Method for transforming filamentous fungus

Abstract

Provided is a method for transforming a filamentous fungus, the method comprising the following steps: providing a host filamentous fungus including a selection marker gene in which the function of a protein encoded thereby is deficient; and transforming the host filamentous fungus using an expression cassette including a coding region of a target gene, a promoter, and a terminator, a transposon sequence, and the selection marker gene having an altered promoter.

Description

Methods for transforming filamentous fungi REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority based on Japanese Patent Application No. 2023-184594, filed on October 27, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a method for transforming filamentous fungi, and more particularly to a method for introducing multiple copies of any gene into the chromosome of a filamentous fungus.

[0003] Filamentous fungi, such as those of the genera Aspergillus, Penicillium, and Trichoderma, generally have high protein production capabilities and are widely used as hosts for producing industrially useful proteins. Filamentous fungi are also known to produce a variety of secondary metabolites, making them highly useful as hosts for producing low-molecular-weight compounds. Therefore, research into protein expression systems using recombinant DNA technology has been actively conducted in filamentous fungi. Among these, the construction of high-expression systems for proteins is considered to be particularly important for achieving high production of target proteins and compounds.

[0004] Known methods for achieving high expression of a target protein generally include using a high-expression promoter, using a plasmid capable of replicating a target gene at high copy numbers, and introducing a target gene at high copy numbers into the genome.

[0005] Many searches for high-expression promoters have been conducted in filamentous fungi as well, and many reports have been published (Patent Documents 1 to 7). High-expression promoters have also been reported that were created by improving existing promoters by adding or deleting specific base sequences (Patent Documents 8 to 11).

[0006] Furthermore, in bacteria and yeast, it is possible to achieve high expression of a target protein by using an autonomously replicating plasmid capable of replicating at a high copy number. Meanwhile, in filamentous fungi, it has been reported that a vector containing AMA1 as a replication origin can function as an autonomously replicating plasmid (Non-Patent Document 5). However, the phenomenon of this plasmid being able to autonomously replicate outside the chromosome has only been observed in some filamentous fungi, and it has also been reported that a vector containing AMA1 introduced into filamentous fungi is quickly lost when cultured in an environment without selective pressure, making it unsuitable for industrial use (Non-Patent Document 6). Therefore, when achieving high protein production in filamentous fungi, a common method is to integrate an expression cassette for a gene encoding the target protein into the chromosome.

[0007] Therefore, one possible strategy for achieving high expression of a target protein is to integrate a high number of copies of the expression cassette encoding the target protein into the chromosome. For example, it is known that using ATP sulfurylase (sC), which is involved in sulfate assimilation, as a selection marker allows multiple copies of the gene to be integrated into the chromosome (Non-Patent Document 7). However, many strains have a copy number of only a few copies at most, and selecting a strain with a higher copy number from a large number of candidate strains requires a great deal of effort. It is also known that using the acetamidase gene (amdS) as a selection marker allows for the insertion of several dozen copies of the gene (Non-Patent Document 2). However, this system is known to be difficult to use with strains that naturally contain the amdS gene, such as the sake koji mold Aspergillus oryzae (Non-Patent Document 3), limiting its applicability. Furthermore, while amdS is essential when amide compounds such as acetamide and acrylamide are used as the sole carbon source, amdS is not required in rich media containing glucose, dextrin, or other carbon sources commonly used in production, and there is also concern that the inserted gene may be easily deleted during culture. Additionally, Non-Patent Document 4 reports that approximately 60 copies of a gene were inserted into an arginine-auxotrophic strain of Aspergillus oryzae by using the ornithine carbamoyltransferase gene (argB) derived from Aspergillus nidulans as a selection marker. Furthermore, Patent Document 12 reports that 64 to 128 copies of a gene were inserted into a leucine-auxotrophic strain of Aspergillus oryzae by using the leu2B gene derived from Aspergillus oryzae as a selection marker. However, these methods are inapplicable to selection marker genes other than argB and leu2B, and therefore are limited in the number of hosts that can be used, resulting in a lack of versatility. In addition, Patent Document 13 reports a highly versatile method for inserting multiple copies of any gene into the chromosome of a filamentous fungus by using a selection marker gene with a modified promoter region and reduced expression level.However, there is a continuing need for more efficient methods of breeding high-producing strains of any protein, and for methods of achieving higher yields of any of the above proteins.

[0008] Japanese Patent No. 3372087 Japanese Patent No. 3759794 Japanese Patent No. 3792467 Japanese Patent No. 4676639 Japanese Patent No. 3743803 International Publication No. 2007 / 071399 Japanese Patent No. 5507062 Japanese Patent No. 3343567 Japanese Patent No. 4495904 Japanese Patent No. 4490284 Japanese Patent Publication No. 2009-254349 Japanese Patent No. 5473179 Japanese Patent Publication No. 2018-68292

[0009] Gems, D., Johnstone, IL, and Clutterbuck, AJ (1991) An autonomously replicating plasmid transforms Aspergillus nidulans at high frequency. Gene, 98(1): 61-67. van den Hondel, CAMJJ, Punt, PJ, and van Gorcom, RFM (1991) 18 - Heterologous Gene Expression in Filamentous Fungi. In Bennett, JW and Lasure, LL (Eds.) More Gene Manipulations in Fungi, Academic Press, pp.396-428. Kitamoto, K. (ed.) (2012) Revised Edition of Molecular Koji Mycelia. Brewing Society of Japan, 269pp. Gomi, K., Iimura, Y., Hara, S. (1987) Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene. Agricultural and Biological Chemistry, 51(9): 2549-2555. Kusumoto, K., Kimura, T., Suzuki, S., Kashiwagi, Y. (2004) Analysis of the intracellular morphology of a plasmid carrying a telomere sequence from Aspergillus oryzae. Food Research Institute Research Report, Vol. 68, pp. 43-47. Katayama, T., Nakamura, H., Zhang, Y., Pascal, A., Fujii, W., Maruyama, J.(2019) Forced recycling of an ama1-based genome-editing plasmid allows for efficient multiple gene deletion / integration in the industrial filamentous fungus Aspergillus oryzae. Applied and Environmental Microbiology, 85(3): e01896-18. Wada, Junpei, and Hirotsune, Masato (2011) Enhanced production of lipase using a koji mold protein expression system. Journal of the Brewing Society of Japan, Vol. 106, No. 6, pp. 336-341.

