Filamentous fungus having enhanced secondary metabolite production

Abstract

In the present invention, xanthoquinodin is produced by using a filamentous fungus that has enhanced xanthoquinodin production as a result of being modified such that one or more genes in a xanthoquinodin-synthesizing gene cluster are overexpressed.

Description

Filamentous fungi with enhanced secondary metabolite production

[0001] The present disclosure relates generally to the microbial industry, and more particularly to filamentous fungi that have been modified to overexpress genes in a xanthoquinodine synthesis cluster, thereby enhancing production of xanthoquinodine, a secondary metabolite of filamentous fungi, and a method for producing xanthoquinodine using the filamentous fungi.

[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] Japanese Patent Application Publication No. 06-116281

[0006] 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.8558-8564

[0007] However, the enzymes and transcriptional regulators involved in the xanthoquinodine synthesis pathway in filamentous fungi have not been elucidated, and the functions of genes, including those encoding enzymes and transcriptional regulators involved in xanthoquinodine synthesis, have not been known until now. Furthermore, a transformed filamentous fungus with improved xanthoquinodine productivity and a method for producing xanthoquinodine using the transformed filamentous fungus have not been known until now.

[0008] Therefore, an object of the present invention is to provide a transformed filamentous fungus with improved productivity of xanthoquinodine, and a method for producing xanthoquinodine using the transformed filamentous fungus.

[0009] As a result of intensive research conducted by the inventors to solve the above-mentioned problems, they succeeded in identifying the genes that constitute the xanthoquinodine synthesis gene cluster of filamentous fungi, and further discovered that xanthoquinodine productivity can be improved by modifying filamentous fungi so that one or more genes in the xanthoquinodine synthesis gene cluster are overexpressed, thereby completing the present invention.

[0010] The present invention provides the following: [1] A filamentous fungus that has been modified to overexpress one or more genes in a xanthoquinodine synthesis gene cluster, thereby enhancing xanthoquinodine production. [2] The filamentous fungus of [1], wherein one or more of the overexpressed genes in the xanthoquinodine synthesis gene cluster are genes whose production of xanthoquinodine is suppressed by gene disruption or expression suppression. [3] The filamentous fungus of [1] or [2], wherein the overexpressed gene in the xanthoquinodine synthesis gene cluster is any one of the following polynucleotides: a-1) a polynucleotide having a nucleotide sequence selected from SEQ ID NOs: 1 to 16, a-2) a polynucleotide that hybridizes under stringent conditions to a complementary sequence of a nucleotide sequence selected from SEQ ID NOs: 1 to 16, a-3) a polynucleotide that encodes a protein having an amino acid sequence selected from SEQ ID NOs: 18 to 33, and a-4) a polynucleotide that encodes a protein having 80% or more sequence identity to an amino acid sequence selected from SEQ ID NOs: 18 to 33. [4] The filamentous fungus according to any of [1] to [3], wherein the genes in the xanthoquinodine synthetic gene cluster are overexpressed by overexpressing a gene encoding a transcriptional regulator that activates the transcription of the genes in the xanthoquinodine synthetic gene cluster. [5] The filamentous fungus according to [4], wherein the gene encoding the transcriptional regulator is any of the following polynucleotides: b-1) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 6; b-2) a polynucleotide that hybridizes under stringent conditions with the complementary sequence of the nucleotide sequence shown in SEQ ID NO: 6 and encodes a protein having transcription factor activity; b-3) a polynucleotide that encodes a protein having the amino acid sequence of SEQ ID NO: 23; b-4) a polynucleotide that encodes a protein having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 23. [6] The filamentous fungus according to any of [1] to [5], wherein the overexpression is achieved by retaining a vector containing one or more genes in the xanthoquinodine synthesis gene cluster extrachromosomally in the filamentous fungus.[7] The filamentous fungus according to any of [1] to [5], wherein the overexpression is achieved by introducing a gene construct containing one or more genes in a xanthoquinodine synthetic gene cluster into the chromosome of the filamentous fungus. [8] The filamentous fungus according to any of [1] to [7], which is a filamentous fungus belonging to the genus Humicola. [9] A vector comprising a gene linked to a promoter in an expressible manner, wherein the gene is one or more of the following polynucleotides: a-1) a polynucleotide having a nucleotide sequence selected from SEQ ID NOs: 1 to 16; a-2) a polynucleotide that hybridizes under stringent conditions with a complementary sequence of a nucleotide sequence selected from SEQ ID NOs: 1 to 16; a-3) a polynucleotide encoding a protein having an amino acid sequence selected from SEQ ID NOs: 18 to 33; and a-4) a polynucleotide encoding a protein having 80% or more sequence identity with an amino acid sequence selected from SEQ ID NOs: 18 to 33.

[10] The vector according to [9], wherein the gene is one or more of the following polynucleotides. b-1) A polynucleotide having the nucleotide sequence shown in SEQ ID NO: 6 b-2) A polynucleotide that hybridizes under stringent conditions with a complementary sequence of the nucleotide sequence shown in SEQ ID NO: 6, and encodes a protein having transcription factor activity a-3) A polynucleotide that encodes a protein having the amino acid sequence of SEQ ID NO: 23 a-4) A polynucleotide that encodes a protein having 80% or more sequence identity to the amino acid sequence of SEQ ID NO: 23

[11] The vector according to [9] or

[10] , wherein the promoter is any of the following polynucleotides: c-1) A polynucleotide having a nucleotide sequence selected from SEQ ID NOs: 34 to 38, 44, 45, and 46 c-2) A polynucleotide that hybridizes under stringent conditions with a complementary sequence of a nucleotide sequence selected from SEQ ID NOs: 34 to 38, 44, 45, and 46, and encodes a protein having promoter activity in filamentous fungal cells

[12] A filamentous fungus transformed with the vector according to any of [9] to

[11] .

[13] The filamentous fungus according to

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

[14] A method for producing xanthoquinodine, comprising culturing the filamentous fungus according to any one of [1] to [7],

[11] and

[12] 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.

[0011] According to the present invention, it is possible to provide a transformed filamentous fungus with improved productivity of xanthoquinodine, and a method for producing xanthoquinodine using the transformed filamentous fungus.

