CN-121991871-A - Genetically engineered bacterium for high yield of colanic acid, construction method and application thereof

Abstract

The invention discloses a genetic engineering bacterium for high yield of colanic acid, a construction method and application thereof, belonging to the technical field of genetic engineering, wherein the construction method comprises the following steps: the waaL gene cluster, lon gene, hns gene, clsA gene, clsB gene, clsC gene, mcbR gene and opgDGH gene on E.coli MG1655 genome are knocked out in sequence, and then rcsA gene, galU gene, manA gene and cpsG gene are overexpressed in sequence. The invention realizes the efficient and stable regulation and control of the synthesis path of the colanic acid by the multiple gene knockout technology and combining exogenous genes and the genome site-specific integration of the enhanced expression element, and the obtained strain has the capability of high yield of the colanic acid and high industrialized application potential.

Inventors

• LIU LEI
• ZHANG MEIXIA
• LI GUANGYU
• SONG ZHE
• Zhu tianze
• ZHENG DEQIANG
• KANG CHUANLI
• Lian Shaojie
• ZHANG WEI
• LIU QIANG
• LI PANPAN
• LIU SHANSHAN

Assignees

• 山东焦点福瑞达生物股份有限公司

Dates

Publication Date

20260508

Application Date

20260202

Claims (9)

1. 1. A construction method of a genetic engineering bacterium for producing the cola acid at high yield is characterized by comprising the following steps of sequentially knocking out a waaL gene cluster, lon genes, hns genes, clsA genes, clsB genes, clsC genes, mcbR genes and opgDGH genes on a escherichia coli E.coli MG1655 genome, and then sequentially overexpressing rcsA genes, galU genes, manA genes and cpsG genes.
2. 2. The construction method of the genetically engineered bacterium for high yield of cola acid according to claim 1, wherein the nucleotide sequence of the waaL gene cluster is shown as SEQ ID No.7 in a sequence table; the nucleotide sequence of the lon gene is shown as SEQ ID No.16 in a sequence table; the nucleotide sequence of the hns gene is shown as SEQ ID No.25 in the sequence table; The nucleotide sequence of clsA gene is shown as SEQ ID No.34 in the sequence table; The nucleotide sequence of the clsB gene is shown as SEQ ID No.43 in the sequence table; the nucleotide sequence of clsC gene is shown as SEQ ID No.52 in the sequence table; the nucleotide sequence of the mcbR gene is shown as SEQ ID No.61 in the sequence table; the nucleotide sequence of opgDGH gene is shown as SEQ ID No.70 in the sequence table; the nucleotide sequence of rcsA gene is shown as SEQ ID No.81 in the sequence table; the nucleotide sequence of the galU gene is shown as SEQ ID No.92 in a sequence table; the nucleotide sequence of the manA gene is shown as SEQ ID No.103 in a sequence table; The nucleotide sequence of the cpsG gene is shown as SEQ ID No.114 in the sequence table.
3. 3. The method for constructing a genetically engineered bacterium capable of producing high yield of colanic acid according to claim 1, wherein when the rcsA gene is overexpressed, a mode of integrating and utilizing a promoter P J23119 to start expression of rcsA gene at the endA gene locus is adopted; When the galU gene is over-expressed, a mode of integrating at yjiP gene locus and utilizing a promoter P J23119 to promote the expression of the galU gene is adopted; When the manA gene is over-expressed, a mode of integrating and utilizing a promoter P J23119 to promote the expression of the manA gene at yhdW gene sites is adopted; In the case of over-expressing the cpsG gene, the expression of the cpsG gene is initiated by integrating the promoter P J23119 at cheW gene locus.
4. 4. The method for constructing genetically engineered bacteria capable of producing high yield of colanic acid according to claim 1, wherein the method for constructing comprises the steps of preparing recombinant DNA fragment, constructing plasmid pGRB, transforming plasmid and recombinant DNA fragment, and eliminating plasmid when knocking out or over-expressing.
5. 5. The method for constructing genetically engineered bacteria capable of producing high yield of colanic acid according to claim 4, wherein the construction plasmid pGRB comprises the steps of preparing transformation competent cells, constructing pGRB plasmid containing target sequence, transferring the recombinant plasmid to E.coli DH5 alpha competent cells, and screening positive transformants.
6. 6. The method for constructing genetically engineered bacteria capable of producing high yield of colanic acid according to claim 4, wherein the transformation plasmid and the preparation of recombinant DNA fragment comprise the steps of transforming pREDCas to prepare a target strain containing pREDCas in electrotransformation competence, transforming pGRB and recombinant DNA fragment.
7. 7. The method for constructing a genetically engineered bacterium capable of producing clavulanic acid at a high yield according to claim 4, wherein the elimination of the plasmid comprises the step of eliminating the plasmid pGRB and the plasmid pREDCas.
8. 8. A genetically engineered bacterium that produces colanic acid at high yield constructed by the construction method of any one of claims 1-7.
9. 9. Use of a genetically engineered bacterium that produces high levels of cola acid constructed by the construction method of any one of claims 1-7 in the fermentative production of cola acid.

