High productivity method of secondary metabolite by organic acid synthesis inhibition

Abstract

To provide a transformed filamentous fungus with improved xanthoquinodin productivity, and a method for producing xanthoquinodin using the transformed filamentous fungus.SOLUTION: A filamentous fungus in which the activity of one or more enzymes selected from the group consisting of an enzyme involved in ethanol synthesis, an enzyme involved in acetic acid synthesis, and an enzyme involved in lactic acid synthesis is modified so as to be reduced in comparison with a non-modified stain, by which production of xanthoquinodin is enhanced.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present disclosure relates generally to the microbial industry, and more particularly to a filamentous fungus that has been modified to reduce the activity of an enzyme involved in the synthesis of a specific organic acid, thereby enhancing the production of xanthoquinodine, a secondary metabolite of the filamentous fungus, and a method for producing xanthoquinodine. [Background technology]

[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. For example, Humicola sp. ( Humicola Xanthoquinodin A and the like, which are produced by the Xanthoquinodin sp. strain FO-888, are known (Patent Document 1 and Non-Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 06-116281 [Non-patent literature]

[0006] [Non-Patent Document 1] Tabata N et al., 1993, the Journal of Antibiotics, Vol.46, No.5, p.749-755 [Non-patent document 2] Tabata N et al., 1993, Journal of the American Chemical Society, Vol.115, No.19, p.8558-8564 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the biosynthetic pathway of xanthoquinodine has not been fully elucidated, and no transformed filamentous fungi with improved xanthoquinodine productivity through genetic recombination, nor any method for producing xanthoquinodine using such transformed filamentous fungi, have been known to date. 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. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above-mentioned problems. As a result of analyzing the whole genome of a filamentous fungus belonging to the genus Humicola and focusing on the organic acid synthesis pathway, they discovered that xanthoquinodine productivity can be improved by modifying the filamentous fungus so that the activity of one or more enzymes selected from the group consisting of enzymes involved in ethanol synthesis, enzymes involved in acetic acid synthesis, and enzymes involved in lactic acid synthesis is reduced, thereby completing the present invention.

[0009] The present invention provides the following: [1] A filamentous fungus, A filamentous fungus that has been modified to reduce the activity of one or more enzymes selected from the group consisting of enzymes involved in ethanol synthesis, enzymes involved in acetate synthesis, and enzymes involved in lactate synthesis, compared to an unmodified strain, thereby enhancing the production of xanthoquinodines. [2] The filamentous fungus described in [1], in which the activity of one or more genes selected from the group consisting of genes encoding enzymes involved in ethanol synthesis, genes encoding enzymes involved in acetic acid synthesis, and genes encoding enzymes involved in lactic acid synthesis is reduced by disrupting or suppressing the expression of the genes. [3] The filamentous fungus according to [1] or [2], wherein the enzyme involved in ethanol synthesis is pyruvate decarboxylase. [4] The enzymes involved in acetate synthesis are acetate kinase and acetyl-CoA hydrolase. The filamentous fungus according to any one of [1] to [3], which is one or more selected from the group consisting of: [5] The filamentous fungus according to any one of [1] to [4], wherein the enzyme involved in lactic acid synthesis is lactate dehydrogenase. [6] The filamentous fungus according to any one of [2] to [5], wherein the gene encoding the enzyme involved in ethanol synthesis is any one of the following polynucleotides: a-1) A polynucleotide having the base sequence shown in SEQ ID NO: 1 a-2) Hybridization under stringent conditions with the complementary sequence of the nucleotide sequence shown in SEQ ID NO: 1 a polynucleotide encoding a protein having pyruvate decarboxylase activity; [7] The filamentous fungus according to any one of [2] to [6], wherein the gene encoding the enzyme involved in acetic acid synthesis is any one of the following polynucleotides: b-1) A polynucleotide having the base sequence shown in SEQ ID NO: 2 b-2) Hybridization under stringent conditions with the complementary sequence of the base sequence shown in SEQ ID NO: 2 a polynucleotide encoding a protein having acetate kinase activity; C-1) Polynucleotide having the base sequence shown in SEQ ID NO: 3 C-2) Hybridization under stringent conditions with the complementary sequence of the base sequence shown in SEQ ID NO: 3 a polynucleotide encoding a protein having acetyl-CoA hydrolase activity; Polynucleotide to be loaded [8] The filamentous fungus according to any one of [2] to [7], wherein the gene encoding the enzyme involved in lactic acid synthesis is any one of the following polynucleotides: d-1) A polynucleotide having a base sequence selected from SEQ ID NOs: 4 and 5 d-2) Complementary sequences of nucleotide sequences selected from SEQ ID NOs: 4 and 5 and stringent conditions a polynucleotide that hybridizes with a polynucleotide encoding a protein having lactate dehydrogenase activity, [9] Fumikora ( Humicola The filamentous fungus according to any one of [1] to [8], which is a filamentous fungus belonging to the genus Bacillus.

[10] A method for producing xanthoquinodine, comprising: A production method comprising culturing the filamentous fungus according to any one of [1] to [9] 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. [Effects of the Invention]

[0010] 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. [Brief explanation of the drawings]

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

[0012] <1> The filamentous fungus of the present invention The filamentous fungus of the present invention is a filamentous fungus that has been modified to reduce the activity of one or more enzymes selected from the group consisting of enzymes involved in ethanol synthesis, enzymes involved in acetic acid synthesis, and enzymes involved in lactic acid synthesis compared to an unmodified strain, thereby enhancing the production of xanthoquinodines.

[0013] 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.

[0014] 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." Xanthoquinodin 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 * -methyl carboxylate" and also known as "xanthoquinodine D." 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 called "xanthoquinodine A." Xanthoquinodin B1 is also known as 1,2,3,5,8,9,15,16-octahydro-1β,4,6,13,14-pentaerythropoietin. It is also called "methyl 5,9,15-trioxo-17aH-8aβ,16β-ethenonaphtho[2',3':5,6]cyclohepta[1,2-c]xanthene-17aα-carboxylate". Xanthoquinodin B2 is also known as 1,2,3,5,8,9,15,16-octahydro-1β,4,6,13,14-pentaerythropoietin. It is also called "methyl penthydroxy-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 "methyl 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."

