Method for increasing copy number of gene menA and increasing yield of MK-7

A yield, open reading frame technology, applied in the field of genetic engineering, can solve problems such as low yield

Active Publication Date: 2019-09-13
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The yield of menadione is very low in the original Bacillus subtilis, and the liquid fermentation is only 9mg / L

Method used

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  • Method for increasing copy number of gene menA and increasing yield of MK-7
  • Method for increasing copy number of gene menA and increasing yield of MK-7
  • Method for increasing copy number of gene menA and increasing yield of MK-7

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: the construction of departure bacterial strain Bacillus subtilis BS14

[0040] (1) Construction of recombinant bacteria BS1

[0041] via the constitutive promoter P 43 Expression of the menaquinone isochorisate synthase (menF, genebank ID: 937190) gene was enhanced by replacing the native promoter of menF on the Bacillus subtilis chromosome. Using a marker-free genetic modification strategy, see the article (Yan, X., Yu, H.-J., Hong, Q., Li, S.P., 2008. Cre / lox system and PCR-based genome engineering in Bacillus subtilis. Appl Environ Microb.74,5556-5562), the specific construction process is as follows:

[0042] (a) Cloning of genes

[0043] ① Using the Bacillus subtilis 168 genome as a template, using primer menF up .FOR and menF up .REV amplifies the upstream homology arm sequence menF of the menF gene up (The sequence is shown in SEQ ID NO.1).

[0044] ② Artificially synthesize the lox71-zeo-lox66 cassette containing the bleomycin gene (sequence...

Embodiment 2

[0098] Embodiment 2: the construction of control bacteria

[0099] On the basis of the recombinant bacteria BS14 obtained in Example 1, the constitutive promoter P 43 Replacement of the native promoter of the 1,4-dihydroxy-2-naphthoate heptapentenyltransferase (menA, genebank ID: 937356) gene on the Bacillus subtilis chromosome. The specific construction process is as follows:

[0100] (1) Cloning of genes

[0101] ① Using the Bacillus subtilis 168 genome as a template, using primer menA up .FOR and menA up .REV amplified to obtain the upstream homology arm sequence menA of the menA gene up .

[0102] ② Artificially synthesize the lox71-zeo-lox66 cassette containing the bleomycin gene (sequence shown in SEQ ID NO.2).

[0103] ③ Using the Bacillus subtilis 168 genome as a template, using primer P 43 .For and P 43 .Rev amplified to get P 43 Promoter sequence (sequence shown in SEQ ID NO.3).

[0104] ④ Use the Bacillus subtilis 168 genome as a template, and use primers ...

Embodiment 3

[0111] Example 3: Amplification of the menA open reading frame

[0112] Amplification of the opening of 1,4-dihydroxy-2-naphthoate heptapentenyltransferase (menA) using the primers menA-orf.FOR and menA-orf.REV using the Bacillus subtilis 168 genome as a template Reading frame, the amplified gene fragment is carried out agarose gel detection size is 1537bp (see figure 1 ), the fragments of the correct size were recovered by gel cutting, and the nucleotide sequence was obtained such as the menA reading frame sequence P of SEQ ID NO.8 mena -menA.

[0113] Table 5 sequence list

[0114]

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Abstract

The invention discloses a method for increasing the copy number of gene menA and increasing the yield of MK-7, and belongs to the field of genetic engineering. The method determines that the menA geneis a key gene in the synthetic pathway of heptaene menadione (MK-7), finds out that the self promoter of the menA gene has an important effect on the activity of menA, and replaces a natural promoterof the menA gene on the bacillus subtilis 168 chromosome with P43 promoter, and the yield of MK-7 produced by fermentation of the obtained strain is obviously reduced compared with that of the original strain. The method integrates and expresses the whole open reading frame of menA at the ganA, thrC and dacA sites of a bacillus subtilis genome to respectively obtain recombinant strains BS15, BS16and BS17, so that the yield of MK-7 is increased to 40mg / L, 59mg / L and 75mg / L respectively.

Description

technical field [0001] The invention relates to a method for increasing the copy number of gene menA to increase the output of MK-7, which belongs to the field of genetic engineering. Background technique [0002] Menadione (MK-7) is an important class of fat-soluble vitamins. As a cofactor of γ-glutamate carboxylase, it regulates the distribution of calcium, promotes bone development, reverses osteoporosis, and protects blood vessels. Plays an important role in preventing atherosclerosis and cardiovascular disease responses. Because menadione has better affinity and longer half-life to the human body, it has attracted more attention. The yield of menadione is very low in the original Bacillus subtilis, and the liquid fermentation is only 9mg / L. [0003] In Bacillus subtilis, chorismate can form a skeleton structure menadione under the action of seven enzymes, menF, menD, ytxM, menC, menE, menB, yuxO, and then in 1,4-dihydroxy-2-naphthoic acid seven Pentenyltransferase (m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N1/21C12P7/66C12R1/125
CPCC12N9/1085C12N15/75C12P7/66C12Y205/01
Inventor 刘龙杨陈亮崔世修吕雪琴李江华堵国成陈坚唐波
Owner JIANGNAN UNIV
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