JP7421615B2 - Transformation method for filamentous fungi - Google Patents

Description

The present invention relates to a method for introducing multiple copies of any gene into filamentous fungal cells using a selectable marker gene whose expression level is reduced by modifying the promoter region.

Filamentous fungi represented by the genus Aspergillus, Penicillim, Trichoderma, etc. generally have high protein production ability and are widely used as production hosts for industrially useful proteins. Furthermore, filamentous fungi are known to produce a variety of secondary metabolites, making them highly useful as hosts for the production of low-molecular-weight compounds. Therefore, research on protein expression systems using genetic recombination technology is being actively conducted in filamentous fungi as well. Among these, construction of a high protein expression system is considered particularly important in order to achieve high production of a target protein or compound.

Generally speaking, methods for achieving high expression of the target protein include using a high-expression promoter, using a plasmid that can replicate the target gene in high copies, or introducing high copies of the target gene into the genome. Methods are known.

In filamentous fungi, many searches for high-expression promoters have been carried out, and there are many reported examples (Patent Documents 1 to 7). Furthermore, high-expression promoters have been reported that are based on existing promoters and are improved by adding or deleting specific base sequences (Patent Documents 8 to 11).

Furthermore, in bacteria and yeast, it is possible to highly express a target protein by using an autonomously replicating plasmid that can replicate in high copies. On the other hand, in filamentous fungi, it has been reported that a vector containing AMA1 as a replication origin can function as an autonomously replicable plasmid. However, this plasmid can autonomously replicate outside the chromosome in some filamentous fungi, and it has also been reported that recombination with chromosomal DNA can occur, albeit at a low frequency, even in such filamentous fungi. For these reasons, it is not suitable for industrial use. Therefore, when producing high amounts of protein in filamentous fungi, a common method is to integrate an expression cassette of a gene encoding the target protein into the chromosome.

Therefore, in order to achieve high expression of the target protein, one possible strategy is to integrate a high copy of the expression cassette of the gene encoding the target protein into the chromosome. For example, it is known that by using ATP sulfurylase (sC), which is involved in sulfate utilization, as a selection marker, multiple copies of the gene can be integrated into the chromosome. However, many strains have a copy number of only a few copies, and it takes a great deal of effort to select strains with a higher copy number. Furthermore, it is known that tens of copies of the gene can be inserted by using the acetamidase gene (amdS) as a selection marker (Non-Patent Document 2). However, this system uses A. koji mold for sake. It is known that it is difficult to use this marker in strains that originally have the amdS gene, such as S. oryzae (Non-Patent Document 3), and there is a drawback that this marker cannot be used in some filamentous fungi. In addition, amdS is essential when an amide compound such as acetamide or acrylamide is the sole carbon source; There is also concern that amdS is not required, and the inserted gene is likely to be deleted during culture. In addition, by using the ornithine carbamoyltransferase gene (argB) derived from Aspergillus nidulans as a selection marker in an arginine auxotrophic strain of Aspergillus oryzae, approximately 60 copies of the gene were inserted in Aspergillus oryzae (Non-patent Document 4). ) and a leucine-auxotrophic strain of Aspergillus oryzae, it has been reported that 64 to 128 copies of the gene were inserted by using the ANleu2B gene derived from Aspergillus nidulans as a selection marker (Patent Document 12). However, these methods cannot be applied to selectable marker genes other than argB and ANleu2B, so the hosts that can be used are limited and lack versatility.

Patent No. 3372087 Patent No. 3759794 Patent No. 3792467 Patent No. 4676639 Patent No. 3743803 International Publication No. 2007/071399 Patent No. 5507062 Patent No. 3343567 Patent No. 4495904 Patent No. 4490284 Japanese Patent Application Publication No. 2009-254349 Patent No. 5473179

Gene. 1991 Feb 1;98(1):61-7 More Gene Manufacturing in Fungi, page 409 Revised edition of Molecular Aspergillus, Japan Brewing Association Agric. Biol. Chem. , 51(9), 2549-2555, 1987

An object of the present invention is to provide a highly versatile method for integrating multiple copies of any gene into the chromosome in filamentous fungi.

As a result of extensive research to solve the above problems, we found that by modifying the promoter region and using a selectable marker gene whose expression level was reduced, we were able to insert multiple copies of any gene into filamentous fungal cells. The present invention was completed based on the discovery that this can be done.

That is, the present invention relates to the following.
[1] The following steps:
providing a host filamentous fungus containing a selectable marker gene that is deficient in the function of the encoded protein;
Transforming the host filamentous fungus using an expression cassette containing the coding region, promoter and terminator of the target gene, and a selectable marker gene in which the promoter has been modified;
A method for transforming filamentous fungi,
[2]
The method according to [1], wherein the filamentous fungus is selected from filamentous fungi belonging to the genus Aspergillus, Neurospora, Penicillium, Fusarium, Trichoderma, Mucor, and Arachnoid;
[3]
The method according to [1] or [2], wherein the filamentous fungus is selected from filamentous fungi belonging to the genus Aspergillus;
[4]
Any one of [1] to [3], wherein the filamentous fungus is Aspergillus sojae, Aspergillus oryzae, Aspergillus niger, Aspergillus tamari, Aspergillus luchiuensis, Aspergillus usami, Aspergillus kawachi, or Aspergillus cytoi. The method described in section
[5]
The method according to any one of [1] to [4], wherein the filamentous fungus is Aspergillus sojae or Aspergillus oryzae;
[6]
The method according to any one of [1] to [5], wherein the modification of the promoter is deletion of a portion of the promoter;
[7]
The selection marker is the pyrG gene, pyrF gene, adeA gene, adeB gene, argB gene, niaD gene, sC gene, amdS gene, trpC gene, leu2 gene, bioDA gene, ptrA gene, or benA gene, [1] The method according to any one of [6],
[8]
The method according to any one of [1] to [6], wherein the selection marker is the pyrG gene,
[9]
The method according to any one of [1] to [8], wherein a length of 40%, 60% or 80% or more of the promoter is deleted;
[10]
The method according to any one of [1] to [8], wherein the promoter length is deleted to 250 bp, 200 bp, 150 bp, 100 bp, 80 bp, or 60 bp or less,
[11]
The method according to any one of [1] to [10], wherein the genes used for transformation are linked in the order of an expression cassette and a selection marker gene.
The present invention also relates to a transformant obtained by the above method, and a method for producing a target protein using the transformant.

