CN1405302A - 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

Novel mutant carbamoyl phosphate synthetase and method for producing carbamoyl phosphate-derived compound

Technical Field

The present invention relates to the microbiology industry, in particular to a method for producing compounds derived from carbamyl phosphate. More particularly, the present invention relates to the production of compounds derived from carbamoyl phosphate, such as arginine, citrulline and pyrimidine derivatives including orotic acid, uridine 5 '-monophosphate (UMP), cytidine and cytidine 5' -monophosphate (CMP), using novel anti-feedback enzymes involved in the arginine and pyrimidine biosynthetic pathways of e.

Background

The carbamoyl phosphate synthetase (CPSase) from E.coli catalyses a complex synthetic process to produce Carbamoyl Phosphate (CP) from bicarbonate, glutamine and two Mg-ATP molecules with the release of glutamate, phosphate and two Mg-ADP [ Meister A., Advan. Enzymol. mol. biol., vol. 62, page 315-374, 1989]. The synthesis of CP is an intermediate step in the two biosynthetic pathways, namely the biosynthetic pathway of pyrimidine nucleotides and arginine. In the first pathway, CP is coupled to aspartate carbamoyltransferase (ATCase), resulting in the formation of orotate via two steps. Orotic acid is an important metabolic intermediate in the biosynthesis of pyrimidine derivatives, including pyrimidines such as uracil; pyrimidine nucleosides such as orotidine, uridine, and cytidine; and pyrimidine nucleotides such as orotidine 5' -monophosphate (OMP), UMP and CMP. The presenceof orotic acid in the culture medium has been shown to contribute significantly to the production and accumulation of the pyrimidine derivative, uracil, during fermentation by a variety of bacteria (US patent 3214344). In the second pathway, CP is coupled to ornithine via an ornithine carbamoyltransferase (OTCase)The sixth step of the arginine biosynthetic pathway (starting from glutamate). CPSase is activated by ornithine and IMP (precursor of purine nucleotides) and is inhibited by UMP. Carbamoyl phosphate synthetase consists of two subunits. For coryneform bacteria (EP1026247A1) and bacteria belonging to the genus Escherichia and Bacillus, it has been demonstrated that the subunits are encoded by the carA and carB genes. The transcription of the carAB operon is cumulatively inhibited by the end products of both pathways [ Charlie D. et al, J. mol. biol., Vol. 226, pp. 367-386, 1992; wang H, et al, J molecular biology, volume 277, page 805-824, 1998; glansdorff N, et al, pyrimidine pathway, Vol.6, pp.53-62, 1998]. The natural Escherichia coli CPSase is composed of a small subunit of 41270Da and a117710Da, encoded by the carA and carB genes, respectively. The small subunit catalyzes the hydrolysis of glutamine and is responsible for NH₃Transfer to the Large subunit and actual Synthesis of CP on the Large subunit. The large subunit includes a binding site for substrates, i.e., ammonium carbonate, ammonia, two independent sites for binding Mg-ATP, and an 18kDa carboxy-terminal region that constitutes the regulatory domain [ Rubio V, et al, biochemistry, Vol.30, pp.1068-1075, 1991; cervera J. et al, biochemistry, Vol.35, pp.7247-7255, 1996]. In addition, it is considered that the large subunit has an activity of catalyzing the synthesis reaction of carbamyl phosphate alone (Stephen D. Rubino et al, J. biol. chem., 206, 4382-4386, 1987).

The crystalline structure of the allosterically activated form of CPSase has recently been disclosed [ Thoden j. et al, biochemistry, vol 36, pp 6305-6316, 1997; thoden j. et al, actacrystallogr.sec.d., volume 55, pages 8-24, 1999]. The first three independent domains on the large subunit, designated A, B, C, are very similar in structure, but the fourth is completely different. The D domain (residue 937-1073) is responsible for binding and for the binding of effector: IMP, UMP and ornithine are allosterically regulated. In addition, it has been shown that two residues, serine 948 and threonine 1042, appear to play a critical role in the allosteric regulation of CPSase [ Delannay S. et al, J. mol. biol., 286, 1217-1228, 1999]. When serine 948 is substituted with phenylalanine, the enzyme becomes insensitive to UMP and IMP, but is still activated by ornithine, although to a reduced extent. The enzyme with the T1042I mutation showed a strong reduction in ornithine activation.

In principle, the anti-feedback (fbr) phenotype of an enzyme occurs as a result of the substitution of the corresponding amino acid residue with another amino acid at one or several positions in the amino acid sequence, and this substitution results in a decrease in the activity of the enzyme. For example, the substitution of native Met256 on Serine Acetyltransferase (SAT) of E.coli (cysE gene) with each of 19 other amino acid residues results in the fbr phenotype in most cases, but the mutant SAT protein fails to restore the activity level of native SAT [ Nakamori S. et al, AEM, Vol.64, p.1607-1611, 1998]. Therefore, the mutant enzyme obtained by the above method has a drawback in that the activity of the mutant enzyme is reduced as compared with the wild-type enzyme.

Disclosure of Invention

The present invention relates to the construction of feedback resistant, highly active enzymes that play a key role in E.coli pyrimidine and arginine or citrulline biosynthesis.

In the present invention, a novel method for synthesizing a large number of mutant carB genes by completely randomizing carB gene fragments has been proposed. Simultaneous substitution of certain amino acid residues on amino acid sequence fragments (where the fbr mutation can be located) can result in mutant proteins that restore activity levels close to the native protein, since the three-dimensional structure of the enzyme is more consistent with the native structure. Thus, the present invention described below was accomplished.

That is, the present invention provides:

(1) the large subunit of carbamoyl phosphate synthetase, corresponding to SEQ ID NO: 20 by the amino acid sequence at position 947-951 as set forth in SEQ ID NO: 1-9, and the feedback inhibition by uridine 5' -monophosphate is desensitized;

(2) the large subunit of carbamoyl phosphate synthetase of (1), wherein the carbamoyl phosphate synthetase is Escherichia coli carbamoyl phosphate synthetase.

(3) The large subunit of carbamoyl phosphate synthetase of (1), wherein SEQ ID NO: 20 by the amino acid sequence at position 947-951 as set forth in SEQ ID NO: 1-9, and the feedback inhibition by uridine 5' -monophosphate is desensitized;

(4) the large subunit of carbamoyl phosphate synthetase of (1), which comprises deletion, substitution, insertion or addition of one or several amino acids at one or more positions other than the 947-951 position, wherein the feedback inhibition by uridine 5' -monophosphate is desensitized;

(5) carbamoyl phosphate synthetase which comprises the large subunit of carbamoyl phosphate synthetase as described in any one of (1) to (4);

(6) the DNA encoding the carbamoyl phosphate synthetase of any one of (1) to (4), wherein the feedback inhibition of uridine 5' -monophosphate is desensitized;

(7) DNA encoding the large subunit of carbamoyl phosphate synthetase in which the feedback inhibition by uridine 5' -monophosphate according to any one of (1) to (4) is desensitized, and the small subunit of E.coli carbamoyl phosphate synthetase;

(8) a bacterium belonging to the genus Escherichia, which has the DNA of (6) or (7);

(9) the bacterium of (8), which has an ability to produce a compound selected from the group consisting of L-arginine, citrulline and a pyrimidine derivative;

(10) the bacterium according to (9), wherein the pyrimidine derivative is orotic acid, uridine, UMP, cytidine, and CMP;

(11) a method of producing a compound selected from the group consisting of L-arginine, citrulline, and a pyrimidine derivative, the method comprising the steps of: culturing the bacterium according to any one of (8) to (10) in a medium so that the compound is produced and accumulated in the medium, and collecting the compound from the medium; and

(12) the method of (11), wherein the pyrimidine derivative is orotic acid, uridine, UMP, cytidine, and CMP.

In the present invention, the term "CPSase activity" means an activity of catalyzing a complex synthesis reaction of producing carbamoyl phosphate from bicarbonate, glutamine and two Mg-ATP molecules. The "CPSase" of the present invention may be a single polypeptide consisting of a large subunit, or a heterodimer comprising a large subunit and a small subunit, as long as the CPSase has CPSase activity. In the present application, the above-mentioned large subunits and heterodimers may be collectively referred to as "CPSase". The DNA encoding the large and small subunits may be referred to as "carAB".