[0012] A diagram showing the predicted synthesis pathway of xanthoquinodine. A diagram showing the results of measuring xanthoquinodine production in Humicola sp. strain FO-888. A diagram showing the expression levels of secondary metabolism genes in Humicola sp. strain FO-888. A diagram showing the chemical structures of xanthoquinodine A1 and beticolin 1. A schematic diagram of the xanthoquinodine synthesis gene cluster in Humicola sp. strain FO-888. A diagram showing the presence or absence of xanthoquinodine production in a wild-type strain and a strain with enhanced expression of the nrPKS (8265_t) gene involved in xanthoquinodine main chain synthesis. A diagram showing the results of measuring xanthoquinodine production in a wild-type strain and a strain with enhanced expression of the transcriptional regulator (8270_t) (ots1 strain). A diagram showing the expression levels of each gene constituting the xanthoquinodine synthesis gene in a wild-type strain and a strain with enhanced expression of the transcriptional regulator (8270_t) (ots1 strain). FIG. 1 shows the amount of xanthoquinodine produced by filamentous fungi carrying extrachromosomal vectors that express transcriptional regulators under the control of various promoters.

[0013] <1> Filamentous fungus of the present invention The filamentous fungus of the present invention is a filamentous fungus that has been modified to overexpress one or more genes in the xanthoquinodine synthesis gene cluster (also referred to as the "xanthoquinodine biosynthesis gene cluster"), thereby enhancing the production of xanthoquinodine.

[0014] The xanthoquinodine 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 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] The filamentous fungi are not particularly limited, but 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, and Cryptococcus. Filamentous fungi belonging to the genus Fusarium, Filamentous fungi belonging to the genus Fusarium, Filamentous fungi belonging to the genus Humicola, Filamentous fungi belonging to the genus Magnaporthe, Filamentous fungi belonging to the genus Mucor, Filamentous fungi belonging to the genus Myceliophthora, Filamentous fungi belonging to the genus 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, filamentous fungi belonging to the genus 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] In particular, the filamentous fungus of the present invention is preferably a filamentous fungus 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: 43). 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] <2> Enhancement of xanthoquinodine production The filamentous fungus of the present invention can be obtained by modifying a filamentous fungus capable of producing xanthoquinodine so that it overexpresses one or more genes in the xanthoquinodine synthetic gene cluster. The filamentous fungus of the present invention can also be obtained by modifying the fungus so that it overexpresses one or more genes in the xanthoquinodine synthetic gene cluster, and then imparting or enhancing xanthoquinodine-producing ability. Furthermore, the filamentous fungus of the present invention may have acquired xanthoquinodine-producing ability by being modified so that it overexpresses one or more genes in the xanthoquinodine synthetic gene cluster.

[0022] The filamentous fungus may be one that inherently has the ability to produce xanthoquinodine, or may be one that has been modified using mutation or DNA recombinant technology to have the ability to produce xanthoquinodine.

[0023] The term "ability to produce xanthoquinodine" means the ability of the 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.

[0024] By modifying a filamentous fungus so that it overexpresses one or more genes in the xanthoquinodine synthesis gene cluster, the xanthoquinodine-producing ability of the filamentous fungus can be improved compared to an unmodified strain, i.e., xanthoquinodine production by the filamentous fungus can be enhanced. "Enhancing xanthoquinodine production" includes improving (increasing) the amount of xanthoquinodine accumulated in the culture medium and / or fungal cells.

[0025] The xanthoquinodine synthetic gene cluster is a group of multiple genes involved in the synthesis of xanthoquinodine. Specific examples of genes constituting the xanthoquinodine synthetic gene cluster include the genes constituting the xanthoquinodine synthetic gene cluster in Humicola sp. FO-888 strain, as shown in Table 1. Note that the "gene numbers" in Table 1 are numbers assigned by the inventors for convenience. As described in the Examples below, each gene in the nucleotide sequence shown in Table 1 shared a relatively high identity with a known beticolin biosynthetic gene in Cercospora sp. JNU001 strain. Therefore, the function of each gene in the nucleotide sequence shown in Table 1 was predicted based on the function of the beticolin biosynthetic gene. The predicted synthetic pathway for xanthoquinodine is shown in Figure 1.

[0026]

[0027] The polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 18, which is presumed to be a non-reducing polyketide synthase (nrPKS) that catalyzes the conversion of acetyl-CoA to octa-β-keto-acyl chain via octa-β-keto-acyl chain to octaketide. The polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 19, which is presumed to be a β-lactamase-type thioesterase (MβL-TE) that catalyzes the conversion of octaketide to atrochrysone carboxylic acid. The polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 20, which is presumed to be an oxidase that catalyzes the conversion of emodin anthrone to emodin. A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 21, which is presumed to be a decarboxylase that catalyzes the conversion of atrochrysone carboxylic acid to emodin anthrone. A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 22, which is presumed to be a ring-cleaving dioxygenase (RCDO) that catalyzes the reaction of synthesizing monodictyphenone by opening the anthraquinone ring of chrysophanol. A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 23, which is presumed to be a transcriptional regulator of the xanthoquinodine synthesis gene cluster.A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 24, which is presumed to be an O-methyltransferase that catalyzes the methylation reaction of a xanthoquinodine precursor. A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 25, which is presumed to be an O-methyltransferase that catalyzes the methylation reaction of a xanthoquinodine precursor. A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 26, which is presumed to be an oxidoreductase that catalyzes the conversion of emodin to emodin dihydroquinone. The polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 10 encodes a protein consisting of the amino acid sequence of SEQ ID NO: 27, which is presumed to be a cytochrome P450 that catalyzes the reaction of combining xanthon and anthraquinone to synthesize a xanthoquinodine precursor. The polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 11 encodes a protein consisting of the amino acid sequence of SEQ ID NO: 28, which is presumed to be a dehydratase that catalyzes the conversion of 3-hydroxy-3,4-dihydroanthracene-1(2H)-one to chrysophanol hydroquinone. A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 12 encodes a protein consisting of the amino acid sequence of SEQ ID NO: 29, which is presumed to be a reductase that catalyzes the conversion of emodin dihydroquinone to 3-hydroxy-3,4-dihydroanthracen-1(2H)-one. A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 13 encodes a protein consisting of the amino acid sequence of SEQ ID NO: 30, which is presumed to be a monooxygenase.A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 14 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 31, which is presumed to be a transcriptional regulator of the xanthoquinodine synthesis gene cluster. A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 15 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 32, which is presumed to be a transporter involved in the transport of compounds that serve as substrates for xanthoquinodine synthesis or the transport of synthesized xanthoquinodine. A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 33, which is presumed to be a dehydrogenase (SDR).