According to the present invention, it is possible to insert multiple copies of any gene into filamentous fungi, and it is possible to achieve high production of proteins and secondary metabolites useful for industrial use.
Furthermore, this method is more versatile and more practical than the previously known methods for inserting multiple copies of genes into filamentous fungi.

(Selectable marker gene with modified promoter)
A selectable marker gene whose promoter has been modified is a promoter that has been modified by deleting or replacing a gene sequence in an arbitrary region from a promoter that has sufficient transcriptional activity to express the function of the selectable marker, or by inserting an arbitrary gene sequence. , refers to a selectable marker gene whose expression level in the coding region is reduced by reducing the function of the promoter and thereby reducing the amount of transcription of the gene in the coding region. The term "promoter with sufficient transcriptional activity for the functional expression of a selectable marker" as used herein refers to the insertion of only one copy of the selectable marker gene onto the chromosome, thereby inhibiting growth on a selective medium (functional expression of the selectable marker). A promoter that has the necessary transcriptional activity to enable transcription.

A decrease in the expression level of the coding region means that the transcription level of the gene in the coding region is 50%, preferably 60%, more preferably 70%, more preferably 80%, compared to the case where the promoter is not modified. More preferably 90%, more preferably 95%, more preferably 98% or more, more preferably 99% or more. The term "unmodified" promoter as used herein refers to a promoter of a selectable marker gene that has transcriptional activity that enables functional expression of the selectable marker gene even if only one copy is inserted onto the chromosome. Note that differences in promoter activity (transcription amount of genes in coding regions) can be investigated by comparing the activities of reporter proteins expressed under each promoter using a reporter protein such as luciferase (Eukaryot Cell ., 7(1), 28-37, 2008).

When a selectable marker gene with a promoter modified to reduce the expression level of such a coding region is used for transformation, a transformed strain in which only one copy of the selectable marker gene has been inserted onto the chromosome will not function as a selectable marker. Although expression is insufficient and growth is not possible, transformed strains in which multiple copies of the selectable marker have been inserted onto the chromosome are able to grow because the reduced expression level of the selectable marker is compensated for by the copy number. Therefore, by using a DNA construct in which a selectable marker gene with a modified promoter and an expression cassette of an arbitrary target gene are linked for transformation, it is possible to easily obtain a strain in which multiple copies of the target gene have been inserted onto the chromosome. It becomes possible to do so.

The method for reducing the function of the promoter of the selectable marker gene is not particularly limited, and various known methods may be used. For example, specific methods include deleting part or all of the promoter sequence of the selectable marker gene, which allows the functional expression of the coding region of the selectable marker gene with only one copy, or deleting the promoter sequence of the selectable marker gene. Examples include replacing or inserting an arbitrary base sequence into part or all of the promoter sequence. Alternatively, a method may be used in which the promoter of the selectable marker gene is completely deleted and replaced with a promoter with weak transcriptional activity or a latent promoter. Various known methods can be used to reduce the transcriptional activity of any promoter. For example, reporter proteins such as GFP, luciferase, and β-galactosidase are expressed under various promoters in which part of the promoter sequence whose transcriptional activity is desired to be deleted/replaced/inserted, and the light intensity and activity of the reporter proteins are compared. Therefore, there is a method of reducing the transcriptional activity of the promoter by identifying the region necessary for transcriptional activity in the promoter sequence, and deleting that region, or substituting/inserting an arbitrary base sequence into that region ( Mol Cell Biol., 7(7), 2352-9, 1987, Appl Environ Microbiol., 72(8), 5266-73, 2006, Yeast., 7(7), 679-89, 1991, Curr Genet., 43(2), 96-102, 2003, J Biol Chem., 272(28), 17802-9, 1997). Alternatively, as a method for finding a promoter with weak transcriptional activity, there is a method of searching for a gene with a low transcription amount using a microarray or the like, and using the upstream sequence of the gene as a promoter with weak transcriptional activity. Alternatively, there is also a method of using a promoter with weak transcriptional activity in the selection medium of the transformed strain using a conditional expression promoter or the like. In addition, although the upstream region adjacent to the coding region is generally used as a promoter, for example, even a latent promoter located far from the coding region may have transcriptional activity. Other promoters can also be used in the present invention.

For example, as an example for deleting a part of the promoter, the length of the promoter of any selectable marker is set to 250 bp, preferably 200 bp, more preferably 150 bp, more preferably 100 bp, more preferably 80 bp, and more preferably 60 bp. There are ways to make it disappear as follows.

For example, a method for reducing the function of the promoter of the pyrG marker gene is to shorten the promoter by deleting a sequence of any length at the 5' end of the promoter of the pyrG marker gene, which has sufficient functionality with one copy. can be mentioned. Preferably, the length of the promoter of the pyrG marker gene is shortened to 229 bp or less upstream of the coding region. More preferably, the promoter length of the pyrG marker gene is shortened to 129 bp or less upstream of the coding region. Most preferably, the length of the promoter of the pyrG marker gene is shortened to 56 bp or less upstream of the coding region. Alternatively, the promoter region of the pyrG marker may be deleted entirely, and then replaced with a weak promoter derived from another gene (for example, a truncated promoter derived from the adeA marker gene, a cryptic promoter derived from the alp terminator, etc.). Also good.

For example, a method for reducing the function of the promoter of the adeA marker gene is to shorten the promoter by deleting a sequence of any length at the 5' end from the promoter of the adeA marker gene, which has sufficient functionality with one copy. can be mentioned. Preferably, the length of the promoter of the adeA marker gene is shortened to 126 bp or less upstream of the coding region. More preferably, the promoter length of the adeA marker gene is shortened to 120 bp or less upstream of the coding region. Most preferably, the length of the promoter of the adeA marker gene is shortened to 80 bp or less upstream of the coding region. Alternatively, the entire promoter region of the adeA marker may be deleted, and then replaced with a weak promoter derived from another gene (for example, a truncated promoter derived from the pyrG marker gene, a latent promoter derived from the alp terminator, etc.). Also good.

