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A kind of recombinant Bacillus subtilis accumulating acetylglucosamine and its application

A technology of Bacillus subtilis and glucosamine, which is applied in the field of genetic engineering, can solve the problems of high price of chitin hydrolase, low production intensity, long transformation time and the like, and achieves the effects of simple construction method, good application prospect and easy use.

Active Publication Date: 2018-03-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the hydrolysis method, the biotransformation method has less pollution to the environment, but due to the high price of chitin hydrolase, it is currently limited to the laboratory research stage
Although direct bioconversion with microbial cells containing chitin hydrolase can reduce production costs, the transformation time is long and production intensity is low

Method used

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  • A kind of recombinant Bacillus subtilis accumulating acetylglucosamine and its application
  • A kind of recombinant Bacillus subtilis accumulating acetylglucosamine and its application

Examples

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Effect test

Embodiment 1

[0023] Example 1 Construction of recombinant plasmid

[0024] According to the nucleotide sequence of the glucosamine acetylase encoding gene (CeGNA1) in Caenorhabditis elegans published on NCBI, as shown in NCBI GenBank: AB017628.1, the codon preference of Bacillus subtilis was optimized to obtain a nuclear The nucleotide sequence is based on SEQ ID NO. 1, and the gene sequence was synthesized by Shanghai Sangon Bioengineering Co., Ltd. Design primers CeGNA1-F: 5'-GGGGTACCATTATAGGTAAGAGAGGAATGTACACATGAGCCATATCTTCGACGCATCTG-3', CeGNA1-R: 5'-CCCAAGCTTTTAAAAGCGCTGGGTCATAAAATTA-3'. The optimized glucosamine acetylase encoding gene (CeGNA1) was amplified from the synthesized nucleotide sequence SEQ ID NO. 1 using the above primers. The amplified fragment was digested with KpnI and HIndIII and then ligated into the pP43NMK expression vector. Enzyme digestion verification and sequencing confirmed that the recombinant plasmid was successfully constructed and named pP43-CeGNA1. For...

Embodiment 2

[0025] Example 2 Construction of recombinant Bacillus subtilis

[0026] The constructed expression vector pP43-CeGNA1 was transformed into recombinant Bacillus subtilis BSGN6-PxylA-glmS. Using CeGNA1-F and CeGNA1-R primers to select transformants for colony PCR, a 498bp band appeared, verifying that the recombinant Bacillus subtilis was successfully constructed.

Embodiment 3

[0027] Embodiment 3 Fermentation produces acetylglucosamine

[0028] The seeds cultured at 37°C and 200 rpm for 12 hours were transferred to the fermentation medium with 5% inoculum and cultured at 37°C and 200 rpm for 30 hours. The content of acetylglucosamine in the final fermentation supernatant reached 7.31 g / L. The control strain took BSGN6-PxylA-glmS as the starting strain, overexpressed the glucosamine acetylase encoding gene GNA1 (nucleotide sequence such as NCBI GenBank: NM_001179949) derived from Saccharomyces cerevisiae S288C, and finally fermented under the same culture conditions The content of acetylglucosamine in the supernatant reached 5.87g / L. By comparison, the production of acetylglucosamine in the fermentation supernatant of recombinant Bacillus subtilis expressing the C. elegans-derived CeGNA1 gene was increased by 24.51% compared with the control strain expressing the Saccharomyces cerevisiae-derived GNA1 gene. The extracellular accumulation of acetylgl...

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Abstract

The invention discloses recombinant bacillus subtilis for accumulating acetylglucosamine and application thereof, and belongs to the field of genetic engineering. According to the recombinant bacillus subtilis for accumulating the acetylglucosamine and the application thereof, recombinant bacillus subtilis BSGN6-PxylA-glmS serves as an original strain, the glucosamine synthesizing route is enhanced by excessively expressing glucosamine histone acetyltransferase coding genes (CeGNA1) from caenorhabditis elegans, and the genetically engineered bacterium bacillus subtilis for accumulating the acetylglucosamine is obtained; the yield on the shake flask level reaches 7.31 g / L, and a foundation is laid for further producing the glucosamine by transforming the bacillus subtilis in metabolic engineering.

Description

technical field [0001] The invention relates to a recombinant Bacillus subtilis for accumulating acetylglucosamine and its application, and belongs to the field of genetic engineering. Background technique [0002] Glucosamine is a compound in which a hydroxyl group of glucose is replaced by an amino group. It is an important functional monosaccharide and the first amino monosaccharide whose structure has been confirmed. N-acetylglucosamine is a derivative of glucosamine with important physiological functions and is widely used in the fields of health food and medicine. Growth is the main component of the new anti-cancer drug chloramphenicol; it can participate in liver and kidney detoxification, and play an anti-inflammatory and liver-protecting role. [0003] The production methods of glucosamine and can be divided into three kinds: chitin hydrolysis method, biotransformation method and microbial fermentation method, among which chitin hydrolysis is the main method for pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/75C12P19/26C12R1/125
Inventor 刘龙陈坚堵国成李江华徐小芳刘延峰马文龙
Owner JIANGNAN UNIV
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