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Plasmid and cell for promoting biotin synthesis and method for promoting synthesis of biotin

A biotin and enzyme synthesis technology, applied in the field of metabolic engineering, can solve the problem of low yield and achieve the effect of improving biotin synthesis efficiency

Active Publication Date: 2017-08-29
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the biosynthesis of biotin still faces the problem of low yield. In the biotin synthesis process, the step from desthiobiotin to biotin has always been the rate-limiting step. Studies have shown that increasing the content of intracellular SAM can strengthen this step. Effectively increase the overall biotin production, and after the biotin production is increased, the absorption rate of the synthetic precursor pimelic acid will become the rate-limiting step of the entire biotin synthesis. These will be the problems that must be solved in the biotin biosynthesis process question

Method used

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  • Plasmid and cell for promoting biotin synthesis and method for promoting synthesis of biotin
  • Plasmid and cell for promoting biotin synthesis and method for promoting synthesis of biotin
  • Plasmid and cell for promoting biotin synthesis and method for promoting synthesis of biotin

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Effect test

Embodiment 1

[0034] Example 1: Related primer design

[0035] According to the published biotin operon gene sequence of Bacillus subtilis in Genebank gi:2627063, primers Bio-EcoRI-up, Bio-XhoI-down and pET-EcoRI-up, pET-NotI-down were designed respectively. According to the Saccharomyces cerevisiae sam2 gene sequence published in Genebank gi:852113, design primers

[0036] pET-EcoRV-up, pET-XhoI-down. According to the Pseudomonas gene sequence published in Genebank gi:1002825811, two pairs of primers pACYC-EcoRV-up, PACYC-XhoI-down and pACYC-EcoRI-up, PACYC-NotI-down were designed respectively. The primer design results are shown in the table 1.

[0037] Table 1 The primers involved in the present invention

[0038] Primer

Embodiment 2

[0039] Example 2: Construction of recombinant plasmid pACYCDuet-bioBFHCD-bioA

[0040] The bioA (1407bp) and bioBFHCD (4489bp) genes were amplified by pACYC-EcoRV up, PACYC-XhoI-down and pACYC-EcoRI-up, PACYC-NotI-down using the P.putida KT2440 genome as a template. The pACYCDuet-1 plasmid and the bioBFHCD fragment were digested with EcoRI and NotI, and then ligated to the multiple cloning site (mcsl) behind T7promoler-1 to obtain the recombinant plasmid pACYCDuet-bioBFHCD.

[0041] Transform the recombinant plasmid pACYCDuet-bioBFHCD into the cloning host DH5a, culture overnight, extract the recombinant plasmid, digest the pACYCDuet-bioBFHCD recombinant plasmid and bioA fragment with EcoRV and XhoI, and enzyme-link to the multi-cloning site behind T7promoler-2 Click on mcs2 to obtain the recombinant plasmid pACYCDuet-bioBFHCD-bioA, the electrophoresis of the recombinant plasmid is as follows figure 1 .

Embodiment 3

[0042] Embodiment 3: Construction of recombinant plasmid pETDuet-bioW-sam2

[0043] Through primers pET-EcoRI-up and pET-NotI-down, using B. subtilis168 genomic DNA as template, carry out bioW (777bp) gene amplification, the amplification result is as follows figure 2 , Purified by PCR, pETDuet-1 plasmid and bioW fragments were digested with EcoRI and NotI, and enzyme-linked to the multiple cloning site mcs1 behind T7promoler-1 to obtain the recombinant plasmid pETDuet-bioW.

[0044] The recombinant plasmid pETDuet-bioW was transformed into the cloning host DH5a, cultivated overnight, and the recombinant plasmid was extracted. Using S.cerevisiae ZJU001 genomic DNA as a template, using pET-EcoRV-up and pET-XhoI-down primers to amplify the sam2 gene (1155bp), the electrophoresis of the amplified product is as follows image 3 . After double-digesting the pETDuet-bioW recombinant plasmid and sam2 gene fragment with restriction endonucleases EcoRV and XhoI, use T 4 The DNA lig...

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Abstract

The invention discloses a method for promoting synthesis of biotin, and bioW gene sequence in B.subtilis168 and S-Adenosyl-L-methionine (SAM) synthase (Sam2) gene sequence in S.cerevisiae ZJU001 are used for promoting synthesis of the biotin by two pieces of biotin operon genes bioBFHCD and bioA. Gene engineering bacteria obtained by the method has efficient biotin synthesis ability, the yield of the biotin produced in a medium with pimelic acid as a substrate by fed-batch fermentation can reach 417mg / L, is 200 times of that of the biotin produced by original Escherichia coli, and the productive rate reaches 10.4mg / (L.h).

Description

technical field [0001] The invention belongs to the technical field of metabolic engineering, and relates to a plasmid, a cell and a method for promoting biotin synthesis. Background technique [0002] Biotin, also known as vitamin H, vitamin B7 and coenzyme R, is one of the water-soluble B vitamins and an indispensable nutrient for animals to maintain normal physiological functions. Biotin is mainly used as a cofactor of many enzymes in the body to participate in the metabolism of the three major nutrients in the body, and participates in the reactions of carboxylation, decarboxylation and transcarboxylation. Biotin is used in cosmetics, food additives, medicine, vitamin preparations, feed, fermentation and other industries. It plays an important role in maintaining the normal growth and development of humans and animals, the health of bone marrow, and protecting skin and feathers. At present, the annual demand in the global market For 300 tons. [0003] Compared with bio...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/63C12P17/00C12R1/19
CPCC12N9/1085C12N15/63C12P17/00C12Y205/01006
Inventor 林建平毕晨光李国四吴绵斌杨立荣
Owner ZHEJIANG UNIV
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