CN-122012359-A - Construction and production method and application of genetically engineered bacterium for synthesizing Gadusol

Abstract

The invention relates to construction and production methods and applications of a synthetic Gadusol genetic engineering bacterium, wherein the synthetic Gadusol genetic engineering bacterium is obtained by taking escherichia coli MG1655 as an initial strain, knocking out a transketolase gene on the escherichia coli MG1655 genome and cooperatively expressing an exogenous EEVS gene and an exogenous MT-Ox gene. According to the invention, EEVS genes and DrMT-Ox genes are integrated into a specific gene locus of a chromosome of the escherichia coli in a multicopy mode, and key enzyme genes of a Pentose Phosphate Pathway (PPP) are integrated and overexpressed on the chromosome, so that the high-efficiency biosynthesis of Gadusol in the escherichia coli is realized for the first time, exogenous plasmids are not needed, an inducer and antibiotics are not relied on, a cheap carbon source can be efficiently utilized, and the high-yield, stable and green manufacturing of Gadusol is realized.

Inventors

• MU WANMENG
• MENG JIAWEI
• Yong Ziyi
• ZHU YINGYING
• ZHANG WENLI

Assignees

• 江南大学

Dates

Publication Date

20260512

Application Date

20260127

Claims (9)

1. 1. A genetically engineered bacterium for synthesizing Gadusol is characterized in that escherichia coli MG1655 is taken as an initial strain, a transketolase gene on the genome of the escherichia coli MG1655 is knocked out, and an exogenous EEVS gene and an exogenous MT-Ox gene are cooperatively expressed to obtain the genetically engineered bacterium.
2. 2. The genetically engineered bacterium of claim 1, wherein the transketolase genes comprise a transketolase a gene talA and a transketolase B gene talB.
3. 3. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium comprises a strain of Gadusol, The exogenous EEVS gene adopts a EEVS gene derived from zebra fish; the exogenous MT-Ox gene adopts MT-Ox gene derived from zebra fish.
4. 4. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium comprises a strain of Gadusol, The exogenous EEVS gene and the exogenous MT-Ox gene are expressed by the promoter P J23119 , respectively.
5. 5. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium comprises a strain of Gadusol, Co-expressing an exogenous EEVS gene and an exogenous MT-Ox gene, comprising: Knocking out nagB gene, gcd gene and edd gene on the escherichia coli MG1655 genome, and respectively integrating exogenous EEVS genes at sites of nagB gene, gcd gene and edd gene; knocking out arsB gene, ybeQ gene and purR gene on the genome of the escherichia coli MG1655, and respectively integrating exogenous MT-Ox genes at the loci of arsB gene, ybeQ gene and purR gene.
6. 6. The genetically engineered strain of claim 1 for the synthesis of Gadusol, wherein the strain is a strain of escherichia coli MG1655, wherein the strain is engineered to knock out the ldhA gene and to integrate into the ldhA gene a gene that is a key enzyme of the pentose phosphate pathway, wherein the gene comprises one or more of pgl gene, zwf gene, gnd gene, rpiA gene, rpe gene, tktA gene, tktB gene.
7. 7. The genetically engineered bacterium of claim 6 for synthesizing Gadusol, The promoter P J23119 is used to start the key enzyme gene for expressing pentose phosphate pathway.
8. 8. The method for producing Gadusol by using the genetically engineered bacterium according to any one of claims 1 to 6, wherein the genetically engineered bacterium is inoculated into a fermentation medium, and is fermented to produce Gadusol by using glycerol carbon source and xylose as a substrate.
9. 9. The use of the genetically engineered bacterium of any one of claims 1-6 in fermentation production Gadusol.