[0015] 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.

[0016] The filamentous fungi are not particularly limited, but for example, Aureobasidium ( Aureobasidium ) genus of filamentous fungi, Bjerkandera ) genus of filamentous fungi, Ceripoliopsis ( Ceriporiopsis ) genus of filamentous fungi, Chrysosporium ( Chrysosporium ) genus of filamentous fungi, Coprinus ( Coprinus) genus of filamentous fungi, Coriolus ( Coriolus ) genus of filamentous fungi, Cryptococcus ( Cryptococcus ) genus of filamentous fungi, and the genus Filibasidium ( Filibasidium ) genus of filamentous fungi, Fusarium ( Fusarium ) genus of filamentous fungi, Humicola ( Humicola ) genus of filamentous fungi, Magnaporthe ( Magnaporthe Mucor () is a filamentous fungus belonging to the genus Mucor ) genus of filamentous fungi, Myceliophthora ( Myceliophthora ), a filamentous fungus belonging to the genus Neocallimastix ( Neocallimastix ) genus of filamentous fungi, Neurospora ( Neurospora ) genus of filamentous fungi, Paecilomyces ( Paecilomyces ) genus of filamentous fungi, Penicillium genus ( Penicillium ), a filamentous fungus belonging to the genus Phanerochaete ( Phanerochaete ) genus of filamentous fungi, Phlebia ( Phlebia ) genus of filamentous fungi, Piromyces ( Piromyces ) genus of filamentous fungi, Pleurotus ( Pleurotus ) genus of filamentous fungi, Rhizopus ( Rhizopus ) genus of filamentous fungi, Schizophyllum ( Schizophyllum ), a filamentous fungus belonging to the genus Talaromyces ( Talaromyces ) genus of filamentous fungi, Thermoascus ( Thermoascus ) genus of filamentous fungi, Thielavia ( Thielavia ) genus of filamentous fungi, Tolypocladium ( Tolypocladium ) genus of filamentous fungi, Trametes ( Trametes ) genus of filamentous fungi, Trichoderma ( Trichoderma ) genus.

[0017] Among these, the filamentous fungus of the present invention is Humicola ( Humicola ), and preferably a filamentous fungus belonging to the genus Humicola ( Humicola The filamentous fungi belonging to the genus Humicola nigrescens ( Humicola nigrescens ), a filamentous fungus belonging to the fungus Humicola allopalonella ( Humicola alopallonella ), a filamentous fungus belonging to the fungus Humicola fuscoata ( Humicolafuscoatra ), a filamentous fungus belonging to the fungus Humicola grisea ( Humicola grisea ), a filamentous fungus belonging to the fungus Humicola insolens ( Humicola insolens ) and other filamentous fungi.

[0018] Fumikora ( Humicola ) genus, especially Humicola sp. Humicola An example is the FO-888 strain (NITE P-03105) of Bacillus sp. The FO-888 strain was deposited on January 15, 2020, at the Fermentation Research Institute of the Agency of Industrial Science and Technology (currently the National Institute of Technology and Evaluation, Patent Microorganism Depositary (NITE NPMD), Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) and 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.

[0019] In addition, the filamentous fungus Humicola sp. Humicola The "substantially equivalent strain" may be a strain substantially equivalent to the FO-888 strain. The "substantially equivalent strain" refers to a strain in which the nucleotide sequence of the internal transcribed spacer 1 (ITS-1) region of the rRNA is the same as or similar to the nucleotide sequence of the ITS-1 region of the rRNA of the FO-888 strain (SEQ ID NO: 5). 9) and 97.5% or more, preferably 98% or more, more preferably 98.7% or more, A microorganism having an identity of 99% or more is more preferable, and particularly preferably 100%. The identity of the nucleotide sequence of the ITS-1 region of rRNA can be calculated using a known program such as BLAST. Furthermore, the filamentous fungus may be a strain bred from the FO-888 strain or a strain substantially equivalent thereto by mutation treatment, genetic recombination, selection of natural mutants, or the like, as long as the effects of the present invention are not impaired.

[0020] <2> Modification of filamentous fungi

[0021] The filamentous fungus of the present invention can be obtained by modifying a xanthoquinodine-producing filamentous fungus so that the activity of one or more enzymes selected from the group consisting of enzymes involved in ethanol synthesis, enzymes involved in acetic acid synthesis, and enzymes involved in lactic acid synthesis is reduced compared to that of an unmodified strain. Herein, these enzymes are sometimes collectively referred to as "target enzymes."

[0022] The filamentous fungus of the present invention can also be obtained by modifying the fungus so that the activity of one or more enzymes selected from the group consisting of enzymes involved in ethanol synthesis, enzymes involved in acetic acid synthesis, and enzymes involved in lactic acid synthesis is reduced compared to an unmodified strain, and then imparting or enhancing xanthoquinodine-producing ability. The filamentous fungus of the present invention may be one that has acquired xanthoquinodine-producing ability by being modified so that the activity of one or more enzymes selected from the group consisting of enzymes involved in ethanol synthesis, enzymes involved in acetic acid synthesis, and enzymes involved in lactic acid synthesis is reduced compared to an unmodified strain.

[0023] Here, 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] The filamentous fungus having the "ability to produce xanthoquinodine" may be one that inherently has the ability to produce xanthoquinodine, or one that has been engineered to produce xanthoquinodine using mutation or DNA recombinant technology. It may also be one to which the ability to produce noggin has been imparted.

[0025] By modifying a filamentous fungus as described above, the xanthoquinodine-producing ability of the filamentous fungus can be improved, i.e., xanthoquinodine production by the filamentous fungus can be enhanced compared to an unmodified strain. "Enhancing xanthoquinodine production" includes improving (increasing) the amount of xanthoquinodine accumulated in the culture medium and / or fungal cells.

[0026] The raw material for xanthoquinodine is octaketide, which is synthesized from acetyl-CoA. On the other hand, as shown in Figure 1, acetyl CoA, or pyruvate, an intermediate of acetyl-CoA, is a substrate for ethanol, acetate, and lactate. Therefore, by modifying the enzymes involved in the synthesis of specific organic acids (ethanol, acetic acid, and lactic acid) to reduce their activity, the production of those organic acids can be suppressed, and more pyruvate and acetyl-CoA can be used for the biosynthesis of xanthoquinodines, i.e., metabolism. It is speculated that the flux will be shifted towards the biosynthesis of xanthoquinodine, resulting in an improvement in the productivity of xanthoquinodine.