[0028] One or more of the genes in the xanthoquinodine synthetic gene cluster that are overexpressed are preferably genes whose production of the xanthoquinodine is suppressed by gene disruption or expression suppression, and examples thereof include any of the following polynucleotides: The genes in the xanthoquinodine synthetic gene cluster that are overexpressed may be one type, two or more types, or all types. a-1) A polynucleotide having a nucleotide sequence selected from SEQ ID NOs: 1 to 16 (preferably a nucleotide sequence selected from SEQ ID NOs: 6 and 14, more preferably the nucleotide sequence shown in SEQ ID NO: 6); a-2) A polynucleotide that hybridizes under stringent conditions with a complementary sequence of a nucleotide sequence selected from SEQ ID NOs: 1 to 16 (preferably a nucleotide sequence selected from SEQ ID NOs: 6 and 14, more preferably the nucleotide sequence shown in SEQ ID NO: 6) (limited to polynucleotides that maintain the function of the encoded protein); a-3) A polynucleotide that encodes a protein having an amino acid sequence selected from SEQ ID NOs: 18 to 33 (preferably an amino acid sequence selected from SEQ ID NOs: 23 and 31, more preferably the amino acid sequence shown in SEQ ID NO: 23); a-4) A polynucleotide that encodes a protein having an amino acid sequence that shares 80% or more, more preferably 90% or more, and particularly preferably 95% or more identity with an amino acid sequence selected from SEQ ID NOs: 18 to 33 (preferably an amino acid sequence selected from SEQ ID NOs: 23 and 31, more preferably the amino acid sequence shown in SEQ ID NO: 23) (limited to polynucleotides that maintain the function of the encoded protein); a-5) A polynucleotide encoding a protein having an amino acid sequence selected from SEQ ID NOs: 18 to 33 (preferably an amino acid sequence selected from SEQ ID NOs: 23 and 31, more preferably the amino acid sequence shown in SEQ ID NO: 23) that includes 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 (limited to polynucleotides that maintain the function of the encoded protein).

[0029] The nucleotide sequences shown in SEQ ID NOs: 1 to 16 are the nucleotide sequences of the coding regions (CDS) of the genes constituting the xanthoquinodine synthesis gene cluster of Humicola sp. FO-888 strain shown in Table 1. The amino acid sequences shown in SEQ ID NOs: 18 to 33 are the amino acid sequences of proteins encoded by polynucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 16, respectively. The nucleotide sequence shown in SEQ ID NO: 17 is the full-length nucleotide sequence of the xanthoquinodine synthesis gene cluster containing the 16 genes constituting the xanthoquinodine synthesis gene cluster of Humicola sp. FO-888 strain.

[0030] In the present disclosure, "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 lower identity, hybridize, but do not hybridize. Examples of such 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.

[0031] For example, a "polynucleotide that hybridizes under stringent conditions to 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. The same applies to polynucleotides that hybridize under stringent conditions to the complementary sequence of the base sequence of a SEQ ID NO: other than SEQ ID NO: 1.

[0032] Furthermore, the overexpressed gene in the xanthoquinodine synthetic gene cluster may be a variant of a gene in the xanthoquinodine synthetic gene cluster (limited to polynucleotides that maintain the function of the encoded protein). Specifically, for example, a gene in the xanthoquinodine synthetic gene cluster may have a nucleotide sequence containing one or several base substitutions, deletions, insertions, and / or additions in the nucleotide sequence of the gene. Here, "one or several" means, for example, 1 to 50, 1 to 30, 1 to 10, or 1 to 5. The gene may also be a partial sequence in which the 5'-end and / or 3'-end of the nucleotide sequence of the gene has been deleted.

[0033] 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. The same applies to polynucleotides that hybridize under stringent conditions with the complementary sequence of the base sequence of any other SEQ ID NO: other than SEQ ID NO: 1.

[0034] The phrase "limited to polynucleotides that maintain the function of the encoded protein" means, for example, in the case of a variant of a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 1, that polynucleotide encodes a protein that maintains the function of the protein encoded by the polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 1 (specifically, the function as an nrPKS shown in Table 1). The same applies to variants of polynucleotides having nucleotide sequences of SEQ ID NOs: 1 other than SEQ ID NO: 1. Whether the function of the encoded protein is maintained can be determined by confirming whether xanthoquinodine production is enhanced when the polynucleotide is overexpressed in a filamentous fungus. Whether the polynucleotide has activity as a transcriptional regulator can be determined by confirming whether the expression level of a target gene controlled by the transcriptional regulator increases when the polynucleotide is overexpressed.

[0035] "Overexpressing one or more genes in the xanthoquinodine synthetic gene cluster" means that the expression level (i.e., amount of expression) of one or more genes in the xanthoquinodine synthetic gene cluster is higher than that in an unmodified strain. Thus, the term "overexpressing one or more genes in the xanthoquinodine synthetic gene cluster" can be used interchangeably or equivalently with the term "enhancing or increasing the expression of one or more genes in the xanthoquinodine synthetic gene cluster." An increase in the expression level of one or more genes in the xanthoquinodine synthetic gene cluster can be measured, for example, as an increase in the expression level of the genes per cell (which may be the average expression level of the genes per cell). Filamentous fungi can be modified so that the expression level of one or more genes in the xanthoquinodine synthetic gene cluster per cell is increased, for example, to 150% or more, 200% or more, or 300% or more of the expression level in an unmodified strain.

[0036] The term "unmodified strain" refers to a filamentous fungal strain that can serve as a control for the above comparison. Examples of unmodified strains 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.

[0037] Methods that can be used to enhance expression of one or more genes in the xanthoquinodine synthetic gene cluster include, but are not limited to, increasing the copy number of one or more genes in the xanthoquinodine synthetic gene cluster, for example, the copy number of the gene in the chromosome of the filamentous fungus and / or the copy number of the gene in a plasmid maintained by the filamentous fungus. The copy number of the gene can be increased, for example, by introducing the gene into the chromosome of the filamentous fungus and / or by maintaining a plasmid containing the gene and capable of autonomously amplifying within the filamentous fungus outside the chromosome of the filamentous fungus. Such modification of filamentous fungi can be carried out by genetic engineering techniques well known to those skilled in the art.

