimidine derivatives is vitamin B13, uridine, UMP, cytidine and CMP;

(11) a kind of production is selected from the L-arginine, the method of the compound of citrulline and pyrimidine derivatives, this method may further comprise the steps: each bacterium in cultivation as (8)-(10) in a kind of substratum, so that in substratum, produce and accumulate described compound, and from described substratum, collect described compound; With

(12) as the method for (11), wherein, described pyrimidine derivatives is vitamin B13, uridine, UMP, cytidine and CMP.

In the present invention, term " CPSase activity " expression catalysis is by the activity of the building-up reactions of the complexity of supercarbonate, glutamine and two Mg-ATP molecule generation carbamyl phosphates.The single polypeptide that " CPSase " of the present invention can be made up of big subunit, or comprise the heterodimer of big subunit and small subunit, as long as CPSase has the CPSase activity.In this application, big subunit and the heterodimer mentioned above can be collectively referred to as " CPSase ".The DNA of big subunit and small subunit of encoding can be called as " carAB ".

According to the present embodiment, CTPase with above-mentioned any one fbr sudden change can be called as " mutant CPSase ", and the DNA of encoding mutant type CPSase can be called as " mutant carB gene " or " mutant carAB gene ", and does not have the CPSase of sudden change can be called as " wild-type CPSase ".

To elaborate to the present invention below.

＜1〉mutant CPSase and mutant carB gene

Subsequently, to carrying the selection and the screening of being cloned into the recombinant clone of the mutant carB gene on the expression vector as the carAB operon, so that allow to select to have the fbr variant of mutant CPSase of the biologic activity of different levels.

According to the data (Delannay S. etc., molecular biology magazine, 286 volumes, 1217-1228,1999) that S.Delannay etc. is obtained, (S948F) is insensitive to UMP for the mutant of intestinal bacteria carbamyl phosphate synthetase.According to these data, select to comprise on the CPSase that the part of No. 948 positions is as the target of modifying.

Described mutant CPSase and mutant carB gene obtain by the random fragment directed mutagenesis.In order on the carB gene, to obtain a plurality of sudden changes, with the fragment randomization (vide infra) of 15 Nucleotide of the carB gene of the part of No. 947 leucine-No. 951 glutaminic acid residues on the aminoacid sequence of coding SEQ ID NO:20.The fragment of randomized 15 Nucleotide can produce 4 ¹²Or near 1.5 * 10 ⁷Individual different dna sequence dna, these sequences 4 * 10 of the pentamer peptide of encoding ⁵Plant different amino-acid residues.The possibility of not introducing terminator codon in the frame of these sequences is approximately 0.95 or 95.Therefore, the encode segmental randomization of carB of 947-951 amino-acid residue of described peptide must produce about 4 * 10 ⁵Kind different aminoacid sequences has diversity at this peptide fragment of CPSase structure.Select subsequently and screen the recombinant clone that carries the mutant carB gene of being cloned on the expression vector, can select to have the fbr varient of mutant CPSase of the biologic activity of different levels.

The present invention has defined the aminoacid sequence of the phenotypic mutant CPSase of fbr of suitable CPSase.Therefore, according to described sequence, import wild-type carB gene by suddenling change and to obtain mutant CPSase with usual way.Wild-type carB gene can be colibacillary carB gene (the 10158-13379 Nucleotide on the sequence of GenBank preserving number AE000113 U00096:SEQ ID NO:19).The carA gene is equivalent to the 8992-10140 Nucleotide on the sequence of GenBank preserving number U00096.

Be used as under the situation of material of the DNA that obtains encoding mutant type CPSase the big subunit of this mutant of mutant carB genes encoding CPSase at the carB gene.Be used as at the carAB gene under the situation of described material, mutant carAB gene is big subunit and the small subunit of encoding mutant type CPSase simultaneously.

947-951 on the mutant CPSase of the present invention number locational aminoacid sequence is any one sequence among the SEQ ID NO:1-9.The amino acid sequence corresponding of known fbr CPSase has been shown in table 1, and wherein, No. 948 locational Serine is replaced by phenylalanine, and the aminoacid sequence of intestinal bacteria wild-type CPSase.In table 1, also show the example of the nucleotide sequence of encode such amino acid sequences.

Table 1

Clone's numbering	The sequence in the proteic randomization of CarB zone (947 → No. 951 amino acid)	SEQ ID NO：	The dna sequence dna of the random fragment of CarB group (5 ' → 3 ')	SEQ ID NO：
					Wt	-Leu-Ser-Val-Arg-Glu-	28	CTTTCCGTGCGCGAA	30
6 (single sudden changes)	-Leu-Phe-Val-Arg-Glu-	29	CTTTTCGTGCGCGAA	31
					10	-Pro-Leu-Arg-Glu-Gly-	1	CCTCTCCGTGAGGGT	10
12	-Ala-Val-Ala-Leu-Lys-	2	GCTGTCGCTTTGAAA	11
					13	-Gly-Val-Phe-Leu-Met-	3	GGTGTCTTCCTAATG	12
27	-Phe-Phe-Cys-Phe-Gly-	4	TTTTTCTGTTTTGGG	13
					31	-Pro-Thr-Gly-Arg-Arg-	5	CCTACCGGTAGGAGA	14
33	-Phe-Ala-Cys-Gly-Val-	6	TTCGCCTGTGGGGTG	15
					34	-Val-Phe-Gly-Ser-Ser-	7	GTTTTCGGTAGTAGT	16
36	-Ala-Ser-Gly-Val-Glu-	8	GCTTCCGGCGTTGAG	17
					37	-Ala-Phe-Cys-Gly-Val-	9	GCCTTCTGTGGGGTG	18

Mutant CPSase can be included in one or more locational one or several amino acid whose disappearance, replacement, insertion or the interpolation except the 947-951 position, only otherwise the activity of infringement CPSase just." several " amino acid whose quantity is according to the position of the amino-acid residue on this protein three-dimensional structure or type and different.This is because following reason.In other words, have higher homology between some amino acid, and these amino acid whose difference can not produce big influence to this proteic three-dimensional structure.Therefore, mutant CPSase of the present invention can be not less than 30-50% with the homology of all amino-acid residues that constitute CPSase, preferred 50-70%, and it has fbr CPSase activity.Under the condition that has uridine 5 '-a phosphoric acid, it is active 25% that the CPSase activity that described mutant CPSase keeps is not less than wild-type CPSase ideally, preferably is not less than 30%, more preferably is not less than 40%.