According to this embodiment, a CTPase having any ofthe above fbr mutations may be referred to as a "mutant CPSase", and DNA encoding the mutant CPSase may be referred to as a "mutant carB gene" or a "mutant carAB gene", and a CPSase having no mutation may be referred to as a "wild-type CPSase".

The present invention will be described in detail below.

<1>mutant CPSase and mutant carB genes

Subsequently, recombinant clones carrying the mutant carB gene cloned as carAB operon into an expression vector were selected and screened in order to allow selection of fbr variants of mutant CPSase with different levels of biological activity.

According to the data obtained by S.Delannay et al (Delannay S.et al, J. mol. biol., Vol. 286, 1217-1228, 1999), the mutant E.coli carbamyl phosphate synthetase (S948F) was not sensitive to UMP. Based on these data, the part of CPSase including the 948 position was selected as the target of modification.

The mutant CPSase and mutant carB genes were obtained by random fragment directed mutagenesis. To obtain multiple mutations in the carB gene, the gene encoding SEQ ID NO: a fragment of 15 nucleotides of the carB gene of the part of the amino acid sequence 947 leucine to 951 glutamic acid residues was randomized (see below). Randomized 15 nucleotide fragments can yield 4¹²Or close to 1.5X 10⁷A different DNA sequence encoding 4X 10 pentameric peptide⁵Different amino acid residues. The probability of not introducing a stop codon in reading frame with these sequences is approximately 0.95 or 95. Due to the fact thatThus, randomization of the carB fragment encoding the 947-951 amino acid residues of the peptide must yield approximately 4X 10⁵This peptide fragment has diversity in CPSase structure, by virtue of its different amino acid sequence. The fbr variants of mutant CPSase with different levels of biological activity can then be selected and screened for recombinant clones carrying the mutant carB gene cloned into an expression vector.

The present invention defines the amino acid sequence of a mutant CPSase suitable for the fbr phenotype of CPSase. Thus, based on the sequence, a mutant CPSase can be obtained by introducing a mutation into the wild-type carB gene by an ordinary method. The wild-type carB gene may be the carB gene of E.coli (nucleotide 10158-13379 of the sequence of GenBank accession AE 000113U 00096: SEQ ID NO: 19). The carA gene corresponds to nucleotide 8992-10140 of the sequence of GenBank accession number U00096.

In the case where the carB gene is used as a material for obtaining a DNA encoding a mutant CPSase, the mutant carB gene encodes the large subunit of the mutant CPSase. In the case where the carAB gene is used as the material, the mutant carAB gene encodes both the large subunit and the small subunit of the mutant CPSase.

The amino acid sequence at position 947-951 of the mutant CPSase of the invention is SEQ ID NO: 1-9. The corresponding amino acid sequence of the known fbr CPSase, in which the serine at position 948 was replaced with phenylalanine, and the amino acid sequence of the wild-type CPSase of e.coli are shown in table 1. Examples of nucleotide sequences encoding the amino acid sequences are also shown in table 1.

TABLE 1

Clone number	Randomization of CarB proteins Sequence of regions (947 → 951 amino acids)	SEQ ID NO：	Randomization of CarB groups DNA sequence of the fragment (5′→3′)	SEQ ID NO：
					Wt	-Leu-Ser-Val-Arg-Glu-	28	CTTTCCGTGCGCGAA	30
6 (Single mutation)	-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

The mutant CPSase may comprise deletion, substitution, insertion or addition of one or several amino acids at one or more positions other than positions 947-951, as long as the activity of CPSase is not impaired. The number of "several" amino acids varies depending on the position or type of amino acid residue on the three-dimensional structure of the protein. This is because of the following reason. That is, some amino acids have high homology to each other, and the difference in these amino acids does not have a large influence on the three-dimensional structure of the protein. Therefore, the mutant CPSase of the present invention may have homology of not less than 30 to 50%, preferably 50 to 70%, with all amino acid residues constituting the CPSase, and it has fbr CPSase activity. The CPSase activity retained by the mutant CPSase in the presence of uridine 5' -monophosphate is desirably not less than 25%, preferably not less than 30%, more preferably not less than 40% of the wild-type CPSase activity.

In the present invention, "the amino acid sequence corresponding to the sequence at position 947-951" means a sequence corresponding to SEQ ID NO: 20 at the amino acid sequence position 947-951. The position of the amino acid residues may vary. For example, if an amino acid residue is inserted into the N-terminal portion, the amino acid residue originally at position 947 becomes position 948. In this case, the amino acid residue corresponding to position 947 of the original sequence is referred to as "the amino acid residue at position 947" in the present invention.

The phrase "the feedback inhibition of uridine 5' -monophosphate is desensitized" means that the degree of feedback inhibition is reduced. The decrease in the degree of feedback inhibition can be measured by the following method: the reduction of CPSase activity in the presence of uridine 5' -monophosphate was determined and compared to a protein having the sequence of SEQ id no: 20, and comparing the activities of the proteins. In addition, the phrase "the feedback inhibition of uridine 5' -monophosphate is desensitized" means that substantial desensitization of the inhibition is sufficient, and complete desensitization is not required. Specifically, in the case of using 5mM glutamine as a substrate, the ratio of the activity of the mutant CPSase in the presence of 10mM uridine 5 '-monophosphate to the activity in the absence of uridine 5'-monophosphate is desirably not less than 50%, preferably not less than 70%, more preferably not less than 90%.

For example, the nucleotide sequence may be modified by a method such as site-directed mutagenesis to obtain a DNA encoding a protein substantially identical to the above-mentioned mutant CPSase, so that one or several amino acid residues located at a specific site are deleted, substituted, inserted or added. The above-mentioned DNA modification can be carried out by commonly used known mutation treatment. The mutation treatment includes a method of treating DNA containing a mutant carB gene in vitro, for example, with hydroxylamine, and a method of treating a microorganism, for example, a bacterium belonging to the genus Escherichia having a mutant carB gene, with ultraviolet irradiation or a mutagen such as N-methyl-N' -nitro-N-guanidine Nitrite (NTG) and nitrous acid which are generally used for such treatment.

The substitution, deletion, insertion or addition of the above-mentioned nucleotide also includes a naturally occurring mutation (mutant or variant), for example, based on the difference of individual bacteria having CPSase or the difference of species or genus.

The DNA encoding a protein substantially identical to the mutant CPSase can be obtained by isolating, from cells having the mutant CPSase which have been subjected to mutation treatment, a DNA which hybridizes with a DNA having a known carB gene sequence or a part thereof as a probe under stringent conditions, and a DNA encoding a protein having CPSase activity.

In this context, the term "stringent conditions" means conditions under which so-called specific hybridization can be formed, but non-specific hybridization cannot be formed. It is difficult to accurately express the condition by any digitized value. However, for example, stringent conditions include conditions under which, for example, DNAs having homology of not less than 50% to each other can hybridize, but DNAs having homology lower than the above standards cannot hybridize. In addition, stringent conditions can be expressed as conditions under which DNAs hybridize to each other at a salt concentration equivalent to ordinary washing conditions in Southern hybridization, for example, 60 ℃, 1XSSC, 0.1% SDS, preferably 0.1XSSC, 0.1% SDS.

Genes that can hybridize under the above conditions include genes that produce a stop codon in the coding region of the gene, and genes that are inactive due to mutation of the active center. However, the above difficulties can be conveniently eliminated by: the gene was ligated to a commercial expression vector and the CPSase activity of the expressed protein was investigated.

When the CPSase of the present invention is a heterodimer comprising a mutant large subunit and a small subunit, the small subunit may be a small subunit of a wild-type CPSase of e.

In the present invention, the small subunit may include deletion, substitution, insertion or addition of one or several amino acids at one or more positions as long as it has CPSase activity when combined with the large subunit. The term "several" has the same meaning as described above for the large subunit.

As the DNA encoding a polypeptide substantially identical to the above-mentioned small subunit, it may be a DNA capable of hybridizing with a DNA containing a carA or a part thereof under stringent conditions. The term "stringent conditions" has the same meaning as described above.