[0038] The plasmid vector is not particularly limited as long as it can be stably maintained and propagated in host cells, and examples thereof include pUC18 / 19, pUC118 / 119, pBR322, pMW218 / 219, pPTR1 / 2 (manufactured by TaKaRa), pRI909 / 910 (manufactured by TaKaRa), pDJB2 (DJBallance et al., 1985, Gene, 36, 321-331), pAB4-1 (van Hartingsveldt W et al., 1987, Mol Gen Genet, 206, 71-75), pLeu4 (MIGRoncero et al., 1989, Gene, 84, 335-343), pPyr225 (CDSkory et al., 2002, Mol Genet Genomics, 268, 397-406), and pFG1 (Gruber, F. et al., Curr Genet, 1990, 18, 447-451).

[0039] One or more genes in the xanthoquinodine synthesis gene cluster can also be introduced into the chromosomal DNA of a filamentous fungus by, for example, homologous recombination. Only one copy, two or more copies of one or more genes in the xanthoquinodine synthesis gene cluster may be introduced. For example, multiple copies of a gene in the xanthoquinodine synthesis gene cluster can be introduced into chromosomal DNA by performing homologous recombination using a sequence having multiple copies in chromosomal DNA. Examples of sequences having multiple copies in chromosomal DNA include, but are not limited to, repetitive DNA and inverted repeats present at the termini of transposable elements. Furthermore, multiple copies of a gene in the xanthoquinodine synthesis gene cluster can be introduced into chromosomal DNA by incorporating the gene into a transposon and transposing the gene.

[0040] Another method that can be used to enhance the expression of one or more genes in the xanthoquinodine synthetic gene cluster is to increase the expression level of the gene by modifying the expression control region of the gene in the xanthoquinodine synthetic gene cluster. The expression control region of a gene in the xanthoquinodine synthetic gene cluster can be modified, for example, by replacing the gene's native expression control region with an endogenous and / or modified foreign expression control region. An "expression control region" is also referred to as an "expression control sequence." Expression control regions include promoters, enhancers, attenuators and termination signals, antitermination signals, ribosome binding sites (RBSs), and other expression control elements (e.g., repressor or inducer binding regions, and / or binding sites for transcriptional and translational regulatory proteins in transcribed mRNAs). Modifying the expression control region of a gene in the xanthoquinodine synthetic gene cluster may be combined with increasing the copy number of the gene in the xanthoquinodine synthetic gene cluster.

[0041] Another method that can be used to enhance the expression of one or more genes in the xanthoquinodine synthetic gene cluster is to overexpress a gene encoding a transcriptional regulator of the xanthoquinodine synthetic gene cluster. Such a transcriptional regulator is not particularly limited as long as it activates the transcription of one or more genes in the xanthoquinodine synthetic gene cluster, and examples thereof include proteins having an amino acid sequence selected from the above-mentioned SEQ ID NOs: 23 and 31, or proteins having a homologous sequence thereof.

[0042] The promoter for enhancing the expression of one or more genes in the xanthoquinodine synthetic gene cluster is not particularly limited as long as it is a promoter that can function in filamentous fungi, and examples thereof include polynucleotides having the base sequences shown in SEQ ID NOs: 34 to 38, 44, 45, and 46.

[0043] Polynucleotides having the nucleotide sequences set forth in SEQ ID NOs: 34 to 38, 44, 45, and 46 are derived from Humicola sp. strain FO-888. As shown in the Examples below, the polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 34 is a promoter located immediately above the start codon of a gene predicted to encode alcohol dehydrogenase. The polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 35 is a promoter located immediately above the start codon of a gene predicted to encode pyruvate decarboxylase based on annotation. The polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 36 is a promoter located immediately above the start codon of a gene predicted to encode glyceraldehyde-3-phosphate dehydrogenase. The polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 37 is a promoter located immediately above the start codon of a gene predicted to encode glutamate dehydrogenase. The polynucleotide having the nucleotide sequence shown in SEQ ID NO: 38 is a promoter located immediately above the start codon of a gene predicted to encode elongation factor 1-alpha. The polynucleotide having the nucleotide sequence shown in SEQ ID NO: 44 is a promoter located immediately above the start codon of a gene predicted to encode actin protein (act). The polynucleotide having the nucleotide sequence shown in SEQ ID NO: 45 is a promoter located immediately above the start codon of a gene predicted to encode a ubiquitin conjugating enzyme. The polynucleotide having the nucleotide sequence shown in SEQ ID NO: 46 is a promoter located immediately above the start codon of a gene predicted to encode a transcription factor (8270_t).

[0044] A promoter for enhancing the expression of one or more genes in the xanthoquinodine synthetic gene cluster may be any of the following polynucleotides: c-1) a polynucleotide having a nucleotide sequence selected from SEQ ID NOs: 34 to 38, 44, 45, and 46; c-2) a polynucleotide that hybridizes under stringent conditions with a complementary sequence of a nucleotide sequence selected from SEQ ID NOs: 34 to 38, 44, 45, and 46, and that encodes a protein having promoter activity in filamentous fungal cells.

[0045] Methods for recombinant DNA molecular manipulation and molecular cloning, such as preparation of plasmid DNA, DNA cleavage, DNA ligation, DNA transformation, selection of oligonucleotides as primers, introduction of mutations, etc., may be conventional methods well known to those skilled in the art.

[0046] Any method can be used as a manipulation method using recombinant DNA, including, for example, conventional methods such as general transformation methods, e.g., electroporation, transformation, transfection, conjugation, protoplast method, particle gun method, and Agrobacterium method.

[0047] In addition to the above-mentioned properties, the filamentous fungi may have specific properties such as various nutritional requirements, drug resistance, drug sensitivity, drug dependence, etc., without departing from the scope of the present invention.

[0048] The above-mentioned techniques for modifying filamentous fungi to overexpress one or more genes in the xanthoquinodine synthesis gene cluster may be used alone or in any combination.

[0049] Enhanced expression of one or more genes in the xanthoquinodine synthesis gene cluster can be confirmed by confirming an increase in the transcription level of the gene or an increase in the amount of protein expressed from the gene.

