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

Quinolinic acid synthase mutant and application thereof

A technology of quinolinic acid and mutants, applied in the field of microorganisms, can solve the problems of little research and utilization on the improvement of NAD ability, and achieve the effect of increasing the level of reducing power, increasing the level of reducing power, and promoting accumulation

Active Publication Date: 2021-10-19
MEIHUA BIOTECH LANGFANG CO LTD
View PDF6 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in the field of microbial metabolic engineering, the means of improving intracellular reducing power (NADH / NADPH) for the production of metabolites such as amino acids mainly focus on providing the regenerative ability of reducing power, however, the improvement of the ability of NAD de novo synthesis has been rarely studied and use

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
  • Quinolinic acid synthase mutant and application thereof
  • Quinolinic acid synthase mutant and application thereof
  • Quinolinic acid synthase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Construction of embodiment 1 recombinant plasmid pK18-nadA (D312N)

[0080] Primers were designed according to the sequence of the nadA (GenBank NO: NCgl1024) gene in the NCBI database, and the primer sequences are shown in Table 1. Using Phusion ultra-fidelity polymerase (New England BioLabs), nadA-UP-1F / nadA-UP-1R, nadA-DN-2F / nadA-DN-2R as a primer pair, Corynebacterium glutamicum MHZ-0112- The genome of 08 was used as a template to prepare recombinant fragments. The PCR program was: denaturation at 98°C for 10s, annealing at 50°C for 20s, extension at 72°C for 15s, cycle 30 times, and thorough extension at 72°C for 10 minutes. (Tiangen) purification, then use nadA-UP-1F / nadA-DN-2R as a primer pair, and the upstream and downstream homology arms as templates to prepare recombinant fragments. The PCR program is: denaturation at 98°C for 10s, renaturation at 50°C for 20s, Extend at 72°C for 30 s, cycle 30 times, and extend thoroughly at 72°C for 10 min. The resulting re...

Embodiment 2

[0081] Example 2 MHZ-0112-8 strain introduces nadA gene point mutation

[0082] MHZ-0112-8 competent cells were prepared according to the classic method of Corynebacterium glutamicum (C. glutamicum Handbook, Charpter 23), and the recombinant plasmid pK18-nadA D312N Transform into Corynebacterium glutamicum MHZ-0112-8 competent cells by electroporation, and select and exchange recombinants on a selection medium containing 15 mg / L kanamycin. Kan was identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with nadA-UP-1F / P85, P82 / nadA-DN-2R primer pair R For cloning, the PCR parameters are: 94°C for 30s, 50°C for 30s, 72°C for 35s, a total of 30 cycles, and 72°C for 10 minutes. The clones with fragments of 1208bp and 1103bp amplified by two pairs of primers were positive clones. The selected positive clones were inoculated into anti-antibody BHI medium and cultured for 12-14h, the bacterial solution was diluted 100-1000 times and spread on solid BHI medium con...

Embodiment 3

[0083] Example 3 Performance Verification of MHZ-0112-9 Mutant Strain Production of Glutamic Acid and Quinolinic Acid

[0084] Ferment the recombinant Corynebacterium glutamicum constructed in embodiment 2, verify the production performance of its glutamic acid and quinolinic acid, specifically as follows:

[0085] Inoculate the strains frozen in -80°C glycerol tubes into the above-mentioned slant medium for activation, and grow a lawn after culturing at 31.5°C for 24 hours. Pick the lawn from the freshly activated slant and inoculate it on the above-mentioned seed culture medium, at 31.5°C and 220rpm, shaking culture to the middle and late stages of logarithmic growth, the culture time is 12h, and the seed liquid is obtained, and the above seed liquid is inoculated into a 500ml shake flask containing 20ml fermentation medium with a 10% inoculum amount. , at 31.5°C, 220rpm shaking culture for 16h. After the glucose was completely consumed, the concentrations of L-glutamic aci...

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 field of microorganisms, and concretely relates to a quinolinic acid synthase mutant and application thereof. The provided quinolinic acid synthase mutant takes an amino acid sequence of corynebacterium glutamicum wild type quinolinic acid synthase as a reference sequence, and the quinolinic acid synthase mutant contains mutation in which aspartic acid at the 312th site is substituted by asparagine. The quinolinic acid synthase mutant has significantly enhanced catalytic activity of quinolinic acid synthase, can enhance the synthesis ability of microbial cells NADH and NADPH, improves the reducing power level of the microbial cells, and further can significantly promote accumulation of quinolinic acid and metabolites synthesized depending on the reducing power. According to the present invention, the reducing power level of the recombinant microorganism expressing the quinolinic acid synthase mutant is significantly improved, such that the yield and the conversion rate of the reducing power dependent metabolites such as quinolinic acid, glutamic acid, lysine and the like can be easily improved;.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a quinolinic acid synthase mutant and its application. Background technique [0002] NADH (reduced nicotinamide adenine dinucleotide, reduced coenzyme I) and NADPH (reduced nicotinamide adenine dinucleotide phosphate, reduced coenzyme II) are the main sources of reducing power in microbial cells, and they are also An important cofactor for the synthesis of various metabolites such as amino acids. At present, in the field of microbial metabolic engineering, the means of improving intracellular reducing power (NADH / NADPH) for the production of metabolites such as amino acids mainly focus on providing the regenerative ability of reducing power, however, the improvement of the ability of NAD de novo synthesis has been rarely studied and exploit. [0003] L-glutamic acid (L-glutamate), the chemical name is α-amino glutaric acid, and the molecular formula is C 5 h 9 NO 4 , ...

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): C12N9/10C12N15/54C12N1/21C12P17/12C12R1/15C12R1/13
CPCC12N9/1085C12P17/12C12Y205/01072
Inventor 牛松峰程江红贾贝贝李岩
Owner MEIHUA BIOTECH LANGFANG CO LTD
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