The selectable marker genes that can be used in the present invention include all selectable marker genes that can be used in filamentous fungi. For example, the pyrG gene (orotidine-5'-phosphate decarboxylase gene) involved in the biosynthesis of uracil, the pyrF gene (orotate phosphoribosyltransferase gene), and the adeA gene N-succinyl-5- involved in the biosynthesis of adenine. aminoimidazole-4-carboxyamide ribotide synthase gene), adeB gene (phosphoribosylaminoimidazole carboxylase gene), argB gene (ornithine carbamoyltransferase gene) involved in arginine biosynthesis, niaD gene involved in nitrate assimilation (nitrate reductase gene), sC gene (ATP sulfurylase gene) involved in sulfate utilization, amdS gene (acetamidase gene) involved in acetamide utilization, trpC gene (glutamine amidotransferase gene) involved in tryptophan biosynthesis. /indoleglycerophosphate synthase gene/phosphoribosylanthranilate isomerase gene), bioDA gene involved in biotin biosynthesis (7,8-diaminopelargonate synthase gene/dethiobiotin synthase gene), involved in leucine biosynthesis Marker genes include the leu2 gene (β-isopropylmalate dehydrogenase gene), the pyrithiamine resistance marker ptrA gene (thiazole synthase gene), and the benomyl resistance marker benA gene (β-tubulin gene).

Furthermore, the origin of the selectable marker gene does not necessarily have to be the same as the host used. Even if a selectable marker gene has a different origin, it can be used in the present invention if it can be expressed and function in its host.

(Constitution of genes used for transformation)
The gene used for transformation in the present invention is constructed by linking a selectable marker gene with a modified promoter, a target gene to be highly expressed, a promoter for expressing the target gene, and a terminator. The promoter for expressing the target gene used in the present invention is not particularly limited, but is preferably a high-expression promoter. Examples of promoters include the promoter region of the TEF1 gene (tef1), which is a translation elongation factor, α- Examples include the promoter region of the amylase gene (amy), the promoter region of the alkaline protease gene (alp), and the promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd). Furthermore, the terminator for expressing the target gene used in the present invention is not particularly limited as long as it can function in the host, but for example, the terminator region of the α-amylase gene (amy), the terminator of the alkaline protease gene (alp), etc. Examples include areas. Note that the expression cassette for expressing the target gene may include genes such as a secretory signal sequence, a translation enhancer sequence, and an organelle localization sequence in addition to the coding region, promoter, and terminator of the target gene.

It is also possible to use a gene in which a selectable marker gene with a modified promoter is linked to an expression cassette for a target gene for transformation, but in that case, the expression cassette for the target gene and then the selectable marker gene should be linked in that order. It is desirable to do so. When a promoter-modified selectable marker gene and a target gene expression cassette are linked in this order, in a strain in which the linked gene is inserted into the promoter region of some gene on the chromosome, the number of copies of the inserted linked gene is Even with one copy, the selectable marker gene may be expressed in a sufficient amount, making it difficult to select a multi-copy strain. Furthermore, even when the target gene expression cassette and the selection marker gene are linked in this order, it is important that the terminator gene for target gene expression does not contain a sequence that can function as a promoter with strong transcriptional activity.

Furthermore, when used for transformation, the selectable marker gene whose promoter has been modified and the expression cassette of the target gene do not necessarily have to be linked. Even if the selectable marker gene with a modified promoter and the expression cassette of the target gene are not linked, it is also possible to design the genes so that they are linked on the chromosome of the host cell after transformation treatment. For example, when a DNA fragment in which a promoter-modified selection marker gene and a homologous recombination sequence are linked together, and a DNA fragment in which a homologous recombination sequence and a target gene expression cassette are used for transformation, the host A selectable marker gene whose promoter has been modified and an expression cassette of a target gene can be linked via a homologous recombination sequence on the chromosome of a cell.

The DNA used for transformation in the present invention may be linear or circular. For example, a DNA fragment in which a selectable marker gene with a modified promoter is linked to an expression cassette of a target gene may be inserted into an arbitrary region of pUC19, etc., and used as the transformation DNA. Alternatively, DNA to which an expression cassette of the target gene has been linked may be circularized by self-ligation.

Furthermore, the DNA used for transformation may contain any DNA sequence other than a selectable marker gene with a modified promoter, a target gene expression cassette, or vector DNA. For example, it may contain a region having a sequence homologous to a DNA sequence present on the chromosome of the host (homologous region). When DNA is inserted into a chromosome, the DNA is inserted by homologous recombination in yeast, so the DNA used for transformation must contain a homologous region. On the other hand, in filamentous fungi, DNA can be inserted by both homologous recombination and non-homologous recombination mechanisms, so the DNA used for transformation does not necessarily need to contain a homologous region. In other words, even if the DNA used for transformation does not contain a homologous region, genes can be randomly inserted onto the chromosome by non-homologous recombination in filamentous fungi, but if the DNA used for transformation does not contain a homologous region, genes can be randomly inserted onto the chromosome by non-homologous recombination. It is assumed that homologous recombination also occurs at the same time. Furthermore, it is more preferable when multiple copies of the homologous region exist on the chromosome, since it is assumed that homologous recombination will occur at each predetermined position.

(host cell)
Host cells that can be used for transformation in the present invention are not particularly limited as long as they are filamentous fungi, but examples include Aspergillus, Neurospora, Penicillium, Fusarium, and Trichoderma. Examples include filamentous fungi belonging to the genus Mucor, genus Rhizopus, and the like.

Specific examples of Aspergillus filamentous fungi include Aspergillus sojae, Aspergillus oryzae, Aspergillus niger, Aspergillus nidorans, Aspergillus fumigatus, Aspergillus flavus, Aspergillus tamari, Aspergillus luchiuensis, Aspergillus usami, Aspergillus spp. Kawachi, Aspergillus saitoi, etc.

Among them, considering safety and ease of culturing, Aspergillus spp. It is preferable that

Furthermore, the host filamentous fungus used for transformation in the present invention needs to be a strain deficient in the function of the protein encoded by the selectable marker gene used. Among these, strains lacking a portion or the entire coding region of the selection marker gene to be used are more preferred.

(Transformant, transformation method)
The transformant of the present invention can be applied to any filamentous fungus that is deficient in the function of the protein encoded by the selectable marker gene to be used. It can be obtained by transformation using the constructed gene.

