WO2018151216A1 - Plant body, food, culturing material, fertilizer, and manufacturing method - Google Patents

Abstract

Provided are a plant body, a food, a culturing substance, and a fertilizer that contain ergothioneine, and a method for producing the plant body. The plant body contains 0.5 µg or more of ergothioneine per 100 g of fresh weight. The food contains 0.5 µg or more of ergothioneine per 100 g (excepting foods containing microorganisms capable of biosynthesis of ergothioneine). The culturing substance is a culturing substance of a microorganism capable of biosynthesis of ergothioneine, and contains 50 mg/L or more of ergothioneine. The fertilizer includes the culturing substance of the microorganism capable of biosynthesis of ergothioneine. The production method is a method for producing the plant body, wherein the method has a cultivation step for cultivating the plant body in a culture medium that contains ergothioneine, the culture medium containing 1 µg/L or more of ergothioneine.

Description

Plant, food, culture, fertilizer and production method

The present invention relates to a plant, food, culture and fertilizer containing ergothioneine, and a method for producing the plant.
This application claims priority based on Japanese Patent Application No. 2017-027866 filed in Japan on February 17, 2017, the contents of which are incorporated herein by reference.

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 has an excellent antioxidant effect, which 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 at least one of mushroom-derived ergothioneine and derivatives thereof. This skin external preparation is used for preventing skin aging and the like. It is said that it is excellent.

Japanese Unexamined Patent Publication No. 2012-211120

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

The present invention has been made in view of the above circumstances, and provides a plant, a culture and a fertilizer containing ergothioneine, and a method for producing the plant.

As a result of intensive studies to achieve the above object, the present inventors have found that the present invention has been completed.

That is, the present invention includes the following aspects.
The plant according to the first aspect of the present invention contains 0.5 μg or more of ergothioneine per 100 g of fresh weight.
The plant according to the first aspect may contain 20 μg or more of ergothioneine per 100 g of fresh weight.
In the plant according to the first aspect, the content of the ergothionein may be in the leaves.
The plant according to the first aspect may belong to the Brassicaceae family.

The food according to the second aspect of the present invention contains 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, and biosynthesis of ergothioneine. Except fermented foods fermented by various microorganisms and processed foods thereof).
The food according to the second aspect may contain 20 μg or more of ergothioneine per 100 g.

The culture according to the third embodiment of the present invention is a culture of a microorganism capable of biosynthesizing ergothioneine and contains 50 mg / L or more of ergothioneine.
In the culture according to the third aspect, the microorganism capable of biosynthesizing ergothioneine may be a microorganism modified so as to increase cysteine-producing ability.

The fertilizer according to the fourth aspect of the present invention includes a culture of a microorganism capable of biosynthesis of ergothioneine.
In the fertilizer according to the fourth aspect, the microorganism capable of biosynthesizing ergothioneine may be a microorganism modified so as to increase cysteine-producing ability.
In the fertilizer according to the fourth aspect, the water content may be 30% by mass or less.

The manufacturing method which concerns on the 5th aspect of this invention is a manufacturing method of the plant body which concerns on the said 1st aspect, Comprising: It is a method which has a cultivation process which grows a plant body with the cultivation medium containing ergothioneine, The said cultivation The medium contains ergothioneine 1 μg / L or more.
In the manufacturing method according to the fifth aspect, the culture medium may contain ergothioneine in an amount of 0.1 mg / L.
In the manufacturing method according to the fifth aspect, the culture medium may include a culture of a microorganism capable of biosynthesis of ergothioneine.
The manufacturing method according to the fifth aspect may further include a harvesting step for harvesting the plant body, and the cultivation step may be performed within 10 days before the harvesting step.

The plant body, food, culture and fertilizer of the above aspect contain ergothioneine. According to the manufacturing method of the said aspect, the plant containing ergothioneine is obtained.

In an Example, it is a calibration curve used for calculation of the amount of ergothioneine. FIG. 5 is an image obtained by photographing plants grown in Examples 5 to 8 and Comparative Examples 5 to 8. FIG. 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 the examples below, the inventors can grow ergothioneine in a culture medium containing ergothioneine, thereby allowing ergothioneine to accumulate in the plant body. It has been found that plants 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 contains 10 μg or more of ergothioneine. It may contain, may contain 20 μg or more ergothioneine, may contain 50 μg or more ergothioneine, may contain 100 μg or more ergothioneine, may contain 150 μg or more ergothioneine, 200 μg or more of ergothioneine may be contained, and 250 μg or more of ergothioneine may be contained.

The upper limit value of ergothioneine contained in the plant according to the embodiment is not particularly limited, but may be, for example, 10000 μg per 100 g fresh weight or 1000 μg.

“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 carried out 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 object to be measured as ergothioneine may be measured by including oxidized ergothioneine in addition to the above-mentioned ergothioneine. In the present invention, the mass of ergothioneine is calculated based on the molecular weight of ergothioneine before oxidation. Shall be.
When the oxidized ergothioneine forms a multimer, it is calculated based on the molecular weight 229.3 of the original monomer ergothioneine.

The ergothionein contained in the plant body of the embodiment may be only one type or two or more types.

The type of the plant body of the embodiment is not particularly limited, but an edible plant is preferable, for example, Brassicaceae, Asteraceae, Solanaceae, Legaceae, Uriaceae Examples include plants such as Cucurbitaceae, Poaceae, Rubiaceae, Theaceae, Amaryllidaceae, Apiaceae, Araceae, and the like. Among these, the plant body of the embodiment is preferably a Brassicaceae, Asteraceae or Eggplant family, and more preferably a Brassicaceae 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, for example, Mizuna (Brassica rapa var. Laciniifolia), Nozawana (Brassica rapa var. Hakabura), Komatsuna (Brassica rapa var. Perviridis), Chinese cabbage (Brassica rapa var.sispekinensis), turnip (Brassica rapa var. Glabra, Brassica rapa var. Rapa), cabbage (Brassica oleracea var. sativus var. longipinnatus).

