A high-yield 5-methyltetrahydrofolate recombinant Bacillus subtilis and its application

A technology of methyltetrahydrofolate and Bacillus subtilis, which is applied in the field of genetic engineering, can solve the problems of low yield of folic acid, a prerequisite substance, and low yield of 5-methyltetrahydrofolate of wild-type strains, and achieves a simple construction method and is easy to use. , the effect of good application prospects

Active Publication Date: 2020-08-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] However, there are many problems in the production of 5-methyltetrahydrofolate by Bacillus subtilis, for example, the synthesis and catabolism of 5-methyltetrahydrofolate proceed simultaneously, and the production of the precursor substance folic acid is also very low, resulting in the wild-type strain 5- Very low production of methyltetrahydrofolate

Method used

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  • A high-yield 5-methyltetrahydrofolate recombinant Bacillus subtilis and its application

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Effect test

Embodiment 1

[0045] The construction of embodiment 1 gene yitJ recombination fragment

[0046] On the Bacillus subtilis (Bacillus subtilis 168) genome, the homology arms on both sides of the yitJ gene were amplified, and the sequences were respectively left (Genbank accession number CP019662.1) Bacillus subtilis subsp.subtilisstr. Accession number CP019662.1) Bacillus subtilis subsp.subtilis str.168 genome: 1177554 to 1178553, design the homology arm primer, the left homology arm primer is yitL_1F:

[0047] CGAAGTCGGTTTGGTGATCTATACTCAAGG; yitJ_1R: TGTGAAATTGTTATCCGCTCTGTCTGCCTCCTTTATTCATCAGCAAGGAA, the right homology arm primer is yitJ_3F: CCTGGCGTTATTTTCCAAAAGACTGCCTGATCGAATCGG; yitJ-3R: CGTGAAAGTCTTGTTTCTCTAAAGGA TGTCAGTTC. According to the lox71-spc-lox66 knockout frame (as shown in SEQ ID NO.6), the primer spc_yitJ_2F was designed: GTGAATAAAGGAGGCAGACAGAGCGGATAACAATTTCACACAGGAAACAG;

[0048] spc_yitJ_2R: CTTTTGGAAATAACGCCAGGGTTTTCCCAGTCA. The amplified three amplified fragments were ...

Embodiment 2

[0049] Construction of embodiment 2 gene metF recombination fragment

[0050] The homology arms on both sides of the metF gene were amplified on the Escherichia coli (Escherichia coli K-12MG1655) genome, and the sequences were the left (Genbank accession number CP019662.1) Bacillus subtilis subsp.subtilisstr.168 genome 1180393 to 1181395 and the right ( Genbank accession number CP032667.1) Escherichiacoli str.K-12substr. MG1655 chromosome, complete genome: 4389561 to 4390451, designed homology arm primers, the left homology arm primer is metF_1F: CGAAGTCGGTTTGGTGATCTATACTCAAG; metF_1R: TTATCCGCTCTGTCTGCCTCGCTTATTC, right homology arm Primers were metF_3F: GAATGTACACATGAGCTTTTTTCACGCCAGCC; metF_3R: TCCACGGAAATGGTTTTGACTTCGAG. According to the sequence information of the P7CP43 expression cassette lox71-crmP43-lox66 (as shown in SEQ ID NO.7), the primers P7CP43_metF_2F: GAGGCAGACAGAGCGGATAACAATTTCACACAGGAAAC; The above three amplified fragments were fused by fusion PCR technolo...

Embodiment 3

[0051] Construction of embodiment 3 gene purU recombination fragment

[0052] On the Bacillus subtilis (Bacillus subtilis 168) genome, the homology arms on both sides of the purU gene were amplified, and the sequences were the left (Genbank accession number CP019662.1) Bacillus subtilis subsp.subtilisstr.168 genome: 1376004 to 1376972 and the right ( Genbank登录号CP019662.1)Bacillussubtilis subsp.subtilis str.168 genome: 1377827 to 1379009,设计同源臂引物,左侧同源臂引物为purU_1F: GGGTAGCGGAGAAAATCGGCATGCATTACA;purU_1R: CCTGTGTGAAATTGTTATCCGCTCTTATGACAGAATTTTTAAACCAACTGCTGAGCATA AAATCAACGCGA,右侧同源臂引物为purU_3F : CCTGGCGTTACGGAAGCGCTGAAAAACATCGGAAGAAC; purU_3R: CTCTCCGCTGCCAGTTCCAATGAATACTTTCA. According to the lox71-spc-lox66 knockout frame (shown in SEQ ID NO.6), the primer spc_putU_2F was designed: TTGGTTTAAAAAATTCTGTCATAAGAGCGGATAACAATTTCACACAGGAAACAGCT;

[0053] spc_purU_2R: CAGCGCTTCCGTAACGCCAGGGTTTTTCCCAGTCACGAC. The 3 amplified fragments were fused by fusion PCR technology to obtain the rec...

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Abstract

The invention discloses a high-yield 5-methyl tetrahydrofolate recombinant bacillus subtilis and an application thereof, and belongs to the field of genetic engineering. The high-yield 5-methyl tetrahydrofolate recombinant bacillus subtilis uses the bacillus subtilis 168 as the expression host; by over-expresseing 5,10-methylene tetrahydrofolate reductase coding gene metF, dihydrofolate reductasecoding gene dfrA, dihydrofolate synthase coding gene folC, knocking-out double function homocysteine S-methyltransferase / 5,10-methylene tetrahydrofolate reductase coding gene yitJ, formyltetrahydrofolate deformylase coding gene purU, the high-yield 5-methyl tetrahydrofolate recombinant bacillus subtilis obtains accumulation of 5-methyl tetrahydrofolate bacillus subtilis gene engineering bacteria;the yield of 5-methyl tetrahydrofolate reached 952.05 ug / L, and the conversion cell dry weight is 342.40ug / g DCW.

Description

technical field [0001] The invention relates to a high-yield 5-methyltetrahydrofolate recombinant bacillus subtilis and its application, belonging to the field of genetic engineering. Background technique [0002] L-5-methyltetrahydrofolate (5-MeTHF) is the only active form of folic acid that works in the human body, and is the only drug that can penetrate the blood-brain barrier among folic acid drugs. 5-MeTHF can prevent neonatal neural tube defects, and has the effect of preventing and treating Alzheimer's disease. In addition, because 5-MeTHF has a high selective protective effect on normal cells, it gradually replaces the antidote 5-formyltetrahydrofolate of the antineoplastic drug methotrexate clinically. 5-MeTHF is also used in the treatment of megaloblastic anemia, rheumatoid arthritis and other diseases. The European Union approved 5-MeTHF series products as safe food supplements at the end of 2005, which further promoted the market demand for 5-MeTHF. At present...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P17/18C12R1/125
CPCC07K14/32C12P17/182
Inventor 刘延峰朱进伟杨晗彭湘屏李江华郑玲辉陈坚
Owner JIANGNAN UNIV
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