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Method for production of polyester copolymer using genetically modified microorganism

A technology of a recombinant microorganism and a manufacturing method, applied in the field of manufacturing copolyester, can solve the problems of unfavorable raw material cost, low yield of productive carbon source and the like

Inactive Publication Date: 2011-05-18
TOYOTA JIDOSHA KK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, the method of synthesizing polyhydroxyalkanoate by adding monomer components such as lactic acid and / or decenoic acid to the medium is disadvantageous in terms of raw material cost
In addition, because the polymer productivity and carbon source yield of copolyester produced by microorganisms are generally low, even in the case of using cheap natural products as carbon sources, improving productivity and yield is very important for reducing production costs. is also an important subject

Method used

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  • Method for production of polyester copolymer using genetically modified microorganism
  • Method for production of polyester copolymer using genetically modified microorganism
  • Method for production of polyester copolymer using genetically modified microorganism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0193] use LA high accumulation Escherichia coli to produce copolyester

[0194] (1) Production of recombinant microorganisms

[0195] Genomic DNA was extracted from M. elsdenii (ATCC17753) using the DNeasy Tissue Kit (Qiagen), and a nucleic acid encoding propionyl-CoA transferase (Accession No. J04987) was synthesized by PCR to include an EcoRI recognition sequence. The primer DNA of the forward primer and the reverse primer containing the PstI recognition sequence. Using the above-mentioned genomic DNA as a template, use iCycler (BioRad), in the environment containing KOD-Plus-DNA polymerase (1U), PCR buffer, 1mM MgSO 4 , 15 pmol of each primer, and 0.2 mM dNTPs (all manufactured by Toyobo Co., Ltd.), one cycle was performed at 94°C for 2 minutes, followed by 1 cycle at 94°C for 15 seconds, 55°C for 30 seconds, and 68°C for 2 minutes. One cycle of PCR reaction was carried out for 30 cycles, and then the amplified fragment of about 1,500 bp was recovered and digested with ...

Embodiment 2

[0242] Production using highly LA-accumulating microorganisms

[0243] (1) Production of polymers

[0244] The pTV118NPCTC1(ST / QK)AB1 prepared in (1) of Example 1 was used to transform Escherichia coli Jw2293 strain, Jw0885 strain, and Jw0886 strain with high accumulation of lactic acid.

[0245] The obtained transformant was inoculated into 1000 ml of LB medium containing 100 μg / ml ampicillin, 2% glucose, and 10 mM pantothenic acid, and cultured at 37° C. for 72 hours. After culturing, the cells were collected by centrifugation at 4°C, 3,100 rpm, and 15 minutes, suspended in 10 mM Tris-hydrochloric acid buffer (pH 7.5), centrifuged again under the same conditions, and then freeze-dried for 2 sky.

[0246] Move the dried bacteria to a glass pressure-resistant reaction tube, add 40ml of chloroform to make a suspension, then keep it in a micro-thermostat at 100°C for 3 hours, then cool it to room temperature, and filter it with a 0.2μm PTFE filter (ADVANTEC) , to separate the ...

Embodiment 3

[0280] Manufacture of copolyester by culturing under anaerobic conditions

[0281] (1) Construction of the carrier

[0282] Using the pTV118NPCTC1(ST / QK)AB produced in (1) of Example 1 as a template, PCR was performed to obtain a linear plasmid in which the gene encoding βKT and the gene encoding AACoA-R were removed, so that the obtained amplified fragment Circularization, thereby making the plasmid pTV118NpctC1(ST / QK) having the gene encoding Pct and the gene encoding PhaCm in pTV118N ( Figure 16 Left).

[0283] The pGEMC1AB prepared in (1) of Example 1 was digested with the restriction enzyme BamHI, and the obtained DNA fragment of about 3,200 base pairs was digested with the linear chain obtained by digesting the pACYC177 DNA, which is a low-copy plasmid, with the restriction enzyme BamHI. Like vector plasmid connection, thereby obtains the plasmid pACYC177AB that the gene encoding βKT and the gene encoding AACoA-R have been introduced into the BamHI site of pACYC177DN...

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Abstract

Disclosed is a method for producing a polyester copolymer comprising 3HB and LA with high efficiency by the microbial fermentation using a sugar as a raw material. Specifically disclosed is a method for producing a polyester copolymer comprising hydroxybutyric acid and lactic acid, which comprises the following steps 1) and 2): 1) culturing a genetically modified microorganism carrying a protein capable of catalyzing the reaction for transferring CoA into propionic acid and / or lactic acid, a protein capable of catalyzing the reaction for producing acetoacetyl-CoA from two acetyl-CoA molecules, a protein capable of catalyzing the reduction reaction of acetoacetyl-CoA, and a protein capable of catalyzing the synthetic reaction of a polyhydroxyalkanoic acid and comprising an amino acid sequence selected from the following amino acid sequences a) and b) in a culture medium containing a carbon source: a) an amino acid sequence having the substitution of at least one of the amino acid residues located at position-130, position-325, position-477 and position-481 by another amino acid residue in the amino acid sequence depicted in SEQ ID NO:2; and b) an amino acid sequence additionally having the deletion or substitution of at least one amino acid residue other than the amino acid residues located at position-130, position-325, position-477 and position-481 or the insertion of at least one amino acid residue in the protein defined in item a); and 2) collecting the polyester copolymer from the culture obtained in step 1). The method enables the efficient production of polyester copolymer composed of 3HB and LA by using an inexpensive carbon source as a raw material, and can achieve the reduction in the cost for production of a biodegradable plastic.

Description

technical field [0001] The present invention relates to methods for producing copolyesters using recombinant microorganisms. Background technique [0002] So far, a large number of microorganisms having the ability to produce polyester using sugar as a carbon source have been reported (Non-Patent Document 1). Polyesters produced by microorganisms are attracting attention as biodegradable plastics that are easily broken down in nature, and "green" plastics that can be synthesized from renewable carbon resources such as sugar and / or vegetable oil. [0003] A representative example of biodegradable plastics produced by microorganisms is polyhydroxybutyrate (polyhydroxybutylate, PHB) in which 3-hydroxybutyrate (3HB) is used as a monomer. PHB is a thermoplastic polymer having a melting temperature of about 180° C., and has an advantage of being excellent in melt processability in addition to biodegradability. On the other hand, PHB has a physical problem of being hard and britt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/21C12P7/62
CPCC12P7/62C12N15/52
Inventor 田口精一田岛健次佐藤康治松本谦一郎山田美和小畑充生神户浩美幸田胜典大野克博嶋村隆
Owner TOYOTA JIDOSHA KK
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