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Recombinant Bacillus subtilis for efficiently synthesizing acetylglucosamine

A technology of Bacillus subtilis and glucosamine, applied in the field of genetic engineering, can solve problems such as the increase of acetyl glucosamine and the inability to obtain the accumulation concentration of acetyl glucosamine

Active Publication Date: 2017-03-08
SHANDONG RUNDE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] It can be seen from the disclosure content of the above patent documents that although the concentration of acetylglucosamine in the extracellular accumulation of the transformed recombinant Bacillus subtilis has increased, there is still room for improvement.
In addition, when the recombinant Bacillus subtilis is cultured, there are still several consecutive fermentations, and there is no growth after inoculation; and the formula of the medium can be used in laboratory shake flasks, but the ideal concentration of acetylglucosamine in the extracellular accumulation cannot be obtained in the tank. and other technical issues

Method used

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  • Recombinant Bacillus subtilis for efficiently synthesizing acetylglucosamine
  • Recombinant Bacillus subtilis for efficiently synthesizing acetylglucosamine
  • Recombinant Bacillus subtilis for efficiently synthesizing acetylglucosamine

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1: Knocking out the non-essential region skin on the genome

[0043] According to the genome sequence of Bacillus subtilis (Bacillus subtilis 168, purchased from the American Type Microorganism Collection, ATCC No.27370) published on NCBI, primers for amplification of the homology arms of the knockout frame were designed, and the primers for the upper and lower arms of the left arm were: skin- L-F (sequence shown in SEQ ID NO.2) and skin-L-R (sequence shown in SEQ ID NO.3); right arm upstream and downstream primers are: skin-R-F (sequence shown in SEQ ID NO.4) and skin-R-R (sequence shown in SEQ ID NO.5).

[0044] The left and right arms contained in the knockout frame were amplified from the Bacillus subtilis 168 genome using the above primers. According to the p7Z6 plasmid sequence published on NCBI (NCBI accession no.EU541492), design primers to amplify the bleomycin resistance gene (zeo), the upstream and downstream primers are: skin-Z-F (sequence such as S...

Embodiment 2

[0053] Embodiment 2: Construction of recombinant plasmid

[0054] According to the nucleotide sequence of the glucosamine acetylase encoding gene (CaGNA 1) in Candida albicans (Candida albicans SC5314) published on NCBI as shown in NCBIGenBank: XM_715990.1, the codon preference optimization of Bacillus subtilis was carried out, and the nucleoside The acid sequence is as SEQ ID NO.1, and the gene sequence was synthesized by Shanghai Sangon Bioengineering Co., Ltd. Design primers CaGNA1-F (sequence shown in SEQ ID NO.8): 5'-GGGGTACCATTATAGGTAAGAGAGGAATGTACACATGCTGCTGCCACAAGGTTATACAT-3', CaGNA1-R (sequence shown in SEQ ID NO.9): 5'-CCCAAGCTTTTAAAAGCGGCATACCATTTCTACG-3'. The glucosamine acetylase encoding gene (CaGNA1) was amplified from the synthetic sequence nucleotide sequence SEQID NO.1 using the above primers. The amplified fragment was digested with KpnI and HindIII and ligated into pP43NMK expression vector. Restriction digestion and sequencing confirmed the successful co...

Embodiment 3

[0055] Embodiment 3: the construction of recombinant Bacillus subtilis

[0056] Transform the constructed expression vector pP43-CaGNA1 into recombinant Bacillus subtilis BSGN6-Δskin-P xylA -glmS and BSGN6-P xylA -glmS. Using CaGNA1-F and CaGNA1-R primers to select transformants for colony PCR, a 450bp band appeared, verifying that the recombinant Bacillus subtilis was successfully constructed, and the recombinant Bacillus subtilis BSGN6Δskin-P was obtained respectively xylA -glmS -P 43 -CaGNA1 and BSGN6-P xylA -glmS -P 43 -CaGNA1.

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Abstract

The invention discloses recombinant Bacillus subtilis for efficiently synthesizing acetylglucosamine and belongs to the field of genetic engineering. Recombinant Bacillus subtilis BSGN6-PxylA-glmS is used as a starting strain, glucosamine synthetic route is enhanced by overexpressing glucosamine acetylase coding gene (CaGNA1) from Candida albicans SC5314 or knocking out unnecessary area skin at the same time, and accumulated acetylglucosamine Bacillus subtilis genetically-engineered bacteria are obtained; the yield is 21% or 94% higher than that of CaGNA1 genetically-engineered bacteria from overexpressed Saccharomyces cerevisiae, and the basis is laid for the further metabolic engineering modification of Bacillus subtilis.

Description

technical field [0001] The invention relates to a recombinant bacillus subtilis capable of increasing the extracellular secretion of acetylglucosamine, which belongs 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 glucosam...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/26C12R1/125
Inventor 卢伟张弘治韩宁
Owner SHANDONG RUNDE BIOTECH CO LTD
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