Methods for transforming filamentous fungi include a method in which genes are introduced using polyethylene glycol and calcium chloride after forming into protoplasts (PEG-protoplast method, Mol. Gen. Genet., 218, 99-104 (1989)); A method is known in which a gene is introduced into conidia immediately after germination by electroporation (Biosci Biotechnol Biochem. 1994 Dec; 58 (12): 2224-7). Desired transformants can be selected by using a selection marker gene, for example, by using auxotrophy, drug resistance, etc. related to the marker gene. For example, when selecting based on auxotrophy, desired transformants can be selected in a medium that does not contain a compound that complements the auxotrophy. Furthermore, when selection is performed based on drug resistance, for example, desired transformants can be selected in a medium containing the drug at an appropriate concentration.

An example of the transformant of the present invention is a filamentous fungal strain lacking the function of the protein encoded by the pyrG gene, in which the pyrG gene whose promoter has been modified and whose expression level has been reduced is linked with an expression cassette of a target gene, etc. Examples include transformants that can be grown on a medium that does not contain uracil/uridine by performing transformation treatment using genes or genes designed to link these genes within cells. Among these, transformants in which multiple copies of the target gene have been inserted are preferred, transformants in which 5 or more copies of the target gene have been inserted are more preferred, and transformants in which 10 or more copies of the target gene have been inserted are even more preferred. , transformants in which 15 or more copies of the target gene have been inserted are more preferred, transformants in which 20 or more copies of the target gene have been inserted are more preferred, and transformants in which 30 or more copies of the target gene have been inserted are more preferred. Preferably, transformants have 40 or more copies of the target gene inserted, more preferably transformants have 50 or more copies of the target gene inserted, and transformants have 60 or more copies of the target gene inserted. Most preferred.

Another example of the transformant of the present invention is a filamentous fungal strain lacking the function of the protein encoded by the adeA gene. Examples include transformants that can grow on adenine-free medium by performing transformation treatment using a gene in which these are linked together, or a gene designed to link them within cells. Among these, transformants in which multiple copies of the target gene have been inserted are preferred, transformants in which 5 or more copies of the target gene have been inserted are more preferred, and transformants in which 10 or more copies of the target gene have been inserted are even more preferred. A transformant in which 15 or more copies of the target gene have been inserted is more preferred, and a transformant in which 20 or more copies of the target gene have been inserted is more preferred.

The copy number can be calculated by comparing the production amount or activity of the target protein in a strain in which one copy of the target gene has been inserted and the obtained transformed strain, or by calculating the amount or activity of the target protein in a strain in which one copy of the target gene has been inserted. There is a method of calculation based on the density of a Southern blot band of the target gene in the obtained transformed strain, a quantitative PCR method using chromosomal DNA of the transformed strain, etc.

(Method for producing industrially useful proteins and secondary metabolites)
In order to produce the target protein or secondary metabolite, a transformant obtained using the present invention into which multiple copies of the gene necessary for producing the target substance have been inserted is cultured, and the transformant obtained using the present invention is extracted from the culture. All you have to do is extract the target substance.

The medium for culturing the above-mentioned transformants may be a conventional medium for culturing filamentous fungi, such as a synthetic medium or a natural medium, as long as it contains carbon sources, nitrogen sources, inorganic substances, and other nutrients in appropriate proportions. Either a liquid medium or a solid medium can be used. As an example, the GPY medium used in the Examples described later can be used, but is not particularly limited. Depending on the target protein or secondary metabolite, a specific substance necessary for producing the substance may be added to the medium.

The culture conditions may be those for filamentous fungi commonly known to those skilled in the art. For example, the initial pH of the medium is adjusted to 5 to 10, the culture temperature is 20 to 40°C, and the culture time is several hours to several hours. The period of time can be set as appropriate, preferably for 1 to 7 days, more preferably for 2 to 5 days. The culturing method is not particularly limited, and aeration-stirring deep culture, shaking culture, static culture, etc. can be employed, but it is generally preferable to culture under conditions that provide sufficient dissolved oxygen. However, depending on the target protein or secondary metabolite, it may be possible that less dissolved oxygen results in higher production. An example of the culture conditions includes, but is not particularly limited to, shaking culture at 30° C. for 3 days using the GPY medium described in the Examples below.

The method for extracting the target protein or secondary metabolite from the culture after completion of the culture is not particularly limited, and any conventional known extraction means may be used.

For example, when extracting a target protein, if the target protein is secreted into the medium, the cultured bacterial cells are removed by filtration or centrifugation, only the medium supernatant is collected, and then the cultured cells are removed by ultrafiltration, etc. Crude enzyme can be obtained by removing and concentrating the culture medium components. When the target protein is produced within the bacterial cells, the bacterial cells can be destroyed using destructive means such as an ultrasonic crusher, French press, Dynomill, or mortar, or the bacterial cells can be destroyed using a cell wall lytic enzyme such as Yatalase. It can be extracted by disrupting the bacterial cells, such as by dissolving the cell wall or by lysing the bacterial cells using a surfactant such as SDS or Triton X-100. The obtained crude enzyme can also be further purified using any known means. To obtain a purified enzyme preparation, for example, gel filtration using Sephadex, Ultrogel or biogel, adsorption/elution using an ion exchanger, electrophoresis using polyacrylamide gel, adsorption using hydroxyapatite, etc. Elution method; purified by appropriately selecting a sedimentation method such as sucrose density gradient centrifugation, an affinity chromatography method, a fractionation method using a molecular sieve membrane or a hollow fiber membrane, or a combination of these methods. Enzyme preparations can be obtained.

For example, when extracting secondary metabolites from a culture, bacterial cells collected from the culture through operations such as filtration or centrifugation may be used as they are, or bacterial cells that have been collected and dried or further crushed. You can also use your body. The method of drying the bacterial cells is not particularly limited, and examples thereof include freeze drying, solar drying, hot air drying, vacuum drying, ventilation drying, reduced pressure drying, and the like. The extraction solvent is not particularly limited as long as the secondary metabolites are dissolved therein, and examples thereof include organic solvents such as methanol, ethanol, isopropanol, and acetone, water-containing organic solvents obtained by mixing these organic solvents with water, and water. , hot water and hot water. After adding the solvent, desired secondary metabolites are extracted while subjecting to bacterial cell crushing treatment, heat treatment, etc., as appropriate. The obtained extract is subjected to centrifugation, filter filtration, ultrafiltration, gel filtration, separation based on solubility difference, solvent extraction, chromatography (adsorption chromatography, hydrophobic chromatography, cation exchange chromatography, anion exchange chromatography) , reverse phase chromatography, etc.), crystallization, activated carbon treatment, membrane treatment, and other purification treatments.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples. However, the technical scope of the present invention is not limited in any way by these examples.

