JP2018130091A - Plant, food, cultured product, fertilizer, and production method - Google Patents

Abstract

An object of the present invention is to provide a plant, food, culture, fertilizer, and method for producing the plant containing ergothioneine. A plant containing 0.5 μg or more of ergothioneine per 100 g of fresh weight. Foods containing 0.5 μg or more of ergothioneine per 100 g (excluding foods containing microorganisms capable of biosynthesis of ergothioneine). A culture of a microorganism capable of biosynthesizing ergothioneine and containing 50 mg / L or more of ergothioneine. A fertilizer containing a culture of microorganisms capable of biosynthesis of ergothioneine. A method for producing the plant body, comprising a cultivation step of cultivating the plant body with a cultivation medium containing ergothioneine, wherein the cultivation medium contains 1 μg / L or more of ergothioneine. [Selection figure] None

Description

  The present invention relates to a plant, a food, a culture, a fertilizer, and a method for producing the plant containing ergothioneine.

Sulfur is an essential component as an element constituting the human body. Human sulfur sources are organic sulfur compounds that are ingested through meals, and microorganisms are responsible for assimilation of inorganic sulfur into organic sulfur compounds in the biological sulfur cycle on earth.
Ergothioneine is a kind of sulfur-containing amino acid and is known to be biosynthesized only by some microorganisms. Ergothioneine is excellent in antioxidant action, and its action is said to be 6000 times that of vitamin E. Therefore, it is a highly useful compound in the fields of health and beauty.
For example, Patent Document 1 describes a skin external preparation containing an extract containing ergothioneine derived from mushrooms and / or a derivative thereof, and this skin external preparation is excellent in terms of preventing skin aging and the like. It is said.

JP 2012-211120 A

  However, commercially available ergothioneine is expensive and hinders the spread of products containing ergothioneine.

  In view of the above background, an object of the present invention is to provide a plant, a culture, a fertilizer, and a method for producing the plant containing ergothioneine.

The plant, food, culture, fertilizer, and method for producing the plant according to the present invention have the following aspects.
[1] A plant containing 0.5 μg or more of ergothioneine per 100 g of fresh weight.
[2] The plant according to [1], which contains 20 μg or more of ergothioneine per 100 g of fresh weight.
[3] The plant according to [1] or [2], wherein the content of the ergothioneine is in leaves.
[4] The plant according to any one of [1] to [3], which belongs to the Brassicaceae family.
[5] A food containing 0.5 μg or more of ergothioneine per 100 g (however, a food containing a microorganism capable of biosynthesizing ergothioneine, a food containing a mushroom capable of biosynthesizing ergothioneine, and a microorganism fermented by a microorganism capable of biosynthesis of ergothioneine Excluding fermented foods and their processed foods).
[6] The food according to [5] above, containing 20 μg or more of ergothioneine per 100 g.
[7] A culture of a microorganism capable of biosynthesizing ergothioneine and containing 50 mg / L or more of ergothioneine.
[8] The culture according to [7], wherein the microorganism capable of biosynthesizing ergothioneine is a microorganism modified to increase cysteine-producing ability.
[9] A fertilizer containing a culture of microorganisms capable of biosynthesis of ergothioneine.
[10] The fertilizer according to [9], wherein the microorganism capable of biosynthesizing ergothioneine is a microorganism modified so as to increase cysteine-producing ability.
[11] The fertilizer according to [9] or [10], wherein the water content is 30% by mass or less.
[12] The method for producing a plant according to any one of [1] to [4],
In the cultivation medium containing ergothioneine, it has a cultivation process to cultivate the plant body,
The said culture medium is a manufacturing method containing 1 microgram / L or more of ergothioneine.
[13] The production method according to [12], wherein the cultivation medium contains 0.1 mg / L or more of ergothioneine.
[14] The production method according to [12] or [13], wherein the cultivation medium includes a culture of a microorganism capable of biosynthesis of ergothioneine.
[15] The production method according to any one of [12] to [14], wherein the microorganism capable of biosynthesizing ergothioneine is a microorganism modified to increase cysteine-producing ability.
[16] A harvesting process for harvesting the plant body,
The production method according to any one of [12] to [15], wherein the cultivation step is performed within 10 days before the harvesting step.

  ADVANTAGE OF THE INVENTION According to this invention, the plant body containing a ergothioneine, a foodstuff, a culture, fertilizer, and the manufacturing method of this plant body can be provided.

In an Example, it is a calibration curve used for calculation of the amount of ergothioneine. It is the image which image | photographed the plant body grown in Examples 5-8 and Comparative Examples 5-8. It is a schematic diagram which shows the structure of the pDES plasmid used in the Example.

  Hereinafter, embodiments of the plant, food, culture, fertilizer and production method of the present invention will be described.

≪Plant body≫
The plant body of the embodiment contains 0.5 μg or more of ergothioneine per 100 g of fresh weight.
Originally, plants did not produce ergothioneine themselves, so the plant body did not contain a large amount of ergothioneine. As shown in Examples described later, the inventors can accumulate ergothioneine in a plant body by cultivating the plant body in a culture medium containing ergothioneine, and 0.5 μg or more per 100 g of fresh weight. It was found that a plant containing ergothioneine can be produced.

  The plant body of the embodiment contains 0.5 μg or more of ergothioneine per 100 g of fresh weight, may contain 1 μg or more of ergothioneine, may contain 5 μg or more of ergothioneine, and may contain 10 μg or more of ergothioneine. May contain 20 μg or more of ergothioneine, may contain 50 μg or more of ergothioneine, may contain 100 μg or more of ergothioneine, and may contain 150 μg or more of ergothioneine. 200 μg or more of ergothioneine may be contained, or 250 μg or more of ergothioneine may be contained.

  Although the upper limit of the ergothioneine contained in the plant body which concerns on embodiment is not restrict | limited in particular, As an example, it may be 10,000 micrograms or less per 100g fresh weight, and may be 1000 micrograms or less.

  “Fresh weight” refers to the weight of a plant that has not been artificially dried. The state includes a state in which the plant is sold in the market as a fresh item or a fresh food.

  Ergothioneine may be naturally occurring, or may be artificially or artificially produced.

  Ergothioneine is a kind of sulfur-containing amino acid, and L-(+)-ergothioneine is known as a compound represented by the following formula (1).

  The measuring method of ergothioneine can be implemented by the method described in the examples described later.

  Ergothioneine has an antioxidant action and can take an oxidized form such as being oxidized to form a disulfide. In the present specification, the target to be measured as ergothioneine contained in the plant body may be measured by including oxidized ergothioneine in addition to the above ergothioneine, and only ergothioneine not containing oxidized ergothioneine. May have been measured.

Ergothionein may be in the form of an ion or a salt, but in the present invention, the mass of ergothioneine is calculated as the molecular weight of 229.3.
The target to be measured as ergothioneine may be one that is measured by including oxidized ergothioneine in addition to the above-mentioned ergothioneine. In the present invention, the mass of ergothioneine is the molecular weight of ergothioneine before oxidation is 229.3. Suppose that it was calculated based on this.
When the oxidized ergothioneine forms a multimer, it is calculated based on the molecular weight 229.3 of the original monomer ergothioneine.

  Only 1 type may be sufficient as the ergothioneine contained in the plant body of embodiment, and 2 or more types may be sufficient as it.

  Although the kind of the plant body of embodiment is not specifically limited, Edible plants are preferable, for example, Brassicaceae, Asteraceae, Solanumaceae (Solanaceae), Legumeaceae (Fabaceae), Uri Plants such as Cucurbitaceae, Poaceae, Rubiaceae, Theaceae, Amaryllidaceae, Apiaceae, Araceae and the like are included. Among these, the plant body of the embodiment is preferably a cruciferous, asteraceae or solanaceous plant, and more preferably a cruciferous plant.

  The Brassicaceae plant is preferably a Brassica plant, more preferably Brassica rapa. It is preferable that the plant body of the embodiment is a cruciferous plant because ergothioneine is easily accumulated in the plant with high efficiency. This is probably because the plant has an ergothioneine transporter for taking in ergothioneine from the outside world, and functions effectively in the Brassicaceae family.

  From the above viewpoint, the plant body of the embodiment is preferably a plant body expressing an ergothioneine transporter.

  Examples of cruciferous plants include Mizuna (Brassica rapa var. Laciniifolia), Nozawana (Brassica rapa var. Hakabura), Komatsuna (Brassica rapa var. Perviridis), Chinese cabbage (Brassica rapa var. Pekinensis), Turnip (Brassica rapa var). glabra (Brassica rapa var. rapa), cabbage (Brassica oleracea var. capitata), medicinal cabbage (Brassica oleracea var. gemmifera), cauliflower (Brassica oleracea var. botrytis), broccoli (B. oleracea var. italica), radish sativus var. longipinnatus).

  Examples of the Asteraceae plants include lettuce (Lactuca sativa), sardana (Lactuca sativa var. Capiata), and garlic (Glebionis coronaria).

The solanaceous plant is preferably a plant of the genus Solanum.
Examples of solanaceous plants include tomato (Solanum lycopersicum), eggplant (Solanum melongena), potato (Solanum tuberosum), and bell pepper (Capsicum annuum).

  It is assumed that the mushrooms are not included in the plant body of the embodiment.

  The plant body of the embodiment may be the whole plant individual or a part of the plant individual. Examples of the part include organs or tissues such as leaves, roots, stems, flowers, fruits, seeds, pollen, and combinations thereof, and among these, leaves are preferable.

  In the plant body of the embodiment, the content of the ergothionein may be in the leaf of the plant body.

  Ergothioneine is excellent in antioxidant action. Since the plant body of the embodiment contains 0.5 μg or more of ergothioneine per 100 g of fresh weight, the plant body is excellent in antioxidant action and has a high utility value.

≪Food≫
The food of the embodiment contains 0.5 μg or more of ergothioneine per 100 g (however, food containing microorganisms capable of biosynthesis of ergothioneine, food containing mushrooms capable of biosynthesis of ergothioneine, and fermentation using microorganisms capable of biosynthesis of ergothioneine. Fermented foods and processed foods are excluded.)

  The food of the embodiment contains 0.5 μg or more of ergothioneine per 100 g, may contain 1 μg or more of ergothioneine, may contain 5 μg or more of ergothioneine, and contains 10 μg or more of ergothioneine. Or 20 μg or more of ergothioneine, 50 μg or more of ergothioneine, 100 μg or more of ergothioneine, 150 μg or more of ergothioneine, or 200 μg or more. Of ergothioneine or 250 μg or more of ergothioneine.

  Although the upper limit of the ergothioneine contained in the foodstuff which concerns on embodiment is not restrict | limited in particular, As an example, it may be 10,000 micrograms or less per 100 g, and may be 1000 micrograms or less.

  It is assumed that the weight of the food is measured in a state of being sold in the market.

  Examples of ergothioneine contained in the food of the embodiment include those exemplified in the above-mentioned << plant body >>, and detailed description thereof is omitted.

  The measuring method of ergothioneine can be implemented by the method described in the examples described later.

  In the present specification, the object to be measured as ergothioneine contained in food may be one that is measured by including oxidized ergothioneine in addition to the above ergothioneine, and only ergothioneine that does not contain oxidized ergothioneine is included. It may be measured.

In the definition of the food of the embodiment, what is excluded from this is a food in which a microorganism that can originally produce ergothioneine is used as a raw material.
Examples of foods containing microorganisms capable of biosynthesis of ergothioneine include, for example, laver containing bacteria such as cyanobacteria capable of biosynthesis of ergothioneine, and mushrooms formed by fungi capable of biosynthesis of ergothioneine. .
Examples of mushrooms that can biosynthesize ergothioneine include shiitake mushroom, oyster mushroom, and eringi.
Examples of fermented food items fermented by microorganisms capable of biosynthesizing ergothioneine include pickles, miso, bread, yogurt, cheese, and the like.

  The food of the embodiment is a concept including an edible plant among the plants of the embodiment of the present invention. In addition, the processed product of the plant body of the embodiment (for example, an extract, crushed product, paste, cooked product, prepared food, canned food, dried food, frozen food, beverage, alcoholic beverage, confectionery obtained from the plant body of the embodiment , Seasonings, fats and oils, jams, spices, food additives, health foods, supplements, etc.) are also included in the food of the embodiment.

  Furthermore, meat, fishery products, eggs, milk, fats and processed products of animals bred by feeding the plant body of the embodiment of the present invention as feed are also included as foods of the embodiment. Examples of meat include beef, pork and chicken.

  Ergothioneine is excellent in antioxidant action. Since the foodstuff of embodiment contains 0.5 microgram or more of ergothioneine per 100g, it is excellent in antioxidant action and is a foodstuff with high utility value.

≪Culture and fertilizer≫
The culture of the embodiment is a culture of a microorganism capable of biosynthesis of ergothioneine, and contains 50 mg / L or more of ergothioneine.

