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A preparation method and application of large particle content in microbial cells

A technology of microorganisms and inclusions, which is applied in the preparation and application of large-scale inclusions in microbial cells, and can solve the problems of high probability of bacterial contamination, high cost, and obstacles to the large-scale application of PHA

Active Publication Date: 2021-03-05
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional PHA production is fermented by Ralstonia eutropha and genetically modified Escherichia coli (E.coli) as production strains. The production has a high probability of contamination, high energy consumption, and low conversion rate of substrates. , high downstream processing costs and other shortcomings, resulting in high costs for a long time, seriously hindering the large-scale application of PHA

Method used

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  • A preparation method and application of large particle content in microbial cells
  • A preparation method and application of large particle content in microbial cells
  • A preparation method and application of large particle content in microbial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Example 1. Obtaining large particle inclusions by knocking out PHA particle surface-binding proteins

[0126] 1. Construction of phaP1 knockout strains

[0127] 1. Insert the DNA molecule shown in the sequence 2 of the sequence table between the BbsI sites of the pHALORNA plasmid, and insert the DNA molecule shown in the sequence 5 of the sequence table between the BsaI sites to obtain the phaP1 gene knockout plasmid pHALORNA-phaP1 (Verified by sequencing). In Sequence 2 of the Sequence Listing, the 17th to 36th positions from the 5' end are gRNA; in Sequence 5 of the Sequence Listing, the 1st to 500th positions from the 5' end are the upstream homology arms of phaP1, and the 505th to 1004th positions are the downstream of phaP1 homology arm.

[0128] 2. The plasmid pQ08 was introduced into Escherichia coli S17-1 to obtain recombinant bacteria S17-1 / pQ08, and then the plasmid was transferred into Halomonas TD01 by conjugation transformation method to obtain recombinan...

Embodiment 2

[0159] Example 2. Large particle inclusions obtained by knocking out PHA particle surface binding protein and overexpressing minCD gene

[0160] First, the acquisition of recombinant strains

[0161] 1. Replace Halomonas TD01 of Example 1 with Halomonas TD-MmP1, and operate according to step 1 of Example 1 to obtain a knockout strain Halomonas TD-MmP1ΔphaP1 with the phaP1 gene knocked out.

[0162] The gene encoding inducible MmP1 RNA polymerase was inserted into the chromosome of Halomonas TD-MmP1.

[0163] Sequencing of Halomonas TD-MmP1 and knockout strain Halomonas TD-MmP1ΔphaP1 showed that the only difference between the two was the deletion of the phaP1 gene on the chromosome.

[0164] 2. The pMmP1 promoter was constructed to induce the plasmid pMmP1-minCD that overexpressed the minCD gene cluster (minC gene and minD gene). After sequencing, the plasmid pMmP1-minCD was inserted between the XbaI and SpeI sites of the pSEVA321 vector. Sequence 8 of the sequence table The...

Embodiment 3

[0177] Example 3. Construction and related characterization of Halomonas phaP1 knockout strain synthesizing P(3HB-co-4HB)

[0178] First, the acquisition of recombinant strains

[0179] 1. Replace Halomonas TD01 of Example 1 with Halomonas TDH4, and operate according to step 1 to obtain a recombinant knockout strain Halomonas TDH4ΔphaP1 with the phaP1 gene knocked out.

[0180] The Roche eutrophic phaCAB operon induced by the pMmP1 promoter and the gene orfZ encoding 4-hydroxybutyrate coenzyme A transferase from Clostridium klebsieri were inserted into the chromosome of Halomonas TDH4. Halomonas TDH4 can utilize Glucose and γ-butyrolactone were used as substrates to synthesize P(3HB-co-4HB).

[0181] The Halomonas TDH4 and the knockout strain Halomonas TDH4ΔphaP1 were sequenced, and the sequencing results showed that the only difference between the two was the deletion of the phaP1 gene on the chromosome.

[0182] 2. The recombinant plasmid pMmP1-minCD obtained in step 1 of ...

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Abstract

The invention discloses a preparation method and application of large particle content in microbial cells. The invention provides a method for increasing the particle size of microbial intracellular content, which is to individually inhibit the expression of the gene encoding the PHA particle surface binding protein in the microorganism, or inhibit the expression of the gene encoding the PHA particle surface binding protein in the microorganism and overexpress the microorganism at the same time The gene encoding the middle cleavage loop inhibitory protein. According to the method of the present invention, large-particle (2-15 μm) inclusions can be obtained in the microbial cells, and the obtained large-particle inclusions effectively reduce the minimum centrifugal speed required for solid-liquid separation, which is beneficial to solving the difficulty of extracting microbial inclusions Large and energy-intensive problems.

Description

technical field [0001] The present invention relates to a preparation method of microbial intracellular large particle inclusions and its application. Background technique [0002] Global plastic production has been increasing, reaching 330 million tons per year in recent years, with China accounting for 130 million tons. The accumulation of so much plastic in the environment has brought about the problem of white pollution, which not only affects the terrestrial environment, but also seriously affects the marine environment. In order to solve the problem of white pollution, the replacement of petroleum-based non-degradable plastics with biodegradable plastics is considered as one of the ways to reduce the burden of plastics on the environment. [0003] Polyhydroxyalkanoate PHA is a general term for a class of biopolyesters, and is the only biobased material that is completely synthesized by microorganisms. Depending on the structure of the monomers that make up polyester,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N15/74C12N1/21C12R1/01
CPCC07K14/195C12N15/74C12N15/902C12N2800/80C12N2810/10
Inventor 陈国强沈睿宁志禹叶健文
Owner TSINGHUA UNIV