Method for producing isoprenoid, and protein, gene and transformant used for same
阅读说明：本技术 类异戊二烯的制造方法以及用于该制造方法的蛋白质、基因和转化体 (Method for producing isoprenoid, and protein, gene and transformant used for same ) 是由 荒木康子 筱原靖智 北洁 于 2018-05-11 设计创作，主要内容包括：本发明要解决的课题在于提供类异戊二烯的制造方法,该制造方法可以以与现有技术相比更高的收率生产类异戊二烯,诸如壳二孢呋喃酮、冬青生菌素A和壳二孢氯素及它们的衍生物,因此能够以工业规模生产类异戊二烯。上述课题是通过冬青生菌素A和壳二孢氯素等的类异戊二烯的制造方法等而得以解决；该制造方法包括使用由壳二孢呋喃酮、冬青生菌素A或壳二孢氯素的生物合成基因进行转化的转化体或者它们的基因敲除生物而获得壳二孢呋喃酮、冬青生菌素A和壳二孢氯素等的类异戊二烯的步骤。(An object of the present invention is to provide a method for producing isoprenoids, which can produce isoprenoids such as ascofuranone, ilicin a, and ascochlorin and derivatives thereof in higher yields than in the prior art, and thus can produce isoprenoids on an industrial scale. The above object is achieved by a method for producing isoprenoids such as ilexolone A and ascochloride; the production method comprises a step of obtaining isoprenoids such as ascofuranone, ilicin A and ascochlorin using a transformant transformed with a biosynthetic gene of ascofuranone, ilicin A or ascochlorin or a knockout organism thereof.)
1. A gene ascI comprising a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing an addition reaction of monatin A epoxide with one atom of oxygen as described in any one of the following (1) to (5):
(1) a base sequence represented by SEQ ID No. 8 of the sequence listing or a base sequence complementary to the base sequence and hybridizing under stringent conditions;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 8;
(3) a base sequence encoding an amino acid sequence of an enzyme having activity to catalyze the one-atom oxygen addition reaction of an epoxide of ilicin A;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence of SEQ ID NO. 18 or 67; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 18 or 67 are deleted, substituted and/or added.
2. A gene ascJ comprising a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction to produce ascofuranol using a compound produced by a reaction of ilexomycin A epoxide through an AscI protein according to any one of the following (1) to (5):
(1) a base sequence that hybridizes to a base sequence represented by SEQ ID No. 9 of the sequence listing or a base sequence complementary to the base sequence under stringent conditions;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 9;
(3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction of producing ascofuranol using a compound produced by a reaction of ilexomycin A epoxide through an AscI protein;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence represented by SEQ ID NO. 19; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 19 are deleted, substituted and/or added.
3. A gene ascK comprising a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing ascofuranone from ascofuranol as described in any one of the following (1) to (5):
(1) a base sequence represented by SEQ ID No. 10 of the sequence listing or a base sequence complementary to the base sequence and hybridizing under stringent conditions;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 10;
(3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction of producing ascofuranone from ascofuranol;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence represented by SEQ ID NO. 20; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 20 are deleted, substituted and/or added.
4. A transformant (wherein, human is excluded) in which any 1 gene or a combination of genes of ascI, ascJ and ascK described in claims 1 to 3 is inserted and the inserted gene is expressed.
5. A transformant (wherein, human is excluded) in which any 1 gene or a combined gene thereof among the genes ascI, ascJ and ascK described in claims 1 to 3 is inserted, and further any 1 gene or a combined gene thereof among the genes ascF, ascE, ascD, ascB and ascC is inserted, and the inserted gene is expressed.
6. A knockout organism (wherein humans are excluded) of a gene ascG derived from a wild-type organism having the gene ascG comprising a base sequence of any one of the following (1) to (5), namely, a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a cyclization reaction of a wintergreen A epoxide:
(1) a base sequence that hybridizes under stringent conditions to a base sequence described in SEQ ID No. 6 or 64 of the sequence Listing or a base sequence complementary to the base sequence;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 6 or 64;
(3) a base sequence encoding an amino acid sequence of an enzyme having activity of catalyzing a cyclization reaction of an epoxide of ilicin A;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence of SEQ ID NO. 16 or 40; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 16 or 40 are deleted, substituted and/or added.
7. A method for producing ascofuranone, comprising the step of obtaining ascofuranone by using the knockout organism of claim 6.
8. A knockout organism (wherein, excluding human) of a gene ascF derived from a wild-type organism having the gene ascF comprising a base sequence of any one of the following (1) to (5), namely, a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing an epoxidation reaction of ilexolone A:
(1) a base sequence represented by SEQ ID No. 5 of the sequence listing or a base sequence complementary to the base sequence and hybridizing under stringent conditions;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 5;
(3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing an epoxidation reaction of ilexolone A;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence of SEQ ID NO. 15 or 39; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 15 or 39 are deleted, substituted and/or added.
9. A method for producing ilexolone A, comprising the step of obtaining ilexolone A using the knockout organism of claim 8.
10. A knockout organism (wherein humans are excluded) of the gene ascI, which is derived from a wild-type organism having the gene ascI described in claim 1.
11. A method for producing ascochyta chloride, comprising the step of obtaining ascochyta chloride by using the knockout organism according to claim 10.
12. A method for producing an ascofuranone analog, an ascofuranone precursor, and an analog thereof, comprising the step of obtaining an ascofuranone analog, an ascofuranone precursor, and an analog thereof using the knockout organism of claim 6.
13. A method of making a wintergreen a analog, a wintergreen a precursor, and analogs thereof, comprising the step of obtaining a wintergreen a analog, a wintergreen a precursor, and analogs thereof using the knockout organism of claim 8.
14. A method for producing an ascochyta analog, an ascochyta precursor, and an analog thereof, comprising the step of obtaining an ascochyta analog, an ascochyta precursor, and an analog thereof using the knock-out organism according to claim 10.
Technical Field
The present invention relates to a gene for synthesizing isoprenoids such as ascofuranone, ascochloride, and ilexolone a, and a method for producing isoprenoids using the gene.
Background
Infection with viruses, protozoa, and the like is often a problem in developed countries and developing countries, including japan where densely populated areas are scattered. In addition, lifestyle-related diseases such as
Therefore, it is desired to develop a substance effective for the treatment or prevention of these diseases. As one of such substances, ascochloride and ascofuranone are known as isoprenoid-based physiologically active substances. Ascochytrium chloride and ascofuranone inhibit the electron transfer system and lower the intracellular ATP concentration, and are thus expected to be useful for the treatment or prevention of, for example, protozoal infections caused by trypanosomes protozoa, i.e., african lethargy, transmitted by tsetse fly (for example, refer to the following
If suffering from African lethargy, the protozoa proliferate in the bloodstream at the initial stage of infection. When entering the chronic stage, protozoa invade the central nerve and cause symptoms such as confusion or general spasm, and finally fall into a state of lethargy to die. African lethargy-induced african deaths are said to be over 1 million per year, with over 7,000 million people at potential infection risk. At present, there is no preventive method using vaccine for African sleeping sickness, and the treatment depends on drug therapy. However, a therapeutic agent effective for African narcolepsy has a problem of great side effects.
Therefore, it is expected that the electron transport system of trypanosoma will be specifically inhibited by ascochytrium chloride or ascofuranone, thereby preventing or treating african lethargy. If protozoa invade the mammalian body, glycolysis is mainly internal to glycolytic enzymeATP synthesis in a lytic system in which Trypanosoma Alteroxidase (TAO) -catalyzed NAD+Regeneration is necessary when ascochloride or ascofuranone inhibits the action of the TAO. Infected mammals do not have the same enzymes as TAO and are therefore able to specifically repel trypanosomes. Furthermore, it has been reported that ascofuranone and derivatives thereof, in particular, inhibit TAO even at very low concentrations.
In addition, it is known that ascochyta chloride, ascofuranone, and derivatives thereof have antitumor activity, hypoglycemic action, hypolipidemic action, glycolytic inhibitory action, antioxidant action, and the like (for example, refer to the following
Among isoprenoids, as a method for producing ascofuranone or ascochyl, a method of culturing a filamentous fungus of the genus Ascochyta (Ascochyta) and separating and extracting ascofuranone accumulated in the hyphae is known (for example, refer to the following
Disclosure of Invention
Problems to be solved by the invention
According to the methods using filamentous fungi known to produce ascochlorin or ascofuranone as described in
For example, in order to realize mass production of isoprenoids such as ascochlorin or ascofuranone and ilexolone a as an intermediate thereof, it is considered that a wild strain which stably produces isoprenoids at a high concentration can be isolated or bred, or a transformant in which a gene involved in isoprenoid biosynthesis is inserted by using a biotechnology can be constructed. However, it has been almost unknown for wild strains that stably produce isoprenoids at high concentrations so far, and there are still many unclear portions regarding the pathway of isoprenoid biosynthesis.
In addition, there are many unknown parts of the biosynthetic genes for ascochylomycete, ascofuranone and iliconcidin A in isoprenoids.
Accordingly, an object of the present invention is to provide a method for producing isoprenoids, which can stably produce isoprenoids such as ascofuranone, ilicin a, and ascochloride and their derivatives in higher yields than in the prior art, and thus can produce isoprenoids on an industrial scale.
Means for solving the problems
The present inventors have conducted extensive studies to solve the above-mentioned problems, and as a result, succeeded in identifying a gene group (3 genes of gene ascI to gene ascK) encoding an enzyme involved in a reaction of biosynthesis of ascofuranone, following a gene group (7 genes of gene ascB to gene ascH) encoding an enzyme involved in a reaction of biosynthesis of ascomycin A in Acremonium sclerotiorum (Acremonium sclerotium) which is a kind of filamentous fungus.
The present inventors have then created a DNA construct for overexpressing a protein encoded by the above-mentioned gene group, introduced the resulting DNA construct as a host organism into a microorganism belonging to the genus Aspergillus (Aspergillus) or the genus Acremonium (Acremonium), which is one of filamentous fungi, and transformed the host organism, thereby successfully creating a transformed filamentous fungus overexpressing a protein encoded by the above-mentioned gene group. In addition, knockout filamentous fungi of ascF, ascG and ascI of Acremonium microorganisms have also been successfully produced.
The transformed filamentous fungus can be cultured by a usual method for culturing filamentous fungus, and the growth rate and the like thereof are not particularly different from those of the host organism. It is thus clear that isoprenoids such as ascofuranone, ilicin A and ascochloride can be produced by using the above transformed filamentous fungus or knockout filamentous fungus.
