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Riboflavin-producing bacillus subtilis and construction method and application thereof

A technology of Bacillus subtilis and riboflavin, which is applied in the field of Bacillus subtilis and its construction, can solve the problems of difficulty in obtaining riboflavin-yielding strains, heavy workload, and the limitation of Bacillus subtilis' ability to increase riboflavin production

Active Publication Date: 2021-03-23
TONGLIAO MEIHUA BIOLOGICAL SCI TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In recent years, although many genetically engineered strains of Bacillus subtilis with high riboflavin production have been obtained through metabolic engineering, due to the limitations of our understanding of microbial physiology and complex metabolic network regulation mechanisms, genetic engineering techniques have been used to continue to transform the strains. It is difficult to obtain strains whose riboflavin production is further greatly improved; and the traditional mutagenesis breeding has a large workload and lacks efficient and rapid screening methods, so that Bacillus subtilis is greatly restricted in improving the riboflavin production capacity. limits

Method used

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  • Riboflavin-producing bacillus subtilis and construction method and application thereof
  • Riboflavin-producing bacillus subtilis and construction method and application thereof
  • Riboflavin-producing bacillus subtilis and construction method and application thereof

Examples

Experimental program
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Embodiment 1

[0029] Embodiment 1. Obtain riboflavin high-yielding bacterial strain by mutagenesis screening

[0030] With Bacillus subtilis 168 (Bacillus subtilis 168) as the original strain, the B. subtilis 168 strain was subjected to conventional mutagenesis treatment with ultraviolet 15W, 30cm, and 20min, and then mutagenized with nitrosoguanidine under the conditions of 0.4mg / mL, 36°C, 20min. Then, spread on the 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, pH7.0-7.2), culture at 36°C for 24 hours. Afterwards, the strain with the best growth was selected for the next round of mutagenesis, and the concentration of 8-azaguanine in the basic medium was increased. After multiple rounds of mutagenesis screening, the B. subtilis MHZ-1908-1 strain was obtained, which ...

Embodiment 2

[0032] Example 2: Construction of B. subtilis168, Δupp strain by gene seamless editing method

[0033] With Bacillus subtilis B.subtilis168 as the starting strain, the gene scarless editing method used in the present invention is based on the two-step integration mediated by the temperature-sensitive plasmid, through chloramphenicol positive screening and 5-fluorouracil (5-FU) reverse screening (Applied Microbiology and Biotechnology, 2014, 98(21):8963-8973. Zhang W, Gao W, Feng J, et al). This screening method needs to delete the upp gene on the genome of the target strain firstly, which encodes uracil phosphoribosyltransferase. When upp and 5-FU exist at the same time, it has a lethal effect on the cells.

[0034] The specific construction process is as follows: using primers upp-1f / 1r, upp-2f / 2r, using the B. subtilis 168 genome as a template, using pfu DNA polymerase to amplify the upstream and downstream homology arms of 888bp and 938bp respectively, and using primers upp...

Embodiment 3

[0035] Example 3: Engineering strain B. subtilis 168, Δupp, purR A148D build

[0036] Using primers purR-1f, purR-1r and purR-2f, purR-2r, and using the B. subtilis 168 genome as a template, use pfu high-fidelity DNA polymerase to amplify to obtain purR A148D Upstream and downstream homology arms; primers purR-1f and purR-2r are used to fuse and amplify the upstream and downstream fragments to obtain purR A148D Fusion fragments (including A148D mutation, complete purR wild-type and mutant nucleotide sequences are shown in SEQ ID No. 1, 2; specific encoded wild-type and mutant protein sequences are shown in SEQ ID No. 7, 8. The fusion fragment and the pKSU plasmid (tool vector) were subjected to SalI and PstI double enzyme digestion respectively, then ligated, and transformed into Trans1 T1 Escherichia coli competent cells. Finally, the recombinant plasmid pKSU-purR was obtained A148D .

[0037] Subsequent transformation and screening methods were the same as in Example 2, a...

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Abstract

The invention belongs to the field of microbial fermentation, and discloses riboflavin-producing bacillus subtilis and a construction method and application thereof. According to the riboflavin-producing bacillus subtilis, at least one gene locus in purR, ribC or pyrE in a bacillus subtilis strain is mutated. The accumulated riboflavin of the bacillus subtilis strain obtained by mutating at leastone gene locus of purR, ribC or pyrE under the fermentation condition is significantly increased. The riboflavin-producing bacillus subtilis is a riboflavin high-yield strain, the produced riboflavinis significantly increased, and a foundation is laid for industrial production of the riboflavin.

Description

technical field [0001] The invention belongs to the field of microbial fermentation, and in particular relates to a riboflavin-producing Bacillus subtilis and a construction method and application thereof. [0002] At present, the riboflavin-producing strains used in production include yeast, bacillus, corynebacterium and Escherichia coli, etc., and most of them are wild strains or mutant strains with poor fermentation performance. Traditional production strains are mainly obtained by multiple rounds of physical and chemical mutagenesis and structural analogue screening. However, due to the uncertainty of the mutation points introduced by random mutagenesis, these strains often have a relatively complex genetic background. The analysis of the role of the relevant mutation points will consume a lot of time and energy, and it will also make the subsequent metabolic engineering face greater challenges. At the same time, the strains obtained by mutagenesis generally have disadva...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/54C12N15/75C12P25/00C12R1/125
CPCC07K14/32C12N9/1085C12N9/1077C12N15/75C12P25/00C12Y205/01009C12Y204/0201
Inventor 吴涛胡丹常利斌龚华李岩赵津津
Owner TONGLIAO MEIHUA BIOLOGICAL SCI TECH CO LTD
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