  The culture according to the embodiment contains 50 mg / L or more of ergothioneine, 100 mg / L or more of ergothioneine, 200 mg / L or more of ergothioneine, and 300 mg / L or more of ergothioneine. The ergothioneine may be contained at 400 mg / L or more, the ergothioneine may be contained at 500 mg / L or more, and the ergothioneine may be contained at 600 mg / L or more.

  The upper limit of ergothioneine contained in the culture according to the embodiment is not particularly limited, but may be 10,000 mg / L as an example.

  The microorganism that can biosynthesize ergothioneine may be a microorganism that originally has the ability to biosynthesize ergothioneine. Examples of such microorganisms include specific bacteria that can biosynthesize ergothioneine such as cyanobacteria and mycobacteria.

  The microorganism capable of biosynthesizing ergothioneine may be a microorganism that has been artificially modified so as to improve the production of ergothioneine. Originally, ergothioneine cannot be biosynthesized, but ergothioneine can be biosynthesized. It may be an artificially modified microorganism. Details of such microorganisms will be described later.

  The culture of the embodiment is obtained by culturing a microorganism capable of biosynthesis of ergothioneine in a medium, and the culture contains 50 mg / L or more of ergothioneine. Regarding the method for obtaining the culture, the following <Method for producing ergothioneine> is also referred to.

The form of the culture is not particularly limited, and may be solid, liquid, or a mixture of both, for example, liquid, powder, granule, tablet, tablet, capsule It may be in the form of an agent or the like.
In addition, mushrooms shall be remove | excluded from the culture of embodiment.
The culture includes a processed product obtained by processing a culture medium after culturing. The medium may be a liquid medium. Here, the processed product includes a product obtained by volatilizing a volatile component contained in a medium and concentrating ergothioneine, a product obtained by extracting ergothioneine and purifying ergothioneine, and the like.

  The fertilizer of the embodiment includes a culture of a microorganism capable of biosynthesis of ergothioneine. That is, the culture can be used as a fertilizer containing ergothioneine, and a plant containing ergothioneine can be cultivated by applying it to a plant.

  The form of the fertilizer is not particularly limited, and may be solid, liquid, or a mixture of both. For example, liquid, powder, granule, tablet, tablet, capsule Or the like.

The moisture content of the fertilizer of the embodiment is preferably small from the viewpoint of transportability, for example, the moisture content may be 30% by mass or less, 25% by mass or less, or 20% by mass or less. It may be 15% by mass or less.
If it is a fertilizer that contains a culture of microorganisms and the water content is in the above range, it usually has a form such as a granule or a powder, and is excellent in transportability as a fertilizer and also has good usability as a fertilizer It is.

The fertilizer of the embodiment may be a microorganism culture itself capable of biosynthesizing ergothioneine, or may be one obtained by adding a diluent such as water or an additive such as nutrients to the culture of the embodiment.
The fertilizer of the embodiment can be manufactured using the culture of the embodiment as a raw material. For example, after the culture of the embodiment is sterilized and crushed, and then extracted with a solvent such as methanol and the solvent is removed, a fertilizer containing ergothioneine at a high concentration and satisfying the above water content can be produced. .

  The content of ergothioneine in the fertilizer is not particularly limited, but may be 0.01 mg to 250 g / L, may be 0.1 mg to 10000 mg / L, and may be 0.5 to 1000 mg / L. L may be sufficient.

≪Plant manufacturing method≫
The manufacturing method of embodiment is a manufacturing method of the plant body of embodiment, Comprising: It has the cultivation process which grows a plant body with the culture medium containing ergothioneine, The said culture medium contains 1 microgram / L or more of ergothioneine. .

  As shown in Examples described later, the inventors can accumulate ergothioneine in a plant body by cultivating the plant body in a culture medium containing ergothioneine, and 0.5 μg or more per 100 g of fresh weight. It was found that the plant body of the embodiment containing the ergothioneine of the present invention can be produced. That is, it discovered that the ergothioneine contained in a cultivation medium is recoverable using a plant body by growing a plant body in the cultivation medium containing an ergothioneine.

The culture medium according to the embodiment contains 1 μg / L or more of ergothioneine, may contain 0.01 mg / L or more of ergothioneine, may contain 0.1 mg / L or more of ergothioneine, 1 mg / L or more may be contained, ergothioneine may be contained 10 mg / L or more, ergothioneine may be contained 50 mg / L or more, ergothioneine may be contained 100 mg / L or more, and ergothioneine 250 mg / L. L or more may be contained, ergothioneine may be contained 500 mg / L or more, and ergothioneine may be contained 600 mg / L or more.
By using a culture medium containing ergothioneine at the lower limit or higher, ergothioneine is likely to be efficiently accumulated in the plant body.

The culture medium according to the embodiment may contain ergothioneine 10000 mg / L or less, ergothioneine 1000 mg / L or less, ergothioneine 800 mg / L or less, and ergothioneine 300 mg / L or less. Ergothioneine may be contained at 100 mg / L or less, ergothioneine may be contained at 10 mg / L or less, and ergothioneine may be contained at 1 mg / L or less.
Even in a culture medium containing ergothioneine below the above upper limit, the plant can accumulate ergothioneine, so that ergothioneine can be recovered or concentrated using a plant even from a culture medium containing a low concentration of ergothioneine. is there.

  The culture medium according to the embodiment may contain ergothioneine in an amount of 1 μg / L to 10000 mg / L, may contain 0.01 mg / L to 1000 mg / L, and may contain 0.1 mg / L to 800 mg / L. It may contain 1 mg / L or more and 300 mg / L or less.

  The measuring method of ergothioneine can be implemented by the method described in the examples described later.

  In this specification, it is assumed that the target to be measured as ergothioneine contained in the culture medium is that only ergothioneine not containing oxidized ergothioneine is measured.

“Grow plant with cultivation medium containing ergothioneine” means cultivating the plant in contact with the cultivation medium containing ergothioneine and the cultivation medium and plant containing ergothioneine It is preferable to cultivate the plant body in a state where the body roots are in contact. The whole plant root may be in contact with the cultivation medium, or only a part may be in contact with the cultivation medium.

  Examples of ergothioneine used in the production method of the embodiment include those exemplified in the above << plant body >>, and detailed description thereof is omitted.

  The ergothioneine used in the production method of the embodiment may be in the form of ions or in the form of a salt as long as it can be taken up by plants.

  Ergothionein used in the cultivation method of the embodiment may be naturally occurring, or may be artificially or artificially produced.

  Examples of naturally occurring ergothioneine include those produced by microorganisms capable of biosynthesis of ergothioneine. Examples of the microorganism include specific bacteria such as cyanobacteria and mycobacteria, and fungi including specific mushrooms such as shiitake mushroom, oyster mushroom, and eringi. Ergothionein recovered from a microorganism capable of biosynthesis of ergothioneine or purified ergothioneine may be used.

Examples of ergothioneine produced artificially or artificially include ergothioneine produced by artificially culturing and producing a microorganism capable of biosynthesis of ergothioneine.
In addition, ergothioneine produced by microorganisms that have been artificially modified to improve the production of ergothioneine, or originally ergothioneine is not biosynthesizeable, but has been artificially modified so that ergothioneine can be biosynthesized Examples include ergothioneine produced by microorganisms, and the details are described in << culture and fertilizer >>.

  In the production method of the embodiment, ergothioneine may be used alone or in combination of two or more.

  The cultivation method of the plant body is not particularly limited, and examples thereof include outdoor cultivation, hydroponics, greenhouse cultivation, house cultivation and the like, and among these, hydroponics is preferable.

“Cultivation medium” refers to a medium that can contain ergothioneine such as soil, soil, and nutrient solution for hydroponics among those that contact the plant body during plant cultivation and provide a plant cultivation environment. Here, carriers such as sponges used in hydroponics and the like, and equipment such as floats do not contain ergothioneine and therefore are not included in the culture medium. The cultivation medium is preferably in contact with the roots of the plant body during plant cultivation.
The cultivation medium of embodiment refers to what contains 1 microgram / L or more of ergothioneine among the said cultivation medium.

  The said culture medium may contain the culture of embodiment, or the fertilizer of embodiment.

The cultivation process in the production method of the embodiment may be a cultivation medium containing a culture of a microorganism capable of biosynthesis of ergothioneine and cultivating a plant body.
The cultivation process in the manufacturing method of embodiment may grow a plant body with the cultivation medium containing the fertilizer containing the culture of the microorganisms which can biosynthesize ergothioneine.

The culture medium containing the fertilizer of the embodiment can be obtained by applying the fertilizer of the embodiment to soil, culture medium, hydroponics nutrient solution, or the like, for example.
The culture medium containing the culture of the embodiment may be obtained, for example, by applying the culture of the embodiment to soil, culture medium, hydroponics nutrient solution, or the like. You may obtain by mixing a liquid etc. and the culture of embodiment.
As the culture medium according to the embodiment, for example, the culture itself of the embodiment may be used, or the culture of the embodiment added with a diluent such as water or an additive such as nutrients may be used. Also good.

In the production method of the embodiment, before the cultivation step, a culture of a microorganism capable of biosynthesis of ergothioneine is added to a culture medium not containing ergothioneine or containing less than 1 μg / L of ergothioneine, and ergothioneine is added at 1 μg / L. You may have the addition process of obtaining the cultivation culture medium contained above.
In the production method of the embodiment, before the cultivation step, a fertilizer containing a culture of a microorganism capable of biosynthesis of ergothioneine is added to a culture medium not containing ergothioneine or containing less than 1 μg / L of ergothioneine, and ergothioneine is added. You may have the addition process of obtaining the culture medium which contains 1 microgram / L or more.

The cultivation process is carried out until the plant contains 0.5 μg or more of ergothioneine per 100 g of fresh weight.
Implementation time of the cultivation process, that is, the time for cultivating the plant in a state where the culturing medium containing ergothioneine is in contact with the plant is the time required for the plant to contain 0.5 μg or more of ergothioneine per 100 g of fresh weight. May be determined as appropriate. As an example, it may be 1 hour or more, 12 hours or more, 24 hours or more, or 3 days or more. A cultivation process may be performed continuously and may be performed in multiple times.

The manufacturing method of the embodiment may include a harvesting step of harvesting the plant body after the cultivation step.
The cultivation process is preferably carried out until just before the harvesting process. As a result, the state in which ergothioneine is accumulated in the plant body can be easily maintained, and ergothioneine can be efficiently accumulated in the harvest target.
From the above viewpoint, the cultivation process is preferably performed within at least 10 days before the harvesting process, and is preferably performed within at least 3 days.

  According to the production method of the embodiment, the plant body of the embodiment containing 0.5 μg or more of ergothioneine per 100 g of fresh weight can be produced.

  Moreover, according to the manufacturing method of embodiment, the ergothioneine contained in a culture medium can be collect | recovered or concentrated to a plant body. The cultivation process in the production method of the embodiment can be considered as a process of refining ergothioneine, and instead of refining and using ergothioneine, it is possible to use ergothioneine as a plant body in which ergothioneine is accumulated. As a result, ergothioneine can be supplied at low cost, and ergothioneine is expected to spread.

<An artificially modified microorganism>
<1> Microorganisms Hereinafter, regarding microorganisms capable of biosynthesizing ergothioneine, microorganisms artificially modified to improve the production of ergothioneine, and microorganisms artificially modified to biosynthesize ergothioneine explain.
These microorganisms can be suitably used as the microorganisms that may be used in the culture, fertilizer, and plant production methods of the embodiments.

  The microorganism capable of biosynthesizing ergothioneine may originally have ergothioneine-producing ability or may be modified to have ergothioneine-producing ability. A microorganism having an ergothioneine-producing ability may be obtained, for example, by imparting an ergothioneine-producing ability to a microorganism or by enhancing the ergothioneine-producing ability of a microorganism.

  Examples of a method for modifying a microorganism so as to have the ability to produce ergothioneine include a method for modifying a microorganism so as to retain a gene involved in ergothioneine synthesis. Modification of a microorganism to retain a gene involved in ergothioneine synthesis can be achieved by introducing a gene involved in ergothioneine synthesis into the microorganism. Moreover, modifying a microorganism so as to retain a gene involved in ergothioneine synthesis can also be achieved by introducing a mutation into a gene possessed by the microorganism by natural mutation or mutagen treatment.

Examples of the genes involved in ergothioneine synthesis include the egtABCDE gene operon encoding the ergothioneine protein group, preferably the mycobacterial-derived egtABCDE gene operon, and more preferably the bacteria belonging to the genus Mycobacterium (Mycobacterium). Further preferred is the egtABCDE gene operon derived from Mycobacterium smegmatis.
In addition, the egtABCDE gene may be a conservative variant (variant in which the original function is maintained) of egtABCDE derived from various organisms such as Mycobacterium smegmatis. “Original function” for egtABCDE refers to ergothioneine synthetic activity. Regarding the conservative variants of genes and proteins, the descriptions regarding the conservative variants of RNA pyrophosphohydrolase gene and RNA pyrophosphohydrolase described later can be applied mutatis mutandis.