<2>the bacterium belonging to the genus Escherichia of the present invention.

The bacterium belonging to the genus Escherichia of the present invention is a bacterium belonging to the genus Escherichia into which a mutant carB gene has been introduced.For example, a bacterium belonging to the genus Escherichia is Escherichia coli. For example, the mutant carB gene may be introduced by transforming a bacterium belonging to the genus escherichia with a recombinant DNA including a vector capable of functioning in a bacterium belonging to the genus escherichia and the mutant carB gene. The mutant carB gene may also be introduced by replacing the carB gene on the chromosome with the mutant carB gene.

For example, the vector for introducing the mutant carB gene may be a plasmid vector such as pBR322, pMW118, or pUC19, a phage vector such as 11059, 1BF101, or M13mp9, and a transposon such as Mu, Tn10, or Tn 5.

For example, the introduction of DNA into a bacterium belonging to the genus Escherichia can be accomplished by: morrison (enzymological methods, 68, 326, 1979) or a method of treating recipient bacterial cells with calcium chloride in order to increase the penetration capacity of DNA (Mandel, m., and Higa, a., journal of molecular biology, 53, 159, 1970), etc.

By introducing the mutant carB gene into the above-mentioned producing bacterium belonging to the genus Escherichia, it is possible to improve the yield of a compound derived from carbamoyl phosphate such as L-arginine, citrulline and pyrimidine derivatives. In addition, the bacteria into which the mutant carB gene has been previously introduced can be imparted with the ability to produce compounds such as L-arginine, citrulline and pyrimidine derivatives. The above pyrimidine derivatives include orotic acid, uridine, UMP, cytidine and CMP.

For example, the bacterium belonging to the genus Escherichia having L-arginine producing activity may be Escherichia coli strains AJ11531 and AF11538(JP56106598A2), AJ11593(FERMP-5616) and AJ11594(FERM P-5617) (Japanese patent laid-open No. 57-5693), VKPM B-7925 (Russian patent application No. 2000117677). The strain VKPM B-7925 has been deposited at Russian national collections of Industrial microorganisms (VKPM) at 10.4.2000.

L-citrulline-producing bacteria belonging to the genus Escherichia, orotic acid-producing bacteria belonging to the genus Escherichia, and uridine 5' -monophosphate (UMP) -producing bacteria belonging to the genus Escherichia are not known at present.

For example, the bacteria belonging to the genus Bacillus having L-citrulline-producing activity may be Bacillus subtilis strains K-X-1A-1(ATCC No. 15561) and K-X-1A-9 (ATCCNO15562) (U.S. Pat. No. 3282794), Bacillus strain cit-70 (Japanese laid-open patent application No. 08-089269). For example, the bacteria belonging to the genus Brevibacterium having L-citrulline-producing activity may be Brevibacterium flavum strains AJ3408(FERM P-1645) (U.S. Pat. No. 5164307) and AJ11677 (Japanese laid-open patent application No. 57-163488). For example, the bacterium belonging to the genus Corynebacterium having an L-citrulline-producing activity may be Corynebacterium glutamicum strain AJ11588(FERM P-5643) (U.S. Pat. No. 5164307).

For example, the bacterium belonging to the genus Bacillus having an orotic acid producing activity may be Bacillus subtilis strain FERM P-11402, which lacks orotate phosphoribosyltransferase (Japanese laid-open patent application No. 04-004891). For example, the bacterium belonging to the genus Corynebacterium having the activity of producing UMP may be Corynebacterium glutamicum strain T-26(FERM BP-1487) resistant to 5-fluorouracil, T-29(FERM BP1488) resistant to 5-fluorouracil and trimethoprim, and T-30(FERM BP1489) resistant to 5-fluorouracil and sulfaguanidine (European patent EP 0312912).

For example, bacteria belonging to the genus Corynebacterium having UMP-producing activity may be Corynebacterium ammoniagenes strains LK40-2(VKPM B-7811), LK75-15(VKPM B-7812) and LK75-66(VKPM B-7813) (Russian patent application No. 99122774).

<3>a method for producing L-arginine, citrulline and pyrimidine derivatives.

The compounds such as L-arginine, citrulline and pyrimidine derivatives can be efficiently produced by the following method: culturing a bacterium into which a mutant carB gene has been introduced and which has an ability to produce the compound, in a medium, producing and accumulating the compound in the medium, and collecting the compound from the medium. The above pyrimidine derivatives include orotic acid, uridine, UMP, cytidine and CMP.

In the method of the present invention, the cultivation of a bacterium belonging to the genus Escherichia, collection and purification of a compound from the liquid medium can be carried out in a manner similar to the conventional method for producing L-arginine by fermentation using a bacterium. The medium used for the culture may be a synthetic medium or a natural medium, as long as the medium contains appropriate amounts of carbon and nitrogen sources and minerals, and, if necessary, appropriate amounts of nutrients required by the bacteria used. The carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids, depending on the assimilation ability of the bacterium used. Alcohols including ethanol and glycerol may be used. As the nitrogen source, ammonia, various ammonium salts such as ammonium sulfate, other nitrogen compounds such as amines, natural nitrogen sources such as peptone, soybean hydrolysate, and digested fermentative microorganisms can be used. As minerals, potassium dihydrogen phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, and calcium carbonate can be used.

The culture is preferably carried out under aerobic conditions such as shake culture, aeration culture and agitation culture. The cultivation is usually carried out at a temperature of 20-40 ℃ and preferably 30-38 ℃. The cultivation is usually carried out at a pH of 5-9, preferably 6.5-7.2. The pH of the medium can be adjusted with ammonia, calcium carbonate, various acids, various bases and buffers. Typically, 1-3 days of culture will result in the accumulation of the compound in the medium.

After the culture, solid materials such as cells can be removed by centrifugation or membrane filtration, the compound can be isolated, and then the compound can be collected and purified by methods such as ion exchange, concentration, and crystallization fractions.

Brief Description of Drawings

FIG. 1 shows a schematic representation of the construction of plasmid pEL-carAB-wt.

FIG. 2 shows a schematic diagram of the construction of a mutant carB gene bank.

Detailed Description

The present invention will be specifically explained with reference to the following examples.Example 1

A plasmid pBScarab-13 carrying the wild-type carAB gene from E.coli was constructed by cloning the AvaIII-Bg1II DNA fragment (4911bp) from the E.coli chromosome into the pBluescript II SK (+) vector (Fermentas, Lithuania) previously digested with BamHI and PstI.

Plasmid pET22-b (+) (Novagen, USA) was modified by replacing the T7 promoter with the lac promoter. The Lac promoter was obtained by PCR amplification using plasmid pUC18 as a template and oligonucleotides 5'-accagatctgcgggcagtgagcaacgc-3' (SEQ ID NO: 21) and 5'-gtttctagatcctgtgtgaaattgttatccgc-3' (SEQ ID NO: 22) as primers. The resulting fragment (0.14kb) with the lac promoter was digested with restriction enzymes Bg1II and XbaI and cloned into pET22-b (+) vector previously digested with the same restriction enzymes. The resulting plasmid pET-Plac was used to clone the promoterless carAB gene from the plasmid pBScarabA-13.

The 5' -end of the carA gene (1.18kb) was obtained by PCR amplification using pBScarabA-13 as template and oligonucleotides 5'-cctctagaaataaagtgagtgaatattc-3' (SEQ ID NO: 23) and 5'-cttagcggttttacggtactgc-3' (SEQ ID NO: 24) as primers. The resulting fragment was digested with XbaI and DraIII, and the XbaI-DraIII fragment (0.61kb) having the 5' -terminal sequence of the carA gene was purified by agarose electrophoresis. This fragment, a mixture of the DraIII-SacI fragment from plasmid pBScarab-13 (having the sequences at the 3' ends of the carA gene and the carB gene) and the vector pET-Plac/XbaI-SacI were ligated and transformed into E.coli TG1 cells. The resulting recombinant plasmid pEL-carAB-wt has the sequence of the wild-type carAB operon under the control of the lac promoter.

TaKaRa La DNA polymerase for PCR amplification is obtained from Takara Shuzo Co., Ltd. (Japan) and used under the conditions recommended by the supplier.