On the other hand, it is considered that the gene ascA in the ascochyrate biosynthesis gene cluster is a transcription factor, and it is considered that the gene ascA does not affect the ascochyrate biosynthesis even if it is not introduced and expressed. Actually, it was revealed that, as described above, ascochloride can be biosynthesized by using a transformed filamentous bacterium in which 7 genes of the genes ascB to ascH are introduced into a microorganism of the genus Aspergillus without the gene ascA.
Despite this fact, the present inventors succeeded in creating a transformed filamentous bacterium which strongly expresses the gene ascA by introducing the gene ascA into Acremonium nucleatum having a biosynthetic gene for ascochyrate or ascofuranone. Then, it has been unexpectedly found that not only ascochylomycete but also ascofuranone can be produced in large quantities by using a transformed filamentous bacterium which strongly expresses the gene ascA.
The present invention has been completed based on the successful examples or findings described above.
Thus, according to one aspect of the present invention, there are provided the following genes [1] to [11], transformants, knock-out organisms, and methods of production.
[1] A gene ascI comprising a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing an addition reaction of monatin A epoxide with one atom of oxygen as described in any one of the following (1) to (5):
(1) a base sequence that hybridizes under stringent conditions to the base sequence described in SEQ ID No. 8 of the sequence listing or a base sequence complementary to the base sequence;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 8;
(3) a base sequence encoding an amino acid sequence of an enzyme having activity to catalyze the one-atom oxygen addition reaction of an epoxide of ilicin A;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence of SEQ ID NO. 18 or 67; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 18 or 67 are deleted, substituted and/or added
[2] A gene ascJ comprising a base sequence of any one of the following (1) to (5), i.e., a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction to produce ascofuranol from ilexomycin A epoxide:
(1) a base sequence that hybridizes to the base sequence shown in SEQ ID No. 9 of the sequence listing or a base sequence complementary to the base sequence under stringent conditions;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 9;
(3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction of producing ascofuranol using a compound produced by a reaction of ilexomycin A epoxide through an AscI protein;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence represented by SEQ ID NO. 19; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 19 are deleted, substituted and/or added
[3] A gene ascK comprising a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing ascofuranone from ascofuranol as described in any one of the following (1) to (5):
(1) a base sequence that hybridizes to the base sequence shown in SEQ ID No. 10 of the sequence listing or a base sequence complementary to the base sequence under stringent conditions;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 10;
(3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction of ascofuranone from ascofuranol;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence represented by SEQ ID NO. 20; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 20 are deleted, substituted and/or added.
[4] A transformant (wherein, human is excluded) in which any 1 gene or a combination of genes of ascI, ascJ and ascK described in the above [1] to [3] is inserted and the inserted gene is expressed.
[5] The transformant according to [4] above, wherein any 1 gene of ascF, ascE, ascD, ascB and ascC or a combination thereof is further inserted and the inserted gene is expressed.
[6] A knockout organism (wherein humans are excluded) of a gene ascG derived from a wild-type organism having the gene ascG, comprising a base sequence of any one of the following (1) to (5), i.e., a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a cyclization reaction of an epoxide of ilicin A:
(1) a base sequence that hybridizes to the base sequence shown in SEQ ID No. 6 of the sequence listing or a base sequence complementary to the base sequence under stringent conditions;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 6;
(3) a base sequence encoding an amino acid sequence of an enzyme having activity of catalyzing a cyclization reaction of an epoxide of ilicin A;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence of SEQ ID NO. 16 or 40; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 16 or 40 are deleted, substituted and/or added.
[7] A method for producing ascofuranone, comprising the step of obtaining ascofuranone by using the knockout organism described in [6] above. A method for producing an ascofuranone analog, an ascofuranone precursor, and an analog thereof, which comprises the step of obtaining an ascofuranone analog, an ascofuranone precursor, and an analog thereof using the knockout organism described in the above [6 ].
[8] A knockout organism (wherein, excluding human) of a gene ascF derived from a wild-type organism having the gene ascF, which comprises the base sequence of any one of the above-mentioned (1) to (5), i.e., a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing an epoxidation reaction of ilexolone A:
(1) a base sequence that hybridizes under stringent conditions to the base sequence described in SEQ ID No. 5 of the sequence listing or a base sequence complementary to the base sequence;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 5;
(3) a base sequence encoding an amino acid sequence having an enzyme that catalyzes an epoxidation reaction of ilexolone A;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence of SEQ ID NO. 15 or 39; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 15 or 39 are deleted, substituted and/or added.
[9] A method for producing ilexolone A, which comprises the step of obtaining ilexolone A using the knockout organism as described in [8] above. A method for producing an analog of ilexolone A, a precursor of ilexolone A, and analogs thereof, comprising the step of obtaining an analog of ilexolone A, a precursor of ilexolone A, and analogs thereof using the knockout organism of [8] above.
[10] A knockout organism (wherein, human is excluded) of the gene ascI derived from a wild-type organism having the gene ascI described in the above [1 ].
[11] A method for producing ascochyta chloride, which comprises a step of obtaining ascochyta chloride by using the knockout organism described in [10] above. A process for producing an ascochyta analog, an ascochyta precursor, and an analog thereof, which comprises the step of obtaining an ascochyta analog, an ascochyta precursor, and an analog thereof by using the knock-out organism described in [10] above.
In another aspect of the present invention, there are provided the genes, transformants and production methods described in [12] to [22 ].
[12] A gene ascF comprising a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing an epoxidation reaction of ilexolone A as described in any one of (1) to (5):
(1) a base sequence that hybridizes to the base sequence shown in SEQ ID No. 5 of the sequence listing or a base sequence complementary to the base sequence under stringent conditions;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 5;
(3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing an epoxidation reaction of ilexolone A;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence of SEQ ID NO. 15 or 39; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 15 or 39 are deleted, substituted and/or added.
[13] A gene ascG comprising a base sequence of any one of the following (1) to (5), namely, a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a cyclization reaction of an epoxide of ilexolone A:
(1) a base sequence that hybridizes to the base sequence shown in SEQ ID No. 6 of the sequence listing or a base sequence complementary to the base sequence under stringent conditions;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 6;
(3) a base sequence encoding an amino acid sequence of an enzyme having activity of catalyzing a cyclization reaction of an epoxide of ilicin A;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence of SEQ ID NO. 16 or 40; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 16 or 40 are deleted, substituted and/or added.
[14] A gene ascH comprising a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction in which ascochloride is produced by dehydrogenation of a compound produced by a reaction of ilexomycin a using an AscF protein and an AscG protein, and having a base sequence of any one of the following (1) to (5):
(1) a base sequence that hybridizes under stringent conditions to the base sequence represented by SEQ ID No. 7 of the sequence listing or to a base sequence complementary to the base sequence;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 7;
(3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction in which ascochloride is produced by dehydrogenation of a compound produced by a reaction of ilicin a using an AscF protein and an AscG protein;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence of SEQ ID NO. 17 or 41; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 17 or 41 are deleted, substituted and/or added.
[15] A gene ascE comprising a base sequence encoding an amino acid sequence of an enzyme having a base sequence catalyzing any one of the above-mentioned (1) to (5), that is, having an activity of catalyzing a reaction to produce ilexolone A from LL-Z1272 β:
(1) a base sequence that hybridizes under stringent conditions to the base sequence described in SEQ ID No. 4 of the sequence listing or a base sequence complementary to the base sequence;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 4;
(3) a nucleotide sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction producing ilexolone A from LL-Z1272 β;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence of SEQ ID NO. 14 or 38; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 14 or 38 are deleted, substituted and/or added.
[16] A gene ascD comprising a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction of producing O-orcinol from acetyl-CoA, which is any one of the following (1) to (5):
(1) a base sequence that hybridizes to the base sequence shown in SEQ ID No. 3 of the sequence listing or a base sequence complementary to the base sequence under stringent conditions;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 3;
(3) a base sequence encoding an amino acid sequence having an activity of catalyzing a reaction of producing O-orcinol from acetyl-coa;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence of SEQ ID NO. 13 or 37; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 13 or 37 are deleted, substituted and/or added.
[17] A gene ascB comprising the base sequence of any one of the above (1) to (5), that is, a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction of producing ilicinic acid B from O-bryoid:
(1) a base sequence that hybridizes to the base sequence shown in SEQ ID No. 1 of the sequence listing or a base sequence complementary to the base sequence under stringent conditions;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 1;
(3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction of generating ilicin B from O-bryoid;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence of SEQ ID NO. 11 or 35; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 11 or 35 are deleted, substituted and/or added.
[18] A gene ascC comprising the base sequence of any one of (1) to (5) above, i.e., a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction to produce LL-Z1272 β from ilexolone B:
(1) a base sequence that hybridizes to the base sequence shown in SEQ ID No. 2 of the sequence listing or a base sequence complementary to the base sequence under stringent conditions;
(2) a nucleotide sequence having 60% or more sequence identity to a gene comprising the nucleotide sequence of SEQ ID NO. 2;
(3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction from ilexolone B to LL-Z1272 β;
(4) a nucleotide sequence encoding an amino acid sequence having 60% or more sequence identity to the amino acid sequence represented by SEQ ID NO. 12 or 36; and
(5) a nucleotide sequence encoding an amino acid sequence in which 1 or several amino acids of the amino acid sequence shown in SEQ ID NO. 12 or 36 are deleted, substituted and/or added.
[19] A transformant (wherein, human is excluded) in which any 1 gene or a combination of genes of ascF, ascG, ascH, ascE, ascD, ascB and ascC described in [12] to [18] is inserted and the inserted gene is expressed.
[20] A method for producing ilexolone A, which comprises a step of obtaining ilexolone A using the transformant described in [19 ].
[21] A process for producing ascochyta chloride, which comprises a step of obtaining ascochyta chloride by using the transformant described in [19 ].
[22] A method for producing ascofuranone, comprising a step of obtaining ascofuranone by using the transformant according to [19 ].
In another aspect of the present invention, there are provided the proteins, genes, transformants and methods described in [23] to [31 ].
[23] An AscA protein comprising an amino acid sequence of any one of the following (a) to (c), and having an activity of enhancing expression of 1 or more genes of any one of [1] to [3] and [12] to [18 ]:
(a) an amino acid sequence described in SEQ ID No. 66 of the sequence Listing;
(b) an amino acid sequence in which 1 to several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID No. 66 of the sequence Listing; and
(c) an amino acid sequence having a sequence identity of 60% or more with the amino acid sequence represented by SEQ ID No. 66 of the sequence Listing.