  The microorganism capable of biosynthesizing ergothioneine may be a microorganism modified so that the cysteine-producing ability is increased. Cysteine is a precursor of ergothioneine, and the productivity of cysteine ergothioneine can be enhanced by increasing the ability to produce cysteine.

<1-1> Microorganism having L-cysteine-producing ability In the present invention, the term “cysteine” means L-cysteine unless otherwise specified. In the present invention, the term “L-cysteine” means free L-cysteine, a salt thereof, or a mixture thereof, unless otherwise specified. The salt will be described later. These explanations concerning cysteine can be applied mutatis mutandis to related substances of L-cysteine. That is, for example, the term “O-acetylserine” means free O-acetyl-L-serine, a salt thereof, or a mixture thereof, unless otherwise specified.

  The microorganism may originally have L-cysteine-producing ability or may be modified so as to have L-cysteine-producing ability. A microorganism having L-cysteine production ability may be obtained, for example, by imparting L-cysteine production ability to a microorganism, or by enhancing L-cysteine production ability of a microorganism.

  L-cysteine-producing ability can be imparted or enhanced by a method that has been conventionally employed for microbial breeding (Amino Acid Fermentation Co., Ltd., Japan Society Publishing Center, May 30, 1986, first edition issued, No. 77- 100 purchases). Examples of such methods include acquisition of auxotrophic mutants, acquisition of L-amino acid analog-resistant strains, acquisition of metabolic control mutants, and recombination with enhanced activity of L-amino acid biosynthetic enzymes. The creation of stocks. In the breeding of L-amino acid-producing bacteria, the auxotrophy, analog resistance, metabolic control mutation, and other properties imparted may be single, or two or more. In addition, L-amino acid biosynthetic enzymes whose activities are enhanced in breeding L-amino acid-producing bacteria may be used alone or in combination of two or more. Furthermore, imparting properties such as auxotrophy, analog resistance, and metabolic control mutation may be combined with enhancing the activity of biosynthetic enzymes.

  An auxotrophic mutant, an analog-resistant mutant, or a metabolically controlled mutant having L-amino acid production ability is subjected to normal mutation treatment of the parent strain or wild strain, and the auxotrophic, analog It can be obtained by selecting those that show tolerance or metabolic control mutations and have the ability to produce L-amino acids. Normal mutation treatments include X-ray and UV irradiation, N-methyl-N'-nitro-N-nitroguanidine (MNNG), ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), etc. Treatment with a mutagen is included. The L-amino acid-producing ability can also be imparted or enhanced by enhancing the activity of an enzyme involved in the target L-amino acid biosynthesis. Enhancing enzyme activity can be performed, for example, by modifying bacteria so that expression of a gene encoding the enzyme is enhanced. Methods for enhancing gene expression are described in pamphlet of W000 / 18935, European Patent Application Publication No. 1010755, and the like. A detailed method for enhancing the enzyme activity will be described later.

  The addition or enhancement of L-amino acid-producing ability can also be achieved by reducing the activity of an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of the target L-amino acid to produce a compound other than the target L-amino acid. It can be carried out. As used herein, “an enzyme that catalyzes a reaction that produces a compound other than the target L-amino acid by branching from the biosynthetic pathway of the target L-amino acid” includes enzymes involved in the degradation of the target amino acid. It is. A method for reducing the enzyme activity will be described later.

  Hereinafter, L-cysteine-producing microorganisms and methods for imparting or enhancing L-cysteine-producing ability are specifically exemplified. In addition, the modification | reformation for providing or enhancing the property and L-cysteine production ability which the L-cysteine production microorganisms which are illustrated below may have may be used independently, and may be used in combination as appropriate.

  Examples of the method for imparting or enhancing L-cysteine production ability include a method of modifying a microorganism so that the L-cysteine biosynthesis system is enhanced. “Enhancing L-cysteine biosynthesis system” means enhancing the activity of one or more enzymes selected from enzymes involved in L-cysteine biosynthesis (also referred to as L-cysteine biosynthesis enzymes) To do. Examples of L-cysteine biosynthetic enzymes include enzymes of the L-cysteine biosynthetic pathway and enzymes involved in the production of compounds that are substrates of the pathway. Specific examples of the L-cysteine biosynthesis enzyme include serine acetyltransferase (SAT) and 3-phosphoglycerate dehydrogenase (PGD). The genes encoding SAT and PGD are also referred to as SAT gene and PGD gene, respectively. As a gene encoding an L-cysteine biosynthesis system enzyme, for example, any of genes derived from bacteria belonging to the genus Escherichia such as Escherichia coli and genes derived from various other organisms can be used. For example, as the SAT gene, the cysE gene was recloned from a wild strain of Escherichia coli and an L-cysteine-secreting mutant, and the nucleotide sequence was revealed (Denk, D. and Boeck, A., '' General Microbiol. , 133, 515-525 (1987)). For example, as a PGD gene, the serA gene of various organisms such as Escherichia coli is known.

  Moreover, since SAT receives feedback inhibition by L-cysteine, SAT in which this feedback inhibition has been reduced or eliminated may be used. “Feedback inhibition is reduced or eliminated” is also referred to as “resistance to feedback inhibition”. SAT in which feedback inhibition by L-cysteine is reduced or eliminated is also referred to as “mutant SAT”. A gene encoding mutant SAT is also referred to as “mutant SAT gene”. That is, examples of a method for imparting or enhancing L-cysteine producing ability include a method of modifying a microorganism so as to retain a mutant SAT gene. That is, the microorganism may be modified to retain the mutant SAT gene. It is possible to enhance the SAT activity by retaining the mutant SAT gene in a microorganism. Mutant SAT includes SAT having a mutation that replaces the methionine residue at position 256 of wild-type SAT with an amino acid residue other than lysine residue and leucine residue, and C from methionine residue at position 256 of wild-type SAT. Examples include SAT having a mutation that deletes the terminal region (Japanese Patent Laid-Open No. 11-155571). Examples of the “amino acid residue other than lysine residue and leucine residue” include 17 types of amino acid residues other than methionine residue, lysine residue, and leucine residue among amino acids constituting ordinary proteins. It is done. Specific examples of the “amino acid residue other than lysine residue and leucine residue” include isoleucine residue and glutamic acid residue. Further, as the mutant SAT, SAT having one or more mutations in amino acid residues 89 to 96 of wild-type SAT (US Patent Publication No. 20050112731 (Al)), valine residue of 95-position of wild-type SAT And SAT (mutant gene name cysE5, U.S. Patent Publication No. 20050112731 (Al)) having mutations that replace the aspartic acid residues at positions 96 and 96 with arginine residues and proline residues, respectively, and wild-type SAT at position 167. Examples include SAT (mutant gene name cysEX, US Pat. No. 6,218,168, US Patent Publication No. 20050112731 (Al)) having a mutation that replaces a threonine residue with an alanine residue. “Wild-type SAT” refers to SAT that does not have the above-described mutation (mutation that is resistant to feedback inhibition by L-cysteine). The “wild type” as used herein is a description for the convenience of distinguishing from the “mutant type”, and is not limited to those obtained in nature unless it has the above-mentioned mutation. Examples of wild-type SAT include SAT derived from various organisms such as Escherichia coli. In addition, examples of wild-type SAT include conservative variants of SAT derived from various organisms such as Escherichia coli (variants in which the original functions are maintained), and those that do not have the above-described mutation. “Original function” for SAT refers to SAT activity. Escherichia coli JM39-8 strain (E. coli JM39-8 (pCEM256E) carrying a plasmid pCEM256E containing a mutant cysE encoding a mutant SAT in which the methionine residue at position 256 is substituted with a glutamic acid residue, private number: AJ13391) is the National Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry on November 20, 1997 (currently Japan Institute for Product Evaluation Technology, Patent Biological Deposit Center, Postal Code: 292-0818, Address: Chiba, Japan) 2-5-8, Kazusa Kamashishi, Mototsuzu City) was deposited under the deposit number of FERM P-16527 and transferred to an international deposit under the Budapest Treaty on July 8, 2002. FERM BP-8112 is granted.

  In addition, the mutant SAT may be modified so as to be resistant to feedback inhibition by L-cysteine as described above, but may be one that is not originally subjected to feedback inhibition. For example, SAT of Arabidopsis thaliana is known not to be subjected to feedback inhibition by L-cysteine and can be suitably used in the present invention. As a plasmid containing the SAT gene derived from Arabidopsis thaliana, pEAS-m (FEMS Microbiol. Lett., 179 (1999) 453-459) is known.

  Moreover, since PGD is subject to feedback inhibition by L-serine, PGD in which this feedback inhibition is reduced or eliminated may be used. In the present invention, PGD in which feedback inhibition by L-serine is reduced or eliminated is also referred to as “mutant PGD”. A gene encoding a mutant PGD is also referred to as a “mutant PGD gene”. That is, as a method for imparting or enhancing L-cysteine production ability, for example, a method of modifying a bacterium so as to retain a mutant PGD gene can also be mentioned. That is, the microorganism may be modified so as to retain the mutant PGD gene. PGD activity can be enhanced by retaining a mutant PGD gene in a microorganism. Examples of mutant PGD include PGD (mutant gene name serA5, US Pat. No. 6,180,373) having a mutation that deletes the tyrosine residue at position 410 (N-terminal) of wild-type PGD. “Wild-type PGD” refers to a PGD that does not have the above-described mutation (mutation that is resistant to feedback inhibition by L-serine). The “wild type” as used herein is a description for the convenience of distinguishing from the “mutant type”, and is not limited to those obtained in nature unless it has the above-mentioned mutation. Examples of the wild-type PGD include PGD derived from various organisms such as Escherichia coli. In addition, examples of wild-type PGD include conservative variants of PGD derived from various organisms such as Escherichia coli (variants in which the original functions are maintained) that do not have the above-described mutation. “Original function” for PGD refers to PGD activity.

  Examples of the method for imparting or enhancing L-cysteine production ability include a method of modifying a microorganism so that the L-cysteine excretion system is enhanced. “Strengthening L-cysteine excretion system” means enhancing the activity of one or more proteins selected from proteins involved in L-cysteine excretion (also referred to as L-cysteine excretion factor). . As L-cysteine excretion factors, proteins encoded by the ydeD gene (eamA gene) (JP 2002-233384), proteins encoded by the yfiK gene (JP 2004-049237), emrAB, emrKY, yojIH, acrEF, Proteins encoded by bcr and cusA genes (Japanese Patent Laid-Open No. 2005-287333) and proteins encoded by yeaS gene (Japanese Patent Laid-Open No. 2010-187552) are known. Alternatively, a YeaS protein having a mutation in the threonine residue at position 28, the phenylalanine residue at position 137, and / or the leucine residue at position 188 of the wild-type YeaS protein may be used. The YeaS protein having the mutation described above is also referred to as “mutant YeaS protein”. A gene encoding a mutant YeaS protein is also referred to as a “mutant yeaS gene”. That is, examples of the method for imparting or enhancing L-cysteine production ability include a method of modifying a microorganism so as to retain a mutant yeaS gene. That is, the microorganism may be modified to retain the mutant yeaS gene. By retaining the mutant yeaS gene in a microorganism, the L-cysteine excretion system can be enhanced (European Patent Publication No. 2218729). Specifically, the mutant YeaS protein is a mutation that replaces the threonine residue at position 28 of the YeaS protein with asparagine, and the phenylalanine residue at position 137 is one of serine, glutamine, alanine, histidine, cysteine, and glycine. There may be a mutation that substitutes and / or a mutation that replaces the leucine residue at position 188 with glutamine (EP-A-2218729). “Wild-type YeaS protein” refers to a YeaS protein that does not have the mutation described above. The “wild type” as used herein is a description for the convenience of distinguishing from the “mutant type”, and is not limited to those obtained in nature unless it has the above-mentioned mutation. Examples of the wild-type YeaS protein include YeaS proteins derived from various organisms such as Escherichia coli. Examples of the wild-type YeaS protein include conservative variants (variants in which the original functions are maintained) of YeaS proteins derived from various organisms such as Escherichia coli, which do not have the above-described mutation. “Original function” for YeaS protein refers to the property of improving the ability of microorganisms to produce L-cysteine when expression is increased in microorganisms.