<1>random fragment directed mutagenesis

Plasmid pBScarAAB-13 was used as template, sense primer P1: 5 '-ggtcgtgcgcctgnN (T/C) N (T/C) CNN (T/C) NNN (G/A) NNggcgataaagaacgcgtggtg-3' (SEQ ID NO: 25) (48 bases) was designed based on the nucleotide sequence of the carB gene and a standard M13 targeting sequence primer was used as an antisense primer. The 5 'end of the 12 nucleotides fixed and the 3' end region of the 21 nucleotides fixed of primer P1 are homologous to the sequences downstream of the Glu951 codon and upstream of the Leu947 codon of the carB gene, respectively.

In the first round of PCR (15 cycles), a 0.75kb DNA fragment (3' -end of the carB gene) was synthesized using primer P1 with a randomized 15 nucleotide region. The first round of PCR was performed as follows. 100 ng of plasmid pBScarab-13 was added as a template to a PCR solution (50. mu.l) containing both primers at a concentration of 10 pmoles. PCR cycles (94 ℃ for 15 seconds, 52 ℃ for 20 seconds, 72 ℃ for 1 minute) were carried out using a model 2400 DNA thermal cycler (Perkin-Elmer, Foster, Calif.).

In the second round of amplification, a further 15 rounds of amplification (94 ℃ for 1 minute, 35 ℃ for 1 minute, 72 ℃ for 2 minutes) were performed, in which the (-) strand of the fragment served as the "primer" for extension to obtain the complete gene sequence.

In the third round of amplification, an aliquot of 10 μ l of this reaction mixture was added to a sample containing 100 pmoles of sense primer: standard M13 targeting sequence primer and P2 as antisense primer: 5'-ccacttcctcgatgacgcgg-3' (SEQ ID NO: 26) and an additional 15 cycles (94 ℃ C. for 0.5 min, 55 ℃ C. for 20 sec, 72 ℃ C. for 2 min).

A1.32 kb DNA fragment of the mutant variant library encoding the 3' -terminal fragment of the carB gene was purified by agarose gel electrophoresis, then digested with Af1II and SacI, and further ligated with pEL-carAB-wt vector previously digested with the same restriction enzymes, so as to obtain pEL-carAB-NN.

In subsequent experiments approximately 150 ng of the ligated DNA was used for the transformation of E.coli TG1(supE hsd. DELTA.5 thi. DELTA. (lac-proAB) F' [ traD36 proAB⁺lacI^qlacZΔM15]) (J.Sambrook et al, molecular cloning, 1989) recipient cells, so that approximately 2000 clones were obtained in each case. The library of recombinant plasmids (pEL-carAB-NN) was purified and transformed into E.coli cells VKPM B-6969 (carB:: Tn10) and used to screen for recombinant plasmids pEL-carAB-NN having a carAB gene encoding an active carAB enzyme.

<2>site-directed mutagenesis

To introduce the single mutant Ser948Phe, PCR was carried out using the plasmid pBScarab-13 as template and the sense primer 5 '-cgtgcttgtttcgtgecgaaggcgataaag-3' (34 bases) (SEQ ID NO: 27) designed on the basis of the nucleotide sequence of the carB gene and the standard M13 guide sequence primer as antisense primer. PCR amplification and cloning of the fragment was performed as described above.

A1.32 kb DNA fragment encoding the 3' -terminal fragment of the carB gene with a single mutation was purified by agarose gel electrophoresis, digested with Af1II and SacI, and then ligated into the pEL-carAB-wt vector previously digested with the same restriction enzymes.

Coli cells VKPM B-6965 were transformed with approximately 100 ng of the resulting DNA plasmid and the recombinant plasmid pEL-caraB-6 carrying the active CarAB enzyme with the single substitution of Ser948Phe was selected.Example 2.The isolation of the carB mutant and the influence of amino acid substitutions on the catalytic Properties in CPSase

CarAB activity and its anti-feedback effect on UMP were first assessed on 40 recombinant B-6969(pEL-carAB-NN) clones in a citrulline biosynthesis reaction catalyzed by carAB and ArgI (ornithine aminomethyltransferase) enzymes and by ornithine.

The formula for the reaction is as follows:

free NH for carbamoyl phosphate synthetase in this reaction₄ ⁺As a substrate

Protein extracts from 40B-6969 (pEL-carAB-NN) strains and TG1(pUC18-argI) cells were prepared by precipitation from crude cell extracts of sonicated cellswith ammonium sulfate (75% saturation). Dissolving the protein precipitate in a buffer having the following formulation: Tris-HCl (50mM), pH7.5, 2-mercaptoethanol (2 mM).

The test system included protein extracts from strains B-6969(pEL-carAB-NN) and TG1(pUC18-argI) and the following reagents: ATP (8mM), magnesium sulfate (8mM), ammonium sulfate (200mM), sodium carbonate (8mM) and ornithine (1mM), pH 7.5. The reaction mixture was analyzed for ornithine and citrulline content by TLC using a liquid phase having the following formulation. Isopropanol/ethyl acetate/ammonium hydroxide/water 40/20/13/27 (v/v).

9 clones expressing the active mutant CPSase and having feedback resistance to UMP and one clone expressing the mutant CPSase with a single Ser948Phe substitution were used to determine the activity of the mutant enzymes.

Plasmids from the above 10 clones were purified and the sequence of the random fragment of the carB gene was determined by the dideoxy chain termination method (Table 1).

Protein extracts from the 9 clones B-6969(pEL-carAB-NN) and one clone B-696 (pEL-carAB-6) were then used to evaluate the activity and fbr of mutant CPSase in the synthesis of Carbamoyl Phosphate (CP) from glutamine or ammonia.

Crude cell extracts from cells were prepared by the following method: 20 mg of wet cell pellet suspended in 0.5 ml of buffer A (200mM potassium hydrogen phosphate/potassium dihydrogen phosphate, pH8.0, 1mM EDTA, 1mM PMSF, 1mM DTT) was sonicated and then treated with solid ammonium sulfate to 65% saturation. After incubation at 4 ℃ for 10 minutes, the suspension was centrifuged at 13000rpm for 10 minutes, and the pellet was dissolved in 1 ml of buffer B (20mM potassium hydrogenphosphate/potassium dihydrogenphosphate, pH8.0, 50mM potassium chloride, 1mM PMSF, 1mM DTT). Aliquots of the protein extracts obtained wereused to assess CPSase activity. The reaction formula is as follows:

I.

II.

50 microliters of each reaction mixture included:

reaction I- -20mM Tris-HCl, pH8.0, 100mM potassium chloride, 5mM sodium carbonate, 10mM ATP, 10mM magnesium chloride, 5mM glutamine, 10. mu.l protein extract;

reaction II- -20mM Tris-HCl, pH8.0, 100mM potassium chloride, 5mM sodium carbonate, 10mM ATP, 10mM magnesium chloride, 200mM ammonium sulfate, 10. mu.l protein extract.

In addition, a series of reactions I were performed in the presence of 10mM UTP in order to assess the level of feedback inhibition by CPSase.

After incubation at 37 ℃ for 10 minutes, the reaction was stopped by adding an equal volume of ethanol, cooled at-20 ℃ for 10 minutes, and centrifuged at 13000rpm for 1 minute at room temperature. The supernatant was cooled at-20 ℃.

The reaction mixture was analyzed for CP content by capillary zone electrophoresis. The separation was performed on a Quanta 4000E capillary electrophoresis system ("Waters", USA) with indirect detection with UV at 254 nm. The injection was performed by hydrostatic pressure for 25 seconds. The separation was performed with uncoated fused silica capillary tubes (75u inner diameter,. about.60 cm, effective length 53 cm) and operated at-25 kV voltage. The temperature was maintained at 20 ℃. The separation buffer consisted of 50mM Tris-base, 25mM benzoic acid (for indirect detection), pH8.5, 0.25mM TTAB (tetraacetyl-trimethyl-ammonium bromide) (for restoring electroosmotic flow).

Table 2 shows the data of the activity and fbr of mutant CPSase measured in the CP synthesis reaction.

