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

A carbamoyl phosphate and synthetase technology, applied in the field of microbiology, can solve the problems of reduced activity of mutant enzymes, decreased enzyme activity, and the inability of mutant SAT proteins to restore natural SAT activity levels, etc.

Inactive Publication Date: 2007-05-16
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In principle, the feedback-resistant (fbr) phenotype of an enzyme occurs as a result of substituting another amino acid for a corresponding amino acid residue at one or several positions in the amino acid sequence, and such substitutions will lead to decreased enzyme activity
For example, substitution of native Met256 on Escherichia coli serine acetyltransferase (SAT) (cysE gene) with each of 19 other amino acid residues results in the fbr phenotype in most cases, however, mutant SAT proteins cannot Restoration of the activity level of native SAT [Nakamori S. et al., AEM, Vol. 64, pp. 1607-1611, 1998]
Therefore, the defect of the mutant enzyme obtained by the above method is that the activity of the mutant enzyme is reduced compared to the wild type enzyme

Method used

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  • New-mutant carbamyl-phosphate synthesized enzyme and method for producing compound derivated from carbamyl-phosphate
  • New-mutant carbamyl-phosphate synthesized enzyme and method for producing compound derivated from carbamyl-phosphate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] CarAB carrying the wild-type carAB gene from E. coli was constructed by cloning the AvaIII-BglII DNA fragment (4911bp) from the E. Plasmid pBScarAB-13.

[0063] Plasmid pET22-b(+) (Novagen, USA) was modified by replacing the T7 promoter with the lac promoter. The Lac promoter was obtained by using the plasmid pUC18 as a template, and using the oligonucleotides 5'-accagatctgcgggcagtgagcaacgc-3' (SEQ ID NO: 21) and 5'-gtttctagatcctgtgtgaaattgttatccgc-3' (SEQ ID NO: 22) as primers, by PCR obtained by amplification. The resulting fragment (0.14 kb) having the lac promoter was digested with restriction enzymes BglII 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 plasmid pBScarAB-13.

[0064] Using pBScarAB-13 as a template, and using oligonucleotides 5'-cctctagaaataaagtgagtgaatattc-3' (SEQ ID NO: 23) and 5'-cttagcggttttacggtactgc-3' (SEQ ID...

Embodiment 2

[0077] Example 2. Isolation of novel carB mutants and the effect of amino acid substitutions on the catalytic properties of CPSase

[0078] First evaluated on 40 recombinant B-6969 (pEL-carAB-NN) clones in the biosynthesis of citrulline from ornithine catalyzed by carAB and ArgI (ornithine aminomethyltransferase) enzymes. carAB activity and its anti-feedback effect on UMP.

[0079] The formula for the reaction is as follows:

[0080] CarAB+ArgI

[0081] NH 3 +HCO 3 - +2MgATP+ornithine------→citrulline+2MgADP+2Pi

[0082] In this reaction carbamyl phosphate synthase uses free NH 4 + as substrate

[0083] Protein extracts from 40 B-6969 (pEL-carAB-NN) strains and TG1 (pUC18-argI) cells, crude cells from sonicated cells by precipitation with ammonium sulfate (75% saturation) prepared from extracts. The protein pellet was dissolved in a buffer with the following formulation: Tris-HCl (50 mM), pH 7.5, 2-mercaptoethanol (2 mM).

[0084] The test system includes protein ...

Embodiment 3

[0102] Example 3. Production of orotic acid by a strain with a mutant carAB gene

[0103] As a mutant strain resistant to 6-azuracil (1 mg / ml), strain 311 was derived from Escherichia coli K12 (VKPM B-3853) with Tn10 inserted into the argA gene. Strain 311 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) on March 5, 2001 under the deposit number VKPM B-8085, and, on July 17, 2002, the original deposit was transferred to International deposit.

[0104] Strain 311 was transformed with plasmids pMW-carAB-wt and pMW-carAB-34, and the orotic acid production of the resulting strains was tested in the presence of different concentrations of uridine.

[0105] The culture conditions of test tube fermentation are as follows:

[0106] 1 / 20 diluted overnight culture, 60 g / L glucose, 25 g / L ammonium sulfate, 2 g / L monopotassium phosphate, 1 g / L magnesium sulfate, 0.1 mg / L thiamine, 5 g / L liter yeast extract Difco, 25 g / l chalk, 1 liter tap wate...

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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

technical field [0001] The present invention relates to the field of microbiology and in particular to methods for the production of compounds derived from carbamyl phosphate. More specifically, the present invention relates to the production of carbamoyl phosphate-derived compounds such as arginine, citrulline, and Pyrimidine derivatives including uridine, uridine 5'-monophosphate (UMP), cytidine and cytidine 5'-monophosphate (CMP). Background technique [0002] The carbamyl phosphate synthase (CPSase) of Escherichia coli can catalyze the complex synthesis process of carbamyl phosphate (CP) from bicarbonate, glutamine and two Mg-ATP molecules, and simultaneously release glutamic acid, phosphate and two Mg-ADPs [Meister A., ​​Advan. Enzymol. Mol. Biol., Vol. 62, pp. 315-374, 1989]. The synthesis of CP is an intermediate step in two biosynthetic pathways, that is, pyrimidine nucleotides and arginine biosynthetic pathways. In the first pathway, CP is coupled to aspartate ca...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N1/21C12N15/52C12P13/10C12P17/12
Inventor L·R·普蒂特斯恩S·V·斯米尔诺夫I·B·奥尔特曼A·E·诺维科瓦V·A·科利亚罗瓦M·M·古斯亚廷尔Y·G·罗斯托瓦T·A·亚珀斯卡亚
Owner AJINOMOTO CO INC
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