[24] A gene ascA comprising a base sequence of any one of the following (A) to (D), that is, a base sequence encoding an amino acid sequence of a protein having an activity of enhancing the expression of 1 or more genes of any one of [1] to [3] and [12] to [18 ]:
(A) a nucleotide sequence encoding the amino acid sequence of the protein according to [23 ];
(B) a base sequence represented by SEQ ID No. 65 of the sequence Listing;
(C) a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence represented by SEQ ID No. 65 of the sequence listing; and
(D) a nucleotide sequence having a sequence identity of 80% or more with a gene consisting of the nucleotide sequence shown in SEQ ID No. 65 of the sequence Listing.
[25] A method for increasing the production of isoprenoids using a filamentous bacterium, comprising the step of increasing the production of isoprenoids using the filamentous bacterium by enhancing the expression of the AscA protein described in [23] or the gene ascA described in [24] in the filamentous bacterium having 1 or more genes described in any one of [1] to [3] and [12] to [18 ].
[26] The method of [25], wherein the isoprenoid is at least 1 compound selected from the group consisting of ascofuranone, ascomycin, and ilicin A.
[27] A transformant (wherein human is excluded) transformed in such a manner that the expression of the gene ascA according to [24] is enhanced.
[28] The transformant according to [27], wherein the host organism of the aforementioned transformant is a microorganism of the genus Acremonium.
[29] A method for producing isoprenoids, comprising a step of obtaining isoprenoids by enhancing the expression of the AscA protein according to [23] or the gene ascA according to [24] in a filamentous fungus having 1 or more genes according to any one of [1] to [3] and [12] to [18 ].
[30] A method for producing an isoprenoid, comprising a step of obtaining an isoprenoid by culturing the transformant according to any one of [27] to [28 ].
[31] The method according to [29] or [30], wherein the isoprenoid is at least 1 compound selected from the group consisting of ascofuranone, ascomycin, and ilexomycin A.
Effects of the invention
According to the present invention, isoprenoids such as ascofuranone, ilicin A, and ascochloride can be stably produced in high yield. Therefore, according to the present invention, isoprenoids such as ascofuranone, ilicin A, and ascochloride can be expected to be produced on an industrial scale.
Drawings
FIG. 1 is a diagram showing the cluster of ascochyrin biosynthesis genes predicted by transcriptome analysis.
FIG. 2 is a graph showing the results of HPLC analysis of an extract of As-DBCE strain and a reference substance for wintergreen A As described in the examples below.
FIG. 3 is a graph showing the results of HPLC analysis of respective extracts of As-DBCE strain, As-DBCEF strain, As-DBCEFG strain and As-DBCEFGH strain As described in the following examples.
FIG. 4A is a graph showing the results of LC/MS analysis of the reactants in the case of using the wild-strain reaction liquid and the As-F reaction liquid, respectively, As described in the following examples.
FIG. 4B is a graph showing the results of LC/MS analysis of the reactants in the case of using an As-F reaction liquid and an As-FG reaction liquid, respectively, As described in the following examples.
FIG. 5 is a graph showing the results of LC/MS analysis of reactants in the case of using As-FG reaction liquid and As-FGH reaction liquid, respectively, As described in the following examples.
FIG. 6 is a schematic diagram showing the relationship of each enzyme reaction to reactants in a reaction system of ilexoside A and ascochloride.
FIG. 7 is a diagram showing the cluster of ascofuranone biosynthesis genes predicted by transcriptome analysis.
FIG. 8 is a graph showing the results of LC/MS analysis of reactants in the case of using an As-F reaction liquid, an As-FI reaction liquid, an As-FIJ reaction liquid, an As-FIK reaction liquid, an As-FJK reaction liquid, an As-IJK reaction liquid and an As-FIJK reaction liquid, respectively, As described in the following examples.
FIG. 9 is a graph showing the results of LC/MS analysis and MS/MS analysis of the reactants in the case of using As-FIJK reaction liquid, As described in the following examples.
FIG. 10 is a graph showing the results of LC/MS analysis of reactants in the case of using an As-F reaction liquid, an As-FI reaction liquid, an As-FIJ reaction liquid, an As-FIK reaction liquid, an As-FJK reaction liquid, an As-IJK reaction liquid and an As-FIJK reaction liquid, respectively, As described in the following examples.
FIG. 11 is a graph showing the relationship of each enzyme reaction to a reactant in a reaction system of ascofuranone, ilicin A and ascochloride.
FIG. 12 is a graph showing the results of HPLC analysis of respective extracts of As-DBCEFIRED strain and As-DBCEFIJKred strain As described in the examples below.
FIG. 13 is a graph showing the results of HPLC analysis of an extract of an ascG-disrupted strain of Acremonium sclerotiorum F-1392 strain as described in the following examples.
FIG. 14 is a graph showing the results of HPLC analysis of extracts of As-Tr-DB strain and As-DB strain As described in the examples below.
FIG. 15 is a graph showing the results of HPLC analysis of extracts of As-DBC-Tr-E strain and As-DBC strain As described in the following examples.
FIG. 16 is a scheme diagram showing the relationship of each enzyme reaction to reactants in a reaction system from Aquifolin A epoxide to ascofuranone.
Fig. 17 is a graph showing the results of HPLC analysis of extracts of Δ ascG strain and Δ ascG-I strain as described in the following examples.
FIG. 18 is a graph showing the results of HPLC analysis of extracts of Δ ascG/Δ ascH strain and Δ ascG/Δ ascH + Nd-ascG strain As well As As-FG reaction liquid As described in the following examples.
Fig. 19 is a graph showing the results of HPLC analysis of the respective extracts of the wild strain and the asci-forced expression strain as described in the examples below.
Detailed Description
The following will describe details of the gene, transformant, knockout organism and production method, which are one embodiment of the present invention, but the technical scope of the present invention is not limited by this item, and various embodiments can be adopted as long as the object of the present invention is achieved. In addition, the technical scope of the present invention is not limited to any guess or inference in the present specification.
(isoprenoid)
The "isoprenoid" in the present specification is not particularly limited if it is a compound having isoprene as a structural unit as is generally known, and examples thereof include ilexolone B (grifol acid), ilexolone a, ilexolone B (LL-Z1272 β), ilexolone a (LL-Z1272 α), ilexolone a epoxide, ilexolone C, ascochloride, hydroxyilexolone a epoxide, ascofuranol, ascofuranone, and derivatives thereof.
The "derivative" as referred to in the present specification includes all modified compounds obtained from ilicinic acid B, ilicinic acid A, ilicin B, ilicin A epoxide, ilicin C, ascochlorin, hydroxyilicin A epoxide, ascofuranol, ascofuranone and the like by chemical synthesis, enzymatic synthesis, fermentation or a method of combining them and the like. The "derivative" includes compounds having a structure similar to that of the above-mentioned compounds and all modified compounds thereof, which can be biosynthesized by any one of the enzymes described in the present specification, even without using ilicinic acid B, ilicinic acid a, ilicinic B, ilicinic a, ilicicin a epoxide, iliconcin C, ascochloride, hydroxyilicin a epoxide, ascofuranol, ascofuranone, etc. Ascofuranone, ascochyl, ilicin A or their precursors are all mixed source terpenoids hybridized by polyketone compounds and terpenoids. As disclosed in the present specification, a mixed source terpenoid is biosynthesized by using polyketide synthase such as ascid to biosynthesize a polyketide backbone, and then converting isoprenoid compounds such as C10, C15, C20, etc. using prenyltransferase such as ascib, thereby hybridizing polyketides with terpenoids. That is, by altering the substrate specificity or by altering a more consistent, but different substrate specificity, AscD or combination of AscB, various ilexolone B-like compounds can be biosynthesized. As an example, colletochlorin B is a similar compound similar to ilicin a in which 1 isoprene skeleton of ilicin a is short, that is, having a monoterpene structure of C10, but the compound may be synthesized by combining reactions of 2 enzymes of AscD of the present specification with AscB of altered specificity, or an enzyme of higher identity with AscB of different substrate specificity, or by organic chemical synthesis to obtain a compound in which the terpene moiety of ilicin B is a monoterpene structure of C10, and then further by reactions of AscC, AscE, and thus may be included in the so-called "derivative" in the present specification.
(amino acid sequences of enzymes (1) to (11))
The gene ascB according to one embodiment of the present invention comprises a nucleotide sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction to produce ilexolone B from O-bryoid (hereinafter also referred to as "enzyme (1)").
The gene ascC according to one embodiment of the present invention comprises a nucleotide sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction to produce LL-Z1272 β from ilexolone B (hereinafter, also referred to as "enzyme (2)").
The gene ascD according to one embodiment of the present invention comprises a nucleotide sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction of producing O-orcinol from acetyl-CoA (hereinafter also referred to as "enzyme (3)").
The gene ascE according to one embodiment of the present invention comprises a nucleotide sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing ilicin A from LL-Z1272 β (hereinafter also referred to as "enzyme (4)"), and the enzyme (4) may have an activity of catalyzing a reaction for producing ilicin acid A from ilicin B.
The gene ascF according to one embodiment of the present invention comprises a nucleotide sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing an epoxidation reaction of ilexolone A (hereinafter also referred to as "enzyme (5)").
The gene ascG according to one embodiment of the present invention comprises a nucleotide sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a cyclization reaction of an epoxide of ilicin A (hereinafter also referred to as "enzyme (6)"). The compound produced from the epoxide of ilexogenin A by the reaction using enzyme (6) is ilexogenin C.
The gene ascH according to one embodiment of the present invention comprises a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction of ilexocin A to produce ascochytrium chloride by dehydrogenation of a compound produced by the reaction of AscF protein and AscG protein (hereinafter, also referred to as "enzyme (7)").
The gene ascI according to one embodiment of the present invention comprises a nucleotide sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing an addition reaction of monatin A epoxide with monatomic oxygen (hereinafter, also referred to as "enzyme (8)"). The one-atom oxygen addition reaction of the epoxide of the ilicin A refers to a reaction in which a hydrogen atom (-H) of the epoxide of the ilicin A is replaced by a hydroxyl group (-OH). The compound produced from the ilexolone A epoxide by the reaction using enzyme (8) is hydroxyilexolone A epoxide.
The genes ascJ and ascK according to one embodiment of the present invention include a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing ascofuranone using a compound produced by a reaction of an AscI protein with an epoxide of ilexolone (hereinafter also referred to as "enzyme (9)" and "enzyme (10)", respectively).
