Method for increasing xanthoquinodine production by pigment synthesis inhibition

Abstract

The xanthoquinodine production method includes culturing a filamentous fungus in culture medium to produce and accumulate xanthoquinodines in the culture medium and / or cells and recovering xanthoquinodines from the culture medium and / or cells. During culture, melanin synthesis is suppressed by the filamentous fungus.

Description

Method for enhancing xanthoquinodine production by inhibiting pigment synthesis

[0001] The present disclosure relates generally to the microbial industry, and more particularly to a method for producing xanthoquinodines using filamentous fungi, which improves xanthoquinodines productivity by suppressing melanin synthesis.

[0002] Coccidiosis is an infectious disease that develops in poultry such as chickens and livestock such as cattle and pigs. Coccidia are protozoan parasites belonging to the order Coccidia in the subclass Coccidiophora of the class Sporozoa. In a narrow sense, they refer to species belonging to the genera Eimeria and Isospora, and primarily parasitize the digestive tracts of chickens, pigs, etc. The symptoms caused by the infestation of these protozoa in the digestive tract, etc., are generally called coccidiosis, and are characterized by diarrhea, bloody stools, and growth inhibition, and in severe cases can even lead to death, making it a major problem for poultry farmers and others.

[0003] Conventionally, sulfa drugs, quinoline drugs, antithiamine drugs, antibiotics, etc. have been put to practical use as anticoccidial agents, and polyether antibiotics such as monensin, salinomycin, and lasalocid are widely used.

[0004] However, in recent years, from the viewpoint of environmental load, there has been an increasing demand for anticoccidial agents derived from microbial metabolites that are highly biodegradable. Known examples of such anticoccidial agents include xanthoquinodin A, which is produced by Humicola sp. FO-888 strain, a type of filamentous fungus (Patent Document 1 and Non-Patent Documents 1 and 2).

[0005] On the other hand, filamentous fungi are known to synthesize melanin using acetyl-CoA as a substrate (Non-Patent Document 3).

[0006] Japanese Patent Application Publication No. 06-116281

[0007] Tabata N et al., 1993, the Journal of Antibiotics, Vol.46, No.5, p.749-755Tabata N et al., 1993, Journal of the American Chemical Society, Vol.115, No.19, p.8558-8564Malika Suthar et al., 2023, Journal of Fungi, 9(9), 891

[0008] However, the biosynthetic pathway of xanthoquinodine in filamentous fungi has not been fully elucidated, and the relationship between the biosynthetic pathway of xanthoquinodine and the melanin synthesis pathway has also been unclear. An object of the present invention is to provide an efficient method for producing xanthoquinodine.

[0009] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, they discovered that the productivity of xanthoquinodines can be improved by suppressing melanin synthesis in filamentous fungi, and thus completed the present invention.

[0010] The present invention provides the following: [1] A method for producing xanthoquinodine, comprising culturing a filamentous fungus in a culture medium to produce and accumulate xanthoquinodine in the culture medium and / or fungal cells, and recovering xanthoquinodine from the culture medium and / or fungal cells, wherein melanin synthesis by the filamentous fungus is suppressed during the culture. [2] The production method according to [1], wherein the melanin is a black pigment. [3] The production method according to [1] or [2], wherein melanin synthesis by the filamentous fungus is suppressed by culturing the filamentous fungus under light shielding. [4] The production method according to [3], wherein the ratio of the culture time under light shielding to the total culture time is 0.85 or more. [5] The production method according to any of [1] to [4], wherein the filamentous fungus is modified so that the activity of a melanin synthase is reduced compared to that of an unmodified strain. [6] The production method according to [5], wherein the activity of the melanin synthase is reduced by disrupting a gene encoding the melanin synthase or by suppressing expression of the gene. [7] The production method according to [6], wherein the gene encoding the melanin synthase is any of the following polynucleotides: a-1) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1 a-2) a polynucleotide that hybridizes under stringent conditions to a complementary sequence of the nucleotide sequence shown in SEQ ID NO: 1 and encodes a protein having melanin synthase activity a-3) a polynucleotide that has 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 and encodes a protein having melanin synthase activity [8] The production method according to [6], wherein the gene encoding the melanin synthase is any of the following polynucleotides: a-1) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1 a-3') a polynucleotide that has 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 and encodes a protein having melanin synthase activity [9] The production method according to any of [1] to [8], wherein the filamentous fungus is a filamentous fungus belonging to the genus Humicola.

[10] A filamentous fungus that has been modified to reduce melanin synthase activity compared to an unmodified strain, thereby enhancing xanthoquinodine production.

[11] The filamentous fungus according to

[10] , wherein the activity of the melanin synthase is reduced by disrupting a gene encoding the melanin synthase or by suppressing expression of the gene.

[12] The filamentous fungus according to

[11] , wherein the gene encoding the melanin synthase is any of the following polynucleotides: a-1) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1 a-2) a polynucleotide that hybridizes under stringent conditions to a complementary sequence of the nucleotide sequence shown in SEQ ID NO: 1, and encodes a protein having melanin synthase activity a-3) a polynucleotide that has 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1, and encodes a protein having melanin synthase activity

[13] The filamentous fungus according to

[11] , wherein the gene encoding the melanin synthase is any of the following polynucleotides. a-1) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1; a-3') a polynucleotide having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 and encoding a protein having melanin synthase activity.

[14] The filamentous fungus according to any of

[10] to

[13] , which is a filamentous fungus belonging to the genus Humicola.

[0011] According to the present invention, a method for producing xanthoquinodine can be provided.

[0012] Diagram showing the synthesis pathways of xanthoquinodine and melanin. The left diagram shows the synthesis pathway when cultured under light, the middle diagram shows the synthesis pathway when the melanin synthesis gene is disrupted, and the right diagram shows the synthesis pathway when cultured in the dark. Photographs of the wild-type strain and the melanin synthesis gene-disrupted strain after plate culture. Diagram showing the results of measuring the amount of xanthoquinodine produced by the wild-type strain and the melanin synthesis gene-disrupted strain. The amount of xanthoquinodine produced is shown as a relative value, with the amount of xanthoquinodine produced by the wild-type strain set to "1". Photographs of the wild-type strain after plate culture under light and in the dark. Photographs of samples 1 to 4 after culture. Diagram showing the expression level of the melanin synthesis gene in the wild-type strain after culture under light and in the dark. The expression level of the melanin synthesis gene is shown as a relative value, with the expression level in culture in the dark set to "1". Diagram showing the amount of xanthoquinodine produced by the wild-type strain after culture under light and in the dark. The amount of xanthoquinodine produced is shown as a relative value when the expression level in culture under light source is set to "1." A diagram showing the expression level of melanin synthesis genes in samples 5 to 7. The expression level of melanin synthesis genes is shown as a relative value when the expression level of melanin synthesis genes in sample 5 is set to "1." A diagram showing the amount of xanthoquinodine produced in samples 5 to 7.

[0013] <1> Method for producing xanthoquinodine of the present invention The method for producing xanthoquinodine of the present invention (also referred to as the "production method of the present invention") comprises culturing a filamentous fungus in a culture medium to produce and accumulate xanthoquinodine in the culture medium and / or fungal cells, and recovering xanthoquinodine from the culture medium and / or fungal cells, wherein melanin synthesis by the filamentous fungus is suppressed during the culture.

[0014] [Xanthoquinodine] The xanthoquinodine produced by the filamentous fungus is not particularly limited, and examples thereof include xanthoquinodine A1, xanthoquinodine A2, xanthoquinodine A3, xanthoquinodine B1, xanthoquinodine B2, and xanthoquinodine B3. The xanthoquinodine may be one or more selected from the group consisting of xanthoquinodine A1, xanthoquinodine A2, xanthoquinodine A3, xanthoquinodine B1, xanthoquinodine B2, and xanthoquinodine B3. The filamentous fungus used in the production method of the present invention may have the ability to produce only one type of xanthoquinodine, or may have the ability to produce two or more types of xanthoquinodine.