In the present invention, " aminoacid sequence that is equivalent to the sequence of 947-951 position " expression is equivalent to the aminoacid sequence of 947-951 number locational aminoacid sequence on the aminoacid sequence of SEQ ID NO:20.The position of amino-acid residue can change.For example, if an amino-acid residue is inserted the N-terminal portions, be positioned at No. 947 locational amino-acid residues originally and just become the position No. 948.In this case, be equivalent to the amino-acid residue of original No. 947 positions, be called as locational amino-acid residue in the present invention No. 947.

The degree of phrase " feedback inhibition of uridine 5 '-a phosphoric acid is desensitized " expression feedback inhibition reduces.The reduction of feedback inhibition degree can be measured by the following method: be determined at the active reduction of CPSase under the condition of uridine 5 '-a phosphoric acid, and itself and the proteic activity with aminoacid sequence of SEQ IDNO:20 are compared.In addition, phrase " feedback inhibition of uridine 5 '-a phosphoric acid is by desensitization " expression restraining effect desensitizes just enough basically, and does not need to desensitize fully.Specifically, when making substrate with the 5mM glutamine, under the condition that 10mM uridine 5 '-a phosphoric acid is arranged, the activity of mutant CPSase is not less than 50% ideally with the ratio of activity under the condition that does not have uridine 5 '-a phosphoric acid, preferably be not less than 70%, more preferably be not less than 90%.

For example, can be by modifying described nucleotide sequence such as the method for site-directed mutagenesis, obtain coding and the essentially identical proteic DNA of said mutation type CPSase, so that make one or several amino-acid residue that is positioned on the specific site lack, replace, insert or add.Above-mentioned dna modification can be handled by known mutations commonly used and realize.The method that extracorporeal treatment contains the DNA of mutant carB gene that is included in is handled in described sudden change, for example, handle with azanol, and handle microorganism with ultraviolet radiation or the mutagenic compound that are normally used for this processing such as N-methyl-N '-nitro-N-guanidine nitrite (NTG) and nitrous acid, for example, the method that has the bacterium that belongs to Escherichia of mutant carB gene.

The replacement of above-mentioned Nucleotide, disappearance, insertion or add the sudden change (mutant or varient) that also comprises natural generation, for example, based on the difference of bacterium individuality or the difference of kind or genus with CPSase.

Can by separation energy from accepted cell that sudden change handles with mutant CPSase under stringent condition with DNA as the known carB gene order of having of probe or its a part of DNA hybridization, have the active proteic DNA of CPSase with coding, obtain the basic proteic DNA identical of coding with mutant CPSase.

In this article, term " stringent condition " expression can form so-called specific hybrid, and can not form the condition of non-specific hybridization.It all is difficult accurately expressing this condition with any digitized value.But, for instance, stringent condition comprises a kind of like this condition, and for example, under this condition, homology each other is not less than 50% DNA can hybridize, and can not hybridize and homology each other is lower than the DNA of above standard.In addition, stringent condition can be expressed as the condition that DNA can be hybridized each other under the salt concn of the common wash conditions in being equivalent to Southern hybridization, for example, 60 ℃, 1XSSC, 0.1%SDS, preferred 0.1XSSC, 0.1%SDS.

The gene that can hybridize is included in the gene that has produced a terminator codon in the coding region of this gene under these conditions, and owing to the sudden change in active centre does not have active gene.But, can eliminate above-mentioned difficulties easily by the following method: described gene is connected on the business-like expression vector, and the CPSase activity of institute's expressed proteins.

When CPSase of the present invention is that described small subunit can be the small subunit of intestinal bacteria wild-type CPSase when comprising the heterodimer of big subunit of mutant and small subunit.

In the present invention, small subunit can comprise and appears at one or more locational one or several amino acid whose disappearances, replacement, insertion or interpolation, as long as it can have the CPSase activity when combining with big subunit.The implication of the implication of term " several " when above describing big subunit is identical.

As the DNA of the coding polypeptide substantially the same with above-mentioned small subunit, it can be the DNA that can hybridize with the DNA that contains carA or its part under stringent condition.The implication of term " stringent condition " is with mentioned above identical.

＜2〉bacterium that belongs to Escherichia of the present invention.

The bacterium that belongs to Escherichia of the present invention is to the bacterium that belongs to Escherichia that has wherein imported mutant carB gene.For example, the bacterium that belongs to Escherichia is intestinal bacteria.For example, can be by transforming the bacterium belong to Escherichia with recombinant DNA, thus importing mutant carB gene, described recombinant DNA comprises the carrier and the described mutant carB gene that can work in belonging to the bacterium of Escherichia.Can also be by importing mutant carB gene with the carB gene on the mutant carB gene substituted dyeing body.

For example, the carrier that is used to import described mutant carB gene can be the plasmid vector such as pBR322, pMW118 or pUC19, phage vector such as 11059,1BF101 or the M13mp9, and the transposon such as Mu, Tn10 or Tn5.

For example, can finish the bacterium that the DNA importing is belonged to Escherichia by the following method: the method for D.A.Morrison (Enzymology method, 68,326,1979) or with calcium chloride handle the acceptor bacterium cell, so that improve method (Mandel, the M. of the penetrativity of DNA, and Higa, A., molecular biology magazine, 53,159,1970) etc.

By mutant carB gene is imported the above-mentioned production bacterium that belongs to Escherichia, can improve output, described compound such as L-arginine, citrulline and pyrimidine derivatives by the carbamyl phosphate derived compounds.In addition, can give the prior bacterium that has imported mutant carB gene and produce such as the L-arginine ability of the compound of citrulline and pyrimidine derivatives.Above-mentioned pyrimidine derivatives comprises vitamin B13, uridine, UMP, cytidine and CMP.

