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
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  • 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

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  • 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...

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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

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Application Information

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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
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