[0015] Xanthoquinodin A1 is also known as "rel-2,3,4,7,8,14,15,17-octahydro-1,4α * ,12,13,16-pentahydroxy-10-methyl-8,14,17-trioxo-4aH-7aβ * ,15β * -Ethenonaphtho[2',3':4,5]cyclohepta[1,2-b]xanthene-4aβ * -methyl carboxylate" and also known as "xanthoquinodine C." Xanthoquinodine A2 is also known as "rel-2,3,4,7,8,14,15,17-octahydro-1,4α * ,12,13,16-pentahydroxy-10-methyl-8,14,17-trioxo-4aH-7aβ * ,15β * -Ethenonaphtho[2',3':4,5]cyclohepta[1,2-b]xanthene-4aα *Xanthoquinodine A3 is also known as "methyl 3,4,6,7,13,14-hexahydro-5,8,9-trihydroxy-11-methyl-4,7,13-trioxo-2-[(tetrahydro-5-oxofuran)-2α-yl]-2H-6α,13aα-ethenonaphtho[2',3':4,5]cyclohepta[1,2-g]-1-benzopyran-2α-carboxylate" and is also known as "xanthoquinodine A." Xanthoquinodine B1 is also known as "methyl 1,2,3,5,8,9,15,16-octahydro-1β,4,6,13,14-pentahydroxy-11-methyl-5,9,15-trioxo-17aH-8aβ,16β-ethenonaphtho[2',3':5,6]cyclohepta[1,2-c]xanthene-17aα-carboxylate." Xanthoquinodine B2 is also known as "methyl 1,2,3,5,8,9,15,16-octahydro-1β,4,6,13,14-pentahydroxy-11-methyl-5,9,15-trioxo-17aH-8aβ,16β-ethenonaphtho[2',3':5,6]cyclohepta[1,2-c]xanthene-17aβ-carboxylate." Xanthoquinodine B3 is also known as "3,4,7,8,14,15-hexahydro-5,10,12,13-tetrahydroxy-10-methyl-4,8,14-trioxo-2-[(tetrahydro-5-oxofuran)-2α-yl]-2H-1-oxa-7aβ,15β-etheno-1H-cyclohepta[1,2-a:4,5-b']dinaphthalene-2α-carboxylate" and is also called "xanthoquinodine E."

[0016] The term "xanthoquinodine" is not limited to xanthoquinodine in its free form, but may also include a salt thereof or an adduct formed by xanthoquinodine and another organic or inorganic compound. That is, the term "xanthoquinodine" means, for example, xanthoquinodine in its free form or an adduct of xanthoquinodine. The term "xanthoquinodine" includes, for example, sodium salts, potassium salts, ammonium salts, etc. of xanthoquinodine.

[0017] [Filamentous fungi] The filamentous fungi are not particularly limited as long as they have the ability to produce xanthoquinodines, and examples thereof include filamentous fungi belonging to the genus Aureobasidium, filamentous fungi belonging to the genus Bjerkandera, filamentous fungi belonging to the genus Ceriporiopsis, filamentous fungi belonging to the genus Chrysosporium, filamentous fungi belonging to the genus Coprinus, filamentous fungi belonging to the genus Coriolus, Filamentous fungi belonging to the genus Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix Filamentous fungi belonging to the genus Neurospora, filamentous fungi belonging to the genus Paecilomyces, filamentous fungi belonging to the genus Penicillium, filamentous fungi belonging to the genus Phanerochaete, filamentous fungi belonging to the genus Phlebia, filamentous fungi belonging to the genus Piromyces, filamentous fungi belonging to the genus Pleurotus, Rhizopus Examples of filamentous fungi include filamentous fungi belonging to the genus Schizophyllum, filamentous fungi belonging to the genus Talaromyces, filamentous fungi belonging to the genus Thermoascus, filamentous fungi belonging to the genus Thielavia, filamentous fungi belonging to the genus Tolypocladium, filamentous fungi belonging to the genus Trametes, and filamentous fungi belonging to the genus Trichoderma.

[0018] Among these, the filamentous fungi are preferably those belonging to the genus Humicola. Examples of filamentous fungi belonging to the genus Humicola include filamentous fungi belonging to Humicola nigrescens, Humicola alopallonella, Humicola fuscoatra, Humicola grisea, and Humicola insolens.

[0019] Examples of filamentous fungi belonging to the genus Humicola include Humicola sp. FO-888 strain (NITE P-03105). The Humicola sp. FO-888 strain is a strain disclosed in Non-Patent Document 1 and can be obtained from the Kitasato Microbial Resource Library (https: / / www.kitasato-u.ac.jp / roics / library / ). The FO-888 strain was deposited on January 15, 2020, at the National Institute of Technology and Evaluation, National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (NITE NPMD), Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, and has been assigned the accession number NITE P-03105. The FO-888 strain inherently has the ability to produce xanthoquinodine, and is therefore suitable as a filamentous fungus capable of producing xanthoquinodine.

[0020] The filamentous fungus may also be a strain substantially equivalent to the Humicola sp. FO-888 strain. A "substantially equivalent strain" refers to a microorganism in which the nucleotide sequence of the internal transcribed spacer 1 (ITS-1) region of its rRNA is 97.5% or more, preferably 98% or more, more preferably 98.7% or more, even more preferably 99% or more, and particularly preferably 100% identical to the nucleotide sequence of the ITS-1 region of rRNA of the FO-888 strain (SEQ ID NO: 3). The identity of the nucleotide sequence of the ITS-1 region of rRNA can be calculated using known programs such as BLAST. Furthermore, the filamentous fungus may be a strain bred from the FO-888 strain or a strain substantially equivalent thereto by mutagenesis, genetic recombination, selection of natural mutants, or the like, as long as the effects of the present invention are not impaired.

[0021] The term "ability to produce xanthoquinodine" means the ability of a filamentous fungus to produce and accumulate xanthoquinodine in the culture medium and / or fungal cells to an extent that it can be recovered from the culture medium and / or fungal cells when cultured in the culture medium.

[0022] A filamentous fungus having "the ability to produce xanthoquinodine" may be one that inherently has the ability to produce xanthoquinodine, or one to which the ability to produce xanthoquinodine has been imparted using mutation or DNA recombinant technology.

[0023] In the production method of the present invention, one type of filamentous fungus may be used, or two or more types of filamentous fungi may be used.

[0024] [Culture Conditions] The culture medium used in the production method of the present invention is not particularly limited as long as it allows the filamentous fungus to grow and xanthoquinodine to be produced. As the medium, a nutrient medium containing a carbon source assimilable by the filamentous fungus, a nitrogen source assimilable by the filamentous fungus, and, if necessary, inorganic acid salts, etc., can be used. As the carbon source, glucose, sucrose, molasses, dextrin, cellulose, etc. can be used alone or in combination.

[0025] Specifically, carbohydrates such as glucose, glycerol, fructose, maltose, mannitol, xylose, galactose, ribose, starch, or hydrolysates thereof can be used as carbon sources. The concentration of these carbohydrates is typically 0.1% to 5% of the medium. Other carbon sources that can be used include organic acids such as gluconic acid, pyruvic acid, lactic acid, and acetic acid; amino acids such as glycine, glutamic acid, and alanine; alcohols such as methanol and ethanol; non-aromatic hydrocarbons such as normal paraffin; and various vegetable or animal fats and oils.

[0026] Examples of nitrogen sources that can be used include various inorganic acids such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, and ammonium nitrate, as well as ammonium salts of organic acids; urea, peptone, NZ-amine, meat extract, yeast extract, dried yeast, corn steep liquor, casein hydrolysate, fish meal or digested products thereof, soybean flour or digested products thereof, and defatted soybeans or digested products or hydrolysates thereof; and various amino acids such as glycine, glutamic acid, and alanine.

[0027] Examples of inorganic substances that can be used include various phosphates, magnesium sulfate, sodium chloride, and even trace amounts of heavy metal salts. When using a mutant strain that exhibits auxotrophy, it is necessary to add substances that satisfy the auxotrophy to the medium. However, when using a medium containing natural substances, it may not be necessary to add such nutrients.