  In the present invention, the “amino acid residue at the X position of wild-type SAT” means an amino acid residue corresponding to the amino acid residue of wild-type SAT unless otherwise specified. In the present invention, the “amino acid residue at the X position of wild-type PGD” means an amino acid residue corresponding to the amino acid residue of wild-type PGD unless otherwise specified. In the present invention, the “amino acid residue at the X position of the wild type YeaS protein” means an amino acid corresponding to the amino acid residue at the X position in the wild type YeaS protein of the Escherichia coli K-12 MG1655 strain, unless otherwise specified. Means residue. The “X position” in the amino acid sequence means the X position from the N terminal of the amino acid sequence, and the amino acid residue at the N terminal is the amino acid residue at the first position. In addition, the position of an amino acid residue shows a relative position, The absolute position may be moved back and forth by deletion, insertion, addition, etc. of an amino acid. For example, “the threonine residue at position 167 of wild-type SAT” means an amino acid residue corresponding to the threonine residue at position 167 in wild-type SAT, and one amino acid residue on the N-terminal side from position 167 Is deleted, it is assumed that the 166th amino acid residue from the N-terminal is “the threonine residue at position 167 of wild-type SAT”. In addition, when one amino acid residue is inserted on the N-terminal side from position 167, the 168th amino acid residue from the N-terminal is assumed to be “the threonine residue at position 167 of wild-type SAT”.

  Which amino acid residue in the amino acid sequence of an arbitrary SAT is “the amino acid residue corresponding to the X-position amino acid residue in wild-type SAT” refers to the amino acid sequence of the SAT and the amino acid sequence of wild-type SAT. This can be determined by performing the alignment. Similarly, with respect to the PGD or YeaS protein, the PGD or YeaS protein may be aligned with the amino acid sequence of the wild type PGD or the amino acid sequence of the wild type YeaS protein of Escherichia coli K-12 MG1655 strain. The alignment can be performed using, for example, a known gene analysis software. Specific software includes DNA Solutions manufactured by Hitachi Solutions and GENETYX manufactured by Genetics (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24 (1), 72-96, 1991; Barton GJ et al., Journal of molecular biology, 198 (2), 327-37. 1987).

  Mutant gene (i.e., mutant SAT gene, mutant PGD gene, or mutant yeaS gene) is a wild type gene (i.e., wild type SAT gene, wild type PGD gene, or wild type yeaS gene) (Ie, mutant SAT, mutant PGD, or mutant YeaS protein) can be obtained by modification. Modification of DNA can be performed by a known method. Specifically, for example, as a site-specific mutation method for introducing a target mutation into a target site of DNA, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press) (1989); Carter, P., Meth. In Enzymol., 154,382 (1987)) and methods using phage (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel , TA et al., Meth. In Enzymol., 154, 367 (1987)). Mutant genes can also be obtained by chemical synthesis. The codon after mutation is not particularly limited as long as it encodes the target amino acid, but it is preferable to use a codon frequently used in the microorganism of the present invention.

  Altering a microorganism to retain a mutant gene can be achieved by introducing the mutant gene into the microorganism. In addition, modifying a microorganism so as to retain a mutant gene can also be achieved by introducing a mutation into the gene of the microorganism by natural mutation or mutagen treatment.

  Further, L-cysteine-producing ability can also be imparted or enhanced by enhancing the expression of a cysPTWAM cluster gene encoding a sulfate / thiosulfate transport system protein group (Japanese Patent Laid-Open No. 2005137369, EP1528108). book).

  In addition, sulfide is incorporated into O-acetyl-L-serine through a reaction catalyzed by O-acetylserine (thiol) -lyase-A and B recoded by the cysK gene and the cysM gene, respectively. -Cysteine is produced. Therefore, these enzymes are included in the enzymes of the L-cysteine biosynthetic pathway, and the ability to produce L-cysteine can also be imparted or enhanced by enhancing the expression of genes encoding these enzymes.

  Examples of a method for imparting or enhancing L-cysteine producing ability include a method of modifying a microorganism so that the degradation system of L-cysteine is suppressed. “Inhibiting the L-cysteine degradation system” means enhancing the activity of one or more proteins selected from proteins involved in the degradation of L-cysteine (also referred to as L-cysteine degrading enzymes). The L-cysteine-degrading enzyme is not particularly limited, but cystathionine-β-lyase encoded by the metC gene (Japanese Patent Laid-Open No. 11-155571, Chandra et.al, Biochemistry, 21 (1982) 3064-3069)), Tryptophanase encoded by tnaA gene (JP 2003-169668, Austin Newton et al., J. Biol. Chem. 240 (1965) 1211-1218), O-acetylserine sulfate encoded by cysM gene Examples include hydrylase B (JP 2005-245311), MalY encoded by malY gene (JP 2005-245311), cysteine desulfhydrase encoded by d0191 gene of Pantoea ananatis (JP 2009-232844) .

  Examples of L-cysteine producing bacteria or parent strains for deriving them include, for example, E. coli JM15 transformed with various cysE alleles encoding mutant SAT (US Pat.No. 6,218,168). E. coli W3110 (US Pat.No. 5,972,663) with enhanced expression of a gene encoding a protein suitable for excretion of toxic substances into cells, E. coli with reduced cysteine desulfhydrase activity (special Kaihei 11-155571), E. coli W3110 (WO01 / 27307), the activity of the positive transcriptional regulator of the cysteine regulon recoded by the cySB gene, ydeD gene, mutant cysE gene (cysEX gene), And E. coli strains such as E. coli carrying plasmid pACYC DES (JP 2005137369 (US20050124049 (Al), EP1528108 (Al))) containing a mutant serA gene (serA5 gene). PACYC-DES is a plasmid obtained by inserting the above three genes into pACYC184, and each gene is controlled by an ompA promoter (PompA).

  Since O-acetylserine is a precursor of L-cysteine, the biosynthetic pathway of O-acetylserine is common with the biosynthetic pathway of L-cysteine. O-acetylserine is easily converted to N-acetylserine by a natural reaction in a neutral to alkaline pH range. In addition, L-cysteine excretion factors as described above are known which can also excrete O-acetylserine. Therefore, the production capacity of these L-cysteine precursors is partially based on methods for imparting or enhancing L-cysteine production capacity such as enhancement of L-cysteine biosynthesis system and enhancement of L-cysteine excretion system. By doing so, it can be imparted or enhanced.

  The ability to produce compounds such as γ-glutamylcysteine, glutathione, cystathionine, homocysteine, L-methionine, and S-adenosylmethionine, which are biosynthesized using L-cysteine as a starting material, is also in the biosynthetic pathway of the target compound. The enzyme activity can be imparted or enhanced by decreasing the activity of an enzyme (including an enzyme that degrades the target compound) in a pathway that branches from the biosynthetic pathway.

  For example, the ability to produce γ-glutamylcysteine can be imparted or enhanced by enhancing γ-glutamylcysteine synthetase activity and / or decreasing glutathione synthetase activity. The ability to produce glutathione can be imparted or enhanced by enhancing γ-glutamylcysteine synthetase activity and / or glutathione synthetase activity. The ability to produce γ-glutamylcysteine or glutathione can also be imparted or enhanced by using a mutant γ-glutamylcysteine synthetase that is resistant to feedback inhibition by glutathione. The production of glutathione is described in detail in a review by Li et al. (Yin Li, Gongyuan Wei, Jian Chen. Appl Microbiol Biotechnol (2004) 66: 233-242).

  The ability to produce L-methionine can be imparted or enhanced by imparting L-threonine requirement or norleucine resistance (Japanese Patent Laid-Open No. 2000-139471). In E. coli, the gene for the enzyme involved in the biosynthesis of L-threonine exists as the threonine operon (thrABC) .For example, by deleting the thrBC part, the biosynthesis ability after L-homoserine is reduced. A lost L-threonine-requiring strain can be obtained. In a norleucine resistant strain, S-adenosylmethionine synthetase activity is weakened and L-methionine-producing ability is imparted or enhanced. In E. coli, S-adenosylmethionine synthetase is encoded by the metK gene. In addition, L-methionine production ability is increased by enhancing the activities of enzymes involved in L-methionine biosynthesis such as methionine repressor deficiency, homoserine transsuccinylase, cystathionine γ-synthase, and aspartokinase-homoserine dehydrogenase II. Can also be imparted or enhanced (Japanese Patent Laid-Open No. 2000-139471). In E. coli, the methionine repressor is encoded by the metJ gene, homoserine transsuccinylase is encoded by the metA gene, cystathionine γ-synthase is encoded by the metB gene, and aspartokinase homoserine dehydrogenase II is encoded by the metL gene. . In addition, the ability to produce L-methionine can be imparted or enhanced by using a mutant homoserine transsuccinylase resistant to feedback inhibition by L-methionine (Japanese Patent Laid-Open No. 2000-139471, US20090029424). Since L-methionine is biosynthesized using L-cysteine as an intermediate, L-methionine production ability can be improved by improving L-cysteine production ability (JP 2000-139471, US20080311632). . Therefore, in order to impart or enhance L-methionine production ability, a method for imparting or enhancing L-cysteine production ability is also effective.

  Specific examples of L-methionine-producing bacteria or parent strains for deriving them include, for example, E. coli AJl1539 (NRRL B-12399), AJ11540 (NRRL B-12400), AJl1541 (NRRL B-12401) , AJ11542 (NRRL B-12402) (British Patent No. 2075055), L-methionine analog 218 strain having resistance to norleucine (VKPM B-8125) (Russian Patent No. 2209248) and 73 strains (VKPM B-8126) ) (Russian Patent No. 2215782) and the like.

  Further, examples of L-methionine-producing bacteria or parent strains for deriving them include, for example, AJ13425 (FERM P-16808) (JP 2000-139471) derived from E. coli W3110. . AJ13425 lacks a methionine repressor, weakens intracellular S-adenosylmethionine synthetase activity, intracellular homoserine transsuccinylase activity, cystathionine γ-synthase activity, and aspartokinase homoserine dehydrogenase II It is an L-threonine-requiring strain with enhanced activity. AJ13425 was launched on May 14, 1998 from the Ministry of International Trade and Industry, Institute of Industrial Science, Biotechnology Institute of Technology (current name: National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center, Address Postal Code 305-8566 Tsukuba City, Ibaraki Prefecture, 1-chome East Deposited at address 1 center 6) and given the deposit number FERM P-16808.

  Since cystathionine and homocysteine are intermediates in the L-methionine biosynthetic pathway, in order to confer or enhance the production ability of these substances, a part of the above-mentioned method for imparting or enhancing L-methionine production ability is used. It is effective to do. As a method for imparting or enhancing cystathionine production ability, specifically, a method using a methionine-requiring mutant (Japanese Patent Application No. 2003-010654), cysteine (or a biosynthetic material thereof) and / or homoserine (or a fermentation medium) And a method of adding the biosynthetic raw material) (JP 2005-168422 A). Since homocysteine uses cystathionine as a precursor, a method of imparting or enhancing cystathionine production ability is also effective for imparting or enhancing homocysteine production ability.

  In addition, the ability to produce compounds such as S-adenosylmethionine biosynthesized using L-methionine as a starting material also enhances the enzymatic activity of the biosynthetic pathway of the target compound or branches off from the biosynthetic pathway It can be imparted or enhanced by reducing the activity of pathway enzymes (including enzymes that degrade target compounds). For example, the ability to produce S-adenosylmethionine can be imparted or enhanced by enhancing methionine adenosyltransferase activity (EP0647712, EP1457569) or enhancing the efflux factor MdfA encoded by the mdfA gene (US7410789). it can.

  The gene and protein used for breeding L-cysteine-producing bacteria may have the base sequence and amino acid sequence of known genes and proteins such as the above-exemplified genes and proteins, respectively. In addition, the genes and proteins used for breeding L-cysteine-producing bacteria may be conservative variants of known genes and proteins such as those exemplified above. Specifically, for example, a gene used for breeding an L-cysteine-producing bacterium is one or several at one or several positions in the amino acid sequence of a known protein as long as the original function is maintained. It may be a gene encoding a protein having an amino acid sequence in which one amino acid is substituted, deleted, inserted or added. Regarding the conservative variants of genes and proteins, the descriptions regarding RNA pyrophosphohydrolase gene and conservative variants of RNA pyrophosphohydrolase described later can be applied mutatis mutandis.

<1-2> Decrease in activity of RNA pyrophosphohydrolase The microorganism may be modified so that the activity of RNA pyrophosphohydrolase is reduced. The microorganism can be obtained by modifying a microorganism having L-cysteine-producing ability so that the activity of RNA pyrophosphohydrolase is reduced. The microorganism can also be obtained by imparting or enhancing L-cysteine production ability after modifying the microorganism so that the activity of RNA pyrophosphohydrolase is reduced. In addition, the microorganism may have acquired L-cysteine producing ability by being modified so that the activity of RNA pyrophosphohydrolase is reduced. The modification for constructing the microorganism can be performed in any order.

  By modifying the microorganism so that the activity of RNA pyrophosphohydrolase decreases, the ability of the microorganism to produce L-cysteine can be improved.

  Below, RNA pyrophosphohydrolase and the gene which codes it are demonstrated.

  “RNA pyrophosphohydrolase” refers to a protein having RNA pyrophosphohydrolase activity. “RNA pyrophosphohydrolase activity” refers to an activity that catalyzes a reaction of hydrolyzing the triphosphorylated 5 ′ end of RNA to liberate diphosphate (pyrophosphate). A gene encoding RNA pyrophosphohydrolase is also referred to as “RNA pyrophosphohydrolase gene”.