[0010] The present invention provides a method for highly efficiently integrating multiple copies of any gene into a chromosome in a filamentous fungus.

[0011] As a result of extensive research aimed at solving the above problems, the present inventors have found that by using a selectable marker gene whose expression level has been reduced by modifying the promoter region and by using a transposon sequence, it is possible to insert any gene into the chromosome of a filamentous fungus with high efficiency and at high copy numbers. The present invention is based on this finding.

[0012] According to one embodiment of the present invention, there is provided a method for transforming a filamentous fungus, comprising the steps of: providing a host filamentous fungus that is deficient in a selection marker gene or that contains a selection marker gene in which the function of the protein it encodes is defective; and transforming the host filamentous fungus with an expression cassette containing a coding region of a target gene, a promoter and a terminator, a transposon sequence, and the selection marker gene with a modified promoter.

[0013] In the above embodiment, it is preferable to transform a host filamentous fungus with a DNA construct in which the coding region of a target gene, an expression cassette containing a promoter and a terminator, a transposon sequence, and a selectable marker gene with a modified promoter are operably linked.

[0014] Furthermore, according to another embodiment of the present invention, there is provided a method for producing a target protein, which comprises culturing a filamentous fungal transformant obtained by the above-mentioned method and producing the target protein encoded by the above-mentioned target gene.

[0015] According to another embodiment of the present invention, there is provided a DNA construct for transforming a host filamentous fungus containing a selectable marker gene that is deleted or whose encoded protein is functionally defective, wherein the DNA construct comprises a coding region of a target gene, an expression cassette containing a promoter and a terminator, a transposon sequence, and the selectable marker gene with a modified promoter, operably linked thereto.

[0016] Furthermore, according to another embodiment of the present invention, there is provided a kit for transforming a host filamentous fungus that lacks a selectable marker gene or that contains a selectable marker gene in which the function of the protein it encodes is defective, the kit comprising a DNA construct to which the coding region of a target gene, an expression cassette containing a promoter and a terminator, a transposon sequence, and the selectable marker gene with a modified promoter are operably linked.

[0017] Furthermore, according to another embodiment of the present invention, there is provided a filamentous fungal transformant in which a DNA construct has been integrated into the chromosome of a host filamentous fungus that contains a selectable marker gene that is deleted or whose encoded protein is defective in function, wherein the DNA construct operably links the coding region of a target gene, an expression cassette containing a promoter and a terminator, a transposon sequence, and the selectable marker gene with a modified promoter.

[0018] According to another embodiment of the present invention, there is provided use of a DNA construct in producing a transformant of a host filamentous fungus that lacks a selection marker gene or that contains a selection marker gene in which the function of the protein it encodes is defective, wherein the DNA construct operably links the coding region of a target gene, an expression cassette containing a promoter and a terminator, a transposon sequence, and the selection marker gene with a modified promoter.

[0019] According to the present invention, it is possible to insert any gene into the chromosome of a filamentous fungus more efficiently and in a larger number of copies than previously known methods, thereby achieving high production of industrially useful proteins and secondary metabolites. Furthermore, the method of the present invention is more versatile and practical than previously known methods for inserting multiple copies of a gene into a filamentous fungus.

[0020] FIG. 1 is a plasmid map of p19-GDH-pyrG3 (a plasmid in which Ptef-GDH-Talp-pyrG3 is inserted into the multi-cloning site of pUC19). FIG. 2 is a plasmid map of p19-RT-GDH-pyrG3 (a plasmid in which RTup (SEQ ID NO: 2)-Ptef-GDH-Talp-pyrG3-RTdown (SEQ ID NO: 3) are inserted into the multi-cloning site of pUC19). FIG. 3 is a plasmid map of p19-rDNA-GDH-pyrG3 (a plasmid in which 18SrDNA-Ptef-GDH-Talp-pyrG3-28SrDNA are inserted into the multi-cloning site of pUC19).

[0021] The present invention will be described in detail below. Note that the following embodiments are presented for the purpose of explanation by way of example, and the present invention is not limited to the following embodiments.

[0022] [Method for transforming filamentous fungi] According to one embodiment of the present invention, a method for transforming filamentous fungi comprises the following steps: providing a host filamentous fungus that contains a selectable marker gene that is deleted or whose encoded protein is functionally defective; and transforming the host filamentous fungus with an expression cassette containing a coding region of a target gene, a promoter and a terminator, a transposon sequence, and the selectable marker gene with a modified promoter.

[0023] (Transposon) A transposon is a base sequence that can transpose its position on the genome within a cell. Transposons are broadly classified into DNA transposons and RNA transposons. In particular, RNA transposons are also called retrotransposons and are distinguished from DNA transposons. DNA transposons transpose when a DNA fragment is excised from the genome by the transposase it encodes and reinserted into an appropriate genome sequence. In contrast, retrotransposons transpose by transcribing themselves into RNA, then reverse-transcription into DNA by the reverse transcriptase it encodes, and reinserting into an appropriate genome sequence. In other words, unlike DNA transposons, the transposition of retrotransposons is accompanied by DNA replication, increasing the copy number of the retrotransposon.

[0024] (RNA Transposon Sequence) RNA transposons are broadly classified into LTR (Long Terminal Repeat) retrotransposons and non-LTR retrotransposons. LTR retrotransposons are characterized by long terminal repeat (LTR) sequences with unidirectional repeats at both ends.

[0025] (Retrotransposon Sequence) LTR-type retrotransposons generate cDNA from an RNA intermediate transcribed by a promoter present in the LTR by the action of reverse transcriptase, and the generated cDNA is introduced into genomic DNA by the action of integrase, resulting in replication movement. Examples of LTR-type retrotransposons include Mg-sine and grasshopper derived from Magnaporthe grisea, AFLAV from Aspergillus flavus, AoLTR from Aspergillus oryzae, and skippy from Fusarium oxysporum.

[0026] Non-LTR retrotransposons are characterized by the presence of a polyA at the 3' end of the transcript RNA, and produce an RNA intermediate transcribed from an adjacent promoter, which generates cDNA through the action of reverse transcriptase, and the resulting cDNA is introduced into genomic DNA through the action of an endonuclease, resulting in replication migration. Non-LTR retrotransposons include those having a "long interspersed nuclear element (LINE)" and those having a "short interspersed nuclear element (SINE)".

