# Patent

## Title
UK-2 biosynthetic gene and method for improving UK-2 productivity using the same

## Abstract

The genomic DNA of Streptoverticillium sp. 3-7, which produces UK-2, was analyzed to identify a region expected to be a UK-2 biosynthetic gene cluster. Moreover, by colony hybridization, DNAs in the region were successfully isolated. Further, the DNAs were used to prepare a strain in which the genes present in the region were disrupted. The strain was found not to produce UK-2. It was verified that the genomic region was the UK-2 biosynthetic gene cluster. Furthermore, Streptoverticillium sp. 3-7 was transformed by introduction of a vector in which the isolated UK-2 biosynthetic gene cluster was inserted. It was also found out that the UK-2 productivity by the transformant was improved about 10 to 60 times or more in comparison with that of the parental strain. Moreover, it was revealed that 2 copies of the UK-2 biosynthetic gene cluster were present per cell in these transformants, respectively.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This is a Divisional of U.S. Application No. 13/936,771, filed Jul. 8, 2013 (now allowed), which claims priority based on Japanese Patent Application No. 2012-153986 filed Jul. 9, 2012.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to a gene necessary for biosynthesis of UK-2 which is a compound useful for rice blast control agents and the like (hereinafter referred to as a “UK-2 biosynthetic gene”) and a method for improving UK-2 productivity. More specifically, the present invention relates to a UK-2 biosynthetic gene, a vector in which the UK-2 biosynthetic gene is inserted, a transformant in which the vector is introduced, a method for determining UK-2 productivity by detecting the presence of the UK-2 biosynthetic gene, a bacterium in which the presence of the UK-2 biosynthetic gene is detected by the method, a bacterium comprising the UK-2 biosynthetic gene inserted in a genome thereof, a bacterium in which one or two or more copies of the UK-2 biosynthetic gene are present per cell, and methods for producing UK-2 and a UK-2A derivative by utilizing these bacteria and so forth.
Related Background Art
UK-2 is a compound produced as a secondary metabolite by actinobacteria, and shows strong antifungal actions similar to antimycin against various fungi including filamentous fungi and yeasts. Further, since having low cytotoxicity to culture cells, UK-2 has been found to be useful for rice blast control agents, agricultural and horticultural fungicides, and medical antifungal agents (Japanese Examined Patent Application Publication No. Hei 07-233165 and International Publication No. WO1999/11127). Moreover, it has been revealed that there naturally exists four analogues, UK-2A to D, based on the difference in structure of their side chains (Ueki M., et al., Journal of antibiotics, Jul. 25, 1996, vol. 49, no. 7, pp. 639 to 643).
UK-2 is produced by culturing actinobacteria (bacteria and the like belonging to the genus Streptoverticillium) and then collecting UK-2 therefrom. However, generally, the amount of UK-2 (all UK-2 factors) produced by microorganisms isolated from nature is very small. Accordingly, in order to use the target (UK-2) industrially at low cost, the productivity has to be improved.
The productivity of the target is improved through investigations on the methods for culturing the microorganisms producing the target, investigations on the medium components, improvement in fermentation conditions by addition of the precursor, and improvement in the bacterial strain utilizing ultraviolet irradiation- or chemical mutagen-induced mutation. Furthermore, in addition to these methods, the productivity has been improved recently by utilizing gene recombination.
A general method for improving the productivity by gene recombination is that including the enhancing of expression of a gene necessary for biosynthesis of the target. For example, International Publication No. WO2001/018179 discloses that this method improves the productivity of PF-1022 in Agonomycetales.
However, when this method is utilized, it is essential to isolate the gene necessary for biosynthesis of the target or the gene synthesized using known techniques, and also to establish the transformation method for microorganisms producing the target (producing microorganisms). Since the UK-2 biosynthetic gene is yet to be elucidated, the transformation using the UK-2 biosynthetic gene cannot be performed. The productivity cannot be improved by gene recombination.
SUMMARY OF INVENTION
The present invention has been made in view of the above-described problems in the conventional techniques. An object of the present invention is to provide a transformant having high UK-2 productivity, obtained by isolating a gene necessary for biosynthesis of UK-2 followed by introduction of the gene. Moreover, another object is to produce a large amount of UK-2 at low cost using the transformant. And a further object is to provide a method for determining UK-2 productivity by detecting the presence of the gene.
UK-2 has a characteristic hydroxypicolinic acid skeleton. Meanwhile, a compound called virginiamycin also has a hydroxypicolinic acid skeleton. Further, it has been revealed that VisA (L-lysine 2-aminotransferase) and VisB (3-hydroxypicolinic acid AMP ligase) are involved in the biosynthesis of virginiamycin (Namwat W., et al, Journal of Bacteriology, September 2002, vol. 184, no. 17, pp. 4811 to 4818).
Thus, in order to achieve the above objects, the present inventors first prepared the genomic DNA library of Streptoverticillium sp. 3-7, which produces UK-2, and comprehensively determined the base sequence of the genomic DNA of the strain. Then, a homology analysis was conducted between the amino acid sequence of a putative protein encoded by the genomic DNA and the amino acid sequences of VisA and VisB to thus find out a genomic site where genes whose products have a high homology with these two amino acid sequences are consecutively located. Furthermore, it was found out that a gene encoding a protein having a homology with a non-ribosomal peptide synthetase (NRPS) and a gene encoding a protein having a homology with a polyketide synthase (PKS) were located near the site.
These enzymes are thought to be necessary to form the UK-2 skeleton. In addition, the secondary metabolite genes of actinobacteria form clusters. Accordingly, the genomic region is expected to be a UK-2 biosynthetic gene cluster.
Then, based on the thus-obtained information on the base sequences of the genes expected to be encoding the enzymes necessary for biosynthesis of UK-2, a probe was prepared. By colony hybridization using the probe, DNAs expected to be in the UK-2 biosynthetic gene cluster (i.e., DNAs contained in the genomic region) were successfully isolated from the above-described genomic DNA library. Moreover, the DNAs were used to prepare Streptoverticillium sp. 3-7 in which the genes present in the genomic region were disrupted. The strain was found not to produce UK-2, It was verified that the genomic region was the UK-2 biosynthetic gene cluster. Further, Streptoverticillium sp. 3-7 was transformed by introduction of a vector in which the isolated UK-2 biosynthetic gene cluster was inserted. It was found out also that the UK-2 productivity by the transformant was improved about 10 to 60 times or more in comparison with that of the parental strain. Furthermore, it was confirmed that 2 copies of the UK-2 biosynthetic gene cluster were present per cell in these transformants, respectively.
Specifically, the present invention relates to a UK-2 biosynthetic gene, a vector in which the UK-2 biosynthetic gene is inserted, a transformant in which the vector is introduced, and methods for producing UK-2 and the like by utilizing the transformant. More specifically, the present invention provides the followings.

    <1> An isolated nucleic acid that induces UK-2 biosynthesis and improves UK-2 productivity, the nucleic acid is at least one nucleic acid selected from the group consisting of the following (a) to (q):


(a) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 3, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 3 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 3, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 2;
(b) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 5, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 5 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 5, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 4;
(c) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 7, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 7 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 7, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 6;
(d) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 9, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 9 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 9, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 8;
(e) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 11, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 11 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 11, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 10;
(f) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 13, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 13 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 13, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 12;
(g) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 15, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 15 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 15, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 14;
(h) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 17, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 17 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 17, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 16;
(i) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 19, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 19 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 19, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 18;
(j) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 21, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 21 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 21, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 20;
(k) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 23, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 23 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 23, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 22;
(l) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 25, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 25 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 25, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 24;
(m) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 27, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 27 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 27, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 26;
(n) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 29, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 29 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 29, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 28;
(o) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 31, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 31 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 31, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 30;
(p) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 33, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 33 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 33, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 32; and
(q) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 35, a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 35 in which one or more amino acids are substituted, deleted, added and/or inserted, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 35, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 34.

    <2> The nucleic acid according to <1>, comprising all the nucleic acids of (a) to (q).
    <3> The nucleic acid according to <2>, comprising a base sequence of SEQ ID NO: 1.
    <4> A vector in which the nucleic acid according to any one of <1> to <3> is inserted, for inducing UK-2 biosynthesis and improving UK-2 productivity.
    <5> A method for determining UK-2 productivity, comprising detecting, in a test bacterium, the presence of a nucleic acid comprising a base sequence of the nucleic acid according to any one of <1> to <3> or a base sequence complementary to the sequence.
    <6> The method according to <5>, wherein a method for detecting the presence of the nucleic acid is a PCR method.
    <7> The method in <5>, wherein the PCR method is a method in which the nucleic acid is amplified using a primer comprising a base sequence of SEQ ID NO: 45 and a primer comprising a base sequence of SEQ ID NO: 46.
    <8> A bacterium in which UK-2 biosynthesis is induced and UK-2 productivity is improved, and in which the presence of the nucleic acid comprising the base sequence of the nucleic acid according to any one of <1> to <3> or the base sequence complementary to the sequence is detected by the method according to any one of <5> to <7>.
    <9> A bacterium in which UK-2 biosynthesis is induced and UK-2 productivity is improved by introducing the vector according to <4>.
    <10> A bacterium in which UK-2 biosynthesis is induced and UK-2 productivity is improved, and in which the nucleic acid according to any one of <1> to <3> is inserted in a genome thereof.
    <11> A bacterium in which one or two or more copies of a nucleic acid comprising a base sequence of the nucleic acid according to any one of <1> to <3> are present per cell.
    <12> The bacterium according to any one of <8> to <11>, which is any one of Streptoverticillium, Streptomyces, Escherichia coli, Bacillus subtilis, yeasts, filamentous fungi and Corynebacterium glutamicum. 
    <13> A method for producing UK-2, comprising the step of:


culturing the bacterium according to any one of <8> to <12>, and collecting UK-2 from a culture of the bacterium.