[0050] Increased transcription of one or more genes in the xanthoquinodine synthetic gene cluster can be confirmed by comparing the amount of mRNA transcribed from the gene with that of an unmodified strain. Methods for assessing mRNA amount include Northern hybridization, RT-PCR, microarray, RNA-seq, etc. The amount of mRNA (e.g., number of molecules per cell) may be increased by, for example, 110% or more, 120% or more, 150% or more, or 200% or more compared to that of an unmodified strain.

[0051] The increased amount of protein expressed by the genes in the xanthoquinodine synthetic gene cluster can be confirmed by Western blotting using an antibody. The amount of protein (e.g., the number of molecules per cell) may be increased by, for example, 110% or more, 120% or more, 150% or more, or 200% or more compared to that of an unmodified strain.

[0052] In addition, an increase in the copy number of genes in the xanthoquinodine synthesis gene cluster can be confirmed, for example, by treating chromosomal DNA with a restriction enzyme and then performing Southern blotting using a probe based on the gene sequence, or fluorescence in situ hybridization (FISH), etc.

[0053] "Enhanced production of xanthoquinodine" means that the amount of xanthoquinodine produced is increased in a filamentous fungus that has been modified to overexpress one or more genes in a xanthoquinodine synthetic gene cluster, compared to an unmodified strain. Specifically, for example, this can mean that a filamentous fungus that has been modified to overexpress one or more genes in a xanthoquinodine synthetic gene cluster produces and accumulates 1.1-fold or more, 1.3-fold or more, or 1.5-fold or more of xanthoquinodine in the culture medium and / or fungal cells, compared to an unmodified strain.

[0054] 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.

[0055] <3> Vector of the Present Invention The vector of the present invention is a vector comprising a gene linked to a promoter in an expressible manner, and the gene is one or more genes in the xanthoquinodine synthetic gene cluster.

[0056] The explanation of the genes in the xanthoquinodine synthetic gene cluster in the vector of the present invention is as described above in "<2> Enhancement of xanthoquinodine production." The explanation of the promoter in the vector of the present invention is as described above in "<2> Enhancement of xanthoquinodine production."

[0057] For example, a method for constructing a vector of the present invention can first involve constructing a DNA fragment containing one or more genes in the xanthoquinodine synthetic gene cluster and a promoter linked upstream thereof in an expressible manner. In addition to the promoter and one or more genes in the xanthoquinodine synthetic gene cluster, the DNA fragment may also contain cis-acting elements that enhance the transcriptional activity of the promoter. Furthermore, the DNA fragment can be constructed to have restriction enzyme recognition sequences at both ends. The promoter can be introduced into the vector using these restriction enzyme recognition sequences. Specifically, a known vector can be cleaved with a restriction enzyme, and a DNA fragment containing the promoter and having a restriction enzyme cleavage sequence at its end can be added to the vector, thereby introducing the promoter into the vector (restriction enzyme method).

[0058] The vector of the present invention may be either a form that is introduced into the chromosome of a host cell or a form that is maintained extrachromosomally. Alternatively, a DNA fragment containing one or more genes in the xanthoquinodine synthetic gene cluster linked to a promoter in an expressible manner may be constructed and directly introduced into the genome of a host cell.

[0059] The vector is not particularly limited as long as it can be stably maintained and propagated in host cells, and examples thereof include pUC18 / 19, pUC118 / 119, pBR322, pMW218 / 219, pPTR1 / 2 (manufactured by TaKaRa), pRI909 / 910 (manufactured by TaKaRa), pDJB2 (DJBallance et al., 1985, Gene, 36, 321-331), pAB4-1 (van Hartingsveldt W et al., 1987, Mol Gen Genet, 206, 71-75), pLeu4 (MIGRoncero et al., 1989, Gene, 84, 335-343), pPyr225 (CDSkory et al., 2002, Mol Genet Genomics, 268, 397-406), and pFG1 (Gruber, F. et al., Curr Genet, 1990, 18, 447-451).

[0060] A transformant transformed with the vector of the present invention can be obtained by introducing the vector of the present invention into a host cell by a known method, for example, by a common transformation method such as electroporation, transformation, transfection, conjugation, protoplast method, particle gun method, or Agrobacterium method.

[0061] By introducing the vector of the present invention into a host cell, a protein encoded by one or more genes in the xanthoquinodine synthetic gene cluster can be expressed in the host cell.

[0062] The host cell is not particularly limited as long as it can express a protein encoded by one or more genes in the xanthoquinodine synthetic gene cluster within the cell, but typically includes a eukaryote, preferably a fungus, more preferably a filamentous fungus. Examples of filamentous fungi include those described above in "<1> Filamentous fungi of the present invention."

[0063] <4> Method for producing xanthoquinodine of the present invention The method for producing xanthoquinodine of the present invention comprises culturing the filamentous fungus of the present invention or a filamentous fungus transformed with the vector of the present invention (also referred to as transformed filamentous fungus) in a culture medium to produce and accumulate xanthoquinodine in the culture medium and / or the fungal cells, and recovering xanthoquinodine from the culture medium and / or the fungal cells. In the method for producing xanthoquinodine of the present invention, one type of filamentous fungus of the present invention or a filamentous fungus transformed with the vector of the present invention may be used, or two or more types of filamentous fungus of the present invention or a filamentous fungus transformed with the vector of the present invention may be used.

[0064] The filamentous fungi can be cultured in the same manner as in conventional filamentous fungal culture methods. The culture medium may be a nutrient medium containing a carbon source assimilable by the microorganism, a nitrogen source assimilable by the microorganism, and, if necessary, inorganic acid salts. Examples of carbon sources that can be used include glucose, sucrose, molasses, dextrin, cellulose, and the like, used alone or in combination.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] Cultivation is usually carried out under aerobic conditions such as shaking or aerated agitation cultivation. Industrially, submerged aerated agitation cultivation is preferred. The pH of the culture is, for example, 5.0 to 8.0, but it is preferable to culture at around neutral. The culture temperature can be between 20 and 40°C, but is usually maintained at 26 to 32°C (preferably around 27°C). The culture time can be 16 to 72 hours, or 20 to 60 hours.

[0069] Culture conditions such as medium composition, liquidity of the medium, culture temperature, and aeration rate can be appropriately adjusted and selected to obtain favorable results depending on the type of strain 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.