[0027] The transposon sequence used for transformation of the present invention may be either a DNA transposon sequence or a retrotransposon sequence, as long as multiple copies are present at different positions on the genome of the filamentous fungus to be transformed (host filamentous fungus). According to one embodiment of the present invention, the transposon sequence used for transformation is a retrotransposon sequence.

[0028] Furthermore, in the method for transforming a filamentous fungus of the present invention, the transposon sequence used for transformation is preferably a sequence homologous to the transposon sequence of the filamentous fungus to be transformed. Furthermore, the criteria for selecting the homologous sequence to be used within the transposon sequence are not particularly limited, and may be, for example, the same criteria as those used when designing a homologous sequence for the purpose of inducing homologous recombination. The transposon sequence may be contained at a single site in the vector used for transformation, or may be split into two sites. For example, when the transposon sequence is split into two sites in the vector, two sites in a single transposon sequence can be identified, and a homologous sequence of the 5' site can be linked to the 5' site of the transgene construct (the construct to be introduced into the filamentous fungus), and a homologous sequence of the 3' site can be identified to be linked to the 3' site of the transgene construct (the construct to be introduced into the filamentous fungus).

[0029] The transposon sequence used in the present invention may be any sequence as long as it is present in multiple locations on the genome. As an example, the LINE present in Aspergillus sojae is used. At least eight of these retrotransposons are present in the Aspergillus sojae NBRC4239 strain. Four of these sequences are completely identical to each other and are represented by SEQ ID NO: 14, and the other retrotransposon sequences share 99% or more identity with the above-mentioned base sequence. According to a preferred embodiment of the present invention, the retrotransposon sequence used is preferably one that hybridizes with SEQ ID NO: 14 under stringent conditions.

[0030] As used herein, the term "a base sequence that hybridizes under stringent conditions" refers to a DNA base sequence that can be obtained by using DNA having the base sequence of a wild-type gene as a probe and employing colony hybridization, plaque hybridization, Southern blot hybridization, or the like.

[0031] As used herein, "stringent conditions" refer to conditions under which signals from specific hybrids can be clearly distinguished from signals from nonspecific hybrids, and these conditions vary depending on the hybridization system used and the type, sequence, and length of the probe. Such conditions can be determined by changing the hybridization temperature and the washing temperature and salt concentration. For example, if even nonspecific hybrid signals are strongly detected, specificity can be improved by increasing the hybridization and washing temperatures and, if necessary, decreasing the washing salt concentration. Furthermore, if signals from specific hybrids are not detected, hybrids can be stabilized by decreasing the hybridization and washing temperatures and, if necessary, increasing the washing salt concentration.

[0032] Specific examples of stringent conditions include hybridization using a DNA probe as the probe, overnight (approximately 8 to 16 hours) using 5xSSC, 1.0% (w / v) blocking reagent for nucleic acid hybridization (Boehringer Mannheim), 0.1% (w / v) N-lauroyl sarcosine, and 0.02% (w / v) SDS. Washing is performed twice for 15 minutes using 0.1 to 0.5xSSC, 0.1% (w / v) SDS, preferably 0.1xSSC, 0.1% (w / v) SDS. Hybridization and washing are performed at a temperature of 65°C or higher, preferably 68°C or higher.

[0033] Examples of DNA having a base sequence that hybridizes under stringent conditions include DNA obtained by hybridizing under the above-mentioned stringent conditions using a filter on which DNA having the base sequence of a wild-type gene derived from a colony or plaque or a fragment of the above DNA is immobilized; and DNA that can be identified by hybridizing in the presence of 0.5 to 2.0 M NaCl at 40 to 75°C, preferably in the presence of 0.7 to 1.0 M NaCl at 65°C, and then washing the filter at 65°C using 0.1 to 1×SSC solution (1×SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). Probe preparation and hybridization methods can be performed in accordance with methods described in, for example, "Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989" and "Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons, 1987-1997" (hereinafter, these documents are also referred to as "reference technical documents"; the entire disclosures of these documents are incorporated herein by reference). Those skilled in the art can appropriately determine conditions for obtaining DNA having a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of a wild-type gene, taking into account not only conditions such as the salt concentration and temperature of the buffer, but also other conditions such as probe concentration, probe length, and reaction time.

[0034] Examples of DNA containing a base sequence that hybridizes under stringent conditions include DNA that has a certain level of sequence identity with the base sequence of DNA containing the base sequence of a wild-type gene used as a probe, such as DNA that has 70% or more, preferably 75% or more, preferably 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with the base sequence of a wild-type gene. Here, the sequence identity of the base sequence (nucleotide sequence) can be determined using an algorithm known in the art, such as BLASTN, with default settings.

[0035] Examples of base sequences that hybridize under stringent conditions with a base sequence complementary to the base sequence of a wild-type gene include, for example, base sequences having deletions, substitutions, additions, etc. of 1 to 40, preferably 1 to 35, more preferably 1 to 30, even more preferably 1 to 25, even more preferably 1 to 20, even more preferably 1 to 15, even more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, even more preferably 1 to several, and even more preferably 1, 2, 3, 4, or 5 bases per unit in the base sequence of the wild-type gene, where "base deletion" means that a base is missing or lost in the sequence, "base substitution" means that a base in the sequence has been replaced with another base, and "base addition" means that a new base has been added so as to be inserted.

[0036] From the viewpoint of transformation efficiency, the length of the transposon sequence ligated to the DNA construct used in the transformation of the present invention is preferably 0.5 kb or more, more preferably 1 kb or more, more preferably 1.5 kb or more, more preferably 2 kb or more, more preferably 2.5 kb or more, more preferably 3 kb or more, more preferably 3.5 kb or more, more preferably 4 kb or more, more preferably 4.5 kb or more, more preferably 5 kb or more, more preferably 6 kb or more, more preferably 7 kb or more, more preferably 8 kb or more, more preferably 9 kb or more, and more preferably 10 kb or more. The upper limit of the length of the transposon sequence ligated to the DNA construct used in the transformation of the present invention is not particularly limited, and can be, for example, 10 kb, but the length of the transposon sequence may be 12 kb or less. Multiple transposon sequences may be ligated to a plasmid. In this case, the length of a single transposon sequence may be 6 kb or less, and the total length of the multiple transposon sequences may be 12 kb or less. Although the retrotransposon sequence used in the examples described below is 5135 bp in length, it is possible to extract sequences of approximately 1.8 kbp each from the sequence so that they do not overlap and ligate them onto a plasmid, or to ligate the entire length of the retrotransposon sequence so that it is 10270 bp in total, sandwiching the expression cassette of the plasmid, or to further increase the number of sequences ligated. Furthermore, transposons other than the retrotransposon with a sequence length of 5135 bp used in the examples can also be used.

