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A method for producing purine ribonucleosides and ribonucleotides

A technology of purine nucleotides and purine nucleosides, applied in the field of producing purine ribonucleosides, which can solve the problems of purine ribonucleoside productivity coding 3-hexulose-6-phosphate synthase gene inactivation and other issues

Active Publication Date: 2011-08-31
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, inactivation of the gene encoding 3-hexulose-6-phosphate synthase for the purpose of improving purine ribonucleoside productivity has not been previously reported

Method used

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  • A method for producing purine ribonucleosides and ribonucleotides
  • A method for producing purine ribonucleosides and ribonucleotides
  • A method for producing purine ribonucleosides and ribonucleotides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Construction of a strain with an inactivated hxlA gene

[0059] In order to inactivate the hxlA gene on the chromosome of the Bacillus subtilis inosine producer KMBS375 (the construction of the strain KMBS375 is described in Reference Example), the upstream and downstream regions of the hxlA gene were cloned into the two multiple cloning sites of the pKS1 plasmid ( Shatalin K.Y. and Neyfakh A.A., FEMS Microbiology Letters, 245:315-9 (2005)) ( figure 1 ). Amplification of the 886 bp fragment in the upstream region of the hxlA gene was performed by PCR using primers P1 (SEQ ID NO: 5) and P2 (SEQ ID NO: 6) and chromosomal DNA of Bacillus subtilis KMBS375 as a template. Primers P1 and P2 contain recognition sites for endonucleases SacII and PstI, respectively. Amplification of the 923 bp fragment in the downstream region of the hxlA gene was performed by PCR using primers P3 (SEQ ID NO: 7) and P4 (SEQ ID NO: 8) and chromosomal DNA of Bacillus subtilis KMBS375...

Embodiment 2

[0060] Example 2. Production of inosine by Bacillus subtilis strain KMBS375Δhxl::Km

[0061] To test the inactivation effect of hxlA on inosine production, Bacillus subtilis strains KMBS375 and KMBS375Δhxl::Km were each cultured in L-broth for 18 hours at 34°C, and then 0.3ml of the culture was inoculated into 3ml of a 20x200mm test tube fermentation medium and incubated for 72 hours at 34°C on a rotary shaker. The results are shown in Table 1.

[0062] Composition of fermentation medium (g / l):

[0063]

[0064]

[0065] After culturing, the amount of inosine accumulated in the medium was determined by HPLC.

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Abstract

The present invention relates to biotechnology, and specifically to a method for producing purine ribonucleosides, which are important as raw materials for synthesis of purine nucleotides. The method uses a bacterium belonging to the genus Bacillus which has been modified to decrease 3-hexulose-6-phosphate synthase activity.

Description

technical field [0001] The present invention relates to the microorganism industry, and in particular to methods for producing purine ribonucleosides, which are important as raw materials in the synthesis of purine nucleotides. The method uses bacteria of the genus Bacillus modified to attenuate the expression of the gene encoding 3-hexulose-6-phosphate. Background technique [0002] Methods for the fermentative production of purine nucleosides using adenine auxotrophic strains are known. Moreover, these strains can be further conferred resistance to various drugs such as purine analogues and sulfaguanidine. Examples of these strains include various Bacillus strains (Japanese Patent Application Laid-Open No. 38-23039 (1963), 54-17033 (1979), 55-2956 (1980) and 55-45199 (1980), Japanese Patent Application Laid-Open No. 56 -162998 (1981), Japanese Patent Publication Nos. 57-14160 (1982) and 57-41915 (1982), and Japanese Patent Application Publication No. 59-42895 (1984)), Br...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N1/20C12P19/32C12P19/34
CPCC12N1/20C12N9/88C12P19/34C12P19/32
Inventor 瑟吉.V.格朗斯基德米特里.V.罗马南科夫纳塔利娅.P.扎卡泰瓦浅原贵之
Owner AJINOMOTO CO INC