[0070] In the method for producing xanthoquinodine 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 allowing the xanthoquinodine within the fungal cells to be eluted into the culture supernatant. Xanthoquinodine can also be recovered by an ion exchange resin method or the like. Xanthoquinodine can be recovered from the culture medium, supernatant, etc. by conventional techniques such as concentration, crystallization, ion exchange chromatography, medium- or high-pressure liquid chromatography, or a combination thereof.

[0071] 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 or two or more types of xanthoquinodine may be produced by the method for producing xanthoquinodine of the present invention.

[0072] 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.

[0073] Examples of the present invention will be described below, but the present invention is not limited to these examples.

[0074] Example 1 Methods (1) Culture Method The culture method was as follows: Humicola sp. FO-888 strain, which had been cultured on CP-added Sabouraud agar medium (Nissui Pharmaceutical Co., Ltd.), was precultured in 100 ml of a preculture medium (glucose 2.0% by weight, yeast extract 0.2% by weight, MgSO4·7H2O 0.05% by weight, Polypepton 0.5% by weight, KH2PO4 0.1% by weight, agar 0.1% by weight, pH 6.0) at 25°C for 3 days. 10 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.

[0075] (2) Whole Genome Analysis of Humicola sp. Strain FO-888: Culture was performed as described in (1) above. After 48 hours, 1 ml of culture was centrifuged (8,000 g for 5 minutes) to collect the 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.

[0076] (3) Quantitative Method for Xanthoquinodine A1: Culture medium of Humicola sp. FO-888 strain 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% by weight 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 detection by UV 400 nm (ultraviolet spectrophotometer). A calibration curve was prepared using purified xanthoquinodine A1 (Cayman, Catalog No. 32892) adjusted to 25 ppm, 50 ppm, 100 ppm, and 200 ppm.

[0077] (4) Gene Expression Analysis (RNAseq Analysis) 48 hours after the start of the main culture described in (1) above, 100 mg of FW bacterial 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 Inc.) to obtain cDNA. RT-qPCR was performed using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Thermo Fisher Scientific) with calibration using an internal standard gene (act1 gene). For RNAseq, 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). The sequence data was mapped to the genome sequence using hisat2 (2.2.1), and the number of reads mapped to each gene (gene expression level) was evaluated using FeatureCounts. Furthermore, the length of intercellular and intracellular gene sequences was corrected using the TPM (Transcripts Per Kilobase Million) method.

[0078] (5) Transformation Method: Humicola sp. FO-888 strain cultured on a PDA agar plate (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 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% PEG 4000, and allowed to stand at room temperature for 30 minutes. 100 μl of recovery medium (glucose 2.0% by weight, yeast extract 0.5% by weight, sorbitol 10.9% by weight, CaCl2 0.2% by weight) was added, and the mixture was allowed to recover and cultured at 25°C with gentle shaking for 2 days. Mycelia were collected from the recovery culture and cultured on PDA medium (Nissui Pharmaceutical Co., Ltd.) containing G418 (100 μg / ml) to obtain transformants.

[0079] <Results> (6) Evaluation of xanthoquinodine productivity (wild strain) After culturing Humicola sp. FO-888 strain using the method described in (1) above, xanthoquinodine A1 was quantified using the method described in (3) above. As a result, production of xanthoquinodine A1 began approximately 48 hours after the start of cultivation and reached approximately 250 ppm after 96 hours of cultivation (Figure 2).

[0080] (7) Search for xanthoquinodine biosynthetic genes. The whole genome information of Humicola sp. strain FO-888 obtained by the method in (2) above was analyzed using antiSMASH, and 48 secondary metabolic genes were extracted (Table 2). The "gene numbers" in Table 2 are numbers assigned by the inventors to each extracted gene for convenience. To select xanthoquinodine biosynthetic genes from Table 2, mRNA extracted from the fungus 48 hours after the start of cultivation, when xanthoquinodine synthesis was observed, was used for RNA sequencing by the method in (4) above. The results confirmed that the gene expression level of gene ID 8265_t was significantly high (Figure 3).

[0081]

[0082] According to Non-Patent Document 2, xanthoquinodine is presumed to be produced by the combination of separately synthesized anthraquinone and xanthone. Furthermore, beticolin, whose biosynthetic gene was recently identified, is also believed to be produced by the combination of anthraquinone and xanthone (Xiaodong Hou et al., 2022, Angewandte Chemie International Edition, 61, e202208772, "Discovery of the Biosynthetic Pathway of Beticolin 1 Reveals a Novel Non-Heme Iron-Dependent Oxygenase for Anthraquinone Ring Cleavage"). The present inventors noted the high structural similarity between xanthoquinodine and beticolin (Figure 4). Using the beticolin biosynthetic genes from Cercospora sp. JNU001 (described in the aforementioned publication by Xiaodong Hou et al.) as a query, they performed a Blast search against the genome information of Humicola sp. FO-888 strain obtained by method (2) above. As a result, a high correlation was observed between the 16 genes in Humicola sp. FO-888 strain shown in Table 1 and the beticolin biosynthetic genes from Cercospora sp. JNU001. Based on these results, the present inventors predicted that the 16 genes in Table 1, including 8265_t, were xanthoquinodine biosynthetic genes (Figure 5).

[0083] Furthermore, based on the results of the Blast search, the xanthoquinodine synthetic pathway described in Non-Patent Document 2, the Emodin family synthetic pathway described on pages 181-182 of Kate MJ de Mattos-Shipley et al., 2023, Natural Product Reports, 40, 174-201, "The 'emodin family' of fungal natural products—amalgamating a century of research with recent genomics-based advances," and the beticolin synthetic pathway described in the aforementioned publication by Xiaodong Hou et al., the functions of the 16 genes in Table 1 and the xanthoquinodine synthetic pathway were predicted. The results are shown in Table 1.

[0084] (8) Functional evaluation of xanthoquinodine biosynthetic genes by gene disruption To experimentally demonstrate the existence of xanthoquinodine biosynthetic genes, the nrPKS (8265_t) gene involved in the synthesis of the xanthoquinodine backbone was disrupted using the procedure described below. As a result, as shown in Figure 6, xanthoquinodine production was completely suppressed. These results indicated that the 16 genes in Table 1, including 8265_t, are xanthoquinodine biosynthetic genes.

