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Transformed filamentous bacteria with enhanced ergothioneine production capacity and preparation method for 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: 2021-09-07
KIKKOMAN CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 filamentous bacteria with enhanced ergothioneine production capacity and preparation method for ergothioneine
  • Transformed filamentous bacteria with enhanced ergothioneine production capacity and preparation method for ergothioneine
  • Transformed filamentous bacteria with enhanced ergothioneine production capacity and preparation method for ergothioneine

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preparation example Construction

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

[0152] The method for producing ergothioneine of the present invention includes at least a step of causing histidine and cysteine ​​to act on the transformed filamentous fungus of the present invention to obtain ergothioneine. As for the method for making histidine and cysteine ​​act on the transformed filamentous bacteria of the present invention, any enzyme possessed by the transformed filamentous bacteria can be used as long as histidine and cysteine ​​can be brought into contact with the transformed filamentous bacteria. The method for producing ergothioneine is not particularly limited. For example, by using a medium containing histidine and cysteine ​​and suitable for the growth of transformed filamentous bacteria, it can be used in various cultures suitable for transformed filamentous bacteria. A method for preparing ergothioneine by culturing transformed filamentous bacteria under conditions. ...

example 1

[0172][Example 1. Preparation of DNA construct inserting gene AsEgtA, AsEgtB or AsEgtC]

[0173] (1) Search for target genes

[0174] In 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 related to the biosynthesis of ergothioneine. Therefore, the genes encoding the above three enzymes of Neurospora crassa were used as query genes, based on Aspergillus sojae (Aspergillus sojae) The genome sequence of the NBRC4239 strain was searched for regions with relatively high sequence identity to the genes encoding NCU04343, NCU04636, and NCU11365, respectively. The genome sequence of Aspergillus sojae NBRC4239 strain was searched using the BLAST program (tblastn) (DDBJ / EMBL / GenBank DNA databases, Accession numbers for the 65 scaffold sequences; DF093557-DF093585, DNA RESEARCH 18, 165-176, 2011).

[0175] As a result, the g...

example 2

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

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

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

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

[0211] Conidia of the pyrG-disrupted strain derived from Aspergillus sojae NBRC4239 strain were inoculated into 100 ml of a polypeptone dextrin liquid medium containing 20 mM uridine in a 500-ml conical flask, and shaken cultured at 30°C for about 20 hours. Then, the bacteria were recovered. Protoplasts were prepared from the recovered bacterial cells. The obtained protoplasts and 20 μg of the ...

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Abstract

The object of the present invention is to provide organisms capable of producing ergothioneine which are simpler and less time-consuming than the prior art and which are capable of producing ergothioneine in high yields, thus enabling the production of ergothioneine on an industrial scale Thioneine. The above objects are solved by a transformed filamentous bacterium that has inserted a gene encoding the following enzyme (1) or genes encoding the following enzymes (1) and (2) and overexpressed the inserted gene. (1) In the presence of S-adenosylmethionine, iron (II) and oxygen, catalyze the formation of histidine trimethyl cysteine ​​sulfoxide from histidine and cysteine The enzyme of reaction; (2) with 5 ' - pyridoxal phosphate as coenzyme, catalyzes the enzyme of the reaction that generates ergothioneine by histidine trimethyl cysteine ​​sulfoxide.

Description

[0001] Cross-referencing 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 disclosed herein by reference in its entirety. technical field [0003] The present invention relates to fungi with approved ergothioneine production capacity and a method for preparing ergothioneine using the fungi. Background technique [0004] The following formula [II]: [0005] [0006] The indicated ergothioneine is a sulfur-containing amino acid, and is a biological substance confirmed to be present in organs such as the liver and blood of animals including humans. [0007] Ergothioneine is known to have antioxidant activity, and has been reported to have, for example, a scavenging action of hydroxyl radicals, an action of inhibiting the production of hydroxyl radicals derived from iron or copper-dependent hydrogen pero...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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