TABLE 2 Activity of mutant CPSase

Cloning Numbering	Activity (CP, nanomole/mg min)
				Substrate: 5Mmgu Glutamine	Substrate: 200mM ammonium sulfate	Substrate: 5mM glutamine; metamorphic Effector(s): 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

Thus, the mutant CPSase was essentially insensitive to UMP, but the single mutation significantly reduced the activity of the enzyme. These results indicate that the peptide fragment of 947-951 amino acid residues determines the feedback inhibition of UMP and determines the catalytic efficiency of mutant CPSase.

The genes encoding wt CarAB and mutant CarAB-34 were cloned into plasmid pMW 119. For this purpose, the plasmids pEL-carAB-wt and pEL-carAB-34 (to be partially digested because the plasmid has two XbaI sites) were digested with the restriction enzymes SacI and XbaI, and the fragment encoding the carAB gene was cloned into the pMW119 vector previously digested with the same restriction enzymes. ResultsLow copy number plasmids pMW119-carAB-wt and pMW119-carAB-34 having the carAB gene under the control of the lac promoter were constructed.Example 3.Production of orotic acid by Strain having mutant CarAB Gene

As a mutant strain against 6-azauracil (1 mg/ml), strain 311 was derived from E.coli K12(VKPM B-3853) having Tn10 inserted with argA gene. Strain 311 has been deposited at Russian national Industrial Collection of microorganisms (VKPM) at 5/3/2001 with the deposit number VKPM B-8085 and the original deposit was converted to an international deposit at 17/7/2002 under the terms of the Budapest treaty.

Strain 311 was transformed with plasmids pMW-carAB-wt and pMW-carAB-34, and the orotic acid production ofthe resulting strains was examined in the presence of various concentrations of uridine.

The culture conditions for the test tube fermentation were as follows:

1/20 diluted overnight culture, 60 g/l glucose, 25 g/l ammonium sulphate, 2 g/l potassium dihydrogen phosphate, 1 g/l magnesium sulphate, 0.1 mg/l thiamine, 5 g/l yeast extract Difco, 25 g/l chalk, 1 l tap water (pH 7.2). Glucose and chalk are sterilized separately. 2 ml of the medium was placed in a test tube and cultured at 32 ℃ for 3 days with shaking. Orotic acid production was assessed by HPLC (table 3).

TABLE 3 strains 311(pMW119), 311(pMW-carAB-wt),

Orotic acid production level of 311(pMW-carAB-34)

Bacterial strains	Uridine, 100mg/l		Uridine, 300mg/l		Uridine, 1000mg/l
							A550， o.u.	Orotic acid Biological box To form, g/l	A550， o.u.	Orotic acid Biological box To form, g/l	A550， o.u.	Orotic acid Biological box To form, 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 strain 311(pMW-carAB-34) having the mutant type carAB gene produced more orotic acid than the parent strain 311(pMW119) and the strain 311(pMW-carAB-wt) having the wild type carAB gene.Example 4.Production of arginine and/or citrulline using a strain having a mutant carAB gene

As mutants against 6-azauracil (1 mg/ml), arginine producing strains 333 and 374 have been selected froma derivative of E.coli strain 57(VKPM B-7386) having transposon Tn5 inserted in the gene ilvA. Strains 333 and 374 have been deposited at Russian national Industrial Collection of microorganisms (VKPM) at 3/5 of 2001 under the numbers VKPM B-8084 and VKPM B-8086, respectively, and the original deposit was subsequently converted to an international deposit at 2002/7 under the provisions of the Budapest treaty.

Strains 333 and 374 were transformed with plasmids pMW-carAB-wt and pMW-carAB-34 and the arginine and citrulline production of these recombinant strains was examined.

The test tube fermentation was carried out in the same manner as in example 3.

Arginine and/or citrulline production levels of strains 333(pMW-carAB-wt) and 333(pMW-carAB-34) in synthetic medium containing 100mg/l uridine are shown in Table 4.

TABLE 4 strains 333(pMW-caraB-wt) and

333(pMW-carAB-34) arginine and/or citrulline production levels

Bacterial strains	The light absorbance of the mixture is measured, A560，u.	arginine production Synthetic water Flat, g/l	Citrulline production Synthetic water Flat, g/l	Citrulline + arginine Acid biosynthesis water Flat, 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

The citrulline production levels of strains 374(pMW-carAB-wt) and 374(pMW-carAB-34) in synthetic medium containing 100mg/l uridine are shown in Table 5.

TABLE 5 strains 374(pMW-caraB-wt) and

citrulline productionlevels of 374(pMW-caraB-34)

Bacterial strains	Absorbance, A566，u.	Citrulline biosynthesis level, g/l
			374(pMW-carAB-wt)	22.3	＜0.01
374(pMW-carAB-34)	15.5	0.26

As shown in table 4, strain 333(pMW-carAB-34) having the mutant carAB gene produced more arginine and citrulline than the strain having the wild-type carAB gene. As shown in Table 5, strain 374(pMW-carAB-34) was able to produce more citrulline than the strain with the wild-type carAB gene.

New mutant carbamyl phosphate synthetase of<120>of Punjaku of<110>in the sequence Listing and production thereof

