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Recombinant halophilus mediterranei and application thereof in preparation of PHBV

A technology of Mediterranean salt-enriched bacteria and recombinant vectors, which is applied in the biological field, can solve the problems of PHBV production that cannot meet the needs of industrialization and high production costs, and achieve the effects of fast glucose consumption rate, high biomass, and shortened fermentation cycle

Pending Publication Date: 2022-05-20
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

[0005] At present, the main factor limiting the application of PHBV is its high production cost. As a strain with great potential for industrial production of PHBV, the production of PHBV cannot meet the needs of industrialization.

Method used

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  • Recombinant halophilus mediterranei and application thereof in preparation of PHBV
  • Recombinant halophilus mediterranei and application thereof in preparation of PHBV
  • Recombinant halophilus mediterranei and application thereof in preparation of PHBV

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Effect test

Embodiment 1

[0078] 1. Knock out the cas3 gene

[0079] Using Haloferax mediterranei DF50-ΔEPS with high PHBV production as the starting strain, the cas3 gene in Haloferax mediterranei DF50-ΔEPS was knocked out to construct Haloferax mediterranei DF50ΔEPSΔcas3. Methods as below:

[0080] (1) Using the genomic DNA of Halobacterium mediterranei DF50-ΔEPS as a template, use primers Cas3-U-F / Cas3-U-R and Cas3-D-F / Cas3-D-R to PCR amplify the upper and lower homology arms of the cas3 gene respectively, and then use the amplified The increased PCR product containing the upper and lower homology arms was used as a template, and the primers Cas3-U-F and Cas3-D-R were used for overlapping PCR. After the product was recovered, it was digested with BamHI and Kpn I, and the large fragment was recovered. The resulting large fragment was used with BamH I and Kpn I pair carrier pHFX (Liu H, Han J, Liu X, et al.Development of pyrF-based gene knockout systems for genome-wide manipulation of the archaea Hal...

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Abstract

The invention discloses a recombinant halophilus mediterranei and an application thereof in preparation of PHBV (Polyhydroxybutyrate Virus). A construction method of the recombinant halophilus mediterranei disclosed by the invention comprises the following steps: 1) knocking out a cas3 gene in the halophilus mediterranei to obtain cas3 knockout bacteria; and 2) introducing an expression cassette of crRNA for transcriptional inhibition of citrate synthase gene expression into the cas3 knockout bacterium to obtain the recombinant halophilus mediterranei. Experiments prove that the recombinant halophilus mediterranei provided by the invention has higher biomass and faster glucose consumption rate, and the dry cell weight and the PHBV accumulation amount are both obviously improved. The recombinant halophilus mediterranei provided by the invention has a good application prospect.

Description

technical field [0001] The invention relates to a recombinant Mediterranean halobacterium and its application in the preparation of PHBV in the field of biotechnology. Background technique [0002] Polyhydroxyalkanoate (polyhydroxyalkanoate, PHA) is an intracellular polymer produced by microorganisms when the carbon source is excessive and other elements such as nitrogen, phosphorus, sulfur, oxygen, etc. are limited, and it is mainly used as a carbon source and storage of energy. According to the number of carbon atoms in the PHA monomer, PHA is mainly divided into short-chain PHA with 3 to 5 carbon atoms, and medium- and long-chain PHA with 6 or more carbon atoms. In addition, it also includes copolymerized PHA composed of short-chain PHA monomers and medium-long chain PHA monomers. According to whether the monomer contained in PHA is single or not, PHA can be divided into homopolymer and copolymer. Homopolymers contain only one monomer, such as poly-3-hydroxybutyrate (P...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12P7/625C12R1/01
CPCC12N15/74C12N9/22C12N9/1025C12Y203/03001C12P7/625Y02E50/10
Inventor 韩静向华林琳
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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