[0085] (Procedure) Using the genomic DNA of Humicola sp. FO-888 as a template, PCR was performed to amplify the 5'-terminal region of xqdA (5-xqdA) using the 5xqdA70-Fw primer (SEQ ID NO: 65) and the 5xqdA-Rv primer (SEQ ID NO: 66). The 3'-terminal region of xqdA (3-xqdA) was also amplified using the 3xqdA-Fw primer (SEQ ID NO: 67) and the 3qdA-Rv primer (SEQ ID NO: 68). Subsequently, 5-xqdA was inserted into the HindIII and EcoRI sites of a synthetic vector (pMK-G418R; SEQ ID NO: 69) containing a G418 resistance gene, and 3-xqdA was inserted into the XbaI and KpnI sites to construct the xqdA disruption vector (pMK-dxqdA). Humicola sp. FO-888 strain was transformed with pMK-dqdA by the method described above in (5) to obtain an xqdA-disrupted strain.

[0086] (9) Estimation of promoter regions in Humicola sp. strain FO-888 RNAseq analysis was performed using the method described in (4) above, and five genes with high gene expression levels were selected. A BLAST search was then performed on each gene. As a result, the five genes were annotated as genes with the functions shown in Table 3. The expression levels of the five genes in the RNAseq analysis are shown in Table 3. The approximately 1,000-bp nucleotide sequence immediately above the start codons of the five genes (i.e., the nucleotide sequences shown in SEQ ID NOS: 34 to 38) was then used as the promoter for the strain with enhanced expression of xanthoquinodine biosynthetic genes, as described below.

[0087]

[0088] (10) Construction of a strain with enhanced expression of xanthoquinodine biosynthetic genes. To predict the functions of the 16 genes constituting the xanthoquinodine biosynthetic gene cluster, a Blast search revealed that the xanthoquinodine biosynthetic gene cluster contains one gene, 8265_t, encoding a PKS and two genes, 8270_t and 8278_t, encoding transcription factors (Figure 5). Generally, transcription factors present in a biosynthetic gene cluster are thought to positively or negatively regulate the overall biosynthetic genes. However, the function of transcription factors in the xanthoquinodine biosynthetic gene cluster remains unknown. Therefore, the present inventors constructed a strain with enhanced expression of transcription factors using the following method.

[0089] First, 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. Next, using the genomic DNA of Humicola sp. strain FO-888 as a template, PCR was performed to amplify the gene 8270_t (nucleotide sequence shown in SEQ ID NO: 6), which is predicted to encode a transcription factor present in the xanthoquinodine biosynthetic gene cluster, with the 8270_t-Fw and 8270_t-Rv primers shown in Table 4. Furthermore, using the genomic DNA of Humicola sp. strain FO-888 as a template, PCR was performed to amplify the promoter region gene 2180_t (nucleotide sequence shown in SEQ ID NO: 36), with the Pro-Fw and Pro-Rv primers shown in Table 4. The amplified 8270_t and 2180_t were then in-fusion ligated into the BamHI-SacI gap of the pHD-G418 vector to construct a transformation vector in which 8270_t and 2180_t were linked to the pHD-G418 vector. The constructed transformation vector was then used to transform Humicola sp. FO-888 by the method described in (5) above, to generate a strain in which 2180_t, 8270_t, and the G418 resistance gene were inserted into the genome of Humicola sp. FO-888 (also referred to as the "ots1 strain").

[0090] In the table, the base sequences are from left to right, from the 5' end to the 3' end.

[0091] (11) Productivity Evaluation of the Strain with Enhanced Expression of Transcriptional Regulators (ots1 Strain) The constructed ots1 strain and a wild-type strain (Humicola sp. FO-888 strain) were cultured using the method described in (1) above. Xanthoquinodine A1 was quantified using the method described in (3) above. The ots1 strain exhibited approximately 2.5-fold increased productivity compared to the wild-type strain (Figure 7). Furthermore, RNA sequencing analysis of the ots1 strain and the wild-type strain was performed using the method described in (4). The results confirmed increased gene expression of almost all genes in the xanthoquinodine biosynthetic gene cluster in the ots1 strain compared to the wild-type strain (Figure 8). These results suggest that enhanced expression of transcriptional regulators in the xanthoquinodine biosynthetic gene cluster enhances the overall gene expression of xanthoquinodine biosynthetic genes, including those encoding the transcriptional regulators, and can thereby improve productivity of the metabolite xanthoquinodine.

[0092] Example 2: The hygromycin resistance gene (SEQ ID NO: 47) was PCR amplified using the hyg-fw primer (SEQ ID NO: 49) and the hyg-rv primer (SEQ ID NO: 50) and in-fusion ligated into the SacI-EcoRI gap of the pUC19 vector to construct pHD-hyg. The ama1 gene (SEQ ID NO: 48) was PCR amplified using the ama1-fw primer (SEQ ID NO: 51) and the ama1-rv primer (SEQ ID NO: 52) and in-fusion ligated into the SphI / HindIII gap of pHD-hyg to construct pHD-amaHyg. Using the genomic DNA of Humicola sp. FO-888 strain as a template, PCR was performed to amplify the codon region (ORF) of a gene (8270_t, nucleotide sequence shown in SEQ ID NO: 6) predicted to encode a transcription factor present in the xanthoquinodine biosynthetic gene cluster. Furthermore, the full-ts1-fw primer (SEQ ID NO: 63) and the full-ts1-rv primer (SEQ ID NO: 64) were used to amplify the full-length 8270_t region including approximately 1,000 bp upstream from the initiation codon of 8270_t (SEQ ID NO: 46).

[0093] The primers used are shown in Table 5.

[0094] Next, using the genomic DNA of Humicola sp. FO-888 strain as a template, the promoter region (2180_t, the nucleotide sequence shown in SEQ ID NO: 36) was amplified by PCR using the Pro-2180-fw primer (SEQ ID NO: 55) and the Pro-2180-rv primer (SEQ ID NO: 56). The promoter region (511_t, the nucleotide sequence shown in SEQ ID NO: 38) was amplified using the Pro-511-fw primer (SEQ ID NO: 57) and the Pro-511-rv primer (SEQ ID NO: 58).