[0037] Furthermore, the transposon sequence used in the transformation of the present invention is preferably one that is present in a larger number of copies at different positions on the genome of the filamentous fungus to be transformed (host filamentous fungus). The copy number of the transposon sequence present at different positions on the genome of such a host filamentous fungus is preferably 2 or more copies, more preferably 4 or more copies, even more preferably 8 or more copies, and particularly preferably 10 or more copies. The upper limit of the copy number of the transposon sequence present at different positions on the genome of the host filamentous fungus is not particularly limited, but may be, for example, 100 copies or less.

[0038] (Selection marker gene with modified promoter) A "selection marker gene with modified promoter" refers to a selection marker gene in which the expression level of the coding region has been reduced by reducing the function of the promoter, for example, by deleting or substituting an arbitrary region from a promoter having sufficient transcription activity for functional expression of the selection marker, or by inserting an arbitrary sequence, thereby reducing the transcription level of the gene in the coding region. Here, a "promoter having sufficient transcription activity for functional expression of the selection marker" refers to a promoter having the transcription activity necessary to enable growth in a selective medium (functional expression of the selection marker) by inserting only one copy of the selection marker gene into a chromosome.

[0039] A reduction in the expression level of the coding region means that the transcription level of the gene in the coding region is reduced by 50%, preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, more preferably 95%, more preferably 98% or more, and more preferably 99% or more compared to when the promoter is not modified. The "unmodified" promoter referred to here refers to a promoter of the selectable marker gene that has transcriptional activity that enables functional expression of the selectable marker gene even when only one copy is inserted into a chromosome. Differences in promoter activity (transcription level of the gene in the coding region) can be investigated by, for example, comparing the activity of a reporter protein such as luciferase expressed under each promoter (Eukaryot Cell., 7(1), 28-37, 2008).

[0040] When a selectable marker gene having 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 is inserted on the chromosome will not be able to grow due to insufficient expression of the selectable marker function. However, a transformed strain in which multiple copies of the selectable marker gene are inserted on the chromosome will be able to grow because the reduced expression level of the selectable marker is compensated for by the increased copy number. Therefore, by using a DNA construct in which a selectable marker gene with a modified promoter is linked to an expression cassette of any target gene, it is possible to easily obtain a strain in which multiple copies of the gene of interest are inserted on the chromosome.

[0041] The method for reducing the function of the promoter of a selectable marker gene is not particularly limited, and various known methods may be used. Specific methods include, for example, deleting part or all of the promoter sequence of the selectable marker gene, which requires only one copy to enable functional expression of the coding region of the selectable marker gene, or substituting or inserting an arbitrary base sequence into part or all of the promoter sequence of the selectable marker gene. Another method includes deleting the entire promoter of the selectable marker gene and replacing it with a promoter with weak transcriptional activity or a cryptic promoter. Various known methods can be used to reduce the transcriptional activity of an arbitrary promoter. For example, a reporter protein such as GFP, luciferase, or β-galactosidase can be expressed under various promoters in which a portion of the promoter sequence whose transcription activity is to be reduced has been deleted, substituted, or inserted, and the amount of luminescence or enzyme activity derived from the reporter protein is compared to identify a region in the promoter sequence necessary for transcription activity. The transcription activity of the promoter can then be reduced by deleting that region or substituting or inserting a desired nucleotide 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, a method for finding a promoter with weak transcriptional activity involves searching for genes with low transcription levels using microarrays or the like, and using the upstream sequence of the gene as a promoter with weak transcriptional activity. Another method involves using an inducible promoter or the like to utilize a promoter with weak transcriptional activity in the selective medium used to obtain a transformant. Generally, an upstream region adjacent to a coding region is often used as a promoter, but even a cryptic promoter located away from the coding region may have transcriptional activity, and such a promoter can also be used in the present invention.

[0042] For example, one example of deleting a portion of a promoter is to delete the length of the promoter of any selection marker to 250 bp, preferably 200 bp, more preferably 150 bp, more preferably 140 bp, more preferably 130 bp, more preferably 120 bp, more preferably 110 bp, more preferably 100 bp, more preferably 90 bp, more preferably 80 bp, more preferably 70 bp, more preferably 60 bp or less. Another example of deleting a portion of a promoter is to delete 40% or more, 60% or more, or 80% or more of the full-length sequence of the promoter sequence of the selection marker gene.

[0043] For example, a method for reducing the function of the promoter of the pyrG marker gene can be achieved by deleting a sequence of any length from the 5'-end of the promoter of the pyrG marker gene, one copy of which is sufficient for function, thereby shortening the promoter. Preferably, in the method for reducing the function of the promoter of the pyrG marker gene, the length of the promoter of the pyrG marker gene is shortened to 229 bp or less upstream of the coding region. More preferably, in the method for reducing the function of the promoter of the pyrG marker gene, the length of the promoter of the pyrG marker gene is shortened to 129 bp or less upstream of the coding region. Most preferably, in the method for reducing the function of the promoter of the pyrG marker gene, the length of the promoter of the pyrG marker gene is shortened to 56 bp or less upstream of the coding region. Alternatively, in a method for reducing the function of the promoter of the pyrG marker gene, the promoter region of the pyrG marker may be deleted entirely, and then replaced with a weak promoter derived from another gene (e.g., a truncated promoter derived from the adeA marker gene or a cryptic promoter derived from the alp terminator).