[0027] 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 Humicola ( Humicola ) genus (e.g., Humicola nigrescens ( Humicola nigrescens )) wild-type strains and parent strains of the filamentous fungi belonging to the is Fumikora Sp.( Humicola sp.) FO-888 strain.

[0028] "Decreased activity of the targeted enzyme" may mean a decrease in expression of the targeted enzyme. Alternatively, "decreased activity of the targeted enzyme" may mean a decrease in the function per molecule of the targeted enzyme. In other words, modifications that "decrease the activity of the targeted enzyme" include modifications that reduce the number of molecules of the targeted enzyme per cell and modifications that reduce the function per molecule of the targeted enzyme. Note that "reduced number of molecules of the targeted enzyme per cell" also includes cases where the targeted enzyme is completely absent. Furthermore, "decreased function per molecule of the targeted enzyme" also includes cases where the function per molecule of the targeted enzyme is completely lost.

[0029] The degree of reduction in target enzyme activity is not particularly limited, as long as the target enzyme activity is reduced compared to that of an unmodified strain. For example, the target enzyme activity may be reduced to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of an unmodified strain.

[0030] Techniques for reducing the activity of a target enzyme will be described later. The activity of a target enzyme can be reduced, for example, by disrupting a gene encoding the target enzyme or by suppressing the expression of the gene encoding the target enzyme. In other words, the activity of the target enzyme encoded by one or more genes (collectively referred to as "target genes") selected from the group consisting of genes encoding enzymes involved in ethanol synthesis, genes encoding enzymes involved in acetic acid synthesis, and genes encoding enzymes involved in lactic acid synthesis can be reduced by disrupting or suppressing the expression of the target genes.

[0031] Examples of target genes and target enzymes include those of the filamentous fungi exemplified above. The nucleotide sequences of target genes derived from various filamentous fungi and the amino acid sequences of the target enzymes encoded by them can be obtained from public databases such as NCBI and technical literature such as patent documents.

[0032] Enzymes involved in ethanol synthesis include pyruvate decarboxylase, alcohol dehydrogenase, aldehyde dehydrogenase, etc. Pyruvate decarboxylase activity refers to the activity of catalyzing the reaction of converting pyruvic acid to acetaldehyde by removing carbon dioxide, and pyruvate decarboxylase refers to an enzyme that catalyzes such a reaction. Alcohol dehydrogenase activity refers to the activity of catalyzing the interconversion reaction between alcohol and aldehyde, and alcohol dehydrogenase refers to an enzyme that catalyzes such a reaction. Aldehyde dehydrogenase activity refers to the activity of catalyzing the interconversion reaction between acetic acid and acetaldehyde, and aldehyde dehydrogenase refers to an enzyme that catalyzes such a reaction.

[0033] Enzymes involved in acetate synthesis include acetate kinase and acetyl-CoA hydrolase. Acetate kinase activity refers to the activity of catalyzing the transfer reaction of a phosphate group between acetate and ADP. Acetate kinase refers to the enzyme that catalyzes this reaction. The enzyme activity refers to the activity of catalyzing the reaction of hydrolyzing acetyl-CoA to convert it into acetic acid and CoA, and acetyl-CoA hydrolase refers to an enzyme that catalyzes such a reaction.

[0034] Enzymes involved in lactic acid synthesis include lactate dehydrogenase, etc. Lactate dehydrogenase activity refers to the activity of catalyzing the interconversion reaction between lactic acid and pyruvic acid, and lactate dehydrogenase refers to an enzyme that catalyzes such a reaction.

[0035] The target enzyme to be modified so as to reduce its activity may be one type or two or more types.

[0036] The "gene encoding an enzyme involved in ethanol synthesis" includes a gene encoding pyruvate decarboxylase, and specifically includes any of the following polynucleotides: a-1) A base sequence selected from SEQ ID NOs: 1 and 11 to 12 (preferably the base sequence shown in SEQ ID NO: 1) a polynucleotide having a base sequence a-2) A base sequence selected from SEQ ID NOs: 1 and 11 to 12 (preferably the base sequence shown in SEQ ID NO: 1) A polynucleotide that hybridizes under stringent conditions with a complementary sequence of the base sequence (a base sequence of the base sequence) and encodes a protein having pyruvate decarboxylase activity. a-3) An amino acid sequence selected from SEQ ID NOs: 6 and 21 to 22 (preferably SEQ ID NO: 6 a polynucleotide encoding a protein having the amino acid sequence shown in a-4) An amino acid sequence selected from SEQ ID NOs: 6 and 21 to 22 (preferably SEQ ID NO: 6 Polynucleotide a-5) A polynucleotide encoding a protein having an amino acid sequence having 80% or more, more preferably 90% or more, and particularly preferably 95% or more identity to an amino acid sequence selected from SEQ ID NOs: 6 and 21 to 22 (preferably SEQ ID NO: 6). a polynucleotide encoding a protein having an amino acid sequence containing substitution, deletion, insertion, and / or addition of one or several (for example, 1 to 50, 1 to 30, 1 to 10, or 1 to 5) amino acid residues in the amino acid sequence shown in

[0037] The base sequences shown in SEQ ID NOs: 1 and 11 to 12 are those of Humicola sp. Humicola The nucleotide sequences are derived from the Bacillus sp. FO-888 strain. The amino acid sequences of the proteins encoded by the polynucleotides having the nucleotide sequences shown in SEQ ID NOS: 1, 11 to 12 are shown in SEQ ID NOS: 6, 21 to 22, respectively. The proteins having the amino acid sequences shown in SEQ ID NOS: 6, 21 to 22 are presumed to be pyruvate decarboxylases based on highly homologous proteins with known functions.

