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Genetically modified microorganism for producing 3-hydroxyhexanedioic acid, (e)-hex-2-enedioic acid and/or hexanedioic acid, and production method for said chemicals

A technology of genetic modification and manufacturing method, applied in the field of genetically modified microorganisms, can solve problems such as no record of pyruvate kinase

Pending Publication Date: 2021-12-10
TORAY IND INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this document, although it is particularly disclosed that the activity of acyltransferase catalyzing the reaction of 3-oxoadipyl-CoA from acetyl-CoA and succinyl-CoA is enhanced to increase 3-hydroxyadipate and α-hydrogenated hexadienate production efficiencies, but nothing about pyruvate kinase

Method used

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  • Genetically modified microorganism for producing 3-hydroxyhexanedioic acid, (e)-hex-2-enedioic acid and/or hexanedioic acid, and production method for said chemicals
  • Genetically modified microorganism for producing 3-hydroxyhexanedioic acid, (e)-hex-2-enedioic acid and/or hexanedioic acid, and production method for said chemicals
  • Genetically modified microorganism for producing 3-hydroxyhexanedioic acid, (e)-hex-2-enedioic acid and/or hexanedioic acid, and production method for said chemicals

Examples

Experimental program
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preparation example Construction

[0099] Preparation of 3-oxididanoyl-COA solution: PCR was carried out in a genomic DNA of Pseudomonas PUTIDA KT2440 strain in accordance with a conventional method, and a nucleic acid (PCAI and PCAJ, NCBI-Gene ID: 1046613) And the full length of 1046612) is amplified. It should be noted that the base sequence of the primer used in this PCR is, for example, serial numbers 194 and 195. The amplification fragment was inserted into a KPNI site of PRSF-1B (manufactured by NOVAGEN company) as the expression vector of E. coli, which becomes the same frame as the histidine tag sequence. The plasmids were introduced into E. coli BL 21 (DE3), in accordance with conventional methods, induced expression of the enzyme using isopropyl-β-thiopylum galactoside (IPTG), followed by the histidine tag from the culture solution Reficient, resulting in a COA transferase solution. The following composition was prepared using this solution to prepare an enzyme reaction solution, and after 3 minutes at 25...

Embodiment

[0297] Hereinafter, the present invention will be specifically described.

reference example 1

[0299] Used to express the reaction of the reaction (reaction a) to form 3-oxidoidanoyl-CoA and coenzyme A, the reaction of 3-hydroxyhexa-CoA, resulting in 3-hydroxy-CoA-COA (reactive E ) And enzymes for catalyzing reactions (reactive f) from 2,3-dehydroxylic acid-CoA, and serial number 1, 2, 3, 4, 5, 6, 7 Made of the plasmid of the polypeptide

[0300] The carrier PBBR1MCS-2 (Me Kovach, (1995), Gene166: 175-176) (Me Kovach, (1995), Gene166: 175-176) were cut off using XHOI to obtain PBBR1MCS-2 / XHOI. In order to assemble the constitutive expression promoter in the carrier, the upstream region 200b (serial number 186) for GAPA (NCBI Gene ID: NC000913.3) is designed in the genomic DNA of Escherichia Coli K-12MG1655. PCR amplification primers (serial number 187, 188), followed by a conventional method. The resulting fragment was connected to the PBBR1 MCS-2 / XHOI using In-Fusion HD Cloning Kit ("" "" "" "" "" "" "" " The plasmid was extracted from the obtained recombinant coliform...

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Abstract

Disclosed is a genetically modified microorganism that produces 3-hydroxyhexanedioic acid, (E)-hex-2-enedioic acid or hexanedioic acid at a high yield. The genetically modified microorganism has been modified so as to introduce a nucleic acid coding for any of the polypeptides described in (a)-(c), below, or to enhance the expression of said peptides, and so as to disrupt the function of pyruvate kinase. (a) A polypeptide including any of the amino acid sequences represented by SEQ ID NO. 1-7. (b) A polypeptide including an amino acid sequence obtained by substituting, deleting inserting and / or adding one or more amino acids in any of said amino acid sequences, said polypeptide having an enzymatic activity that catalyzes a reaction for generating 3-hydroxyadipyl-CoA by reducing 3-oxoadipyl-CoA. (c) A polypeptide having a sequence homology of at least 70% with any of said amino acid sequences, said polypeptide having an enzymatic activity that catalyzes a reaction for generating 3-hydroxyadipyl-CoA by reducing 3-oxoadipyl-CoA.

Description

Technical field [0001] The present invention relates to an introduction of a nucleic acid encoding a polypeptide associated with the production of the object, or enhancing the expression of the polypeptide expression, and a substance manufacturing method using the microorganism. Background technique [0002] 3-hydroxyhexic acid (Iupac name: 3-hydroxyhexanedioic acid), α-hydrogenated hexadiene (Iupac name: (e) -Hex-2-Enedioc acid) and adipic acid (Iupac name: Hexanediic Acid) It is a dicarboxylic acid having a carbon atom having a number of 6. They can be used as a raw material which is polymerized with polyols, or polymerized with polyamine to become polyamide. Further, by adding an amidamination in their end to an amide, a raw material of a polyamide can be alone. [0003] It is known that the following documents are known to produce 3-hydroxyhexic acid or α-hydrogenated hexadiene diacid using microorganisms. [0004] Patent Document 1 describes a method of producing 1,3-butadie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12P7/44C12N1/21C12N15/53C12N15/54C12N9/04
CPCC12P7/44C12P7/6436C12R2001/425C12P19/32C12Y101/01157C12N1/20C12N9/1205C12R2001/19C12N15/70C12P7/42
Inventor 矶部匡平河村健司山田胜成
Owner TORAY IND INC