(Preparation of DNA construct for transformation containing a modified pyrG marker gene with a shortened promoter)
(1) Obtaining modified pyrG1-3 genes with shortened promoters SEQ ID NO: 1 is the sequence of the pyrG marker gene conventionally used in Aspergillus sojae (see WO2014/126186). The pyrG marker gene of SEQ ID NO: 1 is composed of a promoter region of 407 bp, a coding region of 896 bp, and a 535 bp region including a terminator. By inserting one copy of this full-length 1838 bp sequence onto the chromosome, the uridine/uracil auxotrophy is achieved. It is known that they can complement the traits of

For the purpose of creating a modified pyrG marker gene that cannot sufficiently complement the uridine/uracil auxotrophy unless multiple copies are inserted onto the chromosome, we created a modified pyrG marker gene (SEQ ID NO: 1) in which the promoter region of the pyrG marker gene is shortened ( Modified pyrG1-3, SEQ ID NOS: 2-4) were designed as follows. Using the pyrG marker gene of SEQ ID NO: 1 as a template, the modified pyrG1 gene (SEQ ID NO: 2, promoter region 229 bp) was prepared using the primers of SEQ ID NOs: 5 and 6, and the modified pyrG2 gene (SEQ ID NO: 3, promoter region 129 bp) was The modified pyrG3 gene (SEQ ID NO: 4, promoter region 56 bp) was isolated using the primers SEQ ID NO: 7 and 6, and the A. It was obtained by PCR reaction using PrimeSTAR Max DNA polymerase (manufactured by Takara Bio Inc.) using chromosomal DNA of S. sojae NBRC4239 strain as a template. The primers with SEQ ID NOs: 5 to 10 contain a 15 bp homologous sequence required for the subsequent Infusion reaction at their 5' ends.

(2) Preparation of a transformation vector containing a reporter gene expression cassette and each modified pyrG gene marker The glucose dehydrogenase (GDH) gene (patented 4648993) was used. The activity of GDH expressed in filamentous fungi can be easily measured by the activity measuring method described below.
Similarly to WO2014/126186, A. Using chromosomal DNA of S. sojae NBRC4239 strain as a template, the tef1 promoter (Ptef) region for expressing the reporter gene (GDH gene) and the terminator (Talp) region of the alkaline protease gene alp were amplified by PCR reaction. In addition, the GDH gene, which is a reporter gene, was amplified by PCR reaction using pYES2C-Mp plasmid DNA of Patent Document WO2012/169512 as a template and primers of SEQ ID NOs: 9 and 10. Next, using the In-Fusion HD Cloning Kit (Clontech), the linear pUC19, Ptef, GDH genes, Talp, and pyrG genes (SEQ ID NO: 1) and pyrG1 genes (SEQ ID NO: 2) attached to the kit were cloned. ), pyrG2 gene (SEQ ID NO: 3), and pyrG3 gene (SEQ ID NO: 4) DNA fragments were respectively ligated. As a result, transformation plasmid p19 in which Ptef-GDH-Talp-pyrG, Ptef-GDH-Talp-pyrG1, Ptef-GDH-Talp-pyrG2, and Ptef-GDH-Talp-pyrG3 were inserted into the multi-cloning site of pUC19, respectively. -GDH-pyrG, p19-GDH-pyrG1, p19-GDH-pyrG2, and p19-GDH-pyrG3 were obtained.

(Verification of multi-copy insertion effect of modified pyrG in A. sojae)
A. A. sojae pyrG gene-disrupted strain (a strain lacking 48 bp upstream of the pyrG gene, 896 bp of the coding region, and 240 bp downstream of the pyrG gene) was transformed by the protoplast PEG method. sojae transformed strains, 48 As-pyrG strains, 23 As-pyrG1 strains, 26 As-pyrG2 strains, and 18 As-pyrG3 strains were obtained.

The obtained transformed strains As-pyrG strain, As-pyrG1 strain, As-pyrG2 strain, and As-pyrG3 strain were cultured in GPY medium (2% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% phosphoric acid 2 1 potassium hydrogen, 0.05% magnesium sulfate heptahydrate) and cultured at 30°C for 3 days. After the cultured cells were collected on a filter paper, water was removed by suction filtration, and the cells weighing 50 mg in wet weight were placed in a tube. The cells are frozen with liquid nitrogen and disrupted using a multi-bead shocker. 1 mL of 100 mM potassium phosphate buffer (pH 7.0) is added to the crushed cells, and the supernatant after centrifugation is collected to disrupt the cells. A liquid was prepared. The GDH activity of this cell suspension was measured according to the measurement method described below, and the GDH activity per 50 mg of cells was calculated.

In addition, in the same manner as in Japanese Patent No. 4648993, A. A pyrG gene-disrupted strain of S. sojae was transformed to obtain a strain in which one copy of the GDH gene was inserted into the alp gene region on the chromosome. Regarding this 1 copy strain, the GDH activity per 50 mg of wet bacterial weight was calculated in the same manner as above, and based on this value, the copy number of the transformed strain obtained this time was calculated.

<GDH activity measurement method>
Mix 2.05 mL of 100 mM phosphate buffer (pH 7.0), 0.6 mL of 1M D-glucose solution, and 0.15 mL of 2 mM DCIP solution, and incubate at 37° C. for 5 minutes. Next, 0.1 mL of 15 mM PMS solution and 0.1 mL of enzyme sample solution are added to start the reaction. The absorbance at the start of the reaction and over time is measured, the amount of decrease in absorbance at 600 nm per minute (ΔA600) as the enzymatic reaction progresses is determined, and the GDH activity is calculated according to the following formula. At this time, GDH activity is defined as 1 U, which is the amount of enzyme that reduces 1 μmol of DCIP per minute in the presence of D-glucose at a concentration of 200 mM at 37°C.

In addition, 3.0 in the formula is the liquid volume (mL) of the reaction reagent + enzyme reagent, 16.3 is the millimolar molecular extinction coefficient (cm ² /μmol) under this activity measurement condition, and 0.1 is the liquid volume of the enzyme solution. (mL), 1.0 is the optical path length of the cell (cm), and ΔA600 _blank is the absorbance per minute at 600 nm when the reaction is started by adding the buffer used to dilute the enzyme instead of the enzyme sample solution. The amount of decrease, df, represents the dilution factor.