[0038] The "gene encoding an enzyme involved in ethanol synthesis" includes a gene encoding alcohol dehydrogenase, and specifically includes any of the following polynucleotides: a'-1) A polynucleotide having the base sequence shown in SEQ ID NO: 13 a'-2) A polynucleotide that hybridizes under stringent conditions with the complementary sequence of the nucleotide sequence shown in SEQ ID NO: 13, and encodes a protein having alcohol dehydrogenase activity. a'-3) A polynucleotide encoding a protein having the amino acid sequence shown in SEQ ID NO: 23 a'-4) A polynucleotide encoding 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: 23, wherein the protein has alcohol dehydrogenase activity. 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: 23, wherein the protein has alcohol dehydrogenase activity.

[0039] The base sequence shown in SEQ ID NO: 13 is a sequence identified by Humicola sp. Humicola The nucleotide sequence is derived from the FO-888 strain of SEQ ID NO: 13. The amino acid sequence of the protein encoded by the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 13 is shown in SEQ ID NO: 23. The protein consisting of the amino acid sequence shown in SEQ ID NO: 23 was identified as an alcohol dehydrogenase protein from highly homologous proteins with known functions. It is presumed to be a rogenase.

[0040] The "gene encoding an enzyme involved in ethanol synthesis" includes a gene encoding aldehyde dehydrogenase, and specifically includes any of the following polynucleotides: a''-1) A polynucleotide having a base sequence selected from SEQ ID NOs: 14 to 18 a''-2) A complementary sequence of a nucleotide sequence selected from SEQ ID NOs: 14 to 18 under stringent conditions a polynucleotide that hybridizes under the conditions described above and encodes a protein having aldehyde dehydrogenase activity; a''-3) A gene encoding a protein having an amino acid sequence selected from SEQ ID NOs: 24 to 28 Polynucleotides a''-4) 80% or more of an amino acid sequence selected from SEQ ID NOs: 24 to 28, more preferably A polynucleotide encoding a protein having an amino acid sequence with 90% or more, particularly preferably 95% or more identity, wherein the protein has aldehyde dehydrogenase activity. a''-5) In the amino acid sequence selected from SEQ ID NOs: 24 to 28, one or several (e.g., a polynucleotide encoding a protein having an amino acid sequence containing substitutions, deletions, insertions, and / or additions of amino acid residues (e.g., 1 to 50, 1 to 30, 1 to 10, or 1 to 5), wherein the protein has aldehyde dehydrogenase activity.

[0041] The base sequences shown in SEQ ID NOs: 14 to 18 are those of Humicola sp. The base sequences are derived from the Bacillus sp. FO-888 strain. The amino acid sequences of the proteins encoded by the polynucleotides of the base sequences shown in SEQ ID NOS: 14 to 18 are shown in SEQ ID NOS: 24 to 28, respectively. The proteins consisting of the amino acid sequences shown in SEQ ID NOS: 24 to 28 are presumed to be aldehyde dehydrogenases based on highly homologous proteins with known functions.

[0042] The "gene encoding an enzyme involved in acetate synthesis" includes a gene encoding acetate kinase, and specifically includes any of the following polynucleotides: b-1) A polynucleotide having the base sequence shown in SEQ ID NO: 2 b-2) Hybridization under stringent conditions with the complementary sequence of the base sequence shown in SEQ ID NO: 2 a polynucleotide encoding a protein having acetate kinase activity; b-3) A polynucleotide encoding a protein having the amino acid sequence shown in SEQ ID NO: 7 b-4) has a similarity to the amino acid sequence shown in SEQ ID NO: 7 by 80% or more, more preferably 90% or more, particularly Polynucleotide b-5) A polynucleotide encoding a protein having an amino acid sequence with preferably 95% or more identity, wherein the protein has acetate kinase activity. In the amino acid sequence shown in SEQ ID NO: 7, one or several (for example, 1 to 50) amino acids are A polynucleotide encoding a protein having an amino acid sequence containing substitutions, deletions, insertions, and / or additions of 1 to 30, 1 to 10, or 1 to 5 amino acid residues, wherein the protein has acetate kinase activity.

[0043] The base sequence shown in SEQ ID NO: 2 is derived from Humicola sp. Humicola This is a nucleotide sequence derived from the Bacillus sp. FO-888 strain. The amino acid sequence of the protein encoded by the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 7. The protein consisting of the amino acid sequence shown in SEQ ID NO: 7 is predicted to be acetate kinase based on highly homologous proteins with known functions.

[0044] "Genes encoding enzymes involved in acetate synthesis" include acetyl-CoA hydrolase Examples of the gene include a gene encoding the following polynucleotide: can be. C-1) Polynucleotide having the base sequence shown in SEQ ID NO: 3 C-2) Hybridization under stringent conditions with the complementary sequence of the base sequence shown in SEQ ID NO: 3 a polynucleotide encoding a protein having acetyl-CoA hydrolase activity; Polynucleotide to be loaded C-3) A polynucleotide encoding a protein having the amino acid sequence shown in SEQ ID NO: 8 C-4) A sequence identical to the amino acid sequence shown in SEQ ID NO: 8 by 80% or more, more preferably 90% or more, and particularly preferably Preferably, the polynucleotide encodes a protein having an amino acid sequence with 95% or more identity, and the protein has acetyl-CoA hydrolase activity. cleotide C-5) In the amino acid sequence shown in SEQ ID NO: 8, one or several (for example, 1 to 50, a polynucleotide encoding a protein having an amino acid sequence containing substitutions, deletions, insertions, and / or additions of 1 to 30, 1 to 10, or 1 to 5 amino acid residues, wherein the protein has acetyl-CoA hydrolase activity;

[0045] The base sequence shown in SEQ ID NO: 3 is a sequence of Humicola sp. Humicola The base sequence is derived from the FO-888 strain of SEQ ID NO: 3. The amino acid sequence of the protein encoded by the polynucleotide of the base sequence shown in SEQ ID NO: 3 is shown in SEQ ID NO: 8. The protein consisting of the amino acid sequence shown in SEQ ID NO: 8 was identified as an acetyl-CoA hydrolase protein from highly homologous proteins with known functions. It is estimated that...

