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

Method for performing genetic modification on DHDPR (Dihydrodipicolinic Acid Reductase) in corynebacterium glutamicum to increase lysine yield

A Corynebacterium glutamicum and genetic modification technology, applied in the fields of genetic engineering and enzyme engineering, can solve problems such as insufficient NADPH content, and achieve the effect of eliminating insufficient NADPH supply and improving capacity

Inactive Publication Date: 2018-04-17
JIANGNAN UNIV
View PDF2 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during the determination of intracellular redox cofactors during the fermentation of L-lysine in this strain, it was found that the intracellular NADPH content was still insufficient, thus limiting the further increase of L-lysine

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 performing genetic modification on DHDPR (Dihydrodipicolinic Acid Reductase) in corynebacterium glutamicum to increase lysine yield
  • Method for performing genetic modification on DHDPR (Dihydrodipicolinic Acid Reductase) in corynebacterium glutamicum to increase lysine yield
  • Method for performing genetic modification on DHDPR (Dihydrodipicolinic Acid Reductase) in corynebacterium glutamicum to increase lysine yield

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: In vitro site-directed mutagenesis of the DHDPR-encoding gene dapB in C. glutamicum

[0040] Using the C. glutamicum JL-6 genome as a template and dapB-F / dapB-R as primers, a 747bp dapB fragment was amplified by PCR reaction, and EcoRI and HindIII restriction enzymes were introduced at the 5' and 3' ends of the PCR product, respectively. cut site.

[0041] The above dapB fragment was connected to a T vector (Pucm-T), transformed into Escherichia coli, and the recombinant plasmid Pucm-T / dapB was extracted. The plasmid Pucm-T / dapB was used as a template, and the mutant primers MCB1-F / MCB1-R and MCB2-F / MCB2-R were used as primers to carry out PCR reactions, and the DNA fragment purification kit was used to purify and use restriction endonuclease DpnI Enzyme digestion (DpnI only recognizes methylated DNA, but the newly synthesized DNA is not methylated), then the enzyme digestion product is purified and transformed into E.coli JM106 competent cells, and spread o...

Embodiment 2

[0043] Example 2: Expression and purification of DHDPR mutants in E.coli BL21

[0044] According to Table 1, the recombinant plasmid Pucm-T / dapB was cut with restriction enzymes EcoRI and HindIII respectively. A31G,A32C and Pucm-T / dapB C37G,G38C . Subsequently, dapB was recovered using a gel recovery kit A31G,A32C and dapB C37G,G38C fragment. will dapB A31G,A32C and dapB C37G,G38C The fragments were respectively connected with pET28a cut with the same restriction enzyme to construct recombinant plasmid pET28a / dapB A31G,A32C and pET28a / dapB C37G,G38C ; The recombinant plasmid pET28a / dapB A31G,A32C and pET28a / dapB C37G,G38C Transfer to E.coli BL21, pick positive transformants on the kanamycin-resistant LB plate to liquid LB, collect the bacteria after IPTG induction, and after ultrasonic crushing, SDS-PAGE of the supernatant, a molecular weight of about 30kDa was detected The specific band ( figure 2 ), consistent with the reported target protein size, while the cont...

Embodiment 3

[0045] Example 3: Determination of Kinetic Parameters of DHDPR Mutants

[0046] Take the above-mentioned purified mutant DHDPR K11A 、DHDPR R13A Add wild-type DHDPR to the DHDPR enzyme activity assay reaction system, monitor the change of absorbance at 340nm in real time, and calculate the K according to the change of absorbance m and K cat (Table 2).

[0047] Reaction system: 100mmol L -1 MOPS buffer (pH 7.2), 50 μmol L -1 Dihydrodipicolic acid, 0.01~0.04mmol L -1 NADPH or 0.004~0.032mmol L -1 NADH; Reaction temperature: 30°C; Reaction time: ≥300s.

[0048] Table 2. Kinetic parameters of wild-type DHDPR and its mutants

[0049]

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 performing genetic modification on DHDPR (Dihydrodipicolinic Acid Reductase) in corynebacterium glutamicum to increase lysine yield and belongs to the fields of genetic engineering and enzyme engineering. By using the genetic engineering method, DHDPR coding genes dapB in corynebacterium glutamicum JL-6 are subjected to site-specific mutagenesis, and the DHDPRprotein structure is changed, so that the affinity of the DHDPR on different oxidation reduction cofactors is regulated, the defect that the requirement of NADP (H / +) is insufficient in the L-lysine synthesis process is overcome, and the ability of accumulating L-lysine in the strain is improved. According to shaking flask fermentation of recombinant bacteria, the accumulation amount of the L-lysine is 17.6g / L. According to the method disclosed by the invention, the affinity of the oxidation reduction cofactors of the DHDPR in the corynebacterium glutamicum is successfully changed, the defectthat the requirement of the NADP (H / +) is insufficient in the L-lysine synthesis process is overcome, and a new thinking is provided for breeding high-yield L-lysine strains.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and enzyme engineering, and in particular relates to a method for genetically transforming DHDPR in Corynebacterium glutamicum to increase lysine production. technical background [0002] L-Lysine is an amino acid that is necessary for humans and animals and cannot be synthesized by itself, and is one of the eight essential amino acids. Because L-lysine has a variety of physiological functions, such as balancing amino acid composition, regulating internal metabolic balance, improving the body's absorption and utilization of cereal protein, and promoting body growth and development, it is widely used in feed industry, pharmaceutical industry and in the food industry. There are three main methods for industrial production of L-lysine: proteolysis, chemical synthesis and microbial fermentation. Among them, microbial fermentation has the advantages of low production cost, high production intensity...

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): C12N1/21C12N15/53C12N15/77C12P13/08C12R1/15
CPCC12N9/001C12P13/08C12Y103/01026
Inventor 徐建中张伟国
Owner JIANGNAN 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