CN-122012360-A - Serratia marcescens engineering bacteria for high-yield prodigiosin and construction method and application thereof

Abstract

The invention discloses a serratia marcescens engineering bacterium for high yield of prodigiosin, and a construction method and application thereof. The dapB defective engineering strain is constructed by knocking out the dapB gene of Serratia marcescens by using a Red homologous recombination system through a genetic engineering technology. After the strain is shake-cultured in a fermentation medium for 60 hours, the prodigiosin yield reaches 0.91g/L, and is improved by 25.5 percent compared with a wild strain. The engineering bacteria construction method is simple and convenient, has stable heredity and provides a new way for the efficient microorganism production of the prodigiosin.

Inventors

• CHEN TING
• ZHENG YANSHUANG
• GUO JIAQI
• PANG BOWEN

Assignees

• 东华大学

Dates

Publication Date

20260512

Application Date

20260204

Claims (4)

1. 1. The serratia marcescens engineering bacterium for high prodigiosin production is characterized in that a dapB gene for encoding dihydropyridine dicarboxylic acid reductase in the serratia marcescens engineering bacterium is knocked out.
2. 2. The Serratia marcescens engineering bacterium according to claim 1, wherein the dapB gene is replaced by a kanamycin resistance gene by homologous recombination.
3. 3. The method for constructing Serratia marcescens engineering bacteria according to claim 1, which is characterized by comprising the following steps: Step 1), PCR amplifying an upstream homologous arm and a downstream homologous arm of a dapB gene by taking serratia marcescens genome DNA as a template, PCR amplifying a kanamycin resistance gene fragment by taking a plasmid containing a kanamycin resistance gene as a template, linearizing a suicide plasmid pDM4 by inverse PCR; Step 2), adopting a seamless cloning technology to connect the upstream homology arm, the kanamycin resistance gene fragment and the downstream homology arm obtained in the step 1) with the linearized pDM4 to obtain a recombinant suicide plasmid pDM 4-delta dapB; Step 3), the recombinant suicide plasmid pDM 4-DeltadapB obtained in the step 2) is transferred into wild Serratia marcescens through conjugation and transfer; step 4), obtaining dapB knockout bacteria through primary exchange screening and secondary exchange screening, and then obtaining Serratia marcescens engineering bacteria after colony PCR verification and sequencing without errors.
4. 4. Use of the serratia marcescens engineering bacterium according to claim 1 for increasing prodigiosin yield.

Description

Serratia marcescens engineering bacteria for high-yield prodigiosin and construction method and application thereof Technical Field The invention relates to the technical field of microbial genetic engineering and strain transformation, in particular to a serratia marcescens engineering bacterium for high-yield prodigiosin, a construction method and application thereof. Background Prodigiosin (Prodigiosin) is a natural tripyrrole red pigment, which belongs to the quinone class of compounds and can be produced by a variety of bacteria such as Serratia marcescens, actinomycetes and the like. The pigment has biodegradability and environmental friendliness, and has wide application potential in the fields of medicine, agriculture, industry and environment. In the medical aspect, the prodigiosin shows various biological activities such as anticancer, immunosuppression, antimalarial and the like, has broad-spectrum antibacterial, antifungal and algae inhibiting capabilities in the agricultural and environmental fields, and is industrially used as a natural red pigment for dye preparation. The current prodigiosin synthesis method mainly comprises a chemical synthesis method and a microbial synthesis method. The chemical synthesis method has the advantages of high cost, low efficiency and difficult realization of large-scale production, and the microbial synthesis method is represented by Serratia marcescens, has the advantages of mild condition, environmental protection, easy industrialization and the like, and becomes a main production path. The biosynthesis of prodigiosin is mainly controlled by the pig gene cluster (comprising pigA-N and other genes), precursors MBC and MAP are respectively synthesized through two branch paths, and finally the prodigiosin is formed by condensation under the catalysis of PigC. MBC synthetic pathways are heavily metabolically burdened and are one of the key factors limiting yield. Although wild Serratia marcescens can produce prodigiosin, its yield is still insufficient to meet industry needs. Therefore, the strain is modified by genetic engineering means, the prodigiosin yield is improved, and the method has important research value and application prospect. Disclosure of Invention In order to solve the technical problems, the invention provides a serratia marcescens engineering bacterium for high yield of prodigiosin, a construction method and application thereof, and the metabolic pathway of the strain is optimized by knocking out a dapB gene, so that the prodigiosin synthesis efficiency is improved. The invention provides a serratia marcescens engineering bacterium for high yield of prodigiosin, and a construction method and application thereof, wherein the technical scheme is as follows: In a first aspect of the present invention, there is provided a serratia marcescens engineering bacterium that produces prodigiosin in which dapB gene encoding dihydrodipicolinate reductase is knocked out. Preferably, the dapB gene is replaced by a kanamycin resistance gene by homologous recombination. The second aspect of the invention provides a construction method of the serratia marcescens engineering bacteria, which comprises the following steps: Step 1) PCR amplifying the upstream homology arm and the downstream homology arm of the dapB gene by taking serratia marcescens genome DNA as a template, PCR amplifying a KanR fragment by taking a plasmid containing a kanamycin resistance gene (KanR) as a template, linearizing a suicide plasmid pDM4 by inverse PCR; Step 2), adopting a seamless cloning technology to connect the upstream homology arm, the kanamycin resistance gene fragment and the downstream homology arm obtained in the step 1) with the linearized pDM4 to obtain a recombinant suicide plasmid pDM 4-delta dapB; Step 3), the recombinant suicide plasmid pDM 4-DeltadapB obtained in the step 2) is transferred into wild Serratia marcescens through conjugation and transfer; Step 4), obtaining dapB knockout bacteria through primary exchange screening and secondary exchange screening, and then obtaining Serratia marcescens engineering bacteria after colony PCR verification and sequencing without errors. The invention provides an application of the serratia marcescens engineering bacteria in improving the yield of prodigiosin. The colonies were picked up and inoculated into LB medium for seed culture, and inoculated into fermentation medium (fructose 16.29g/L, peptone 11.76g/L, tween 80 2.64g/L, initial pH 6.0) at 5% inoculum size, and cultured at 30℃and 160rpm for 60 hours. The result shows that the prodigiosin yield of the constructed serratia marcescens engineering bacteria is improved by 25.5 percent compared with that of a wild control strain. The invention knocks out the dapB gene in Serratia marcescens for the first time, obviously improves the yield of the prodigiosin, and provides a new technical scheme for efficiently producing the prodigiosin by a microbiological method. Compared