Examples of the Asteraceae plants include lettuce (Lactuca sativa var. サ ラ ダ capiata), sengoku (Glebionis coronaria), and the like.

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), bell pepper (Capsicum annuum) and the like.

Suppose 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 ergothioneine may be in the leaf of the plant body.

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

≪Food≫
The food of the embodiment contains 0.5 μg or more of ergothioneine per 100 g (however, a food containing a microorganism that can biosynthesize ergothioneine, a food containing a mushroom that can biosynthesize ergothioneine, and a microorganism that can biosynthesize ergothioneine. Fermented foods and processed foods).

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. It may contain 20 μg or more of ergothioneine, may contain 50 μg or more of ergothioneine, may contain 100 μg or more of ergothioneine, may contain 150 μg or more of ergothioneine, and may contain 200 μg or more. Ergothioneine may be contained, and 250 μg or more of ergothioneine may be contained.

The upper limit of ergothioneine contained in the food according to the embodiment is not particularly limited, but may be, for example, 10000 μg per 100 g or 1000 μg.

Suppose that the weight of the food is measured in the 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 carried out 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 that includes an edible plant among the plants of the embodiment of the present invention. Moreover, the processed product of the plant body of embodiment is also included in the foodstuff of embodiment. Examples of processed products include extracts, crushed materials, pastes, cooked products, canned foods, canned foods, dried foods, frozen foods, beverages, alcoholic beverages, confectionery, seasonings, fats and oils, jams obtained from the plant body of the embodiment. , Spices, food additives, health foods, supplements and the like.

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.

Ergothionein is excellent in antioxidant effect. Since the food of the embodiment contains 0.5 μg or more of ergothioneine per 100 g, the food is excellent in antioxidant action and has high utility value.

≪Culture and fertilizer≫
The culture according to 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 ergothioneine at 50 mg / L or more, may contain ergothioneine at 100 mg / L or more, may contain ergothioneine at 200 mg / L or more, and ergothioneine at 300 mg / L or more. It may be contained, ergothioneine may be contained at 400 mg / L or more, ergothioneine may be contained at 500 mg / L or more, and 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 10000 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 in “<Artificially modified microorganisms” below.

The culture of the embodiment is obtained by culturing a microorganism capable of biosynthesizing ergothioneine in a medium, and the culture contains 50 mg / L or more of ergothioneine. For the method of obtaining the culture, the following “<Method for producing ergothionein>” 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 water content of the fertilizer of the embodiment is preferably small from the viewpoint of transportability, for example, the water content may be 30% by mass or less, 25% by mass or less, and 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 powder, is excellent in transportability as a fertilizer, and is also usable as a fertilizer. It is good.

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 / L or more and 250 g / L or less, 0.1 mg / L or more and 10000 mg / L or less, 0.5 mg / L or more and 1000 mg / L or less may be sufficient.

≪Plant manufacturing method≫
The production method of the embodiment is a production method of the plant body of the embodiment, and has a cultivation step of cultivating the plant body in a cultivation medium containing ergothioneine, and the cultivation medium contains 1 μg / L or more of ergothioneine. .

As shown in the examples below, the inventors can grow ergothioneine in a culture medium containing ergothioneine, thereby allowing ergothioneine to accumulate in the plant body. It has been found that the plant body of the embodiment containing ergothioneine 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 cultivation 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 1 mg / L or more of ergothioneine, and may contain 1 mg / L of ergothioneine. May contain more than 10 mg / L ergothioneine, more than 50 mg / L ergothioneine, more than 100 mg / L ergothioneine, more than 250 mg / L ergothioneine Alternatively, ergothioneine may be contained in an amount of 500 mg / L or more, and ergothioneine may be contained in an amount of 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, may contain ergothioneine 1000 mg / L or less, may contain ergothioneine 800 mg / L or less, and ergothioneine 300 mg / L or less. It may be contained, 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 1 μg / L or more and 10000 mg / L or less, may contain 0.01 mg / L or more and 1000 mg / L or less, and contains 0.1 mg / L or more and 800 mg / L or less. It may be contained in an amount of 1 mg / L to 300 mg / L.

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 in a state where the roots of the body 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-mentioned “<< 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 details are as described above in “<< culture and fertilizers >>”.

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 that does not contain ergothioneine or contains 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, fertilizer containing a culture of microorganisms 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 containing 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 the state where the cultivation 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 longer, 12 hours or longer, 24 hours or longer, or 3 days or longer. A cultivation process may be performed continuously and may be performed in multiple times.

The manufacturing method of the embodiment may further include a harvesting step for 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 that can biosynthesize ergothioneine, microorganisms that have been artificially modified to improve the production of ergothioneine, and microorganisms that have been artificially modified so that ergothioneine can be biosynthesized Will be described.
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.

The genes involved in ergothioneine synthesis include the egtABCDE gene operon encoding the ergothioneine protein group, the egtABCDE gene operon derived from mycobacteria is preferred, and the egtABCDE gene operon derived from bacteria belonging to the genus Mycobacterium is more preferred The egtABCDE gene operon derived from Mycobacterium smegmatis is more preferable.
The egtABCDE gene may be a conservative variant of egtABCDE derived from various organisms such as Mycobacterium smegmatis (a variant in which the original function is maintained). “Original function” for egtABCDE refers to ergothioneine synthetic activity. 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.

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-producing ability may be obtained, for example, by imparting L-cysteine-producing ability to a microorganism, or by enhancing L-cysteine-producing ability of a microorganism.