  Examples of RNA pyrophosphohydrolase include NudH protein encoded by the nudH gene. The nudH gene is also called rppH gene or ygdP gene. Similarly, NudH protein is also called RppH protein, YgdP protein, and the like. The base sequence of the nudH gene possessed by the microorganism and the amino acid sequence of the NudH protein encoded by them can be obtained from a public database such as NCBI. The nudH gene of Escherichia coli K-12MG1655 strain corresponds to a complementary sequence of sequences 2968447 to 2968977 in the genome sequence registered as GenBank accession NC_000913 (VERSI0N NC_000913.3 GI: 556503834) in the NCBI database. The NudH protein of Escherichia coli K-12 MG1655 strain is registered as GenBank accession NP_417307 (version NP_417307.l GI: 16130734). The nudH gene of Pantoea ananatis AJ13355 strain corresponds to a complementary sequence of positions 2891727 to 2892254 in the genome sequence registered as GenBank accession NC_017531 (VERSI0N NC 017531.l GI: 386014600) in the NCBI database. Moreover, the NudH protein of Pantoea ananatis AJ13355 strain is registered as GenBank accession WP_013026988 (version WP_013026988.l GI: 502792012). In addition, the expression “having an (amino acid or base) sequence” includes the case of “including the (amino acid or base) sequence” and the case of “consisting of the (amino acid or base) sequence”.

  The RNA pyrophosphohydrolase gene may be a variant of the RNA pyrophosphohydrolase gene exemplified above, for example, the nudH gene exemplified above, as long as the original function is maintained. Similarly, the RNA pyrophosphohydrolase may be a variant of the RNA pyrophosphohydrolase exemplified above, for example, the NudH protein exemplified above, as long as the original function is maintained. Such a variant in which the original function is maintained may be referred to as a “conservative variant”. The term “nudH gene” is intended to encompass conservative variants thereof in addition to the nudH gene exemplified above. Similarly, the term “NudH protein” is intended to encompass those conservative variants in addition to the NudH proteins exemplified above. Examples of conservative variants include the above-described RNA pyrophosphohydrolase gene, RNA pyrophosphohydrolase homologues, and artificially modified variants.

  “The original function is maintained” means that the variant of the gene or protein has a function (activity or property) corresponding to the function (activity or property) of the original gene or protein. “The original function is maintained” for a gene means that the variant of the gene encodes a protein in which the original function is maintained. “The original function is maintained” for an RNA pyrophosphohydrolase gene means that the variant of the gene encodes a protein having RNA pyrophosphohydrolase activity. In addition, “the original function is maintained” for RNA pyrophosphohydrolase means that the variant of the protein has RNA pyrophosphohydrolase activity.

  The RNA pyrophosphohydrolase activity of a protein is determined by incubating the protein with a substrate (e.g., triphosphorylated mono- or oligoribonucleotide) and measuring the production of the protein and substrate-dependent diphosphate (pyrophosphate). (Vasilyev N, Serganov A. Structures of RNA complexes with the Escherichia coli RNApyrophosphohydrolase RppH unveil the basis for specific 5'-end-dependent mRNA decay. '' Biol Chem. 2015 Apr 10; 290 (15): 9487-99 .).

  Hereinafter, the conservative variant will be exemplified.

  RNA pyrophosphohydrolase gene homologs or RNA pyrophosphohydrolase homologs are, for example, BLAST searches and FASTAs using the base sequence of the RNA pyrophosphohydrolase gene exemplified above or the amino acid sequence of the RNA pyrophosphohydrolase exemplified above as a query sequence. It can be easily obtained from public databases by searching. In addition, RNA pyrophosphohydrolase gene homologs can be obtained, for example, by PCR using chromosomes of various organisms as templates and oligonucleotides prepared based on the base sequences of these known RNA pyrophosphohydrolase genes as primers. Can do.

  The RNA pyrophosphohydrolase gene is an amino acid sequence in which one or several amino acids at one or several positions are substituted, deleted, inserted or added in the above amino acid sequence as long as the original function is maintained. It may be a gene encoding a protein having the same. For example, the encoded protein may have its N-terminus and / or C-terminus extended or shortened. The “one or several” is different depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, and specifically, for example, 1 to 50, 1 to 40, 1 to 30 It means 1 to 20, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 to 3.

  One or several amino acid substitutions, deletions, insertions, or power days described above are conservative mutations that maintain the protein function normally. A typical conservative mutation is a conservative substitution. Conservative substitution is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In this case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, CyS to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Thr to Ser or Ala, substitution from Trp to Phe or Tyr, substitution from Tyr to His, Phe or Trp, and substitution from Val to Met, Ile or Leu. In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences of the organism from which the gene is derived. Also included are those that occur.

  In addition, the RNA pyrophosphohydrolase gene is sealed in the entire amino acid sequence as long as the original function is maintained, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably May be a gene encoding a protein having a homology of 95% or more, more preferably 97% or more, particularly preferably 99% or more. In the present specification, “homology” means “identity”.

  In addition, the RNA pyrophosphohydrolase gene hybridizes under stringent conditions with a probe that can be prepared from the base sequence, for example, a complementary sequence to the whole or a part of the base sequence, as long as the original function is maintained. It may be DNA. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 99%. % At 60 ° C, 1 x SSC, 0.1% SDS, which is a condition in which DNAs having homology of at least% hybridize and DNAs having lower homology do not hybridize, or washing conditions of normal Southern hybridization Preferably at 60 ° C., 0.1 × SSC, 0.1% SDS, more preferably at a salt concentration and temperature corresponding to 68 ° C., 0.1 × SSC, 0.1% SDS, and conditions for washing once, preferably 2-3 times. Can be mentioned.

  As described above, the probe used for the hybridization may be a part of a complementary sequence of a gene. Such a probe can be prepared by PCR using an oligonucleotide prepared based on a known gene sequence as a primer and a DNA fragment containing the above gene as a template. For example, a DNA fragment having a length of about 300 bp can be used as the probe. When a DNA fragment having a length of about 300 bp is used as the probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

  In addition, since the degeneracy of the codon varies depending on the host, the RNA pyrophosphohydrolase gene may be an arbitrary codon substituted with an equivalent codon. For example, the RNA pyrophosphohydrolase gene may be modified to have optimal codons depending on the codon usage of the host to be used.

  The percentage sequence identity between two sequences can be determined, for example, using a mathematical algorithm. Non-limiting examples of such mathematical algorithms include the Myers and Miller (1988) CAB10S 4:11 17 algorithm, the Smith et al (1981) Adv.Appl, Math. 2: 482 local homology algorithm, Needleman and Wunsch. (1970) J. Mol. Biol. 48: 443 453 homology alignment algorithm, Pearson and Lipman (1988) Proc, Natl. Acad. Sci. 85: 2444 A method for searching similarities of 2448, Karlin and Altschul (1993) An improved algorithm of Karlin and Altschul (1990) Proc.Natl.Acad.Sci.USA 872264, as described in Proc.Natl.Acad.Sci.USA 90: 5873 5877.

  A program based on these mathematical algorithms can be used to perform sequence comparison (alignment) to determine sequence identity. The program can be appropriately executed by a computer. Such programs include, but are not limited to, the PC / Gene program CLUSTAL (available from Intelligents, Mountain View, Calif.), The ALIGN program (Version 2.0), and the Wisconsin Genetics Software Package, Version 8 (Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA) GAP, BESTFIT, BLAST, FASTA, and TFASTA. Alignment using these programs can be performed using initial parameters, for example. For the CLUSTAL program, HigGlns et al. (1988) Gene 73: 237244 (1988), HigGlns et al. (1989) CABIOS 5: 151 153, Corpet et al. (1988) Nucleic Acids Res. 16: 1088190, Huang et al. (1992) CABIOS 8: 155 65, and Pearson et al. (1994) Metho Mol. Biol. 24: 307 331.

  In order to obtain a nucleotide sequence having homology with the nucleotide sequence encoding the protein of interest, specifically, for example, a BLAST nucleotide search can be performed with the BLASTN program, score = 100, word length = 12. In order to obtain an amino acid sequence having homology with the protein of interest, specifically, for example, a BLAST protein search can be performed with the BLASTX program, score = 50, word length = 3. Please refer to http://www.ncbi.nlm.nih.gov for BLAST nucleotide search and BLAST protein search. In addition, Gapped BLAST (BLAST 2.0) can be used to obtain an alignment with a gap added for the purpose of comparison. PSI-BLAST (BLAST 2.0) can also be used to perform an iterated search that detects distant relationships between sequences. For Gapped BLAST and PSI-BLAST, see Altschul et al. (1997) Nucleic Acids Res. 25: 3389. When utilizing BLAST, Gapped BLAST, or PSI-one BLAST, for example, the initial parameters of each program (eg, BLASTN for nucleotide sequences, BLASTX for amino acid sequences) can be used. The alignment may be performed manually.

  Sequence identity between two sequences is calculated as the percentage of residues that match between the two sequences when the two sequences are aligned for maximum matching.

  In addition, the description regarding the conservative variant of said gene and protein can be applied mutatis mutandis to arbitrary proteins, such as L-cysteine biosynthesis system enzyme, and the gene which codes them.

<1-3> Method for Increasing Protein Activity A method for increasing the activity of proteins such as SAT and PGD will be described below.

  “Increasing the activity of a protein” means that the activity per cell of the protein is increased relative to an unmodified strain. As used herein, “unmodified strain” means a control strain that has not been modified to increase the activity of the target protein. Non-modified strains include wild strains and parent strains. Note that “increasing protein activity” is also referred to as “enhancing protein activity”. Specifically, “the protein activity is increased” means that the molecular teaching per cell of the protein is increased and / or the function per molecule of the protein compared to the unmodified strain. Is increasing. In other words, “activity” in the case of “increasing the activity of a protein” means not only the catalytic activity of the protein but also the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. May be. In addition, “the protein activity increases” means not only to increase the activity of the protein in a strain that originally has the activity of the target protein, but also to the activity of the protein in a strain that does not originally have the activity of the target protein. Including granting. Further, as long as the activity of the protein increases as a result, the activity of the target protein inherent in the host may be reduced or eliminated, and the activity of a suitable target protein may be imparted.

  The activity of the protein is not particularly limited as long as it is increased as compared with the non-modified strain. For example, the protein activity may be increased 1.5 times or more, 2 times or more, or 3 times or more compared to the non-modified strain. In addition, when the non-modified strain does not have the activity of the target protein, it is sufficient that the protein is generated by introducing a gene encoding the protein. For example, the protein has an enzymatic activity. It may be produced to the extent that it can be measured.

  Modifications that increase the activity of the protein are achieved, for example, by increasing the expression of the gene encoding the protein. “Gene expression is increased” means that the expression level of the gene per cell is increased as compared to a non-modified strain such as a wild strain or a parent strain. Specifically, “gene expression increases” means that the amount of gene transcription (mRNA amount) increases and / or the amount of gene translation (protein amount) increases. Good. Note that “increasing gene expression” is also referred to as “enhanced gene expression”. The expression of the gene may increase, for example, 1.5 times or more, 2 times or more, or 3 times or more as compared with the unmodified strain. In addition, “increasing gene expression” means not only increasing the expression level of a target gene in a strain that originally expresses the target gene, but also in a strain that originally does not express the target gene. Including expressing a gene. That is, “increasing gene expression” includes, for example, introducing the gene into a strain that does not hold the target gene and expressing the gene.

  An increase in gene expression can be achieved, for example, by increasing the copy number of the gene.

  Increasing the copy number of a gene can be achieved by introducing the gene into the host chromosome. Introduction of a gene into a chromosome can be performed, for example, using homologous recombination (Miller I, J. H. Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). As a gene introduction method utilizing homologous recombination, for example, the Red-driven integration method (Datsenko, KA, and Wanner, BL, Proc. Natl. Acad. Sci. USA, 97: 6640-6645 (2000 )), Etc., a method using a linear DNA, a method using a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugation transfer, a method using a suspension vector that does not have an origin of replication functioning in the host, and a phage. The transduction method used is mentioned. Only one copy of the gene may be introduced, or two copies or more may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination with a sequence having multiple copies on the chromosome as a target. Examples of sequences having multiple copies on a chromosome include repetitive DNA sequences and inverted repeats present at both ends of a transposon. Alternatively, homologous recombination may be performed by targeting an appropriate sequence on a chromosome such as a gene unnecessary for production of the target substance. The gene can also be randomly introduced onto the chromosome using transposon or Mini-Mu (Japanese Patent Laid-Open No. 2-109985, US Pat. No. 5,882,888, EP805867Bl).

  Confirmation of the introduction of the target gene on the chromosome was performed using Southern hybridization using a probe having a sequence complementary to all or part of the gene, or a primer prepared based on the sequence of the gene. It can be confirmed by PCR.