Description

Construction and production method and application of genetically engineered bacterium for synthesizing Gadusol Technical Field The invention belongs to the technical field of genetic engineering and fermentation, and in particular relates to a construction and production method and application of genetically engineered bacteria for synthesizing Gadusol. Background Gadusol is a cyclohexenone compound naturally occurring in a variety of aquatic organisms (e.g., zebra fish, sea urchin eggs, artemia, etc.). The molecular structure is simple, but has strong ultraviolet absorption capacity (especially in UVB/UVC wave band) and remarkable antioxidant activity, so that the molecular structure has wide application prospect in high-end cosmetics, functional foods and pharmaceutical preparations. Researches show that Gadusol can be directly used as a photo-protective agent to relieve DNA damage induced by ultraviolet rays, can be used as a precursor to synthesize a series of mycosporine-like amino acids (MAAs) with UVA protection function, and further expands the application range. Currently, gadusol production is mainly dependent on extraction from marine tissues. However, the content of the extract in organisms is extremely low (the extract is usually distributed in specific tissues such as fish eggs, embryos and the like in a trace amount), the extraction process is complex, the cost is high, the pressure is caused on ocean resources, and the large-scale and sustainable production requirements are difficult to meet. In terms of microbial synthesis, there have been studies attempting to construct Gadusol synthetic pathways in yeast systems. For example, de novo synthesis of Gadusol was achieved by heterologous expression of zebra fish-derived EEVS and MT-Ox genes, and knockout of the competing transaldolase gene in saccharomyces cerevisiae (s. Cerevisiae) or pichia pastoris (Komagataella phaffii). However, the yield reported in the prior art is still low, the maximum shake flask culture is 315.5 mg/L, and large-scale fermentation verification is not yet achieved. More importantly, the systems depend on plasmid expression exogenous genes, antibiotics are required to be continuously added to maintain plasmid stability, and induction agents such as IPTG and the like are used for promoting gene expression, so that the production cost is increased, potential risks of drug resistance gene diffusion and toxicity of induction agents are brought, and the feasibility of industrial application of the systems is limited. Therefore, developing a recombinant escherichia coli system which does not need exogenous plasmid, does not depend on inducer and antibiotics and can efficiently utilize cheap carbon source, realizes high-yield, stable and green manufacturing of Gadusol, and becomes a technical problem to be solved in the field. Disclosure of Invention The invention aims to overcome the defects in the prior art and provides a construction and production method and application of the synthetic Gadusol genetic engineering bacteria, which not only greatly improves the yield of Gadusol, but also does not need exogenous plasmid, does not depend on inducer and antibiotics, can efficiently utilize cheap carbon sources, and realizes high-yield, stable and green manufacture of Gadusol. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: A genetically engineered bacterium for synthesizing Gadusol is prepared from the Escherichia coli MG1655 through knocking out the ketolase gene on the genome of Escherichia coli MG1655, and expressing exogenous EEVS gene and exogenous MT-Ox gene. As a further technical scheme, the transketolase genes comprise a transketolase A gene talA and a transketolase B gene talB. As a further technical scheme, the exogenous EEVS gene adopts a EEVS gene (gene ID. LOC100003999, the nucleotide sequence of which is shown in SEQ ID NO. 1) derived from zebra fish; As a further technical scheme, the exogenous MT-Ox gene adopts a zebra fish-derived MT-Ox gene (gene ID. zgc:113054, the nucleotide sequence of which is shown in SEQ ID NO. 3). As a further technical scheme, the exogenous EEVS gene and the exogenous MT-Ox gene are respectively expressed by the promoter P J23119. As a further technical scheme, the nagB gene, the gcd gene and the edd gene on the escherichia coli MG1655 genome are knocked out, and exogenous EEVS genes are respectively integrated at the sites of the nagB gene, the gcd gene and the edd gene. As a further technical scheme, nagB gene: annogtion: NC_000913.3 (702811.. 703611, complex). As a further technical scheme, the gcd gene is Annogtion NC_000913.3 (138835.. 141225, complex). As a further technical scheme, edd gene: annogtion: NC_000913.3 (1932793.. 1934604, complex). As a further technical scheme, arsB genes, ybeQ genes and purR genes on the genome of the escherichia coli MG1655 are knocked out, and exogenous MT-Ox genes are respectively integrated