Description

Genetically engineered bacterium for high yield of colanic acid, construction method and application thereof Technical Field The invention relates to the technical field of genetic engineering, in particular to a genetic engineering bacterium for high yield of colanic acid, and a construction method and application thereof. Background Colanic acid (Colanic acid, CA) is an anionic heteropolysaccharide, normally secreted by the enterobacteriaceae family. The colanic acid is a white fibrous substance which can be dissolved in water and dilute salt solution, and the solution is acidic. The polysaccharide of the colanic acid is polymerized by hexasaccharide repeating units consisting of D-galactose, D-glucose, D-glucuronic acid and L-fucose, and the corresponding molar ratio of the four monosaccharides is 2:1:1:2, wherein the fucose and galactose are modified by nonstoichiometric oxyacetyl and pyruvic acid. The clavulanic acid not only can improve the growth state of the host cells under severe environment and maintain the normal physiological functions of the host cells, but also has important potential application value in different industries as a polysaccharide with unique physiological activity. For example, the kola acid polysaccharide has a porous cellulose structure and a large amount of hydrophilic groups exist on the surface, so that the kola acid is an excellent natural hydrogel, has excellent water retention capacity and soft texture, and is a good candidate for future cosmetic market. The content of fucose in the colanic acid is high, and the fucose has various known biological activities, including anti-inflammatory, anti-tumor, anticancer, immunity enhancing, whitening and anti-aging activities. The lacosac acid can also regulate mitochondrial dynamics and unfolded protein reactions in a host, so that mitochondrial aging and cell rupture are delayed, mitochondrial reactions and longevity effects caused by the lacosac acid are ubiquitous among different species, and therefore, the lacosac acid also has wide application prospects in the fields of functional nutrition and health. At present, the synthesis of the cola acid in the escherichia coli is mainly influenced by using a mode of genetically engineering to modify the escherichia coli, but the existing modification method mainly surrounds a metabolic path of the cola acid, partial genes are knocked out to realize the synthesis of the cola acid, the enhancement effect on the yield of the cola acid is limited, although the bypass metabolic path is modified at present, the metabolic flux is redistributed by disturbing the global metabolic network of cells, so that the yield of the cola acid is further enhanced, at present, the related research of genetically engineering bacteria for constructing high-yield cola acid by modifying the bypass metabolic path is less, most of the existing genetically engineering bacteria for high-yield cola acid depend on plasmids, the problem of plasmid loss occurs in a solution in passage, the passage is unstable, and further, the industrialization application potential is lower. Disclosure of Invention The invention provides a genetic engineering bacterium for high yield of the corrasion, a construction method and application thereof, and the invention combines exogenous genes and enhanced genome site-directed integration of expression elements through a multiple gene knockout technology, thereby realizing the efficient and stable regulation and control of a corrasion synthesis path, and the obtained strain has the capacity of high yield of the corrasion and high industrialized application potential. In order to achieve the above object, the present invention provides the following technical solutions: in a first aspect, the invention provides a construction method of genetically engineered bacteria for high yield of colanic acid, comprising the following steps: Sequentially knocking out waaL gene cluster, lon gene, hns gene, clsA gene, clsB gene, clsC gene, mcbR gene and opgDGH gene on E.coli E. coliMG1655 genome, and then sequentially overexpressing rcsA gene, galU gene, manA gene and cpsG gene; the nucleotide sequence of the waaL gene cluster is shown as SEQ ID No.7 in a sequence table; the nucleotide sequence of the lon gene is shown as SEQ ID No.16 in a sequence table; the nucleotide sequence of the hns gene is shown as SEQ ID No.25 in the sequence table; The nucleotide sequence of clsA gene is shown as SEQ ID No.34 in the sequence table; The nucleotide sequence of the clsB gene is shown as SEQ ID No.43 in the sequence table; the nucleotide sequence of clsC gene is shown as SEQ ID No.52 in the sequence table; the nucleotide sequence of the mcbR gene is shown as SEQ ID No.61 in the sequence table; the nucleotide sequence of opgDGH gene is shown as SEQ ID No.70 in the sequence table; the nucleotide sequence of rcsA gene is shown as SEQ ID No.81 in the sequence table; the nucleotide sequence of the galU gene is show