Method for raising S-adenosyl-L-methionine production level by saccharomyces cerevisiae metabolic engineering

A technology of metabolic engineering transformation and adenosylmethionine, which is applied in microorganism-based methods, biochemical equipment and methods, fermentation, etc., can solve problems such as insufficiency, and achieve improved knockout efficiency, good technical effects, and excellent food safety. The effect of biological properties

Active Publication Date: 2016-04-13
SHANDONG JINCHENG BIO PHARMA CO LTD +1
View PDF3 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the natural Saccharomyces cerevisiae has a strong ability to produce adenosylmethionine, it still cannot meet the growing demand of the market, and it is urgent to find more ways to continue to increase the production of adenosylmethionine

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
  • Method for raising S-adenosyl-L-methionine production level by saccharomyces cerevisiae metabolic engineering
  • Method for raising S-adenosyl-L-methionine production level by saccharomyces cerevisiae metabolic engineering
  • Method for raising S-adenosyl-L-methionine production level by saccharomyces cerevisiae metabolic engineering

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Taking diploid Saccharomyces cerevisiae strain HD as an example, construct homozygous knockout SPE2 gene and compare SAM yield with HD.

[0050] 1. PCR construction of knockout box. The method is as follows: using the plasmid pUG6 as a template, using primers to perform PCR reactions on SPE2-up and SPE2-down, the PCR products are purified and recovered by a DNA purification kit, confirmed by DNA electrophoresis, and finally used as a knockout frame for SPE2 gene knockout.

[0051] 2. LiAC conversion. The method is as follows: Pick a single colony of the original strain HD and place it in a 20mLYPD shake flask, rotate at 200rpm, cultivate overnight at 30°C, then transfer 2mL to a 50mLYPD shake flask and continue to cultivate for 3-4 hours, OD 600 Centrifuge to collect the bacteria at about 1. Wash once with 20mL sterile water, centrifuge, and remove the supernatant; then wash once with 0.1M LiAC, centrifuge, and remove the supernatant; resuspend the bacteria with ...

Embodiment 2

[0059] Example 2: Knock out the SPE2 gene in the model strain BY4741 and verify its yield.

[0060] Methods as below:

[0061] 1. PCR construction of knockout box. The method is as follows: using the plasmid pUG6 as a template, using primers to perform PCR reactions on SPE2-up and SPE2-down, the PCR products are purified and recovered by a DNA purification kit, confirmed by DNA electrophoresis, and finally used as a knockout frame for SPE2 gene knockout.

[0062] 2. LiAC transformation and PCR verification. The method is as follows: Pick a single colony of the original strain BY4741 and place it in a 20mLYPD shake flask, rotate at 200rpm, cultivate overnight at 30°C, then transfer 2mL to a 50mLYPD shake flask and continue to cultivate for 7-8 hours, OD 600 Centrifuge to collect the bacteria at about 1. Wash once with 20mL sterile water, centrifuge, and remove the supernatant; then wash once with 0.1M LiAC, centrifuge, and remove the supernatant; resuspend the bacteria with 1mL0.1ML...

Embodiment 3

[0066] Example 3, taking the diploid Saccharomyces cerevisiae strain HD as an example, a strain with both GLC3 and SPE2 knocked out was constructed, and SAM yields were compared.

[0067] 1. Similar to the first step of the example, first knock out the GLC3 gene: use pUG6 as a template PCR to construct a GLC3 knockout box. After LiAC transformation, G418 plate screening and PCR verification, the GLC3 knockout heterozygotes are obtained, and then the heterozygotes are cultured for sporulation , Isolate the spores, screen on the G418 plate to obtain the homozygous GLC3 knockout, and perform PCR verification and confirmation.

[0068] 2. Recover the marker gene. The method is as follows: place the homozygous single colony knocked out of GLC3 in a 20mLYPD shake flask, rotate at 200 rpm, cultivate overnight at 30°C, then transfer to a 2mL 50mLYPD shake flask and continue to incubate for 3-4 hours, when the OD600 is about 1. Collect the bacteria by centrifugation. Wash once with 20mL s...

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 discloses a method for raising S-adenosyl-L-methionine production level by saccharomyces cerevisiae metabolic engineering. By means of saccharomyces cerevisiae gene knockout, an S-adenosylmethionine decarboxylase coding gene SPE2 on a chromosome of saccharomyces cerevisiae is subjected to mutation inactivation to obtain SPE2 mutant strains. Compared with an original saccharomyces cerevisiae strain, the mutant strains have the advantages of high SAM (S-adenosyl-L-methionine) accumulation capability and evident improvement of SAM yield. Particularly, on the basis that the saccharomyces cerevisiae mutant strains are obtained after knockout of a glycogen branching enzyme gene GLC3, knockout of the gene SPE2 is further carried out, or knockout of the gene SPE2 is carried out before knockout of the gene GLC3, yield of ademetionine produced with the saccharomyces cerevisiae strains is evidently increased to be higher than 55.1%.

Description

Technical field [0001] The invention belongs to the field of microbial technology and metabolic engineering, and relates to a method for improving the ability of Saccharomyces cerevisiae strains to produce S-adenosyl-L-methionine through metabolic pathway modification. Background technique [0002] S-adenosyl-L-methionine, or SAM for short, is widely present in animal, plant and microbial cells and is an important metabolic intermediate product. As an intracellular methyl donor, SAM plays an important role in the methylation modification of nucleic acids, proteins and lipids. At the same time, SAM also participates in important biochemical reactions such as intracellular transsulfide reaction and polyamine synthesis. SAM is also a very valuable medical molecule, playing an important role in the treatment of liver disease, depression and rheumatoid arthritis. [0003] SAM is catalyzed by adenosylmethionine synthetase in the cell and is produced by the combination of methionine and...

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): C12N15/81C12N1/19C12P19/40C12R1/865
Inventor 徐志南赵伟军杨修亮杭宝建黄磊李江涛蔡谨
Owner SHANDONG JINCHENG BIO PHARMA CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap