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Codon-optimized N-acetylglucosamine transferase gene and application thereof

A technology of glucotransferase and codon optimization, applied in application, genetic engineering, plant genetic improvement and other directions, to achieve the effect of high level, huge application value and industrialization potential

Active Publication Date: 2021-10-22
YANGZHOU RIXING BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzyme SmNodc has not been reported and verified by any journals.

Method used

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  • Codon-optimized N-acetylglucosamine transferase gene and application thereof
  • Codon-optimized N-acetylglucosamine transferase gene and application thereof
  • Codon-optimized N-acetylglucosamine transferase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1. Codon optimization of N-acetylglucosamine transferase

[0029] An N-acetylglucosamine transferase derived from Sinorhizobium medicae, the amino acid sequence of which is shown in SEQ ID NO.1. According to the codon preference of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens DSM7), the N-acetylglucosamine transferase is optimized and the whole gene is synthesized. Its nucleotide sequence is shown in SEQ ID NO.2. The target gene codon is optimized and the whole The gene synthesis was completed by General Biosystems (Anhui) Co., Ltd.

[0030] 2. Construction of recombinant Bacillus amyloliquefaciens

[0031] The nucleotide sequence shown in SEQ ID NO.2 was cloned into the expression plasmid pMA5 to obtain pMA5-nodC, and then the recombinant plasmid pMA5-nodC was transformed into Bacillus amyloliquefaciens by electroporation to obtain The engineering strain of sugar, the specific process is as follows:

[0032] (1) Using primer F and primer R to amplify the n...

Embodiment 2

[0041] Using genetically engineered strains to ferment and produce chitosan oligosaccharides, the steps are as follows:

[0042] (1) Activate the genetically engineered bacteria obtained in Example 1 at a culture temperature of 32°C to 37°C, inoculate the strain into the seed culture medium, and cultivate at 32°C to 37°C for 10 to 16 hours at a shaking table speed of 180 to 220 rpm to OD 660 Greater than 5.0, as a seed solution. The components of the seed medium are: glucose 50g / L, ammonium sulfate 4g / L, yeast powder 8g / L, peptone 4g / L, K 2 HPO 4 ·3H 2 O6g / L, KH 2 PO 4 2g / L, MgSO 4 ·7H 2 O 1.5g / L, the rest is water, pH 6.0-8.0.

[0043] (2) the seed liquid obtained in the step (1) is inoculated on the fermentation medium with an inoculum size of 2% to 6% to carry out aerobic fermentation, and the detection shows that the extracellular fermentation liquid is rich in chitosan oligosaccharides ( image 3 ). The components of the fermentation medium are: glucose 50g / L, a...

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Abstract

The invention discloses a codon-optimized N-acetylglucosamine transferase gene and an application thereof, and belongs to the technical field of bioengineering. According to the invention, a Nodc gene from Sinorhizobium meliloti is taken as a starting sequence, the N-acetylglucosamine transferase is subjected to codon optimization according to codon preference of bacillus amyloliquefaciens and then is subjected to whole-gene synthesis, and a nucleotide sequence of the N-acetylglucosamine transferase is shown as SEQ ID NO.2. A recombinant vector is constructed aby using the codon-optimized N-acetylglucosamine transferase, and the recombinant vector is transferred into host bacillus amyloliquefaciens to obtain the genetically engineered bacterium capable of producing the chitosan oligosaccharide by fermentation. The concentration of the chitooligosaccharide in extracellular fermentation liquor of the genetically engineered bacterium reaches 676mg / L, and the contents of the chitotetraose and the chitopentaose are 246mg / L and 430mg / L respectively, so that a new fermentation process for extracellular synthesis of the chitooligosaccharide such as the chitotetraose and the chitopentaose can be realized by utilizing the recombinant strain.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a codon-optimized N-acetylglucosamine transferase gene and application thereof. Background technique [0002] Chitooligosaccharides (Chitooligosaccharides, COs), also known as chitosan oligosaccharides, oligomeric glucosamine, and chitooligosaccharides, are low-carbon compounds formed by connecting 2 to 10 N-acetylglucosamines through β-1,4 glycosidic bonds. Polymer ( figure 1 ). Compared with the basic polysaccharide chitosan, oligochitosan has higher water solubility, absorption availability and biological activity, which makes it widely used in antibacterial activity, tumor suppression, and regulation of plant immunity. [0003] At present, the preparation methods of chitosan oligosaccharide mainly include three categories: physical method, chemical method and enzymatic method. The controllability of preparing specific molecular weight chitosan oligosaccharides by phys...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/75C12N1/21C12P19/26C12R1/07
CPCC12N9/1051C12N15/75C12P19/26C12N2800/22
Inventor 邱益彬丁振中张超朱萌方祥高小燕徐俊山张万宏
Owner YANGZHOU RIXING BIO TECH
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