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

Novel o-succinyl homoserine transferase mutant and method for producing o-succinyl homoserine using same

一种琥珀酰高丝氨酸、转移酶的技术，应用在O-琥珀酰高丝氨酸转移酶突变体领域，能够解决变异稳定性、低稳定性、恶化等问题

Active Publication Date: 2020-04-17
CJ CHEILJEDANG CORP
View PDF9 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the homoserine O-succinyltransferase encoded by the metA gene (even as a wild-type protein) has low stability, and its stability can be further deteriorated by introducing mutations to relieve feedback

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
  • Novel o-succinyl homoserine transferase mutant and method for producing o-succinyl homoserine using same
  • Novel o-succinyl homoserine transferase mutant and method for producing o-succinyl homoserine using same
  • Novel o-succinyl homoserine transferase mutant and method for producing o-succinyl homoserine using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Example 1: Preparation of metX plasmid with O-acetyl homoserine transferase activity

[0097] To amplify the gene encoding O-acetyl homoserine transferase (MetX), based on a reporter sequence derived from wild-type (WT), a BamHI restriction enzyme site was inserted into the 300 bp) to the two ends of each primer (SEQ ID NO:5 and 6) for amplification of the stop region (located about 100 bp downstream of the stop codon).

[0098] Table 1

[0099] SEQ ID NO: Primer sequence (5'-3') 5 Primer 1 GGATCCCCTCGTTGTTCACCCAGCAACC 6 Primer 2 GGATCCCAAAGTCACAACTACTTATGTTAG

[0100] PCR was performed under the following conditions. After denaturation at 95°C for 5 minutes, the cycle (denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 90 seconds) was repeated 30 times, and then polymerization was performed at 72°C for 7 minutes . As a result, a DNA fragment of 1546 bp was obtained as the coding r...

Embodiment 2

[0101] Embodiment 2: the preparation of the variant metX plasmid with O-succinyl homoserine transferase activity

[0102] A new metX mutation site was selected, and the 176th and 313th amino acids of the amino acid sequence of SEQ ID NO: 1 were respectively replaced with another amino acid.

[0103] More specifically, Q176N and L313R mutations were made. Using the pECCG117-metX WT plasmid prepared in Example 1 as a template, a primer pair (SEQ ID NO: 7 and 8) for the 176th mutation and a primer pair (SEQ ID NO: 8) for the 313rd mutation were designed. :9 and 10), to prepare a mutant vector in which the 176th amino acid of O-acetylhomoserine transferase was substituted by another amino acid and the 313th amino acid thereof was substituted by arginine.

[0104] Table 2

[0105] SEQ ID NO: Primer sequence (5'-3') 7 Primer 3 ACGCGCCAGCGCCTGGAACATCGGCATTCAATCCG 8 Primer 4 CGGATTGAATGCCGATGTTCCAGGCGCTGGCGCGT 9 Primer 5 GTAGATACCGATATTCGGTACC...

Embodiment 3

[0107] Example 3: Substrate specificity and activity of variant metX with O-succinyl homoserine transferase activity comparative test

[0108] In order to compare the activity of mutated metX that produces excess O-succinyl homoserine, a strain in which homoserine was accumulated and utilization of the produced O-succinyl homoserine was absent was prepared. Deletion of metB gene encoding cystathionine gamma synthase in O-succinyl homoserine degradation pathway and metY deletion encoding O-acetyl homoserine (thiol)-lyase in O-succinyl homoserine degradation pathway Genetic strains. First, in order to delete the metB gene, based on the nucleotide sequence information of the WT-derived metB gene, a primer pair (SEQ ID NO: 11 and 12) for amplifying the 5' upstream region of the metB gene and a primer pair for amplifying the metB gene were designed. Primer pair for the 3' downstream region of the gene (SEQ ID NO: 13 and 14). An XbaI restriction enzyme site (underlined) was in...

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 present application relates to an O-succinyl homoserine transferase mutant, a polynucleotide for coding same, a microorganism comprising the mutant, and a method for producing O-succinyl homoserine using the microorganism.

Description

technical field [0001] The present disclosure relates to an O-succinyl homoserine transferase mutant, a polynucleotide encoding the same, a microorganism comprising the mutant, and a method of producing O-succinyl homoserine using the microorganism. Background technique [0002] O-succinyl homoserine acts as a precursor of methionine, one of the essential amino acids for the human body. Methionine has been used as a synthetic raw material for medical solutions and medical supplies as well as feed and food additives. [0003] Methionine is produced chemically or biosynthetically. Meanwhile, a two-step process for producing L-methionine from an L-methionine precursor produced by fermentation through an enzymatic conversion reaction has been reported (International Publication No. WO / 2008 / 013432). [0004] In a two-step process, O-succinyl homoserine and O-acetyl homoserine are used as methionine precursors, and the production of O-succinyl homoserine in high yield is importa...

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): C12N9/10C12N9/12C12N15/77C12P13/06C12P13/12C07C319/08
CPCC12N15/77C12P13/06C12P13/12C12N15/1034C40B40/08C12N9/1029C07C319/14C12Y203/01031C12R2001/15C07C323/58C12N9/1025C12N9/1217C07C319/08C12Y207/02004C12N9/10C12N1/205
Inventor 金径林沈知炫金贤雅申容旭P·李
Owner CJ CHEILJEDANG CORP
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