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Process for prepn. of polyhydroxy alkanoate by using maoC gene

A chain polyhydroxyalkanoate and gene technology, applied in the field of preparing PHA, can solve problems such as no results

Inactive Publication Date: 2004-10-27
KOREA ADVANCED INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since MCL-PHA can be efficiently produced by using recombinant E.coli lacking fadB gene, efforts are still being made to find the enzyme that provides PHA monomer in recombinant E.coli lacking fadB gene, but no specific results have been seen so far

Method used

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  • Process for prepn. of polyhydroxy alkanoate by using maoC gene
  • Process for prepn. of polyhydroxy alkanoate by using maoC gene
  • Process for prepn. of polyhydroxy alkanoate by using maoC gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Preparation of recombinant E.coli producing PHA with maoC gene

[0032] A mutant E.coli with deletion of fadB gene and maoC gene, a recombinant vector containing E.colimaoC gene, and a recombinant vector containing MCL-PHA synthetase gene were constructed. These recombinant vectors were used to produce recombinant E. coli for the production of MCL-PHA.

Embodiment 1-1

[0033] Example 1-1: Preparation of mutant E.coli with deletion of fadB gene and maoC gene

[0034] According to a conventional method, the maoC gene was removed from E. coli using the red operon of bacteriophage λ (Jeong and Lee, Appl. Environ. Microbiol., 68:4979-85, 2002).

[0035] In other words, the mutant E.coli WB101 with deletion of the fadB gene was transformed with the vector pTrcEBG containing the red operon of λ phage (Jeong and Lee, Appl. Environ. Microbiol., 68:4979-85, 2002), and 1 mM IPTG to induce the expression of the red operon in E.coliWB101 transformed with pTrcEBG, and this E.coli was used to prepare cells capable of electroporation. E. coli WB101 was obtained by removing the fadB gene from E. coli W3110 (KCTC 2223) (Park et al., Enzyme Micro. Technol., 33:62-70, 2003).

[0036] At the same time, since the 60 bp at the 5' end and 60 bp at the 3' end of the maoC gene contain DNA sequences similar to the 60 bp at the 5' end and 60 bp at the 3' end of the ...

Embodiment 1-2

[0038] Example 1-2: Construction of p10499A and p10499613C2

[0039] Chromosomal DNA (JCM 10015) of Pseudomonas sp.61-3 containing the PHA synthase gene (Matsusaki et al., J. Bacteriol., 180:6459-67, 1998) was used as a template, and the primer was phaC2 Ps -1 (SEQ ID NO: 5) and phaC2 Ps -2 (SEQ ID NO: 6) Performed 30 cycles of PCR including denaturation at 94°C for 50 seconds, annealing at 52°C for 50 seconds, and extension at 72°C for 2 minutes, thereby obtaining a PCR product. 5'-cgcggatccaataaggagatatctagatgagagagaaaccaacgccg-3' (SEQ ID NO: 5) 5'-cggatccccgggtaccgagctcgaattctcagcgcacgcgcacgtaggta-3' (SEQ ID NO: 6)

[0040] The PCR product was analyzed by agarose gel electrophoresis, and the 1.7 kb gene fragment corresponds to the phaC2Ps gene.

[0041] Then, amplify the gntT104 promoter to continue to express the PHA synthase gene, use the chromosomal gene of E.coliW3110 (ATCC 39936) as a template, and the primers are gntT104-1 (SEQ ID NO: 7) and gntT104-2 (SEQ ID NO: ...

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Abstract

The present invention relates to a method for producing middle-chain-length polyhydroxyalkanoate (MCL-PHA) using a maoC gene. The producing method of MCL-PHA according to the present invention comprises the steps of transforming a microorganism with the maoC gene to give a transformant, the microorganism being deleted of a fadB gene and containing a PHA synthase gene; culturing the transformant in medium containing a C6-10 carbon source; and obtaining PHA consisting of monomers with 6-10 carbon atoms. When the maoC gene whose function has not yet been established is used according to the present invention, high quality PHA with a higher number of carbon atoms than the prior PHA can be produced at a higher efficiency.

Description

technical field [0001] The present invention relates to a method for preparing polyhydroxyalkanoate (PHA described below) using maoC gene. More specifically, the present invention relates to a maoC gene, a protein having enoyl-CoA hydratase activity, and a method for producing PHA using a recombinant vector containing the maoC gene and a microorganism transformed with a recombinant vector containing a PHA synthesis gene method. Background technique [0002] PHA is a polyester-type compound that accumulates in microorganisms to serve as a carbon source when excess carbon exists in microorganisms and lacks other nutrients such as phosphorus, nitrogen, magnesium, and oxygen. Since PHA has similar physical properties to gasoline-derived synthetic polymers and is completely biodegradable, it is considered as a substitute for synthetic plastics that were previously used. [0003] Generally, PHAs are classified into short-chain PHAs containing several carbon atoms (SCL-PHA) and m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C08G63/06C12N1/15C12N1/19C12N1/21C12N9/00C12N9/88C12N15/11C12P7/62
CPCC12N9/93C12Y402/01017C12N9/88C08G63/06C12P7/625C12N15/113
Inventor 李相烨朴时载
Owner KOREA ADVANCED INST OF SCI & TECH
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