The technical scope of the present invention is not limited to any guess or inference, but the following possibilities exist: the enzyme (1) has the same function as prenyltransferase; the enzyme (2) has the same function as the oxidoreductase; the enzyme (3) has the same function as polyketide synthase; the enzyme (4) has the same function as the halogenase; the enzyme (5) has the same function as a p450/p450 reductase which is an epoxidase; the enzyme (6) has the same function as terpene cyclase; the enzyme (7) has the same function as p450 which is a dehydrogenase; the enzyme (8) has the same function as p450 which is a monooxygenase; the enzyme (9) has the same function as terpene cyclase; the enzyme (10) has the same function as dehydrogenase. However, as described in the examples below, the enzyme (9) and the enzyme (10) were able to synthesize ascofuranone from a reaction product of AscI protein by expressing two genes of the genes encoding these enzymes. In the present specification, regardless of the specific mechanism of action, in the case where a specific reaction occurs by expressing genes encoding 2 enzymes, one enzyme and the other enzyme are said to act "together". Wherein the enzyme (9) is an enzyme having a function of catalyzing a reaction for producing ascofuranol from hydroxyilicin A epoxide. The enzyme (10) is an enzyme having an activity of catalyzing a reaction of ascofuranone from ascofuranol.
The AscA protein in one embodiment of the present invention is a protein having an activity of enhancing the expression of 1 or more genes among the genes encoding the enzymes (1) to (10). The AscA protein enhances the expression of 1 or more genes among the genes encoding the enzymes (1) to (10), thereby promoting the biosynthesis of isoprenoids in an organism containing these genes and also increasing the production of isoprenoids using the organism. The AscA protein can function as a positive transcription factor of a gene encoding the enzymes (1) to (10). Furthermore, the gene encoding the AscA protein may be contained in a ascochyta biosynthesis gene or an ascofuranone biosynthesis gene. The AscA protein is strictly a transcription factor and not an enzyme, but the AscA protein is classified as an enzyme, sometimes referred to as "enzyme (11)" for convenience in the present specification.
The amino acid sequences of the enzymes (1) to (11) are not particularly limited if they have the above-mentioned enzymatic activities.
For example, there are the amino acid sequences shown in SEQ ID Nos. 11, 35 and 47 as one form of the enzyme (1) having the above-mentioned enzymatic activity; the amino acid sequences shown in SEQ ID Nos. 12, 36 and 48 exist as one form of the enzyme (2) having the above-mentioned enzymatic activity; the amino acid sequences shown in SEQ ID Nos. 13, 37 and 49 exist as one form of the enzyme (3) having the above-mentioned enzymatic activity; the amino acid sequences shown in SEQ ID Nos. 14, 38 and 50 exist as one form of the enzyme (4) having the above-mentioned enzymatic activity; the amino acid sequences shown in SEQ ID Nos. 15 and 39 exist as one form of the enzyme (5) having the above-mentioned enzymatic activity; the amino acid sequences shown in SEQ ID Nos. 16 and 40 exist as one form of the enzyme (6) having the above-mentioned enzymatic activity; the amino acid sequences shown in SEQ ID Nos. 17 and 41 exist as one form of the enzyme (7) having the above-mentioned enzymatic activity; the presence of the amino acid sequence shown in SEQ ID NO. 18 as one form of the enzyme (8) having the above-mentioned enzymatic activity; the presence of the amino acid sequence shown in SEQ ID NO. 19 as one form of the enzyme (9) having the above-mentioned enzymatic activity; an amino acid sequence represented by SEQ ID NO. 20 as one embodiment of the enzyme (10) having the above-mentioned enzymatic activity; there is an amino acid sequence shown in SEQ ID NO. 66 as one form of the enzyme (11) having the above-mentioned enzymatic activity.
The enzymes having the amino acid sequences shown in SEQ ID Nos. 11 to 20 and 66 were all derived from Acremonium sclerotiorum (Acremonium sclerotiogenum), which is a kind of filamentous fungus of the genus Acremonium, and they were named as AscA, AscB, AscC, AscD, AscE, AscF, AscG, AscH, AscI, AscJ and AscK proteins, respectively, by the present inventors. Further, the base sequences of the genes encoding these enzymes are the base sequences shown in SEQ ID Nos. 1 to 10 and 65.
The enzymes having the amino acid sequences shown in SEQ ID Nos. 35 to 41 and 67, all derived from the species Neonectria pit Viridis (Neonectria ditisma), were named Nd-AscB, Nd-AscC, Nd-AscD, Nd-AscE, Nd-AscF, Nd-AscH and Nd-AscI proteins, respectively, by the present inventors. The nucleotide sequence of the gene encoding Nd-AscG protein is the nucleotide sequence shown in SEQ ID NO. 64.
The enzymes having the amino acid sequences shown in SEQ ID Nos. 47 to 50 were all derived from Trichoderma reesei (Trichoderma reesei), and they were named Tr-AscB, Tr-AscC, Tr-AscD and Tr-AscE proteins by the present inventors, respectively. In addition, the base sequences of the genes encoding Tr-AscC, Tr-AscD, and Tr-AscB proteins are the base sequences shown in SEQ ID Nos. 53, 57, and 60, respectively.
AscA, AscB, AscC, AscD, AscE, AscF, AscG, AscH, AscI, AscJ and AscK proteins are encoded by genes encoding these enzymes present on the chromosomal DNA of Acremonium, Chitosa or Trichoderma. In the present specification, a gene present on the chromosomal DNA of an organism having such a source and a protein or enzyme encoded by the gene are sometimes referred to as "wild-type gene" and "wild-type protein" or "wild-type enzyme", respectively.
The amino acid sequences of the enzymes (1) to (11) may be composed of amino acid sequences having deletions, substitutions, additions, and the like of 1 to several amino acids in the amino acid sequence of the wild-type enzyme, if they have the enzymatic activities of the above-mentioned enzymes (1) to (11), respectively. Here, the range of "1 to several" in "deletion, substitution, and addition of 1 to several amino acids" of an amino acid sequence is not particularly limited, and for example, if the number of amino acids in an amino acid sequence is 100 as one unit, this means 1,2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1,2, 3, 4, 5, 6, 7, 8,9, or 10, and more preferably 1,2, 3, 4, or 5 in the one unit on average. The term "deletion of an amino acid" means the absence or disappearance of an amino acid residue in a sequence, "substitution of an amino acid" means the substitution of an amino acid residue in a sequence with another amino acid residue, and "addition of an amino acid" means the addition of a new amino acid residue to a sequence.
Specific examples of "deletion, substitution, and addition of 1 to several amino acids" include those in which 1 to several amino acids are substituted with another chemically similar amino acid. For example, when a hydrophobic amino acid is substituted with another hydrophobic amino acid, a polar amino acid may be substituted with another polar amino acid having the same charge. Each of such chemically similar amino acids is known in the art. Specific examples of the nonpolar (hydrophobic) amino acid include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of the polar (neutral) amino acid include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of the positively charged basic amino acid include arginine, histidine, and lysine. Examples of the negatively charged acidic amino acid include aspartic acid and glutamic acid.
Examples of the amino acid sequence having deletion, substitution, addition or the like of 1 to several amino acids in the amino acid sequence of the wild-type enzyme include amino acid sequences having a certain degree or more of sequence identity with the amino acid sequence of the wild-type enzyme, and for example, amino acid sequences having 60% or more, preferably 65% or more, preferably 70% or more, preferably 75% or more, preferably 80% or more, preferably 85% or more, more preferably 90% or more, and further preferably 95% or more of sequence identity with the amino acid sequence of the wild-type enzyme.
The method for obtaining the enzymes (1) to (11) is not particularly limited, and examples thereof include a method comprising culturing a transformant transformed so as to enhance the expression of genes encoding the enzymes (1) to (11), and then recovering the enzymes (1) to (11) from the culture. The method for recovering the enzymes (1) to (11) from the culture is not particularly limited, and for example, the enzymes (1) to (11) can be separated by a common method, for example, by obtaining a protein concentrate containing the enzymes (1) to (11) from the culture supernatant from which impurities have been removed by ammonium sulfate precipitation or the like, and then using gel filtration chromatography or SDS-PAGE using the molecular weights of the enzymes (1) to (11) as an index. Further, the theoretical molecular weights calculated based on the constituent elements of AscB, AscC, AscD, AscE, AscF, AscG, AscH, AscI, AscJ, AscK, and AscA proteins having the amino acid sequences shown in SEQ ID Nos. 11 to 20 and 66 were about 37000, 120000, 230000, 61000, 31000, 61000, 57000, 42000, 32000, and 55000, respectively.
(genes encoding enzymes (1) to (11))
The genes ascB, ascC, ascD, ascE, ascF, ascG, ascH, ascI, ascJ, ascK, and ascA (hereinafter, these are sometimes collectively referred to as "genes encoding enzymes (1) to (11)") are not particularly limited if they contain a base sequence encoding an amino acid sequence possessed by the enzymes (1) to (11) having the above-mentioned enzymatic activities. The genes encoding the enzymes (1) to (11) produce the enzymes (1) to (11) by expression in an organism. The expression "expression of a gene" as used herein means production by transcription, translation or the like in a form in which a protein or an enzyme encoded by the gene has an original function or activity (particularly, enzymatic activity). In addition, the expression of "gene" includes: the protein or enzyme encoded by the gene is produced in such a way that the expression of the gene is high, i.e., the amount of the gene is more than the amount originally expressed by the host organism by insertion.
The genes encoding the enzymes (1) to (11) may be those capable of producing the enzymes (1) to (11) by splicing after transcription of the genes, or those capable of producing the enzymes (1) to (11) without splicing after transcription of the genes, when they are introduced into a host organism, and both of these genes may be used.
The genes encoding the enzymes (1) to (11) may be completely the same as the genes originally possessed by the source organism (i.e., wild-type genes), and as long as they encode the enzymes having the above-mentioned enzymatic activities, they may be DNAs having a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence of the wild-type gene.
The term "nucleotide sequence to be hybridized under stringent conditions" as used herein refers to a nucleotide sequence of a DNA obtained by colony hybridization, plaque hybridization, southern blotting, etc., using a DNA having a nucleotide sequence of a wild-type gene as a probe.
The term "stringent conditions" as used herein refers to conditions under which a specific hybridization signal is clearly distinguished from a non-specific hybridization signal; the hybridization system used varies depending on the type, sequence and length of the probe. Such conditions can be determined by varying the temperature of hybridization, varying the temperature of washing and the salt concentration. For example, in the case where a nonspecific hybridization signal is detected until it is strongly detected, the specificity can be improved by increasing the temperature of hybridization and washing while decreasing the washing salt concentration as necessary. In addition, when no specific hybridization signal is detected, hybridization can be stabilized by lowering the temperature for hybridization and washing and, if necessary, increasing the salt concentration for washing.