  An increase in gene copy number can also be achieved by introducing a vector containing the gene into a host. For example, a DNA fragment containing a target gene can be linked to a vector that functions in the host to construct an expression vector for the gene, and the host can be transformed with the expression vector to increase the copy number of the gene. it can. A DNA fragment containing a target gene can be obtained, for example, by PCR using a gnome DNA of a microorganism having the target gene as a template. As the vector, a vector capable of autonomous replication in a host cell can be used. The vector is preferably a multicopy vector. In order to select a transformant, the vector preferably has a marker such as an antibiotic resistance gene. Moreover, the vector may be equipped with a promoter or terminator for expressing the inserted gene. The vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid. Specifically, vectors that can autonomously replicate in bacteria such as Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all available from Takara Bio Inc.), pACYC184, pMW219. (Nippon Gene), pTrc99A (Pharmacia), pPROK vector (Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Qiagen), pACYC vector, wide host Area vector RSF1010.

  When a gene is introduced, the gene only needs to be retained in the microorganism so that it can be expressed. Specifically, the gene may be introduced so as to be expressed under the control of a promoter sequence that functions in a microorganism. The promoter may be a host-derived promoter or a heterologous promoter. The promoter may be a circular promoter of the gene to be introduced, or may be a promoter of another gene. As the promoter, for example, a stronger promoter as described later may be used.

  A terminator for termination of transcription can be arranged downstream of the gene. The terminator is not particularly limited as long as it functions in microorganisms. The terminator may be a host-derived terminator or a heterogeneous terminator. The terminator may be a terminator specific to the gene to be introduced, or may be a terminator of another gene.

  Vectors, promoters, and terminators that can be used in various microorganisms are described in detail in, for example, “Basic Course of Microbiology 8 Genetic Engineering, Kyoritsu Shuppan, 1987”, and these can be used.

  In addition, when two or more genes are introduced, each gene may be retained in a microorganism so that it can be expressed. For example, all the genes may be held on a single expression vector, or all may be held on a chromosome. Moreover, each gene may be separately hold | maintained on several expression vector, and may be separately hold | maintained on the single or several expression vector and chromosome. Moreover, an operon may be constituted by two or more genes and introduced. “When introducing two or more genes”, for example, when introducing genes encoding two or more proteins, respectively, two or more subunits constituting a single protein complex Examples thereof include a case where a gene encoding each is introduced, and a combination thereof.

  The introduced gene is not particularly limited as long as it encodes a protein that functions in the host. The introduced gene may be a host-derived gene or a heterologous gene. The gene to be introduced can be obtained by PCR using, for example, a primer designed based on the base sequence of the gene, and using a genomic DNA of an organism having the gene or a plasmid carrying the gene as a template. In addition, the introduced gene may be totally synthesized based on the base sequence of the gene (Gene, 60 (1), H5127 (1987)), for example. The acquired gene can be used as it is or after being appropriately modified.

  When the protein functions as a complex composed of a plurality of subunits, all of the plurality of subunits may be modified or only a part may be modified as long as the activity of the protein increases as a result. . That is, for example, when the activity of a protein is increased by increasing the expression of a gene, the expression of a plurality of genes encoding those subunits may be enhanced, or only a part of the expression may be enhanced. Also good. Usually, it is preferable to enhance the expression of all of a plurality of genes encoding these subunits. In addition, each subunit constituting the complex may be derived from one organism or two or more different organisms as long as the complex has the function of the target protein. That is, for example, genes derived from the same organism encoding a plurality of subunits may be introduced into the host, or genes derived from different organisms may be introduced into the host.

  Moreover, the increase in gene expression can be achieved by improving the transcription efficiency of the gene. Moreover, the increase in gene expression can be achieved by improving the translation efficiency of the gene. Improvement of gene transcription efficiency and translation efficiency can be achieved, for example, by altering an expression regulatory sequence. “Expression regulatory sequence” is a general term for sites that affect gene expression. Examples of expression control sequences include promoters, Shine-Dalgarno (SD) sequences (also referred to as ribosome binding sites (RBS)), and spacer regions between RBS and the start codon. The expression regulatory sequence can be determined using a promoter search vector or gene analysis software such as GENETYX. These expression regulatory sequences can be modified by, for example, a method using a temperature sensitive vector or a Red driven integration method (W02005 / 010175).

  Improvement of gene transcription efficiency can be achieved, for example, by replacing a promoter of a gene on a chromosome with a stronger promoter. By “stronger promoter” is meant a promoter that improves transcription of the gene over the native wild-type promoter. Examples of stronger promoters include the known high expression promoters T7 promoter, trp promoter, lac promoter, thr promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, PR promoter, and PL promoter. Can be mentioned. As a more powerful promoter, a highly active promoter of a conventional promoter may be obtained by using various reporter genes. For example, promoter activity can be increased by bringing the -35 and -10 regions in the promoter region closer to the consensus sequence (WO 00/18935). Examples of highly active promoters include various tac-like promoters (Katashkina JI et al. Russian Federation Patent application 2006134574) and pnlp8 promoter (W02010 / 027045). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

  Improvement of gene translation efficiency can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence (also referred to as ribosome binding site (RBS)) of the gene on the chromosome with a stronger SD sequence. By “a stronger SD sequence” is meant an SD sequence that improves the translation of mRNA over the native wild-type SD sequence. An example of a stronger SD sequence is RBS of gene 10 derived from phage T7 (Olins P.0. Et al, Gene, 1988, 73, 227-235). In addition, substitution of several nucleotides in the spacer region between the RBS and the start codon, especially in the sequence immediately upstream of the start codon (5'-UTR), or insertion or deletion increases mRNA stability and translation efficiency. It is known to have a great influence, and the translation efficiency of a gene can also be improved by modifying these.

  Improvement of gene translation efficiency can also be achieved, for example, by codon modification. For example, the translation efficiency of a gene can be improved by replacing a rare codon present in the gene with a synonymous codon that is used more frequently. That is, the introduced gene may be modified to have an optimal codon according to, for example, the codon usage frequency of the host to be used. Codon substitution can be performed, for example, by a position-specific mutation method in which a target mutation is introduced into a target site of DNA. As site-directed mutagenesis, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989), Carter, P., Meth. In Enzymol., 154, 382 (1987) )) And methods using phage (Kramer, W. and Frits, H. ", Meth, in Enzymol., 154, 350 (1987), Kunkel, TA et al., Meth. In Enzymol., 154,367 (1987 )). Alternatively, gene fragments in which codons have been replaced may be fully synthesized. The frequency of codon usage in various organisms is described in the “Codon Usage Database” (http://wwv.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)). Is disclosed.

  An increase in gene expression can also be achieved by amplifying a regulator that increases gene expression, or by deleting or weakening a regulator that decreases gene expression.

  The methods for increasing gene expression as described above may be used alone or in any combination.

  Modifications that increase protein activity can also be achieved, for example, by enhancing the specific activity of the protein. Specific activity enhancement also includes the reduction and elimination of feedback inhibition. Proteins with enhanced specific activity can be obtained by searching for various organisms, for example. Alternatively, a highly active protein may be obtained by introducing a mutation into a conventional protein. The introduced mutation may be, for example, a substitution, deletion, insertion or addition of one or several amino acids at one or several positions of the protein. Mutation can be introduced by, for example, the site-specific mutation method as described above. Moreover, you may introduce | transduce a variation | mutation by a mutation process, for example. Mutation treatments include X-ray irradiation, ultraviolet irradiation, and N-methyl-N′-nitro-N-nitroguanidine (MNNG), ethyl methane sulfonate (EMS), and methyl methane sulfonate (MMS). ) And the like. Alternatively, DNA may be treated directly with hydroxylamine in vitro to induce random mutations. The enhancement of specific activity may be used alone or in any combination with the above-described technique for enhancing gene expression.

  The method for transformation is not particularly limited, and a conventionally known method can be used. Microorganism transformation is, for example, protoplast method (Gene, 39, 281286 (1985)), electroporation method (Bio / Techn01ogy, 7,10671070 (1989)), electric pulse method (Japanese Patent Laid-Open No. 2-207791) Can be performed.

  An increase in protein activity can be confirmed by measuring the activity of the protein.

  The increase in the activity of the protein can also be confirmed by confirming that the expression of the gene encoding the protein has increased. An increase in gene expression can be confirmed by confirming that the transcription amount of the gene has increased, or by confirming that the amount of protein expressed from the gene has increased.

  Confirmation that the amount of gene transcription has increased can be carried out by comparing the amount of mRNA transcribed from the gene with an unmodified strain such as a wild strain or a parent strain. Methods for assessing the amount of mRNA include Northern hybridization, RT-PCR, etc. (Sambrook, J., et al., Molecular Cloning A Laboratory Manua1 / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA ), 2001). The amount of mRNA may be increased by, for example, 1.5 times or more, 2 times or more, or 3 times or more as compared to the unmodified strain.

  Confirmation that the amount of protein has increased can be performed by Western blotting using an antibody (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of protein may be increased by, for example, 1.5 times or more, 2 times or more, or 3 times or more as compared to the unmodified strain.

  The above-described technique for increasing the activity of a protein can be used to enhance the activity of an arbitrary protein, such as an L-cysteine biosynthetic enzyme, or to enhance the expression of an arbitrary gene, such as a gene encoding the arbitrary protein.

<1-4> Technique for reducing protein activity A technique for reducing the activity of a protein such as RNA pyrophosphohydrolase will be described below.

  “Protein activity is decreased” means that the activity per cell of the protein is decreased as compared to an unmodified strain, and includes the case where the activity is completely lost. As used herein, “unmodified strain” refers to a control strain that has not been modified so that the activity of the target protein is reduced. Non-modified strains include wild strains and parent strains. Specifically, “the protein activity is decreased” means that the number of molecules per cell of the protein is decreased and / or the function per molecule of the protein compared to the unmodified strain. Means that it is decreasing. In other words, “activity” in the case of “decrease in protein activity” is not limited to the catalytic activity of the protein, but means the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. May be. Note that “the number of molecules per cell of the protein is decreased” includes a case where the protein does not exist at all. Moreover, “the function per molecule of the protein is reduced” includes the case where the function per molecule of the protein is completely lost. The activity of the protein is not particularly limited as long as it is lower than that of the unmodified strain.For example, it is 50% or less, 20% or less, 10% or less, 5% or less, or 0, compared to the non-modified strain. May drop to%.

  The modification that decreases the activity of the protein can be achieved, for example, by decreasing the expression of a gene encoding the protein. “Gene expression decreases” means that the expression level of the gene per cell decreases as compared to an unmodified strain such as a wild strain or a parent strain. The phrase “gene expression decreases” specifically means that the amount of gene transcription (mRNA amount) decreases and / or the amount of gene translation (protein amount) decreases. Good. “Gene expression decreases” includes the case where the gene is not expressed at all. In addition, “the expression of the gene is reduced” is also referred to as “the expression of the gene is weakened”. Gene expression may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to an unmodified strain.

  The decrease in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof. For example, gene expression can be reduced by modifying expression control sequences such as the promoter of the gene, Shine-Dalgarno (SD) sequence (also called ribosome binding site (RBS)), spacer region between RBS and start codon, etc. Can be achieved. When modifying the expression control sequence, the expression control sequence is preferably modified by 1 base or more, more preferably 2 bases or more, particularly preferably 3 bases or more. Further, part or all of the expression regulatory sequence may be deleted. In addition, reduction of gene expression can be achieved, for example, by manipulating factors involved in expression control. Factors involved in expression control include small molecules (such as inducers and inhibitors), proteins (such as transcription factors), and nucleic acids (such as siRNA) involved in transcription and translation control. In addition, reduction of gene expression can be achieved, for example, by introducing a mutation that reduces gene expression into the coding region of the gene. For example, gene expression can be reduced by replacing codons in the coding region of the gene with synonymous codons that are used less frequently in the host. In addition, for example, gene expression itself may be reduced by gene disruption as described below.

  Moreover, the modification that decreases the activity of the protein can be achieved, for example, by destroying a gene encoding the protein. “Gene is disrupted” means that the gene is modified so that it does not produce a normally functioning protein. “Does not produce a protein that functions normally” includes the case where no protein is produced from the same gene, or the case where a protein whose function (activity or property) per molecule is reduced or lost is produced from the same gene. It is.

  Gene disruption can be achieved, for example, by deleting part or all of the coding region of the gene on the chromosome. Furthermore, the entire gene including the sequences before and after the gene on the chromosome may be deleted. The region to be deleted may be any region such as an N-terminal region, an internal region, or a C-terminal region as long as the reduction in protein activity can be achieved. Usually, the longer region to be deleted can surely inactivate the gene. Moreover, it is preferable that the reading frames of the sequences before and after the region to be deleted do not match.

