Recombinant bacillus subtilis for accumulating acetylglucosamine and application thereof

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

Active Publication Date: 2015-12-23
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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  • Recombinant bacillus subtilis for accumulating acetylglucosamine and application thereof
  • Recombinant bacillus subtilis for accumulating acetylglucosamine and application thereof

Examples

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Embodiment 1

[0023] The construction of embodiment 1 recombinant plasmid

[0024] According to the nucleotide sequence of the glucosamine acetylase coding gene (CeGNA1) in Caenorhabditis legans published on NCBI as shown in NCBIGenBank: AB017628.1, the codon preference of Bacillus subtilis was optimized to obtain the nucleotide The sequence is based on SEQ ID NO.1, and the gene sequence was synthesized by Shanghai Sangon Bioengineering Co., Ltd. Primers CeGNA1-F: 5'-GGGGTACCATTATAGGTAAGAGAGGAATGTACACATGAGCCATATCTTCGACGCATCTG-3', CeGNA1-R: 5'-CCCAAGCTTTTAAAAGCGCTGGGTCATAAAATTA-3' were designed. The optimized glucosamine acetylase encoding gene (CeGNA1) was amplified from the synthetic sequence nucleotide sequence SEQ ID NO.1 using the above primers. The amplified fragment was digested with KpnI and HindIII and then ligated into pP43NMK expression vector. Restriction digestion verification and sequencing confirmed the successful construction of the recombinant plasmid, which was named pP43...

Embodiment 2

[0025] The construction of embodiment 2 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] Example 3 Fermentative production of acetylglucosamine

[0028] The seeds cultivated at 37° C. and 200 rpm for 12 hours were transferred to the fermentation medium at an inoculum size of 5%, and cultivated at 37° C. and 200 rpm for 30 hours. The content of acetylglucosamine in the final fermentation supernatant reached 7.31g / L. The control strain was BSGN6-PxylA-glmS as the starting strain, overexpressing the glucosamine acetylase encoding gene GNA1 (nucleotide sequence such as NCBI GenBank: NM_001179949) derived from Saccharomycescerevisiae S288C, and the final fermentation supernatant under the same culture conditions The content of acetylglucosamine in the medium reaches 5.87g / L. By comparison, the yield of acetylglucosamine in the fermentation supernatant of the recombinant Bacillus subtilis expressing the CeGNA1 gene derived from Caenorhabditis elegans was 24.51% higher than that of the control strain expressing the GNA1 gene derived from Saccharomyces cerevisiae....

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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 accumulating acetylglucosamine and an application thereof, belonging to the field of genetic engineering. Background technique [0002] Glucosamine is a compound in which one 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-acetyl glucosamine is a derivative of glucosamine, which has important physiological functions and is widely used in the fields of health food and medicine. For example, it can specifically act on articular cartilage and effectively treat rheumatoid arthritis; it can inhibit the production of leukemia cells K562 Growth, is the main component of the new anticancer drug chlorurecin; it can participate in liver and kidney detoxification, play the role of anti-inflammation, liver protection, etc. [0003] The production methods of glucosamine and glucosami...

Claims

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Application Information

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Patent Type & Authority Applications(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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