[0044] For example, a method for reducing the function of the promoter of the adeA marker gene can be achieved by deleting a sequence of any length from the 5'-end of the promoter of the adeA marker gene, a promoter for which one copy is sufficient for function, thereby shortening the promoter. Preferably, in a method for reducing the function of the promoter of the adeA marker gene, the length of the promoter of the adeA marker gene is shortened to 126 bp or less upstream of the coding region. More preferably, in a method for reducing the function of the promoter of the adeA marker gene, the length of the promoter of the adeA marker gene is shortened to 120 bp or less upstream of the coding region. Most preferably, in a method for reducing the function of the promoter of the adeA marker gene, the length of the promoter of the adeA marker gene is shortened to 80 bp or less upstream of the coding region. Alternatively, in a method for reducing the function of the promoter of the adeA marker gene, the promoter region of the adeA marker may be deleted entirely, and then replaced with a weak promoter derived from another gene (e.g., a truncated promoter derived from the pyrG marker gene or a cryptic promoter derived from the alp terminator).

[0045] The selectable marker gene usable in this embodiment may be any selectable marker gene usable in filamentous fungi. For example, the pyrG gene (orotidine-5'-phosphate decarboxylase gene) and pyrF gene (orotate phosphoribosyltransferase gene) involved in the biosynthesis of uracil, the adeA gene (N-succinyl-5-aminoimidazole-4-carboxyamide ribotide synthase gene) and adeB gene (phosphoribosylaminoimidazole carboxylase gene) involved in the biosynthesis of adenine, the argB gene (ornithine carbamoyltransferase gene) and argC / argE gene (argininosuccinate synthase gene) involved in the biosynthesis of arginine, the niaD gene (nitrate reductase gene) involved in the assimilation of nitrate, the sC gene (ATP sulfurylase gene) involved in the assimilation of sulfate, and the acetonitrile gene are examples of selectable marker genes usable in filamentous fungi. Examples of marker genes include the amdS gene (acetamidase gene) involved in amide assimilation, the trpC gene (glutamine amidotransferase gene / indoleglycerophosphate synthase gene / phosphoribosylanthranilate isomerase gene) involved in tryptophan biosynthesis, the bioDA gene (7,8-diaminopelargonic acid synthase gene / dethiobiotin synthase gene) involved in biotin biosynthesis, the leu2 gene (β-isopropylmalate dehydrogenase gene) involved in leucine biosynthesis, the pyrithiamine resistance marker ptrA gene (thiazole synthase gene), the benomyl resistance marker benA gene (β-tubulin gene), and the hygromycin resistance hph marker gene (hygromycin phosphotransferase gene). In this embodiment, in order to introduce more copies of a gene, multiple combinations of any of these selection marker genes may be used in the same host. When multiple selection marker genes are combined, one type of selection marker gene may be used per transformation, or multiple marker genes may be used simultaneously in a single transformation.In this case, it is also preferable to carry out transformation using a combination of multiple selection marker genes that show auxotrophy for the same substance, such as the pyrG selection marker gene and the pyrF selection marker gene, both of which show auxotrophy for uracil / uridine.

[0046] (Configuration of Gene Used for Transformation) In this embodiment, the gene used for transformation is constructed by linking a transposon sequence, a selectable marker gene with a modified promoter, a target gene to be highly expressed, and a promoter and terminator for expressing the target gene. The promoter and terminator are not limited to the promoter and terminator native to the target gene, and any promoter and terminator can be used. The promoter for expressing the target gene used in this embodiment 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, the promoter region of the α-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 used in this embodiment for expressing the target gene is not particularly limited as long as it can function in the host, and examples thereof include the terminator region of the α-amylase gene (amy), the terminator region of the alkaline protease gene (alp), etc. Note that the expression cassette for expressing the target gene may also include sequences such as a secretory signal sequence, a translation enhancer sequence, and an organelle-localized sequence in addition to the coding region, promoter, and terminator of the target gene.

[0047] In this embodiment, the DNA used for transformation may be linear or circular. For example, a DNA fragment in which a transposon sequence, a selectable marker gene with a modified promoter, and an expression cassette for a target gene are linked may be inserted into an arbitrary region of pUC19 or the like and used as the DNA for transformation.

[0048] Furthermore, the DNA used for transformation may contain any DNA sequence other than a transposon sequence, a selectable marker gene with a modified promoter, an expression cassette for a target gene, and vector DNA.

[0049] (Host Cells) Host cells that can be used for transformation in this embodiment are not particularly limited as long as they are filamentous fungi, and examples include filamentous fungi belonging to the genera Aspergillus, Neurospora, Penicillium, Fusarium, Trichoderma, Talaromyces, Acremonium, Mucor, and Rhizopus.

[0050] Specific examples of filamentous fungi of the genus Aspergillus include Aspergillus sojae, Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus flavus, Aspergillus tamarii, Aspergillus ruuchiuensis, Aspergillus usamii, Aspergillus kawachii, and Aspergillus saitoi.

[0051] Among these, taking into consideration safety and ease of culture, at least one species selected from the group consisting of Aspergillus sojae, Aspergillus oryzae, Aspergillus niger, Aspergillus tamarii, Aspergillus ruuchiuensis, Aspergillus usamii, Aspergillus kawachi, and Aspergillus saitoi is preferred, any one species selected from the group consisting of these is more preferred, any one species selected from Aspergillus sojae and Aspergillus oryzae is more preferred, and Aspergillus sojae is even more preferred.

[0052] Without being bound by theory, it is believed that Aspergillus oryzae, Aspergillus flavus, Aspergillus sojae, and Aspergillus parasiticus are closely related in the Aspergillus genus section Flavi (e.g., Evolutionary relationships among Aspergillus oryzae and related species based on the sequences of 18S rRNA genes and internal transcribed spacers. J Gen Appl Microbiol. 1998 Jun;44(3):225-230.), and therefore these filamentous fungi in particular are believed to exhibit similar effects as host cells in the transformation of embodiments of the present invention. This can be understood by those skilled in the art from the disclosure in Patent Document 13 that multiple copies can be introduced into both Aspergillus sojae and Aspergillus oryzae using a selection marker with a shortened promoter.

[0053] Furthermore, the host filamentous fungus used for transformation in this embodiment must be a strain that lacks at least one selection marker gene used in the transformation or that lacks the function of the protein encoded by the selection marker gene used. Among these, a strain that lacks a part or all of the coding region of the selection marker gene used is more preferred.

[0054] Furthermore, as described above, the host filamentous fungus used for transformation in this embodiment preferably has multiple transposon sequences at different positions on its genome. The preferred copy number of the transposon sequence in such a host filamentous fungus is as described above.