[0095] The promoter region (3257_t, the nucleotide sequence shown in SEQ ID NO: 44) was amplified using the Pro-3257-fw primer (SEQ ID NO: 59) and the Pro-3257-rv primer (SEQ ID NO: 60). The promoter region (4945_t, the nucleotide sequence shown in SEQ ID NO: 45) was amplified using the Pro-4945-fw primer (SEQ ID NO: 61) and the Pro-4945-rv primer (SEQ ID NO: 62).

[0096] The primers used are shown in Table 6.

[0097] The amplified promoter regions, the 8270_t codon region (ORF), and the full-length 8270_t region including 1,000 bp upstream were then in-fusion ligated into the SphI-KpnI gap of the pHD-amaHyg vector to construct a transformation vector in which gene cassettes consisting of the 2180_t, 511_t, 3257_t, and 4945_t promoter regions linked to 8270_t and the full-length 8270_t region including 1,000 bp upstream were inserted into the pHD-amaHyg vector. Next, the constructed transformation vector was used to transform Humicola sp. FO-888 strain by the method described above in (5), yielding filamentous fungi P2180t-ts1, P511t-ts1, P3257t-ts1, P4945t-ts1, and Pts1-ts1, which carry extrachromosomal vectors that express transcription factors under the control of various promoters. These fungi were then cultured by the method described above in (1), and xanthoquinodine A1 was quantified by the method described above in (3).

[0098] The results are shown in Figure 9. In a strain transformed with an independently replicating plasmid (extrachromosomal retention) carrying the promoter and coding regions of 8270_t, a gene encoding a transcriptional regulator, we confirmed increased gene expression of 8270_t and 8265_t, the key gene for xanthoquinodine synthesis, as well as improved xanthoquinodine A1 productivity. These results led to the invention of a method for improving xanthoquinodine production through enhanced expression of transcriptional regulatory genes within the xanthoquinodine biosynthetic gene cluster. Next, to confirm that switching to a promoter with high gene expression levels enhances xanthoquinodine production, we transformed an independently replicating plasmid (intracellular retention) carrying a gene construct in which endogenous promoters (34-38, 44-45) selected from RNAseq data were linked to 8270_t. We confirmed that xanthoquinodine A1 productivity increased in proportion to the expression level of the endogenous promoter, with 2180_t exhibiting the highest productivity.

Claims

1. A filamentous fungus that has been modified to overexpress one or more genes in a xanthoquinodine synthesis gene cluster, thereby enhancing the production of xanthoquinodine.

2. The filamentous fungus according to claim 1, wherein one or more of the overexpressed genes in the xanthoquinodine synthesis gene cluster are genes whose production of xanthoquinodine is suppressed by gene disruption or expression suppression.

3. The filamentous fungus according to claim 1 or 2, wherein the gene in the xanthoquinodine synthetic gene cluster that is overexpressed is any one of the following polynucleotides: a-1) a polynucleotide having a nucleotide sequence selected from SEQ ID NOs: 1 to 16; a-2) a polynucleotide that hybridizes under stringent conditions with a complementary sequence of a nucleotide sequence selected from SEQ ID NOs: 1 to 16; a-3) a polynucleotide that encodes a protein having an amino acid sequence selected from SEQ ID NOs: 18 to 33; and a-4) a polynucleotide that encodes a protein having 80% or more sequence identity with an amino acid sequence selected from SEQ ID NOs: 18 to 33.

4. A filamentous fungus described in any one of claims 1 to 3, wherein the genes in the xanthoquinodine synthesis gene cluster are overexpressed by overexpressing a gene encoding a transcriptional regulator that activates the transcription of the genes in the xanthoquinodine synthesis gene cluster.

5. The filamentous fungus according to claim 4, wherein the gene encoding the transcriptional regulator is any one of the following polynucleotides: b-1) a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 6; b-2) a polynucleotide that hybridizes under stringent conditions with the complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 6 and encodes a protein having transcriptional regulator activity; b-3) a polynucleotide that encodes a protein having the amino acid sequence set forth in SEQ ID NO: 23; and b-4) a polynucleotide that encodes a protein having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:

23.

6. A filamentous fungus described in any one of claims 1 to 5, wherein the overexpression is achieved by maintaining a vector containing one or more genes in the xanthoquinodine synthesis gene cluster extrachromosomally in the filamentous fungus.

7. A filamentous fungus described in any one of claims 1 to 5, wherein the overexpression is achieved by introducing a gene construct containing one or more genes in the xanthoquinodine synthesis gene cluster onto the chromosome of the filamentous fungus.

8. The filamentous fungus according to any one of claims 1 to 7, which is a filamentous fungus belonging to the genus Humicola.

9. A vector comprising a gene linked to a promoter in an expressible manner, wherein the gene is one or more of the following polynucleotides: a-1) a polynucleotide having a nucleotide sequence selected from SEQ ID NOs: 1 to 16; a-2) a polynucleotide that hybridizes under stringent conditions with a complementary sequence of a nucleotide sequence selected from SEQ ID NOs: 1 to 16; a-3) a polynucleotide that encodes a protein having an amino acid sequence selected from SEQ ID NOs: 18 to 33; and a-4) a polynucleotide that encodes a protein having 80% or more sequence identity with an amino acid sequence selected from SEQ ID NOs: 18 to 33.

10. The vector according to claim 9, wherein the gene is one or more of the following polynucleotides: b-1) a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 6; b-2) a polynucleotide that hybridizes under stringent conditions with the complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 6 and encodes a protein having transcription factor activity; a-3) a polynucleotide that encodes a protein having the amino acid sequence set forth in SEQ ID NO: 23; and a-4) a polynucleotide that encodes a protein having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:

23.

11. The vector according to claim 9 or 10, wherein the promoter is any one of the following polynucleotides: c-1) a polynucleotide having a nucleotide sequence selected from SEQ ID NOs: 34 to 38, 44, 45, and 46; and c-2) a polynucleotide that hybridizes under stringent conditions with a complementary sequence of a nucleotide sequence selected from SEQ ID NOs: 34 to 38, 44, 45, and 46, and that encodes a protein having promoter activity in filamentous fungal cells.

12. A filamentous fungus transformed with the vector according to any one of claims 9 to 11.

13. The filamentous fungus according to claim 12, which is a filamentous fungus belonging to the genus Humicola.

14. A method for producing xanthoquinodine, comprising culturing the filamentous fungus according to any one of claims 1 to 8, 12 and 13 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.

Images