Table 1 shows the number of copies of the inserted GDH gene and the number of strains obtained in the As-pyrG strain, As-pyrG1 strain, As-pyrG2 strain, and As-pyrG3 strain.

From Table 1, the percentage of strains with GDH activity of 10 copies or more is 33% for the As-pyrG3 strain, 19% for the As-pyrG2 strain, 17% for the As-pyrG1 strain, and 2% for the As-pyrG strain. You can see that it's getting higher. This indicates that the GDH gene can be efficiently inserted in multiple copies by shortening the promoter of the pyrG gene. Furthermore, among the 18 As-pyrG3 strains analyzed in Table 1, some strains had 30 or more copies of the GDH gene inserted. Furthermore, when more transformants were obtained using the pyrG3 gene, it was found that strains having GDH activity equivalent to 60 copies or more could also be obtained. In addition, when we investigated the copy number of the GDH gene existing on the chromosome by quantitative PCR for the obtained strain equivalent to 60 copies and strain equivalent to 30 copies, it was found that approximately 60 copies and approximately 30 copies of the GDH gene were actually present on the chromosome, respectively. It was found that it was inserted into.

From the above, it was found that the method of shortening the promoter of a selectable marker and reducing its transcriptional activity is a very effective method for inserting multiple copies of any gene. Furthermore, the multicopy body maintains GDH activity even after being subcultured multiple times in a minimal medium, shows the same value with good reproducibility even when cultured in a rich nutrient medium used in actual production, and has high activity. Therefore, it was found that the strain obtained by this method has excellent stability and is an industrially practical strain.

(Verification of multi-copy insertion effect of modified pyrG in A. oryzae)
As mentioned above, it is possible to efficiently insert multiple copies of a gene by using a selectable marker with a shortened promoter. confirmed in sojae. Therefore, in order to verify whether this method could be applied to other filamentous fungi, A. A pyrG gene-disrupted strain of S. oryzae RIB40 strain was transformed using the above p19-GDH-pyrG and p19-GDH-pyrG3. After culturing the obtained transformants Ao-pyrG strain and Ao-pyrG3 strain in GPY medium at 30°C for 3 days, the pelleted bacterial bodies in the culture solution were sheared with a Hiscotron, and the cells were further cultured with a multibead shocker. The body was crushed. This cell suspension was centrifuged and the supernatant was collected to obtain crude enzyme solutions of Ao-pyrG strain and Ao-pyrG3 strain. Then, by measuring the GDH activity of these crude enzyme solutions, the GDH production amount (U/mL culture) in the Ao-pyrG strain and Ao-pyrG3 strain was calculated (Table 2).

From Table 2, the Ao-pyrG3 strain obtained using the pyrG marker gene pyrG3 with a shortened promoter has higher GDH activity than the Ao-pyrG strain obtained using the pyrG marker gene pyrG without a shortened promoter. It turns out that the percentage of companies that can acquire stocks is increasing.

From this, a method for efficiently inserting multiple copies of a gene using a selectable marker with a shortened promoter and reduced transcription level is proposed by A. It was shown that the method can be applied to other filamentous fungi besides S. sojae.

(Verification of multi-copy insertion effect using modified adeA marker with shortened promoter)
(1) Obtaining modified adeA1-3 genes with shortened promoters As mentioned above, it is possible to efficiently insert genes in multiple copies by shortening the promoter and using the pyrG marker with a reduced transcription amount. sojae and A. oryzae. Therefore, we verified whether similar effects could be obtained with selection markers other than pyrG. Specifically, similar to the pyrG marker, the adeA marker (FEMS Microbiol Lett., 239(1), 79-85, 2004), which is often used as a selection marker for filamentous fungi, was used to shorten the promoter and perform selection. We decided to verify the effect of multiple copy insertion of the target gene by reducing the expression level of the marker gene. Sequence number 11 is A. This is the sequence of the adeA marker gene derived from S. sojae NBRC4239, and is composed of 241 bp including a promoter region of 642 bp, a coding region of 1128 bp, and a terminator. By inserting one copy of this full-length 2011 bp adeA marker gene sequence onto the chromosome, A. It has been found that this gene can complement the traits of the adeA gene-disrupted strain (258 bp upstream of the adeA gene, 1128 bp coding region, and 83 bp downstream of the adeA gene) of S. sojae NBRC4239.

For the purpose of creating a modified adeA marker gene that cannot sufficiently complement the adenine auxotrophy unless multiple copies are inserted onto the chromosome, we created a modified adeA marker gene (modified version) in which the promoter region of the adeA marker gene of SEQ ID NO: 11 is shortened. adeA1-3, SEQ ID NOS: 12-14) were designed as follows. A. sojae NBRC4239 strain as a template, the modified adeA1 gene (SEQ ID NO: 12, promoter region 126 bp) was prepared using the primers SEQ ID NOs: 15 and 16, and the modified adeA2 gene (SEQ ID NO: 13, promoter region 120 bp) was prepared using the sequence The modified adeA3 gene (SEQ ID NO: 14, promoter region 80 bp) was obtained by PCR reaction using PrimeSTAR Max DNA polymerase (manufactured by Takara Bio Inc.) using primers SEQ ID NO: 18 and 16 using primers Nos. 17 and 16. did. Next, based on the amino acid sequence of Heike firefly-derived luciferase described in JP-A-2-171189, in which a heat-resistant mutation of A217L was introduced, a luciferase gene codon-modified for Aspergillus expression was transferred to each promoter derived from adeA and adeA1 to 3. Below A. The promoter activities of adeA and adeA1-3 were compared by expressing them in the A. sojae NBRC4239 strain and measuring the luminescence intensity caused by luciferase. We confirmed that only one copy of the expression cassette of each luciferase gene was inserted into the chromosome, and measured the luciferase activity according to the method described in JP-A-2007-325546. ) was found to have a promoter activity of about 1/90 and about 1/5000, respectively, compared to the adeA promoter (642 bp). Furthermore, no luminescence due to luciferase could be detected under the promoter region (80 bp) of adeA3. Note that when shortened adeA promoters with promoter regions of 128 bp and 127 bp were used, approximately 40% and 20% of the luciferase activity of the adeA promoter (642 bp) was maintained. From this, we decided to conduct a multi-copy insertion study using the adeA1 promoter (126 bp), adeA2 promoter (120 bp), and adeA3 promoter (80 bp).

