Gene and expression of a codon-optimized n-acetylglucosamine isomerase

A technology of acetylglucosamine and codon optimization, which is applied in the field of microorganisms and can solve problems that are not suitable for large-scale applications

Active Publication Date: 2014-10-01
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical catalysis method is to isomerize GlcNAc to ManNAc under strong alkaline conditions, but this method has a strong degradation effect on downstream substrates and enzymes, so it is not suitable for large-scale applications[1]

Method used

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  • Gene and expression of a codon-optimized n-acetylglucosamine isomerase
  • Gene and expression of a codon-optimized n-acetylglucosamine isomerase
  • Gene and expression of a codon-optimized n-acetylglucosamine isomerase

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: Construction of recombinant Escherichia coli E. coli Rosseta-pET28a-anAGEAGE.

[0034] 1.1 Codon optimization of N-acetylglucosamine isomerase gene:

[0035] AnAGE codon usage was analyzed according to the gene sequence of anAGE in the NCBI database (Genbank ID DQ661858) and the codon usage frequency distribution table of Pichia pastoris in the codon usage database (http: / / www.kazusa.or.jp / codon / ) Condition. The rare codons in P. pastoris whose usage frequency is less than 15% are defined as rare codons, and the codon optimization process is based on the following principles:

[0036] 1) Do not change the amino acid sequence encoded by anAGE;

[0037] 2) Replace the rare codons in anAGE with codons with higher usage frequency, and assign the usage of different codons of the same amino acid according to the ratio of codon usage frequency.

[0038] 3) Reduce the possible stable structural regions in the secondary structure of mRNA corresponding to the gene s...

Embodiment 2

[0049] Example 2: Inducible expression of codon-optimized N-acetylglucosamine isomerase anAGE.

[0050] 2.1 Induced expression of recombinant bacteria Rosetta-pET28a-anAGE

[0051] Activation of strains: In the ultra-clean work, the pET28a-Nal-Rosetta recombinant bacteria were separated by four-section lines on the LB solid plate containing kanamycin and chloramphenicol, and cultured in a constant temperature incubator at 37°C for 12 hours.

[0052] Seed culture: Under sterile conditions, pick a single colony on the plate, insert it into 20mL LB liquid medium (250mL Erlenmeyer flask) supplemented with kanamycin and chloramphenicol, and culture it on a shaker at 37°C for 12h at a speed of 200rpm.

[0053] Induced expression: The seed liquid was transferred into 200ml LB medium containing kanamycin and chloramphenicol at an inoculum size of 2 (v / v). Proliferate at 220rpm on a shaker at 37°C for 2.5-3h to OD600=06-0.8, and add a final concentration of 0.5mM IPTG. Then placed at...

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Abstract

The invention discloses gene of codon optimized N-acetyl glucosamine isomerase and the expression thereof, and the nucleotide sequence of the gene is shown as SEQ ID NO:1. The gene has the advantages that anAGE gene is optimized and synthesized according to Pichia pastoris codon preference, the homology of the optimized anAGE gene is 79 percent of that of a wild gene, the enzyme activity of 6.29 U / mg crude enzyme is shown, and the gene can be applied to the synthesis of N-acetylmannosamine.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and relates to a codon-optimized N-acetylglucosamine isomerase (N-acetylglucosamine2-epimerase, AGE) gene and its expression. Background technique [0002] N-acetyl-mannosamine (N-acetyl-D-mannosamine, ManNAc) is an important intermediate, which can be used as anti-influenza drug precursor and infant milk powder additive N-acetylneuraminic acid (N-acetyl-D- neuraminic acid, Neu5Ac) synthesis. [0003] N-acetyl-D-glucosamine (GlcNAc) is used as substrate to synthesize ManNAc, and ManNAc and pyruvate are used as substrates to pass N-acetylneuraminic acid lyase (N-acetylneuraminic acid lyase, Nal) catalyzed synthesis of N-acetylneuraminic acid is currently the most important way to synthesize Neu5Ac. The isomerization of the cheap substrate GlcNAc to its expensive isomer ManNAc is the key step in the two-step reaction. [0004] So far, there have been many reports on the reaction of cataly...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/61C12N15/63C12N1/21C12N9/88C12R1/19
Inventor 谢婧婧孙梧进应汉杰郭亭季雯燕朱晨杰陈晓春陈勇吴菁岚
Owner NANJING TECH UNIV
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