For example, has the arginic active bacterium that belongs to Escherichia of the L-of generation, can be coli strain AJ11531 and AF11538 (JP56106598A2), AJ11593 (FERMP-5616) and AJ11594 (FERM P-5617) (Japanese patent application publication No. 57-5693), VKPM B-7925 (Russ P application number 2000117677).Bacterial strain VKPM B-7925 had been deposited in Russian state-run industrial microorganism preservation center (VKPM) already on April 10th, 2000.

Also do not know at present to belong to the L-citrulline production bacterium of Escherichia, the vitamin B13 that belongs to Escherichia is produced bacterium, and uridine 5 '-a phosphoric acid (UMP) the production bacterium that belongs to Escherichia.

For example, has the active bacterium that belongs to bacillus of the L-citrulline of generation, can be bacillus subtilis strain K-X-1 A-1 (ATCC No 15561) and K-X-1 A-9 (ATCCNo15562) (United States Patent (USP) 3282794), Bacillus strain cit-70 (Japanese Laid-Open Patent Application 08-089269).For example, having the active bacterium that belongs to brevibacterium sp of the L-citrulline of generation, can be brevibacterium flavum strains A J3408 (FERM P-1645) (United States Patent (USP) 5164307) and AJ11677 (Japan later stage publication application number 57-163488).For example, having the active bacterium that belongs to Corynebacterium of the L-citrulline of generation, can be Corynebacterium glutamicum strain AJ11588 (FERM P-5643) (United States Patent (USP) 5164307).

For example, having the active bacterium that belongs to bacillus of the vitamin B13 of generation, can be bacillus subtilis strain FERM P-11402, and it lacks orotate phosphoribosyl-transferase (Japanese Laid-Open Patent Application 04-004891).For example, has the active bacterium that belongs to Corynebacterium of the UMP of generation, can be the Corynebacterium glutamicum strain T-26 (FERMBP-1487) of anti-5 FU 5 fluorouracil, the T-29 (FERMBP1488) of its anti-5 FU 5 fluorouracil and Trimethoprim BP, and the T-30 of anti-5 FU 5 fluorouracil and Sulphaguanidine (FERM BP1489) (European patent EP 0312912).

For example, having the active bacterium that belongs to Corynebacterium of the UMP of generation, can be to produce ammonia coryneform bacterial strains LK40-2 (VKPM B-7811), LK75-15 (VKPM B-7812) and LK75-66 (VKPM B-7813) (Russ P application number 99122774).

＜3〉method of production L-arginine, citrulline and pyrimidine derivatives.

Can effectively produce the L-arginine by the following method, compound such as citrulline and pyrimidine derivatives: in substratum, cultivate to wherein having imported mutant carB gene, and bacterium with the ability that produces described compound, in described substratum, produce and accumulate described compound, and from described substratum, collect described compound.Above-mentioned pyrimidine derivatives comprises vitamin B13, uridine, UMP, cytidine and CMP.

In the method for the invention, belong to the cultivation of the bacterium of Escherichia, from liquid nutrient medium, collect and purifying compounds, can utilize bacterium to finish with being similar to by the mode of the arginic ordinary method of fermentative production L-.The substratum that is used to cultivate can be synthetic medium or natural medium, as long as this substratum comprises an amount of carbon source and nitrogenous source and mineral substance, if necessary, also has the needed an amount of nutrient of employed bacterium just.Carbon source can comprise various carbohydrate, as dextrose plus saccharose, and various organic acid, this depends on the assimilative capacity of the bacterium of using.Can use the alcohol that comprises ethanol and glycerine.As nitrogenous source, can use ammonia, such as the various ammonium salts of ammonium sulfate, such as other nitrogen compound of amine, such as the natural nitrogenous source of peptone, soybean hydrolyzate and the organism of fermentation that digested.As mineral substance, can use potassium primary phosphate, sal epsom, sodium-chlor, ferric sulfate, manganous sulfate, lime carbonate.

Cultivate and preferably under aerobic conditions, carry out, as shaking table cultivation, aerated culture and stir culture.Cultivation is normally carried out under 20-40 ℃ temperature, preferred 30-38 ℃.Cultivate normally at pH5-9, carry out under the condition of preferred 6.5-7.2.The pH of substratum can use ammonia, lime carbonate, various acid, various alkali and damping fluid to regulate.Usually, cultivation will cause described compound to accumulate in substratum in 1-3 days.

After cultivating, can remove such as solid matters such as cells by centrifugal or membrane filtration, separate described compound, collect then and the described compound of method purifying by ion-exchange, concentrated and crystallization stage part and so on.

The accompanying drawing summary

Fig. 1 represents to make up the synoptic diagram of plasmid pEL-carAB-wt.

Fig. 2 represents to make up the synoptic diagram of mutant carB gene pool.

Embodiment

To do the present invention in conjunction with the following examples and specify.

Embodiment 1

By being cloned into prior pBluescript II SK (+) carrier (Fermentas that digested with BamHI and PstI from the chromosomal AvaIII-BglII dna fragmentations of intestinal bacteria (4911bp), Lithuania) on, make up the plasmid pBScarAB-13 that carries from colibacillary wild-type carAB gene.

By replacing the T7 promotor with the lac promotor, (Novagen USA) modifies to plasmid pET22-b (+).The Lac promotor is by doing template with plasmid pUC18, and do primer with oligonucleotide 5 '-accagatctgcgggcagtgagcaacgc-3 ' (SEQ ID NO:21) and 5 '-gtttctagatcctgtgtgaaattgttatccgc-3 ' (SEQ ID NO:22), obtain by pcr amplification.With the resulting fragment (0.14kb) of restriction enzyme BglII and XbaI digestion, and be cloned on pET22-b (+) carrier that the identical restriction enzyme of prior usefulness digested with lac promotor.Resulting plasmid pET-Plac is used to clone the carAB gene that is not activated son from plasmid pBScarAB-13.

