The invention provides recombinant corynebacterium glutamicum with high yield of acetylglucosamine, which is characterized in that acetylglucosamine deacetylase-encoding gene NagA and the acetylglucosamine deaminase-encoding gene GamA are knocked out, and the glucosamine deacetylase-encoding gene GNA1 derived from caenorhabditis elegans is expressed. The recombinant corynebacterium glutamicum canachieve extracellular accumulation of acetylglucosamine, the concentration can achieve up to 24.7 g/L, which is 84.2% higher than that of the unmodified strain, and the recombinant corynebacterium glutamicum establishes the foundation for reconstruction of corynebacterium glutamicum by metabolic engineering to produce glucosamine. The recombinant corynebacterium glutamicum provided by the invention has simple and convenient construction method and has good application prospect.

Recombinant corynebacterium glutamicum with high yield of acetylglucosamine and application thereof

The invention relates to an extracellular expression L-aspartate-alpha-decarboxylase engineering bacterium constructing method and an application of the bacterium to production of beta-alanine A high-activity L-aspartate-alpha-decarboxylase gene is cloned and co-expressed with a signal peptide gene, a competent escherichia coli cell is transformed, an extracellular secretion engineering bacteriumfor the signal peptide mediated L-aspartate-alpha-decarboxylase is constructed, the extracellular secretion amount of the L-aspartate-alpha-decarboxylase is increased with the matching of the fermenting and regulating measures, and the obtained L-aspartate-alpha-decarboxylase is used for transforming the L-aspartate so as to produce beta-alanine The implementation of the extracellular release ofan enzyme reduces the toxicity of the excessively stacked protein in the cell on the cell and is favorable for the contact between a substrate and the enzyme; a fermentation liquid is directly used to catalyze the L-aspartate to produce beta-alanine, the reaction is carried out without breaking the cell and extracting the pure enzyme; the beta-alanine is catalytically synthesized by means of substrate fed-batch, the catalytic process is prevented from producing excessive salt, and the product purity is high

Extracellular expression L-aspartate-alpha-decarboxylase engineering bacterium constructing method and application thereof

Belonging to the technical field of genetic engineering, the invention discloses a repetitive extragenic palindromic sequence for improving exogenous gene mRNA and application thereof. The repetitiveextragenic palindromic sequence and a spacer region sequence thereof are shown as SEQ ID NO, 1, the signal peptide can improve the stability of mRNA by changing the secondary structure, so that the extracellular enzyme activity of the target protein cyclodextrin glycosyltransferase can be improved by 14%, the recombinant escherichia coli modified by the signal peptide has strengthened extracellular protein production capacity, therefore the repetitive extragenic palindromic sequence can be used for escherichia coli to express exogenous protein.

Repetitive extragenic palindromic sequence for improving exogenous gene mRNA and application thereof

The invention relates to a genetically engineered bacterium for synthesizing N-acetylglucosamine and an application of the genetically engineered bacterium. The genetically engineered bacterium is recombinant corynebacterium glutamicum containing a glucosamine synthetase gene glmS and a glucosamine transacetylase gene gna1, and is modified by a chassis microorganism of which related genes of a GlcNAc reverse transport pathway, a catabolism pathway and a competitive metabolism pathway are knocked out; the genetically engineered bacterium also contains a 6p-GlcNAc specific phosphatase gene yqaBused for increasing the concentration of extracellular GlcNAc, and the genetically engineered bacterium expresses the glucosamine synthetase gene glmS, the glucosamine transacetylase gene gna1 and the6p-GlcNAc specific phosphatase gene yqaB. The genetically engineered bacterium is safe and non-toxic, can produce the GlcNAc at high yield by utilizing a microbial fermentation method, and is stablein batch, relatively low in production cost and wide in application prospect.

Genetically engineered bacterium for synthesizing N-acetylglucosamine and application of genetically engineered bacterium

The invention discloses a secretory expression method of carboxypeptidase B. The secretory expression method of the carboxypeptidase B includes: (1) adopting sequences of signal peptides including OmpA, PelB, Endoxylanase and OmpT as starting sequences to perform site-specific mutagenesis; (2) preparing a nucleotide sequence SDB-pCPB-6xHis; (3) preparing recombinant expression vectors; (4) transforming the recombinant expression vectors into host escherichia coli E.coli BL21 and induce to express pCPB; (5) enzyme-digesting and purifying the pCPB. The secretory expression method of the carboxypeptidase B has the advantages that through transformation of the signal peptides, the procarboxypeptidase B can be directly secreted into a culture medium, compared with intracellular and periplasmicexpressions, the secretory expression method does not need smudge cells, and recovery and purification of the carboxypeptidase B are easier to operate. In addition, the culture medium with enough space is beneficial to overexpression of recombinant protein.

Secretory expression method of carboxypeptidase B

The invention discloses corynebacterium glutamicum for producing N-acetylglucosamine and application of the corynebacterium glutamicum and belongs to the field of metabolic engineering. According to the corynebacterium glutamicum, a corynebacterium glutamicum strain S9114 is adopted as an original strain, and a key enzyme gene in a synthesis way of acetylglucosamine is expressed by using a shuttleexpression vector pJYW-4 of the corynebacterium glutamicum and escherichia coli; with glucosamine-6-bacillus stearothermophilus phosphotransacetylase GNAl derived from caenorhabditis elegans, the synthesis way of the acetylglucosamine is enhanced by inserting repeated palindrome into a 3'-non-translation region of the GNAl gene, the yield of the acetylglucosamine can be increased, acetylglucosamine production recombinant corynebacterium glutamicum can be prepared, the yield can be up to 6.1g/L, and a basis for further transforming corynebacterium glutamicum to produce acetylglucosamine in metabolic engineering is made.

Corynebacterium glutamicum for producing N-acetylglucosamine and application of corynebacterium glutamicum

The invention discloses a pelB signal peptide mutant capable of improving the protein secretion efficiency and an application of the pelB signal peptide mutant and belongs to the technical field of gene engineering A sequence of a pelB mutant secretive expression signal peptide is as shown in SEQ ID NO1 The signal peptide is capable of improving the extracellular enzyme activity of objective protein cyclodextrin glycosyltransferase by 16 times The extracellular protein production capability of recombinant escherichia coli transformed by using the signal peptide is strengthened, and industrialized production is facilitated

PelB signal peptide mutant capable of improving protein secretion efficiency and application of pelB signal peptide mutant

The invention discloses a signal peptide mutant for improving secretion efficiency of protein and application thereof, and belongs to the technical field of gene engineering. The known signal peptide PelB is modified; the tyrosine at the site 3 of the PelB signal peptide is mutated into histidine, so that the activity of cyclodextrin glucanotransferase and the extracellular secretion efficiency are obviously improved. The signal peptide mutant for improving secretion efficiency of protein has the advantages that the activity of the target protein cyclodextrin glucanotransferase is improved by 1.4 times, the ability of extracellular secretion protein of recombinant bacteria is improved, and the extracellular expression of conventional protein is facilitated.

Signal peptide mutant for improving secretion efficiency of protein and application thereof

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Corynebacterium glutamicum for producing N-acetylglucosamine and application of corynebacterium glutamicum

PelB signal peptide mutant capable of improving protein secretion efficiency and application of pelB signal peptide mutant

Signal peptide mutant for improving secretion efficiency of protein and application thereof

Repetitive extragenic palindromic sequence for improving exogenous gene mRNA and application thereof