L-cysteine-producing ability can be imparted or enhanced by a method conventionally used for microbial breeding (for example, `` Amino Acid Fermentation, Inc., Society Publishing Center, May 30, 1986, first edition issued, See pages 77-100). 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, properties such as auxotrophy, analog resistance, and metabolic control mutation that are 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 strain, 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 resistance or metabolic control mutations and have the ability to produce L-amino acids. Common mutation treatments include X-ray and ultraviolet irradiation, N-methyl-N'-nitro-N-nitroguanidine (MNNG), ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), etc. Treatment with a mutagen is included. Further, the L-amino acid-producing ability can 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 International Publication No. 00/18935, European Patent Application Publication No. 1010755, and the like. A detailed method for enhancing the enzyme activity will be described later.

Furthermore, L-amino acid-producing ability can be imparted or enhanced 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 will be 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. “Enhance L-cysteine biosynthesis system” means that the activity of one or more enzymes selected from enzymes involved in L-cysteine biosynthesis (also called L-cysteine biosynthesis enzymes) is enhanced. 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 L-cysteine biosynthetic enzymes include serine acetyltransferase (SAT) and 3-phosphoglycerate dehydrognase (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 biosynthetic enzyme, for example, any of genes derived from Escherichia bacteria such as Escherichia coli and genes derived from various other organisms can be used. For example, as the SAT gene, the cysE gene has been recloned from a wild strain of Escherichia coli and an L-cysteine secreting mutant, and the nucleotide sequence has been clarified (for example, “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.

Also, since SAT is subject to 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 (see, for example, Japanese Patent Application Laid-Open No. 11-155571). Examples of the above-mentioned “amino acid residues other than lysine residues and leucine residues” include 17 types of amino acid residues other than methionine residues, lysine residues and leucine residues among amino acids constituting ordinary proteins. . Specific examples of the “amino acid residue other than lysine residue and leucine residue” include isoleucine residue and glutamic acid residue. As the mutant SAT, SAT having one or more mutations at amino acid residues 89 to 96 of wild-type SAT (see, for example, US Patent Application Publication No. 2005/0112731), wild-type SAT SAT (mutant gene name cysE5; for example, US Patent Application Publication No. 2005/0112731) having a mutation that replaces the valine residue at position 95 and the aspartic acid residue at position 96 with an arginine residue and a proline residue, respectively. SAT) having a mutation that substitutes the threonine residue at position 167 of wild-type SAT with an alanine residue (mutant gene name cysEX; for example, US Pat. No. 6,218,168, US Patent Application Publication No. 2005 / 0112731). “Wild-type SAT” refers to a 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) that do not have the above-described mutation. “Original function” for SAT refers to SAT activity. Escherichia coli JM39-8 strain (E.coli39JM39-8 (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) was established on November 20, 1997 by the Institute of Biotechnology, Ministry of International Trade and Industry, Ministry of International Trade and Industry (currently, National Institute for Product Evaluation and Technology, Patent Biological Deposit Center, Postal Code: 292-0818, Address: Chiba, Japan. 2-5-8, Kazusa-Kamashita, Motosarazu City), deposited under the accession number of FERM P-16527, transferred to an international deposit under the Budapest Treaty on July 8, 2002. FERM BP-8112 is granted.

Further, 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 (see, for example, “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 altering bacteria 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 having a mutation that deletes the tyrosine residue at position 410 (N-terminal) of wild-type PGD (mutant gene name serA5; see, for example, US Pat. No. 6,180,373).
“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 a method for imparting or enhancing L-cysteine production ability include a method of modifying a microorganism so that an L-cysteine excretion system is enhanced. “Strengthening the 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). . Examples of L-cysteine excretion factors include proteins encoded by the ydeD gene (eamA gene) (see, for example, Japanese Patent Application Laid-Open No. 2002-233384), proteins encoded by the yfiK gene (for example, Japanese Patent Application Laid-Open No. 2004-233). 049237), emrAB, emrKY, yojIH, acrEF, bcr, and each protein encoded by the cusA gene (for example, refer to Japanese Unexamined Patent Publication No. 2005-2873334), protein encoded by the yeaS gene (for example, Japan) JP 2010-1875524 A) is known. Alternatively, a YeaS protein having a mutation in at least one selected from the group consisting of a threonine residue at position 28, a phenylalanine residue at position 137, and a 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, as a method for imparting or enhancing L-cysteine production ability, for example, a method of modifying a microorganism so as to retain a mutant yeaS gene can also be mentioned. 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 (see, for example, 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 replaced with one of serine, glutamine, alanine, histidine, cysteine, and glycine. And at least one mutation selected from the group consisting of a mutation in which the leucine residue at position 188 is substituted with glutamine (see, for example, European Patent Application Publication No. 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. The “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 Escherichia coli K-12 MG1655 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, for the PGD or YeaS protein, the PGD or YeaS protein may be aligned with the wild-type PGD amino acid sequence or the amino acid sequence of the wild-type YeaS protein of Escherichia coli K-121MG1655 strain. The alignment can be performed using, for example, a known gene analysis software. Specific software includes DNA solutions manufactured by Hitachi Solutions, GENETYX manufactured by GENETICS, etc. (for example, “Elizabeth® C. Tyler® et al., Computers® and Biomedical Research, 24 (1), 72-96, 1991). ", BartonBarGJ et al., Journal of molecular biology, 198 (2), 327-37. 1987").

Mutant genes (i.e., mutant SAT genes, mutant PGD genes, or mutant yeaS genes) are wild-type genes (i.e., wild-type SAT genes, wild-type PGD genes, or wild-type yeaS genes) are mutated proteins (i.e., Mutated SAT, mutated PGD, or mutated YeaS protein). 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 (for example, `` Higuchi, R., 61, in, PCR technology, Erlich, H. A Eds., Stockton press (1989), CarCarter, P., Meth. In Enzymol., 154,382 (1987)) and methods using phage (Kramer, W. and Frits, HJ, Meth.in Enzymol ., 154, 350 (1987) ”and“ Kunkel, T. A. 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.

Modification of a microorganism so as 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 (for example, Japanese Patent Application Laid-Open No. 2005-137369). No., European Patent No. 1528108).