    <14> A method for producing a derivative of UK-2, comprising the steps of:


culturing the bacterium according to any one of <8> to <12>, and collecting UK-2 from a culture of the bacterium; and
synthesizing a derivative of UK-2 represented by any one of the following formulae (1) from the collected UK-2




[in the formula (1),

R represents any one of a 2-methylpropanoyl group, a trans-2-methyl-2-butenoyl group, a 3-methylbutanoyl groupand a 2-methylbutanoyl group.
R1 represents any one of a C1-6 alkyl group, a benzyl group, a C1-10 alkylcarbonyl group (the C1-10 alkylcarbonyl group may be substituted with any one of a carboxyl group, a benzyloxycarbonyl group, a C1-4 alkyloxycarbonyl group and benzyloxycarbonyl amino group) , a benzoyl group, a C1-4 alkyloxycarbonyl group, a (C1-4) alkyloxycarbonyl (C1-4) alkyl group, a benzyloxycarbonyl (C1-4) alkyl group may be substituted with a nitro group, a C1-6 alkylsulfonyl, di(C1-6)alkylphosphoryl group, a diphenylphosphoryl group and a substituent represented by the following formula (2);




(in the formula (2),

Q is selected from the group consisting of H, CH3, CH2CH3, CF3, Ph, CH═CH2 and a cyclopropyl.
M is selected from the group consisting of H, CH3, CH2CH3, CF3, Ph, CH═CH2 and a cyclopropyl.
T is selected from the group consisting of O, OC(O), OC(O)O, S, SC(O), SC(O)O and a substituent represent ed by the following formula (3);




G is selected from the group consisting of H, C1-6 alkyl group, a C1-6alkyloxy C1-6 alkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, a C3-6 cycloalkyl group, an aryl group and a heteroaryl group.
G and M may form an isobenzofuran ring optionally having an oxo group.
M and Q may form a 3-8 membered carbocyclic system.]. <15> A method for producing a derivative of UK-2A, comprising the steps of:
culturing the bacterium according to any one of <8> to <12>, and collecting UK-2A from a culture of the bacterium; and
synthesizing a derivative of UK-2A represented by any one of the following formulae (4) to (7) from the collected UK-2A.








Note that, as used herein, the term “acyl” shall mean a residue RCO— provided by removing OH from a carboxylic acid R—COOH, wherein R represents a hydrocarbon group. As used herein, the term “aryl” shall mean phenyl or naphthyl. As used herein, the term “heteroaryl” shall mean any 5 or 6 membered aromatic ring, containing one or more heteroatoms, where such heteroatoms are selected from the group consisting of O, N, and S, and where the remaining atoms of the aromatic ring are carbon atoms. Suitable examples include, but are not limited to a pyridine, a pyridazine, a pyrimidine, a pyrazine, a pyrrole, a pyrazole, an imidazole, a furan, a thiophene, an oxazole, an isoxazole, a thiazole, an isothiazole, a quinoline, a quinoxoline and a thiadiazole.
The present invention makes it possible to provide a transformant having high UK-2 productivity by introducing a UK-2 biosynthetic gene into a host cell such as a bacterium. Further, mass production of UK-2 at low cost is also possible using the transformant. Moreover, it is also made possible to provide a method for determining UK-2 productivity by detecting the presence of the gene.


DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
<UK-2 Biosynthetic Gene>
The present invention provides a UK-2 biosynthetic gene. As described in Examples later, the present inventors have isolated, as novel UK-2 biosynthetic genes, genes shown in Table 2 from a genomic DNA of Streptoverticillium sp. 3-7.
Thus, one embodiment of the UK-2 biosynthetic gene of the present invention is an “isolated nucleic acid that induces UK-2 biosynthesis and improves UK-2 productivity, the nucleic acid is a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35,” and typically an “isolated nucleic acid that induces UK-2 biosynthesis and improves UK-2 productivity, the nucleic acid is a nucleic acid comprising a base sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 or 34.”
In the present invention, the phrase “improvement in UK-2 productivity” and related phrases mean not only improvement in UK-2 productivity that a bacterium or the like naturally has, but also the acquisition of a UK-2 production ability by a bacterium or the like that does not naturally have the UK-2 production ability.
In the present invention, the term “isolation” and related terms mean an artificial treatment which allows the nucleic acid to exist under a condition different from the originally existing condition. The UK-2 biosynthetic gene of the present invention can be isolated, for example, by first synthesizing an appropriate primer on the basis of the information on the base sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 or 34, and then carrying out PCR using the primer with a template of the genomic DNA of Streptoverticillium sp. 3-7, Alternatively, as described in Example later, the UK-2 biosynthetic gene of the present invention can also be isolated from a genomic DNA library or cDNA library of Streptoverticillium sp. 3-7 by carrying out colony hybridization using the amplification product obtained by the PCR as a probe. Besides, the UK-2 biosynthetic gene of the present invention can also be prepared by total chemical synthesis based on the base sequence information.
In the present invention, “UK-2” is a compound represented by the following formula (8):




wherein R represents a linear or branched saturated aliphatic acyl group or a linear or branched unsaturated aliphatic acyl group. Preferably, “UK-2” is a compound wherein R is an isobutyryl group (2-methylpropanoyl group) (UK-2A), a compound wherein R is a tigloyl group (trans-2-methyl-2-butenoyl group) (UK-2B), a compound wherein R is an isovaleryl group (3-methylbutanoyl group) (UK-2C) and a compound wherein R is a 2-methylbutanoyl group (UK-2D).