[0028] The culture can be carried out using a liquid medium. During the culture, the filamentous fungus may be pre-cultured in a solid medium such as an agar medium and then directly inoculated into the liquid medium, or the filamentous fungus may be pre-cultured in a liquid medium and then inoculated into the liquid medium for the main culture. That is, the culture may be carried out separately into a pre-culture and a main culture. In this case, the culture conditions for the pre-culture and the main culture may or may not be the same. There is no particular limitation on the amount of filamentous fungus cells contained in the medium at the start of the culture. The main culture may be carried out, for example, by inoculating the pre-culture solution into the medium for the main culture at 1 to 50% (v / v).

[0029] The culture can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. When the culture is divided into a preculture and a main culture, for example, both the preculture and the main culture may be carried out by batch culture. Alternatively, for example, the preculture may be carried out by batch culture, and the main culture may be carried out by fed-batch culture or continuous culture.

[0030] Cultivation can be carried out under aerobic conditions, for example, by conventional shaking or aerated agitation cultivation. Submerged aerated agitation cultivation is preferred for industrial applications. The pH of the culture is, for example, 5.0 to 8.0, but it is preferable to cultivate at a pH close to neutral. The culture temperature can be between 20 and 40°C, but is usually maintained at 20 to 32°C (preferably around 25°C).

[0031] The culture time (when the culture is divided into a preculture and a main culture, the culture time for the main culture) may be, for example, 16 hours or more, 20 hours or more, 24 hours or more, 2 days or more, 4 days or more, 6 days or more, 8 days or more, or 10 days or more, or 30 days or less, 25 days or less, 20 days or less, 15 days or less, 10 days or less, 8 days or less, 6 days or less, 4 days or less, 2 days or less, or 24 hours or less, or a compatible combination thereof. Specifically, for example, it may be 16 hours to 30 days, 20 hours to 25 days, 24 hours to 20 days, 2 days to 15 days, 4 days to 10 days, 6 days to 8 days, 8 days to 30 days, 10 days to 25 days, 16 hours to 6 days, 20 hours to 4 days, 24 hours to 2 days, or 16 hours to 24 hours.

[0032] Culture conditions such as medium composition, medium properties, culture temperature, aeration rate, and culture time can be appropriately adjusted and selected to obtain favorable results depending on the type of filamentous fungus used, external conditions, etc. If foaming occurs during liquid culture, antifoaming agents such as silicone oil, vegetable oil, and surfactants can be used as appropriate.

[0033] [Recovery of Xanthoquinodine] In the production method of the present invention, xanthoquinodine can be recovered from the culture medium and / or fungal cells by known techniques used for separating and purifying compounds. When xanthoquinodine accumulates within the fungal cells, for example, the fungal cells can be treated with cellulase or the like commonly used for protoplasting filamentous fungi, followed by disruption of the fungal cells by ultrasonication or other mechanical disruption methods, thereby eluting the xanthoquinodine from the fungal cells into the culture supernatant. Xanthoquinodine can then be recovered by an ion exchange resin method or the like. Recovery of xanthoquinodine from the culture medium, supernatant, etc. can be carried out by conventional techniques such as concentration, crystallization, ion exchange chromatography, medium- or high-pressure liquid chromatography, or a combination thereof.

[0034] The recovered xanthoquinodine may be xanthoquinodine in a free form, a salt or hydrate thereof, an adduct formed by xanthoquinodine and another organic or inorganic compound, or a mixture thereof. One type of xanthoquinodine may be produced by the production method of the present invention, or two or more types may be produced.

[0035] The recovered xanthoquinodine may contain, in addition to xanthoquinodine, for example, fungal cells, medium components, water, metabolic by-products of the filamentous fungus, etc. The xanthoquinodine may be purified to a desired degree. The purity of the recovered xanthoquinodine may be, for example, 50% or more, 85% or more, or 95% or more.

[0036] The type and amount of xanthoquinodine can be determined by known methods, for example, xanthoquinodine accumulated in the culture medium or bacterial cells can be measured by HPLC analysis.

[0037] [Inhibition of Melanin Synthesis] In the culture according to the production method of the present invention, melanin synthesis by the filamentous fungus is inhibited. Melanin is a brown to black pigment biosynthesized by the polymerization of phenolic substances. The melanin is preferably a black pigment, more preferably allomelanin.

[0038] It is known that the raw material for xanthoquinodine is octaketide, which is synthesized from acetyl-CoA (Non-Patent Document 2). Meanwhile, acetyl-CoA is also a substrate for melanin synthesis (Non-Patent Document 3). Therefore, the inventors speculate that by inhibiting melanin synthesis, more acetyl-CoA is used for xanthoquinodine biosynthesis, i.e., metabolic flux shifts toward xanthoquinodine biosynthesis, resulting in improved xanthoquinodine productivity (Figure 1).

[0039] In the production method of the present invention, the inhibition of melanin synthesis can be achieved by culturing under conditions in which melanin synthesis is suppressed, or by culturing using a modified filamentous fungus in which melanin synthesis is suppressed. Specifically, the method for producing xanthoquinodine of the present invention includes the following [Aspect 1] (also referred to as "First Aspect") and [Aspect 2] (also referred to as "Second Aspect"). Aspect 1 and Aspect 2 may be combined. [Aspect 1] A method for producing xanthoquinodine, comprising culturing a filamentous fungus in a culture medium to produce and accumulate xanthoquinodine in the culture medium and / or fungal cells, and recovering xanthoquinodine from the culture medium and / or fungal cells, wherein the culture is performed under conditions in which melanin synthesis by the filamentous fungus is suppressed. [Aspect 2] A method for producing xanthoquinodine, comprising culturing a filamentous fungus in a culture medium to produce and accumulate xanthoquinodine in the culture medium and / or fungal cells, and recovering xanthoquinodine from the culture medium and / or fungal cells, wherein the filamentous fungus is a filamentous fungus that has been modified so that the activity of melanin synthase is reduced compared to an unmodified strain.

[0040] In the first embodiment of the production method of the present invention, for example, culturing can be performed under conditions in which melanin synthesis is suppressed by culturing in the dark (i.e., by providing a step of culturing in the dark). Furthermore, the first embodiment of the production method of the present invention may also be a production method comprising culturing a filamentous fungus in a culture medium in the dark to produce and accumulate xanthoquinodine in the culture medium and / or fungal cells, and recovering xanthoquinodine from the culture medium and / or fungal cells. Here, apart from culturing in the dark, the filamentous fungus described in the above section [Filamentous Fungus] may be cultured under normal conditions, for example, under the conditions described above.

[0041] In the present invention, "under light shielding" means under conditions where light is shielded. Examples of methods for shielding light include culturing in a light-shielding container such as a fermenter or culturing in a dark room.

[0042] When the culture is divided into a preculture and a main culture, the preculture may be performed under light irradiation and the main culture may be performed under shading, or both the preculture and the main culture may be performed under shading, but it is preferable that both the preculture and the main culture are performed under shading.

[0043] In the first embodiment of the production method of the present invention, the culture is carried out under light shielding, but as long as the effects of the present invention are obtained, the culture period may include culturing the filamentous fungus in a culture medium under light irradiation for a certain period of time. Here, the culture under light irradiation may be carried out continuously or by intermittent light irradiation in multiple batches.

[0044] In a first aspect of the production method of the present invention, the ratio of the culture time spent culturing in the dark to the total culture time during culture (main culture, when the culture is divided into a preculture and a main culture) (culture time spent culturing in the dark / total culture time) may be 0.85 or more, 0.90 or more, or 0.95 or more, or may be 1.00 or less, 0.95 or less, or 0.90 or less, or any combination thereof that is compatible. By setting the ratio of the culture time spent culturing in the dark to the total culture time to 0.85 or more, the production amount of xanthoquinodine can be improved. Specifically, the ratio of the culture time spent culturing in the dark to the total culture time may be, for example, 0.85 to 1.00, 0.90 to 0.95, 0.95 to 1.00, or 0.85 to 0.90. Here, the "culture time spent culturing in the dark" may refer to the total culture time during culture in the dark when the culture is performed by intermittently shading the light in multiple sessions.

