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Gene overexpression and obtained strains, applications

A gene overexpression and overexpression technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problem of undiscovered de novo biosynthesis pathway of MK-7

Active Publication Date: 2021-03-09
TIANJIN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, no comprehensive and systematic study on the de novo biosynthesis pathway of MK-7 has been found

Method used

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  • Gene overexpression and obtained strains, applications
  • Gene overexpression and obtained strains, applications
  • Gene overexpression and obtained strains, applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Overexpression of menA

[0067] The yxlA locus of B. subtilis chromosome was selected to overexpress the menA gene. First, using the chromosome of B. subtilis 168 as a template, primers yxlA-menA-U1 / yxlA-menA-U2q, yxlA-menA-1q / yxlA-menA-2, yxlA-menA-D1q / yxlA-menA-D2, yxlA-menA-G1q / yxlA-menA-G2 amplified fragment U (as shown in SEQ ID No.59, 1115bp), A (as shown in SEQ ID No.61, 1057bp), D (as shown in SEQ ID No.62 Shown, 1053bp), G (shown in SEQ ID No.64, 806bp); With plasmid pUC57-1.8k-P1 as template, with primer yxlA-menA-P1 / yxlA-menA-P2 amplification containing promoter P lapS Fragment P (as shown in SEQ ID No.60, 442bp); With the chromosome of BS168NUm preserved in the laboratory as a template, amplify fragment CR with primer yxlA-menA-CR1q / CR2 (as shown in SEQ ID No.63, 2069bp). Then by overlapping PCR method, using primer yxlA-menA-U1 / yxlA-menA-2, fragment U, P and A are spliced ​​into fragment UPA (2614bp); Fragment UPA and fragment D were spliced ​...

Embodiment 2

[0068] Example 2 Replacement of the promoter of the hepS-menG-hepT operon in situ

[0069] constitutive promoter P lapS In situ replacement of the promoter of the hepS-menG-hepT operon on the BS168NU chromosome. Firstly, using the chromosome of B. subtilis 168 as a template, the SGT-U1m / SGT-U2qm amplified fragment U(1319) was obtained with primers, and connected to the vector pSS by BglII and XhoI enzyme digestion, and transformed into E.coli Trans T1 competent to obtain Recombinant plasmid pSS-SGT-U. Then, using the plasmid pUC57-1.8k-P1 as a template, primer P lapS 1 / P lapS 2 Amplified containing promoter P lapS Fragment P (443bp) of B. subtilis 168; use the chromosome of B. subtilis 168 as a template, and use primers to amplify fragment D (1359bp) of SGT-D1q / SGT-D2. lapS 1qm / SGT-D2m was spliced ​​into a fragment PD (952bp), which was finally digested with BamHI and KpnI and connected to the plasmid pSS-SGT-U to transform E. coli Trans T1 competent to obtain the recombi...

Embodiment 3

[0072] Embodiment 3 shake flask fermentation culture and the mensuration of thalline growth situation

[0073] Table 3 fermentation medium and fermentation conditions

[0074] parameter scope glycerin 20~80mL / L soy peptone 60~180g / L Yeast extract 0~20g / L K 2 HPO 4

1~5g / L MgSO 4 ·7H 2 o

0.1~0.8g / L pH 6.5～7.5 Inoculation amount 1％～6％ temperature 35～45℃ Rotating speed 100~250r / min fermentation time 72-144h

[0075] Shake flask fermentation: Pick a single colony from a newly activated plate and insert it into a test tube containing 5 mL of LB medium, culture it with shaking at 200 r / min at 37°C for 14 hours; Shaped flasks (three parallels), 37 ° C, dark conditions, 200r / min shaking culture 144h.

[0076] Determination of biomass: First, at an interval of 6 hours, at an interval of 12 hours, and at an interval of 24 hours, take an appropriate amount of fermentation broth, centrifuge at 13...

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Abstract

The invention relates to the technical field of biology, in particular to gene overexpression, an obtained strain and application. Bacillus subtilis uses glycerin as the carbon source to synthesize MK-7 sequentially through a shikimic acid path, a methyl erythritol-4-phosphoric acid (MEP) path and a methyl naphthoquinone (MK-7) path. By overexpressing the gene menA of the MK-7 path, replacing thepromoter of hepS-menG-hepT operon in an in-situ manner and overexpressing the dxs, dxr and yacM-yacN of the MEP path, the influence of the gene menA, the promoter and the dxs, dxr and yacM-yacN on MK-7 synthesis is observed, the rate-limiting step affecting the MK-7 synthesis is determined, and a fundamental research and theoretical foundation is provided for the building of the high-yield strainsof bacillus natto MK-7.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to gene overexpression and obtained bacterial strains and applications. Background technique [0002] Menaquinone-7 is a fat-soluble vitamin K 2 Naturally occurring forms of vitamin K include plant sources of vitamin K 1 (also known as phylloquinone, PK) and vitamin K from bacterial sources 2 (Also known as Menaquinone, MK). According to the number of isoprene units in the side chain, there are a total of 14 kinds of menaquinones, which are recorded as MK-n, and the common ones are MK-4 and MK-7. In prokaryotes, MK-n participates in the electron transport of the respiratory chain. For humans and other mammals, since vitamin K is an important cofactor for the translation of glutamic acid residues in specific proteins in blood and bones into gamma-carboxyglutamic acid (Gla), it is used to maintain calcium homeostasis, inhibit blood vessels Wall calcification, supports endothelial int...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/31C12P7/66C12P7/00C12R1/125
CPCC07K14/32C12P7/00C12P7/66
Inventor 宋浩杨绍梅曹英秀张国银蔡志刚
Owner TIANJIN UNIV
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