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Transformed fungus having enhanced ergothioneine productivity and method for producing ergothioneine

A technology of ergothioneine and filamentous bacteria, applied in the direction of biochemical equipment and methods, methods based on microorganisms, transferase, etc., can solve the problems of no ergothioneine production, no ergothioneine production capacity, etc., and achieve simple production Effect

Active Publication Date: 2017-10-13
KIKKOMAN CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, in the following non-patent documents 1 and 2 (the entire contents of which are disclosed here by reference), it is described that most bacteria do not have ergothioneine production ability, and it is also described that among fungi, although Aspergillus niger (Aspergillus niger , Aspergillus niger) or Neurospora crassa (Pink bread mold) also have the ability to produce ergothioneine, but Saccharomyces cerevisiae ( Saccharomyces cerevisiae , baker's yeast) have little ability to produce ergothioneine

Method used

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  • Transformed fungus having enhanced ergothioneine productivity and method for producing ergothioneine
  • Transformed fungus having enhanced ergothioneine productivity and method for producing ergothioneine
  • Transformed fungus having enhanced ergothioneine productivity and method for producing ergothioneine

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preparation example Construction

[0151] (the preparation method of ergothioneine of the present invention)

[0152] The preparation method of ergothioneine of the present invention at least comprises: the step of making histidine and cysteine ​​act on the transformed filamentous bacteria of the present invention to obtain ergothioneine. Regarding the method of allowing histidine and cysteine ​​to act on the transformed filamentous bacterium of the present invention, as long as the histidine and cysteine ​​can be brought into contact with the transformed filamentous bacterium, the enzyme possessed by the transformed filamentous bacterium can be utilized. The method for producing ergothioneine is not particularly limited, for example, it can be enumerated by utilizing a medium that contains histidine and cysteine ​​and is suitable for the growth of transformed filamentous bacteria, in various cultures suitable for transformed filamentous bacteria A method for preparing ergothioneine by cultivating and transform...

example 1

[0172][Example 1. Production of DNA constructs inserting genes AsEgtA, AsEgtB or AsEgtC]

[0173] (1) Search for target genes

[0174] In Neurospora crassa (Neurospora crassa), NCU04343 and NCU1136 are known as enzymes involved in ergothioneine biosynthesis (see Non-Patent Documents 3 and 4). In addition, Non-Patent Document 3 suggests the possibility that NCU04636 is involved in the biosynthesis of ergothioneine. Therefore, the genes encoding the above three enzymes of Neurospora crassa were used as query genes, and based on Aspergillus sojae (Aspergillus sojae) For the genome sequence of the NBRC4239 strain, regions with relatively high sequence identities to the genes encoding NCU04343, NCU04636, and NCU11365 were searched. The genome sequence of Aspergillus sojae NBRC4239 strain was searched using BLAST program (tblastn) (DDBJ / EMBL / GenBank DNA databases, Accession numbers for the 65 scaffold sequences; DF093557-DF093585, DNA RESEARCH18, 165-176, 2011).

[0175] As a re...

example 2

[0207] [Example 2. Production of transformed Aspergillus sojae (1)]

[0208] (1) pyrG disrupted strain from Aspergillus sojae NBRC4239 strain

[0209] After each DNA construct was precipitated with ethanol, it was dissolved in TE, and the DNA solution prepared into the required concentration was used in the following order for the pyrG disrupted strain from Aspergillus sojae NBRC4239 strain (upstream 48bp of pyrG gene, coding region 896bp, downstream 240bp ) conversion.

[0210] (2) Transformation of pyrG disruption strain derived from Aspergillus sojae NBRC4239 strain

[0211] Inoculate conidia derived from the pyrG disrupted strain of Aspergillus sojae NBRC4239 strain in 100 ml of polypeptone dextrin liquid medium containing 20 mM uridine in a 500 ml Erlenmeyer flask, and perform shaking culture at 30°C for about 20 hours Thereafter, the bacterial cells were recovered. Protoplasts were prepared from the recovered bacterial cells. Utilize the obtained protoplast and 20 μ ...

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Abstract

The purpose of the present invention is to provide an organism having an ergothioneine productivity that is capable of easily producing ergothioneine within a short period of time at a high yield, as compared with a conventional technology, and, therefore, enables ergothioneine production on an industrial scale. This purpose can be achieved by a transformed fungus into which a gene encoding enzyme (1) or genes encoding enzymes (1) and (2) have been inserted and in which the inserted gene(s) are overexpressed. (1) An enzyme catalyzing a reaction of synthesizing hercynyl cysteine sulfoxide from histidine and cysteine in the presence of S-adenosyl methionine, iron (II) and oxygen. (2) An enzyme catalyzing a reaction of synthesizing ergothioneine from hercynyl cysteine sulfoxide using pyridoxal 5'-phosphate as a coenzyme.

Description

[0001] Cross-reference of related applications [0002] This application claims the priority of Japanese Patent Application No. 2015-17328 filed on January 30, 2015 and Japanese Patent Application No. 2015-157444 filed on August 7, 2015 , and is incorporated herein by reference in its entirety. technical field [0003] The present invention relates to a fungus with recognized ergothioneine production capacity and a method for preparing ergothioneine using the fungus. Background technique [0004] The following formula [II]: [0005] [0006] The indicated ergothioneine is a sulfur-containing amino acid and is a biological substance that has been confirmed to exist in organs such as the liver or blood of animals including humans. [0007] Ergothioneine is known to have antioxidant activity, and is reported to have, for example, a hydroxyl radical trapping action, a hydroxyl radical generation inhibitory action from iron or copper-dependent hydrogen peroxide, a copper-dep...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N9/10C12N15/09C12P13/04C12R1/66C12R1/665C12R1/685C12R1/69
CPCC12N9/10C12N15/09C12P13/04C12N9/1007C12N9/13C12Y201/01C12Y208/01007C12R2001/665C12N1/145C12R2001/685C12R2001/69C12R2001/66
Inventor 原精一黑泽惠子市川惠一
Owner KIKKOMAN CORP
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