Moreover, in the present invention, the “UK-2 biosynthetic gene” is a gene encoding a protein having an activity capable of inducing UK-2 biosynthesis. The “activity capable of inducing UK-2 biosynthesis” can be evaluated by, for example, a method described in Example 9 later. Specifically, after a nucleic acid for encoding the test protein is inserted into a vector which is subjected to introduction or the like into a host cell (for example, Streptoverticillium sp. 3-7), the amount of UK-2 produced in the host cell is measured by forced expression of the test protein in the host cell. If the amount produced is larger than that in a host cell in which the test protein is not expressed, it can be evaluated that the test protein has an activity capable of inducing UK-2 biosynthesis.
In the state of the art, if the information on the base sequence of the UK-2 biosynthetic gene is available, those skilled in the art can modify the base sequence and obtain a nucleic acid encoding a protein involved in UK-2 biosynthesis, although the amino acid sequence of the protein is different from one that is encoded from the base sequence. Meanwhile, in nature also, the amino acid sequence of a protein to be encoded may undergo mutation by a mutation of the base sequence. Thus, another embodiment of the UK-2 biosynthetic gene of the present invention is an “isolated nucleic acid that is a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 in which one or more amino acids are substituted, deleted, added and/or inserted.” Here, “more than one” refers to the number of amino acids modified in a protein involved in UK-2 biosynthesis after the modification, provided that the protein still has an activity of inducing UK-2 biosynthesis. The number is normally 1 to 50, preferably 1 to 40, and more preferably 1 to several (for example, 1 to 20, 1 to 10, 1 to 8, and 1 to 4).
Those skilled in the art can prepare the nucleic acid encoding such a variant by known methods such as site-directed mutagenesis on the basis of the information on the base sequence of the UK-2 biosynthetic gene.
Further, in the state of the art, if the information on the base sequence of the UK-2 biosynthetic gene is available, those skilled in the art can obtain nucleic acids (homologous genes) encoding a protein having an activity of inducing UK-2 biosynthesis from strains other than Streptoverticillium sp. 3-7 and other bacteria by a hybridization technique or a polymerase chain reaction (PCR) technique. Thus, another embodiment of the UK-2 biosynthetic gene of the present invention is an “isolated nucleic acid that is a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 or 34.”
To isolate such a homologous gene, normally a hybridization reaction is carried out under stringent conditions. The “stringent conditions” mean that under which the membrane washing procedure following the hybridization is carried out at high temperature in a solution having a low salt concentration. The “stringent conditions” include washing conditions, for example, at a 2×SSC concentration (1×SSC: 15 mM trisodium citrate, 150 mM sodium chloride) in a 0.5% SDS solution at 60° C. for 20 minutes. Additionally, the hybridization can be carried out, for example, according to a method described in the instruction attached to known ECL Direct DNA/RNA Labeling and Detection System (manufactured by Amersham Pharmacia Biotech Inc.).
Moreover, the protein encoded by the homologous gene obtained by such a method normally has a high homology with an amino acid sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35, Thus, another embodiment of the UK-2 biosynthetic gene of the present invention is an “isolated nucleic acid that is a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35.”
The homology of the sequences can be determined using, for example, a program of BLASTX (amino acid level) from NCBI.
As described in Examples later, the UK-2 biosynthetic gene of the present invention can be used for preparing a transformant having high UK-2 productivity, and can also be used effectively for screening for the UK-2 biosynthetic gene cluster.
In preparing such a transformant and screening for the UK-2 biosynthetic gene cluster, the use of the above-described UK-2 biosynthetic genes in combination is preferable to the individual use of the UK-2 biosynthetic genes. The number of the UK-2 biosynthetic genes in combination is not particularly limited, as long as the UK-2 biosynthesis can be induced by the combination. For example, the number is 2 or larger, preferably 5 or larger, further preferably 10 or larger, and more preferably 15 or larger. The number of the UK-2 biosynthetic genes in combination is most preferably 17 because the UK-2 productivity in the transformant can be significantly improved.
The UK-2 biosynthetic genes in combination may exist as a single nucleic acid or as separate nucleic acids.
The present invention provides a “nucleic acid comprising a base sequence of SEQ ID NO: 1” as a single nucleic acid (UK-2 biosynthetic gene cluster) comprising the 17 UK-2 biosynthetic genes. The locations of open reading frames (ORFs) of the genes in the nucleic acid comprising the base sequence of SEQ ID NO: 1 are as shown in Table 1 described later.
As described in Example later, the “nucleic acid comprising the base sequence of SEQ ID NO: 1” can be isolated by first synthesizing an appropriate primer on the basis of the information on the base sequence of the UK-2 biosynthetic gene, and the like, and then carrying out PCR using the primer with a template of a cosmid genomic DNA library of Streptoverticillium sp. 3-7 prepared independently, followed by colony hybridization using the obtained amplification product as a probe.
<Vector>
The present invention provides a vector in which the UK-2 biosynthetic gene of the present invention is inserted. The vector of the present invention can be constructed based on a self-replicating vector, i.e., for example, a plasmid which exists as an extrachromosomal element, and which replicates independently of the replication of the chromosome. Alternatively, the vector of the present invention may be replicated together with the chromosome of a host cell such as a bacterium, after introduced into the host cell and incorporated into the genome thereof. As a procedure and a method for constructing the vector of the present invention, any procedure and method commonly used in the field of genetic engineering can be used.
Those skilled in the art can select as appropriate the vector of the present invention from known vectors according to the type of the host cell to be introduced. Examples of the known vectors include cosmid vectors (SuperCos 1 cosmid vector and the like), phage vectors, pUC based plasmids (pCR2.1-TOPO plasmid vector and the like), pBluescript based plasmids, and pBR322 plasmids.
To express the protein encoded by the UK-2 biosynthetic gene of the present invention in the host cell, the “vector” of the present invention preferably comprises, in addition to the gene, a DNA sequence for regulating the expression, a marker gene for selecting the transformed host cell, and the like.
Examples of the “DNA sequence for regulating the expression” include a promoter, an enhancer and a terminator. The example also includes a lactose operon capable of inducing expression of the gene located downstream by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to the bacteria. The vector of the present invention can be constructed, for example, by operably ligating a promoter and a terminator respectively upstream and downstream of the UK-2 biosynthetic gene of the present invention.
The “marker gene” can be selected as appropriate according to the method for selecting the transformed host cell (transformant). For example, a gene encoding drug resistance or a gene complementing the auxotrophy can be used. In a case where the host cell used is a bacterium, examples of the marker gene include an ampicilin resistance gene, a kanamycin resistance gene, and a tetracycline resistance gene. Particularly, in a case of an actinobacterium, the examples include an apramycin resistance gene, a thiostrepton resistance gene, a hygromycin resistance gene, a kanamycin resistance gene, a streptomycin resistance gene, a viomycin resistance gene, and the like. In a case of a yeast, the examples include a tryptophan biosynthetase gene (TRP1), a uracil biosynthesis gene (URA3), a leucine biosynthesis gene (LEU2), and the like. In a case of a mold, the examples include a hygromycin resistance gene, a bialaphos resistance gene, a bleomycin resistance gene, an aureobasidin resistance gene, and the like. In a case of a plant, the examples include a kanamycin resistance gene, a bialaphos resistance gene, and the like.
<Transformant etc.>
The present invention provides a transformant in which the vector of the present invention is introduced (for example, a bacterium in which UK-2 biosynthesis is induced and UK-2 productivity is improved by introducing the vector of the present invention).
Moreover, the present invention provides a transformant in which UK-2 biosynthesis is induced and UK-2 productivity is improved, and in which the UK-2 biosynthetic gene of the present invention is inserted in a genome thereof.
The host cell which is transformed by the introduction of the vector of the present invention or the host cell in the genome of which the UK-2 biosynthetic gene of the present invention is not particularly limited. Examples thereof include actinobacteria, Escherichia coli, Bacillus subtilis, yeasts, filamentous fungi, Corynebacterium glutamicum, plant cells, insect cells, and animal cells. From the viewpoint of UK-2 productivity, actinobacteria are preferable, bacteria belonging to the genus Streptoverticillium and bacteria belonging to the genus Streptomyces are more preferably, bacteria belonging to the genus Streptoverticillium are even more preferably, and Streptoverticillium sp. 3-7 is particularly preferable.
The method for introducing the vector of the present invention into the host cell is not particularly limited. It can be selected and employed as appropriate by those skilled in the art from known transformation methods such as conjugal transfer, phage transduction, a calcium ion method, a lithium ion method, an electroporation method, a PEG method, an Agrobacterium method, and a particle gun method, depending on the type of the host cell under test. Moreover, in a case where the vector comprising the “marker gene” is introduced to the host cell, the transformant of the present invention can be efficiently prepared by culturing in a medium to which an antibiotic corresponding to the drug resistance gene is added or in a medium which is deficient in a nutrient corresponding to the gene complementing the auxotrophy.
Further, the present invention provides a bacterium in which UK-2 biosynthesis is induced and UK-2 productivity is improved by improvement in fermentation conditions, mutation induction, or the like. Furthermore, it has been revealed as described in Examples later that comprising at least two copies of the UK-2 biosynthetic gene of the present invention induces UK-2 biosynthesis and significantly improves UK-2 productivity. Thus, the present invention also provides a bacterium in which one or two or more copies of the UK-2 biosynthetic gene of the present invention are present per cell. From the viewpoint of UK-2 productivity, such bacteria are preferably actinobacteria, more preferably bacteria belonging to the genus Streptoverticillium and bacteria belonging to the genus Streptomyces, and further preferably bacteria belonging to the genus Streptoverticillium, Additionally, from the viewpoint of UK-2 productivity, the number of copies of the UK-2 biosynthetic gene of the present invention per cell is preferably two or larger. Note that the number of copies of the UK-2 biosynthetic gene of the present invention per cell can be identified, for example, by a PCR method as described in Examples later.
<Method for Determining UK-2 Productivity>
The present inventors have isolated and identified genes necessary for biosynthesis of UK-2, and therefore have made it possible to determine UK-2 productivity by detecting the presence of the genes. Thus, the present invention also provides a method for determining UK-2 productivity, comprising detecting, in a test bacterium, the presence of a nucleic acid comprising a base sequence of the UK-2 biosynthetic gene of the present invention or a base sequence complementary to the sequence.
In the method of the present invention, the “test bacterium” is not particularly limited. Examples thereof include actinobacteria (bacteria belonging to the genus Streptoverticillium, bacteria belonging to the genus Streptomyces, and the like), Escherichia coli, Bacillus subtilis, yeasts, filamentous fungi, and Corynebacterium glutamicum. 
In the method for determining UK-2 productivity of the present invention, the base sequence of the UK-2 biosynthetic gene of the present invention to be detected, that is, the base sequence of the nucleic acid of the present invention, is a base sequence of at least one nucleic acid selected from the group consisting of the above-described (a) to (q).
The nucleic acid and so forth can be detected directly by targeting a genomic DNA including the nucleic acid and so forth or a transcription product from the genomic DNA. Alternatively, the nucleic acid and so forth can also be detected indirectly by targeting a translation product from the transcription product (a protein encoded by the UK-2 biosynthetic gene of the present invention). Further, the detection of the nucleic acid and so forth can employ any of known methods. In a case of targeting the genomic DNA, it is possible to employ, for example, an in situ hybridization (ISH) method, a genomic PCR method, a direct sequencing method, a southern blotting method, and an analysis method using a genome microarray. In a case of targeting the transcription product, it is possible to employ, for example, a PCR method, a direct sequencing method, a northern blotting method, a dot plot method, and an analysis method using a cDNA microarray. In a case of targeting the translation product, examples of the known methods include immunological methods using an antibody against a protein encoded by the UK-2 biosynthetic gene of the present invention (a western blotting method, an ELISA method, flow cytometry, immunohistochemical staining, imaging cytometry, radioimmunoassay, immunoprecipitation, an analysis method using an antibody array, and the like). Among these methods, preferable is a PCR method, and more preferable is a PCR method in which the nucleic acid is amplified using a primer comprising a base sequence of SEQ ID NO: 45 and a primer comprising a base sequence of SEQ ID NO: 46.
Additionally, in the method of the present invention, from the viewpoint of achieving more accurate determination of UK-2 productivity, it is preferable to detect the presence of multiple nucleic acids (the UK-2 biosynthetic genes of the present invention) described above, rather than detecting the presence of one of the nucleic acids. The number of the nucleic acids to be detected is, for example, two or larger, preferably five or larger, more preferably 10 or larger, and even more preferably 15 or larger. Detecting all of the 17 nucleic acids is particularly preferable, and detecting a single nucleic acid comprising all the 17 nucleic acids (the nucleic acid comprising the base sequence of SEQ ID NO: 1) is the most preferable. Furthermore, besides the entire length of the nucleic acid, a portion thereof is targeted in a normal practice for detecting the presence of the nucleic acid. Thus, in the method of the present invention also, the detection of the nucleic acid and so forth may be detection of a portion of the nucleic acid and so forth. Those skilled in the art can select as appropriate the length of the portion of the nucleic acid to be detected by the method of the present invention, depending on the detection method.
Then, if the presence of the nucleic acid in the test bacterium can be detected by such a method, the test bacterium is determined to have UK-2 productivity. Additionally, the method of the present invention may further comprises culturing the test bacterium in which the presence of the nucleic acid can be detected, in conditions that allow UK-2 to be produced.
In addition, the present invention also provides a bacterium in which UK-2 biosynthesis is induced and UK-2 productivity is improved, and in which the presence of the nucleic acid comprising the base sequence of the nucleic acid of the present invention or the base sequence complementary to the sequence is detected by the method for determining UK-2 productivity of the present invention. From the viewpoint of UK-2 productivity, such bacteria are preferably actinobacteria, more preferably bacteria belonging to the genus Streptoverticillium and bacteria belonging to the genus Streptomyces, and even more preferably bacteria belonging to the genus Streptoverticillium. 
Note that, as used herein, the above-described bacteria and so forth having the UK-2 biosynthetic gene of the present invention, that is, the bacterium in which UK-2 biosynthesis is induced and UK-2 productivity is improved, and in which the presence of the nucleic acid is detected by the method for determining UK-2 productivity of the present invention, the transformant in which UK-2 biosynthesis is induced and UK-2 productivity is improved by introducing the vector of the present invention, the transformant in which UK-2 biosynthesis is induced and UK-2 productivity is improved, and in which the UK-2 biosynthetic gene of the present invention is inserted in a genome thereof, the bacterium in which one or two or more copies of the UK-2 biosynthetic gene of the present invention are present per cell, and the bacterium in which UK-2 biosynthesis is induced and UK-2 productivity is improved by improvement in fermentation conditions, mutation induction, or the like, are hereinafter collectively referred to as “bacteria etc. of the present invention.”
<Method for Producing UK-2>
The present invention provides a method for producing UK-2, comprising the step of:
culturing the bacteria etc. of the present invention, and collecting UK-2 from a culture of the bacteria etc.
The bacteria etc. can be cultured by selecting the medium, the culture condition, and the like as appropriate according to a conventional method. As the medium, commonly used components can be used. For example, as the carbon source, it is possible to use glucose, sucrose, cellulose, starch syrup, dextrin, starch, glycerol, molasses, animal and vegetable oils, or the like. Moreover, as the nitrogen source, it is possible to use soybean flour, wheat germ, pharmamedia, corn steep liquor, cottonseed meal, broth, peptone, polypeptone, malt extract, yeast extract, ammonium sulfate, sodium nitrate, urea, or the like. Besides, if necessary, it is effective to add inorganic salts which can produce sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other ions; examples of the inorganic salts include potassium chloride, calcium carbonate, dipotassium hydrogen phosphate, magnesium sulfate, monopotassium phosphate, zinc sulfate, manganese sulfate, and copper sulfate. Additionally, if necessary, it is also possible to add various vitamins such as thiamine (thiamine hydrochloride and the like), amino acids such as glutamic acid (sodium glutamate and the like) and asparagine (DL-asparagine and the like), trace nutrients such as nucleotide, and selective drugs such as antibiotics. Further, organic and inorganic substances to promote growth of the bacterium and the UK-2 production can be added as appropriate. The pH of the medium is not particularly limited, and may be adjusted according to the type of the bacteria etc. to be cultured. For example, the pH is approximately 6 to 8.
Those skilled in the art can select and set as appropriate the culture conditions according to the type of the bacteria etc. to be cultured, the type of the medium to be used, and so forth. For example, the culture method can be selected from known culture methods such as a shaking culture method under an aerobic condition, an aerated and agitated culture method and an aerobic submerged culture method. The aerated and agitated culture method is preferable. An appropriate culture temperature is 15° C. to 40° C. In many cases, the culture temperature is set around 26° C. to 37° C. Moreover, the culture period is preferably 2 days to 25 days when the maximum accumulation of UK-2 is achieved.
In the present invention, the “culture” refers to a medium obtained by culturing the bacteria etc. of the present invention, the medium containing the proliferated bacteria etc., a secretion and a metabolite of the bacteria etc., and the like. The culture also includes a dilution and a concentrate of these.
In the culture, UK-2 is accumulated in both of the bacteria etc. and the medium. Thus, an example of the method for collecting UK-2 from the medium of the culture is an extraction method using an organic solvent such as ethyl acetate, chloroform, and dichloromethane which do not mix with water freely, and which are capable of effectively extracting UK-2, Meanwhile, from the bacteria etc. of the culture, for example, UK-2 can be collected by extraction, with an organic solvent such as acetone, on the bacteria etc. which has been obtained by means such as filtration or centrifugation. Further, UK-2 can be collected by extraction in the same way as the above-described extraction from the medium, after the bacteria etc. of the culture has been disrupted using glass beads or the like.
Moreover, in collecting UK-2 from the culture, UK-2 can be isolated and purified by subjecting a thus-prepared extraction fraction such as organic solvent to known purification techniques such as solvent transfer dissolution, normal-phase and reverse-phase chromatographies, gel filtration chromatography, and crystallization in combination.
<Method for Producing UK-2 Derivative>
As described above, the present invention makes mass production of UK-2 at low cost possible. Accordingly, mass production of UK-2 derivatives at low cost is also made possible using UK-2 obtained by the production method of the present invention as the material thereof.
Thus, the present invention can also provide a method for producing a derivative of UK-2, comprising the steps of:
culturing the bacteria etc. of the present invention, and collecting UK-2 (UK-2A, UK-2B, UK-2C or UK-2D) from a culture of the bacteria etc.; and
synthesizing a derivative of UK-2 represented by any one of the following formulae (1) from the collected UK-2