Specific examples of stringent conditions include hybridization using a DNA probe as a probe, 5 XSSC, 1.0% (w/v) nucleic acid hybridization blocker (manufactured by Boehringer Mannheim Co., Ltd.), 0.1% (w/v) N-lauroylsarcosine, and 0.02% (w/v) SDS at about 8 to 16 hours. Washing is carried out for 15 minutes and 2 times using 0.1 to 0.5 XSSC, 0.1% (w/v) SDS, preferably 0.1 XSSC, 0.1% (w/v) SDS. The temperature at which hybridization and washing are carried out is 65 ℃ or higher, preferably 68 ℃ or higher.
Examples of the DNA having a nucleotide sequence that hybridizes under stringent conditions include: DNA obtained by hybridizing under the above-mentioned stringent conditions or in the presence of 0.5 to 2.0M NaCl using DNA having a base sequence of a wild-type gene derived from a colony or plaque or a filter having a fragment of the DNA immobilized thereon, at 40 to 75 ℃, and preferably in the presence of 0.7 to 1.0M NaCl at 65 ℃, and then washing the filter with 0.1 to 1 XSSC solution (1 XSSC solution is 150mM sodium chloride or 15mM sodium citrate) at 65 ℃ to thereby identify the DNA or the like. The method of probe preparation or hybridization can be carried out according to the methods described in Molecular Cloning, A Laboratory Manual,2nd-Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.,1989, Current protocols Molecular Biology, Supplement 1-38, John Wiley & Sons,1987-1997 (these documents are also referred to below as "reference documents" the entire contents of which are incorporated herein by reference in a published manner), and the like. Furthermore, according to the person skilled in the art, conditions for obtaining a DNA having a nucleotide sequence that hybridizes to a nucleotide sequence complementary to the nucleotide sequence of the wild-type gene under stringent conditions can be appropriately set by adding various conditions such as probe concentration, probe length, reaction time, and the like to the conditions such as the salt concentration and temperature of the buffer.
Examples of the DNA containing a base sequence that hybridizes under stringent conditions include a DNA having a sequence identity to some extent or more with the base sequence of a DNA of a base sequence of a wild-type gene used as a probe, and examples of the DNA include a DNA having a sequence identity to 60% or more, preferably 65% or more, preferably 70% or more, preferably 75% or more, preferably 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more with the base sequence of a wild-type gene.
As the base sequence which hybridizes under stringent conditions to a base sequence complementary to the base sequence of the wild type gene, for example, if the number of bases in the base sequence is 100 as one unit, a base sequence having, on average, 1 to several, preferably 1 to 40, preferably 1 to 35, preferably 1 to 30, preferably 1 to 25, preferably 1 to 20, more preferably 1 to 15, further preferably 1,2, 3, 4, 5, 6, 7, 8,9 or 10, further preferably 1,2, 3, 4 or 5 bases deleted, substituted, added, or the like of the one unit is contained in the base sequence of the wild type gene. Here, the term "deletion of a base" means the presence of a lack or disappearance of a base in a sequence, "substitution of a base" means the base in a sequence is substituted with another base, and "addition of a base" means the addition of a new base by insertion.
An enzyme encoded by a base sequence that hybridizes under stringent conditions to a base sequence that is complementary to the base sequence of the wild-type gene may have an amino acid sequence having a deletion, substitution, addition, or the like of 1 to several amino acids in the amino acid sequence of the enzyme encoded by the base motif of the wild-type gene, but has the same activity as the enzyme encoded by the base sequence of the wild-type gene.
The genes encoding the enzymes (1) to (11) are nucleotide sequences encoding amino acid sequences identical or similar to the amino acid sequence of the enzyme encoded by the wild-type gene, using the fact that there are several codons corresponding to 1 amino acid, and may include nucleotide sequences different from the wild-type gene. Examples of the nucleotide sequence in which codon changes are performed for the nucleotide sequence of such a wild-type gene include nucleotide sequences shown in SEQ ID Nos. 21 to 24, 28 to 30, and 61. As the base sequence for carrying out the codon change, for example, a base sequence for carrying out the codon change in such a manner that it can be easily expressed in a host organism is preferable.
(method for calculating sequence identity)
The method for determining the sequence identity of a base sequence or an amino acid sequence is not particularly limited, and can be determined, for example, by aligning base sequences or amino acid sequences of proteins or enzymes encoded by a wild-type gene or a wild-type gene by a generally known method and using a program for calculating the sequence identity ratio of the two.
As a program for calculating the consensus among 2 amino acid sequences or base sequences, for example, there are known algorithms of Karlin and Altschul (Proc. Natl. Acad. Sci. USA,87: 2264-. In addition, a program that more sensitively determines sequence identity than BLAST is also known, i.e., Gapped BLAST (Nucleic Acids Res.25:3389-3402,1997, the entire contents of which are incorporated herein by reference in a published manner). Thus, one skilled in the art can retrieve sequences from a database that exhibit high sequence identity with respect to a given sequence using, for example, the procedures described above. These sequences can be obtained, for example, at the Internet site of the national center for Biotechnology information (http:// blast. ncbi. nlm. nih. gov/BLAST. cgi).
The above-mentioned methods can be generally used for searching a database for sequences showing sequence identity, but homology analysis of Genetyx network 12.0.1 (GenetYX) can be used as a method for determining sequence identity of individual sequences. This method is also based on the Lipman-Pearson method (Science 227: 1435-. In analyzing the sequence identity of the base sequences, protein-encoding domains (CDS or ORF) are used, if possible.
(sources of genes encoding enzymes (1) to (11))
The genes encoding the enzymes (1) to (11) are derived from, for example, a biological species having an isoprenoid-producing ability such as ilexolone A, ascofuranone, ascochlorin, or the like, or a biological species in which the expression of the enzymes (1) to (11) is observed, or the like. Examples of the source organism of the gene encoding the enzyme of the enzymes (1) to (11) include microorganisms. Among microorganisms, many species having an ability to produce ascochyrin or ascofuranone are known among filamentous fungi, and are therefore preferable. Specific examples of filamentous fungi having an ability to produce a chlorin or a chlorin analog include Acremonium, Chitosan, Fusarium, Cylindrocarpon, Verticillium, Nectria, Sclerotium, Cylindrocladium, Colletotrichum, Cephalosporium, Nigrospora, and Nigrospora, more specifically, Acremonium nucleatum, apple branch ulcer, Verticillium hemipterorum, and Nicotrichum. Specific examples of filamentous fungi having a furanone-producing ability include filamentous fungi of Acremonium, Paecilomyces, and Verticillium, and more specifically, Acremonium sclerotiorum, Acremonium applierum, Trichoderma reesei, Paecilomyces variotii, and Verticillium hemipterigenum. Specific examples of the filamentous fungus having an ability to produce ilexolone A include Trichoderma filamentous fungi, and more specifically Trichoderma reesei. Specific examples of the filamentous bacterium having an ascochyrin-producing ability and the filamentous bacterium having an ascochyrin-producing ability may include those having an L-ilicin A-producing ability.
As described above, the source organism of the genes encoding the enzymes (1) to (11) is not particularly limited, but the enzymes (1) to (11) expressed in the transformant are preferably inactivated without being affected by the growth conditions of the host organism and can exhibit their activities. Accordingly, the source organism of the gene organism encoding the enzymes (1) to (11) is preferably a microorganism whose growth conditions are similar to those of the host organism transformed by inserting the genes encoding the enzymes (1) to (11).
(cloning of genes encoding enzymes (1) to (11) by genetic engineering method)
The genes encoding the enzymes (1) to (11) can be inserted into various known vectors as appropriate. Furthermore, the vector can be introduced into an appropriate known host organism to prepare a transformant into which a recombinant vector (recombinant DNA) containing the genes encoding the enzymes (1) to (11) is introduced. The method for obtaining the genes encoding the enzymes (1) to (11), the method for obtaining the nucleotide sequences of the genes encoding the enzymes (1) to (11), the amino acid sequence information of the enzymes (1) to (11), the method for producing various vectors, the method for producing transformants, and the like can be appropriately selected by those skilled in the art. In addition, in the present specification, the transformation or the transformant contains gene transduction or a gene transductant, respectively. An example of cloning of the genes encoding the enzymes (1) to (11) is described below without limitation.
For cloning the genes encoding the enzymes (1) to (11), generally, a generally employed gene cloning method can be suitably employed. For example, chromosomal DNA or mRNA can be extracted from a microorganism or various cells having the productivity of the enzymes (1) to (11) by a commonly used method, for example, a method described in reference technical literature. cDNA can be synthesized using the extracted mRNA as a template. The chromosomal DNA or cDNA obtained in this manner can be used to prepare a library of chromosomal DNA or cDNA.
For example, the genes encoding the enzymes (1) to (11) can be obtained by cloning, as a template, chromosomal DNA or cDNA of an organism derived from the genes. The organisms from which the genes encoding the enzymes (1) to (11) are derived are as described above, and specific examples thereof include Acremonium sclerotiorum and the like. For example, Acremonium nucleatum is cultured, water is removed from the obtained cell body, the cell body is physically pulverized with a mortar or the like while being cooled in liquid nitrogen to prepare a fine powdery cell sheet, and a chromosomal DNA portion is extracted from the cell sheet by a conventional method. For the chromosomal DNA extraction procedure, a commercially available chromosomal DNA extraction Kit such as DNeasy Plant Mini Kit (manufactured by QIAGEN) can be used.
Then, using the chromosomal DNA as a template, a polymerase chain reaction (hereinafter referred to as "PCR") is performed using synthetic primers complementary to the 5 '-end sequence and the 3' -end sequence, thereby amplifying the DNA. The primer is not particularly limited as long as it can amplify a DNA fragment containing the gene. Alternatively, DNA containing a target gene fragment may be amplified by appropriate PCR such as the 5 'RACE method or the 3' RACE method, and these may be ligated to obtain DNA containing the full length of the target gene.
The method for obtaining the genes encoding the enzymes (1) to (11) is not particularly limited, and the genes encoding the enzymes (1) to (11) can be constructed by, for example, chemical synthesis methods, even without employing a genetic engineering method.
