Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Gene modification application and bacterial strain obtained through gene modification

A gene and strain technology, applied in the field of application and obtained strains, can solve the problem of undiscovered de novo biosynthesis pathway of MK-7 and the like

Active Publication Date: 2018-10-19
TIANJIN UNIV +1
View PDF9 Cites 8 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Gene modification application and bacterial strain obtained through gene modification
  • Gene modification application and bacterial strain obtained through gene modification
  • Gene modification application and bacterial strain obtained through gene modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Overexpression of glycerol-3-phosphate dehydrogenase gene glpD

[0082] Select the pksJ site of B. subtilis chromosome to overexpress the glpD gene. This site has no relationship with the MK-7 synthesis pathway, and the study of the minimal genome of Bacillus subtilis shows that its knockout does not affect the normal growth characteristics of cells. First, using the chromosome of the intermediate bacterium YqiD-CR preserved in our laboratory as a template, the fragment UP was amplified with primers pksJ-glpD-U1 / pksJ-glpD-U2q and pksJ-glpD-D1q / pksJ-glpD-G2 respectively (such as SEQ Shown in ID No.15, 1806bp) and DCRG (shown in SEQ ID No.17, 3917bp); With the chromosome of starting bacterium B.subtilis MK3-MEP123 as template, use primer pksJ-glpD-1 / pksJ-glpD- 2. Amplify the fragment D of the glpD gene (as shown in SEQ ID No.16, 1749bp); and then splice it into UPDDCRG (7472bp) by overlapping PCR method. The competent cells of the recipient strain MK3-MEP123 we...

Embodiment 2

[0106] Example 2 Knockout of the gene dhbB encoding lactate dehydrogenase

[0107] Using the chromosome of the starting bacterium B. subtilis MK3-MEP123 as a template, primers dhbB-U1 / dhbB-U2, dhbB-D1q / dhbB-D2 and dhbB-G1q / dhbB-G2 were used to amplify fragment U (such as SEQ ID No. 18, 1003bp), D (as shown in SEQ ID No.19, 815bp) and G (as shown in SEQ ID No.21, 605bp); the chromosome of the intermediate bacterium YqiD-CR preserved in this laboratory is used as a template , the fragment CR (as shown in SEQ ID No. 20, 2069bp) with a selection cassette (cat-araR) was amplified with primer dhbB-CR1q / CR2. First, by overlapping PCR, using primer dhbB-U1 / dhbB-D2, fragments U and D are spliced ​​into UD (1818bp); then using primer dhbB-U1 / dhbB-G2, the above three fragments UD, GR and G were spliced ​​into UDCRG (4492bp). The UDCRG fragment was transformed into the competent cells of the recipient strain MK3-MEP123-glpD, and the recombinant strain MK3-MEP123-glpD-Δldh in which the g...

Embodiment 3

[0117] Embodiment 3 fermentation culture

[0118] In order to investigate the influence of the expansion of fermentation conditions on the growth of recombinant bacteria MK3-MEP123-glpD-ΔdhbB and the synthesis ability of MK-7, the present invention enlarged the fermentation conditions in the 250mL Erlenmeyer flask to a 2L baffled Erlenmeyer flask. As can be seen from Table 7, the same medium and fermentation conditions, when fermented in a 250mL Erlenmeyer flask, the OD at 120h 600 Reached the maximum, then the growth decreased; but when fermented in 2L shake flask, at 48h OD 600 Just reached the maximum, and then declined rapidly, that is, the stable period of bacterial growth was very short, indicating that the carbon source or nitrogen source was insufficient at this time, resulting in a decline in growth (see Table 9). It can be seen from Table 10 that although the bacterial growth decreased rapidly at 72 hours of fermentation, a large amount of MK-7 was synthesized at th...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to the technical scheme of organisms, in particular to gene modification application and a bacterial strain obtained through gene modification. MK3-MEP123, sourced from B.subtilis168 and established in a laboratory, is used as an original strain, at first, glycerinum-3-phosphate dehydrogenase (GlpD) is subjected to overexpression, the transformation from glycerinum-3-phosphate to dihydroxyacetone phosphate is improved, and the influence on MK-7 synthesis is inspected; then, dhbB genes are knocked out, consumption of isochorismate is reduced, and the influence on MK-7 synthesis is inspected; and finally the influence of the supply of an oxygen and carbon and nitrogen source on MK-7 synthesis is inspected through 2L shake flask fermentation. A fundamental research and theoretical basis is provided for establishing a high-yield MK-7 bacterial strain of bacillus natto by a metabolic engineering method industrially.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of genetic modification and the strain obtained therefrom. 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 endotheli...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/66C12R1/125
CPCC12N9/0006C12N9/88C12P7/66C12Y101/05003C12Y402/99021
Inventor 宋浩杨绍梅曹英秀张国银蔡志刚
Owner TIANJIN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com