Modifications to lysine decarboxylase enzymes

A technology of lysine residues and amino acids, applied in the field of modification of lysine decarboxylase enzymes, can solve the problem that there is no literature describing CadA

Active Publication Date: 2019-12-06
CATHAY R&D CENT CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no literature describing how CadA higher oligomers are formed, and the amino acid residues that play a role in their formation

Method used

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  • Modifications to lysine decarboxylase enzymes
  • Modifications to lysine decarboxylase enzymes
  • Modifications to lysine decarboxylase enzymes

Examples

Experimental program
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preparation example Construction

[0067] Preparation of recombinant vector

[0068] The recombinant vector for expressing the variant CadA protein can be prepared using methods well known in the art. For example, the DNA sequence encoding the CadA variant polypeptide can be combined with transcription and other regulatory sequences that will direct the sequence from the gene in the desired cell (e.g., bacterial cells such as Hafnia alves, Escherichia coli, or glutamine). Corynebacterium acidi). In some embodiments, the expression vector comprising an expression cassette further comprises a promoter operably linked to the nucleic acid sequence encoding the CadA variant polypeptide, the expression cassette comprising a gene encoding the CadA variant polypeptide. In other embodiments, the promoter and / or other regulatory elements that direct the transcription of the cadA polynucleotide encoding the variant Cada polypeptide are endogenous to the host cell, and the expression of the cadA gene is introduced, for examp...

Embodiment 1

[0098] Example 1: Construction of a plasmid vector encoding CadA

[0099] A plasmid vector containing wild-type E. coli cadA (SEQ ID NO: 1) (which encodes the lysine decarboxylase CadA (SEQ ID NO: 2)) uses PCR primers cadA-F and cadA-R ( figure 1 ) Amplify from Escherichia coli MG1655 K12 genomic DNA, digest the plasmid with restriction enzymes Sad and XbaI, and ligate into pUC18 to generate plasmid pCIB60. The PCR primers cadA-F2 and cadA-R2 were used to optimize the 5'sequence upstream of the cadA gene to generate pCIB71.

Embodiment 2

[0100] Example 2: Construction of a plasmid vector encoding CadA with a cysteine ​​mutation at the predicted interface amino acid residue.

[0101] A primer pair was designed to introduce mutations into the CadA gene of pCIB71 to modify the amino acid at positions 287, 290, 319, 320, 325, 346, 357, 381, 477, or 500 of CadA to cysteine ​​using Quickchange PCR. DNA sequencing was used to verify the mutation, and the plasmids carrying cysteine ​​mutations were labeled pCIB71-K287C, pCIB71-K290C, pCIB71-K319C, pCIB71-K320C, pCIB71-K325C, pCIB71-K346C, pCIB71-K357C, pCIB71-K381C, pCIB71 -K477C or pCIB71-K500C.

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Abstract

Provided are CadA polypeptides with mutations that increase activity in alkaline pH compared to the wild-type lysine decarboxylase. Methods of generating such mutant polypeptides, microorganisms genetically modified to over-express the mutant polypeptides, and methods of generating such microorganisms are also provided.

Description

Background technique [0001] Most enzymes function best in a narrow pH range because they are facultative molecules. The pH of the surrounding environment directly affects the charges on the acidic and basic groups of the amino acids constituting the enzyme. These charge changes affect the net charge of the enzyme, the pKa of the active site and the charge distribution on the surface of the enzyme. As a result, changes in pH will affect enzyme activity, solubility and stability. [0002] A type of protein called acid decarboxylase is a group of enzymes that catalyze the decarboxylase reaction of basic amino acids (such as lysine, arginine, ornithine) to produce polyamines as the acid stress reaction in many microorganisms. Part. E. coli has several PLP-dependent acid decarboxylases: CadA, LdcC, AdiA, SpeA, SpeC, SpeF, GadA and GadB. All these enzymes function in a narrow pH range, and the enzyme activity decreases significantly outside this pH range (Kanjee et al., Biochemistry...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/09C12P13/00C12N1/21
CPCC12Y401/01018C12N9/88C12P13/001C12N15/70C12N15/77
Inventor 周豪宏陆文强陈玲于丽珺刘修才
Owner CATHAY R&D CENT CO LTD
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