Make template with pBScarAB-13, and make primer with oligonucleotide 5 '-cctctagaaataaagtgagtgaatattc-3 ' (SEQ ID NO:23) and 5 '-cttagcggttttacggtactgc-3 ' (SEQ ID NO:24), obtain 5 ' terminal (1.18kb) of carA gene by pcr amplification.Digest resulting fragment with XbaI and DraIII, and, the XbaI-DraIII fragment (0.61kb) of 5 ' end sequence of carA gene had by the agarose electrophoresis purifying.Connect this fragment, from the mixture of the DraIII-SacI fragment of plasmid pBScarAB-13 (3 ' terminal sequence) and carrier pET-Plac/XbaI-SacI with carA gene and carB gene, and be transformed into the e. coli tg1 cell.The recombinant plasmid pEL-carAB-wt that is obtained has the sequence of the wild-type carAB operon that is subjected to the control of lac promotor.

The TaKaRa La archaeal dna polymerase that is used for pcr amplification obtains from Takara Shuzo company (Japan), and uses under the condition that the supplier recommends.

＜1〉random fragment directed mutagenesis

PBScarAB-13 makes template with plasmid, sense primer P1:5 '-ggtcgtgcgctgNN (T/C) N (T/C) CNN (T/C) NNN (G/A) NNggcgataaagaacgcgtggtg-3 ' (SEQ ID NO:25) (48 bases) is according to the design of the nucleotide sequence of carB gene, and makes antisense primer with standard M13 targeting sequencing primer.3 ' end region of 5 ' terminal and 21 Nucleotide of fixed of 12 Nucleotide of fixed of primer P1 is respectively with the sequence homology of carB gene Glu951 codon downstream and Leu947 codon upstream.

In first round PCR (15 circulations), with the dna fragmentation (3 '-end of carB gene) of the synthetic 0.75kb of the primer P1 with randomization 15 Nucleotide districts.First round PCR carries out by the following method.100 nanogram plasmid pBScarAB-13 are added to as template contain in the PCR solution of two kinds of primers that concentration is 10 picomole (50 microlitre).With 2400 type DNA thermal cyclers (Perkin-Elmer company, Foster city, markon Fu Niya) carry out 15 PCR circulation (94 ℃ 15 seconds, 52 ℃ 20 seconds, 72 ℃ 1 minute).

Take turns in the amplification second, carry out again 15 take turns amplification (94 ℃ 1 minute, 35 ℃ 1 minute, 72 ℃ 2 minutes), wherein, this segmental (-) chain plays " primer " effect, extends, so that obtain complete gene order.

In the third round amplification, the aliquot sample of 10 these reaction mixtures of microlitre added to contain 100 picomole sense primers: in standard M13 targeting sequencing primer and the fresh 90 microlitre reaction mixtures as the P2:5 '-ccacttcctcgatgacgcgg-3 ' (SEQ ID NO:26) of antisense primer, and carry out 15 circulations (94 ℃ 0.5 minute again, 55 ℃ 20 seconds, 72 ℃ 2 minutes).

The 1.32kb dna fragmentation in the mutant varient library of 3 '-terminal fragment by agarose gel electrophoresis purifying coding carB gene, then with AflII and SacI digestion, and the pEL-carAB-wt carrier that digested of further identical with prior usefulness restriction enzyme connects, so that obtain pEL-carAB-NN.

The DNA that in experiment subsequently about 150 nanograms is connected is used for transformed into escherichia coli TG1 (supE hsd Δ 5 thi Δ (lac-proAB) F ' [traD36 proAB ⁺LacI ^qLacZ Δ M15]) (J.Sambrook etc., molecular cloning, 1989) recipient cell, so that all obtain about 2000 clones each time.Purification of Recombinant plasmid (pEL-carAB-NN) library, and change Bacillus coli cells VKPM B-6969 (carB::Tn10) over to, use it for the recombinant plasmid pEL-carAB-NN that screening has the carAB gene of the active CarAB enzyme of coding.

＜2〉site-directed mutagenesis

In order to import single sudden change Ser948Phe, pBScarAB-13 makes template with plasmid, and uses sense primer 5 '-cgtgcgctgcttttcgtgcgcgaaggcgataaag-3 ' (34 bases) (SEQ ID NO:27) and standard M13 targeting sequencing primer according to the design of the nucleotide sequence of carB gene to carry out PCR as antisense primer.Pcr amplification and this segmental clone carry out as stated above.

By agarose gel electrophoresis, purifying has the 1.32kb dna fragmentation of 3 ' terminal fragment of the coding carB gene of single sudden change, with AflII and SacI digestion, is connected to then on the pEL-carAB-wt carrier that the identical restriction enzyme of prior usefulness digested.

With the resulting DNA plasmid transformation escherichia coli of about 100 nanograms cell VKPM B-6969, and the recombinant plasmid pEL-carAB-6 of the active CarAB enzyme with single replacement Ser948Phe is carried in screening.

Embodiment 2.The separation of new carB mutant, and in the influence of CPSase upper amino acid replacement to catalysis characteristics

At first on 40 kinds of reorganization B-6969 (pEL-carAB-NN) clones, in by carAB and the enzymatic reaction of ArgI (ornithine aminomethyl transferring enzyme) by ornithine biosynthesizing citrulline, assessment carAB activity and to the anti-retroactive effect of UMP.

The formula of reaction is as follows:

CarAB+ArgI

NH ₃+ HCO ₃ ^-+ 2MgATP+ ornithine------→ citrulline+2MgADP+2Pi

The free NH of carbamyl phosphate synthetase in this reaction ₄ ⁺Make substrate

From the protein extract of 40 kinds of B-6969 (pEL-carAB-NN) bacterial strains and TG1 (pUC18-argI) cell, be to prepare the rough cell extract by the cell crossed from ultrasonication with ammonium sulfate (75% saturation ratio) precipitation.Described albumen precipitation is dissolved in the damping fluid with following prescription: Tris-HCl (50mM), pH7.5,2 mercapto ethanol (2mM).