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 and cysM genes, 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.

Further, as a method for imparting or enhancing L-cysteine production ability, for example, a method of modifying a microorganism so that the degradation system of L-cysteine is suppressed can also be mentioned. “Inhibiting the L-cysteine degradation system” refers to 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 (for example, Japanese Patent Application Laid-Open No. 11-155571, “Chandra et.al ,, Biochemistry, 21 (1982) 3064-3069)), tryptophanase encoded by the tnaA gene (e.g., Japanese Patent Application Laid-Open No. 2003-169668, `` Austin Newton et al., J. Biol. Chem. 240 (1965) 1211- 1218 "), O-acetylserine sulfhydrylase B encoded by the cysM gene (see, for example, Japanese Patent Application Laid-Open No. 2005-245311), MalY encoded by the malY gene (for example, Japanese Patent Application Laid-Open No. 2005-2005). -245311) Cysteine desulfhydrase encoded by the d0191 gene of Pantoea ananatis (for example, see Japanese Patent Application Laid-Open No. 2009-232844).

Examples of L-cysteine-producing bacteria or parent strains for deriving them include, specifically, E. coli JM15 (e.g., U.S. Pat.No. 6,218,168 transformed with various cysE alleles encoding mutant SAT). E. coli W3110 (U.S. Pat.No. 5,972,663), expression of a gene encoding a protein suitable for excretion of toxic substances into cells, decreased cysteine desulfhydrase activity E.coli (see, for example, Japanese Patent Application Laid-Open No. 11-155571), E.coli W3110 (for example, WO 01/155) having an increased activity of a positive transcriptional regulator of cysteine regulon recoded by the cySB gene. 27307), a plasmid pACYC DES containing a ydeD gene, a mutant cysE gene (cysEX gene), and a mutant serA gene (serA5 gene) (for example, Japanese Patent Application Laid-Open No. 2005-137369 (corresponding US patent application) Publication No. 2005/0124049, E.coli strain E.coli such as to hold the states Patent Publication No. 1528108 Pat)) can be mentioned. 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 that 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 that are biosynthesized using L-cysteine as a starting material is also an enzyme in the biosynthetic pathway of the target compound The activity can be imparted or enhanced by decreasing the activity of an enzyme (including an enzyme that degrades the target compound) in a pathway branched from the biosynthetic pathway.

For example, the ability to produce γ-glutamylcysteine can be imparted or enhanced by at least one of enhancement of γ-glutamylcysteine synthetase activity and reduction of glutathione synthetase activity. The ability to produce glutathione can be imparted or enhanced by enhancing at least one of γ-glutamylcysteine synthetase activity and glutathione synthetase activity. The ability to produce γ-glutamylcysteine or glutathione can also be imparted or enhanced by using a mutant γ-glutamylcysteine synthetase 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 (for example, see Japanese Patent Application 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-producing ability is also due to enhanced activity of enzymes involved in L-methionine biosynthesis such as deficiency of methionine repressor, homoserine transsuccinylase, cystathionine γ-synthase and aspartokinase-homoserine dehydrogenase II. Can be imparted or enhanced (see, for example, Japanese Patent Application Laid-Open No. 2000-139471). In E.coli, the methionine repressor is encoded by the metJ gene, the homoserine transsuccinylase is encoded by the metA gene, the cystathionine γ-synthase is encoded by the metB gene, and the aspartokinase homoserine dehydrogenase II is encoded by the metL gene. . In addition, L-methionine-producing ability can also be imparted or enhanced by using a mutant homoserine transsuccinylase resistant to feedback inhibition by L-methionine (for example, Japanese Patent Application Laid-Open No. 2000-139471, US (See Japanese Patent Application Publication No. 2009/0029424). Since L-methionine is biosynthesized using L-cysteine as an intermediate, L-methionine production ability can be improved by improving L-cysteine production ability (for example, Japanese Patent Application Laid-Open No. 2000-139471). No., US Patent Application Publication No. 2008/0311632). 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) (see, for example, British Patent No. 2075055), 218 strain (VKPM B-8125) having norleucine resistance, which is an analog of L-methionine (see, for example, Russian Patent No. 2209248) E. coli strains such as 73 strains (VKPM B-8126) (see, for example, the specification of Russian Patent No. 2215782).

Further, as examples of L-methionine-producing bacteria or parent strains for inducing them, specifically, for example, AJ13425 (FERM P-16808) derived from E. coli W3110 (for example, Japanese Patent Application Laid-Open No. 2000-139471) (See the publication).
AJ13425 lacks methionine repressor, weakens intracellular S-adenosylmethionine synthetase activity, intracellular homoserine transsuccinylase activity, cystathionine γ-synthase activity and aspartokinase homoserine dehydrogenase II activity Is an enhanced L-threonine-requiring strain. 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 method for imparting or enhancing the above-mentioned production ability of L-methionine is used. It is effective to do. As a method for imparting or enhancing cystathionine-producing ability, specifically, a method using a methionine-requiring mutant (see, for example, Japanese Patent Application Laid-Open No. 2004-22518), cysteine (or a biosynthetic raw material thereof) in a fermentation medium ) And homoserine (or a biosynthetic raw material thereof) are added (for example, see Japanese Patent Application Laid-Open No. 2005-168422). 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 that is 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 enhances methionine adenosyltransferase activity (see, for example, EP 0647712 and EP 1457569), an excretion factor encoded by the mdfA gene It can be imparted or enhanced by strengthening MdfA (see, eg, US Pat. No. 7,410,789).

The gene and protein used for breeding L-cysteine-producing bacteria may have known gene and protein base sequences and amino acid sequences 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 L-cysteine-producing bacteria may be 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. In addition, the microorganism can 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 production 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 is reduced, the L-cysteine production ability of the microorganism can be improved.

The RNA pyrophosphohydrolase and the gene encoding it will be described below.

“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”.