[0046] The "gene encoding an enzyme involved in lactic acid synthesis" includes a gene encoding lactate dehydrogenase, and specifically includes any of the following polynucleotides: d-1) A base sequence selected from SEQ ID NOs: 4 to 5 and 19 to 20 (preferably, SEQ ID NOs: a polynucleotide having a base sequence selected from the group consisting of 4 and 5 d-2) A base sequence selected from SEQ ID NOs: 4 to 5 and 19 to 20 (preferably, SEQ ID NOs: A polynucleotide that hybridizes under stringent conditions with a complementary sequence of the base sequence selected from the base sequences of 4 and 5, and encodes a protein having lactate dehydrogenase activity. d-3) An amino acid sequence selected from SEQ ID NOs: 9 to 10 and 29 to 30 (preferably, A polynucleotide encoding a protein having an amino acid sequence selected from SEQ ID NOs: 9 and 10 d-4) An amino acid sequence selected from SEQ ID NOs: 9 to 10 and 29 to 30 (preferably, a polynucleotide encoding a protein having an amino acid sequence having 80% or more, more preferably 90% or more, particularly preferably 95% or more identity with an amino acid sequence selected from sequences 9 and 10, wherein the protein has lactate dehydrogenase activity. d-5) An amino acid sequence selected from SEQ ID NOs: 9 to 10 and 29 to 30 (preferably, a polynucleotide encoding a protein having an amino acid sequence containing substitutions, deletions, insertions, and / or additions of one or several (e.g., 1 to 50, 1 to 30, 1 to 10, or 1 to 5) amino acid residues in an amino acid sequence selected from sequences 9 and 10, wherein the protein has lactate dehydrogenase activity.

[0047] The nucleotide sequences shown in SEQ ID NOs: 4-5 and 19-20 are those of Humicola sp. Humicola The amino acid sequences of the proteins encoded by the polynucleotides of the nucleotide sequences shown in SEQ ID NOS: 4-5 and 19-20 are shown in SEQ ID NOS: 9-10 and 29-30, respectively. The proteins consisting of the amino acid sequences shown in SEQ ID NOS: 9-10 and 29-30 were obtained by isolating lactate dehydrogenase from highly homologous proteins with known functions, respectively. It is presumed to be genase.

[0048] In the present disclosure, "stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, conditions are exemplified in 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, particularly preferably two DNAs with identity of 95% or more, hybridize with each other, but two DNAs with lower identity do not hybridize. For example, conditions are exemplified in which two DNAs with high identity, preferably two DNAs with identity of 80% or more, more preferably two DNAs with higher identity do not hybridize with each other. For example, conditions are exemplified in which two DNAs with high identity are hybridized with each other, for example, conditions are 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS, and a salt concentration and temperature corresponding to the above, for example, 60°C, 0.1×SSC, 0.1% SDS, and a salt concentration and temperature corresponding to the above, for example, 68°C, 0.1×SSC, 0.1% SDS, are hybridized once. Preferably, washing is performed two to three times.

[0049] For example, a "polynucleotide that hybridizes under stringent conditions to the complementary sequence of the nucleotide sequence shown in SEQ ID NO: 1" may be a polynucleotide having a nucleotide sequence that is 80% or more, more preferably 90% or more, and particularly preferably 95% or more identical to the nucleotide sequence shown in SEQ ID NO: 1. The same applies to polynucleotides that hybridize under stringent conditions to the complementary sequence of the nucleotide sequence of any other SEQ ID NO: 1.

[0050] Furthermore, the target gene may be a variant of the target gene, so long as it encodes a target enzyme that maintains its original activity. Specifically, for example, the target gene may have a nucleotide sequence that includes one or several nucleotide substitutions, deletions, insertions, and / or additions in the nucleotide sequence of the target gene, so long as it encodes a target enzyme that maintains its original activity. Here, "one or several" means, for example, 1 to 50, 1 to 30, 1 to 10, or 1 to 5 nucleotides. Furthermore, the target gene may be a partial sequence of the nucleotide sequence of the target gene, in which the 5'-end and / or the 3'-end are deleted, so long as it encodes a target enzyme that maintains its original activity.

[0051] For example, a "polynucleotide that hybridizes under stringent conditions to the complementary sequence of the nucleotide sequence shown in SEQ ID NO: 1" may be a polynucleotide having a nucleotide sequence that includes substitution, deletion, insertion, and / or addition of one or several nucleotides in the nucleotide sequence shown in SEQ ID NO: 1. The same applies to polynucleotides that hybridize under stringent conditions to the complementary sequence of the nucleotide sequence of any other SEQ ID NO: other than SEQ ID NO: 1.

[0052] Modifications that reduce the activity of a target enzyme can be achieved, for example, by suppressing the expression of a target gene encoding the target enzyme. "Target gene expression is suppressed" means that the expression of the target gene is reduced compared to an unmodified strain such as a wild-type strain or a parent strain. "Target gene expression is suppressed" specifically means that the expression level of the target gene per cell is reduced compared to an unmodified strain. "Target gene expression is suppressed" may more specifically mean that the transcription level (mRNA level) of the target gene is reduced and / or the translation level (protein level) of the target gene is reduced. "Target gene expression is suppressed" also includes cases where the target gene is not expressed at all. Target gene expression 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.

[0053] Suppression of target gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof. Suppression of target 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 initiation 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. A decrease in gene transcription efficiency can be achieved, 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 deletion of an expression regulatory sequence may be performed. Reduction of gene expression can also be achieved, for example, by manipulating factors involved in expression regulation. Factors involved in expression regulation include small molecules (inducers, inhibitors, etc.), proteins (transcription factors, etc.), and nucleic acids (siRNA, etc.) involved in transcription and translation regulation. Suppression of target gene expression can also be achieved, for example, by introducing a mutation into the coding region of the target gene that suppresses the expression of the target gene. For example, the expression of the target gene can be suppressed by replacing a codon in the coding region of the target gene with a synonymous codon that is used less frequently in the host. Furthermore, for example, the expression of the target gene itself can be suppressed by disrupting the target gene as described below.

[0054] Furthermore, a modification that reduces the activity of a target enzyme can be achieved, for example, by disrupting the target gene that encodes the target enzyme. "Disruption of the target gene" means that the target gene is modified so that it does not produce a target enzyme that functions normally. "Not producing a target enzyme that functions normally" includes cases where the target gene does not produce any target enzyme at all, or cases where the target gene produces a target enzyme with reduced or lost function (e.g., activity or properties) per molecule.

