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Pyruvate dehydrogenase mutant and method for producing L-amino acid by using mutant

A technology of pyruvate dehydrogenase and mutants, applied in the direction of microorganism-based methods, biochemical equipment and methods, botany equipment and methods, etc., can solve problems such as no new mutation sites and achieve improved conversion rate , the effect of increasing production

Active Publication Date: 2021-08-13
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no new mutation sites have been reported

Method used

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  • Pyruvate dehydrogenase mutant and method for producing L-amino acid by using mutant
  • Pyruvate dehydrogenase mutant and method for producing L-amino acid by using mutant
  • Pyruvate dehydrogenase mutant and method for producing L-amino acid by using mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1. Editing plasmid pCas9gRNA- aceE 217 builds

[0082] The present invention has carried out in-depth analysis on the AceE enzyme, and it is predicted that position 217 may be a site that affects its activity, so the site is mutated for follow-up research. Subsequently, according to the Goldengate cloning method reported in the literature (WANG, Yu, et al. Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum . Biotechnology and bioengineering, 2019, 116: 3016-3029) to construct targeted aceE The pCas9gRNA plasmid of the 217th amino acid residue codon of the gene, the target DNA binding region of the sgRNA is CCAACTGTGTCCATGGGTCT. The specific method is as follows: 217-F / 217-R is denatured and annealed to obtain a DNA double-stranded product with sticky ends, and then combined with pCas9gRNA- ccdB The plasmid (reference CN112111469B) was cloned with Goldengate (NEB® Golden Gate Assembly Kit, ...

Embodiment 2

[0087] Example 2. Construction of Corynebacterium glutamicum aceE The mutant of the 217th amino acid codon mutation in the gene

[0088] Since wild-type Corynebacterium glutamicum cannot produce lysine, the introduction of lysC , pyc and hom Lysine can be produced after the point mutation. In this embodiment, ATCC13032 is used as the starting strain, and a lysine-producing bacterial strain is constructed at first, that is, the aspartokinase gene in Corynebacterium glutamicum ATCC13032 strain lysC Introduced the T311I point mutation (relieves feedback inhibition of the enzyme) in the pyruvate carboxykinase gene pyc Introduction of the P458S point mutation (relieves feedback inhibition of the enzyme) in the homoserine dehydrogenase gene hom Introduce the V59A point mutation (weaken the activity of the enzyme) to obtain the lysine high-yielding strain AHP-3.

[0089] Subsequently, using the CRISPR / Cas9 genome editing system based on single-strand recombination (refer ...

Embodiment 3

[0092] Example 3. Corynebacterium glutamicum aceE The effect of gene 217 mutation on L-lysine synthesis

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Abstract

The invention provides a pyruvate dehydrogenase mutant, on the basis of an amino acid sequence as shown in SEQ ID NO: 1, the 217 site of the sequence is mutated into any one of alanine, aspartic acid, glutamate, leucine and proline. The mutant can improve the yield and conversion rate of L-amino acids in a strain, growth of the strain is not inhibited while the yield is improved, a new way is provided for large-scale production of the L-amino acids, and the mutant has high application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a mutant of pyruvate dehydrogenase, a polynucleotide encoding the mutant, a host cell containing the mutant, and a method for producing L-amino acid using the mutant. Background technique [0002] Pyruvate dehydrogenase multienzyme complex (PDHC) is a group of rate-limiting enzymes. In Corynebacterium glutamicum, PDHC catalyzes the irreversible oxidative decarboxylation of pyruvate produced during glycolysis into acetyl-CoA, which converts sugar Aerobic oxidation is linked with the Krebs cycle and oxidative phosphorylation, and is a key enzyme of the Krebs cycle. PDHC consists of pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3p). Unlike phosphorylases, enzymes have both inactive (phosphorylated) and active (dephosphorylated) forms. There are various allosteric modulators that regulate the switch between the tw...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N1/21C12P13/04C12P13/08C12P13/12C12P13/06C12R1/19C12R1/15
CPCC12N9/0008C12P13/04C12P13/08C12P13/12C12P13/06C12Y102/01051
Inventor 孙际宾刘娇郑平周文娟马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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