First, DNA whose sequence is to be determined is inserted into an appropriate vector according to a commonly used method to prepare a recombinant DNA, for example, commercially available kits such as TA Cloning Kit (Invitrogen), commercially available plasmid DNA such as pUC19 (Takara-bio), pUC18 (Takara-bio), pBR322 (Takara-bio), pBluescript SK + (Stratagene), pYES2/CT (Invitrogen), commercially available phage vector DNA such as λ EMBL3 (Stratagene), and the like are used to purify a recombinant DNA obtained by transforming a host organism (for example, Escherichia coli (Escherichia coli), preferably Escherichia coli strain JM109 (Takara-bio) or Escherichia
The determination of the base sequence of each gene inserted into the recombinant DNA is carried out by dideoxy method (method in Enzymology,101,20-78,1983, the entire contents of which are incorporated herein by reference in a published manner) or the like. The sequence analyzer used for determining the nucleotide sequence is not particularly limited, and examples thereof include a Li-COR MODEL4200L sequencer (manufactured by Aloka), a 370DNA sequencing system (manufactured by Perkinelmer), and a
(construction of recombinant vector comprising genes encoding enzymes (1) to (11))
A recombinant vector (recombinant DNA) comprising the genes encoding the enzymes (1) to (11) can be constructed by combining a PCR amplification product comprising any one of the genes encoding the enzymes (1) to (11) with various vectors in a form enabling the expression of the genes encoding the enzymes (1) to (11). For example, it can be constructed by excising a DNA fragment containing any one of the genes encoding the enzymes (1) to (11) with an appropriate restriction enzyme and ligating a plasmid obtained by cleaving the DNA fragment with an appropriate restriction enzyme. Alternatively, it can be obtained by ligating a DNA fragment containing the gene, to which the same sequence as that of the plasmid has been added at both ends, with a DNA fragment derived from the plasmid amplified by inverse PCR, using a commercially available recombinant vector preparation kit such as a seamless ligation HD cloning kit (Clontech).
(method for producing transformant)
The method for producing the transformant is not particularly limited, and examples thereof include a method of inserting the gene encoding the enzymes (1) to (11) into a host organism in a form in which the gene is expressed according to a conventional method. Specifically, a DNA construct in which any one of the genes encoding the enzymes (1) to (11) is inserted between an expression-inducing promoter and a terminator is prepared, and then the host organism is transformed in the DNA construct containing the genes encoding the enzymes (1) to (11), thereby obtaining a transformant in which the genes encoding the enzymes (1) to (11) are overexpressed. In the present specification, a DNA fragment comprising an expression-inducing promoter prepared for transformation of a host organism, a gene terminator encoding the enzymes (1) to (11), and a recombinant vector comprising the DNA fragment will be collectively referred to as a DNA construct.
The method of inserting the gene encoding the enzymes (1) to (11) into the host organism in the form in which it is expressed is not particularly limited, and examples thereof include a method of inserting the gene directly into the chromosome of the host organism by homologous recombination or nonhomologous recombination; a method of introducing the vector into a host organism by ligation to a plasmid vector, and the like.
The high expression promoter is not particularly limited, and examples thereof include a promoter domain of TEF1 gene (TEF1), a promoter domain of α -amylase gene (amy), a promoter domain of alkaline protease gene (alp), and a promoter domain of glyceraldehyde-3-phosphate dehydrogenase (gpd) as a translation elongation factor.
In the method using non-homologous recombination, the DNA construct used for transformation may be linear or circular, and the high expression promoter is not particularly limited, and examples thereof include a promoter domain of TEF1 gene (TEF1), a promoter domain of α -amylase gene (amy), a promoter domain of alkaline protease gene (alp), and a promoter domain of glyceraldehyde-3-phosphate dehydrogenase (gpd) as a translation elongation factor.
In the method using a vector, the DNA construct may be incorporated into a plasmid vector used in transformation of a host organism by a conventional method, and the corresponding host organism may be transformed by a conventional method.
Such a suitable vector-host system is not particularly limited as long as it can produce the enzymes (1) to (11) in the host organism, and examples thereof include pUC19 and the system of filamentous fungi, pSTA14(mol.Gen.Genet.218,99-104,1989, the entire contents of which are incorporated herein by reference) and the system of filamentous fungi.
The DNA construct is preferably used by introducing it into the chromosome of the host organism, but as a further method it can also be used in a form which is not introduced into the chromosome by incorporating the DNA construct into an autonomously replicating vector (Ozeki et al, Biosci.Biotechnol.biochem.59,1133(1995), the entire content of which is incorporated herein by reference in a published manner).
In the DNA construct, a marker gene for enabling selection of transformed cells may also be included. The marker gene is not particularly limited, and examples thereof include genes that supplement the nutritional requirements of the host organism, such as pyrG, pyrG3, niaD, and adeA; drug resistance genes aiming at drugs such as thiamine resistance, hygromycin B, oligomycin and the like. In addition, it is preferred that the DNA construct comprises a promoter, a terminator, other control sequences (e.g., enhancers, polyadenylation sequences, etc.) capable of overexpressing the genes encoding the enzymes (1) to (11) in the host organism. The promoter is not particularly limited, but may be an appropriate expression-inducing promoter or a structural promoter, and examples thereof include a tef1 promoter, an alp promoter, an amy promoter, and a gpd promoter. The terminator is not particularly limited, and examples thereof include an alp terminator, an amy terminator, and a tef1 terminator.
In the DNA construct, the expression control sequence of the gene encoding the enzymes (1) to (11) is indispensable in the case where the DNA fragment containing the inserted gene encoding the enzymes (1) to (11) has a sequence having an expression control function. In the case of transformation by the co-transformation method, the DNA construct may not have a marker gene.
There may be a tag in the DNA construct for purification. For example, purification using a nickel column can be made possible by appropriately ligating a linker sequence upstream or downstream of the genes encoding the enzymes (1) to (11) and
The same sequences necessary for marker recovery may also be included in the DNA construct. For example, the pyrG tag can be detached on a medium containing 5-fluoroorotic acid (5FOA) by adding a sequence identical to the sequence upstream of the insertion site (5 'homologous recombination domain) downstream of the pyrG tag or a sequence identical to the sequence downstream of the insertion site (3' homologous recombination domain) upstream of the pyrG tag. Preferably, the length of the identical sequence suitable for marker recovery is 0.5kb or more.
One form of the DNA construct is, for example, a DNA construct in which Ptef as a promoter of tef1 gene, genes encoding enzymes (1) to (11), a Tref or alp gene terminator as a terminator of tef1 gene, and a pyrG marker gene are ligated to a seamless cloning site located at the multiple cloning site of pUC 19.
One form of the DNA construct in the case of inserting a gene by homologous recombination is a DNA construct in which a 5 'homologous recombination sequence, tef1 gene promoter, genes encoding enzymes (1) to (11), alp gene terminator and pyrG marker gene, 3' homologous recombination sequence are ligated.
One form of the DNA construct in the case where the gene is inserted by homologous recombination and the marker is recovered is a DNA construct in which a 5 'homologous recombination sequence, tef1 gene promoter, genes encoding enzymes (1) to (11), alp gene terminator, marker recovery are linked with the same sequence, pyrG marker gene, 3' homologous recombination sequence.
In the case where the host organism is a filamentous fungus, as a method for transformation into a filamentous fungus, a method known to those skilled in the art may be appropriately selected, and for example, a protoplast PEG method using polyethylene glycol and calcium chloride after preparation of protoplasts of the host organism can be employed (for example, refer to mol. Gen. Genet.218,99-104,1989, Japanese patent laid-open No. 2007-222055, etc., the entire contents of which are incorporated herein by reference in a published manner). The medium used for regenerating the transformant is a medium suitable for the host organism and the transformation marker gene used. For example, in the case of using Aspergillus oryzae (A.oryzae), Aspergillus sojae (A.sojae) as a host organism and pyrG gene as a transformation marker gene, regeneration of the transformant may be performed in Czapek-Dox minimal medium (manufactured by Difco) containing 0.5% agar and 1.2M sorbitol, for example.
In addition, for example, in order to obtain a transformant, the promoter of the gene encoding the enzymes (1) to (11) originally present on the chromosome of the host organism may be replaced with a high-expression promoter such as tef1 by homologous recombination. In this case, it is preferable that a transformation marker gene such as pyrG be inserted in addition to the high expression promoter. For this purpose, for example, with reference to the examples described in Japanese patent laid-open No. 2011-239681 or FIG. 1, a module for transformation or the like composed of the whole or part of the upstream region of the genes encoding the enzymes (1) to (11) -the transformation marker gene-the high expression promoter-the genes encoding the enzymes (1) to (11) may be used. In this case, the upstream region of the gene encoding the enzymes (1) to (11) and all or part of the gene encoding the enzymes (1) to (11) are applied to homologous recombination. All or part of the genes encoding the enzymes (1) to (11) may be a gene comprising an intermediate domain derived from the start codon. In the case of filamentous fungi, the domain suitable for homologous recombination is preferably 0.5kb or more in length.
The confirmation of the production of the transformant can be performed by the following method: the transformant is cultured under conditions in which the activity of the enzymes (1) to (11) is confirmed, followed by detecting the product of interest (e.g., isoprenoid such as ascochyl, ilicin A, ascofuranone) in the culture obtained after the culture, or confirming that the amount of the product of interest detected is larger than that in the culture of the host organism cultured under the same conditions.
Further, the determination of the production of the transformant can be performed as follows: chromosomal DNA was extracted from the transformant and PCR was performed using the chromosomal DNA as a template, and it was confirmed that an amplifiable PCR product was produced when transformation occurred. In this case, for example, PCR is performed in a combination of a forward primer corresponding to the nucleotide sequence of the promoter to be used and a reverse primer corresponding to the nucleotide sequence of the transformation marker gene, and the generation of a product having an expected length is confirmed.
Preferably, in the case of transformation by homologous recombination, PCR is performed in a combination of a forward primer located at an upstream position with respect to the same domain on the upstream side used and a reverse primer located at a downstream position with respect to the same domain on the downstream side used, and it is preferable to confirm that a product of an expected length is produced when homologous recombination occurs.
(method for producing Gene knock-out organism)
"knockout" refers to loss of functional expression of a protein encoded by a gene by deletion, mutation, introduction of an arbitrary sequence into the gene, deletion of a promoter necessary for expression of the gene, or the like of a part or all of the gene. Strictly speaking, functional expression of a protein encoded by the gene is not completely lost, that is, even if there is a possibility that the protein encoded by the gene is functionally expressed, it is included in the so-called "gene knockout" in the present specification as long as the functional expression is largely lost. In the present specification, the "knockout organism" may be referred to as a "disrupted strain" or a "deleted strain" or the like.