RNA pyrophosphohydrolase includes NudH protein encoded by 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 2968777 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-12KMG1655 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 GenBankBIaccession NC_017531 (VERSI0N NC 017531.l GI: 386014600) in the NCBI database. 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 a 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). (E.g., `` VasilyevN, Serganov A. Structures of RNA complexes with the Escherichia coli RNApyrophosphohydrolase RppH unveil the basis for specific 5'-end-dependent mRNA decay. Biol Chem. 99.)).

The following are examples of conservative variants.

RNA pyrophosphohydrolase gene homologs or RNA pyrophosphohydrolase homologs include, 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 the 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.

As long as the original function of the RNA pyrophosphohydrolase is maintained, 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. It may be a gene encoding a protein having the same. For example, at least one of the N-terminal and C-terminal of the encoded protein may be extended or shortened. The above “one or several” differs depending on the position and type of the protein in the three-dimensional structure of the amino acid residue, and specifically, for example, 1 to 50, 1 to 40 1 or more and 30 or less, preferably 1 or more and 20 or less, more preferably 1 or more and 10 or less, still more preferably 1 or more and 5 or less, and particularly preferably 1 or more and 3 or less.

The above substitution, deletion, insertion or addition of one or several amino acids is a conservative mutation in which the function of the protein is maintained 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 hydroxy 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 are caused by 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. It also includes what happens.

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”.

The RNA pyrophosphohydrolase gene hybridizes under stringent conditions with a probe that can be prepared from the above base sequence, for example, a complementary sequence to the whole or a part of the above 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%. The conditions under which DNAs having a homology of at least% are hybridized and DNAs having lower homology are not hybridized with each other, or normal Southern hybridization washing conditions are “60 ° C., 1 × SSC, 0.1 % SDS ”, preferably“ 60 ° C., 0.1 × SSC, 0.1% SDS ”, more preferably“ 68 ° C., 0.1 × SSC, 0.1% SDS ”at a salt concentration and temperature corresponding to once, preferably twice. The conditions for washing 3 times or less 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, as the probe, a DNA fragment having a length of about 300 bp can be used. When a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

Also, since the codon degeneracy varies depending on the host, the RNA pyrophosphohydrolase gene may be one in which an arbitrary codon is replaced 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 using, for example, a mathematical algorithm. Non-limiting examples of such mathematical algorithms include Myers and Miller (1988) CAB10S 4:11 17 algorithm, smith et al (1981) Adv.Appl, Math. 2: 482 local homology algorithm, Needleman and Wunsch. (1970) J. ア ラ イ メ ン ト Mol. Biol. 48: 443 453 homology alignment algorithm, Pearson and Lipman (1988) Proc, Natl. Acad. Sci. 85: 24442448 Similarity search method, Karlin and Altschul (1993) Proc A modified algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, as described in. Natl. Acad. Sci. USA 90: 5873-5877.

Using a program based on these mathematical algorithms, sequence comparison (alignment) for determining sequence identity can be performed. The program can be appropriately executed by a computer. Such programs include, but are not limited to, PC / Gene program CLUSTAL (available from Intelligents, MountainMoView, Calif.), ALIGN program (Version 2.0), and Wisconsin Genetics Software Package, Version 8 (Genetics Computer GAP, BESTFIT, BLAST, FASTA and TFASTA from Group (GCG), 575 Science Drive, Madison, Wis., USA. Alignment using these programs can be performed using initial parameters, for example. For the CLUSTAL program, see `` HigGlns et al. (1988) Gene 73: 237-244 (1988) '', `` HigGlns et al. (1989) CABIOS 5: 151-153 '', `` Corpet et al. (1988) Nucleic Acids Res. 16: 10881-10890 ”,“ Huang etal. (1992) CABIOS 8: 155-165 ”and“ Pearson et al. (1994) Metho Mol. Biol.24: 307-331 ” Yes.

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. See “Altschul et al. (1997) Nucleic Acids Res. 25: 3389” for Gapped BLAST and PSI-BLAST. When using BLAST, Gapped BLAST, or PSI-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 said gene and protein conservative variant can be applied mutatis mutandis to arbitrary proteins, such as L-cysteine biosynthesis system enzyme, and the gene which codes them.

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

"" Protein activity increases "means that the activity per cell of the protein is increased relative to the 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 increases” means that the molecular teaching per cell of the protein is increased and the function per molecule of the protein is increased compared to the unmodified strain. This means at least one of the states. That is, “activity” in the case of “increasing protein activity” 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. Furthermore, 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 then the activity of a suitable target protein may be imparted.

The protein activity is not particularly limited as long as it is increased compared to the unmodified strain, but may be increased 1.5 times or more, 2 times or more, or 3 times or more compared to the non-modified strain, for example. 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 '' specifically means that the amount of gene transcription (mRNA amount) increases and the amount of gene translation (protein amount) increases. It may mean the state of
Note that “increasing gene expression” is also referred to as “enhanced gene expression”. The expression of the gene may be increased 1.5 times or more, 2 times or more, or 3 times or more, for example, as compared to 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.

Increase in gene copy number 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 (see, for example, “MillerI, J. H. Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory) ''. Examples of gene introduction methods utilizing homologous recombination include, for example, the Red-driven integration method (e.g., `` Datsenko, KA, and Wanner, BL, Proc. Natl. Acad. Sci. USA, 97: 6640- 6645 (2000))), 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, and a suspension vector that does not have a replication origin and functions in a host. Examples thereof include a method used and a transduction method using a phage. Only 1 copy of the gene may be introduced, or 2 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 many 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 (for example, Japanese Patent Laid-Open No. 2-109985, US Pat. No. 5,882,888, European Patent No. 805867) Refer to the book).

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

An increase in the copy number of a gene 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. As vectors that can autonomously replicate in bacteria such as Escherichia coli, specifically, 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 range Vector RSF1010 etc. are mentioned.