Methods for derivatizing compounds<130>OP1367<140><141>2002-08-<150>RU 2001121697<151>2001-08-03<160>33<170>PatentIn Ver.2.1<210>1<211>5<212>PRT<213>Artificial sequence<220><223>Artificial sequence description: the partial amino acid sequence<400>1Pro Leu Arg Glu Gly 15<210>2<211>5<212>PRT<213>artificial sequence<220><223>encoded by the random sequence is described: the partial amino acid sequence<400>2Ala Val Ala Leu Lys 15<210>3<211>5<212>PRT<213>artificial sequence<220><223>encoded by the random sequence is described: the partial amino acid sequence<400>3Gly Val Phe Leu Met 15<210>4<211>5<212>PRT<213>artificial sequence<220><223>encoded by the random sequence is described: the partial amino acid sequence<400>4Phe Phe Cys Phe Gly 15<210>5<211>5<212>PRT<213>artificial sequence<220><223>encoded by the random sequence is described: the partial amino acid sequence<400>5Pro Thr Gly Arg Arg 15<210>6<211>5<212>PRT<213>artificial sequence<220><223>encoded by the random sequence is described: the partial amino acid sequence<400>6Phe Ala Cys Gly Val 15<210>7<211>5<212>PRT<213>artificial sequence<220><223>encoded by the random sequence is described: the partial amino acid sequence<400>7Val Phe Gly Ser Ser 15<210>8<211>5<212>PRT<213>artificial sequence<220><223>encoded by the random sequence is described: the partial amino acid sequence<400>8Ala Ser Gly Val Glu 15<210>9<211>5<212>PRT<213>artificial sequence<220><223>encoded by the random sequence is described: partial amino acid sequence<400>9Ala Phe CysGly Val 15<210>10<211>15<212>DNA<213>artificial sequence<220><223>artificial sequence description encoded by a random sequence: random sequence<400>10cctctccgtg agggt 15<210>11<211>15<212>DNA<213>artificial sequence<220><223>artificial sequence description: random sequence<400>11gctgtcgctt tgaaa 15<210>12<211>15<212>DNA<213>artificial sequence<220><223>artificial sequence description: random sequence<400>12ggtgtcttcc taatg 15<210>13<211>15<212>DNA<213>artificial sequence<220><223>artificial sequence description: random sequence<400>13tttttctgtt ttggg 15<210>14<211>15<212>DNA<213>artificial sequence<220><223>artificial sequence description: random sequence<400>14cctaccggta ggaga 15<210>15<211>15<212>DNA<213>artificial sequence<220><223>artificial sequence description: random sequence<400>15ttcgcctgtg gggtg 15<210>16<211>15<212>DNA<213>artificial sequence<220><223>artificial sequence description: random sequence<400>16gttttcggta gtagt 15<210>17<211>15<212>DNA<213>artificial sequence<220><223>artificial sequence description: random sequence<400>17gcttccggcg ttgag 15<210>18<211>15<212>DNA<213>artificial sequence<220><223>artificial sequence description: random sequence<400>18gccttctgtg gggtg<210>19<211>3222<212>DNA<213>E.coli (Escherichia coll)<220>CDS<222>(1).<3222)<400>19atgccaaaac gtacagatat aaaaagtatc ctgattctgg gtgcgggccc gattgttatc ggtcaggcgt gtgagtttga ctactctggc gcgcaagcgt gtaaagccct gcgtgaagag 120ggttaccgcg tcattctggt gaactccaac ccggcgacca tcatgaccga cccggaaatg gctgatgcaa cctacatcga gccgattcac tgggaagttg tacgcaagat tattgaaaaa 240gagcgcccgg acgcggtgct gccaacgatg ggcggtcaga cggcgctgaa ctgcgcgctg gagctggaac gtcagggcgt gttggaagag ttcggtgtca ccatgattgg tgccactgcc gatgcgattg ataaagcaga agaccgccgt cgtttcgacg tagcgatgaa gaaaattggt 420ctggaaaccg cgcgttccgg tatcgcacac acgatggaag aagcgctggc ggttgccgct 480gacgtgggct tcccgtgcat tattcgccca tcctttacca tgggcggtag cggcggcggt 540atcgcttata accgtgaaga gtttgaagaa atttgcgccc gcggtctgga tctctctccg accaaagagt tgctgattga tgagtcgctg atcggctgga aagagtacga gatggaagtg 660gtgcgtgata aaaacgacaa ctgcatcatc gtctgctcta tcgaaaactt cgatgcgatg ggcatccaca ccggtgactc catcactgtc gcgccagccc aaacgctgac cgacaaagaa tatcaaatca tgcgtaacgc ctcgatggcg gtgctgcgtg aaatcggcgt tgaaaccggt 840 63900 aacccacgcg tgtcccgttc ttcggcgctg gcgtcgaaag cgaccggttt cccgattgct 63960 aaagtggcgg cgaaactggc ggtgggttac accctcgacg aactgatgaa cgacatcact 1020ggcggacgta ctccggcctc cttcgagccg tccatcgact atgtggttac taaaattcct 1080cgcttcaact tcgaaaaatt cgccggtgct aacgaccgtc tgaccactca gatgaaatcg 1140gttggcgaag tgatggcgat tggtcgcacg cagcaggaat ccctgcaaaa agcgctgcgc 1200ggcctggaag tcggtgcgac tggattcgac ccgaaagtga gcctggatga cccggaagcg 1260ttaaccaaaa tccgtcgcga actgaaagac gcaggcgcag atcgtatctg gtacatcgcc 1320gatgcgttcc gtgcgggcct gtctgtggac ggcgtcttca acctgaccaa cattgaccgc 1380tggttcctgg tacagattga agagctggtg cgtctggaag agaaagtggc ggaagtgggc 1440atcactggcc tgaacgctga cttcctgcgc cagctgaaac gcaaaggctt tgccgatgcg 1500cgcttggcaa aactggcggg cgtacgcgaa gcggaaatcc gtaagctgcg tgaccagtat 1560gacctgcacc cggtttataa gcgcgtggat acctgtgcgg cagagttcgc caccgacacc 1620gcttacatgt actccactta tgaagaagag tgcgaagcga atccgtctac cgaccgtgaa 1680aaaatcatgg tgcttggcgg cggcccgaac cgtatcggtc agggtatcga attcgactac 1740tgttgcgtac acgcctcgct ggcgctgcgc gaagacggtt acgaaaccat tatggttaac 1800tgtaacccgg aaaccgtctc caccgactac gacacttccg accgcctcta cttcgagccg 1860gtaactctgg aagatgtgct ggaaatcgtg cgtatcgaga agccgaaagg cgttatcgtc 1920cagtacggcg gtcagacccc gctgaaactg gcgcgcgcgc tggaagctgc tggcgtaccg 1980gttatcggca ccagcccgga tgctatcgac cgtgcagaag accgtgaacg cttccagcat 2040gcggttgagc gtctgaaact gaaacaaccg gcgaacgcca ccgttaccgc tattgaaatg 2100gcggtagaga aggcgaaaga gattggctac ccgctggtgg tacgtccgtc ttacgttctc 2160ggcggtcggg cgatggaaat cgtctatgac gaagctgacc tgcgtcgcta cttccagacg 2220gcggtcagcg tgtctaacga tgcgccagtg ttgctggacc acttcctcga tgacgcggta 2280gaagttgacg tggatgccat ctgcgacggc gaaatggtgc tgattggcgg catcatggag 2340catattgagc aggcgggcgt gcactccggt gactccgcat gttctctgcc agcctacacc 2400ttaagtcagg aaattcagga tgtgatgcgc cagcaggtgc agaaactggc cttcgaattg 2460caggtgcgcg gcctgatgaa cgtgcagttt gcggtgaaaa acaacgaagt ctacctgatt 2520gaagttaacc cgcgtgcggc gcgtaccgtt ccgttcgtct ccaaagccac cggcgtaccg 2580ctggcaaaag tggcggcgcg cgtgatggct ggcaaatcgc tggctgagca gggcgtaacc 2640aaagaagtta tcccgccgta ctactcggtg aaagaagtgg tgctgccgtt caataaattc 2700ccgggcgttg acccgctgtt agggccagaa atgcgctcta ccggggaagt catgggcgtg 2760ggccgcacct tcgctgaagc gtttgccaaa gcgcagctgg gcagcaactc caccatgaag 2820aaacacggtc gtgcgctgct ttccgtgcgc gaaggcgata aagaacgcgt ggtggacctg 2880gcggcaaaac tgctgaaaca gggcttcgag ctggatgcga cccacggcac ggcgattgtg 2940ctgggcgaag caggtatcaa cccgcgtctg gtaaacaagg tgcatgaagg ccgtccgcac 3000attcaggacc gtatcaagaa tggcgaatat acctacatca tcaacaccac ctcaggccgt 3060cgtgcgattg aagactcccg cgtgattcgt cgcagtgcgc tgcaatataa agtgcattac 3120gacaccaccc tgaacggcgg ctttgccacc gcgatggcgc tgaatgccga tgcgactgaa 3180aaagtaattt cggtgcagga aatgcacgca cagatcaaat aa 3222<210>20<211>1074<212>PRT<213>E.coli (Escherichia coli)<400>20Met Pro Lys Arg Thr Asp Ile Lys Ser Ile Leu Ile Leu Gly Ala Gly 151015 Pro Ile Val Ile Gly Gln Ala Cys Glu Phe Asp Tyr Ser Gly Ala Gln.

20 25 30Ala Cys Lys Ala Leu Arg Glu Glu Gly Tyr Arg Val Ile Leu Val Asn

35 40 45Ser Asn Pro Ala Thr Ile Met Thr Asp Pro Glu Met Ala Asp Ala Thr

50 55 60Tyr Ile Glu Pro Ile His Trp Glu Val Val Arg Lys Ile Ile Glu Lys 65 7075 80Glu Arg Pro Asp Ala Val Leu Pro Thr Met Gly Gly Gln Thr Ala Leu

85 90 95Asn Cys Ala Leu Glu Leu Glu Arg Gln Gly Val Leu Glu Glu Phe Gly

100 105 110Val Thr Met Ile Gly Ala Thr Ala Asp Ala Ile Asp Lys Ala Glu Asp

115 120 125Arg Arg Arg Phe Asp Val Ala Met LysLys Ile Gly Leu Glu Thr Ala

130 135 140Arg Ser Gly Ile Ala His Thr Met Glu Glu Ala Leu Ala Val Ala Ala145 150 155 160Asp Val Gly Phe Pro Cys Ile Ile Arg Pro Ser Phe Thr Met Gly Gly

165 170 175Ser Gly Gly Gly Ile Ala Tyr Asn Arg Glu Glu Phe Glu Glu Ile Cys

180 185 190Ala Arg Gly Leu Asp Leu Ser Pro Thr Lys Glu Leu Leu Ile Asp Glu

195 200 205Ser Leu Ile Gly Trp Lys Glu Tyr Glu Met Glu Val Val Arg Asp Lys

210 215 220Asn Asp Asn Cys Ile Ile Val Cys Ser Ile Glu Asn Phe Asp Ala Met225 230 235 240Gly Ile His Thr Gly Asp Ser Ile Thr Val Ala Pro Ala Gln Thr Leu

