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Preparing method and application of large-grained inclusion in microorganism cells

A technology of microorganisms and inclusions, which is applied in the preparation and application of large-particle inclusions in microbial cells, and can solve the problems of high downstream processing costs, high energy consumption, and high probability of bacterial contamination

Active Publication Date: 2019-07-12
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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  • Preparing method and application of large-grained inclusion in microorganism cells
  • Preparing method and application of large-grained inclusion in microorganism cells
  • Preparing method and application of large-grained inclusion in microorganism cells

Examples

Experimental program
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Effect test

Embodiment 1

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

[0126] 1. Construction of phaP1 gene knockout strain

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

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

Embodiment 2

[0159] Example 2. Obtaining large particle inclusions by knocking out the PHA particle surface binding protein and overexpressing the minCD gene

[0160] 1. Acquisition of recombinant strains

[0161] 1. Replace Halomonas TD01 in Example 1 with Halomonas TD-MmP1, and perform the operation according to Step 1 of Example 1 to obtain the knockout strain Halomonas TD-MmP1ΔphaP1 in which the phaP1 gene is knocked out.

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

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

[0164] 2. Construct pMmP1 promoter to induce overexpression of the plasmid pMmP1-minCD of the minCD gene cluster (minC gene and minD gene). After sequencing, the plasmid pMmP1-minCD is the sequence 8 inserted between the XbaI and SpeI ...

Embodiment 3

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

[0178] 1. Acquisition of recombinant strains

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

[0180] The pMmP1 promoter-induced phaCAB operon of Eutropha roseri and the 4-hydroxybutyrate-CoA transferase gene orfZ of Clostridium krusei were inserted into the chromosome of Halomonas TDH4, and Halomonas TDH4 can utilize Glucose and γ-butyrolactone are used as substrates to synthesize P(3HB-co-4HB).

[0181] 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. Introduce the recombinant plasmid pMmP1-minCD obtained in Step 1 ...

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Abstract

The invention discloses a preparing method and application of large-grained inclusion in microorganism cells. According to the method for improving the granular size of the inclusion in the microorganism cells, the encoding gene expression of PHA grain surface binding protein in microorganisms is independently restrained, or the encoding gene expression of the PHA grain surface binding protein inthe microorganisms is restrained and meanwhile an encoding gene of cleaved ring restrained protein in the microorganisms is over-expressed. According to the method, large-grained (2-15 micron) inclusion can be obtained in the microorganism cells, and the obtained large-grained inclusion effectively reduces the minimum centrifugal rotating speed required for solid-liquid separation and easily solves the problem that the inclusion in the microorganisms is high in extraction difficulty and energy consumption.

Description

technical field [0001] The invention relates to a preparation method and application of large particle content in microbial cells. 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 white pollution, not only affecting the terrestrial environment, but also seriously affecting the marine environment. In order to solve the problem of white pollution, the replacement of petroleum-based non-degradable plastics by biodegradable plastics is considered to be one of the ways to reduce the environmental burden of plastics. [0003] Polyhydroxyalkanoate (PHA) is a general term for a class of biopolyesters and is the only bio-based material that is completely synthesized by microorganisms. Depending on the structure of the monomers that make up the polyester, PHA can exhibit a variety of mecha...

Claims

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Application Information

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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