[0055] (Transformation method) The transformant in this embodiment can be obtained by transforming any filamentous fungus lacking the function of a protein encoded by a selection marker gene to be used, using a plasmid or a DNA fragment comprising a transposon sequence, the selection marker gene with a modified promoter, and an expression cassette for a target gene, etc.

[0056] Known methods for transforming filamentous fungi include a method in which a DNA construct is introduced into protoplasts using polyethylene glycol and calcium chloride (PEG-protoplast method, Mol. Gen. Genet., 218, 99-104 (1989)), and a method in which a DNA construct is introduced into immediately germinated conidia by electroporation (Biosci Biotechnol Biochem. 1994 December; 58(12):2224-7). Desired transformants can be selected using a selectable marker gene, for example, by using auxotrophy or drug resistance associated with the marker gene. For example, when selection is based on auxotrophy, the desired transformant can be selected in a medium that does not contain a compound that complements the auxotrophy. Furthermore, when selection is based on drug resistance, the desired transformant can be selected in a medium containing an appropriate concentration of the drug.

[0057] According to one embodiment of the present invention, the number of copies of the gene introduced into the genome of the host filamentous fungus may, for example, exceed 20 copies, preferably 30 copies or more, more preferably 40 copies or more, and even more preferably 60 copies or more.

[0058] According to one embodiment of the present invention, the percentage of host filamentous fungal cells in which the copy number of the target gene introduced into the genome of the host filamentous fungus is 5 or more is preferably 90.0% or more, more preferably 95.0% or more. The percentage of host filamentous fungal cells in which the copy number of the target gene introduced into the genome of the host filamentous fungus is 20 or more is preferably 20.0% or more, more preferably 25.0% or more, and even more preferably 50.0% or more. The percentage of host filamentous fungal cells in which the copy number of the target gene introduced into the genome of the host filamentous fungus is 40 or more is preferably 20.0% or more.

[0059] [Method for Producing a Transformant of Filamentous Fungus / Transformant] According to the transformant of this embodiment, a transformant of filamentous fungus can be produced by the transformation method described above. Thus, according to an embodiment of the present invention, there is provided a method for producing a transformant of filamentous fungus, the method comprising the steps of: providing a host filamentous fungus that is deficient in a selectable marker gene or that contains a selectable marker gene in which the function of the protein it encodes is defective, and transforming the host filamentous fungus with a DNA construct in which a coding region of a target gene, an expression cassette including a promoter and a terminator, a transposon sequence, and the selectable marker gene with a modified promoter are operably linked.

[0060] Furthermore, according to an embodiment of the present invention, there is provided a filamentous fungal transformant obtained by the above method. Furthermore, according to a preferred embodiment of the present invention, the filamentous fungal transformant is a filamentous fungal transformant obtained by integrating a DNA construct into the chromosome of a host filamentous fungus, the DNA construct comprising a selection marker gene whose encoded protein is defective in function, wherein the DNA construct comprises a target gene coding region, an expression cassette comprising a promoter and a terminator, a transposon sequence, and the selection marker gene with a modified promoter operably linked thereto. Furthermore, according to one embodiment, the DNA construct comprises a DNA unit comprising the expression cassette and the selection marker gene with a modified promoter operably linked thereto, and the transposon sequence split into two portions, wherein a portion of the split transposon sequence is linked to the 5' end of the DNA unit, and another portion of the split transposon sequence is linked to the 3' end of the DNA unit.

[0061] An example of a transformant in this embodiment is a filamentous fungal strain lacking the function of the protein encoded by the pyrG gene, which has been transformed with a pyrG gene whose promoter has been modified to reduce its expression level and a gene in which an expression cassette for a target gene or the like is linked, or a gene designed to be linked intracellularly with these, and which can grow on a medium not containing uracil / uridine. Among such transformants, a transformant in which multiple copies of the target gene have been inserted is preferred, a transformant in which 5 or more copies of the target gene have been inserted is more preferred, a transformant in which 10 or more copies of the target gene have been inserted is even more preferred, a transformant in which 15 or more copies of the target gene have been inserted is even more preferred, a transformant in which 20 or more copies of the target gene have been inserted is even more preferred, a transformant in which 30 or more copies of the target gene have been inserted is even more preferred, a transformant in which 40 or more copies of the target gene have been inserted is even more preferred, a transformant in which 50 or more copies of the target gene have been inserted is even more preferred, and a transformant in which 60 or more copies of the target gene have even more preferred. It is also preferred that the target gene be inserted with high efficiency and in a large number of copies. In this embodiment, the percentage of transformants having 5 or more copies of the target gene inserted is preferably 90% or more, more preferably 95% or more. The percentage of transformants having 20 or more copies of the target gene inserted is preferably 20% or more, more preferably 25% or more, and even more preferably 50% or more. Furthermore, the percentage of transformants having 40 or more copies of the target gene inserted is preferably 20% or more.

[0062] Methods for determining the copy number include a method of calculating the copy number by comparing the production amount or activity of the target protein in a strain into which one copy of the target gene has been inserted with that in the obtained transformed strain, a method of calculating the copy number based on the density of the band in a Southern blot of the target gene in a strain into which one copy of the target gene has been inserted with that in the obtained transformed strain, and a quantitative PCR method using the chromosomal DNA of the transformed strain.

[0063] [Method for Producing Industrially Useful Proteins and Secondary Metabolites] To produce a target protein or secondary metabolite, a transformant obtained according to this embodiment into which multiple copies of a gene necessary for producing the target substance have been inserted is cultured, and the target substance is extracted from the culture. Therefore, according to an embodiment of the present invention, there is provided a method for producing a target protein, comprising: culturing the filamentous fungal transformant obtained by the above method, and producing the target protein encoded by the target gene.

[0064] The target proteins produced using this technology refer to proteins or polypeptides originally produced in animal or plant cells, bacteria, fungi, or viruses. The present invention is not limited to these, and those skilled in the art can select the target proteins as appropriate. These proteins typically include proteins that need to be produced in large quantities. For example, they may be enzymes used in food additive applications, such as proteases and cellulases, enzymes that can be used for diagnostic purposes, such as glucose dehydrogenase, or enzymes involved in secondary metabolism, or non-enzymatic proteins, such as antibodies and collagen, or peptides with some functionality, such as flavor peptides or antimicrobial peptides. The target proteins and peptides are not particularly limited and can be selected as appropriate by those skilled in the art. Examples include cellulase, protease, amylase, lipase, glucose dehydrogenase, glutamate oxidase, alkaline phosphatase, catalase, cholesterol oxidase, creatinase, glutaminase, urease, luciferase, fluorescent proteins such as GFP (green fluorescent protein), and other animal-derived proteins.