245 250 255Thr Asp Lys Glu Tyr Gln Ile Met Arg Asn Ala Ser Met Ala Val Leu

260 265 270Arg Glu Ile Gly Val Glu Thr Gly Gly Ser Asn Val Gln Phe Ala Val

275 280 285Asn Pro Lys Asn Gly Arg Leu Ile Val Ile Glu Met Asn Pro Arg Val

290 295300Ser Arg Ser Ser Ala Leu Ala Ser Lys Ala Thr Gly Phe Pro Ile Ala305 310 315 320Lys Val Ala Ala Lys Leu Ala Val Gly Tyr Thr Leu Asp Glu Leu Met

325 330 335Asn Asp Ile Thr Gly Gly Arg Thr Pro Ala Ser Phe Glu Pro Ser Ile

340 345 350Asp Tyr Val Val Thr Lys Ile Pro Arg Phe Asn Phe Glu Lys Phe Ala

355 360 365Gly Ala Asn Asp Arg Leu Thr Thr Gln Met Lys Ser Val Gly Glu Val

370 375 380Met Ala Ile Gly Arg Thr Gln Gln Glu Ser Leu Gln Lys Ala Leu Arg385 390 395 400Gly Leu Glu Val Gly Ala Thr Gly Phe Asp Pro Lys Val Ser Leu Asp

405 410 415Asp Pro Glu Ala Leu Thr Lys Ile Arg Arg Glu Leu Lys Asp Ala Gly

420 425 430Ala Asp Arg Ile Trp Tyr Ile Ala Asp Ala Phe Arg Ala Gly Leu Ser

435 440 445Val Asp Gly Val Phe Asn Leu Thr Asn Ile Asp Arg Trp Phe Leu Val

450 455 460Gln Ile Glu Glu Leu Val Arg Leu Glu Glu Lys Val Ala Glu Val Gly465 470 475 480Ile Thr Gly Leu Asn Ala Asp Phe Leu Arg Gln Leu Lys Arg Lys Gly

485 490 495Phe Ala Asp Ala Arg Leu Ala Lys Leu Ala Gly Val Arg Glu Ala Glu

500 505 510Ile Arg Lys Leu Arg Asp Gln Tyr Asp Leu His Pro Val Tyr Lys Arg

515 520 525Val Asp Thr Cys Ala Ala Glu Phe Ala Thr Asp Thr Ala Tyr Met Tyr

530 535 540Ser Thr Tyr Glu Glu Glu Cys Glu Ala Asn Pro Ser Thr Asp Arg Glu545 550 555 560Lys Ile Met Val Leu Gly Gly Gly Pro Asn Arg Ile Gly Gln Gly Ile

565 570 575Glu Phe Asp Tyr Cys Cys Val His Ala Ser Leu Ala Leu Arg Glu Asp

580 585 590Gly Tyr Glu Thr Ile Met Val Asn Cys Asn Pro Glu Thr Val Ser Thr

595 600 605Asp Tyr Asp Thr Ser Asp Arg Leu Tyr Phe Glu Pro Val Thr Leu Glu

610 615 620Asp Val Leu Glu Ile Val Arg Ile Glu Lys Pro Lys Gly Val Ile Val625 630 635 640Gln Tyr Gly Gly Gln Thr Pro Leu Lys Leu Ala Arg Ala Leu Glu Ala

645 650 655Ala Gly Val Pro Val Ile Gly Thr Ser Pro Asp Ala Ile Asp Arg Ala

660 665 670Glu Asp Arg Glu Arg Phe Gln His Ala Val Glu Arg Leu Lys Leu Lys

675 680 685Gln Pro Ala Asn Ala Thr Val Thr Ala Ile Glu Met Ala Val Glu Lys

690 695 700Ala Lys Glu Ile Gly Tyr Pro Leu Val Val Arg Pro Ser Tyr Val Leu705 710 715 720Gly Gly Arg Ala Met Glu Ile Val Tyr Asp Glu Ala Asp Leu Arg Arg

725 730 735Tyr Phe Gln Thr Ala Val Ser Val Ser Asn Asp Ala Pro Val Leu Leu

740 745 750Asp His Phe Leu Asp Asp Ala Val Glu Val Asp Val Asp Ala Ile Cys

755 760 765Asp Gly Glu Met Val Leu Ile Gly Gly Ile Met Glu His Ile Glu Gln

770 775 780Ala Gly Val His Ser Gly Asp Ser Ala Cys Ser Leu Pro Ala Tyr Thr785 790 795 800Leu Ser Gln Glu Ile Gln Asp Val Met Arg Gln Gln Val Gln Lys Leu

805 810 815Ala Phe Glu Leu Gln Val Arg Gly Leu Met Asn Val Gln Phe Ala Val

820 825 830Lys Asn Asn Glu Val Tyr Leu Ile Glu Val Asn Pro Arg Ala Ala Arg

835 840 845Thr Val Pro Phe Val Ser Lys Ala Thr Gly Val Pro Leu Ala Lys Val

850 855 860Ala Ala Arg Val Met Ala Gly Lys Ser Leu Ala Glu Gln Gly Val Thr 865 870 875 880Lys Glu Val Ile Pro Pro Tyr Tyr Ser Val Lys Glu Val Val Leu Pro

885 890 895Phe Asn Lys Phe Pro Gly Val Asp Pro Leu Leu Gly Pro Glu Met Arg

900 905 910Ser Thr Gly Glu Val Met Gly Val Gly Arg Thr Phe Ala Glu Ala Phe

915 920 925Ala Lys Ala Gln Leu Gly Ser Asn Ser Thr Met Lys Lys His Gly Arg

930 935 940Ala Leu Leu Ser Val Arg Glu Gly Asp Lys Glu Arg Val Val Asp Leu945 950 955 960Ala Ala Lys Leu Leu Lys Gln Gly Phe Glu Leu Asp Ala Thr His Gly

965 970 975Thr Ala Ile Val Leu Gly Glu Ala Gly Ile Asn Pro Arg Leu Val Asn

980985 990Lys Val His Glu Gly Arg Pro His Ile Gln Asp Arg Ile Lys Asn Gly

995 1000 1005Glu Tyr Thr Tyr Ile Ile Asn Thr Thr Ser Gly Arg Arg Ala Ile Glu 1010 1015 1020Asp Ser Arg Val Ile Arg Arg Ser Ala Leu Gln Tyr Lys Val His Tyr1025 1030 1035 1040Asp Thr Thr Leu Asn Gly Gly Phe Ala Thr Ala Met Ala Leu Asn Ala