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 transcription terminator can be placed 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 heterologous terminator. The terminator may be a terminator specific to the gene to be introduced, or may be a terminator of another gene.

The 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 Publishing, 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. Further, an operon may be constituted by two or more genes and introduced. In the case of introducing two or more genes, for example, when genes encoding two or more proteins are introduced, two or more subunits constituting a single protein complex are selected. When introducing the gene which codes each, and those combinations are mentioned.

The gene to be introduced 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, for example, the base sequence of the gene (see, for example, “Gene, 60 (1), H5127 (1987)”). 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 (see, for example, International Publication No. 2005/010175).

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. More powerful promoters include, for example, known high expression promoters such as T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, PR promoter, and PL promoter. Is mentioned. As a more powerful promoter, a highly active promoter of a conventional promoter may be obtained by using various reporter genes. For example, the activity of the promoter can be increased by bringing the −35 and −10 regions in the promoter region closer to the consensus sequence (see, for example, International Publication No. 00/18935). Examples of highly active promoters include various tac-like promoters (see, for example, Russian Patent Application Publication No. 2418069 (inventor: Katashkina ZI et al.)) And pnlp8 promoter (see, eg, International Publication No. 2010/027045). It is done. An evaluation method of promoter strength and examples of strong promoters are described in Goldstein et al.'S paper “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. Examples of a stronger SD sequence include RBS of gene 10 derived from phage T7 (see, for example, “Olins P.0.et al, Gene, 1988, 73, 227-235”). Furthermore, several nucleotide substitutions, insertions or deletions in the spacer region between the RBS and the start codon, especially in the sequence immediately upstream of the start codon (5'-UTR), greatly contribute to mRNA stability and translation efficiency. It is known to have an effect, 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 (for example, `` Higuchi, R., 61, in PCR technology, rlErlich, H. A. Eds., Stockton press (1989) '', `` Carter, P., Meth in Enzymol., 154, 382 (1987)) and methods using phages (for example, `` Kramer, W. and Frits, H., Meth, in Enzymol., 154, 350 (1987) '', `` Kunkel, T . A. 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 can be found in the `` Codon Usage Database '' (http://wwv.kazusa.or.jp/codon; `` Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000) For example).

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

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

Further, the modification that increases the activity of the protein can 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, UV 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 directly treated 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 of transformation is not particularly limited, and a conventionally known method can be used. The transformation of the microorganism is, for example, a protoplast method (see, for example, `` Gene, 39,281286 (1985) ''), an electroporation method (see, for example, `` Bio / Techn01ogy, 7, 1067-1070 (1989) ''), It can be performed by an electric pulse method (for example, see Japanese Patent Application Laid-Open No. 2-207791).

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

The increase in protein activity 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.

It can be confirmed that the transcription amount of the gene has been increased by comparing the amount of mRNA transcribed from the gene with an unmodified strain such as a wild strain or a parent strain. Examples of methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR and the like (for example, `` 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 with the unmodified strain.

Confirmation that the amount of protein has increased can be performed by Western blotting using an antibody (see, for example, “Molecular cloning (Cold Spring Spring Laboratory, Cold Spring Spring (USA), 2001”). 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 method for increasing the activity of a protein can be used to enhance the activity of an arbitrary protein, such as an L-cysteine biosynthesis enzyme, and to enhance the expression of an arbitrary gene, such as a gene encoding the arbitrary protein.

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

“The protein activity decreases” means that the activity per cell of the protein is decreased as compared to the 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 decreases” means that the number of molecules per cell of the protein is decreased and the function per molecule of the protein decreases compared to the unmodified strain. This means at least one of the states. That is, “activity” in the case of “decrease in protein activity” means not only the catalytic activity of the protein, but also the transcription amount (mRNA amount) of the gene encoding the protein or the translation amount (protein amount). 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 reduced compared to the non-modified strain, for example, 50% or less, 20% or less, 10% or less, 5% or less or 0% compared to the non-modified strain. It may decline.

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.
Specifically, `` gene expression decreases '' specifically means at least one of a decrease in gene transcription amount (mRNA amount) and a decrease in gene translation amount (protein amount). It may mean the state of “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”. The expression of the gene 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.

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, a 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 (inducer, inhibitor, etc.), proteins (transcription factors, etc.), nucleic acids (siRNA, etc.), etc. 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.

Further, 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 can be achieved, for example, by introducing an amino acid substitution (missense mutation) into a coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or adding or deleting 1 to 2 bases. (E.g., `` Journa1 of Biological Chemistry 272: 8611-8617 (1997) '', `` Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998)), (See "Journa 1 of Biological Chemistry 26 116, 20833-20839 (1991)").

Also, 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 '' (e.g., `` Datsenko, K. A, and Wanner, B. L. Proc. Natl. Acad. Sci. U S A. 97: 6640-6645 (2000)), Red-driven integration method and λ phage-derived excision system (e.g., `` Cho, EH, Gumport, R. I ., Gardner, J. FJ Bacteriol. 184: 5200-5203 (2002))) (see, for example, International Publication No. 2005/010175) There are a method using a plasmid containing an origin of replication, a method using a plasmid capable of conjugation transfer, a method using a suicide vector that does not have an origin of replication functioning in a host (for example, U.S. Pat. (See Kaihei 05007491).

Further, the modification that reduces the activity of the protein may be performed by, for example, a mutation treatment. Mutation treatments include X-ray irradiation, UV 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 protein activity 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.

It can be confirmed that the amount of transcription of the gene has been reduced by comparing the amount of mRNA transcribed from the same gene with that of the unmodified strain. Examples of methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR and the like (for example, see “Molecular cloning (Cold Spring Spring Laboratory, 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 (see, for example, “Molecular cloning (Cold Spring Spring Laboratory, 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 the unmodified strain.