[0055] Disruption of a target gene can be achieved, for example, by disrupting (also referred to as deleting or missing) the target gene on a chromosome. In the present invention, "disrupting a target gene" means that the target gene has been modified to reduce or eliminate its original function. "Disruption of a target gene" refers to the deletion of part or all of the coding region of the target gene. Furthermore, the entire target gene may be deleted, including the sequences before and after the coding region of the target gene on the chromosome. The sequences before and after the coding region of the target gene may include, for example, an expression regulatory sequence for the target gene. As long as the activity of the target enzyme 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 a protein), an internal region, or a C-terminal region (the region encoding the C-terminal side of a 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 target gene. Furthermore, it is preferable that the sequences before and after the region to be deleted do not have the same reading frame. A mismatch in the reading frame may cause a frameshift downstream of the region to be deleted.

[0056] Disruption of a target gene can also be achieved, for example, by introducing an amino acid substitution (missense mutation) into the coding region of the target gene on a chromosome, by introducing a stop codon (nonsense mutation), or by adding or deleting one to two bases (frameshift mutation).

[0057] Disruption of a target gene can also be achieved, for example, by inserting another base sequence into the coding region of the target gene on a chromosome. The insertion site may be any region of the target gene, but the longer the inserted base sequence, the more reliably the target gene can be 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. There are no particular limitations on the other base sequence as long as it reduces or eliminates the activity of the encoded target enzyme, but for example, a marker gene such as an antibiotic resistance gene or a base sequence effective for the production of a target substance can be used. Examples of useful genes include:

[0058] Disruption of a target gene may be carried out particularly so as to delete (delete) the amino acid sequence of the encoded target enzyme. In other words, modification to reduce the activity of a target enzyme can be achieved, for example, by deleting the amino acid sequence of the target enzyme (a part or all of the region of the amino acid sequence), specifically by modifying the gene so as to encode a protein from which the amino acid sequence (a part or all of the region of the amino acid sequence) has been deleted. The term "deletion of the amino acid sequence of a target enzyme" refers to the deletion of a part or all of the region of the amino acid sequence of a target enzyme. The term "deletion of the amino acid sequence of a target enzyme" also refers to the absence of the original amino acid sequence in the protein, and also encompasses cases in which 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 may be considered a deleted region. While deletion of the amino acid sequence of a target enzyme typically shortens the overall length of the protein, it may also be the case that the overall length of the protein remains unchanged or is extended. For example, by deleting part or all of the coding region of a target gene, the region encoded by the deleted region can be deleted in the amino acid sequence of the encoded target enzyme. Furthermore, by introducing a stop codon into the coding region of a target gene, the region encoded by the region downstream of the introduction site can be deleted in the amino acid sequence of the encoded target enzyme. Furthermore, by frameshifting in the coding region of a target gene, the region encoded by the frameshift site can be deleted. The same explanations as for the position and length of the region deleted in the deletion of an amino acid sequence can be applied mutatis mutandis.

[0059] The deletion, substitution, or insertion of all or part of the base sequence of the target gene can be achieved using homologous recombination techniques, gene editing nucleases, zinc finger nuclease proteins (ZFN), TAL effector nucleases (TALEN), or a combination of CRISPER-Cas9. This can be done using genome editing technology, etc.

[0060] Furthermore, a modification that reduces the activity of a target enzyme may be performed, for example, by a mutation treatment. Examples of the mutation treatment include irradiation with X-rays, irradiation with ultraviolet light, and N-methyl-N'-diaminobenzylamine. These include treatment with mutagens such as tro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0061] In addition, when a target enzyme functions as a complex consisting of multiple subunits, all or only a portion of the multiple subunits may be modified, as long as the activity of the target enzyme is reduced as a result. That is, for example, all or only a portion of the multiple genes encoding those subunits may be disrupted, etc. Furthermore, when a target enzyme has multiple isozymes, the activities of all or only a portion of the multiple isozymes may be reduced, as long as the activity of the target enzyme is reduced as a result. That is, for example, all or only a portion of the multiple genes encoding those isozymes may be disrupted, etc.

[0062] The above-mentioned methods for reducing the activity of a target enzyme may be used alone or in any combination.

[0063] The decrease in the activity of the target enzyme can be confirmed by measuring the activity of the target enzyme.

[0064] A decrease in the activity of a target enzyme can also be confirmed by confirming a decrease in the expression of a target gene encoding the target enzyme, which can be confirmed by confirming a decrease in the transcription level of the target gene or a decrease in the amount of the target enzyme expressed from the target gene.

[0065] The reduction in the transcription level of the target gene can be confirmed by comparing the amount of mRNA transcribed from the target 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., 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.

[0066] The reduction in the amount of the target enzyme can be confirmed by Western blotting using an antibody. The amount of the target enzyme (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 the unmodified strain.

[0067] Disruption of the target gene can be confirmed by determining the base sequence, restriction enzyme map, or full length of a part or all of the target gene, depending on the means used for disruption.

[0068] The above-mentioned methods for reducing the activity of a target enzyme can be used to reduce the activity of any protein or the expression of any gene.

[0069] "Enhanced production of xanthoquinodine" means that in a filamentous fungus that has been modified so that the activity of one or more enzymes selected from the group consisting of enzymes involved in ethanol synthesis, enzymes involved in acetic acid synthesis, and enzymes involved in lactic acid synthesis is reduced compared to an unmodified strain, the amount of xanthoquinodine produced is increased compared to an unmodified 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 an unmodified strain.

[0070] 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.

[0071] <3> Method for producing xanthoquinodines of the present invention The method for producing xanthoquinodine of the present invention includes culturing the filamentous fungus of the present invention 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 the filamentous fungus of the present invention may be used, or two or more types of the filamentous fungus of the present invention may be used.

[0072] 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.

[0073] Specifically, carbohydrates such as glucose, glycerol, fructose, maltose, mannitol, xylose, galactose, ribose, starch, or their hydrolysates can be used as carbon sources. Their concentrations are typically 0.1% to 5% of the medium. Other usable carbon sources 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.