(2) Preparation of a transformation vector containing a reporter gene expression cassette and each modified adeA marker As a reporter gene for verifying how much gene has been inserted onto the chromosome, the glucose dehydrogenase (GDH) gene is used as described above. was used. Using In-Fusion HD Cloning Kit (Clontech), pUC19, gene, Ptef, gene, GDH gene, Talp gene, adeA1 gene (SEQ ID NO: 12), adeA2 gene (SEQ ID NO: 13), The DNA fragments of the adeA3 gene (SEQ ID NO: 14) were ligated. As a result, transformation plasmids p19-GDH-adeA1 and p19-GDH in which Ptef-GDH-Talp-adeA1, Ptef-GDH-Talp-adeA2, and Ptef-GDH-Talp-adeA3 were inserted into the multi-cloning site of pUC19, respectively, were created. -adeA2 and p19-GDH-adeA3 were obtained.

(3) Analysis of transformed strains into which the GDH expression cassette and each modified adeA marker were inserted ．． sojae NBRC4239 adeA gene-disrupted strain (258 bp upstream of the adeA gene, 1128 bp coding region, and 83 bp downstream deletion strain) was transformed by the protoplast PEG method, resulting in 19 As-adeA1 strains, 12 As-adeA2 strains, Seven As-adeA3 stocks were acquired.

The obtained transformed strains As-adeA1 strain, As-adeA2 strain, and As-adeA3 strain were cultured in GPY medium (2% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% monopotassium dihydrogen phosphate, 0 05% magnesium sulfate heptahydrate) and cultured at 30°C for 3 days. After the cultured cells were collected on a filter paper, the moisture was thoroughly removed by suction filtration, and the cells weighing 50 mg in wet weight were placed in a tube. The cells were frozen with liquid nitrogen and crushed using a multi-bead shocker, 1 mL of 100mM potassium phosphate buffer (pH 7.0) was added to the crushed cells, and the supernatant after centrifugation was collected. A crushing solution was prepared. The GDH activity of this cell suspension was measured according to the measurement method described below, and the GDH activity per 50 mg of cells was calculated. Thereafter, the copy number of the obtained transformed strain was calculated based on the GDH activity value of the 1-copy strain in the same manner as above.

Table 3 shows the number of copies of the inserted GDH gene and the number of strains obtained in the As-adeA1 strain, As-adeA2 strain, and As-adeA3 strain.

From Table 3, the percentage of strains with GDH activity of 5 copies or more is higher in the following order: 43% for As-adeA3 strain, 25% for As-adeA2 strain, and 21% for As-adeA1 strain. It was found that the GDH gene could be efficiently inserted in multiple copies by shortening the promoter of the adeA gene and reducing its promoter activity. Furthermore, among the transformed strains analyzed in Table 3, the strains with the highest number of copies were As-adeA1 strain with 10 copies, As-adeA2 strain with 12 copies, and As-adeA3 strain with 21 copies. It was found that by shortening the promoter, strains with higher copy numbers could be obtained.

From the above, it was found that the method of reducing the expression level of a selection marker gene and inserting multiple copies of an arbitrary target gene in the present invention is effective for various selection markers.

(Verification of multi-copy insertion effect using pyrG marker gene linked to promoter derived from adeA marker)
(1) Preparation of a transformation vector containing a pyrG marker in which the reporter gene expression cassette and the promoter region derived from the adeA marker are linked As described above, both the pyrG marker and the adeA marker have their own promoter regions shortened. It was found that multiple copies of any target gene can be inserted by reducing the promoter activity and, as a result, reducing the expression level of the selectable marker protein. In order to insert multiple copies of any target gene, it is important to reduce the expression level of the selectable marker protein, and methods for this purpose include shortening the promoter region of the selectable marker gene itself. Alternatively, a method of replacing the promoter with a promoter derived from another gene known to have weak promoter activity may also be considered. Therefore, we created a modified pyrG marker gene (pyrG4, SEQ ID NO: 19) in which the entire promoter region (643 bp) of the pyrG marker gene was deleted and a promoter (80 bp) derived from As-adeA3 was linked upstream of the coding region of the pyrG marker gene. was created. Next, using In-Fusion HD Cloning Kit (Clontech), the DNA fragments of pUC19, Ptef, GDH gene, Talp, and pyrG4 gene (SEQ ID NO: 19) were respectively ligated in the same manner as above. As a result, a transformation plasmid p19-GDH-pyrG4 in which Ptef-GDH-Talp-pyrG4 was inserted into the multi-cloning site of pUC19 was obtained.

(2) Analysis of transformed strains into which the GDH expression cassette and pyrG4 marker have been inserted A. A pyrG gene-disrupted strain of S. sojae (a strain lacking 48 bp upstream of the pyrG gene, 896 bp of the coding region, and 240 bp downstream) was transformed by the protoplast PEG method, and 32 As-pyrG4 strains were obtained.

The obtained transformed strain As-pyrG4 strain was cultured in GPY medium (2% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% monopotassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate). ) and cultured at 30°C for 3 days. After the cultured cells were collected on a filter paper, the moisture was thoroughly removed by suction filtration, and the cells weighing 50 mg in wet weight were placed in a tube. The cells were frozen with liquid nitrogen and crushed using a multi-bead shocker, 1 mL of 100mM potassium phosphate buffer (pH 7.0) was added to the crushed cells, and the supernatant after centrifugation was collected. A crushing solution was prepared. The GDH activity of this cell suspension was measured according to the measurement method described below, and the GDH activity per 50 mg of cells was calculated. Thereafter, the copy number of the As-pyrG4 strain was calculated based on the GDH activity value of the 1-copy strain in the same manner as above.

Table 4 shows the number of copies of the inserted GDH gene and the number of strains obtained in the As-pyrG4 strain.

From Table 4, it was found that among the As-pyrG4 strains, the percentage of strains having GDH activity of 10 copies or more was 13%, which was higher than 2% of the As-pyrG strains. Furthermore, the percentage of strains having 5 or more copies of GDH activity was 4% for the As-pyrG strain, whereas it was 22% for the As-pyrG4 strain. From these results, it was found that the GDH gene can be efficiently inserted in multiple copies by lowering the expression level of the selectable marker gene by replacing it with a promoter with low promoter activity. Furthermore, among the As-pyrG4 strains shown in Table 4, the strain with the highest GDH activity was 18 copies.

