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Method for producing purine nucleosides and nucleotides by fermentation using bacterium belonging to the genus bacillus or escherichia

A purine nucleotide and purine nucleoside technology, applied in the field of purine nucleoside production, can solve the problems of no Bacillus amyloliquefaciens, no effect of YdhL, and no effect yet

Active Publication Date: 2012-05-23
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the effect of YdhL overexpression on nucleoside secretion and / or nucleoside production has not been shown
[0005] In addition, neither YdhL from Bacillus amyloliquefaciens has been reported nor the effect of YdhL overexpression on nucleoside secretion and / or nucleoside production has been shown

Method used

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  • Method for producing purine nucleosides and nucleotides by fermentation using bacterium belonging to the genus bacillus or escherichia
  • Method for producing purine nucleosides and nucleotides by fermentation using bacterium belonging to the genus bacillus or escherichia
  • Method for producing purine nucleosides and nucleotides by fermentation using bacterium belonging to the genus bacillus or escherichia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1. Cloning of the Bacillus subtilis ydhL gene

[0100] The entire nucleotide sequence of the Bacillus subtilis subsp. 168 strain has been determined (Kunst et al., Nature 390, 249-256 (1997)). Based on the reported nucleotide sequence, primers ydhL_N (SEQ ID No. 5) and ydhL_C (SEQ ID NO: 6) were synthesized for PCR amplification of the ydhL gene from Bacillus subtilis strain 168. Primer ydhL_N is consistent with the sequence of 489 to 467 bp upstream of the start codon of ydhL gene, and has a restriction enzyme SalI recognition site introduced at its 5'-end. Primer ydhL_C is complementary to the 58 to 80 bp sequence downstream of the stop codon of ydhL gene, and has a restriction enzyme BglII recognition site introduced at its 5'-end.

[0101] Chromosomal DNA of the Bacillus subtilis 168 strain was prepared by the usual method. PCR was carried out on "Perkin Elmer GeneAmp PCR System 2400" according to the following conditions: 94°C for 30 seconds, 61°C for 45 ...

Embodiment 2

[0114] Embodiment 2. Cloning of Bacillus amyloliquefaciens ydhL gene

[0115] The data shown in Example 1 show that when simultaneously introduced into M9 glucose minimal agar (Table 1), the Bacillus subtilis ydhL gene (hereinafter referred to as ydhL Bs ) confers resistance to 2,6-diaminopurine and 6-mercaptopurine in cells. Based on this fact, the clone from Bacillus amyloliquefaciens ydhL Bs The orthologue of the gene (hereinafter referred to as ydhL Ba ). Chromosomal DNA of B. amylobacter strain BA1 (IAM1523) was prepared by the general method. The chromosomal DNA was cut with EcoRI enzyme and ligated with the pMW118 vector which had been treated with the same enzyme before. The resulting DNA was used to transform E. coli strain TG1. Transformants were selected on minimal agar containing 400 μg / ml 2,6-diaminopurine, 400 μg / ml 6-mercaptopurine and 100 μg / ml ampicillin. Plasmid DNA was isolated from transformants and analyzed. Thus, a hybrid plasmid pYDHL2 containing ...

Embodiment 3

[0116] Example 3.ydhL Bs and ydhL Ba Effect of Gene Amplification on Inosine Production of Escherichia coli Inosine-producing Strains

[0117] The inosine-producing E. coli strain FADRaddedd (pMWKQ) was transformed with the pMW18 vector, and the YDHL1 and pYDHL2 plasmids, respectively. Transformants were selected on L-agar containing 100 mg / l ampicillin and 75 mg / l kanamycin. Strains FADRaddedd (pMWKQ, pMW18), FADRaddedd (pMWKQ, pYDHL1) and FADRaddedd (pMWKQ, pYDHL2) were thus obtained. These strains were cultured in L-broth containing 100 mg / l ampicillin and 75 mg / l kanamycin, respectively, at 37°C for 18 hours, and 0.3 ml of the resulting culture was inoculated into 3 ml containing 100 mg / l ampicillin and 75 mg / l kanamycin. 75mg / l kanamycin in the fermentation medium, the fermentation medium was placed in a 20x200mm test tube, and cultured at 37° C. for 72 hours with a rotary shaker.

[0118] Composition of fermentation medium (g / l):

[0119] Glucose 40.0

[0120] (NH 4...

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Abstract

The invention provides methods for producing purine base analogues, purine nucleosides, and purine nucleotides, such as inosine and 5'-inosinic acid, which include using a bacterium belonging to the genus Bacillus or to the genus Escherichia wherein the purine productivity of said bacterium is enhanced by increasing an activity of the YdhL protein. The invention also provides the amino acid sequence of the YdhL protein from Bacillus amyloliquefaciens and the gene encoding it.

Description

technical field [0001] The present invention relates to a method for producing purine nucleosides, which are important raw materials in the synthesis of 5'-inosinic acid and 5'-guanylic acid. The invention also relates to novel microorganisms used in the production method. The present invention also relates to novel DNA and protein species that confer resistance to purine base analogs and purine nucleosides in bacteria expressing said novel DNA. Background technique [0002] Nucleosides such as inosine are conventionally produced industrially by fermentation methods using adenine auxotrophic strains, or strains conferred drug resistance against various drugs such as purine analogs and sulfaguanidine. Examples of these strains include strains belonging to the genus Bacillus (Japanese Patent Laid-Open Nos. 54-17033 (1979), 55-2956 (1980), and 55-45199 (1980), Japanese Patent Application Laid-Open (laid-open) No. 56-162998 (1981), Japanese Patent Publication Nos. 57-14160 (19...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12P19/00C12N15/31C07K14/24C07K14/245C12N1/21C12P19/28
Inventor 纳塔利亚・P・扎卡泰瓦维塔利・A・利夫希茨瑟盖・V・格朗斯基埃卡特里纳・A・库图科瓦安娜・E・诺维科瓦尤里・I・科兹洛夫
Owner AJINOMOTO CO INC
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