[0074] Examples of nitrogen sources that can be used include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, and ammonium nitrate; nitrogen-containing organic substances such as urea, peptone, NZ-amine, meat extract, yeast extract, dry 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.

[0075] 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.

[0076] 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 cultivation is, for example, 5.0 to 8.0, but it is preferable to cultivate at around neutral. The cultivation temperature can be 20 to 40°C, but is usually maintained at 26 to 32°C (preferably around 27°C). The cultivation time can be 16 to 72 hours, or 20 to 60 hours.

[0077] 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.

[0078] 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. 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.

[0079] 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.

[0080] The recovered xanthoquinodine may contain, in addition to xanthoquinodine, for example, fungal cells, medium components, water, and metabolic by-products of the filamentous fungus. 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. [Example]

[0081] Examples of the present invention will be described below, but the present invention is not limited to these examples.

[0082] [Example 1] <Method> (1)Culture method The cultivation method is as follows. Cultured on CP-added Sabouraud agar medium (Nissui Pharmaceutical Co., Ltd.) sp. FO-888 strain in 100 ml Preculture medium (glucose 2.0% by weight, yeast extract 0.2% by weight, MgSO4·7H2O 0.05% by weight) , Polypepton 0.5 wt%, KH2PO4 0.1 wt%, agar 0.1 wt%, pH 6.0) at 25°C for 3 days. Preculture: 10 ml of the preculture solution is inoculated into 100 ml of the main culture medium (sucrose 2.0% by weight, glucose 1.0% by weight, corn steep liquor 1.0% by weight, meat extract 0.5% by weight, KH2PO4 0.1% by weight, CaCO3 0.3% by weight, agar 0.1% by weight, pH 6.0), and the main culture is carried out at 25°C.

[0083] (2) Humicola Whole genome analysis of the sp. FO-888 strain The culture was performed according to the method (1) above. After 48 hours from the start of the main culture, the bacterial cells were collected from 1 ml of the culture medium by centrifugation (8,000 g, 5 minutes) and washed with 5 ml of sterile water. Genomic DNA was extracted from the bacterial cells using a Quick-DNA Fungal / Bacterial Kit (ZYMO RESEARCH) to prepare a library. The resulting library was subjected to NovaSeq™ 6000 (Illumina) to analyze the genomic DNA. After obtaining the data, the gene regions are predicted after assembly.

[0084] (3) Quantitative method for xanthoquinodine A1 Humicola The culture medium of the sp. FO-888 strain is mixed with an equal volume of ethyl acetate and shaken at room temperature overnight. 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 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.

[0085] (4) Expression analysis method for organic acid synthesis-related genes (RNA sequencing analysis) After 48 hours from the start of the main culture in (1) above, 100 mg FW of bacterial cells were collected from 2 ml of culture medium by centrifugation (8,000 g, 10 minutes) and disrupted with a TisseLyzer (Qiagen). mRNA was extracted using an RNeasy Plus Mini Kit (Qiagen). Subsequently, reverse transcription was performed using a PrimeScript™ RT Reagent Kit (Takara Bio) to obtain cDNA. RT-qPCR was performed using PowerUp ( SYBR™ Green Master Mix (Applied Biosystems, Thermo Fisher Scientific) was used, and correction was performed 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). 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, correction was performed using the TPM (Transcripts Per Kilobase Million) method to correct for intercellular and intracellular gene sequence length.

[0086] (5) Transformation method Cultured on PDA agar plates (Nissui Pharmaceutical Co., Ltd.) HumicolaThe sp. FO-888 strain was inoculated into 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 then 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.

[0087] (6)Organic acid quantitative method Humicola The supernatant obtained by centrifugation (10,000 rpm / 10 minutes) from the culture medium of the sp. FO-888 strain is used for organic acid analysis. The ethanol concentration is determined by diluting the obtained supernatant with distilled water and Measurement was performed using Osensor BF-9 (Oji Scientific Instruments Co., Ltd.), and the ethanol in the supernatant was determined based on the dilution ratio. The lactic acid concentration is measured by diluting the obtained supernatant with distilled water and using a Lactate Assay Kit-WST (manufactured by Wako), and the ethanol concentration in the supernatant is calculated from the dilution ratio. The acetic acid concentration is measured by diluting the obtained supernatant with distilled water and using an EnzyChrom Acetate Assay Kit (manufactured by Bio Assay Systems), and the ethanol concentration in the supernatant is calculated from the dilution ratio.

[0088] <Result> (7) Organic acid analysis results By the method (1) above Humicola After culturing the sp. FO-888 strain, The concentrations of various organic acids in the culture supernatant were quantified, and it was found that acetic acid, lactic acid, and ethanol were produced 48 hours after the start of main culture (Fig. 2). These organic acids are produced from pyruvate and acetyl-CoA, which are the main intermediates of glycolysis (Fig. 1), and are therefore key raw materials. It is thought to compete with santoquinodine production.

[0089] (8) Search for genes related to organic acid synthesis Obtained by the method (2) above Humicola A Blast search was performed based on the whole genome data of the Bacteroides sp. strain FO-888 to identify genes that share homology with genes of known function. For each identified gene, the predicted function (function of the encoded protein), the SEQ ID NO of the nucleotide sequence of the coding region (CDS), and the SEQ ID NO of the amino acid sequence of the translation product are shown in Table 1. Table 1 also shows the GenBank accession numbers and biological species of origin of the known genes used as queries in the Blast search. In Table 1, the "gene numbers" are numbers assigned by the inventors for convenience.

[0090] Next, using the method described in (4) above, gene expression levels were compared 48 hours after the start of main cultivation, when xanthoquinodine synthesis began, and organic acid synthesis-related genes with relatively high expression levels were selected as the main organic acid synthesis-related genes. The gene expression levels of each gene are shown in Table 1. As a result, the pyruvate decarboxylase gene (735_t), the aldehyde dehydrogenase gene (735_t), and the ethanol synthesis-related genes were selected. The following genes were selected: the acetate kinase gene (8192_t) and the acetyl-coA hydrolase gene (9413_t) as genes related to acetate synthesis; and the lactate dehydrogenase gene (9851_t and 4166_t) as genes related to lactate synthesis.