This test macro comprises from the protein extract of bacterial strain B-6969 (pEL-carAB-NN) and TG1 (pUC18-argI) and following reagent: ATP (8mM), sal epsom (8mM), ammonium sulfate (200mM), yellow soda ash (8mM) and ornithine (1mM), pH7.5.Use has the liquid phase of following prescription, by the content of ornithine and citrulline in the TLC analyze reaction mixture.Virahol/ethyl acetate/ammonium hydroxide/water=40/20/13/27 (v/v).

With expression activity and mutant CPSase that UMP is had anti-retroactive effect 9 clones with express the activity that a clone with mutant CPSase that single S er948Phe replaces is used to measure mutant enzyme.

Purifying is from above-mentioned 10 clones' plasmid, and the sequence (table 1) of the random fragment by double deoxidating chain termination measuring carB gene.

To be used at the activity and the fbr that assess mutant CPSase by the reaction of glutamine or ammonia synthesis carbamyl phosphate (CP) from the protein extract of above-mentioned 9 clone B-6969 (pEL-carAB-NN) and a clone B-6969 (pEL-carAB-6) then.

Rough cell extract from cell prepares by the following method: to being suspended in 0.5 milliliter of buffer A (200mM potassium hydrogen phosphate/potassium primary phosphate, pH8.0,1mM EDTA, 1mMPMSF, 1mM DTT) 20 milligrams of wet cell precipitations carry out ultrasonication, then, handle, reach 65% saturation ratio with solid ammonium sulfate.After 4 ℃ are down cultivated 10 minutes, with the speed of 13000rpm with centrifugal 10 minutes of suspension, then with resolution of precipitate in 1 milliliter of buffer B (20mM potassium hydrogen phosphate/potassium primary phosphate, pH8.0,50mM Repone K, 1mM PMSF, 1mM DTT).Aliquot sample assessment CPSase activity with the protein extract that is obtained.The reaction formula is as follows:

I. glutamine+CO ₂+ 2MgATP+H ₂O → NH ₂COOPO ₃ ^2-+ 2 MgADP+ L-glutamic acid+Pi

II.NH ₃+CO ₂+2MgATP+H ₂O→NH ₂COOPO ₃ ^2-+2 MgADP+Pi

Each reaction mixture of 50 microlitres comprises:

Reaction I--20mM Tris-HCl, pH8.0,100mM Repone K, 5mM yellow soda ash, 10mM ATP, 10mM magnesium chloride, 5mM glutamine, 10 microlitre protein extracts;

Reaction II--20mM Tris-HCl, pH8.0,100mM Repone K, 5mM yellow soda ash, 10mM ATP, 10mM magnesium chloride, 200mM ammonium sulfate, 10 microlitre protein extracts.

In addition, under the situation that 10mM UTP is arranged, carry out a series of reaction I, so that the feedback inhibition level of assessment CPSase.

After 37 ℃ of following incubations 10 minutes, by adding isopyknic ethanol termination reaction ,-20 ℃ of coolings 10 minutes down, and at room temperature with the speed of 13000rpm centrifugal 1 minute.Cool off supernatant liquor down at-20 ℃.

Content by CP in the capillary zone electrophoresis analyze reaction mixture.Separation is that (" Waters " carries out on USA), uses the UV indirect detection under 254 nanometers at Quanta 4000E capillary electrophoresis system.Implement injection 25 seconds by hydrostaticpressure.Described separation is (useful length is 53 centimetres for 75u internal diameter, 60 centimetres of *) of carrying out with the fused silica kapillary of dressing not, and works under-25kV voltage.Temperature is remained on 20 ℃.Dissociating buffer is by 50mM Tris-alkali, 25mM phenylformic acid (being used for indirect detection), and pH8.5,0.25mM TTAB (tetrem acyl-trimethylammonium-brometo de amonio) (being used to recover electroosmotic flow) forms.

In table 2, show the activity of the mutant CPSase that in the CP building-up reactions, measures and the data of fbr.

The activity of table 2. mutant CPSase

Clone's numbering	Active (CP, nmole/milligram * minute)
				Substrate: 5Mmgu glutamine	Substrate: 200mM ammonium sulfate	Substrate: 5mM glutamine; Allosteric effector: 10mM UMP
	Wt	1350	425				170
6				320	220	320
	10	690	225				625
12				540	95	540
	13	350	60				350
27				730	400	670
	31	1120	375				810
33				510	150	510
	34	765	345				765
36				390	90	390
	37	475	205				475

Therefore, mutant CPSase is insensitive to UMP basically, and still, described single sudden change can significantly reduce the activity of enzyme.These results show, the feedback inhibition of the peptide fragment decision UMP of 947-951 amino-acid residue, and the catalytic efficiency of decision mutant CPSase.

With the gene clone of coding wt CarAB and mutant CarAB-34 to plasmid pMW119.For this reason, with restriction enzyme SacI and XbaI digested plasmid pEL-carAB-wt and pEL-carAB-34 (because described plasmid has two XbaI sites, carry out part digestion), and the fragment cloning of the carAB gene of will encoding is to prior pMW119 carrier with the same restrictions enzymic digestion.As a result, made up low copy number plasmid pMW119-carAB-wt and pMW119-carAB-34 with the carAB gene that is subjected to the control of lac promotor.

Embodiment 3.Produce vitamin B13 with bacterial strain with mutant carAB gene

As the saltant of anti-6-azauracil (1 mg/ml), bacterial strain 311 derives from the e. coli k12 (VKPM B-3853) with the Tn10 that inserts the argA gene.Bacterial strain 311 was deposited in Russian state-run industrial microorganism preservation center (VKPM) in March 5 calendar year 2001 already, and preserving number is VKPM B-8085, and, according on July 17th, 2002 that is specified in of budapest treaty original preservation having been changed into international preservation.

Transform bacterial strain 311 with plasmid pMW-carAB-wt and pMW-carAB-34, and under the condition of the uridine that different concns is arranged, detect the vitamin B13 output of resulting bacterial strain.