[in the formula (1),

R represents any one of a 2-methylpropanoyl group, a trans-2-methyl-2-butenoyl group, a 3-methylbutanoyl groupand a 2-methylbutanoyl group.
R1 represents any one of a C1-6 alkyl group, a benzyl group, a C1-10 alkylcarbonyl group (the C1-10 alkylcarbonyl group may be substituted with any one of a carboxyl group, a benzyloxycarbonyl group, a C1-4 alkyloxycarbonyl group and benzyloxycarbonylamino group), a benzoyl group, a C1-4 alkyloxycarbonyl group, a (C1-4) alkyloxycarbonyl (C1-4) alkyl group,a benzyloxycarbonyl (C1-4) alkyl group may be substituted with a nitro group, a C1-6alkylsulfonyl, di(C1-6)alkylphosphoryl group, a diphenylphosphoryl group and a substituent represented by the following formula (2);




(in the formula (2),

Q is selected from the group consisting of H, CH3, CH2CH3, CF3, Ph, CH═CH2 and a cyclopropyl.
M is selected from the group consisting of H, CH3, CH2CH3, CF3, Ph, CH=CH2 and a cyclopropyl.
T is selected from the group consisting of O, OC(O), OC(O)O, S, SC(O), SC(O)O and a substituent represent ed by the following formula (3);




G is selected from the group consisting of H, C1-6 alkyl group, a C1-6alkyloxy C1-6 alkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, a C3-6 cycloalkyl group, an aryl group and a heteroaryl group.
G and M may form an isobenzofuran ring optionally having an oxo group.
M and Q may form a 3-8 membered carbocyclic system.].
In the substituent represented by the formula (2), the alkyl group, the alkynyl group, the alkenyl group, the cycloalkyl group, the aryl group and the heteroaryl group may be substituted with at least one substituent selected from the group consisting of the following substituent groups;
a C1-6 alkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, a C3-6 cycloalkyl group, a C5-6 cycloalkenyl group, an aryl group, a heteroaryl group, a halogen atom, a nitro group, a hydroxy group, a cyano group, a C1-6 alkoxy group, a C2-6 alkenoxy group, a C3-6 cycloalkoxy group, an aryloxy group, a heteroaryloxy group, an acyloxy group, a C1-6 alkylacyloxy group, a C3-6 cycloalkylacyloxy group, an arylacyloxy group, a heteroarylacyloxy group, a C1-6 alkyloxyacyl group, a C3-6 cycloalkyloxyacyl group, an aryloxyacyl group, a heteroaryloxyacyl group, a C1-6 alkylacyl group, a C3-6 cycloalkylacyl group, an arylacyl group, a heteroaryl acyl group, a C1-6 alkyl acylamino group, a C3-6 cycloalkylacylamino group, an arylacylamino group, a heteroarylacylamino group, a C1-6 alkylaminoacyl group, a C3-6 cycloalkylaminoacyl group, an arylaminoacyl group, a heteroarylaminoacyl group, a C1-6alkylthio group, a C3-6 cycloalkylthio group, an arylthio group, a heteroarylthio group, a C1-6 alkylsulfonyl group, a C3-6 cycloalkylsulfonyl group, an arylsulfonyl group, a heteroarylsulfonyl group, a C1-6 alkylsulfinyl group, a C3-6 cycloalkylsulfinyl group, an arylsulfinyl group, a heteroarylsulfinyl group and —C(NORx)RY wherein RY and Rx are independently any one of H, a C1-6 alkyl group, a C2-6 alkenyl group, a C3-6 cycloalkyl group, an aryl group and a heteroaryl group in which any alkyl or cycloalkyl containing substituent may be substituted with one or more halogens.
Note that, the substituent may also be substituted with at least one substituent selected from the group consisting of the following substituent groups;
a C1-6 alkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, a C3-6 cycloalkyl group, a C5-6 cycloalkenyl group, an aryl group, a heteroaryl group, a halogen atom, a nitro group, a hydroxy group, a cyano group, a C1-6 alkoxy group, a C2-6 alkenoxy group, a C3-6 cycloalkoxy group, an aryloxy group, a heteroaryloxy group, an acyloxy group, a C1-6 alkylacyloxy group, a C3-6 cycloalkylacyloxy group, an arylacyloxy group, a heteroarylacyloxy group, a C1-6 alkyloxyacyl group, a C3-6 cycloalkyloxyacyl group, an aryloxyacyl group, a heteroaryloxyacyl group, a C1-6 alkylacyl group, a C3-6 cycloalkylacyl group, an arylacyl group, a heteroaryl acyl group, a C1-6 alkyl acyl amino group, a C3-6 cycloalkylacylamino group, an arylacylamino group, a heteroarylacylamino group, a C1-6 alkylaminoacyl group, a C3-6 cycloalkylaminoacyl group, an arylaminoacyl group, a heteroarylaminoacyl group, a C1-6 alkylthio group, a C3-6 cycloalkylthio group, an arylthio group, a heteroarylthio group, a C1-6 alkylsulfonyl group, a C3-6 cycloalkylsulfonyl group, an arylsulfonyl group, a heteroarylsulfonyl group, a C1-6 alkylsulfinyl group, a C3-6 cycloalkylsulfinyl group, an arylsulfinyl group, a heteroarylsulfinyl group and —C(NORx)RY wherein RY and Rx are independently any one of H, a C1-6 alkyl group, a C2-6 alkenyl group, a C3-6 cycloalkyl group, an aryl group and a heteroaryl group in which any alkyl or cycloalkyl containing substituent may be substituted with one or more halogens.
In addition, the present invention can also provide a method for producing a UK-2A derivative, comprising the steps of:
culturing the bacteria etc. of the present invention, and collecting UK-2A from a culture of the bacteria etc.; and
synthesizing a UK-2A derivative represented by any one of the following formulae (4) to (7) from the collected UK-2A.