[0091] [Table 1]

[0092] (9) Construction of disruptants related to organic acid synthesis The selected strains in which the organic acid synthesis-related genes were disrupted were prepared by the following procedure. first, Humicola The genomic DNA of the sp. FO-888 strain was used as a template for PCR to detect selected organic acids. Genomic regions upstream and downstream of the synthesis-related gene were amplified using the primers shown in Table 2. Next, the SphI / BamHI fragment of the G418 resistance gene was ligated into the SphI-BamHI gap of the pUC19 vector. By gating the vector, the pHD-G418 vector was generated. 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 treated with restriction enzymes BamHI, SpeI, SacI, and SphI, to construct a gene disruption plasmid vector (gene disruption vector). Humicola By transforming the sp. FO-888 strain, a G418 resistance gene was inserted into the target organic acid synthesis-related gene by homologous recombination, and an organic acid synthesis-related gene disruptant was obtained.

[0093] [Table 2] In the table, the base sequences are from left to right, from the 5' end to the 3' end.

[0094] (10) Evaluation of xanthoquinodine productivity in organic acid synthesis-related gene disruptants The disruptant strains of each organic acid synthesis-related gene obtained by the method (9) above and the wild-type strain ( Humicola Humicola Humicola strain FO-888) were cultured by the method described in (1) above, and the xanthoquinodine production and organic acid concentration 120 hours after the start of the main culture were quantified by the methods described in (3) and (6) above.

[0095] (i) Evaluation of xanthoquinodine productivity in ethanol synthesis-related gene disruptants As a result, the pyruvate decarboxylase gene (735_t), which is related to ethanol synthesis, The disruptant of the α-pyruvate decarboxylase gene (735_t) and the disruptant of the alcohol dehydrogenase gene (10012_t) showed a decrease in ethanol production compared to the wild-type strain (Fig. 3). In addition, the disruptant of the pyruvate decarboxylase gene (735_t) showed an increase in xanthoquinodine production compared to the wild-type strain. These results suggest that disruption of 735_t or 10012_t inhibits the synthesis of acetaldehyde from pyruvate or acetate, thereby improving the efficiency of xanthoquinodine synthesis from pyruvate or acetyl-CoA, thereby increasing the productivity of xanthoquinodine. It was confirmed that it is possible to improve

[0096] (ii) Evaluation of xanthoquinodine productivity in acetic acid synthesis-related gene disruptants Furthermore, in the mutants lacking the acetate kinase gene (8192_t) and the acetyl-coA hydrolase gene (9413_t), which are acetate synthesis-related genes, increased xanthoquinodine production was observed, but decreased acetate production was also observed compared to the wild-type strain (Fig. 4). These results confirmed that disruption of 8192_t or 9413_t inhibits acetate synthesis from acetyl-coA, improving the efficiency of xanthoquinodine synthesis from pyruvate, thereby enabling increased xanthoquinodine productivity.

[0097] (iii) Evaluation of xanthoquinodine productivity in lactate synthesis-related gene disruptants In addition, the lactate dehydrogenase gene (9851_t, 4166_t), which is a gene related to lactate synthesis, Compared to the wild-type strain, the disrupted strain showed increased xanthoquinodine production and decreased lactate production (Fig. 5). These results confirmed that disruption of 9851_t or 4166_t inhibits lactate synthesis from pyruvate, improving the efficiency of xanthoquinodine synthesis from pyruvate, thereby enabling increased xanthoquinodine productivity.

Claims

1. A filamentous fungus, A filamentous fungus that has been modified to reduce the activity of one or more enzymes selected from the group consisting of enzymes involved in ethanol synthesis, enzymes involved in acetic acid synthesis, and enzymes involved in lactic acid synthesis compared to an unmodified strain, thereby enhancing the production of xanthoquinodines.

2. The filamentous fungus according to claim 1, wherein the activity of one or more genes selected from the group consisting of genes encoding enzymes involved in ethanol synthesis, genes encoding enzymes involved in acetic acid synthesis, and genes encoding enzymes involved in lactic acid synthesis is reduced by disrupting or suppressing the expression of the genes.

3. The filamentous fungus according to claim 1 or 2, wherein the enzyme involved in ethanol synthesis is pyruvate decarboxylase.

4. The enzymes involved in acetate synthesis consist of acetate kinase and acetyl-CoA hydrolase. The filamentous fungus according to any one of claims 1 to 3, wherein the filamentous fungus is one or more selected from the group consisting of:

5. The filamentous fungus according to any one of claims 1 to 4, wherein the enzyme involved in lactic acid synthesis is lactate dehydrogenase.

6. The filamentous fungus according to any one of claims 2 to 5, wherein the gene encoding an enzyme involved in ethanol synthesis is any one of the following polynucleotides: a-1) A polynucleotide having the base sequence shown in SEQ ID NO: 1 a-2) Hybridization under stringent conditions with the complementary sequence of the base sequence shown in SEQ ID NO: 1 a polynucleotide encoding a protein having pyruvate decarboxylase activity;

7. The filamentous fungus according to any one of claims 2 to 6, wherein the gene encoding the enzyme involved in acetic acid synthesis is any one of the following polynucleotides: b-1) A polynucleotide having the base sequence shown in SEQ ID NO: 2 b-2) Hybridization under stringent conditions with the complementary sequence of the base sequence shown in SEQ ID NO: 2 a polynucleotide encoding a protein having acetate kinase activity; C-1) A polynucleotide having the base sequence shown in SEQ ID NO: 3 C-2) Hybridize under stringent conditions with the complementary sequence of the base sequence shown in SEQ ID NO: 3 a polynucleotide encoding a protein having acetyl-CoA hydrolase activity; Polynucleotide to be loaded

8. The filamentous fungus according to any one of claims 2 to 7, wherein the gene encoding the enzyme involved in lactic acid synthesis is any one of the following polynucleotides: d-1) A polynucleotide having a base sequence selected from SEQ ID NOs: 4 and 5 d-2) Complementary sequences of the base sequences selected from SEQ ID NOs: 4 and 5 and stringent conditions a polynucleotide that hybridizes with a polynucleotide encoding a protein having lactate dehydrogenase activity,

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

10. A method for producing xanthoquinodines, comprising: A production method comprising culturing the filamentous fungus according to any one of claims 1 to 9 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.

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