1045 1050 1055Asp Ala Thr Glu Lys Val Ile Ser Val Gln Glu Met His Ala Gln Ile

106010651070 Lys<210>21<211>29<212>DNA<213>Artificial sequence<220><223>Artificial sequence description: primer<400>21accagatctg cgggcagtga gcgcaacgc 29<210>22<211>32<212>DNA<213>artificial sequence<220><223>artificial sequence description: primer<400>22gtttctagat cctgtgtgaa attgttatcc gc 32<210>23<211>28<212>DNA<213>artificial sequence<220><223>artificial sequence description: primer<400>23cctctagaaa taaagtgagt gaatattc 28<210>24<211>22<212>DNA<213>artificial sequence<220><223>artificial sequence description: primer<400>24cttagcggtt ttacggtact gc 22<210>25<211>48<212>DNA<213>artificial sequence<220><223>artificial sequence description: the primer<220><221>is not defined as<222>(13, 14, 16, 19, 20, 21, 22, 23, 25, 26)<223>n ═ a or g or c or t<400>25ggtcgtgcgc tgnnynycnn ynnnrnnggc gataaagaac gcgtggtg 48<210>26<211>20<212>DNA<213>artificial sequence<220><223>artificial sequence description: primer<400>26ccacttcctc gatgacgcgg 20<210>27<211>34<212>DNA<213>artificial sequence<220><223>artificial sequence description: primer<400>27cgtgcgctgc ttttcgtgcg cgaaggcgat aaag 34<210>28<211>5<212>PRT<213>artificial sequence<220><223>artificial sequence description: the partial amino acid sequence<400>28Leu Ser Val Arg Glu 15<210>29<211>5<212>PRT<213>artificial sequence<220><223>which is encoded by the wild-type carB gene is described: the partial amino acid sequence<400>29Leu Phe Val Arg Glu 15<210>30<211>15<212>DNA<213>artificial sequence<220><223>encoded by the carB gene having a single mutation is described: primer<400>30ctttccgtgc gcgaa 15<210>31<211>15<212>DNA<213>artificial sequence<220><223>artificial sequence description: primer<400>31cttttcgtgc gcgaa 15<210>32<211>1149<212>DNA<213>E.coli (Escherichia coli)<400>32ttgattaagt cagcgctatt ggttctggaa gacggaaccc agtttcacgg tcgggccata 60ggggcaacag gttcggcggt tggggaagtc gttttcaata cttcaatgac cggttatcaa 120gaaatcctca ctgatccttc ctattctcgt caaatcgtta ctcttactta tccccatatt 180ggcaatgtcg gcaccaatga cgccgatgaa gaatcttctc aggtacatgc acaaggtctg 240 39240 240gtgattcgcg acctgccgct gattgccagc aacttccgta ataccgaaga cctctcttct 300tacctgaaac gccataacat cgtggcgatt gccgatatcg atacccgtaa gctgacgcgt 360ttactgcgcg agaaaggcgc acagaatggc tgcattatcg cgggcgataa cccggatgcg 420gcgctggcgt tagaaaaagc ccgcgcgttc ccaggtctga atggcatgga tctggcaaaa 480gaagtgacca ccgcagaagc ctatagctgg acacaaggga gctggacgtt gaccggtggc 540ctgccagaag cgaaaaaaga agacgagctg ccgttccacg tcgtggctta tgattttggt 600gccaagcgca acatcctgcg gatgctggtg gatagaggct gtcgcctgac catcgttccg 660gcgcaaactt ctgcggaaga tgtgctgaaa atgaatccag acggcatctt cctctccaac 720ggtcctggcg acccggcccc gtgcgattac gccattaccg ccatccagaa attcctcgaa 780accgatattc cggtattcgg catctgtctc ggtcatcagc tgctggcgct ggcgagcggt 840gcgaagactg tcaaaatgaa atttggtcac cacggcggca accatccggt taaagatgtg 900gagaaaaacg tggtaatgat caccgcccag aaccacggtt ttgcggtgga cgaagcaaca 960ttacctgcaa acctgcgtgt cacgcataaa tccctgttcg acggtacgtt acagggcatt 1020catcgcaccg ataaaccggc attcagcttc caggggcacc ctgaagccag ccctggtcca 1080cacgacgccg cgccgttgtt cgaccacttt atcgagttaa ttgagcagta ccgtaaaacc 1140 gctaaagtaa 1149<210>33<211>382<212>PRT<213>Escherichia coli (Escherichia coli)<400>33Leu Ile Lys Ser Ala Leu Leu Val Leu Glu Asp Gly Thr Gln Phe His 151015 Gly Arg Ala Ile Gly Ala Thr Gly Ser Ala Val Gly Glu Val Val Phe

20 25 30Asn Thr Ser Met Thr Gly Tyr Gln Glu Ile Leu Thr Asp Pro Ser Tyr

35 40 45Ser Arg Gln Ile Val Thr Leu Thr Tyr Pro His Ile Gly Asn Val Gly

50 55 60Thr Asn Asp Ala Asp Glu Glu Ser Ser Gln Val His Ala Gln Gly Leu 65 70 75 80Val Ile Arg Asp Leu Pro Leu Ile Ala Ser Asn Phe Arg Asn Thr Glu

85 90 95Asp Leu Ser Ser Tyr Leu Lys Arg His Asn Ile Val Ala Ile Ala Asp

100 105 110Ile Asp Thr Arg Lys Leu Thr Arg Leu Leu Arg Glu Lys Gly Ala Gln

115 120 125Asn Gly Cys Ile Ile Ala Gly Asp Asn Pro Asp Ala Ala Leu Ala Leu

130 135 140Glu Lys Ala Arg Ala Phe Pro Gly Leu Asn Gly Met Asp Leu Ala Lys145 150 155 160Glu Val Thr Thr Ala Glu Ala Tyr Ser Trp Thr Gln Gly Ser Trp Thr

165 170 175Leu Thr Gly Gly Leu Pro Glu Ala Lys Lys Glu Asp Glu Leu Pro Phe

180 185 190His Val Val Ala Tyr Asp Phe Gly Ala Lys Arg Asn Ile Leu Arg Met

195 200 205Leu Val Asp Arg Gly Cys Arg Leu Thr Ile Val Pro Ala Gln Thr Ser

210 215 220Ala Glu Asp Val Leu Lys Met Asn Pro Asp Gly Ile Phe Leu Ser Asn225 230 235 240Gly Pro Gly Asp Pro Ala Pro Cys Asp Tyr Ala Ile Thr Ala Ile Gln

245 250 255Lys Phe Leu Glu Thr Asp Ile Pro Val Phe Gly Ile Cys Leu Gly His

260 265 270Gln Leu Leu Ala Leu Ala Ser Gly Ala Lys Thr Val Lys Met Lys Phe

275 280 285Gly His His Gly Gly Asn His Pro Val Lys Asp Val Glu Lys Asn Val

290 295 300Val Met Ile Thr Ala Gln Asn His Gly Phe Ala Val Asp Glu Ala Thr305 310 315 320Leu Pro Ala Asn Leu Arg Val Thr His Lys Ser Leu Phe Asp Gly Thr

325 330 335Leu Gln Gly Ile His Arg Thr Asp Lys Pro Ala Phe Ser Phe Gln Gly

340 345 350His Pro Glu Ala Ser Pro Gly Pro His Asp Ala Ala Pro Leu Phe Asp

355 360 365His Phe Ile Glu Leu Ile Glu Gln Tyr Arg Lys Thr Ala Lys

370 375 380

Claims (12)

1. The large subunit of carbamoyl phosphate synthetase, corresponding to SEQ ID NO: 20 by the amino acid sequence at position 947-951 as set forthin SEQ ID NO: 1-9, and the feedback inhibition of uridine 5' -monophosphate is desensitized.

2. The large subunit of carbamoyl phosphate synthetase in accordance with claim 1, wherein the wild-type carbamoyl phosphate synthetase is E.coli carbamoyl phosphate synthetase.

3. The large subunit of carbamoyl phosphate synthetase of claim 1, wherein SEQ ID NO: 20 by the amino acid sequence at position 947-951 as set forth in SEQ ID NO: 1-9, and the feedback inhibition of uridine 5' -monophosphate is desensitized.

4. The large subunit of carbamoyl phosphate synthetase in accordance with claim 1, which comprises deletion, substitution, insertion or addition of one or several amino acids at one or more positions other than the 947-951 position, wherein feedback inhibition of uridine 5' -monophosphate is desensitized.

5. A carbamoyl phosphate synthetase comprising the large subunit of the carbamoyl phosphate synthetase of any one of claims 1 to 4.

6. A DNA encoding the large subunit of carbamoyl phosphate synthetase according to any one of claims 1 to 4, wherein the feedback inhibition of uridine 5' -monophosphate is desensitized.

7. A DNA encoding the large subunit of carbamoyl phosphate synthetase with desensitized feedback inhibition by uridine 5' -monophosphate according to any one of claims 1 to 4 and the small subunit of carbamoyl phosphate synthetase of Escherichia coli.

8. A bacterium belonging to the genus Escherichia having the DNA of claim 6 or 7.

9. The bacterium of claim 8, which has an ability to produce a compound selected from the group consisting of L-arginine, citrulline, and a pyrimidine derivative.

10. The bacterium of claim 9, wherein said pyrimidine derivative is orotic acid, uridine 5 '-monophosphate, cytidine, and cytidine 5' -monophosphate.

11. A method of producing a compound selected from the group consisting of L-arginine, citrulline, and a pyrimidine derivative, the method comprising the steps of: culturing the bacterium of any one of claims 8-10 in a culture medium to produce and accumulate said compound in the culture medium, and collecting said compound from the culture medium.

12. The method of claim 11, wherein said pyrimidine derivative is orotic acid, uridine 5 '-monophosphate, cytidine, and cytidine 5' -monophosphate.

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