The method for preparing a gene knockout is not particularly limited, and any method can be used, for example, as shown in the following examples, in which a part or all of a gene is deleted by homologous recombination, or in which a gene is deleted, inserted, or substituted by a genome editing technique such as TALEN and CRISPR-Cas 9. One form of the DNA construct in the case of gene knockout of a gene by homologous recombination is a DNA construct in which a 5 'homologous recombination sequence, a pyrG marker gene, and a 3' homologous recombination sequence are linked, but is not limited thereto.
One form of DNA construct in the case where a gene is inserted by homologous recombination and a marker is recovered is a DNA construct in which a 5 'homologous recombination sequence, the same sequence for marker recovery, a pyrG marker gene, a 3' homologous recombination sequence are ligated, and the like.
(host organism)
As the host organism, if the enzymes (1) to (11) or the organism producing isoprenoid can be produced by transformation using a DNA construct containing a gene encoding the enzymes (1) to (11) or a DNA construct containing a gene encoding the enzymes (1) to (11), there are no particular limitations, and examples thereof include microorganisms or plants, and examples thereof include microorganisms of the genus Aspergillus, Acremonium, Chitosa, Fusarium, Escherichia, Saccharomyces, Pichia, Schizosaccharomyces, Trichoderma, Penicillium, Rhizopus, Neurospora, Saccharomyces, and Neotorula, Microorganisms of the genus Chlamydomonas (Byssochlamys), microorganisms of the genus Talaromyces (Talaromyces), microorganisms of the genus Histoplasma (Ajinomyces), microorganisms of the genus Paracoccidioides (Paracoccus), microorganisms of the genus Ascomyces (Uncinococcus), microorganisms of the genus Coccidioides (Coccidioides), microorganisms of the genus Arthroderma (Arthroderma), microorganisms of the genus Trichophyton (Trichophyton), microorganisms of the genus Exophyces (Exophiala), microorganisms of the genus Capnocladium (Capnonia), microorganisms of the genus Cladosporium (Cladophyllum), microorganisms of the genus Septoria (Macrophoma), microorganisms of the genus Micrococcus (Leptospira), microorganisms of the genus Paphioleracea (Paphioleracea), microorganisms of the genus Paphioleracea (Diphyllum), microorganisms of the genus Micrococcus (Micrococcus), microorganisms of the genus Penicillium, microorganisms of the genus Micrococcus (Micrococcus, microorganisms of the genus Micrococcus (Micrococcus), microorganisms of the genus Micrococcus (Microchaetocercosporus), microorganisms of the genus Microchaetomium (Microchaetobacter), microorganisms of the genus Microchaetocercosporus, microorganisms of the genus Microchaetomium (Microchaetobacter), microorganisms of the genus Microchaetomium (Microchaetomium), microorganisms of the genus Microchaetobacter), microorganisms of the genus Microchaetomium (Microchaetobacter), microorganisms of the genus Sepiella, Microchaetomium (Micro, Microorganisms of the genus Sclerotinia (Sclerotinia), microorganisms of the genus Ascophyllum (Mmagnaporthe), microorganisms of the genus Verticillium, microorganisms of the genus Pseudocercospora (Pseudocercospora), microorganisms of the genus anthrax (Colletotrichum), microorganisms of the genus Ophiostoma, microorganisms of the genus Metarhizium (Metarhizium), microorganisms of the genus Sporotrichum, microorganisms of the genus Sordaria, plants of the genus Arabidopsis (Arabidopsis), etc., with preference given to microorganisms and plants. The filamentous fungus may be a filamentous fungus which has been confirmed to have an ability to produce isoprenoids such as ilexolone A, ascochloride, ascofuranone, etc., or a filamentous fungus which has genes encoding enzymes (1) to (11) on its genomic DNA.
When the host organism does not have any of the ascochyrin biosynthesis genes or ascofuranone biosynthesis genes, transformation with the genes encoding the enzymes (1) to (11) is carried out without confirming the isoprenoid-producing ability. That is, a transformant (e.g., a transformed filamentous fungus) into which a gene for biosynthesis of ascochyrin or a gene for biosynthesis of ascofuranone has been introduced and which has been transformed to heterologously express isoprenoid can also be used as the host organism. However, in any case, humans are excluded from the host organism.
Examples of organisms for confirming the ability to produce isoprenoid include filamentous bacteria of Acremonium, Trichoderma, Fusarium, Cylindrocarpon, Verticillium, Neurospora, Pectinophyta, and Paecilomyces, and more specifically, Acremonium sclerotiorum, apple branch canker, Trichoderma reesei, Paecilomyces varioti, and Verticillium hemipteri.
Among filamentous fungi, microorganisms belonging to the genus aspergillus such as aspergillus oryzae, aspergillus sojae, aspergillus niger (a.niger), aspergillus tamarii (a.tamarii), aspergillus awamori (a.awamori), aspergillus usamii (a.usami), aspergillus kawachi (a.kawachi), and aspergillus saitoi (a.saitoi) are preferable in view of safety and ease of culture.
Since filamentous fungi typified by Acremonium microorganisms and Aspergillus microorganisms tend to have a low homologous recombination frequency, it is preferable to use transformed filamentous fungi in which the Ku gene, such as Ku70 or Ku80, involved in the mechanism of non-homologous recombination, is suppressed when preparing transformants by homologous recombination.
Such suppression of the Ku gene can be carried out by any method known to those skilled in the art, and can be achieved, for example, by disrupting the Ku gene using a Ku gene disruption vector, or inactivating the Ku gene by an antisense RNA method using an antisense expression vector for the Ku gene. The transformed microorganism of the genus Aspergillus obtained in this manner has a significantly increased homologous recombination frequency as compared with the original microorganism of the genus Aspergillus in which gene manipulation involving Ku gene suppression is carried out. In particular, the increase is at least 2-fold, preferably at least 5-fold, preferably at least 10-fold, preferably at least about 50-fold.
In addition, as the host organism, it is preferable to use a transformed filamentous bacterium in which a marker gene such as pyrG is suppressed. The marker gene to be suppressed can be appropriately set according to the marker gene contained in the DNA construct.
(specific examples of the genes encoding the enzymes (1) to (11))
Examples of the genes encoding the enzymes (1) to (11) derived from Acremonium nucleatum include genes ascB, ascC, ascD, ascE, ascF, ascG, ascH, ascI, ascJ, ascK, and ascA having the nucleotide sequences shown in SEQ ID Nos. 1 to 10 and 65, respectively. In addition, the amino acid sequences of AscB, AscC, AscD, AscE, AscF, AscG, AscH, AscI, AscJ, AscK, and AscA proteins are represented as sequence Nos. 11-20 and 66, respectively.
The method for obtaining the genes encoding the enzymes (1) to (11) from organisms other than the sterilized Acremonium nucleatum and Acremonium sclerotiorum is not particularly limited, and can be obtained, for example, by: based on the base sequences (SEQ ID NOS: 1 to 10 and 65) of the genes ascB, ascC, AscD, ascE, ascF, ascG, ascH, ascI, ascJ, and ascK, BLAST homology search was performed with respect to the genomic DNA of the target organism, thereby identifying genes having base sequences with high sequence identity to the base sequences of the genes ascA, ascB, ascC, ascD, ascE, ascF, ascG, ascH, ascI, ascJ, ascK, and ascA. In addition, it can be obtained by: based on the total protein of the subject organism, a protein having an amino acid sequence with high sequence identity to the amino acid sequences (SEQ ID NOS: 11 to 20 and 66) of the AscB, AscC, AscD, AscE, AscF, AscG, AscH, AscI, AscJ, AscK, and AscA proteins is identified, and a gene encoding the protein is identified. For example, the amino acid sequences having high sequence identity to the amino acid sequences of AscB, AscC, AscD, AscE, AscF, AscG, AscH and AscI proteins derived from Acremonium sclerotiorum include the amino acid sequences of SEQ ID Nos. 35 to 41 and 67 derived from the genus Erythrophyllum; examples of the amino acid sequence having high sequence identity with the amino acid sequences of AscB, AscC, AscD and AscE proteins derived from Acremonium sclerotiorum include the amino acid sequences of SEQ ID Nos. 47 to 50 derived from Trichoderma.
The genes encoding the enzymes (1) to (11) obtained from Acremonium sclerotiorum or the genes encoding the enzymes having sequence identity to the enzymes (1) to (11) can be introduced into any host cell of a host organism, such as an Aspergillus microorganism or an Acremonium microorganism, and transformed.
(transformant)
One form of the transformant is a transformant in which a filamentous fungus, a plant or the like is used as a host organism, any one of genes ascA, ascB, ascC, ascD, ascE, ascF, ascG, ascH, ascI, ascJ and ascK, or a combination thereof is inserted, and the inserted gene is expressed (hereinafter, also referred to as "transformant (1)"). When the host organism is an organism that is confirmed to have an ability to produce ascochyrin or ascofuranone, such as Acremonium sclerotiorum, it is preferable that the inserted gene is forcibly expressed constantly or expressed at a higher level than the intrinsic expression, or is expressed conditionally at the late stage of culture after cell growth. Such transformants may be produced not substantially in the host organism by the action of the expressed AscA, AscB, AscC, AscD, AscE, AscF, AscG, AscH, AscI, AscJ and/or AscK, or may produce ilexin A, ascomycin or ascofuranone in detectable amounts or amounts above that, even if only in minute amounts.
Another form of the transformant is a transformant (hereinafter, also referred to as "transformant (2)") which comprises a host organism such as a filamentous fungus or a plant, a biosynthetic gene cluster gene derived from a wild type (which may contain a promoter sequence other than ORF) and which contains all or a part of the genes ascB, ascC, ascD, ascE, ascF, ascG, ascH, ascI, ascJ and ascK, and which is transformed so as to express a gene that is designed to control transcription of the biosynthetic gene cluster such as ascI at a high or low level by inserting a DNA construct into the host organism. When the host organism is an organism such as Acremonium sclerotiorum, which is confirmed to have an ability to produce ascochloride or ascofuranone, it is preferable that the inserted gene is forcibly expressed constantly or expressed at a higher level than the intrinsic expression, or is expressed conditionally at the late stage of culture after cell growth. Such a transformant can be cultured or grown under conditions suitable for the host organism or the transformant by utilizing the action of a transcription factor whose expression amount varies, whereby ilexolone A, ascochytrid or ascochytrid, even if produced, is not substantially produced or produced in a detectable amount or more in the host organism.
A specific form of the transformant is a transformant in which a host organism such as aspergillus sojae is used, genes ascF, ascE, ascD, ascB, and ascC are inserted in addition to the genes ascI, ascJ, and ascK, and the inserted genes are expressed; a transformant in which a gene ascF is inserted in addition to the genes ascI, ascJ and ascK and the inserted gene is expressed, and the like, but is not limited thereto.