From the above, the method of reducing the expression level of a marker gene by deleting all the promoters of the original selectable marker gene and replacing them with promoters derived from another gene with low promoter activity is to insert multiple copies of any gene. This method was found to be effective.

(Verification of multi-copy insertion effect using pyrG marker gene without promoter region)
(1) Preparation of a transformation vector containing a reporter gene expression cassette and a pyrG marker without the a promoter region From Example 4, it was possible to insert a multicopy gene even using the adeA3 marker, which has extremely low promoter activity. . From this, it was considered that a selectable marker gene without a promoter region could also be used for multi-copy insertion. Therefore, a modified pyrG marker gene (pyrG5, SEQ ID NO: 20) was created in which the promoter region (643 bp) of the pyrG marker gene was completely deleted. Next, using In-Fusion HD Cloning Kit (Clontech), the DNA fragments of pUC19, Ptef, GDH gene, Talp, and pyrG5 gene (SEQ ID NO: 20) were respectively ligated in the same manner as above. As a result, a transformation plasmid p19-GDH-pyrG5 in which Ptef-GDH-Talp-pyrG5 was inserted into the multi-cloning site of pUC19 was obtained.

(2) Analysis of transformed strains into which the GDH expression cassette and pyrG5 marker have been inserted A. A pyrG gene-disrupted strain of S. sojae (a strain lacking 48 bp upstream of the pyrG gene, 896 bp of the coding region, and 240 bp downstream of the pyrG gene) was transformed by the protoplast PEG method, and 19 As-pyrG5 strains were obtained.

The obtained transformed strain As-pyrG4 strain was cultured in GPY medium (2% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% monopotassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate). ) and cultured at 30°C for 3 days. After the cultured cells were collected on a filter paper, the moisture was thoroughly removed by suction filtration, and the cells weighing 50 mg in wet weight were placed in a tube. The cells were frozen with liquid nitrogen and crushed using a multi-bead shocker, 1 mL of 100mM potassium phosphate buffer (pH 7.0) was added to the crushed cells, and the supernatant after centrifugation was collected. A crushing solution was prepared. The GDH activity of this cell suspension was measured according to the measurement method described below, and the GDH activity per 50 mg of cells was calculated. Thereafter, the copy number of the As-pyrG5 strain was calculated based on the GDH activity value of the 1-copy strain in the same manner as above.

Table 5 shows the number of copies of the inserted GDH gene and the number of strains obtained in the As-pyrG5 strain.

From Table 5, it was found that among the 5 As-pyrG strains, the percentage of strains having GDH activity of 10 copies or more was 21%, which was higher than 2% of the As-pyrG strains. Furthermore, the percentage of strains having 5 or more copies of GDH activity was 4% for the As-pyrG strain, while it was 32% for the As-pyrG5 strain. These results indicate that the GDH gene can be efficiently inserted in multiple copies by using a selectable marker gene that does not have a promoter region. Note that among the As-pyrG5 strains shown in Table 5, the strain with the highest GDH activity was 21 copies.

From the above, it was found that it is possible to efficiently insert multiple copies of any gene using pyrG5 in which the entire promoter region has been deleted. In the DNA sequence used for transformation in this example, the upstream of pyrG5 is the alp terminator (Talp). The Talp region does not contain any ORF, and A. Although there is a coding region of another gene in the same orientation as the alp coding region downstream of Talp on the chromosome of Sojae NBRC4239, the start codon of this coding region is approximately 1.2 kb away from the 3' end of Talp. Therefore, it was predicted that the Talp region alone would be unlikely to function as an effective promoter. However, since complementation of uridine/uracil requirement was possible even using pyrG5, which does not have a promoter region, Talp functions as a potential weak promoter, and by introducing multiple copies, the level of complementation can be increased. It was suggested that the marker gene may have been expressed up to that point.

From the above, it was found that the method of reducing the expression level of the marker gene by deleting the entire original promoter region of the selectable marker gene is effective as a method for inserting multiple copies of any gene.

From the above, it was shown that the method of using a selection marker in which the expression level of the coding region is reduced by modifying the promoter is effective for efficiently inserting multiple copies of any gene in filamentous fungi. In addition, this time we showed an example using the pyrG marker gene and the adeA marker gene, but by modifying the promoter and lowering the expression level of the marker gene in the same way for other selection marker genes, it is possible to select any gene. It is possible to efficiently insert multiple copies of . This method is more versatile and more practical than the previously known methods for inserting multiple copies of genes into filamentous fungi, and is very useful industrially.

Claims (6)

A method for producing a filamentous fungal transformant into which multiple copies of a target gene are inserted, the method comprising:
an expression cassette for the target gene, and
Transforming a host filamentous fungus using a selectable marker gene operably linked to a modified promoter;
a step in which a target gene is inserted by a non-homologous recombination mechanism, and a step in which the transformant capable of growing on a selective medium is selected using the selection marker gene,
The modified promoter is modified so that the expression level of the protein encoded by the selectable marker gene is reduced compared to when an unmodified promoter is used,
The unmodified promoter is a promoter of the selectable marker gene that has transcriptional activity that enables functional expression of the selectable marker gene even if only one copy of the selectable marker gene is inserted into the chromosome,
The modified promoter is encoded by a base sequence that has a deletion of some or all bases in the base sequence of the unmodified promoter, so that the promoter function is reduced compared to the unmodified promoter. As a result, the expression level of the selectable marker gene has decreased.
The promoter of the selectable marker gene is deleted to a length of 100 bp or less,
The host filamentous fungus is a filamentous fungus that lacks the function of the protein encoded by the selection marker gene,
The method, wherein the selection marker gene is an auxotrophic marker gene. The method according to claim 1, wherein the filamentous fungi are selected from filamentous fungi belonging to the genus Aspergillus, Neurospora, Penicillium, Fusarium, Trichoderma, Mucor, and Arachnoid. A transformant obtained by the method according to claim 1 or 2 . A method for producing a target substance using the transformant according to claim 3 . 5. The method according to claim 4 , comprising the step of extracting a target substance from a culture obtained by culturing the transformant. 5. The method according to claim 4 , wherein the target substance is a protein expressed by the target gene or a metabolite thereof.