In collecting UK-2A, UK-2B, UK-2C or UK-2D from the culture, UK-2A, UK-2B, UK-2C or UK-2D can be isolated and purified, for example, as described above, by subjecting the extraction fraction such as organic solvent to known purification techniques such as solvent transfer dissolution, normal-phase and reverse-phase chromatographies, gel filtration chromatography, and crystallization in combination. More specifically, the extraction fraction such as organic solvent is concentrated under reduced pressure. The resultant is transferred to and dissolved in chloroform, and subjected to silica gel chromatography, which is then eluted stepwise with chloroform/methanol. Thus, a fraction which contains UK-2A and UK-2D at a ratio of approximately 3:1, and which also contains trace amounts of UK-2B and UK-2C can be obtained. Further, the fraction is treated by reverse-phase high performance liquid chromatography (HPLC) using a C-18 column, and thus UK-2A, UK-2B, UK-2C or UK-2D can be isolated (see Japanese Examined Patent Application Publication No. Hei 07-233165).
Then, the derivative of UK-2A, UK-2B, UK-2C or UK-2D represented by any one of the formulae (1) and (4) to (7) can be synthesized using UK-2A, UK-2B, UK-2C or UK-2D thus collected as the material thereof by, for example, the synthesis method described in International Publication No. 2003/035617 or International Publication No. 1999/40081.
EXAMPLES
Hereinafter, the present invention will be more specifically described based on Example. However, the present invention is not to be limited to Examples below.
Note that the microorganism described in the present Examples is deposited as follows. Streptoverticillium sp. 3-7 was deposited at International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki, postal code 305-8566, Japan) on November 9, Heisei 23 (2011) under the accession number of FERM BP-11437, Incidentally, the deposit of the patent microorganisms by International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (former name: IPOD) was succeeded by National Institute of Technology and Evaluation (NITE, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, postal code 292-0818) on April, 2012.
Streptoverticillium sp. 3-7 was established from SAM 2084 strain described in Japanese Examined Patent Application Publication No. Hei 07-233165, which was artificially mutated through a single ultraviolet irradiation by the present inventors. The SAM 2084 strain is a UK-2-producing bacterial strain obtained from soil in Kyoto prefecture of Japan and identified under the international deposit number FERM BP-6446.
Example 1
<Preparation of Genomic DNA Library>
To isolate genes necessary for biosynthesis of UK-2, first, the genomic DNA library of Streptoverticillium sp. 3-7 capable of producing UK-2 was prepared by a method described below.
Streptoverticillium sp. 3-7 was inoculated into 50 ml of modified YEME (0.3% Difco yeast extract, 0.5% Difco bacto peptone, 0.3% Oxoid malt extract, 3.0% sucrose, 1.0% glucose, 5 mmol/L MgCl2.6H2O) and shake-cultured at 220 rpm at 30° C. for 18 hours. After the culturing was complete, the bacterial cells were collected by centrifugation at 7500 rpm for 10 minutes. From the bacterial cells thus obtained, the genomic DNA was prepared employing the salting out method [see “Practical Streptomyces Genetics,” The John Innes Foundation, (UK), Norwich, 2000].
The obtained genomic DNA was partially digested with a restriction enzyme MboI, and then treated with alkaline phosphatase to dephosphorylate the terminal of the DNA. This DNA fragment was ligated to a commercially available cosmid vector SuperCosl (manufactured by Stratagene Corporation) which had been subjected in advance to digestion with a restriction enzyme XbaI, an alkaline phosphatase treatment for dephosphorylation and further digestion with a restriction enzyme BamHI. Thus, a recombinant cosmid vector was prepared. This recombinant cosmid vector was subjected to in vitro packaging using MAXPLAX Lambda Packaging Extracts manufactured by Epicentre Biotechnologies. Escherichia coli XLI-Blue MRA was infected therewith to prepare the cosmid genomic DNA library.
Example 2
<Estimation of UK-2 Biosynthetic Gene>
Based on the genomic DNA prepared by the method described in Example 1, construction of the mate-pair library for Roche GS FLX Titanium sequencer was entrusted to Genaris, Inc. Then, this sequencer was used to determine the sequence. Separately from this, based on the genomic DNA, the fragment library for this sequencer was constructed. Then, this sequencer was used to determine the sequence. The sequence obtained from the mate-pair library and the sequence obtained from the fragment library were co-assembed together to obtain the contig sequence and the scaffold sequence.
UK-2 has a characteristic 3-hydroxypicolinic acid skeleton. Meanwhile, virginiamycin also has a hydroxypicolinic acid skeleton. Two genes (visA, visB) involved in the biosynthesis of virginiamycin have been disclosed (see Namwat W., et al, Journal of Bacteriology, September 2002, vol. 184, no. 17, pp. 4811 to 4818). Thus, a homology analysis was conducted between the amino acid sequence of the proteins encoded by these two genes and the proposed amino acid sequence obtained from the genome of the UK-2 producing bacterium to examine the existence of genes involved in formation of the hydroxypicolinic acid skeleton. Tables 1 and 2 show the obtained result.









TABLE 1









Protein encoded by ORF













SEQ
Location in


The number


ORF
ID
base sequence of
ORF
SEQ
of amino


name
NO:
SEQ ID NO: 1
direction
ID NO:
acid residues















ORF1
2
 1-681
+
3
226


ORF2
4
 674-2560
−
5
628


ORF3
6
2590-4200
−
7
536


ORF4
8
4377-4559
−
9
60


ORF5
10
4550-5686
−
11
378


ORF6
12
5800-7485
−
13
561


ORF7
14
7637-8884
+
15
415


ORF8
16
9109-9654
+
17
181


ORF9
18
 9671-10201
−
19
176


ORF10
20
10302-11078
−
21
258


ORF11
22
11121-12422
−
23
433


ORF12
24
12814-16644
−
25
1276


ORF13
26
16649-26383
−
27
3244


ORF14
28
26814-27986
−
29
390


ORF15
30
28051-29112
−
31
353


ORF16
32
29275-29904
+
33
209


ORF17
34
29978-31318
+
35
446



















TABLE 2









Known protein having high homology with protein encoded by ORF














ORF 
SEQ ID


GenBank 
Homology 
Putative functional protein 


name
NO.
Protein name
Species
accession number
(%)
to be encoded by ORF
















ORF1
2
LuxR family transcriptional regulator
Streptomyces
NP_821584.1
60
Transcriptional regulator





Avermitilis





ORF2
4
Long-chain-fatty-acid--CoA ligase
Pseudonocardia
YP_004335893.1
53
Long-chain fatty-acid-CoA ligase





Dioxanivorans





ORF3
6
Histidine ammonia-lyase
Rubrobacter
YP_644511.1
55
Phenylalanine/histidine ammonia-





Xylanophilus


lyase


ORF4
8
4-oxalocrotonate tautomerase
Streptomyces
CBW45760.1
47
4-oxalocrotonate tautomerase





pristinaespiralis





ORF5
10
L-lysin 2-aminotransferase
Streptomyces
ZP_06913862.1
58
L-Lysine 2-aminotransferase





pristinaespiralis





ORF6
12
3-hydroxypicolinic acid:AMP ligase
Streptomyces
AEF33098.1
60
3-hydroxypicolinic acid AMP ligase





Pyridomyceticus





ORF7
14
Cytochrome P450
Streptomyces
ZP_06593022.1
46
Cytochrome P450





Albus





ORF8
16
Thioesterase type II
Verrucosispora
YP_004406133.1
41
Thioesterase





Maris





ORF9
18
Ribosomal-protein-serine acetyltransferase
Streptomyces
ZP_07300520.1
90
Acetyltransferase





Hygroscopicus





ORF10
20
Oxidoreductase, short-chain 
Streptomyces
ZP_07314911.1
58
Short chain dehydrogenase




dehydrogenase/reductase
Griseoflavus





ORF11
22
Putative polyketide associated protein
Streptomyces
CAJ89364.1
40
Condensation domain protein





Ambofaciens





ORF12
24
Putative polyketide synthase related protein
Streptomyces
CAJ89362.1
58
Polyketide synthase





Ambofaciens





ORF13
26
Putative peptide synthetase
Streptomyces
CAJ89363.1
52
Non-ribosomal peptidesynthetase





Ambofaciens





ORF14
28
Sarcosine oxidase
Streptomyces
ZP_07310135.1
49
N-methyltryptophan oxidase





Griseoflavus





ORF15
30
Favin-dependent oxidoreductase
Streptomyces
AEF33076.1
53
Flavin-dependent oxidoreductase





Pyridomyceticus





ORF16
32
O-methyltransferase
Mycobacterium
YP_001848635.1
52
O-methyltransferase





Marinum





ORF17
34
Crotonyl-CoA reductase
Streptomyces
AAR32675.1
67
Crotonyl-CoA reductase