The culture condition of test tube fermentation is as follows:

1/20 overnight culture of diluting, 60 grams per liter glucose, 25 grams per liter ammonium sulfate, 2 grams per liter potassium primary phosphates, 1 grams per liter sal epsom, 0.1 mg/litre VitB1,5 grams per liter yeast extract Difco, 25 grams per liter chalks, 1 liter of tap water (pH7.2).Glucose and chalk are the difference disinfectant.2 milliliters of substratum are put into test tube, and 32 ℃ of following wave and culture 3 days.By HPLC assessment vitamin B13 output (table 3).

The vitamin B13 yield level of table 3. bacterial strain 311 (pMW119), 311 (pMW-carAB-wt), 311 (pMW-carAB-34)

Bacterial strain	Uridine, 100mg/l		Uridine, 300mg/l		Uridine, 1000mg/l
							A 550， o.u.	The vitamin B13 biosynthesizing, g/l	A 550， o.u.	The vitamin B13 biosynthesizing, g/l	A 550， o.u.	The vitamin B13 biosynthesizing, g/l
	311 (pMW119)	13.1	0.12	13.8	0.11	9.0							0.01
311 (pMW-carAB-wt)							11.4	0.27	12.2	0.18	9.8	0.03
	311 (pMW-carAB-34)	12.6	0.66	12.7	0.40	10.3							0.11

As shown in table 3, the bacterial strain 311 (pMW-carAB-34) with mutant carAB gene can produce more vitamin B13 than parent strain 311 (pMW119) with wild-type carAB gene and bacterial strain 311 (pMW-carAB-wt).

Embodiment 4.Utilization has the bacterial strain of mutant carAB gene and produces arginine and/or citrulline

As the mutant of anti-6-azauracil (1 mg/ml), from the derivative of the bacterial strain intestinal bacteria 57 (VKPM B-7386) that inserted transposon Tn5 at gene ilvA, screened arginine already and produced bacterial strain 333 and 374.Bacterial strain 333 and 374 had been deposited in Russian state-run industrial microorganism preservation center (VKPM) already in March 5 calendar year 2001, preserving number is respectively VKPM B-8084 and VKPM B-8086, in the July, 2002 that is specified in according to budapest treaty original preservation has been changed into international preservation subsequently.

Transform bacterial strain 333 and 374 with plasmid pMW-carAB-wt and pMW-carAB-34, and detect the arginine and the citrulline output of these recombinant bacterial strains.

Test tube fermentation is to carry out according to the method identical with example 3.

In the synthetic medium that contains 100 mg/litre uridines, arginine and/or the citrulline yield level of bacterial strain 333 (pMW-carAB-wt) and 333 (pMW-carAB-34) are as shown in table 4.

The arginine of table 4. bacterial strain 333 (pMW-carAB-wt) and 333 (pMW-carAB-34) and/or citrulline yield level

Bacterial strain	Absorbancy, A 560，u.	Arginine biosynthesizing level, g/l	Citrulline biosynthesizing level, g/l	Citrulline+arginine biosynthesizing level, g/l
					333 (pMW-carAB-wt)	24.1	0.60	0.39	0.99
333 (pMW-carAB-34)	20.5	1.01	0.51	1.52

In the synthetic medium that contains 100 mg/litre uridines, the citrulline yield level of bacterial strain 374 (pMW-carAB-wt) and 374 (pMW-carAB-34) is as shown in table 5.

The citrulline yield level of table 5. bacterial strain 374 (pMW-carAB-wt) and 374 (pMW-carAB-34)

Bacterial strain	Absorbancy, A 560，u.	Citrulline biosynthesizing level, g/l
			374(pMW-carAB-wt)	22.3	＜0.01
374(pMW-carAB-34)	15.5	0.26

As shown in table 4, the bacterial strain 333 (pMW-carAB-34) with mutant carAB gene can produce more arginine and citrulline than the bacterial strain with wild-type carAB gene.As shown in table 5, bacterial strain 374 (pMW-carAB-34) can produce more citrulline than the bacterial strain with wild-type carAB gene.

＜110〉Ajincomoto Co., Inc

＜120〉New-mutant carbamyl-phosphate synthesized enzyme and production are by the method for carbamyl phosphate derived compounds

<130>OP1367

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<141>2002-08-

<150>RU 2001121697

<151>2001-08-03

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cgcttggcaa aactggcggg cgtacgcgaa gcggaaatcc gtaagctgcg tgaccagtat 1560

gacctgcacc cggtttataa gcgcgtggat acctgtgcgg cagagttcgc caccgacacc 1620

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aaaatcatgg tgcttggcgg cggcccgaac cgtatcggtc agggtatcga attcgactac 1740

tgttgcgtac acgcctcgct ggcgctgcgc gaagacggtt acgaaaccat tatggttaac 1800

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Glu Phe Asp Tyr Cys Cys Val His Ala Ser Leu Ala Leu Arg Glu Asp

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Gly Tyr Glu Thr Ile Met Val Asn Cys Asn Pro Glu Thr Val Ser Thr

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Asp Tyr Asp Thr Ser Asp Arg Leu Tyr Phe Glu Pro Val Thr Leu Glu

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Asp Val Leu Glu Ile Val Arg Ile Glu Lys Pro Lys Gly Val Ile Val

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Gln Tyr Gly Gly Gln Thr Pro Leu Lys Leu Ala Arg Ala Leu Glu Ala

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Ala Gly Val Pro Val Ile Gly Thr Ser Pro Asp Ala Ile Asp Arg Ala

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Glu Asp Arg Glu Arg Phe Gln His Ala Val Glu Arg Leu Lys Leu Lys

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Gln Pro Ala Ash Ala Thr Val Thr Ala Ile Glu Met Ala Val Glu Lys

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Ala Lys Glu Ile Gly Tyr Pro Leu Val Val Arg Pro Ser Tyr Val Leu

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Gly Gly Arg Ala Met Glu Ile Val Tyr Asp Glu Ala Asp Leu Arg Arg

