CN-121975766-A - Mutant GlpQ protein and strain expressing high-yield L-tryptophan thereof

Abstract

The invention relates to a mutant GlpQ protein and a strain expressing the mutant GlpQ protein and producing L-tryptophan with high yield, belonging to the technical field of genetic engineering. The amino acid sequence of the mutant GlpQ protein comprises a sequence with R101C mutation based on the sequence shown in SEQ ID NO. 1. The invention digs a mutant GlpQ protein in the mutagenesis bacteria of high-yield L-tryptophan, verifies that the mutant GlpQ protein can promote the strain to produce L-tryptophan, further constructs the genetic engineering bacteria of high-yield L-tryptophan, realizes the improvement of the yield of L-tryptophan, and provides a new idea for constructing the high-yield L-tryptophan strain.

Inventors

• LI GUANGYU
• LI XUEPENG
• WANG KUIZHI
• WANG ZHONGKAI
• WANG JUNCHENG
• FANG NING
• SUN GUANGLING
• ZHAO JIE
• CAO LINYUAN

Assignees

• 新疆阜丰生物科技有限公司

Dates

Publication Date

20260505

Application Date

20260311

Claims (10)

1. 1. A mutant GlpQ protein, wherein the amino acid sequence of the mutant GlpQ protein comprises a sequence in which an R101C mutation occurs based on the sequence shown in SEQ ID No. 1.
2. 2. A nucleic acid molecule encoding the mutant GlpQ protein of claim 1.
3. 3. A recombinant vector comprising the nucleic acid molecule of claim 2.
4. 4. Use of a mutant GlpQ protein according to claim 1, a nucleic acid molecule according to claim 2 or a recombinant vector according to claim 3 to promote the synthesis of L-tryptophan by a strain.
5. 5. An engineered tryptophan-producing bacterium, wherein the engineered bacterium expresses the mutant GlpQ protein of claim 1.
6. 6. The tryptophan-producing engineering bacterium according to claim 5, wherein the starting strain of the engineering bacterium is escherichia coli.
7. 7. The tryptophan-producing engineering bacterium according to claim 5 or 6, wherein the genome of the tryptophan-producing engineering bacterium lacks the wild-type GlpQ protein gene sequence and has inserted therein the mutant GlpQ protein gene sequence of claim 1.
8. 8. A method for preparing the tryptophan-producing engineering bacterium according to any one of claims 5 to 7, comprising: Removing the strain to prepare competent cells, knocking out the wild-type GlpQ protein gene, and inserting the mutant GlpQ protein gene sequence according to claim 1.
9. 9. A method for producing tryptophan, the method comprising: Culturing the tryptophan-producing engineering bacteria of any one of claims 5-7, and taking the culture solution for product purification to obtain tryptophan.
10. 10. The method for producing tryptophan according to claim 9, wherein the culture medium contains glucose, yeast extract, ammonium sulfate, magnesium sulfate, potassium dihydrogen phosphate, sodium citrate, sodium chloride, L-tyrosine, L-phenylalanine and calcium carbonate.

Description

Mutant GlpQ protein and strain expressing high-yield L-tryptophan thereof Technical Field The invention belongs to the technical field of genetic engineering, and relates to a mutant GlpQ protein and a strain expressing the mutant GlpQ protein and producing L-tryptophan with high yield. Background L-tryptophan is an important aromatic amino acid, and has a plurality of physiological functions in organisms and wide application in the fields of foods, medicines, feeds and the like. Tryptophan is one of the essential amino acids that cannot be synthesized by itself in humans and animals, and must be ingested through diet or feed, and the market demand for tryptophan continues to grow as the food, pharmaceutical and feed industries develop. Traditional tryptophan production processes rely primarily on chemical synthesis and microbial fermentation, which is of great interest because of its environmental friendliness and cost effectiveness. The escherichia coli has multiple advantages as industrial microorganisms for producing tryptophan, has clear genetic background, high-efficiency metabolic network and mature industrial application, becomes an ideal host for tryptophan biological production, and is one of the most commonly used tryptophan engineering bacteria at present. With the increasing global demand for L-tryptophan, the construction and modification of high-yield L-tryptophan strains is particularly important. The production strain is modified through genetic engineering, the tryptophan synthesis path is optimized, the feedback inhibition is released, the precursor substance supply is improved, and the product secretion is enhanced, so that the method is an effective method for producing L-tryptophan at present. The construction method of the E.coli strain for producing tryptophan and the constructed strain are disclosed in CN101812426A, the tryptophan enzyme gene of the E.coli is knocked out, and a temperature control vector is utilized to express the 3-deoxy-D-arabino-heptulose 7-phosphate synthase gene and the anthranilate synthase gene which are not regulated and controlled by tryptophan feedback inhibition, so that the E.coli strain for producing tryptophan at high yield is obtained. CN111154706A discloses recombinant escherichia coli with improved L-tryptophan yield and a construction method thereof, escherichia coli CICC 10303 is used as an initial strain, error-prone PCR and CRISPR-Cas9 gene editing technology are adopted, a key gene trpR is knocked out to remove feedback repression regulation on tryptophan synthesis and transport key enzyme, competition of phenylalanine synthesis pathway is removed after a pre-benzene dehydrogenase encoding gene pheA is knocked out, and a gene random mutation kit is utilized to randomly mutate a promoter sequence ParoK of an L-tryptophan synthesis related gene aroK. In summary, the existing studies have been mainly conducted to improve tryptophan yield by modifying related genes in tryptophan anabolic pathways. The development of a novel genetic transformation strategy provides a new idea for constructing high-yield L-tryptophan strains, and has important significance for the field of producing L-tryptophan by a microbial fermentation method. Disclosure of Invention Aiming at the defects and actual demands of the prior art, the research of the invention improves the permeability of the cell membrane of the escherichia coli by introducing glpQ mutation sites of genes, accelerates the excretion of tryptophan from the inside to the outside of the cell, and reduces the accumulation of intracellular tryptophan, thereby enhancing the yield of the tryptophan. In order to achieve the above purpose, the invention adopts the following technical scheme: In a first aspect, the invention provides a mutant GlpQ protein, the amino acid sequence of which mutant GlpQ protein comprises a sequence in which an R101C mutation occurs based on the sequence shown in SEQ ID NO. 1. The invention digs a mutant GlpQ protein in the mutagenesis bacteria of high-yield L-tryptophan, verifies that the mutant GlpQ protein can promote the strain to produce L-tryptophan, and provides a new idea for constructing engineering bacteria of high-yield L-tryptophan. In the invention, the glycerophosphoryl diester phosphodiesterase (GlpQ) is related to membrane lipid metabolism and is responsible for degrading glycerol-3-phosphate derivatives (such as deacylated glycerophosphoric diester) and can catalyze the hydrolysis of the glycerophosphoric diester into glycerol 3-phosphate and alcohols (such as choline, ethanolamine and the like), thereby providing a carbon source or energy for cells. It will be appreciated that, on the basis of the mutant GlpQ protein of the present invention, functionally similar GlpQ proteins obtained by modification such as amino acid substitution, deletion or addition by genetic modification means in the art should be within the scope of the present invention, and the number of