It can be confirmed that the gene has been destroyed 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 generates 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 may 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>
As 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 the carbon source include saccharides, organic acids, alcohols, and fatty acids. Examples of the saccharide include glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, molasses, starch hydrolyzate, biomass hydrolyzate, and the like. Examples of organic acids include acetic acid, fumaric acid, citric acid, succinic acid and the like. Examples of alcohols include glycerol, crude glycerol, ethanol and the like.
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, degradation products of these plant organs, and the like. 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 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, organic nitrogen sources, ammonia, urea, and the like. Examples of the ammonium salt include ammonium sulfate, ammonium chloride, and ammonium phosphate. Examples of the organic nitrogen source include peptone, yeast extract, meat extract, soybean protein degradation product, and the like. 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 and phosphoric acid polymers. Examples of the phosphate include potassium dihydrogen phosphate and dipotassium hydrogen phosphate. Examples of the phosphoric acid polymer include 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 other various organic and inorganic components include, for example, inorganic salts, trace metals, vitamins, amino acids, nucleic acids, and peptone, casamino acid, yeast extract, and soy proteolysis containing these. Organic components such as products. Examples of inorganic salts include sodium chloride and potassium chloride. Examples of trace metals include iron, manganese, magnesium, calcium, and the like. Examples of vitamins include vitamin Bl, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12. As other various organic components and inorganic components, one component may be used, or two or more components may be used in combination.

Moreover, when using an auxotrophic mutant strain that requires an amino acid or the like for growth, 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.

Cultivation 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 the culture medium of the seed culture with 1 to 50% (v / v) of the seed culture solution.

Culture 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, feeding 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.

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 be, for example, 5% to 50%, preferably about 10% of the saturated oxygen concentration. Specifically, the 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 or more and 10 or less, preferably pH 5 or more and 8 or less. 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 ° C. or higher and 40 ° C. or lower, preferably 25 ° C. or higher and 37 ° C. or lower. 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 microorganisms are no longer active. By culturing the microorganism under such conditions, the target substance accumulates in the medium.

The formation of the target substance can be confirmed by a known method used for detection or identification of a 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 method used for separation and purification of compounds. Examples of such techniques include ion exchange resin methods (see, for example, `` Nagai, H. et al., Separation Science and Technology, 39 (16), 3691-3710 ''), precipitation methods, membrane separation methods (e.g. , Japanese Laid-Open Patent Publication No. 9-164323, Japanese Laid-Open Patent Publication No. 9-173792) and crystallization methods (see, for example, International Publication No. 2008/078448 and International Publication No. 2008/078646). These methods can be used alone or in appropriate combination. When the target substance accumulates in the microbial cells, for example, the microbial cells are crushed by ultrasonic waves, etc., and the microbial cells are 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.

In addition, 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 (for example, Japan No. 1214636, U.S. Pat.No. 5,431,933, U.S. Pat.No. 4,956,471, U.S. Pat.No. 4,775,151, U.S. Pat.No. 4,494,654, U.S. Pat.No. 5,840,358, U.S. Pat. Description, see US Patent Application Publication No. 2005/0025878).

Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.

[Example 1]
(Cultivation of plants)
Mizuna (Brassica rapa var. Laciniifolia) seeds were sown in a hydroponics pot and cultivated in a hydroponic culture solution with an ergothioneine content of 223 μg / 10 mL (100 μM). Hydroponic broth is liquid fertilizer (liquid fertilizer UH-ZK020, manufactured by OAT Aguri Co., Ltd., diluted 133 times), ergothioneine (L-(+)-ergothioneine, Item No. 14905, Cayman Chemical, CAS 497-30- 3) is added. 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 document “Journal of Bioscience and Bioengineering VOL. 119 No. 3, 310-313, 2015”. To 1 mg of the seedlings obtained above, excluding the root, 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 / After adding 9.9 μL of methanol from w) and grinding with a mortar and pestle, this was centrifuged at 15000 rpm at 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 Monobromobimane (mBBr) is further added, and the mixture is stirred for 10 minutes, under conditions of 15000 rpm, 4 ° C., 3 minutes And centrifuged. 80 μL of the supernatant was recovered 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, and the amount of ergothioneine (not oxidized) was measured.

(Calculation of ergothioneine amount)
Using an ergothioneine sample with ultrapure water as the solvent, measure the ergothioneine by LC-MS / MS analysis similar to the above, obtain the relative peak area value, and plot 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 amount per 100 g of fresh weight / total addition amount 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 seedling 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. Hakabura) 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 to 4]
In the above Examples 1 to 4, the plant body was cultivated in the same manner as in Examples 1 to 4 except that a hydroponic culture solution containing no ergothioneine was used instead of the hydroponic culture solution containing ergothioneine. Then, the amount of ergothioneine contained in the seedling and the accumulation rate of ergothioneine were determined. Hydroponic culture solution that does not contain ergothioneine is liquid fertilizer (liquid fertilizer UH-ZK020, manufactured by OAT Agri Corporation, diluted 133 times). 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 broth containing no ergothioneine.
As a hydroponic culture solution, liquid fertilizer (liquid fertilizer UH-ZK020, manufactured by OAT Agri Corporation, diluted 133 times) was used.
On the 20th day after sowing, instead of the above-described hydroponic culture solution not containing ergothioneine, the plant was cultivated for 24 hours using a hydroponic culture solution having an ergothioneine content of 568 μg / 225 mL (11 μM), and then planted on the 21st day after sowing. The body was harvested (Figure 2). Hydroponic culture solution with ergothioneine content of 568μg / 225mL (11μM) is liquid fertilizer (liquid fertilizer UH-ZK020, manufactured by OAT Agri Co., Ltd., diluted 133 times) to 568μg / 225mL (11μM) ergothioneine (L- (+)-ergothioneine, Item No. 14905, Cayman Chemical, CAS 497-30-3).

(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, except that Nozawana (Brassica rapa var. Hakabura) was used instead of Mizuna, the plant 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.