725 730 735

Tyr Phe Gln Thr Ala Val Ser Val Ser Asn Asp Ala Pro Val Leu Leu

740 745 750

Asp His Phe Leu Asp Asp Ala Val Glu Val Asp Val Asp Ala Ile Cys

755 760 765

Asp Gly Glu Met Val Leu Ile Gly Gly Ile Met Glu His Ile Glu Gln

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Ala Gly Val His Ser Gly Asp Ser Ala Cys Ser Leu Pro Ala Tyr Thr

785 790 795 800

Leu Ser Gln Glu Ile Gln Asp Val Met Arg Gln Gln Val Gln Lys Leu

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Ala Phe Glu Leu Gln Val Arg Gly Leu Met Asn Val Gln Phe Ala Val

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Lys Asn Asn Glu Val Tyr Leu Ile Glu Val Asn Pro Arg Ala Ala Arg

835 840 845

Thr Val Pro Phe Val Ser Lys Ala Thr Gly Val Pro Leu Ala Lys Val

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Ala Ala Arg Val Met Ala Gly Lys Ser Leu Ala Glu Gln Gly Val Thr

865 870 875 880

Lys Glu Val Ile Pro Pro Tyr Tyr Ser Val Lys Glu Val Val Leu Pro

885 890 895

Phe Asn Lys Phe Pro Gly Val Asp Pro Leu Leu Gly Pro Glu Met Arg

900 905 910

Ser Thr Gly Glu Val Met Gly Val Gly Arg Thr Phe Ala Glu Ala Phe

915 920 925

Ala Lys Ala Gln Leu Gly Ser Asn Ser Thr Met Lys Lys His Gly Arg

930 935 940

Ala Leu Leu Ser Val Arg Glu Gly Asp Lys Glu Arg Val Val Asp Leu

945 950 955 960

Ala Ala Lys Leu Leu Lys Gln Gly Phe Glu Leu Asp Ala Thr His Gly

965 970 975

Thr Ala Ile Val Leu Gly Glu Ala Gly Ile Asn Pro Arg Leu Val Asn

980 985 990

Lys Val His Glu Gly Arg Pro His Ile Gln Asp Arg Ile Lys Asn Gly

995 1000 1005

Glu Tyr Thr Tyr Ile Ile Asn Thr Thr Ser Gly Arg Arg Ala Ile Glu

1010 1015 1020

Asp Ser Arg Val Ile Arg Arg Ser Ala Leu Gln Tyr Lys Val His Tyr

1025 1030 1035 1040

Asp Thr Thr Leu Asn Gly Gly Phe Ala Thr Ala Met Ala Leu Asn Ala

1045 1050 1055

Asp Ala Thr Glu Lys Val Ile Ser Val Gln Glu Met His Ala Gln Ile

1060 1065 1070

Lys

<210>21

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<400>21

accagatctg cgggcagtga gcgcaacgc 29

<210>22

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<400>22

gtttctagat cctgtgtgaa attgttatccgc 32

<210>23

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<400>23

cctctagaaa taaagtgagt gaatattc 28

<210>24

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<400>24

cttagcggtt ttacggtact gc 22

<210>25

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<220>

＜221〉uncertain

<222>(13，14，16，19，20，21，22，23，25，26)

<223>n＝a or g or c or t

<400>25

ggtcgtgcgc tgnnynycnn ynnnrnnggc gataaagaac gcgtggtg 48

<210>26

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<400>26

ccacttcctc gatgacgcgg 20

<210>27

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<400>27

cgtgcgc tgcttttcgtgcg cgaaggcgat aaag 34

<210>28

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: by the partial amino-acid series of wild-type carB genes encoding

<400>28

Leu Ser Val Arg Glu

1 5

<210>29

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: by the partial amino-acid series of carB genes encoding with single sudden change

<400>29

Leu Phe Val Arg Glu

1 5

<210>30

<211>15

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<400>30

ctttccgtgc gcgaa 15

<210>31

<211>15

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<400>31

cttttcgtgc gcgaa 15

<210>32

<211>1149

<212>DNA

＜213〉intestinal bacteria (Escherichia coli)

<400>32

ttgattaagt cagcgctatt ggttctggaa gacggaaccc agtttcacgg tcgggccata 60

ggggcaacag gttcggcggt tggggaagtc gttttcaata cttcaatgac cggttatcaa 120

gaaatcctca ctgatccttc ctattctcgt caaatcgtta ctcttactta tccccatatt 180

ggcaatgtcg gcaccaatga cgccgatgaa gaatcttctc aggtacatgc acaaggtctg 240

gtgattcgcg acctgccgct gattgccagc aacttccgta ataccgaaga cctctcttct 300

tacctgaaac gccataacat cgtggcgatt gccgatatcg atacccgtaa gctgacgcgt 360

ttactgcgcg agaaaggcgc acagaatggc tgcattatcg cgggcgataa cccggatgcg 420

gcgctggcgt tagaaaaagc ccgcgcgttc ccaggtctga atggcatgga tctggcaaaa 480

gaagtgacca ccgcagaagc ctatagctgg acacaaggga gctggacgtt gaccggtggc 540

ctgccagaag cgaaaaaaga agacgagctg ccgttccacg tcgtggctta tgattttggt 600

gccaagcgca acatcctgcg gatgctggtg gatagaggct gtcgcctgac catcgttccg 660

gcgcaaactt ctgcggaaga tgtgctgaaa atgaatccag acggcatctt cctctccaac 720

ggtcctggcg acccggcccc gtgcgattac gccattaccg ccatccagaa attcctcgaa 780

accgatattc cggtattcgg catctgtctc ggtcatcagc tgctggcgct ggcgagcggt 840

gcgaagactg tcaaaatgaa atttggtcac cacggcggca accatccggt taaagatgtg 900

gagaaaaacg tggtaatgat caccgcccag aaccacggtt ttgcggtgga cgaagcaaca 960

ttacctgcaa acctgcgtgt cacgcataaa tccctgttcg acggtacgtt acagggcatt 1020

catcgcaccg ataaaccggc attcagcttc caggggcacc ctgaagccag ccctggtcca 1080

cacgacgccg cgccgttgtt cgaccacttt atcgagttaa ttgagcagta ccgtaaaacc 1140

gctaagtaa 1149

<210>33

<211>382

<212>PRT

＜213〉intestinal bacteria (Escherichia coli)

