Pyridoxine phosphate synthase pdxj mutant and its application in the preparation of vitamin b6

A technology of pyridoxine phosphate and mutant, applied in the biological field, can solve problems such as limiting fermentation yield, and achieve the effect of improving the ability

Active Publication Date: 2022-06-17
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

In Escherichia coli, pyridoxine 5-phosphate synthase (pyridoxine 5-phosphate synthase = PdxJ) catalyzes the synthesis of pyridoxine 5-phosphate ( PNP), whose Kcat = 0.07 s -1 , Km=26.9 μM, Kcat / Km=0.002s -1 μM -1 , whose catalytic constant is much lower than other enzymes in the synthetic pathway, so the activity of pyridoxine phosphate synthase PdxJ severely limits vitamin B 6 Catalytic efficiency of biosynthetic pathways, vitamin B 6 Rate-limiting enzyme of the synthetic pathway, which greatly limits vitamin B 6 Increased Fermentation Yield

Method used

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  • Pyridoxine phosphate synthase pdxj mutant and its application in the preparation of vitamin b6
  • Pyridoxine phosphate synthase pdxj mutant and its application in the preparation of vitamin b6
  • Pyridoxine phosphate synthase pdxj mutant and its application in the preparation of vitamin b6

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of the original gene of pyridoxine phosphate synthase PdxJ

[0054] 1. Amplify the original gene of pyridoxine phosphate synthase PdxJ whose nucleotide sequence is as shown in SEQ ID NO: 2, the nucleotide sequence of which is shown in SEQ ID NO: 2, and the primers used For liulx-1 / liulx-2, the wild-type pRSFDuet-1 plasmid was used as a template to amplify the backbone (primers liulx-3 / liulx-4), and the PdxJ gene was linked to the plasmid backbone by Gibson assembly, transformed into DH5α E. coli, and coated On the LB plate (containing 50 μg / mL kanamycin), screen the positive clones and verify that the bands (primers DuetUP1 / Duetdown1) are correct and sequenced to confirm the correct recombinant plasmid pRSFDuet-1_pdxJ original gene vector (for the map, see figure 1 ), and the plasmid was extracted for use.

[0055] Table 1 Primers constructed by PdxJ original gene vector

[0056]

Embodiment 2

[0057] Example 2: Design of pyridoxine phosphate synthase PdxJ mutation site

[0058] First, the homology alignment analysis was performed between the pyridoxine phosphate synthase PdxJ derived from Escherichia coli and the amino acid sequence of pyridoxine phosphate synthase reported in the Genbank database. 1M5W) analysis and understanding of the catalytic mechanism, simulate the docking mode between the substrate and the enzyme, and select a binding mode similar to the catalytic binding mechanism according to the docking results. For the designed residues, enzyme design was performed using Rosetta to design the amino acid sequence of the mutant.

Embodiment 3

[0059] Example 3: Construction of pyridoxine phosphate synthase PdxJ mutants

[0060] 1. A site-directed mutagenesis site was introduced by a one-step PCR method, and the recombinant plasmid pRSFDuet-1_pdxJ obtained in Example 1 was originally used as a template for single-site-directed mutagenesis. Three point mutants are plasmid templates. The basic process is to first design mutation primers (see Table 2 for primers), introduce mutation sites into the primers, perform overlap PCR, and then use Dpnl enzyme to identify methylation sites and digest them with the template. PCR treated with Dpnl enzyme The product was transformed, and finally the bacteria were selected for sequencing verification, and the correct plasmid was extracted for use.

[0061] Primers used in table 2 embodiment 3

[0062]

[0063] 2. A total of 34 PdxJ mutants were obtained and named as PdxJ1-pdxJ34. The amino acid differences of PdxJ1-pdxJ34 compared with the pRSFDuet-1_pdxJ original gene are sho...

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Abstract

The invention discloses a pyridoxine phosphate synthase PdxJ mutant and its use in the preparation of vitamin B 6 in the application. The present invention introduces mutations at specific sites on the basis of wild-type pyridoxine phosphate synthase PdxJ, and produces vitamin B by overexpressing the mutant gene of pyridoxine phosphate synthase in Escherichia coli 6 The ability has been greatly improved, and it has great application and promotion value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a pyridoxine phosphate synthase PdxJ mutant and its method for preparing vitamin B 6 applications in . Background technique [0002] Vitamin B 6 It has a wide range of applications in the pharmaceutical, food and feed industries, and is an indispensable vitamin for humans or other animals. It includes three natural forms: pyridoxine, pyridoxal, and pyridoxamine, which exist in the body as phosphate derivatives. . Vitamin B 6 The active form of pyridoxal phosphate is mainly involved in nearly a hundred enzymatic reactions, most of which are related to amino acid metabolism, such as transamination, decarboxylation, dehydration and transsulfation reactions. [0003] Current B vitamins 6 The product form is pyridoxine hydrochloride, which is mainly synthesized by chemical method using the 4-methyl-5-ethoxyoxazole route. In the synthesis process of the intermediate oxazol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P17/12C12R1/19
CPCC12N9/1096C12N15/70C12P17/12C12Y206/99002C12N2800/101
Inventor 张大伟刘林霞王岩岩李金龙
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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