  In addition, gene disruption is, for example, introducing an amino acid substitution (missense mutation) into the coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or adding or deleting 1 to 2 bases. (Journa1 of Biological Chemistry 272: 8611-8617 (1997), Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journa1 of Biological Chemistry 26 116, 20833-20839 (1991)).

  In addition, gene disruption can be achieved, for example, by inserting another sequence into the coding region of the gene on the chromosome. The insertion site may be any region of the gene, but the longer the inserted sequence, the more inactive the gene can be. Moreover, it is preferable that the reading frames of the sequences before and after the insertion position do not match. The other sequence is not particularly limited as long as it reduces or eliminates the activity of the encoded protein, and examples thereof include marker genes such as antibiotic resistance genes and genes useful for the production of target substances.

  To modify a gene on a chromosome as described above, for example, a deletion type gene modified so as not to produce a normally functioning protein is prepared, and a host is transformed with a recombinant DNA containing the deletion type gene. This can be accomplished by replacing the wild-type gene on the chromosome with the deletion-type gene by converting and causing homologous recombination between the deletion-type gene and the wild-type gene on the chromosome. In that case, it is easy to operate the recombinant DNA if a marker gene is included according to the traits such as auxotrophy of the host. Deletion-type genes include genes in which all or part of the gene has been deleted, genes introduced with missense mutations, genes introduced with nonsense mutations, genes introduced with frameshift mutations, transposon and marker genes, etc. Examples include genes into which an insertion sequence has been introduced. Even if the protein encoded by the deletion-type gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function is reduced or lost. Gene disruption by gene replacement using such homologous recombination has already been established, and a method called `` Red-driven integration '' (Datsenko, K.A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97: 6640-6645 (2000)), Red-driven integration method and lambda phage-derived excision system (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002) ) And the like (see W02005 / 010175), a method using linear DNA, a method using a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugation transfer, replication that functions in the host There is a method using a suicidal vector having no origin (US Pat. No. 6,303,383, Japanese Patent Laid-Open No. 05007491).

  Further, the modification that decreases the activity of the protein may be performed by, for example, a mutation treatment. Mutation treatments include X-ray irradiation, ultraviolet irradiation, and N-methyl-N′-nitro-N-nitroguanidine (MNNG), ethyl methane sulfonate (EMS), and methyl methane sulfonate (MMS). ) And the like.

  When the protein functions as a complex composed of a plurality of subunits, all of the plurality of subunits may be modified or only a part may be modified as long as the activity of the protein decreases as a result. . That is, for example, all of a plurality of genes encoding these subunits may be destroyed, or only a part of them may be destroyed. In addition, when a plurality of isozymes are present in a protein, as long as the activity of the protein is reduced as a result, all the activities of the plurality of isozymes may be reduced, or only a part of the activities may be reduced. That is, for example, all of a plurality of genes encoding these isozymes may be destroyed, or only a part of them may be destroyed.

  The decrease in the activity of the protein can be confirmed by determining the activity of the protein.

  The decrease in the activity of the protein can also be confirmed by confirming that the expression of the gene encoding the protein has decreased. The decrease in gene expression can be confirmed by confirming that the transcription amount of the gene has decreased, or confirming that the amount of protein expressed from the gene has decreased.

  Confirmation that the amount of gene transcription has been reduced can be confirmed by comparing the amount of mRNA transcribed from the same gene with an unmodified strain. Examples of methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR and the like (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of mRNA may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the unmodified strain.

  Confirmation that the amount of protein has decreased can be performed by Western blotting using an antibody (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of protein may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to an unmodified strain.

  Whether or not a gene has been destroyed can be confirmed by determining part or all of the base sequence, restriction enzyme map, full length, etc. of the gene according to the means used for the destruction.

  In addition to reducing the activity of RNA pyrophosphohydrolase, the above-described method for reducing the activity of a protein catalyzes a reaction that produces a compound other than L-cysteine by branching from the biosynthetic pathway of any protein, such as L-cysteine. It can be used to reduce the activity of the enzyme, and to reduce the expression of any gene, for example, a gene encoding any of these proteins.

  In addition, although the microorganism modified so that cysteine production ability might increase as described above was demonstrated, use of the said microorganism is not essential and it may replace with the use of the said microorganism and may add cysteine.

<Method for producing ergothioneine>
In one embodiment, the present invention provides a method for producing ergothioneine, comprising culturing a microorganism capable of biosynthesis of ergothioneine in a medium, and collecting ergothioneine from the medium. In this method, “ergothioneine” is also referred to as “target substance”.

  The medium to be used is not particularly limited as long as the microorganism can grow and the target substance is produced. As the medium, for example, a normal medium used for culturing microorganisms can be used. As the medium, for example, a medium containing a carbon source, a nitrogen source, a phosphate source, a sulfur source, and other components selected from various organic components and inorganic components as necessary can be used. The type and concentration of the medium component may be appropriately set according to various conditions such as the type of microorganism used.

  Specific examples of carbon sources include glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, waste molasses, starch hydrolysate, biomass hydrolyzate, and other sugars, acetic acid, fumaric acid, citric acid, Examples include organic acids such as succinic acid, alcohols such as glycerol, crude glycerol, and ethanol, and fatty acids. In addition, as a carbon source, a plant-derived raw material can be used suitably. Examples of plants include corn, rice, wheat, soybean, sugar cane, beet, and cotton. Examples of plant-derived materials include organs such as roots, stems, trunks, branches, leaves, flowers, seeds, plants containing them, and degradation products of these plant organs. The form of use of the plant-derived raw material is not particularly limited, and for example, any form such as a raw product, juice, pulverized product, or product can be used. Moreover, pentoses such as xylose, hexoses such as glucose, or a mixture thereof can be obtained and used from, for example, plant biomass. Specifically, these saccharides can be obtained by subjecting plant biomass to treatment such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis with enzymes such as cellulase, and alkali treatment. Since hemicellulose is generally more easily hydrolyzed than cellulose, hemicellulose in plant biomass is hydrolyzed in advance to release pentose, and then cellulose is hydrolyzed to produce hexose. Good. In addition, xylose may be supplied by conversion from hexose, for example, by allowing the microorganism of the present invention to have a conversion pathway from hexose such as glucose to xylose. As the carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

  Specific examples of the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract, and soybean protein degradation product, ammonia, and urea. Ammonia gas or ammonia water used for pH adjustment may be used as a nitrogen source. As the nitrogen source, one kind of nitrogen source may be used, or two or more kinds of nitrogen sources may be used in combination.

  Specific examples of the phosphoric acid source include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphoric acid. As the phosphate source, one type of phosphate source may be used, or two or more types of phosphate sources may be used in combination.

  Specific examples of the sulfur source include inorganic sulfur compounds such as sulfate, thiosulfate, and sulfite. As the sulfur source, one kind of sulfur source may be used, or two or more kinds of sulfur sources may be used in combination.

  Specific examples of various other organic and inorganic components include inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium and calcium; vitamin B1, vitamin B2, vitamin B6 and nicotine. Examples include vitamins such as acids, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components such as peptone, casamino acid, yeast extract, and soybean protein degradation products containing these. As other various organic components and inorganic components, one component may be used, or two or more components may be used in combination.

  In addition, when an auxotrophic mutant strain that requires an amino acid or the like for growth is used, it is preferable to supplement nutrients required for the medium.

  Culture conditions are not particularly limited as long as microorganisms can grow and target substances are produced. The culture can be performed, for example, under normal conditions used for culturing microorganisms. The culture conditions may be appropriately set according to various conditions such as the type of microorganism used.

  The culture can be performed using a liquid medium. In culturing, a microorganism cultured in a solid medium such as an agar medium may be directly inoculated into a liquid medium, or a microorganism cultured with a seed in a liquid medium may be inoculated into a liquid medium for main culture. Good. That is, the culture may be performed separately for seed culture and main culture. In that case, the culture conditions of the seed culture and the main culture may or may not be the same. The amount of microorganisms contained in the medium at the start of culture is not particularly limited. The main culture may be performed, for example, by inoculating 1-50% (v / v) of the seed culture solution into the medium of the main culture.

  Culturing can be performed by batch culture, fed-batch culture, continuous culture, or a combination thereof. The culture medium at the start of the culture is also referred to as “initial culture medium”. A medium supplied to a culture system (fermentor) in fed-batch culture or continuous culture is also referred to as “fed-batch medium”. In addition, supplying a feeding medium to a culture system in fed-batch culture or continuous culture is also referred to as “fed-batch”. In addition, when culture | cultivation is performed by dividing into seed culture and main culture, for example, both seed culture and main culture may be performed by batch culture. Further, for example, seed culture may be performed by batch culture, and main culture may be performed by fed-batch culture or continuous culture.

  The culture can be performed, for example, under aerobic conditions. The aerobic condition means that the dissolved oxygen concentration in the liquid medium is 0.33 ppm or more, which is the detection limit by the oxygen membrane electrode, and preferably 1.5 ppm or more. The oxygen concentration may be controlled to, for example, about 5 to 50%, preferably about 10% of the saturated oxygen concentration. Specifically, culture under aerobic conditions can be performed by aeration culture, shaking culture, agitation culture, or a combination thereof. The pH of the medium may be, for example, pH 3 to 10, preferably pH 5 to 8. During the culture, the pH of the medium can be adjusted as necessary. The pH of the medium is adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, etc. can do. The culture temperature may be, for example, 20 to 40 ° C, preferably 25 to 37 ° C. The culture period may be, for example, 10 hours to 120 hours. The culture may be continued, for example, until the carbon source in the medium is consumed or until the activity of the microorganism is lost. By culturing the microorganism under such conditions, the target substance accumulates in the medium.

  Generation of the target substance can be confirmed by a known method used for detection or identification of the compound. Examples of such methods include HPLC, LC / MS, GC / MS, and NMR. These methods can be used alone or in appropriate combination.

  Recovery of the target substance from the fermentation broth can be performed by a known technique used for separation and purification of compounds. Examples of such techniques include ion exchange resin method (Nagai, H. et al., Separation Science and Technology, 39 (16), 3691-3710), precipitation method, membrane separation method (Japanese Patent Laid-Open No. 9-164323). And JP-A-9737737) and crystallization methods (W02008 / 078448, W02008 / 078646). These methods can be used alone or in appropriate combination. In addition, when the target substance accumulates in the microbial cell, for example, the microbial cell is crushed by ultrasonic waves, etc., and the microbial cell is removed by centrifugation. The material can be recovered. The target substance to be recovered may be a free form, a salt thereof, or a mixture thereof. Examples of the salt include sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, and potassium salt.

  The target substance to be recovered may contain components such as microorganisms, medium components, moisture, and microorganism metabolic byproducts in addition to the target substance. The target substance may be purified to a desired degree. The purity of the target substance to be recovered may be, for example, 50% (w / w) or more, preferably 85% (w / w) or more, particularly preferably 95% (w / w) or more (JP1214636B, USP5, 431,933, USP4,956,471, USP4,777,051, USP4,946,654, USP5,840,358, USP6,238,714, US2005 / 0025878)).

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.

[Example 1]
(Cultivation of plants)
Mizuna (Brassica rapa var. Laciniifolia) seeds were sown in hydroponics pots, and ergothioneine content 223 μg / 10 mL (100 μM) hydroponic culture solution [Liquid fertilizer (liquid fertilizer (UH-ZK020), manufactured by OAT Aguri Co., Ltd.) , 133-fold diluted) with ergothioneine (L-(+)-ergothioineine, Item No. 14905, Cayman Chemical, CAS 497-30-3)]. On the 8th day after sowing, the seedlings cultivated above were harvested.

(Measurement of ergothioneine)
The amount of ergothioneine was measured according to the method described in the literature (Journal of Bioscience and Bioengineering VOL. 119 No. 3, 310-313, 2015). To 1 mg of the portion obtained by removing the root from the seedling obtained above, 10 μL of the extract (final concentration of 5 μM D-camphor-10-sodium sulfonate 0.1 μL diluted with ultrapure water, final concentration 99% (w / W) methanol (9.9 μL) was added and ground using a mortar and pestle, and then centrifuged at 15000 rpm, 4 ° C. for 3 minutes. 10 μL of 2M Tris-HCl buffer (pH 8.8) is added to 100 μL of the collected supernatant, and 10 μL of 20 mM Monobrombiane (mBBr) is further added, and the mixture is stirred for 10 minutes at 15000 rpm, 4 ° C. for 3 minutes. And centrifuged. 80 μL of the supernatant was collected and dried with a centrifugal evaporator for about 2 hours to dry the supernatant. After adding 60 μL of ultrapure water to the dried supernatant and resuspending it, the mixture was centrifuged at 15000 rpm, 4 ° C. for 3 minutes, and 50 μL of the resulting supernatant was transferred to a sample cup, of which 5 μL was transferred. LC-MS / MS analysis was performed to determine the amount of ergothioneine (not oxidized).