[0045] The photon flux density under light irradiation is, for example, 10 μmol m －２ s －１ Above, 50 μmol m －２ s －１ Above, 100 μmol m －２ s －１ Above, 200 μmol m －２ s －１ Above, 500 μmol m －２ s －１ or more, or 1000 μmol m －２ s －１ It may be 2000 μmol m or more, －２ s －１ Below, 1500 μmol m －２ s －１ Below, 1000 μmol m －２ s －１ Below, 500 μmol m －２ s －１ Below, 200 μmol m －２ s －１ or less, or 100 μmol m －２ s －１ The photon flux density under light irradiation may be, for example, 10 μmol m－２ s －１ 200μB Q －２ s －１ 、50μs Q －２ s －１ 1500μl Q －２ s －１ 、100μl Q －２ s －１ 1000μl Q －２ s －１ 、200μB Q －２ s －１ 500μl Q －２ s －１ 、500μs Q －２ s －１ 200μB Q －２ s －１ 、1000μB －２ s －１ 1500μl Q －２ s －１ 、10μs Q －２ s －１ 200μB Q －２ s －１ 、50μs Q －２ s －１ 100μB Q －２ s －１ であってもよい。

[0046] In the first aspect of the production method of the present invention, the culture time for culturing under light-shading (when the culture is divided into a preculture and a main culture, the culture time for culturing under light-shading in the main culture) may be 16 hours or more, 20 hours or more, 24 hours or more, 2 days or more, 4 days or more, 6 days or more, 8 days or more, or 10 days or more, or 30 days or less, 25 days or less, 20 days or less, 15 days or less, 10 days or less, 8 days or less, 6 days or less, 4 days or less, 2 days or less, or 24 hours or less, or any combination thereof that is not inconsistent. Specifically, the culture time for culturing under light-shading may be, for example, 16 hours to 30 days, 20 hours to 25 days, 24 hours to 20 days, 2 days to 15 days, 4 days to 10 days, 6 days to 8 days, 8 days to 30 days, 10 days to 25 days, 16 hours to 6 days, 20 hours to 4 days, 24 hours to 2 days, or 16 hours to 24 hours. Here, "culture time in the dark" may mean the total culture time in the dark when the culture is carried out by intermittently shading the light in multiple sessions.

[0047] Furthermore, in the first aspect of the production method of the present invention, the culture time for culturing under light irradiation (when the culture is divided into a preculture and a main culture, the culture time for culturing under light irradiation in the main culture) may be 5 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 3 hours or more, 5 hours or more, or 10 hours or more, or 20 hours or less, 16 hours or less, 12 hours or less, 10 hours or less, or 5 hours or less, or any combination thereof that is not inconsistent. Specifically, the culture time for culturing under light irradiation may be, for example, 5 minutes to 20 hours, 10 minutes to 16 hours, 30 minutes to 12 hours, 1 hour to 10 hours, 3 hours to 5 hours, 5 hours to 20 hours, 10 hours to 16 hours, or 5 minutes to 5 hours. Here, the "culture time for culturing under light irradiation" may refer to the total culture time under light irradiation when the culture under light irradiation is performed by intermittently irradiating light multiple times.

[0048] In the second embodiment of the production method of the present invention, inhibition of melanin synthesis can be achieved, for example, by using a filamentous fungus that has been modified so that the activity of melanin synthase is reduced compared to that of an unmodified strain. For example, the filamentous fungi described above in the section [Filamentous Fungi] can be used for modification.

[0049] By modifying the fungus so that the activity of melanin synthase is reduced compared to that of an unmodified strain, melanin synthesis can be suppressed compared to that of the unmodified strain, thereby improving the xanthoquinodine-producing ability, i.e., increasing xanthoquinodine production by the filamentous fungus compared to that of the unmodified strain. "Enhancing xanthoquinodine production" includes improving (increasing) the amount of xanthoquinodine accumulated in the culture medium and / or fungal cells.

[0050] The term "unmodified strain" refers to a filamentous fungal strain that can serve as a control for the above comparison. An unmodified strain may be any strain prior to modification to reduce melanin synthase activity, and examples thereof include wild-type strains and parent strains. Specific examples of unmodified strains include the filamentous fungi exemplified in the description of filamentous fungi. Specific examples of unmodified strains include wild-type strains and parent strains of filamentous fungi belonging to the genus Humicola (e.g., filamentous fungi belonging to Humicola nigrescens). Specific examples of unmodified strains include Humicola sp. FO-888 strain.

[0051] "Decreased activity of melanin synthase" may mean a decrease in expression of melanin synthase. Furthermore, "decreased activity of melanin synthase" may mean a decrease in the function per molecule of melanin synthase. In other words, modifications that "decrease activity of melanin synthase" include modifications that reduce the number of melanin synthase molecules per cell and modifications that reduce the function per molecule of melanin synthase. Note that "reduced number of melanin synthase molecules per cell" also includes the complete absence of melanin synthase. Furthermore, "decreased function per molecule of melanin synthase" also includes the complete loss of the function per molecule of melanin synthase.

[0052] The degree of reduction in melanin synthase activity is not particularly limited as long as the melanin synthase activity is reduced compared to that of a non-modified strain, and may be, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of a non-modified strain.

[0053] Techniques for reducing melanin synthase activity will be described later. The activity of melanin synthase can be reduced, for example, by disrupting the gene encoding the melanin synthase (also referred to as the "melanin synthase gene") or by suppressing the expression of the melanin synthase gene. In other words, the activity of melanin synthase can be reduced by disrupting the melanin synthase gene or by suppressing the expression of the melanin synthase gene.

[0054] Examples of melanin synthase genes and melanin synthases include those of the filamentous fungi exemplified above in the [Filamentous Fungi] section. The nucleotide sequences of melanin synthase genes and amino acid sequences of melanin synthases derived from various filamentous fungi can be obtained, for example, from public databases such as NCBI and technical literature such as patent documents. The melanin synthase is preferably a polyketide synthase, and the melanin synthase gene is preferably a gene encoding a polyketide synthase. The gene encoding a polyketide synthase is more preferably a gene encoding a polyketide synthase that catalyzes the reaction synthesizing 1,3,6,8-tetrahydroxynaphthalene from acetyl-CoA.

[0055] Specific examples of melanin synthase genes include any of the following polynucleotides: a-1) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1 a-2) a polynucleotide that hybridizes under stringent conditions with the complementary sequence of the nucleotide sequence shown in SEQ ID NO: 1 and encodes a protein having melanin synthase activity (specifically, for example, the activity of catalyzing the reaction of synthesizing 1,3,6,8-tetrahydroxynaphthalene from acetyl-CoA) a-3) a polynucleotide that encodes a protein having the amino acid sequence shown in SEQ ID NO: 2 a-4) a polynucleotide that encodes a protein having an amino acid sequence that is 80% or more, more preferably 90% or more, and particularly preferably 95% or more identical to the amino acid sequence shown in SEQ ID NO: 2, and the protein has melanin synthase activity (specifically, for example, the activity of catalyzing the reaction of synthesizing 1,3,6,8-tetrahydroxynaphthalene from acetyl-CoA) a-5) A polynucleotide encoding a protein having an amino acid sequence containing substitution, deletion, insertion, and / or addition of one or several (e.g., 1 to 50, 1 to 30, 1 to 10, or 1 to 5) amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, wherein the protein has melanin synthase activity (specifically, for example, the activity of catalyzing the reaction of synthesizing 1,3,6,8-tetrahydroxynaphthalene from acetyl-CoA).

[0056] The nucleotide sequence shown in SEQ ID NO: 1 is derived from Humicola sp. strain FO-888. As described in the Examples, the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1 is presumed to be a melanin synthase gene encoding a polyketide synthase that catalyzes the synthesis of 1,3,6,8-tetrahydroxynaphthalene from acetyl-CoA, based on highly identical genes with known functions. The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1 encodes a protein having the amino acid sequence shown in SEQ ID NO: 2.

