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

An archaeal polyhydroxyalkanoate and polyhydroxyalkanoate technology, which is applied to the extreme halophilic archaea polyhydroxyalkanoate synthase and its encoding gene and application fields, 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: 2008-03-12
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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033]Example 1, the acquisition of polyhydroxyalkanoate (PHA) synthase and its coding gene (the schematic diagram of the cloning strategy is shown in Figure 1)

[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 AS 1.2049 has a very close relationship with the extreme halophilic archaea Haloarcula marismortui ATCC 43049 (GenBank number: AY596297) whose complete genome sequence has been published. There is a PHA synthase subunit gene annotated as phaC in the 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′CTA GGATCC ATGTCCAGCAACCCCTTC3′...

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 AS1.2049 Hh , phaE Hh and phaC HhThe 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 CAACTCGAAGAAGTGCAG3′( BamHI )

[0060] PE2: 5, GGC GGTACC TTACTCTTTCCAGGTGTTC3′( KpnI )

[0061] PE3:5, CGC CCATGG CAACTCGAAGAAGTGCAG3′( NCOI )

[0062] PE4: 5′ATA GGATCC AATAGTACCTCGGCGGCG3′( BamHI )

[0063] PC1:5, CTA GGATCC ATGTCCAGCAACCCGTTT3′( BamHI )

[0064] PC2: 5′CGT GGTACC TTACAGTTGGTCGAGCCA3′( KpnI )

[0065] 1) Using Haloarcula hispanica AS1.2049 genomic D...

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 shown in Figure 2, and 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. USA 87: 6772-6 for the sequence information and construction method of pUBP2). 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 ar...

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PUM

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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 amino acid residue sequence in sequence 1 in the sequence table or amino acid residue sequence got by substituting and / or deleting and / or adding one or more amino acid residue for the amino acid residue sequence in sequence 1 in the sequence table; the PhaCHh subunit has amino acid residue sequence in sequence 2 in the sequence table or amino acid residue sequence got by substituting and / or deleting and / or adding one or more amino acid residue for the amino acid residue sequence in sequence 2 in the sequence table. The polyhydroxyalkanoates enzyme is of very high PHA enzyme activity; introducing the polyhydroxyalkanoates enzyme gene phaECHh into a host bacterial strain will make possible high-efficient expression of PHA enzyme, and can improve the activity of the extremely halophilic archaea polyhydroxyalkanoates enzyme and PHA output.

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 Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N1/21
Inventor 向华韩静陆秋鹤
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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