Hygroscopicus









As a result of the examination, the position where genes having a high homology with VisA and VisB were consecutively located was found out as a single position on the genome derived from Streptoverticillium sp. 3-7 (see Table 1). Moreover, it was found out that genes associated with a non-ribosomal peptide synthetase (NRPS) and a polyketide synthase (PKS) which were thought to be necessary to form the UK-2 skeleton were located near these genes (see Table 2). A region around the genes was expected to be a UK-2 biosynthetic gene cluster because the secondary metabolite genes of actinobacteria form a cluster. Further, there is an alignment between the genes (ORFs) located in the UK-2 biosynthetic gene cluster and putative functions of proteins encoded by the respective genes as follows.
ORF1 is a gene potentially involved in the regulation of the biosynthetic gene cluster. ORF5, ORF6, ORF7, and ORF16 are genes involved in the biosynthesis of the 3-hydroxypicolinic acid skeleton. ORF2, ORF3, and ORF17 are genes involved in the biosynthesis of a benzylmalonic acid skeleton. ORF13 is a gene involved in the biosynthesis of a picolinic acid skeleton, serine, and lactic acid. ORF11 and ORF12 are genes involved in the biosynthesis of benzylmalonic acid and metabolism of picolinic acid, serine, and lactic acid. ORF8 is a gene involved in the cleavage of a thioester bond of a polyketide synthase (PKS) and metabolism of picolinic acid, serine, lactic acid, and benzylmalonic acid.
Example 3
<Screening of Genomic DNA Library>
A portion of the sequence of ORF5 located upstream of the UK-2 biosynthetic genes was used as a probe for screening of the genomic DNA library prepared in Example 1, and prepared by PCR as described below.
PCR was carried out using the genomic DNA described in Example 1 as a template and oligo DNAs of visA′-F: 5′-GGGGCAGCCTGCTCGGCGAG-3′ (SEQ ID NO: 36) and visA'-R: 5′-GGTGAGCTCCCCGATCAGGG-3′ (SEQ ID NO: 37) as primers. The PCR was performed using LA Taq DNA polymerase (manufactured by Takara Bio Inc.) as a DNA polymerase and PERKIN ELMER GeneAmp PCR System 9700, The amount of the reaction solution was adjusted to 50 μl by addition of: 0.5 μl (corresponding to 0.5 μg in amount) of the genomic DNA, 25 μl of a buffer for two-fold concentration reaction accompanying the enzyme, 2.5 μl of a DMSO solution, 5 μl of a 2.5-mM dNTP solution, 0.25 μl of each of the primers whose concentration was adjusted to 100 μmol/μl, 0.3 μl of the enzyme, and 16.2 μl of sterilized water. The reaction was carried out as follows: the pretreatment at 95° C. for 10 minutes; incubation in 30 cycles each consisting of 95° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes; further incubation at 72° C. for 5 minutes. After the reaction was complete, a portion of the reaction solution was electrophoresed on an agarose gel. As a result, it was confirmed that approximately 1.3 kbp of a DNA fragment was specifically amplified. Then, the remaining reaction solution was subjected to extraction with a mixture solution (phenol:chloroform:isoamyl alcohol=25:24:1, V/V) for nucleic acid purification, followed by ethanol precipitation. The precipitate was dissolved again in sterilized water, and electrophoresed on an agarose gel. Approximately 1.3 kbp of a band was cut out according to a conventional method, and a DNA fragment was collected.
Colony hybridization was carried out using the DNA fragment as a probe and ECL Direct DNA/RNA Labeling and Detection System (manufactured by Amersham Pharmacia Biotech Inc.), and approximately 5000 colonies were screened. Several positive clones were obtained. A plasmid pUK2-B44 was isolated from one of the clones.
Further, a portion of ORF13 located downstream of the UK-2 biosynthetic genes was used as a probe, and prepared by PCR as described below.
PCR was carried out using the genomic DNA described in Example 1 as a template and oligo DNAs of caiC-F: 5′-GCGCTCGTACGCCTCGCTGAT-3′ (SEQ ID NO: 38) and caiC-R: 5′-CGGGCTCGGTGGTGAGCAGG-3′ (SEQ ID NO: 39) as primers. The PCR was performed using LA Taq DNA polymerase (manufactured by Takara Bio Inc.) as a DNA polymerase and PERKIN ELMER GeneAmp PCR System 9700, The amount of the reaction solution was adjusted to 50 μl by addition of: 0.5 μl (corresponding to 0.5 μg in amount) of the genomic DNA, 25 μl of a buffer for two-fold concentration reaction accompanying the enzyme, 2.5 μl of a DMSO solution, 5 μl of a 2.5-mM dNTP solution, 0.25 μl of each of the primers whose concentration was adjusted to 100 pmol/μl, 0.3 μl of the enzyme, and 16.2 μl of sterilized water. The reaction was carried out as follows: the pretreatment at 95° C. for 10 minutes; incubation in 30 cycles each consisting of 95° C. for 30 seconds, 59° C. for 30 seconds, and 72° C. for 2 minutes and 20 seconds. After the reaction was complete, a portion of the reaction solution was electrophoresed on an agarose gel. As a result, it was confirmed that approximately 2.3 kbp of a DNA fragment was specifically amplified. Then, the remaining reaction solution was subjected to extraction with the above-described mixture solution for nucleic acid purification, followed by ethanol precipitation. The precipitate was dissolved again in sterilized water, and electrophoresed on an agarose gel. Approximately 2.3 kbp of a band was cut out according to a conventional method, and a DNA fragment was collected.
Colony hybridization was carried out using the DNA fragment as a probe and ECL Direct DNA/RNA Labeling and Detection System (manufactured by Amersham Pharmacia Biotech Inc.), and approximately 5000 colonies were screened. Several positive clones were obtained. A plasmid pUK2-E4 was isolated from one of the clones.
Example 4
<Construction of Plasmid pUK2-3 Comprising Biosynthetic Gene Cluster>
Using the thus-obtained plasmids pUK2-B44 and pUK2-E4 respectively comprising the upstream region 1 to 21531 and the downstream region 16211 to 34641 of the expected biosynthesis cluster, a plasmid comprising the entire biosynthesis cluster region was constructed. First, both of the plasmids were digested with restriction enzymes ClaI and PspXI, followed by electrophoresis on agarose gels, and approximately 28 kbp and approximately 19 kbp of bands were respectively cut out according to a conventional method, and DNA fragments were collected. The DNA fragments were ligated using DNA Ligation Kit<Mighty Mix>(manufactured by Takara Bio Inc.) to prepare pUK2-16.
Next, using the redirect technology described in [Gust, B., et al, “Proceedings of the National Academy of Sciences of the United States of America, ” (US), 2003, vol. 100, pp. 1541-1546], the plasmid pUK2-16 was used as a vector capable of conjugal transfer to actinobacteria. First, the plasmid pUK2-16 was introduced in an E. coli BW25113/pIJ790 strain by electroporation, and an E. coli BW25113/pIJ790/pUK2-16 strain was obtained. This strain was inoculated into 100 ml of an LB liquid medium (1% bacto tryptone, 0.5% yeast extract, 0.5% sodium chloride) containing chloramphenicol, kanamycin and ampicillin respectively at concentrations of 25 μg/ml, 50 μg/ml and 50 μg/ml, and cultured at 30° C. overnight. Then, 100 μl of the culture solution was inoculated into 10 ml of an SOB medium (2% bacto tryptone, 0.5% yeast extract, 0.05% sodium chloride, 0.0186% potassium chloride) prepared in a 65-ml test tube containing chloramphenicol, kanamycin, ampicillin and L-arabinose respectively at concentrations of 25 μg/ml, 50 μg/ml, 50 μg/ml and 10 mM. The resulting culture was shake-cultured at 30° C. for 4 hours. The bacterial cells were collected from all of the culture solution, washed twice with an ice-cooled 10% glycerin solution, and resuspended to 100 μl of the 10% glycerin solution as cells for electroporation. Meanwhile, 5.2 kb of an SspI fragment containing oriT, attP, IntφC31 and an apramycin resistance gene derived from a plasmid pMJCOS1 (John Innes Centre (Norwich)) was purified. The DNA fragment (approximately 100 ng) and 50 μl of the cells thus prepared were transferred to an already ice-cooled cuvette with a gap of 2 mm, and subjected to electroporation (using Electro Cell Manipulator 600: manufactured by BM Equipment Co., Ltd.). After the treatment, 1 ml of a cooled LB liquid medium was added to the resultant, which was allowed to stand at 37° C. for 1 hour for culturing. This was then applied to an LB agar medium containing ampicillin and apramycin, and cultured at 37° C. overnight. The grown strain was cultured in an LB liquid medium containing ampicillin and apramycin, and a plasmid pUK2-3 was isolated. This pUK2-3 is a plasmid which is capable of conjugal transfer to actinobacteria, and which has oriT, attP, IntφC31 and the apramycin resistance gene in the vector portion and the entire region expected to be the UK-2 biosynthesis cluster.
Example 5
<Construction of Biosynthetic Gene-Deficient Vector>
A gene disrupted strain deficient in approximately 7.5 kbp corresponding to portions of ORF12 and ORF13 from the genomic DNA of Streptoverticillium sp. 3-7 was prepared by the method described below.
PCR was carried out using the genomic DNA described in Example 1 as a template and oligo DNAs of caiC-F: 5′-GCGCTCGTACGCCTCGCTGAT-3′ (SEQ ID NO: 38) and 41c29-R: 5′-GTCCGTGGCGCCGCCGGATT-3′ (SEQ ID NO: 40) as primers. The PCR was performed using LA Taq DNA polymerase (manufactured by Takara Bio Inc.) as a DNA polymerase and PERKIN ELMER GeneAmp PCR System 9700, The amount of the reaction solution was adjusted to 50 μl by addition of: 0.5 μl (corresponding to 0.5 μg in amount) of the genomic DNA, 25 μl of a buffer for two-fold concentration reaction accompanying the enzyme, 2.5 μl of a DMSO solution, 5 μl of a 2.5-mM dNTP solution, 0.25 μl of each of the primers whose concentration was adjusted to 100 pmol/μl, 0.3 μl of the enzyme, and 16.2 μl of sterilized water. The reaction was carried out as follows: the pretreatment at 95° C. for 10 minutes; incubation in 30 cycles each consisting of 95° C. for 30 seconds, 60° C. for 5 seconds, and 72° C. for 7 minutes. After the reaction was complete, a portion of the reaction solution was electrophoresed on an agarose gel. As a result, it was confirmed that approximately 7.5 kbp of a DNA fragment was specifically amplified. The DNA fragment was inserted into a pCR2.1-TOPO plasmid vector using TOPO TA cloning kit (manufactured by Invitrogen Corporation) in accordance with the protocol attached thereto. Thereby, a plasmid TOPO-41c29 was obtained.
Subsequently, an apramycin resistance gene was inserted into the inserted fragment of the plasmid TOPO-41c29 to prepare a plasmid TOPO-41c29-Am as follows.
A plasmid pIJ773 [Gust, B., et al., “Proceedings of the National Academy of Sciences of the United States of America,” (US), 2003, vol. 100, pp. 1541-1546] was double-digested with HindIII and EcoRI, followed by electrophoresis on an agarose gel. Then, a DNA fragment was cut out according to a conventional method and collected. Thus, approximately 1.3 kb of a DNA fragment comprising the target apramycin resistance gene was obtained. PCR was carried out using this fragment as a template and two types of synthetic primers of 41c30-apraF:

    5′-GTCACCGTCCCCGCCTACGGCGACGGCGTCGTCCTGGTGATTCCGGGGATC CGTCGACC-3′ (SEQ ID NO: 41) and 41c30-apraR: 5′-GGTCGCGGGCGAAGGCGTAGCCGGGCAGGTCGGGCAGGATGTAGGCTGGAG CTGCTTC-3′ (SEQ ID NO: 42). The PCR was performed using LA Taq DNA polymerase (manufactured by Takara Bio Inc.)