[0057] "Stringent conditions" refer to conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. For example, these conditions include conditions under which two DNAs with high identity, preferably two DNAs with identity of 80% or more, more preferably two DNAs with identity of 90% or more, and particularly preferably two DNAs with identity of 95% or more, hybridize, but two DNAs with lower identity do not hybridize. Examples of stringent conditions include washing once, preferably two to three times, at a salt concentration and temperature equivalent to 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0058] For example, a "polynucleotide that hybridizes under stringent conditions with the complementary sequence of the base sequence shown in SEQ ID NO: 1" may be a polynucleotide having a base sequence that is 80% or more, more preferably 90% or more, and particularly preferably 95% or more identical to the base sequence shown in SEQ ID NO: 1.

[0059] Furthermore, the melanin synthase gene may be a variant of the melanin synthase gene, so long as it encodes a protein that maintains melanin synthase activity. Specifically, for example, the melanin synthase gene may have a nucleotide sequence that includes one or several nucleotide substitutions, deletions, insertions, and / or additions in the nucleotide sequence of the original melanin synthase gene, so long as it encodes a melanin synthase that maintains melanin synthase activity. Here, "one or several" means, for example, 1 to 50, 1 to 30, 1 to 10, or 1 to 5 nucleotides. Furthermore, the melanin synthase gene may be a partial sequence in which the 5'-end and / or 3'-end of the nucleotide sequence of the original melanin synthase gene is deleted, so long as it encodes a melanin synthase that maintains melanin synthase activity.

[0060] For example, a "polynucleotide that hybridizes under stringent conditions with the complementary sequence of the base sequence shown in SEQ ID NO: 1" may be a polynucleotide having a base sequence that includes substitution, deletion, insertion, and / or addition of one or several bases in the base sequence shown in SEQ ID NO: 1.

[0061] Modifications that reduce melanin synthase activity can be achieved, for example, by suppressing the expression of the melanin synthase gene. "Suppression of melanin synthase gene expression" means that the expression of the melanin synthase gene is reduced compared to an unmodified strain such as a wild-type strain or a parent strain. "Suppression of melanin synthase gene expression" specifically means that the expression level per cell of the melanin synthase gene is reduced compared to an unmodified strain. "Suppression of melanin synthase gene expression" may more specifically mean a reduction in the transcription level (mRNA level) of the melanin synthase gene and / or a reduction in the translation level (protein level) of the melanin synthase gene. "Suppression of melanin synthase gene expression" also includes cases where the melanin synthase gene is not expressed at all. The expression of the melanin synthase gene may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of an unmodified strain.

[0062] Suppression of melanin synthase gene expression may be due to, for example, reduced transcription efficiency, reduced translation efficiency, or a combination thereof. Suppression of melanin synthase gene expression can be achieved, for example, by modifying expression regulatory sequences such as the gene promoter, the Shine-Dalgarno (SD) sequence (also known as the ribosome binding site (RBS)), or the spacer region between the RBS and the start codon. When modifying an expression regulatory sequence, preferably one or more bases, more preferably two or more bases, and particularly preferably three or more bases are modified. Gene transcription efficiency can be reduced, for example, by replacing the promoter of a gene on a chromosome with a weaker promoter. A "weaker promoter" refers to a promoter that weakens gene transcription compared to the native wild-type promoter. Examples of weaker promoters include inducible promoters. In other words, inducible promoters can function as weaker promoters under non-inducing conditions (e.g., in the absence of an inducer). Alternatively, a partial or complete region of the expression regulatory sequence may be deleted (deleted). Reduction of gene expression can also be achieved, for example, by manipulating factors involved in expression control. Factors involved in expression control include small molecules (inducers, inhibitors, etc.), proteins (transcription factors, etc.), and nucleic acids (siRNA, etc.) involved in transcription and translation control. Suppression of melanin synthase gene expression can also be achieved, for example, by introducing a mutation into the coding region of the gene that suppresses expression of the melanin synthase gene. For example, the expression of the melanin synthase gene can be suppressed by replacing a codon in the coding region of the melanin synthase gene with a synonymous codon that is used less frequently in the host. Furthermore, for example, disruption of the melanin synthase gene, as described below, can suppress the expression of the gene itself.

[0063] Furthermore, a modification that reduces the activity of melanin synthase can be achieved, for example, by disrupting the melanin synthase gene. "Disruption of the melanin synthase gene" means that the melanin synthase gene is modified so that it does not produce a normally functioning melanin synthase. "Not producing a normally functioning melanin synthase" includes cases where no melanin synthase is produced from the melanin synthase gene at all, and cases where the melanin synthase gene produces a melanin synthase with reduced or lost function per molecule (e.g., activity or properties).

[0064] Disruption of a melanin synthase gene can be achieved, for example, by disrupting (also referred to as deleting or deficiency) the melanin synthase gene on a chromosome. In the present invention, "disrupting a melanin synthase gene" means that the melanin synthase gene has been modified, resulting in a reduction or deletion of the gene's original function. "Disruption of a melanin synthase gene" refers to the deletion of a portion or the entire coding region of the melanin synthase gene. Furthermore, the entire melanin synthase gene may be deleted, including the sequences before and after the coding region of the melanin synthase gene on the chromosome. The sequences before and after the coding region of the melanin synthase gene may include, for example, an expression regulatory sequence for the melanin synthase gene. As long as the activity of the melanin synthase can be reduced, the region to be deleted may be any region, such as the N-terminal region (the region encoding the N-terminal side of the protein), an internal region, or a C-terminal region (the region encoding the C-terminal side of the protein). Generally, the longer the region to be deleted, the more reliably the gene can be inactivated. The region to be deleted may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the total length of the coding region of the melanin synthase gene. Furthermore, it is preferable that the sequences before and after the region to be deleted do not have the same reading frame. Reading frame mismatches can cause frameshifts downstream of the region to be deleted.

[0065] Disruption of the melanin synthase gene can also be achieved by, for example, introducing an amino acid substitution (missense mutation) into the coding region of the melanin synthase gene on the chromosome, introducing a stop codon (nonsense mutation), or introducing an addition or deletion of 1 to 2 bases (frameshift mutation).

[0066] Disruption of the melanin synthase gene can also be achieved, for example, by inserting another base sequence into the coding region of the melanin synthase gene on the chromosome. The insertion site may be anywhere in the melanin synthase gene, but the longer the inserted base sequence, the more reliably the melanin synthase gene is inactivated. Furthermore, it is preferable that the sequences before and after the insertion site do not match in reading frame. A mismatch in reading frame can cause a frameshift downstream of the insertion site. The other base sequence is not particularly limited as long as it reduces or eliminates the activity of the encoded melanin synthase, but examples include marker genes such as antibiotic resistance genes and genes useful for producing target substances.

[0067] Disruption of a melanin synthase gene may be carried out, in particular, so as to delete (delete) the amino acid sequence of the encoded melanin synthase. In other words, a modification that reduces melanin synthase activity can be achieved, for example, by deleting the amino acid sequence of the melanin synthase (a part or all of the amino acid sequence), specifically by modifying the gene to encode a protein lacking the amino acid sequence (a part or all of the amino acid sequence). Note that "deletion of the amino acid sequence of the melanin synthase" refers to the deletion of a part or all of the amino acid sequence of the melanin synthase. Furthermore, "deletion of the amino acid sequence of the melanin synthase" refers to the absence of the original amino acid sequence in the protein, and also includes cases where the original amino acid sequence is changed to a different amino acid sequence. For example, a region that has been changed to a different amino acid sequence due to frameshifting can be considered a deleted region. Deletion of the amino acid sequence of the melanin synthase typically shortens the overall length of the protein, but it may also remain unchanged or be extended. For example, by deleting part or all of the coding region of the melanin synthase gene, the region encoded by the deleted region can be deleted in the amino acid sequence of the melanin synthase. Furthermore, by introducing a stop codon into the coding region of the melanin synthase gene, the region encoded by the region downstream of the introduction site can be deleted in the amino acid sequence of the encoded melanin synthase. Furthermore, by causing a frameshift in the coding region of the melanin synthase gene, the region encoded by the frameshift site can be deleted. The same explanations as for the position and length of the region to be deleted in the deletion of the amino acid sequence can be applied mutatis mutandis.

