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Mutant polyhydroxyalkanoic acid synthase, gene and transformant thereof, and method for producing polyhydroxyalkanoic acid

A technology of polyhydroxyalkanoate and enzyme synthesis, applied in the direction of biochemical equipment and methods, enzymes, bacteria, etc., can solve the problem of low melt processability

Pending Publication Date: 2021-02-12
KANEKA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] As PHA, polyhydroxybutyrate (Poly-3-hydroxybutyrate; hereinafter abbreviated as "PHB") which is a homopolymer of 3-hydroxybutyrate (3-hydroxybutyrate; hereinafter abbreviated as "3HB") is known, but PHB High crystallinity, hard and brittle due to high crystallinity, and low melt processability

Method used

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  • Mutant polyhydroxyalkanoic acid synthase, gene and transformant thereof, and method for producing polyhydroxyalkanoic acid
  • Mutant polyhydroxyalkanoic acid synthase, gene and transformant thereof, and method for producing polyhydroxyalkanoic acid

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Embodiment

[0097] Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

[0098] The genetic manipulation described below can be carried out with reference to the description of Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). In addition, enzymes, cloning hosts, and the like used in genetic manipulation can be purchased from commercial suppliers and used according to their instructions. In addition, as said enzyme, if it can be used for gene manipulation, it will not specifically limit.

manufacture example 1

[0099] (Manufacturing Example 1) Preparation of H16ΔphaC1 Ptrc-phaJ4b dZ1,2,6 strains

[0100] First, a plasmid for disrupting the phaC1 gene was prepared. A DNA fragment (SEQ ID NO: 12) having the nucleotide sequences upstream and downstream of the phaC1 gene was obtained by PCR using synthetic oligo DNA. The resulting DNA fragment was digested with restriction enzyme SwaI. This DNA fragment was ligated with the vector pNS2X-sacB described in JP 2007-259708 A, which was also digested with SwaI, to prepare a plasmid vector pNS2X-sacB-phaC1UL for gene disruption having the nucleotide sequences upstream and downstream of phaC1. .

[0101] Next, ΔphaC1 Ptrc-phaJ4b dZ1,2,6 strains were prepared using pNS2X-sacB-phaC1UL. pNS2X-sacB-phaC1UL was introduced into Escherichia coli S17-1 strain (ATCC47055). The obtained transformants and KNK005 trc-phaJ4bΔphaZ1, 2, and 6 strains (see International Publication No. 2015 / 115619) were mixed on Nutrient Agar medium (manufactured by Difco)...

manufacture example 2

[0104] (Production Example 2) Preparation of pCUP2-Ptrp-NSDG

[0105] A DNA fragment having the base sequence shown in SEQ ID NO: 13 was amplified by PCR using synthetic oligo DNA or the like. The obtained DNA fragment was ligated with a DNA fragment obtained by digesting the pCUP2 vector described in JP-A-2007-259708 with MunI and SpeI using the In-fusion HD Cloning Kit (TAKARA BIO), to obtain pCUP2-Ptrp -NSDG. pCUP2-Ptrp-NSDG is a plasmid expressing NSDG under the trp promoter.

[0106] NSDG is a mutant PHA synthetase consisting of the amino acid sequence represented by SEQ ID NO: 14, in which asparagine at the 149th position from the N-terminus is replaced with serine and asparagine at the 171st position is introduced into the amino acid sequence represented by SEQ ID NO: 1 A mutant PHA synthetase obtained by replacing amino acid with glycine.

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Abstract

A mutant polyhydroxyalkanoic acid synthase having an amino acid sequence that shows 85% or higher sequence identity with an amino acid sequence represented by SEQ ID NO: 1 and is such that at least one amino acid from among amino acids 27-33, 39, 56, 106, 129, 144, 165, 170, and 172-187 from the N-end of the amino acid sequence represented by SEQ ID NO: 1 is substituted by another amino acid.

Description

technical field [0001] The present invention relates to a mutant polyhydroxyalkanoate synthetase, a gene encoding the enzyme, a transformant having the gene, and a method for producing polyhydroxyalkanoate using the transformant. Background technique [0002] Polyhydroxyalkanoate (Polyhydroxyalkanoate; hereinafter referred to as "PHA") is a thermoplastic polyester produced and accumulated as an energy storage substance in many microbial cells. PHA produced by microorganisms using various natural carbon sources is an environmentally friendly plastic that can be completely biodegraded by microorganisms in soil and water. [0003] As PHA, polyhydroxybutyrate (Poly-3-hydroxybutyrate; hereinafter abbreviated as "PHB") which is a homopolymer of 3-hydroxybutyrate (3-hydroxybutyrate; hereinafter abbreviated as "3HB") is known, but PHB It is highly crystalline, and since the degree of crystallinity is high, it is hard and brittle, and has a problem of low melt processability. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N1/21C12N9/00C12P7/62
CPCC12N15/52C12N9/1029C12P7/625C12P7/62C12N1/20
Inventor 小林新吾佐藤俊辅田冈直明
Owner KANEKA CORP
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