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Extremely halophilic archaea polyhydroxy fatty acid ester synthases and encoding gene and application

A technology of archaea polyhydroxyalkanoate and extreme halophilic archaea, which is applied to the polyhydroxyalkanoate synthase of extreme halophilic archaea and its encoding gene and application field, and can solve deletion mutants and non-highly expressed strains and other problems, to achieve the effect of increasing the production of PHA and increasing the activity of PHA synthase.

Inactive Publication Date: 2012-01-04
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the international research work on PHA in extreme halophilic archaea is still mainly focused on the research on the fermentation process. So far, the functional verification of the gene encoding PHA synthase has not been reported, let alone the high-efficiency expression of PHA synthase similar to that in bacteria. strains and deletion mutants

Method used

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  • Extremely halophilic archaea polyhydroxy fatty acid ester synthases and encoding gene and application
  • Extremely halophilic archaea polyhydroxy fatty acid ester synthases and encoding gene and application
  • Extremely halophilic archaea polyhydroxy fatty acid ester synthases and encoding gene and application

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1, the acquisition of polyhydroxyalkanoate (PHA) synthase and its coding gene (the schematic diagram of cloning strategy is as follows figure 1 shown)

[0034] 1. Two subunits of polyhydroxyalkanoate (PHA) synthase (PhaE Hh and PhaC Hh ) and the acquisition of its coding gene

[0035] The extreme halophilic archaea Haloarcula hispanica AS1.2049 has a very close relationship with the extreme halophilic archaea Haloarcula marismortui ATCC 43049 (GenBank number: AY596297) whose complete genome sequence has been published. According to the There is a PHA synthase subunit gene annotated as phaC in the whole genome sequence Hm , this laboratory study proved that the ORF of an unannotated gene function adjacent to its upstream encoded another subunit of PHA synthase, and named it phaE Hm . According to phaC in the extreme halophilic archaea Haloarcula marismortui ATCC 43049 Hm and phaE Hm Two pairs of primers, P1 / P2 and P3 / P4, were designed.

[0036] P1: 5′...

Embodiment 2

[0056] Embodiment 2, PHA synthase gene phaEC Hh and phaEC Hh The gene encoding the two subunits of phaE Hh and phaC Hh functional verification of

[0057] 1. Overexpression of the PHA synthase gene phaEC in the extreme halophilic archaea Haloarcula hispanica AS1.2049Hh , phaE Hh and phaC Hh The construction of recombinant plasmids and the construction of overexpression recombinant engineering bacteria

[0058] The following primers were designed according to the cloned sequence information of the extreme halophilic archaea Haloarcula hispanica AS1.2049:

[0059] PE1: 5′ATT GGATCC CAACTCGAAGAAGTGCAG 3′( BamHI )

[0060] PE2: 5'GGC GGTACC TTACTCTTCCAGGTGTTC 3'( KpnI )

[0061] PE3: 5'CGC CCATGG CAACTCGAAGAAGTGCAG 3′( NCOI )

[0062] PE4: 5′ATA GGATCC AATAGTACCTCGGCGGCG 3′( BamHI )

[0063] PC1: 5′CTA GGATCC ATGTCCAGCAACCCGTTT 3′( BamHI )

[0064] PC2: 5′CGT GGTACC TTACAGTTGGTCGAGCCA 3′( KpnI )

[0065] 1) Using Haloarcula hispanica AS1.2049 gen...

Embodiment 3

[0072] Example 3, PHA synthase gene deletion mutant strain Haloarcula hispanica PHB - 1 construction and its functional verification

[0073] 1. Destruction of phaEC Hh Construction of the homologous recombination integration plasmid pUBPHL

[0074] The construction process of pUBPHL is as follows figure 2 As shown, the specific implementation is as follows:

[0075] 1) Construction of departure plasmid pUBP

[0076]pUBP2 is a shuttle plasmid for extreme halophilic archaea and Escherichia coli (see the literature Proc. Natl. Acad. Sci. USA87: 6772-6 for pUBP2 sequence information and construction method). First, pUBP2 was digested with EcoRI to remove the replicon pHH9 of the halophilic archaea in pUBP2 to obtain a 6.6 kb linear fragment, and then the 6.6 kb linear fragment was self-ligated to form plasmid pUBP, which was used to construct the integration vector pUBPHL.

[0077] 2) Used to destroy phaEC Hh Amplification of the homologous recombination double crossover a...

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Abstract

The invention discloses an extremely halophilic archaea polyhydroxyalkanoates enzyme and the code gene and application of the enzyme. The polyhydroxyalkanoates enzyme comprises PhaEHh subunit and PhaCHh subunit. The PhaEHh subunit has an amino acid residue sequence in sequence 1 in the sequence table; The PhaCHh subunit has an amino acid residue sequence in sequence 2 in the sequence table. The polyhydroxyalkanoates enzyme of the invention has very high PHA enzyme activity. The PHA enzyme can be high-efficiently expressed, and the activity of the extremely halophilic archaea polyhydroxyalkanoates enzyme and PHA output can be improved by introducing the polyhydroxyalkanoates enzyme gene phaECHh into a host bacterial strain.

Description

technical field [0001] The invention relates to an extreme halophilic archaea polyhydroxyalkanoate synthase, its coding gene and application. Background technique [0002] Polyhydroxyalkanoate (PHA) is a linear polyester composed of hydroxy fatty acid monomers. As a carbon source and energy reserve, it widely exists in a variety of prokaryotic microorganisms in nature, including extreme halophilic archaea. The public's increasing concern about the "white pollution" problem caused by petroleum plastics has promoted the strong interest of chemists, microbiologists, and molecular biologists in the synthesis and application of biodegradable plastics produced by microorganisms (Biochem.J.376: 15-33, 2003). Since PHAs are synthesized from renewable resources, are biodegradable, and possess tissue compatibility, their commercial applications are gaining increasing attention. Currently known application fields include packaging materials such as biodegradable films and containers,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N1/21C12R1/01C12N9/00
Inventor 向华韩静陆秋鹤
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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