[0065] As a medium for culturing the above-mentioned transformant, any medium commonly used for culturing filamentous fungi, i.e., any medium containing a carbon source, a nitrogen source, inorganic substances, and other nutrients in appropriate proportions, can be used, including synthetic and natural media, liquid and solid media. An example of a medium for culturing the above-mentioned transformant is the GPY (glucose-peptone-yeast) medium used in the Examples described below, but is not particularly limited thereto. Depending on the target protein or secondary metabolite, it may be necessary to add special substances required for producing the substance to the medium.

[0066] Culture conditions may be those commonly known to those skilled in the art for culturing filamentous fungi. For example, the initial pH of the medium may be adjusted to 5-10, the culture temperature may be 20-40°C, and the culture time may be set as appropriate, for example, from several hours to several days, preferably 1-7 days, and more preferably 3-7 days. The culture method is not particularly limited, and submerged culture with aeration and agitation, shaking culture, static culture, and the like may be used. However, it is generally preferable to culture under conditions that ensure sufficient dissolved oxygen. However, depending on the target protein or secondary metabolite, it is expected that lower dissolved oxygen levels may result in higher production. An example of culture conditions is, but is not limited to, shaking culture at 30°C for 3 days using GPY medium, as described in the Examples below.

[0067] There are no particular limitations on the method for extracting the target protein or secondary metabolite from the culture after the culture is completed, and any commonly known extraction method may be used.

[0068] For example, when extracting a target protein, if the target protein is secreted into the medium, the cultured bacterial cells can be removed by filtration, centrifugation, or the like, and only the medium supernatant can be recovered, followed by removal of medium components by ultrafiltration or the like and concentration to obtain a crude enzyme. When the target protein is produced intracellularly, the target protein can be extracted by disrupting the bacterial cells using disruption means such as a bead shocker, ultrasonic disrupter, French press, Dynomill, or mortar; by dissolving the bacterial cell walls using a cell wall-lytic enzyme such as Yatalase; or by dissolving the bacterial cells using a surfactant such as SDS (sodium dodecyl sulfate) 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, Biogel, etc., adsorption elution using an ion exchanger, electrophoresis using polyacrylamide gel, etc., adsorption elution using hydroxyapatite, sedimentation methods such as sucrose density gradient centrifugation, affinity chromatography, fractionation methods using molecular sieve membranes or hollow fiber membranes, etc. may be appropriately selected or a combination of these may be used to obtain a purified enzyme preparation.

[0069] For example, when extracting secondary metabolites from a culture, bacterial cells recovered from the culture by filtration, centrifugation, or other procedures may be used as is, or they may be dried or further pulverized after recovery. The method for drying the bacterial cells is not particularly limited, and examples 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 it dissolves the secondary metabolite. Examples include organic solvents such as methanol, ethanol, isopropanol, and acetone, aqueous organic solvents obtained by mixing these organic solvents with water, water, warm water, and hot water. After adding the solvent, the target secondary metabolite is extracted by, as appropriate, disrupting the bacterial cells or heat treatment. The resulting extract can be purified by purification procedures such as centrifugation, filter filtration, ultrafiltration, gel filtration, solubility separation, solvent extraction, chromatography (e.g., adsorption chromatography, hydrophobic chromatography, cation exchange chromatography, anion exchange chromatography, and reverse-phase chromatography), crystallization, activated carbon treatment, and membrane treatment.

[0070] [DNA Construct / Kit / Use] In embodiments of the present invention, transformation of the filamentous fungus can be carried out using a DNA construct having a specific configuration. Thus, according to another preferred embodiment of the present invention, there is provided a DNA construct for transforming a host filamentous fungus containing a selectable marker gene that is deficient in the selectable marker gene or in which the function of the protein encoding it is deficient, the DNA construct comprising a coding region of a target gene, an expression cassette including a promoter and a terminator, a transposon sequence, and the selectable marker gene with a modified promoter operably linked thereto.

[0071] In an embodiment of the present invention, transformation of a filamentous fungus can be carried out using a kit containing the above-mentioned DNA construct. Therefore, according to another embodiment of the present invention, there is provided a kit for transforming a host filamentous fungus that is deficient in a selectable marker gene or a selectable marker gene whose encoded protein is functionally defective, the kit comprising a DNA construct operably linked to an expression cassette including a promoter, a terminator, and a coding region of a target gene, a transposon sequence, and the selectable marker gene whose promoter has been modified. Furthermore, according to another more preferred embodiment, the kit preferably further comprises a host filamentous fungus that contains a selectable marker gene whose encoded protein is functionally defective. The kit may further comprise other components such as an experimental protocol, experimental equipment, etc.

[0072] As described above, the DNA construct can be suitably used for transforming a host filamentous fungus that is deficient in a selectable marker gene or that contains a selectable marker gene whose encoded protein is functionally defective. Therefore, according to another preferred embodiment of the present invention, there is provided use of the DNA construct or kit in producing a transformant of a host filamentous fungus that is deficient in a selectable marker gene or that contains a selectable marker gene whose encoded protein is functionally defective. Furthermore, according to yet another preferred embodiment of the present invention, there is provided use of the DNA construct or kit for transforming a host filamentous fungus that is deficient in a selectable marker gene or that contains a selectable marker gene whose encoded protein is functionally defective.

[0073] The above-mentioned methods for producing filamentous fungal transformants, the filamentous fungal transformants, the DNA constructs, the kits, and the respective embodiments of their uses can be carried out by those skilled in the art in accordance with the description of the filamentous fungal transformation method of the present invention.

