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Method for improving yield of vernine from bacillus amyloliquefaciens

A technology of amylolytic spores and bacilli, applied in the field of bioengineering, to achieve stable production performance and high genetic stability

Inactive Publication Date: 2014-12-24
STAR LAKE BIOSCI CO INC ZHAOQING GUANGDONG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the feedback inhibition of key enzymes is not lifted

Method used

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  • Method for improving yield of vernine from bacillus amyloliquefaciens
  • Method for improving yield of vernine from bacillus amyloliquefaciens
  • Method for improving yield of vernine from bacillus amyloliquefaciens

Examples

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Embodiment 1

[0024] Construction of a guanosine-producing engineering bacterium B. amyloliquefaciens XH-△po based on Bacillus amyloliquefaciens XH7

[0025] (1) Obtaining the truncated fragment containing the purine promoter: the DNA fragment containing the purine promoter gene of the Bacillus amyloliquefaciens DSM7 strain (Bacillus amyloliquefaciens XH7: CP002927) was obtained by PCR using the chromosomal DNA of the bacterium as a template, and the primer was SEQ ID No.3 and SEQ ID No.4 amplify the left arm of the homology arm, primers are SEQ ID No.5 and SEQ ID No.6 amplify the right arm of the homology arm, and the product of gene PCR amplification is recovered by gel, and then taken The amplified product with an equimolar ratio is used as a template, and the forward primer and reverse primer used are respectively SEQ ID No.3 and SEQ ID No.6, and the gene size truncated from -193 to -29 bp at the start codon of the purE gene is amplified Matching DNA fragments such as figure 1 As shown...

Embodiment 2

[0029] Construction of B. amyloliquefaciens XH7-△po-purF-217, a guanosine-producing engineered strain, based on Bacillus amyloliquefaciensXH-△po

[0030] (1) PurF gene amplification and acquisition of mutant fragments: the DNA fragment containing the purine promoter gene of Bacillus amyloliquefaciens XH7 strain (Bacillus amyloliquefaciens XH7: CP002927) was obtained by PCR using the chromosomal DNA of the bacterium as a template, and the primers were SEQ ID No7, SEQ ID No8 amplify the left arm of the homologous arm, primers are SEQIDNo9, SEQIDNo10 amplify the right arm of the homologous arm, the product of gene PCR amplification is recovered from the gel, and then the amplified product in equimolar ratio is taken as a template. The forward primer and the reverse primer are respectively SEQ ID No.7 and SEQ ID No.10, and a DNA fragment of the same size containing the L217I mutation site of the purF gene is amplified. like figure 2 As shown, lane 1 in the figure is a DNA marker...

Embodiment 3

[0034] Detection of guanosine-producing ability of Bacillus amyloliquefaciensXH-△po and B. amyloliquefaciensXH7-△po-purF-217 constructed by shake flask fermentation

[0035] (1) From the preserved glycerol tube (glycerol concentration 18%, -80 ℃ preservation) streaked on the preferred solid activation medium (weight percentage) (ingredients: 1% peptone, 1% sodium chloride, 0.5% yeast extract extract, 1% glucose, 1.5% agar, and the balance is water), cultivated at 37°C for 24 to 36 hours; pick a single colony grown on a solid plate, and inoculate it on a shaker flask seed medium (percentage by weight) (ingredients: Glucose 1.5%, peptone 1%, yeast extract 0.5%, NaCl 0.5%, pH 7.2), cultured at 37°C for 6-7h, inoculated into shake flask fermentation medium (weight percentage) with an inoculum size of 10% by volume (8% glucose, 0.6% magnesium sulfate, 0.8% yeast powder, 0.15% potassium dihydrogen sulfate; 1.0% ammonium sulfate, 0.15% potassium chloride, 1.6% monosodium glutamate, 0...

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Abstract

The invention discloses a method for improving the yield of vernine from bacillus amyloliquefaciens. The method comprises the following steps: by taking thermo-sensitive type plasmid pKS2 as a carrier, removing a promoter partial segment of a purine operon (purEKBCSQLFMNHD) through adopting a homologous recombination method, wherein the preferable sequence segment is -193bp to -29bp segment of the purE-deleted gene, and the repression effect of purR (repressor protein of the purine operon) and the transcription attenuation action caused by the palindrome structure of the segment of a strain with the segment deleted are removed; and desensitizing transamidase gene of purF coded phosphoribosyl pyrophosphate (PRPP) through point mutation, wherein preferably, leucine at 217th site is mutated into isoleucine (L217I), and the feedback inhibition from products adenosine and vernine can be removed. The production capability of the transformed genetically engineered bacterium vernine is obviously improved.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for increasing the yield of guanosine produced by bacillus amyloliquefaciens. technical background [0002] Guanine nucleoside, namely guanosine, is a kind of purine nucleoside, which is formed by connecting N-9 of guanine and C-1 of D-ribose through β-glycosidic bond. Guanosine has a very wide range of uses in the food and pharmaceutical industries. In the food industry, guanosine phosphate is guanylic acid, which is composed of guanylic acid and inosinic acid. One of the main taste ingredients of instant noodle seasoning packets and condiments (such as chicken essence, soy sauce, etc.). In the pharmaceutical industry, guanosine is used as an intermediate for many highly effective drugs, such as antiviral drugs such as influenza virus, HIV and herpes virus. For example, many hepatitis B virus drugs, such as lamivudine and entecavir, have intermediates of gua...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N1/21C12P19/40C12R1/07
Inventor 廖瑜玲谢畅丰李红潘力谢清华
Owner STAR LAKE BIOSCI CO INC ZHAOQING GUANGDONG
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