as a DNA polymerase and PERKIN ELMER GeneAmp PCR System 9700.
The amount of the reaction solution was adjusted to 50 μl by addition of: 0.5 μl (corresponding to 0.5 μg in amount) of the genomic DNA, 25 μl of a buffer for two-fold concentration reaction accompanying the enzyme, 2.5 μl of a DMSO solution, 5 μl of a 2.5-mM dNTP solution, 0.25 μl of each of the primers whose concentration was adjusted to 100 pmol/μl, 0.3 μl of the enzyme, and 16.2 μl of sterilized water. The reaction was carried out as follows: the pretreatment at 94° C. for 2 minutes; incubation in 10 cycles each consisting of 94° C. for 45 seconds, 50° C. for 45 seconds, and 72° C. for 1 minute and 30 seconds; then, incubation in 15 cycles each consisting of 94° C. for 45 seconds, 55° C. for 45 seconds, and 72° C. for 1 minute and 30 seconds; a further reaction at 72° C. for 5 minutes. After the reaction was complete, a portion of the reaction solution was electrophoresed on an agarose gel. As a result, it was confirmed that approximately 1.4 kbp of a DNA fragment was specifically amplified.
Next, TOPO-41c29 was introduced in E. coli BW25113/pIJ790 [Gust, B., et al., “Proceedings of the National Academy of Sciences of the United States of America,” (US), 2003, vol. 100, pp. 1541-1546] to obtain an E. coli BW25113/pIJ790/TOPO-41c29 strain. This strain was inoculated into 100 ml of an LB liquid medium containing chloramphenicol, kanamycin and ampicillin respectively at concentrations of 25 μg/ml, 25 μg/ml and 50 μg/ml, and cultured at 30° C. overnight. Then, 10 ml of an SOB medium was fed into a 65-ml test tube supplemented with chloramphenicol, kanamycin, ampicillin, and L-arabinose respectively at concentrations of 25 μg/ml, 25 μg/ml, 50 μg/ml, and 10 mM. To this, 100 μl of a culture solution of the E. coli BW25113/pIJ790/TOPO-41c29 strain cultured overnight was transferred, and shake-cultured at 30° C. for 4 hours. All of the culture solution was centrifuged at 3000 rpm at 4° C. for 5 minutes to collect the bacterial cells which were then suspended in 10 ml of an ice-cooled 10% glycerin solution. After this operation was repeated, the resulting bacterial cells were resuspended in 100 μl of a cooled 10% glycerin solution. Next, 50 μl of the bacterial cell-suspension was collected into an Eppendorf tube to which 5 μl of a solution of approximately 1.4 kb of a DNA fragment containing the above-described apramycin resistance gene derived from pIJ773 was added. The mixture was transferred to an already ice-cooled electroporation cuvette with a gap of 2 mm (BM6200: manufactured by BM Equipment Co., Ltd.). Electroporation was conducted using Electro Cell Manipulator 600 (manufactured by BM Equipment Co., Ltd.) under conditions of 12.5 kV, 25 pF, and 128Ω. After the treatment, 1 ml of an already ice-cooled LB liquid medium was added to the bacterial cells, which were then allowed to stand at 37° C. for 1 hour for culturing. This was applied to an LB agar medium supplemented with ampicillin and apramycin each at a concentration of 50 μg/ml. The resultant was cultured at 37° C. all the night to obtain a strain having resistance to both of ampicillin and apramycin. This strain was cultured in an LB liquid medium supplemented with ampicillin and apramycin each at a concentration of 50 pg/ml. Thus, a plasmid TOPO-41c29-Am was isolated.
Example 6
<Creation of Biosynthetic Gene-Deficient Strains>
The plasmid TOPO-41c29 was introduced in an E. coli ET12567/pUZ8002 strain [“Practical Streptomyces Genetics,” The John Innes Foundation, (UK), Norwich, 2000] according to a conventional method to obtain E. coli ET12567/pUZ8002/TOPO-41c29.
Streptoverticillium was conjugated to E. coli ET12567/pUZ8002/TOPO-41c29 as follows. First, the Streptoverticillium strain was inoculated into 10 ml of a liquid medium (S#1) [Ueki, M, et al, “The Journal of Antibiotics,” (Japan), 1996, vol. 49, pp. 639-643] prepared in a 65-ml test tube, and cultured at 30° C. for 24 hours. The resultant was applied to an MS agar medium (2% soybean flour, 2% mannitol, 2% agar), and cultured at 30° C. for 2 days. After the culturing, mycelia were collected by scraping with 3 ml of 20% glycerol to prepare a host mycelium solution.
After the bacterial cells were collected by centrifugation at 3000 rpm for 5 minutes, the bacterial cells were suspended in 3 ml of a 20% glycerin solution. Meanwhile, E. coli ET12567/pUZ8002/TOPO-41c29-Am was cultured at 37° C. for 18 hours in an LB liquid medium supplemented with ampicillin and apramycin each at a concentration of 50 μg/ml. Then, 1 ml of the culture solution was transferred to 100 ml of an LB liquid medium (containing ampicillin and apramycin each at a concentration of 50 μg/ml), and cultured at 37° C. for 4 hours. Subsequently, 50 ml of the culture solution was centrifuged at 3000 rpm for 5 minutes to collect the bacterial cells. The bacterial cells were suspended in 20 ml of an LB liquid medium. After this operation was repeated twice, the bacterial cells were suspended in 2 ml of an LB liquid medium.
Next, 100 μl of the Streptoverticillium cell-suspension and 100 μl of a bacterial cell-suspension of E. coli ET12567/pUZ8002/cosmid203-7 were combined together in a 1.5-ml tube, and centrifuged to collect bacterial cells. After suspended in 100 μl of a 20% glycerin solution, this was applied to an MS agar medium having a volume of 20 ml and containing 10 mM MgCl2. After the culturing at 30° C. for 18 hours, 1 ml of sterilized water containing 400 μg of apramycin and 1500 μg of nalidixic acid was overlaid thereon. After cultured at 30° C. for 5 days, Streptoverticillium colonies grown on the agar medium were subjected to pure culture and cultured at 30° C. for 2 days in a 1⁄2 MS agar medium (agar: 2%, mannitol: 1%, soybean flour: 1%, 10 mM MgCl2) supplemented with 250 μg/ml of apramycin and 250 μg/ml of kanamycin. A colony grew in any plate and was subcultured for several passages by: inoculation into an S#1 medium, followed by culturing at 30° C. for 24 hours, inoculation into an modified YEME medium (10 ml in a 65-ml test tube), followed by shake-culturing at 30° C. for 1 day, and further inoculation of 1 ml of the resulting culture into another fresh modified YEME medium (50 ml in a 250-ml Erlenmeyer flask). After this operation was repeated five times, the resulting culture was diluted in such a manner as to obtain an appropriate number of living bacterial cells. This culture solution was applied to a 1⁄2 MS agar medium containing 250 μg/ml of apramycin, and cultured at 30° C. for 4 days. A colony thus grown was replicated in a 1⁄2 MS agar medium supplemented with 250 μg/ml of apramycin and 250 μg/ml of kanamycin. Two kanamycin-susceptible strains (D1 strain, D2 strain) were selected which did not grow in a kanamycin-containing medium but grew in an apramycin-containing medium.
The genomic DNAs of the obtained two strains were prepared, and a PCR reaction was carried out using a combination of primers of 41c30F4: 5′-CGTGACCGAGGTGGCGCG-3′ (SEQ ID NO: 43) and 41c30RR2: 5′-GTCGTCGGATGCGCCGTGCG-3′ (SEQ ID NO: 44). It was confirmed that the two strains were disrupted strains as designed because approximately 0.5 kbp of an amplified DNA fragment was not obtained.
Example 7
<Culturing of Biosynthetic Gene-Deficient Strains, and Quantification of UK-2A in Culture Solution>
The disrupted strains, D1 strain and D2 strain, were each inoculated into 50 ml of an S#1 medium [Ueki, M, et al, “The Journal of Antibiotics,” (Japan), 1996, vol. 49, pp. 639-643] prepared in a 250-ml Erlenmeyer flask, and shake-cultured at 30° C. for 24 hours. Then, 1 ml of the culture solution was inoculated into a production medium, and shake-cultured at 30° C. for 4 days. Then, 4 ml of acetone was added to 1 ml of the resulting culture solution to thereby extract UK-2A which was then filtered to obtain an extraction liquid. Of this, 5 μl was subjected to HPLC analysis. In the HPLC analysis, HPLC System LC-2010C (manufactured by Shimadzu Corporation) was used for the analysis. As the analysis conditions, the column was Inertsil ODS-3 4.6×250 mm, the mobile phase was acetonitrile:water:phosphoric acid=60:40:0.1, the flow rate was 1.1 ml/min, the column temperature was 40° C., and the UV wavelength was 340 nm. The obtained pattern was compared with that of the UK-2A reference standard. The peak derived from UK-2A was identified. Based on the area thereof, UK-2A was quantified.
At the same time, the same culturing and quantification of UK-2A in a culture solution were carried out also for Streptoverticillium sp. 3-7, which was the parental strain of the transformants. As a result, the UK-2A productivity by the D1 and D2 strains was 0 μg/ml.
Example 8
<Creation of Biosynthetic Gene Cluster-Introduced Transformant>
Constructed pUK2-3 was introduced in Streptoverticillium sp. 3-7 according to a method generally used for actinobacteria [“Practical Streptomyces Genetics,” The John Innes Foundation, (UK), Norwich, 2000, pp. 311-338], First, the plasmid pUK2-3 was introduced in an E. coli ET12567/pUZ8002 strain by electroporation according to a conventional method to obtain E. coli ET12567/pUZ8002/pUK2-3, This strain was cultured at 37° C. for 18 hours in an LB liquid medium supplemented with chloramphenicol, kanamycin and apramycin respectively at concentrations of 25 μg/ml, 50 μg/ml and 50 μg/ml. Then, 1 ml of the culture solution was transferred to 100 ml of an LB liquid medium (containing chloramphenicol, kanamycin and apramycin respectively at concentrations of 25 μg/ml, 25 μg/ml and 50 μg/ml), and cultured at 37° C. for 4 hours. Subsequently, 50 ml of the culture solution was centrifuged at 3000 rpm for 5 minutes to collect the bacterial cells. The bacterial cells were suspended in 50 ml of an LB liquid medium. After this operation was repeated twice, the bacterial cells were suspended in 100 μL of an LB liquid medium.
Meanwhile, Streptoverticillium sp. 3-7 was applied to an MS agar medium, and cultured at 30° C. for 2 days. After the culturing, mycelia were scraped with 1 ml of 20% glycerol to prepare a host mycelium solution.
Next, 500 μl of the host mycelium solution and 500 μl of the Escherichia coli solution comprising the plasmid pUK2-3 prepared as described above were mixed together, and the bacterial cells were collected. Then, the bacterial cells were applied to an MS agar medium which had been diluted by addition of 10 mM MgCl2 in such a manner as to bring the final concentration to 10 mmol/L. After the culturing at 30° C. for 20 hours, 0.5 ml of sterilized water containing 6 mg of apramycin and 0.5 mg of nalidixic acid was overlaid thereon. After further cultured at 30° C. for 5 days, a transformant was obtained as an apramycin-resistant strain.
Example 9
<Culturing of Gene-Introduced Transformant, and Quantification of UK-2A in Culture Solution>
The gene-introduced transformant was cultured by the method described in Example 7, As a result, as shown in Table 3, the UK-2A productivity of the gene-introduced transformant was improved 58 to 77 times in comparison with that of the parental strain.











