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Bacillus subtilis and construction method and application thereof

The technology of a Bacillus subtilis and its construction method is applied in the field of adenosine-producing Bacillus subtilis and its construction, which can solve the problems of poor fermentation performance, low conversion rate of adenosine, and inability to meet large-scale industrial production, so as to increase yield, The effect of increasing the accumulation of adenosine

Active Publication Date: 2019-09-20
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fermentation performance of adenosine strains is still poor, and the conversion rate of adenosine is still low, which still cannot meet the needs of large-scale industrial production.

Method used

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  • Bacillus subtilis and construction method and application thereof
  • Bacillus subtilis and construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1: Mutagenesis and screening to obtain adenosine-producing strains

[0039] Using B.subtilis 168 as the original strain, the B.subtilis 168 strain was first subjected to conventional mutagenesis treatment with UV 15W, 30cm, 20min, and then mutagenized with nitrosoguanidine under the conditions of 0.4mg / mL, 36℃, 20min; Then spread on a minimal medium containing 0.2g / L 8-azaguanine (g / L: glucose 20, ammonium sulfate 2, magnesium sulfate 0.4, calcium chloride 0.02, ferrous sulfate 0.02, disodium hydrogen phosphate 1.5, Zinc sulfate 0.01, manganese sulfate 0.01, potassium dihydrogen phosphate 1.5, agar 18, pH value 7.0~7.2), culture at 36°C for 24h, select the best growing strain for the next round of mutagenesis, and increase the 8 -Concentration of azaguanine, after multiple rounds of mutagenesis and screening, the B.subtilisMHA strain was obtained, which can grow on the medium containing 1g / L 8-azaguanine, and B.subtilisMHA shake flask produces adenosine levels It ...

Embodiment 2

[0041] Example 2: Construction of B.subtilis168 (Δupp) strain by the method of seamless gene editing

[0042] Taking Bacillus subtilis B. subtilis168 as the starting strain, the method of gene traceless editing used in the present invention is based on the principle of two-step integration mediated by a temperature-sensitive plasmid and the principle of upp reverse screening. The transformation used is the Spizize transformation method. The editing process can be referred to A markerless gene replacement method for B.amyloliquefaciensLL3and its usein genome reduction and improvement of poly-γ-glutamic acid production[J].(Applied Microbiology and Biotechnology,2014,98(21):8963-8973.Zhang W,Gao W,Feng J, et al).

[0043] Use primers upp-1f / 1r, upp-2f / 2r, use B.subtilis168 genome as template, use pfu DNA polymerase to amplify the upstream and downstream homology arms of 888bp and 938bp, respectively, and use primer upp-1f / 2r to amplify Obtain the upstream and downstream fusion fragme...

Embodiment 3

[0044] Example 3: Engineering strain B.subtilisA1 (purD P116L ), A2(purR A65D ), A3(guaB G279R ) Build

[0045] Use primers purD-1f / 1r and purD-2f / 2r, purR-1f / 1r and purR-2f / 2r, guaB-1f / 1r and guaB-2f / 2r, use B.subtilis 168 genome as template, use pfu high security True DNA polymerase amplification obtains the upstream and downstream homology arms of purD, purR and guaB respectively; the upstream and downstream fragments are fused with primers purD-1f / 2r, purR-1f / 2r and guaB-1f / 2r to obtain purD* homologous fragments, respectively (Containing the P116L mutation, the nucleotide sequence of the complete purD* gene is shown in SEQ ID No. 17, a total of 1269 bp; the amino acid sequence is shown in SEQ ID No. 18, a total of 422 amino acids), purR* homologous fragments (including A65D mutation, the nucleotide sequence of the complete purR* gene is shown in SEQ ID No. 19, a total of 858 bp; the amino acid sequence is shown in SEQ ID No. 20, a total of 285 amino acids) and the guaB* ho...

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Abstract

The invention belongs to the field of microorganisms and discloses bacillus subtilis and a construction method and application thereof. At least one of purDP116L, purRA65D or guaBG279R in bacillus subtilis strains is in site mutation. B. subtilis A1, A2 and A3 strains are obtained respectively through single-site mutation of purDP116L, purRA65D or guaBG279R. Three single-site mutation plasmids sequentially convert a B. subtilis 168 (Delta upp) strain to obtain a B. subtilis A5 strain with three sites all in site mutation. The bacillus subtilis is an adenosine high-yield strain, adenosine can be accumulated effectively, yield of adenosine is increased, and a foundation is laid for industrialized production of adenosine.

Description

Technical field [0001] The invention belongs to the field of microorganisms, and specifically relates to a Bacillus subtilis and a construction method and application thereof, in particular to a Bacillus subtilis that produces adenosine, and a construction method and application thereof. Background technique [0002] Adenosine is adenosine, its chemical name is 6-amino-9-β-D-ribofuranosyl-9-hydropurine. It is the product of adenine nucleotide dephosphorylation and is an important nucleotide derivative Things. Adenosine is an endogenous nucleoside that is distributed throughout human cells. It can directly enter the myocardium to generate adenylate through phosphorylation, and participate in myocardial energy metabolism. At the same time, it also participates in expanding coronary blood vessels and increasing blood flow. Adenosine has physiological effects on the cardiovascular system and many other systems and tissues of the body. In addition to being used as a special medicine...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12P19/40C12R1/125
CPCC07K14/32C12N9/0006C12N9/1014C12N15/75C12P19/40C12Y101/01205
Inventor 胡丹袁辉吴涛李岩
Owner MEIHUA BIOTECH LANGFANG CO LTD
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