One specific example of the transformant is a transformant in which a host organism is selected from the group consisting of Acremonium sclerotiorum, apple branch canker, Trichoderma reesei, etc., and a transformant in which at least 1 arbitrary gene selected from the group consisting of ascA to ascI is inserted and the inserted gene is expressed, but the present invention is not limited thereto.
(Gene knock-out organism)
One form of the gene-knockout organism is a gene-knockout organism obtained by gene-knocking out the gene ascB, ascC, ascD, ascE, ascF, ascG, and ascI from a wild-type organism having both the genes ascB, ascC, ascD, ascE, ascF, ascG, and ascI that produce ascochytin and ascofuranone, such as Acremonium sclerotium (hereinafter also referred to as "gene-knockout organism (1)"). Such a knockout organism does not express the AscG protein as an enzyme involved in the biosynthesis of ascochytrium chloride, and therefore can produce ascofuranone or an ascochytrium furanone precursor alone instead of ascochytrium chloride, for example, can produce ascochytrium furanone or an ascochytrium furanone precursor in a larger amount than in a wild-type organism.
Another form of the gene-knocked-out organism is a gene-knocked-out organism obtained by knocking out the AscF gene from a wild-type organism such as Acremonium nucleatum or Chitosa rubrum having the genes ascB, ascC, ascD, ascE and ascF which produce ascochlorin or a precursor of ascochlorin (hereinafter also referred to as "gene-knocked-out organism (2)"). By culturing or growing such a knockout organism under conditions suitable for the wild-type organism, there is the possibility that the amount of ilexolone A is produced in greater amounts than in the wild-type organism.
Another form of the gene-knocked-out organism is a gene-knocked-out organism obtained by gene-knocking out the gene ascI from a wild-type organism such as Sclerotium sclerotiorum having the genes ascB, ascC, ascD, ascE, ascF, ascG, and ascI that produce both ascochytrid chloride and ascofuranone, or a wild-type organism such as Sclerotium rubrum having the genes ascB, ascC, ascD, ascE, ascF, ascG, and ascI (hereinafter also referred to as "gene-knocked-out organism (3)"). Such a knockout organism does not express the AscI protein as an enzyme involved in the biosynthesis of ascofuranone, and therefore only ascochlorous chloride can be produced instead of ascofuranone, for example, there is a possibility that ascochlorous chloride is produced in a larger amount than in a wild-type organism.
Another example of the knockout organism is a knockout organism obtained by knocking out a gene involved in the biosynthesis after wintergreen A from a wild-type organism such as Trichoderma reesei having genes involved in the biosynthesis after wintergreen A and ascB, ascC, ascD, ascE and wintergreen A which are genes involved in the production of the derivative of wintergreen A (hereinafter also referred to as "knockout organism (4)"). By culturing or growing such a knockout organism under conditions suitable for the wild-type organism, there is the possibility of producing ilexolone A in greater amounts as compared to the wild-type organism.
(production method)
The manufacturing method of one embodiment of the invention is a manufacturing method of the ilexolone A, the ascochloride or the ascofuranone; the production method at least comprises a step of obtaining ilicin A, ascomycin or ascofuranone by culturing the transformant (1) or the transformant (2) under conditions suitable for the host cell.
The method for producing ilicin A, ascochloride or ascofuranone according to another embodiment of the present invention is a method for producing ilicin A, ascochloride or ascofuranone, which comprises at least a step of allowing a transformant (1) or a transformant (2) to act on a precursor of ilicin A, ascochloride or ascofuranone such as LL-Z1272 β or ilicin A (LL-Z1272 α) or the like to obtain ilicin A, ascochloride or ascofuranone.
Another embodiment of the present invention is a method for producing ilicin A, ascochloride or ascofuranone, which comprises at least a step of obtaining ilicin A, ascochloride or ascofuranone by allowing an enzyme extracted from the transformant (1) or the transformant (2) to act on a precursor of ilicin A, ascochloride or ascofuranone such as LL-Z1272 β or ilicin A.
The process according to another embodiment of the present invention is a process for producing ascofuranone or an ascofuranone precursor; the production method comprises at least a step of obtaining ascofuranone or an ascofuranone precursor by culturing the knockout organism (1) under conditions suitable for a wild-type organism.
Another embodiment of the present invention relates to a method for producing ilexolone A; the manufacturing method comprises at least a step of obtaining the ilexolone A by culturing or growing the knockout organism (2) or (4) under conditions suitable for the wild-type organism.
The process according to another embodiment of the present invention is a process for producing ascochyta chloride or an ascochyta chloride precursor; the production method comprises at least a step of obtaining ascochyta or an ascochyta precursor by culturing or growing the knockout organism (3) under conditions suitable for a wild-type organism.
The following description mainly describes the production method in the case where the host organism or the wild-type organism is a filamentous fungus, but the production method of each embodiment of the present invention is not limited to the following description.
The medium may be any of a synthetic medium and a natural medium, as long as it is a general medium for culturing a host organism or a wild-type organism (hereinafter, these are also collectively referred to as "host organism or the like"), that is, a medium containing a carbon source, a nitrogen source, an inorganic substance, and other nutrients in an appropriate ratio. In the case where the host organism or the like is a microorganism of the genus Acremonium or a microorganism of the genus Aspergillus, a GPY medium or the like as described in the following examples can be used, but is not particularly limited.
The culture conditions for the transformant or the knockout organism (hereinafter, they are also collectively referred to as "transformant or the like") may be those for a host organism or the like generally known to those skilled in the art, and for example, in the case where the host organism or the like is a filamentous fungus such as Acremonium or Aspergillus, the culture conditions may be appropriately set: adjusting the initial pH value of the culture medium to 5-10, the culture temperature to 20-40 ℃, and the culture time to be several hours to several days, preferably 1-7 days, more preferably 2-4 days, etc. The culture means is not particularly limited, and aeration-agitation submerged culture, shaking culture, static culture, or the like can be used, but culture under conditions where dissolved oxygen becomes sufficient is preferable. For example, as an example of a medium and culture conditions for culturing an acremonium microorganism or an aspergillus microorganism, shaking culture using a GPY medium at 30 ℃ and 160rpm for 3 to 5 days as described in the following examples can be given.
The method for extracting a target product (isoprenoid) such as ascochlorin, ascofuranone, or ilicin A from the culture after the culture is completed is not particularly limited. In the extraction, the cells collected from the culture by filtration, centrifugation or the like may be used as they are, or cells dried after collection or cells further pulverized may be used. The method for drying the bacterial cells is not particularly limited, and examples thereof include freeze drying, sun drying, hot air drying, vacuum drying, aeration drying, and reduced pressure drying.
The extraction solvent is not particularly limited as long as the target product is dissolved therein, and examples thereof include organic solvents such as methanol, ethanol, isopropanol, and acetone; an aqueous organic solvent in which these organic solvents are mixed with water; water, warm water, hot water, and the like. After the solvent is added, the objective product is extracted while appropriately performing cell disruption treatment.
In place of the above-mentioned heating treatment, a method of destroying the cells by using a destroying means such as an ultrasonic crusher, a French press, a high-efficiency horizontal sand mill, or a mortar; a method for dissolving cell walls of thalli by using cell wall lytic enzymes such as fungus wall-breaking enzyme and the like; and a method of disrupting the cells such as a method of dissolving the cells with a surfactant such as SDS or TritonX-100. These methods may be used alone or in combination.
The desired product can be purified by subjecting the obtained extract to purification treatments such as centrifugation, membrane filtration, ultrafiltration, gel filtration, separation by a difference in solubility, solvent extraction, chromatography (adsorption chromatography, hydrophobic chromatography, cation exchange chromatography, anion exchange chromatography, reverse phase chromatography, etc.), crystallization, activated carbon treatment, membrane treatment, etc.
Qualitative analysis or quantitative analysis can be performed by LC-MS, LC-ICP-MS, MS/MS, or the like. The conditions for these analyses can be appropriately selected by those skilled in the art and can be carried out, for example, under the conditions described in the following examples.
In the manufacturing method of each embodiment of the present invention, various steps or operations may be added to the preceding or subsequent steps or steps of the above steps as long as the object of the present invention can be achieved.
(method)
The method of one embodiment of the present invention is a method for increasing the production of isoprenoids using filamentous fungi; the method comprises a step of enhancing the expression of AscA protein or gene ascA in a filamentous bacterium having any 1 or more genes of ascB to ascK or a gene for biosynthesis of ascochylomicron and/or a gene for biosynthesis of ascofuranone, thereby increasing the production of isoprenoids using the filamentous bacterium. The method of another embodiment of the present invention is a method for producing isoprenoids; the production method comprises a step of obtaining isoprenoid by enhancing expression of AscA protein or gene ascA in a filamentous fungus having an ascochyrin biosynthesis gene and/or an ascofuranone biosynthesis gene. The method of another embodiment of the present invention is a method for producing isoprenoids; the production method comprises a step of obtaining isoprenoids by culturing a transformant transformed so that the expression of the gene ascA is enhanced.
Means for enhancing the expression of the AscA protein or the gene ascA is not particularly limited, and for example, a transformant transformed so as to enhance the expression of the gene ascA can be used as the filamentous bacterium to be used; means for enhancing the expression of the gene ascA originally possessed by a filamentous fungus having the gene ascA for biosynthesis of ascofuranone and/or ascochyrate comprising the gene ascA by adjusting the culture conditions or introducing other transcription factors.
Whether or not the production of isoprenoid using a filamentous fungus is increased can be confirmed, for example, by comparing the isoprenoid production amount of a filamentous fungus having an ascochylomycin biosynthesis gene and/or an ascofuranone biosynthesis gene which does not employ a means for enhancing the expression of an AscA protein or a gene ascA with the isoprenoid production amount of a filamentous fungus having an ascochylomycin biosynthesis gene and/or an ascofuranone biosynthesis gene which employs a means for enhancing the expression of an AscA protein or a gene ascA.
(use)
Ascochylomycetin, ascofuranone, ilexolone a and the like obtained by using the gene, transformant, knock-out organism and production method of one embodiment of the present invention are functional living substances expected to have various physiological activities such as antiprotozoal activity, antitumor activity, hypoglycemic activity, hypolipidemic activity, glycolytic inhibitory activity, antioxidant activity and the like, and can be used as raw materials for producing drugs, quasi drugs and the like or products thereof by effectively utilizing the characteristics thereof.
The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples, and various forms can be adopted as long as the object of the present invention can be achieved.
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