<400>33

Leu Ile Lys Ser Ala Leu Leu Val Leu Glu Asp Gly Thr Gln Phe His

1 5 10 15

Gly Arg Ala Ile Gly Ala Thr Gly Ser Ala Val Gly Glu Val Val Phe

20 25 30

Asn Thr Ser Met Thr Gly Tyr Gln Glu Ile Leu Thr Asp Pro Ser Tyr

35 40 45

Ser Arg Gln Ile Val Thr Leu Thr Tyr Pro His Ile Gly Asn Val Gly

50 55 60

Thr Asn Asp Ala Asp Glu Glu Ser Ser Gln Val His Ala Gln Gly Leu

65 70 75 80

Val Ile Arg Asp Leu Pro Leu Ile Ala Ser Asn Phe Arg Asn Thr Glu

85 90 95

Asp Leu Ser Ser Tyr Leu Lys Arg His Asn Ile Val Ala Ile Ala Asp

100 105 110

Ile Asp Thr Arg Lys Leu Thr Arg Leu Leu Arg Glu Lys Gly Ala Gln

115 120 125

Asn Gly Cys Ile Ile Ala Gly Asp Asn Pro Asp Ala Ala Leu Ala Leu

130 135 140

Glu Lys Ala Arg Ala Phe Pro Gly Leu Asn Gly Met Asp Leu Ala Lys

145 150 155 160

Glu Val Thr Thr Ala Glu Ala Tyr Ser Trp Thr Gln Gly Ser Trp Thr

165 170 175

Leu Thr Gly Gly Leu Pro Glu Ala Lys Lys Glu Asp Glu Leu Pro Phe

180 185 190

His Val Val Ala Tyr Asp Phe Gly Ala Lys Arg Asn Ile Leu Arg Met

195 200 205

Leu Val Asp Arg Gly Cys Arg Leu Thr Ile Val Pro Ala Gln Thr Ser

210 215 220

Ala Glu Asp Val Leu Lys Met Asn Pro Asp Gly Ile Phe Leu Ser Asn

225 230 235 240

Gly Pro Gly Asp Pro Ala Pro Cys Asp Tyr Ala Ile Thr Ala Ile Gln

245 250 255

Lys Phe Leu Glu Thr Asp Ile Pro Val Phe Gly Ile Cys Leu Gly His

260 265 270

Gln Leu Leu Ala Leu Ala Ser Gly Ala Lys Thr Val Lys Met Lys Phe

275 280 285

Gly His His Gly Gly Asn His Pro Val Lys Asp Val Glu Lys Asn Val

290 295 300

Val Met Ile Tbr Ala Gln Asn His Gly Phe Ala Val Asp Glu Ala Thr

305 310 315 320

Leu Pro Ala Asn Leu Arg Val Thr His Lys Ser Leu Phe Asp Gly Thr

325 330 335

Leu Gln Gly Ile His Arg Thr Asp Lys Pro Ala Phe Ser Phe Gln Gly

340 345 350

His Pro Glu Ala Ser Pro Gly Pro His Asp Ala Ala Pro Leu Phe Asp

355 360 365

His Phe Ile Glu Leu Ile Glu Gln Tyr Arg Lys Thr Ala Lys

370 375 380

Claims (12)

1. the big subunit of carbamyl phosphate synthetase, wherein, the aminoacid sequence of the 947-951 position of SEQ ID NO:20 is replaced by any one aminoacid sequence among the SEQ ID NO:1-9, and the feedback inhibition of uridine 5 '-a phosphoric acid is desensitized.

2. the big subunit of the carbamyl phosphate synthetase of claim 1, wherein, described carbamyl phosphate synthetase is from colibacillary wild-type carbamyl phosphate synthetase.

3. the big subunit of a carbamyl phosphate synthetase, its aminoacid sequence by the 947-951 position among the SEQ ID NO:20 is formed by the aminoacid sequence that any one aminoacid sequence among the SEQ ID NO:1-9 is replaced the back gained, and the feedback inhibition of uridine 5 '-a phosphoric acid is desensitized.

4. the big subunit of the carbamyl phosphate synthetase of claim 1, it is included in locational one or several amino acid whose disappearance, replacement, insertion or interpolation except the 947-951 position, and wherein, the feedback inhibition of uridine 5 '-a phosphoric acid is desensitized.

5. carbamyl phosphate synthetase, it comprises the big subunit of carbamyl phosphate synthetase any among the claim 1-4.

6. the DNA of each the big subunit of carbamyl phosphate synthetase among the claim 1-4 that encodes, wherein, the feedback inhibition of uridine 5 '-a phosphoric acid is desensitized.

7. the big subunit of carbamyl phosphate synthetase that each uridine 5 '-phosphoric acid feedback inhibition is desensitized among the claim 1-4 that encodes and the DNA of intestinal bacteria carbamyl phosphate synthetase small subunit, wherein, the albumen formed by the aminoacid sequence shown in the SEQ ID NO:33 of described small subunit.

8. bacterium that belongs to Escherichia, it has the DNA of claim 6 or 7.

9. the bacterium of claim 8, it has the ability of producing the compound that is selected from L-arginine, citrulline and pyrimidine derivatives.

10. the bacterium of claim 9, wherein, described pyrimidine derivatives is vitamin B13, uridine, uridine 5 '-a phosphoric acid, cytidine and cytidine 5 '-a phosphoric acid.

11. a production is selected from the method for the compound of L-arginine, citrulline and pyrimidine derivatives, this method may further comprise the steps: cultivate any one bacterium among the claim 8-10 in substratum, so that in this substratum, produce and accumulate described compound, and from described substratum, collect described compound.

12. the method for claim 11, wherein, described pyrimidine derivatives is vitamin B13, uridine, uridine 5 '-a phosphoric acid, cytidine and cytidine 5 '-a phosphoric acid.

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