[0068] In addition, deletion, substitution, or insertion of all or part of the base sequence of the melanin synthase gene can be performed using homologous recombination technology or genome editing technology using a combination of gene editing nucleases, zinc finger nuclease proteins (ZFN), TAL effector nucleases (TALEN), or CRISPER-Cas9.

[0069] Alternatively, a modification to reduce the activity of melanin synthase may be performed by, for example, mutation treatment, such as irradiation with X-rays, ultraviolet light, or treatment with a mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), or methyl methanesulfonate (MMS).

[0070] In addition, when melanin synthase has multiple isozymes, the activities of all or only some of the isozymes may be reduced as long as the resulting melanin synthase activity is reduced. That is, for example, all or only some of the genes encoding those isozymes may be disrupted.

[0071] The above-mentioned methods for reducing the activity of melanin synthase may be used alone or in any combination.

[0072] A decrease in melanin synthase activity can be confirmed by measuring the activity of melanin synthase. A decrease in melanin synthase activity can also be confirmed by confirming a decrease in expression of the melanin synthase gene. A decrease in expression of the melanin synthase gene can be confirmed by confirming a decrease in the transcription level of the melanin synthase gene or a decrease in the amount of melanin synthase expressed from the melanin synthase gene.

[0073] The reduction in the transcription level of the melanin synthase gene can be confirmed by comparing the amount of mRNA transcribed from the melanin synthase gene with that of an unmodified strain. Methods for assessing the amount of mRNA include Northern hybridization, RT-PCR, microarray, RNA-seq, etc. The amount of mRNA (e.g., number of molecules per cell) may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of an unmodified strain.

[0074] The reduction in the amount of melanin synthase can be confirmed by Western blotting using an antibody. The amount of melanin synthase (e.g., the number of molecules per cell) may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of an unmodified strain.

[0075] Disruption of the melanin synthase gene can be confirmed by determining the base sequence, restriction enzyme map, or full length of a part or all of the melanin synthase gene, depending on the means used for the disruption.

[0076] In the present invention, "enhanced production of xanthoquinodine" or "improved productivity of xanthoquinodine" means, for example, that the amount of xanthoquinodine produced is increased when the culture is performed in the dark compared to when the culture is performed under light irradiation. Specifically, it can mean, for example, that 1.1-fold or more, 1.3-fold or more, or 1.5-fold or more of xanthoquinodine is produced and accumulated in the culture medium and / or fungal cells compared to when the culture is performed under light irradiation. In the present invention, "enhanced production of xanthoquinodine" or "improved productivity of xanthoquinodine" means, for example, that when a filamentous fungus that has been modified so that the activity of melanin synthase is reduced compared to a non-modified strain is used, the amount of xanthoquinodine produced is increased compared to the non-modified strain. Specifically, it can mean, for example, that 1.1-fold or more, 1.3-fold or more, or 1.5-fold or more of xanthoquinodine is produced and accumulated in the culture medium and / or fungal cells compared to the non-modified strain.

[0077] The amount of xanthoquinodine produced can be measured by known methods, for example, xanthoquinodine accumulated in the culture medium or cells can be measured by HPLC analysis.

[0078] In the present invention, "melanin synthesis is suppressed" means, for example, that when culture is performed under light shielding, the amount of melanin synthesis is reduced compared to when culture is performed under light irradiation, and specifically, for example, that melanin is produced and accumulated in the culture medium and / or fungal cells in an amount that is 0.6-fold or less, 0.4-fold or less, or 0.2-fold or less compared to when culture is performed under light irradiation. Furthermore, in the present invention, "melanin synthesis is suppressed" means, for example, that when a filamentous fungus that has been modified so that the activity of melanin synthase is reduced compared to an unmodified strain is used, the amount of melanin synthesis is reduced compared to an unmodified strain, and specifically, for example, that melanin is produced and accumulated in the culture medium and / or fungal cells in an amount that is 0.6-fold or less, 0.4-fold or less, or 0.2-fold or less compared to an unmodified strain.

[0079] <2> Filamentous Fungus of the Present Invention The filamentous fungus of the present invention is a filamentous fungus that has been modified to reduce melanin synthase activity compared to an unmodified strain, thereby enhancing xanthoquinodine production. The explanation of the filamentous fungus of the present invention is based on the explanation of the filamentous fungus that has been modified to reduce melanin synthase activity compared to an unmodified strain, described above in "Suppression of Melanin Synthesis."

[0080] Examples of the present invention will be described below, but the present invention is not limited to these examples.

[0081] Reference Example 1: Search for secondary metabolism genes of Humicola sp. strain FO-888 (1-1) Cultivation Humicola sp. strain FO-888, which had been cultured in the dark on CP-added Sabouraud agar medium (Nissui Pharmaceutical Co., Ltd.), was pre-cultured in 100 ml of a pre-culture medium (glucose 2.0 wt %, yeast extract 0.2 wt %, MgSO.7H0 0.05 wt %, Polypepton 0.5 wt %, KHPO 0.1 wt %, agar 0.1 wt %, pH 6.0) at 25°C for 3 days in the dark. Ten ml of the preculture solution was inoculated into 100 ml of the main culture medium (sucrose 2.0 wt%, glucose 1.0 wt%, corn steep liquor 1.0 wt%, meat extract 0.5 wt%, KH2PO4 0.1 wt%, CaCO3 0.3 wt%, agar 0.1 wt%, pH 6.0), and the main culture was carried out at 25°C in the dark.

[0082] (1-2) Whole Genome Analysis of Humicola sp. Strain FO-888 After 48 hours from the start of the main culture, 1 ml of culture medium was centrifuged (8,000 g, 5 minutes) to collect cells, which were then washed with 5 ml of sterile water. Genomic DNA was extracted from the cells using a Quick-DNA Fungal / Bacterial Kit (ZYMO RESEARCH) to prepare a library. The resulting library was run on a NovaSeq™ 6000 (Illumina) to obtain genome data, which was then assembled and used to predict gene regions.

[0083] The whole genome information of the obtained Humicola sp. strain FO-888 was analyzed using antiSMASH, and 48 secondary metabolic genes were extracted (Table 1). Note that the "gene numbers" in Table 1 are numbers assigned by the inventors for convenience.

[0084]

[0085] (1-3) RNAseq: RNAseq was performed using the following method, and highly expressed genes were selected from the secondary metabolism genes listed in Table 1. 48 hours after the start of the main culture in (1-1), 100 mg of FW bacterial cells were harvested from 2 ml of culture medium by centrifugation (8,000 g, 5 minutes). They were then disrupted using a TisseLyzer (Qiagen) and mRNA was extracted using an RNeasy Plus Mini Kit (Qiagen). Subsequently, reverse transcription was performed using the PrimeScript™ RT Reagenet Kit (Takara Bio Inc.) to obtain cDNA. RT-qPCR was performed using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Thermo Fisher Scientific) and normalization was performed using an internal standard gene (act1 gene). For RNA-seq, libraries were prepared using the MGEIE Easy RNA Directional Library Prep Set (MGI Tech), and sequencing analysis was performed using a DNBSEQ-G400 (MGI Tech). Sequence data was mapped to the genome sequence using hisat2 (2.2.1), and the number of reads mapped to genes (gene expression levels) was evaluated using FeatureCounts. The length of intercellular and intracellular gene sequences was corrected using the TPM (Transcripts Per Kilobase Million) method.

[0086] As a result, gene ID 2276_t, which showed high gene expression, was selected. As shown in Table 1, 2276_t is classified as a T1PKS (Type I Polyketide Synthase). A Blast search was performed based on the nucleotide sequence of the 2276_t gene (SEQ ID NO: 1). As shown in Table 2, 2276_t had high identity to a known gene encoding a melanin synthesis polyketide synthase (a polyketide synthase that catalyzes the synthesis of 1,3,6,8-tetrahydroxynaphthalene, an intermediate in melanin synthesis, from acetyl-CoA). Therefore, the present inventors predicted that 2276_t is a gene encoding a melanin synthesis polyketide synthase.

[0087] In the table, "Accession No." indicates the GenBank accession number of the protein encoded by each known gene, and "Organism Name" indicates the name of the organism from which each known gene is derived.