TABLE 3








Productivity in culture





solution (μg/ml)
Relative



Strains
UK-2A
productivity




















Parental strain (3-7)
2
1



Transformant 1 (3-7-1)
116
58



Transformant 2 (3-7-2)
153
77










Example 10
<Culturing of Gene-Introduced Transformant, and Quantifications of UK-2A, UK-2B, and sum of UK-2C and UK-2D in Culture Solution>
The gene-introduced transformant was cultured by the method described in Example 7, Specifically, 4 ml of acetone was added to 1 ml of the resulting culture solution to thereby extract UK-2A, UK-2B, UK-2C and UK-2D which were then filtered to obtain an extraction liquid. Of this, 5 μl was subjected to HPLC analysis. In the HPLC analysis, HPLC solution system (manufactured by Shimadzu Corporation) was used for the analysis. As the analysis conditions, the column was Inertsil ODS-3 4.6×150 mm; the mobile phase was a solution obtained by dissolving 7.8 g of sodium dihydrogen phosphate dihydrate in approximately 800 mL of water, adjusting the pH of the resultant to 4.0 using phosphoric acid, adding water thereto to prepare 1000 ml of a phosphoric acid aqueous solution, and adding 650 mL of acetonitrile for liquid chromatography to 350 mL of the phosphoric acid aqueous solution; the flow rate was 1.0 ml/min; the column temperature was 40° C.; and the UV wavelength was 230 nm. The obtained pattern was compared with those of the UK-2A, UK-2B, and UK-2C and UK-2D reference standards. The respective peaks derived from UK-2A, UK-2B, UK-2C and UK-2D were identified. Based on the areas thereof, the amount of UK-2A, the amount of UK-2B, and the sum of UK-2C and UK-2D were determined.
As a result, as shown in Table 4, the productivities of UK-2A, UK-2B, and the sum of UK-2C and UK-2D of the gene-introduced transformant were respectively improved 37 to 57 times, 10 to 11 times, and 12 to 13 times in comparison with those of the parental strain.










TABLE 4








UK-2A
UK-2B
UK-2C and UK-2D (sum)














Productivity

Productivity

Productivity




in culture solution
Relative
in culture solution
Relative
in culture solution
Relative


Strains
(μg/ml)
productivity
(μg/ml)
productivity
(μg/ml)
productivity
















Strain (3-7)
10
1
1
1
7
1


Transformant
368
37
11
11
86
12


(3-7-1)








Transformant
565
57
10
10
89
13


(3-7-2)









Example 11
<Quantification of Number of Copies of UK-2 Biosynthetic Gene Cluster in Transformant>
Genomic DNAs of the two strains of the transformant confirmed in Example 9 to have the UK-2 productivity improved and Streptoverticillium sp. 3-7, which was the host cell, were prepared by the method described in Example 1, PCR reactions were carried out using the genomic DNAs as templates and StepOnePlus Real-Time PCR System (manufactured by Applied BioSystems Inc.) in accordance with the protocol attached thereto. Amplified fragments thus obtained were quantified. Table 5 shows the obtained result.
Note that, in the PCR reactions, the following primer set was designed, synthesized and used to amplify a region in the introduced UK-2 biosynthetic gene cluster.









(SEQ ID NO: 45)










UK-2 F2 (RT):
5′-GCACCTTCATGTCCGGGTTG-3′



 









(SEQ ID NO: 46)










UK-2 R2 (RT):
5′-ATCGCCGCGTACACCATGAC-3′.






Further, the following primer set was designed, synthesized and used as an internal control to amplify a region other than the UK-2 biosynthetic gene cluster.









(SEQ ID NO: 47)










cont F1 (RT):
5′-CGAAGGTCCGGTTGATGGTG-3′



 









(SEQ ID NO: 48)










Cont R1 (RT): 
5′-ATCGCTGCGACACCCTGGAG-3′
















TABLE 5







Strains
Number of copies









Parental strain (3-7)
1.00



Transformant (3-7-1)
2.35



Transformant (3-7-2)
2.08










As shown in Table 5, it was revealed that the number of copies of the UK-2 biosynthetic gene cluster in the transformant was double that of the parental strain sp. 3-7.
As described hereinabove, the present invention makes it possible to provide a transformant having high UK-2 productivity by introduction of a UK-2 biosynthetic gene or a UK-2 biosynthetic gene cluster.
Therefore, by using the transformant of the present invention, mass production of UK-2 at low cost is made possible. Accordingly, the present invention is useful in producing rice blast control agents, agricultural and horticultural fungicides, and medical antifungal agents.
REFERENCE TO DEPOSITED BIOLOGICAL MATERIAL
[Accession Number]1.


    (1) Indication for identification: Streptoverticillium sp. 3-7
    (2) Accession number: FERM BP-11437
    (3) Date of deposition: Nov. 9, 2011
    (4) Depository institution: International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology
    (5) The deposit of the patent microorganisms by International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (former name: IPOD) was succeeded by National Institute of Technology and Evaluation (NITE) on April, 2012.

## Claims

1. A method for producing a derivative of UK-2, comprising the steps of:
culturing a bacterium, and collecting UK-2 from a culture of the bacterium; and
synthesizing a derivative of UK-2 represented by any one of the following formulae (1) from the collected UK-2





in the formula (1),
R represents any one of a 2-methylpropanoyl group, a trans-2-methyl-2-butenoyl group, a 3-methylbutanoyl group and a 2-methylbutanoyl group;
R1 represents any one of a C1-6 alkyl group, a benzyl group, a C1-10 alkylcarbonyl group(the C1-10 alkylcarbonyl group may be substituted with any one of a carboxyl group, a benzyloxycarbonyl group, a C1-4 alkyloxycarbonyl group and benzyloxycarbonylamino group), a benzoyl group, a C1-4 alkyloxycarbonyl group, a (C1-4)alkyloxycarbonyl(C1-4)alkyl group, a benzyloxycarbonyl(C1-4)alkyl group may be substituted with a nitro group, a C1-6 alkylsulfonyl, di(C1-6)alkylphosphoryl group, a diphenylphosphoryl group and a substituent represented by the following formula (2);





in the formula (2),
Q is selected from the group consisting of H, CH3, CH2CH3, CF3, Ph, CH═CH2 and a cyclopropyl;
M is selected from the group consisting of H, CH3, CH2CH3, CF3, Ph, CH═CH2 and a cyclopropyl;
T is selected from the group consisting of O, OC(O), OC(O)O, S, SC(O), SC(O)O and a substituent represented by the following formula (3);




G is selected from the group consisting of H, C1-6 alkyl group, a C1-6 alkyloxy C1-6 alkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, a C3-6 cycloalkyl group, an aryl group and a heteroaryl group;
G and M may form an isobenzofuran ring optionally having an oxo group;
M and Q may form a 3-8 membered carbocyclic system,
wherein the bacterium is a bacterium transformant comprising a nucleic acid, in which UK-2 biosynthesis is induced and UK-2 production is increased in comparison to a parental strain bacterium, and
the nucleic acid is an isolated nucleic acid that induces UK-2 biosynthesis and improves UK-2 productivity, and is at least one nucleic acid selected from the group consisting of the following (a) to (q):
(a) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 3, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 3, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 2;
(b) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 5, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 5, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 4;
(c) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 7, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 7, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 6;
(d) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 9, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 9, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 8;
(e) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 11, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 11, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 10;
(f) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 13, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 13, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 12;
(g) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 15, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 15, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 14;
(h) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 17, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 17, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 16;
(i) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 19, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 19, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 18;
(j) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 21, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 21, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 20;
(k) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 23, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 23, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 22;
(1) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 25, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 25, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 24;
(m) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 27, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 27, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 26;
(n) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 29, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 29, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 28;
(o) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 31, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 31, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 30;
(p) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 33, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 33, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 32; and
(q) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 35, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 35, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 34.

2. A method for producing a UK-2A derivative, comprising the steps of:
culturing a bacterium, and collecting UK-2A from a culture of the bacterium; and
synthesizing a UK-2A derivative represented by any one of the following formulae (4) to (7) from the collected UK-2A,




wherein the bacterium is a bacterium transformant comprising a nucleic acid, in which UK-2 biosynthesis is induced and UK-2 production is increased in comparison to a parental strain bacterium, and
the nucleic acid is an isolated nucleic acid that induces UK-2 biosynthesis and improves UK-2 productivity, and is at least one nucleic acid selected from the group consisting of the following (a) to (q):
(a) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 3, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 3, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 2;
(b) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 5, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 5, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 4;
(c) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 7, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 7, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 6;
(d) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 9, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 9, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 8;
(e) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 11, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 11, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 10;
(f) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 13, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 13, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 12;
(g) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 15, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 15, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 14;
(h) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 17, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 17, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 16;
(i) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 19, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 19, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 18;
(j) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 21, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 21, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 20;
(k) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 23, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 23, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 22;
(l) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 25, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 25, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 24;
(m) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 27, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 27, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 26;
(n) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 29, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 29, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 28;
(o) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 31, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 31, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 30;
(p) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 33, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 33, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 32; and
(q) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 35, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 35, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 34.