[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, a plant body was cultivated in the same manner as in Examples 5 to 8 except that a hydroponic culture solution not containing ergothioneine was used instead of the hydroponic culture solution containing ergothioneine. Then, the amount of ergothioneine contained in the leaves and the accumulation rate of ergothioneine were determined. Hydroponic broth without ergothioneine is liquid fertilizer (liquid fertilizer UH-ZK020), manufactured by OAT Aguri Co., Ltd., diluted 133 times. The results are shown in Table 1.

Plants cultivated by a conventional hydroponics method (Comparative Examples 1 to 8) did not contain ergothioneine, but plants cultivated with a hydroponics solution containing ergothioneine (Examples 1 to 8). ) 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 broth containing no ergothioneine.
As a hydroponic culture solution, liquid fertilizer (liquid fertilizer UH-ZK020, manufactured by OAT Agri Corporation, diluted 133 times) was used.
On the 27th day after sowing, instead of the hydroponic culture solution containing no ergothioneine, cultivated for 5 days using the hydroponic culture solution containing ergothioneine at the values shown in Table 3 below, and then on the 32nd day after sowing. Plants were harvested. Hydroponic culture liquid containing ergothioneine is liquid fertilizer (liquid fertilizer UH-ZK020, manufactured by OAT Aguri Co., Ltd., 133-fold diluted) with ergothioneine (L-(+)-ergothioneine, Item No. 14905, Cayman Chemical, CAS 497-30-3).

(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.

In plants (Examples 9 to 10) cultivated with a hydroponic liquid containing ergothioneine, ergothioneine was contained.

[Example 11]
(Production of recombinant E. coli strain)
An E. 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 (see, for example, International Publication No. 2012/137689) has a strong expression promoter (ompA), a feedback inhibition sensitive mutant SerA, a feedback inhibition sensitive mutant CysE, and a wild type YdeD (cysteine). The excretion carrier) has three genes linked to each other, and cysteine can be mass-produced extracellularly by forcibly expressing them.
The pQE88-Ms-egtABCDE plasmid 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. Since the egtABCDE gene does not have Escherichia coli, it is an essential gene group for ergothioneine synthesis in the Escherichia coli used this time.
The nucleotide sequence of the above-mentioned Mycobacterium smegmatis egtABCDE gene operon and the nucleotide sequence of pQE88-Ms-egtABCDE are shown in SEQ ID NOs: 1 and 2, respectively.

(culture)
The E. coli strain (pDES pQE88-Ms-egtABCDE) prepared above was cultured in jar fermenter under the following culture conditions.
Temperature: 30 ° C
Stirring: Stirring blade 490rpm
pH: Control between 6.9 and 7 (add ammonia water when pH is 6.9 or less, add sulfuric acid when pH is 7 or more)
Ventilation: 1L / min
Inoculation: Preculture was LB medium containing antibiotics, and cultured overnight (16 hours) in an Erlenmeyer flask with baffle while swirling at 30 ° C. Of this pre-culture solution, the culture solution containing “the amount of cells corresponding to 400 mL for OD660 = 1” is centrifuged, the supernatant is discarded, and the resulting cell pellet is reconstituted with a new 20 mL LB medium. The suspension was resuspended, and the entire amount was put into an initial medium, and other components were sequentially added and cultured for 144 hours under the above conditions.
In addition, the following feed 1 liquid and feed 2 liquid were added to the culture solution intermittently at a constant pace from 24 hours to 72 hours after the start of culture.
Tetracycline, Ampicillin, and antifoaming agent were all added at the start of the culture, and IPTG and Pyrydoxine · HCl were all added 24 hours after the start of the culture.

・ Initial medium (1) Basic medium: Basic ingredients (1L)
(2) Additional added carbon source: 40% (w / v) glucose (100 mL)
(3) Feed liquid (0.9L)
(3-1) Feed 1 liquid (total 600mL)
40% (w / v) glucose (400 mL) + 1 M Na ₂ S ₂ O ₃ or 2 M Na ₂ SO ₄ (100 mL) + H ₂ O (100 mL)
(3-2) Feed 2 liquid (300mL)
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 m / z corresponding to ergothioneine and the area of the mass chromatography peak at the elution time were 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 in the culture supernatant was confirmed.

Claims (16)

1. A plant containing 0.5 μg or more of ergothioneine per 100 g of fresh weight.
2. 2. The plant according to claim 1, which contains 20 μg or more of ergothioneine per 100 g of fresh weight.
3. The plant according to claim 1 or 2, wherein the content of the ergothioneine is in leaves.
4. The plant according to any one of claims 1 to 3, which belongs to the Brassicaceae family.
5. Food containing 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, fermented food fermented by microorganisms capable of biosynthesis of ergothioneine, and the like Except processed foods).
6. 6. The food according to claim 5, comprising 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 claim 7, wherein the microorganism capable of biosynthesizing ergothioneine is a microorganism modified so as to increase cysteine-producing ability.
9. Fertilizer containing a culture of microorganisms capable of biosynthesis of ergothioneine.
10. The fertilizer according to claim 9, wherein the microorganism capable of biosynthesizing ergothioneine is a microorganism modified so as to increase cysteine production ability.
11. The fertilizer according to claim 9 or 10, wherein the water content is 30% by mass or less.
12. 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 which contains 1 microgram / L or more of ergothioneine.
13. The production method according to claim 12, wherein the cultivation medium contains ergothioneine in an amount of 0.1 mg / L or more.
14. The production method according to claim 12 or 13, wherein the cultivation medium contains a culture of a microorganism capable of biosynthesis of ergothioneine.
15. The production method according to any one of claims 12 to 14, wherein the microorganism capable of biosynthesis of ergothioneine is a microorganism modified so as to increase cysteine-producing ability.
16. Furthermore, it has a harvesting process for harvesting the plant body,
    The production method according to any one of claims 12 to 15, wherein the cultivation step is performed within 10 days before the harvesting step.

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