[0088] Example 1: Examination of the effect of melanin synthesis gene disruption on xanthoquinodine productivity (2-1) Creation of a melanin synthesis gene-disrupted strain A melanin synthesis gene-disrupted strain (2276_t-disrupted strain) was created as follows: First, using the genomic DNA of Humicola sp. FO-888 strain as a template, the genomic regions upstream and downstream of 2276_t (SEQ ID NO: 1) were amplified by PCR using the primers shown in Table 3.

[0089] In the table, the base sequences are from left to right, from the 5' end to the 3' end.

[0090] Next, the pHD-G418 vector was constructed by ligating a SphI / BamHI fragment of the G418 resistance gene into the SphI-BamHI gap of the pUC19 vector. The upstream and downstream fragments amplified by PCR were then inserted into both sides of the drug resistance gene (G418) of the pHD-G418 vector, which had been digested with BamHI, SpeI, SacI, and SphI restriction enzymes, to construct a gene disruption plasmid vector (gene disruption vector). The constructed gene disruption vector was used to transform Humicola sp. FO-888 using the method described below in (2-2). The G418 resistance gene was inserted into the target 2276_t gene by homologous recombination, resulting in a melanin synthesis gene disruption strain.

[0091] (2-2) Transformation Method: Humicola sp. FO-888 strain cultured on PDA agar plates (Nissui Pharmaceutical Co., Ltd.) was transferred to GPY medium (Glucose 2.0 wt%, Yeast Extract 0.5 wt%, MgSO4·7H2O 0.05 wt%, Polypepton 0.5 wt%, KH2PO4 0.1 wt%) and cultured with shaking at 25°C for 2 days. The mycelia were collected from the culture medium using a cell strainer (PLS) and washed with sterilized water and 2M MgSO4 aqueous solution. The mycelia were incubated in protoplasting solution (Lysing Enzyme (Sigma) 1.5 wt%, Yatalase (Takara) 0.5 wt%, Cellulase (Onozuka) 0.5 wt%) at 25°C for 8 hours to obtain protoplasts. 50 μl of protoplast solution and 5 μg of plasmid vector were mixed, overlaid with 50 μl of 60% (wt%) PEG 4000, and allowed to stand at room temperature for 30 minutes. 100 μl of recovery medium (glucose 2.0 wt%, yeast extract 0.5 wt%, sorbitol 10.9 wt%, CaCl2 0.2 wt%) was added, and the mixture was allowed to recover for 2 days with gentle shaking at 25°C. Mycelia were collected from the recovery medium and cultured on PDA medium (Nissui Pharmaceutical Co., Ltd.) containing G418 (100 μg / ml) to obtain transformants.

[0092] (2-3) Examination of the effect of disruption of melanin synthesis gene on black pigment synthesis. The obtained melanin synthesis gene disruptant and the wild-type strain (Humicola sp. FO-888 strain) were inoculated onto CP-added Sabouraud agar medium (Nissui Pharmaceutical Co., Ltd.) and incubated under light irradiation (also referred to as "under light source") (photon flux density 100 μmol m -2 s -1 The plates were cultured under a medium temperature of 25°C for 14 days. The results are shown in Figure 2. It was confirmed that the wild-type strain produced black pigment, while the melanin synthesis gene disruptant produced almost no black pigment.

[0093] (2-4) Evaluation of Xanthoquinodine Productivity in the Melanin Synthesis Gene-Disrupted Strain The resulting melanin synthesis gene-disrupted strain and a wild-type strain (Humicola sp. FO-888 strain) were cultured under the following conditions: The melanin synthesis gene-disrupted strain and the wild-type strain were cultured in CP-added Sabouraud agar medium (Nissui Pharmaceutical Co., Ltd.) in the dark, and then pre-cultured in 100 ml of pre-culture medium (glucose 2.0 wt%, yeast extract 0.2 wt%, MgSO4·7H2O 0.05 wt%, Polypepton 0.5 wt%, KH2PO4 0.1 wt%, agar 0.1 wt%, pH 6.0) at 25°C for 2 days in the dark. Ten ml of the preculture was inoculated into 100 ml of the main culture medium (sucrose 2.0 wt%, glucose 1.0 wt%, corn steep liquor 1.0 wt%, meat extract 0.5 wt%, KH2PO4 0.1 wt%, CaCO3 0.3 wt%, agar 0.1 wt%, pH 6.0), and the main culture was carried out at 25°C in the dark.

[0094] Next, the production of xanthoquinodine A1 was measured using the culture medium 96 hours after the start of main cultivation. The culture medium was mixed with an equal volume of ethyl acetate and shaken overnight at room temperature. After separating the supernatant and precipitate by centrifugation (10,000 rpm, 5 minutes), the supernatant was subjected to HPLC analysis. The HPLC system used was a Vanquish (Thermo Fisher) and the Symmetry C18 (Waters) HPLC column. The mobile phase was an acetonitrile:water (containing 0.05 wt% phosphoric acid) gradient of 60:40 to 80:20, with a flow rate of 0.2 m / min, a column temperature of 40°C, and UV detection at 400 nm (ultraviolet spectrophotometer). A calibration curve was prepared using purified xanthoquinodine A1 (Cayman, Catalog No. 32892) at concentrations of 25 ppm, 50 ppm, 100 ppm, and 200 ppm.

[0095] The results are shown in Figure 3. The melanin synthesis gene disruptant showed higher xanthoquinodine production than the wild-type strain. These results suggest that disruption of the 2276_t gene inhibits melanin synthesis from acetyl-CoA, thereby improving the efficiency of xanthoquinodine synthesis from acetyl-CoA and increasing xanthoquinodine productivity.

[0096] Example 2: Examination of the effect of shading culture on xanthoquinodine productivity 1 (3-1) Examination of the effect of plate culture under shading on black pigment synthesis A wild-type strain (Humicola sp. FO-888 strain) was inoculated onto CP-added Sabouraud agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and incubated under light irradiation (also referred to as "under a light source") (photon flux density 100 μmol m -2 s -1 The plates were cultured at 25°C for 14 days under light or shade. The results are shown in Figure 4. It was confirmed that black pigment was produced when cultured under light, but almost no black pigment was produced when cultured in the dark.

[0097] (3-2) Examination of the effect of cultivation under shading on black pigment synthesis. A wild-type strain (Humicola sp. FO-888 strain) plate-cultured on CP-added Sabouraud agar medium (Nissui Pharmaceutical Co., Ltd.) under shading at 25°C for 14 days was incubated in 100 ml of pre-culture medium (glucose 2.0 wt%, yeast extract 0.2 wt%, MgSO4·7H2O 0.05 wt%, Polypepton 0.5 wt%, KH2PO4 0.1 wt%, agar 0.1 wt%, pH 6.0) under light irradiation (photon flux density 100 μmol m -2 s -1 The mixture was pre-cultured for 2 days at 25°C under dark conditions. Ten ml of the pre-culture solution was transferred to 100 ml of the main culture medium (sucrose 2.0 wt%, glucose 1.0 wt%, corn steep liquor 1.0 wt%, meat extract 0.5 wt%, KH2PO4 0.1 wt%, CaCO3 0.3 wt%, agar 0.1 wt%, pH 6.0) and incubated under light irradiation (photon flux density 100 μmol m -2 s -1) or in the dark, the main culture was carried out at 25°C for 6 days (Samples 1 to 4). For Sample 1, pre-culture and main culture were carried out in the dark. For Sample 2, pre-culture was carried out in the dark, and main culture was carried out under a light source. For Sample 3, pre-culture was carried out under a light source, and main culture was carried out in the dark. For Sample 4, pre-culture and main culture were carried out under a light source.

[0098] The results are shown in Figure 5. It was confirmed that black pigment was produced when the main culture was cultivated under a light source (samples 2 and 4). On the other hand, it was confirmed that almost no black pigment was produced when the main culture was cultivated in the dark, regardless of whether the light conditions for the pre-culture were under a light source or in the dark (samples 1 and 3).