(Calculation of ergothioneine amount)
Using an ergothioneine sample with ultrapure water as the solvent, ergothioneine is measured by LC-MS / MS analysis similar to the above, and the relative peak area value is obtained, and an approximation obtained by plotting the average value of three independent measurements A straight calibration curve was created (FIG. 1). The ergothioneine content (μg) was estimated from the obtained relative peak area value. The results are shown in Table 1.
Further, the accumulation rate of ergothioneine = (ergothioneine accumulation per 100 g of fresh weight / total addition of ergothioneine to hydroponic culture medium) × 100 was estimated. The results are shown in Table 2.

[Example 2]
In Example 1 above, except that lettuce (Lactuca sativa) was used instead of Mizuna, the plant body was cultivated in the same manner as in Example 1 above, and the amount of ergothioneine contained in the sprout and the accumulation rate of ergothioneine were determined. . The results are shown in Tables 1 and 2.

[Example 3]
In Example 1 above, except that Nozawana (Brassica rapa var. Hababura) was used instead of Mizuna, the plant body was cultivated in the same manner as in Example 1 above, and the amount of ergothioneine contained in the seedling and the accumulation rate of ergothioneine Asked. The results are shown in Tables 1 and 2.

[Example 4]
In Example 1 above, except that Komatsuna (Brassica rapa var. Perviridis) was used instead of Mizuna, the plant body was cultivated in the same manner as in Example 1 above, and the amount of ergothioneine contained in the seedling and the accumulation rate of ergothioneine Asked. The results are shown in Tables 1 and 2.

[Comparative Examples 1-4]
In Examples 1-4 above, instead of the hydroponic culture solution containing ergothioneine, a hydroponic culture solution not containing ergothioneine [liquid fertilizer (liquid fertilizer (UH-ZK020), manufactured by OAT Aguri Co., Ltd., 133-fold dilution] )] Was used in the same manner as in Examples 1 to 4 above, and the amount of ergothioneine contained in the seedling and the accumulation rate of ergothioneine were determined. The results are shown in Table 1.

[Example 5]
(Cultivation of plants)
Mizuna (Brassica rapa var. Laciniifolia) seeds were sown in a hydroponics pot and cultivated in a hydroponic culture solution not containing ergothioneine.
Liquid fertilizer (liquid fertilizer (UH-ZK020), manufactured by OAT Aguri Co., Ltd., diluted 133 times) was used as the hydroponic culture solution.
On the 20th day after sowing, instead of the above-mentioned hydroponic broth without ergothioneine, hydroponic broth with liquid content of 568 μg / 225 mL (11 μM) [liquid fertilizer (liquid fertilizer (UH-ZK020), manufactured by OAT Aguri Co., Ltd.) , 133-fold dilution) and ergothioneine (L-(+)-ergothionine, Item No. 14905, Cayman Chemical, CAS 497-30-3)] for 24 hours, then 21 days after sowing Plants were harvested in the eyes (FIG. 2).

(Measurement of ergothioneine)
In Example 1 described above, instead of 1 mg of the part obtained by removing the root from the seedling, 1 g of the leaf tip of the plant obtained above was used, and 1000 μL of the extract (D- with a final concentration of 5 μM diluted with ultrapure water) was used. Ergothioneine was measured in the same manner as in Example 1 except that 10 μL of sodium camphor-10-sulfonate and 990 μL of methanol having a final concentration of 99% (w / w) were added.

(Calculation of ergothioneine amount)
In the same manner as in Example 1 above, the amount of ergothioneine contained in the leaves of the plant body and the accumulation rate of ergothioneine were determined. The results are shown in Tables 1 and 2.

[Example 6]
In Example 5 above, except that lettuce (Lactuca sativa) was used instead of Mizuna, the plant was grown and harvested in the same manner as in Example 5 above (FIG. 2), and the amount of ergothioneine contained in the leaves The accumulation rate of ergothioneine was determined. The results are shown in Tables 1 and 2.

[Example 7]
In Example 5 above, plants were grown and harvested in the same manner as in Example 5 above, except that Nozawana (Brassica rapa var. Hababura) was used instead of Mizuna (Figure 2), and contained in the leaves The amount of ergothioneine and the accumulation rate of ergothioneine were determined. The results are shown in Tables 1 and 2.

[Example 8]
In Example 5 above, except that Komatsuna (Brassica rapa var. Perviridis) was used instead of Mizuna, the plant body was cultivated and harvested in the same manner as in Example 5 above (FIG. 2) and contained in the leaves The amount of ergothioneine and the accumulation rate of ergothioneine were determined. The results are shown in Tables 1 and 2.

[Comparative Examples 5 to 8]
In the above Examples 5 to 8, instead of the above-mentioned hydroponic culture solution containing ergothioneine, hydroponic culture solution not containing ergothioneine [Liquid fertilizer (liquid fertilizer (UH-ZK020), manufactured by OAT Agri Corporation, diluted 133 times] )] Was used, and the plants were grown and harvested in the same manner as in Examples 5 to 8 above (FIG. 2), and the amount of ergothioneine contained in the leaves and the ergothioneine accumulation rate were determined. The results are shown in Table 1.

  Although the ergothioneine was not contained in the plant body (Comparative Examples 1-8) cultivated by the normal hydroponics method, the plant body (Examples 1-8) cultivated with the hydroponics solution containing ergothioneine. ) Contained ergothioneine. Therefore, the plant used in the experiment does not synthesize ergothioneine, and the plant used in the experiment accumulates ergothioneine in the body and can recover even a small amount of ergothioneine contained in the hydroponics medium. It has been shown.

  Moreover, in the cruciferous plants, Mizuna (Examples 1 and 5), Nozawana (Examples 3 and 7), and Komatsuna (Examples 4 and 8), while high accumulation of ergothioneine was observed, In the cruciferous lettuce (Examples 2 and 6), only low levels of ergothioneine accumulated.

[Examples 9 to 10]
(Cultivation of plants)
Nozawana (Brassica rapa var. Hakabura) seeds were sown in a hydroponics pot and cultivated in a hydroponic culture solution not containing ergothioneine.
Liquid fertilizer (liquid fertilizer (UH-ZK020), manufactured by OAT Aguri Co., Ltd., diluted 133 times) was used as the hydroponic culture solution.
On the 27th day after sowing, instead of the above-mentioned hydroponic broth without ergothioneine, hydroponic broth containing ergothioneine with the values shown in Table 3 below [Liquid fertilizer (liquid fertilizer (UH-ZK020), OAT Aguri Co., Ltd.) Cultivated for 5 days using ergothioneine (L-(+)-ergothionine, Item No. 14905, Cayman Chemical, CAS 497-30-3)] and then seeded 32 Plants were harvested on the day.

(Measurement of ergothioneine)
Ergothioneine was measured in the same manner as in Example 5 above.

(Calculation of ergothioneine amount)
In the same manner as in Example 1 above, the amount of ergothioneine contained in the leaves of the plant body was determined. The results are shown in Table 3.

  Ergothioneine was contained in the plants (Examples 9 to 10) cultivated with the hydroponics solution containing ergothioneine.

[Example 11]
(Production of recombinant E. coli strain)
An Escherichia coli strain (pDES pQE88-Ms-egtABCDE) that was modified to produce ergothioneine and further modified to increase the ability to produce ergothioneine was prepared.
More specifically, a pDES plasmid was retained by transformation in an Escherichia coli metJ gene disruption strain, and a pQE88-Ms-egtABCDE plasmid was retained by transformation.
As shown in FIG. 3, the pDES plasmid (WO2012 / 137689) is linked to a strong expression promoter (ompA) with feedback inhibition sensitive mutant SerA, feedback inhibition sensitive mutant CysE, and wild type YdeD (cysteine excretion carrier). In addition, the cysteine can be mass-produced extracellularly by forced expression of these three genes.
The plasmid pQE88-Ms-egtABCDE encodes the egtABCDE gene operon from Mycobacterium smegmatis. These genes are a group of genes necessary for biosynthesis of ergothioneine from substrates such as histidine, S-adenosylmethionine, and cysteine. This operon gene was linked to a promoter capable of inducing expression when IPGT was added. The egtABCDE gene is an essential gene group for ergothioneine synthesis in Escherichia coli, which is not used in E. coli.
The nucleotide sequence of the above mycobacterium smegmatis egABCDE gene operon and the nucleotide sequence of pQE88-Ms-egtABCDE are shown in SEQ ID NOS: 1 and 2, respectively.

(culture)
The Escherichia coli strain (pDES pQE88-Ms-egtABCDE) prepared above was cultured in jar fermenter under the following culture conditions.
Temperature: 30 ° C
Stirring: Stirring blade 490 rpm
pH: Control to 6.9-7 (add ammonia water when pH 6.9 or lower, add sulfuric acid when pH 7 or higher)
Ventilation: 1L / min
Inoculation: The preculture was an LB medium containing antibiotics, and cultured overnight (16 hours) in a conical flask with baffles while swirling at 30 ° C. Of this pre-culture solution, the amount of the culture solution containing “the amount of cells corresponding to 400 mL for OD ₆₆₀ = 1” was centrifuged, the supernatant was discarded, and the resulting cell pellet was replaced with a new 20 mL LB medium. The whole amount was put into an initial medium, and other components were added sequentially, followed by culturing for 144 hours under the above conditions.
In addition, the following 1st feed liquid and 2 feed liquids were added to the culture solution intermittently at constant pace over 24 to 72 hours after the start of culture.
Tetracycline, Ampicillin, and antifoaming agent were all added at the beginning of the culture, and IPTG and Pyridoxine.HCl were added all 24 hours after the start of the culture.

-Initial medium (1) Basic medium: Basic components (1 L)
(2) Separate added carbon source: 40% (w / v) glucose (100 mL)
(3) Feed liquid (0.9L)
(3-1) Feed 1 liquid (total 600 mL)
40% (w / v) glucose (400 mL) + 1M Na ₂ S ₂ O ₃ or 2M Na ₂ SO ₄ (100 mL) + H ₂ O (100 mL)
(3-2) Feed 2 liquid (300 mL)
15.2 g / L histidine / HCl / H ₂ O, 11.5 g / L serine / other ingredients (expression induction, antibiotics, enzyme activation, growth inhibition relaxation, metabolic control substances, etc.)

  Table 4 shows the composition of the initial medium and other components.

(Quantitative determination of ergothioneine)
A sample obtained by diluting the culture supernatant after the culture was subjected to LC-MS / MS analysis, and the area area of the mass chromatography peak at m / z corresponding to ergothioneine and the elution time was measured.
In addition, the sample in which the ergothioneine sample at a constant concentration was added to the sample was similarly analyzed, and the peak area value representing the ergothioneine content was converted into the ergothioneine concentration using the obtained calibration curve.
As a result of the measurement, 523 mg / L and 700 mg / L ergothioneine fermentation production into the culture supernatant was confirmed.

Claims (16)

  A plant containing 0.5 μg or more of ergothioneine per 100 g of fresh weight.   The plant body according to claim 1, which contains 20 μg or more of ergothioneine per 100 g of fresh weight.   The plant body of Claim 1 or 2 whose content of the said ergothionein is in a leaf.   The plant body as described in any one of Claims 1-3 which belongs to the Brassicaceae.   A food containing 0.5 μg or more of ergothioneine per 100 g (however, a food containing a microorganism capable of biosynthesis of ergothioneine, a food containing a mushroom capable of biosynthesis of ergothioneine, a fermented food fermented by a microorganism capable of biosynthesis of ergothioneine, And their processed foods are excluded).   The food according to claim 5, which contains 20 μg or more of ergothioneine per 100 g.   A culture of a microorganism capable of biosynthesizing ergothioneine and containing 50 mg / L or more of ergothioneine.   The culture according to claim 7, wherein the microorganism capable of biosynthesizing ergothioneine is a microorganism modified so that cysteine-producing ability is increased.   A fertilizer containing a culture of microorganisms capable of biosynthesis of ergothioneine.   The fertilizer according to claim 9, wherein the microorganism capable of biosynthesizing ergothioneine is a microorganism modified so as to increase cysteine-producing ability.   The fertilizer according to claim 9 or 10, wherein the water content is 30% by mass or less. A method for producing a plant according to any one of claims 1 to 4,
In the cultivation medium containing ergothioneine, it has a cultivation process to cultivate the plant body,
The said culture medium is a manufacturing method containing 1 microgram / L or more of ergothioneine.   The production method according to claim 12, wherein the cultivation medium contains 0.1 mg / L or more of ergothioneine.   The manufacturing method of Claim 12 or 13 in which the said culture medium contains the culture of the microorganisms which can biosynthesize ergothioneine.   The production method according to any one of claims 12 to 14, wherein the microorganism capable of biosynthesizing ergothioneine is a microorganism modified so that cysteine-producing ability is increased. A harvesting step for harvesting the plant body;
The manufacturing method according to any one of claims 12 to 15, wherein the cultivation step is performed within 10 days before the harvesting step.