[0099] (3-3) Examination of the effect of shading on the expression level of melanin synthesis genes. Cultures were conducted under shading under the following conditions: A wild-type strain (Humicola sp. FO-888) was plate-cultured on CP-added Sabouraud agar medium (Nissui Pharmaceutical Co., Ltd.) for 14 days at 25°C under shading. Then, the wild-type strain was pre-cultured in 100 ml of pre-culture medium (glucose 2.0 wt%, yeast extract 0.2 wt%, MgSO4·7H2O 0.05 wt%, Polypepton 0.5 wt%, KH2PO4 0.1 wt%, agar 0.1 wt%, pH 6.0) under shading for 2 days at 25°C. Ten ml of the preculture was transferred to 100 ml of the main culture medium (sucrose 2.0 wt%, glucose 1.0 wt%, corn steep liquor 1.0 wt%, meat extract 0.5 wt%, KH2PO4 0.1 wt%, CaCO3 0.3 wt%, agar 0.1 wt%, pH 6.0), and the main culture was performed at 25°C for 6 days in the dark. Separately, the preculture and main culture were exposed to light (photon flux density 100 μmol m -2 s -1 The cells were cultured under light irradiation under the same conditions as above, except that the temperature was 100°C.

[0100] RT-qPCR was then performed to measure the expression level of the 2276_t gene. 100 mg of FW cells were harvested from 2 ml of culture medium by centrifugation (8,000 g, 5 minutes) and disrupted using a TisseLyzer (Qiagen). mRNA was extracted using an RNeasy Plus Mini Kit (Qiagen). Subsequently, reverse transcription was performed using a PrimeScript™ RT reagenet Kit (Takara Bio) to obtain cDNA. RT-qPCR was performed using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Thermo Fisher Scientific) and normalization was performed using an internal standard gene (act1 gene).

[0101] The results of RT-qPCR are shown in Figure 6. When cultured under shading, the expression level of the 2276_t gene was significantly lower than when cultured under light. This indicates that shading suppresses the expression of melanin synthesis genes.

[0102] (3-4) Evaluation of xanthoquinodine productivity in dark culture After the completion of the culture in (3-3), the amount of xanthoquinodine A1 produced was measured using the culture medium. The amount of xanthoquinodine A1 produced was measured in the same manner as in (2-4) of Example 1.

[0103] The results are shown in Figure 7. When cultured under shading, the amount of xanthoquinodine produced was significantly higher than when cultured under light. The results of (3-3) and (3-4) suggest that shading culture suppresses melanin synthesis, thereby improving xanthoquinodine productivity.

[0104] Example 3: Examination of the effect of culture in the dark on xanthoquinodine productivity 2 (4-1) Examination of the effect of culture in the dark on the expression level of melanin synthesis genes A wild-type strain (Humicola sp. FO-888 strain) that had been plate-cultured on CP-added Sabouraud agar medium (Nissui Pharmaceutical Co., Ltd.) at 25°C in the dark for 14 days was pre-cultured in 100 ml of pre-culture medium (glucose 2.0 wt%, yeast extract 0.2 wt%, MgSO4·7H2O 0.05 wt%, Polypepton 0.5 wt%, KH2PO4 0.1 wt%, agar 0.1 wt%, pH 6.0) in the dark for 2 days at 25°C. Add 10 ml of preculture solution to 100 ml of main culture medium (sucrose 2.0 wt%, glucose 1.0 wt%, corn steep liquor 1.0 wt%, meat extract 0.5 wt%, KH2PO4 0.1 wt%, CaCO3 0.3 wt%, agar 0.1 wt%, pH 6.0) and under light irradiation (photon flux density 100 μmol m -2 s -1 ) or in the dark, the main culture was carried out at 25°C for 6 days (samples 5 to 7). In the case of sample 5, the main culture was carried out in the dark. In the case of sample 6, the main culture was carried out under light for 16 hours from the start of the main culture, then switched to dark conditions and continued for 128 hours. In the case of sample 7, the main culture was carried out under light for 24 hours from the start of the main culture, then switched to dark conditions and continued for 120 hours.

[0105] The expression levels of the 2276_t gene were measured using Sample 5, which was cultured after the completion of the main culture; Sample 6, which was cultured 16 hours after the start of the main culture (i.e., after the end of the culture under light irradiation); and Sample 7, which was cultured 24 hours after the start of the main culture (i.e., after the end of the culture under light irradiation). The measurement of the expression level of the 2276_t gene was performed using the same method as in Example 2 (3-3) above. The results are shown in Figure 8. The expression levels of the 2276_t gene were significantly lower in Samples 5 and 6 than in Sample 7. These results indicate that even when cultured under light for 16 hours, the expression of melanin synthesis genes was significantly suppressed, similar to when cultured constantly in the dark.

[0106] (4-2) Evaluation of xanthoquinodine productivity in dark culture After completion of the main culture in (4-1), samples 5 to 7 were used to measure the amount of xanthoquinodine A1 produced. The measurement of the amount of xanthoquinodine A1 produced was performed in the same manner as in (2-4) of Example 1.

[0107] The results are shown in Figure 9. Samples 5 and 6 produced approximately the same amount of xanthoquinodine. Furthermore, Samples 5 and 6 produced significantly higher amounts of xanthoquinodine than Sample 7. This indicates that even when culturing was carried out under light for 16 hours during the 6-day main culture, approximately the same amount of xanthoquinodine was produced as when culturing was always carried out in the dark.

Claims

1. A method for producing xanthoquinodine, comprising culturing a filamentous fungus in a culture medium to produce and accumulate xanthoquinodine in the culture medium and / or fungal cells, and recovering xanthoquinodine from the culture medium and / or fungal cells, wherein melanin synthesis by the filamentous fungus is suppressed during the culture.

2. The method of claim 1, wherein the melanin is a black pigment.

3. The method according to claim 1 or 2, wherein the culture is carried out in the dark, thereby suppressing melanin synthesis by the filamentous fungus.

4. The method according to claim 3, wherein the ratio of the time spent culturing under light shielding to the total culture time is 0.85 or more.

5. The production method according to any one of claims 1 to 4, wherein the filamentous fungus is a filamentous fungus that has been modified so that the activity of melanin synthase is reduced compared to an unmodified strain.

6. The method according to claim 5, wherein the activity of the melanin synthase is reduced by disrupting the gene encoding the melanin synthase or by suppressing the expression of the gene.

7. The production method according to claim 6, wherein the gene encoding the melanin synthase is any one of the following polynucleotides: a-1) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1; a-2) a polynucleotide that hybridizes under stringent conditions with the complementary sequence of the nucleotide sequence shown in SEQ ID NO: 1 and encodes a protein having melanin synthase activity; and a-3) a polynucleotide that has 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 and encodes a protein having melanin synthase activity.

8. The production method according to claim 6, wherein the gene encoding the melanin synthase is any one of the following polynucleotides: a-1) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1; a-3') a polynucleotide having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 and encoding a protein having melanin synthase activity.

9. The method according to any one of claims 1 to 8, wherein the filamentous fungus is a filamentous fungus belonging to the genus Humicola.

10. A filamentous fungus that has been modified to reduce the activity of a melanin synthase compared to an unmodified strain, thereby enhancing the production of xanthoquinodines.

11. The filamentous fungus according to claim 10, wherein the activity of the melanin synthase is reduced by disrupting the gene encoding the melanin synthase or by suppressing the expression of the gene.

12. The filamentous fungus according to claim 11, wherein the gene encoding the melanin synthase is any one of the following polynucleotides: a-1) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1; a-2) a polynucleotide that hybridizes under stringent conditions with the complementary sequence of the nucleotide sequence shown in SEQ ID NO: 1 and encodes a protein having melanin synthase activity; and a-3) a polynucleotide that has 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 and encodes a protein having melanin synthase activity.

13. The filamentous fungus according to claim 11, wherein the gene encoding the melanin synthase is any one of the following polynucleotides: a-1) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1; and a-3') a polynucleotide having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 and encoding a protein having melanin synthase activity.

14. The filamentous fungus according to any one of claims 10 to 13, which is a filamentous fungus belonging to the genus Humicola.

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