[0074] According to one embodiment of the present invention, the following are provided: [1] A method for transforming a filamentous fungus, comprising the steps of: providing a host filamentous fungus that is deficient in a selectable marker gene or that contains a selectable marker gene in which the function of the protein encoding it is defective; and transforming the host filamentous fungus with an expression cassette comprising a coding region of a target gene, a promoter and a terminator, a transposon sequence, and the selectable marker gene with a modified promoter. [2] The method according to [1], comprising transforming the host filamentous fungus with a DNA construct in which an expression cassette comprising a coding region of a target gene, a promoter and a terminator, a transposon sequence, and the selectable marker gene with a modified promoter are operably linked. [3] The method according to [1] or [2], wherein the transposon sequence used for transformation is a sequence homologous to a transposon sequence of the host filamentous fungus present in multiple copies at different positions on the genome. [4] The method according to any of [1] to [3], wherein the transposon sequence used for transformation is a retrotransposon sequence. [5] The method according to any one of [1] to [4], wherein the filamentous fungus is selected from filamentous fungi belonging to the genus Aspergillus, Neurospora, Penicillium, Fusarium, Trichoderma, Talaromyces, Acremonium, Mucor, or Rhizopus. [6] The method according to any one of [1] to [5], wherein the filamentous fungus is selected from filamentous fungi belonging to the genus Aspergillus. [7] The method according to any one of [1] to [6], wherein the filamentous fungus is at least one species selected from the group consisting of Aspergillus sojae, Aspergillus oryzae, Aspergillus niger, Aspergillus tamarii, Aspergillus ruuchiuensis, Aspergillus usamii, Aspergillus kawachii, and Aspergillus saitoi. [8] The method according to any one of [1] to [7], wherein the filamentous fungus is any one species selected from Aspergillus sojae and Aspergillus oryzae. [9] The method according to any one of [1] to [8], wherein the filamentous fungus is Aspergillus sojae.

[10] The method according to any one of [1] to [9], wherein the modification of the promoter of the selectable marker gene is a deletion of a portion of the promoter.

[11] The method according to any one of [1] to

[10] , wherein the selectable marker gene is the pyrG gene, pyrF gene, adeA gene, adeB gene, argB gene, argC / E gene, argA gene, niaD gene, sC gene, amdS gene, trpC gene, leu2 gene, bioDA gene, ptrA gene, hph gene, or benA gene.

[12] The method according to any one of [1] to

[11] , wherein the selectable marker gene is the pyrG gene.

[13] The method according to any one of [1] to

[12] , wherein 40% or more, 60% or more, or 80% or more of the full-length sequence of the promoter sequence of the selectable marker gene is deleted.

[14] The method according to any one of [1] to

[13] , wherein the length of the promoter of the selectable marker gene is 250 bp, 200 bp, 150 bp, 100 bp, 80 bp, or 60 bp or less.

[0075]

[15] A method for producing a transformant of a filamentous fungus, comprising the steps of: providing a host filamentous fungus that is deficient in a selectable marker gene or that contains a selectable marker gene in which the function of the protein encoding it is defective, and transforming the host filamentous fungus with a DNA construct in which an expression cassette containing a coding region of a target gene, a promoter and a terminator, a transposon sequence, and the selectable marker gene with a modified promoter are operably linked.

[16] The method of

[15] , wherein the transposon sequence used for transformation is a sequence homologous to a transposon sequence of the host filamentous fungus present in multiple copies at different positions on the genome.

[17] The method of

[15] or

[16] , wherein the transposon sequence used for transformation is a retrotransposon sequence.

[18] The method according to any one of

[15] to

[17] , wherein the filamentous fungus is selected from filamentous fungi belonging to the genus Aspergillus, Neurospora, Penicillium, Fusarium, Trichoderma, Talaromyces, Acremonium, Mucor, or Rhizopus.

[19] The method according to any one of

[15] to

[18] , wherein the filamentous fungus is selected from filamentous fungi belonging to the genus Aspergillus.

[20] The method according to any one of

[15] to

[19] , wherein the filamentous fungus is at least one species selected from the group consisting of Aspergillus sojae, Aspergillus oryzae, Aspergillus niger, Aspergillus tamarii, Aspergillus ruuchiuensis, Aspergillus usamii, Aspergillus kawachii, and Aspergillus saitoi.

[21] The method according to any one of

[15] to

[20] , wherein the filamentous fungus is any one species selected from Aspergillus sojae and Aspergillus oryzae.

[22] The method according to any one of

[15] to

[21] , wherein the filamentous fungus is Aspergillus sojae.

[23] The method according to any one of

[15] to

[22] , wherein the modification of the promoter of the selectable marker gene is a deletion of a part of the promoter.

[24] The method according to any one of

[15] to

[23] , wherein the selectable marker gene is the pyrG gene, pyrF gene, adeA gene, adeB gene, argB gene, argC / E gene, argA gene, niaD gene, sC gene, amdS gene, trpC gene, leu2 gene, bioDA gene, ptrA gene, hph gene, or benA gene.

[25] The method according to any one of

[15] to

[24] , wherein the selectable marker gene is the pyrG gene.

[26] The method according to any one of

[15] to

[25] , wherein 40% or more, 60% or more, or 80% or more of the full-length promoter sequence of the selectable marker gene is deleted.

[27] The method according to any one of

[15] to

[26] , wherein the length of the promoter of the selectable marker gene is 250 bp, 200 bp, 150 bp, 100 bp, 80 bp, or 60 bp or less.

[0076]

[28] A method for producing a target protein, comprising culturing a transformant of a filamentous fungus obtained by the method according to any one of

[15] to

[27] , and producing the target protein encoded by the target gene.

[29] The method according to

[28] , wherein the target protein is a protein or polypeptide that can be produced in animal or plant cells, bacteria, fungi, or viruses.

[30] The method according to

[28] or

[29] , wherein the target protein is cellulase, protease, amylase, lipase, glucose dehydrogenase, glutamate oxidase, alkaline phosphatase, catalase, cholesterol oxidase, creatinase, glutaminase, urease, luciferase, a fluorescent protein such as GFP (green fluorescent protein), or another animal-derived protein.

[0077]

[31] A DNA construct for transforming a host filamentous fungus containing a selectable marker gene that is deficient in the selectable marker gene or in which the function of the protein encoding it is deficient, the DNA construct comprising an expression cassette containing a coding region of a target gene, a promoter and a terminator, a transposon sequence, and the selectable marker gene with a modified promoter operably linked thereto.

[32] The DNA construct according to

[31] , wherein the transposon sequence used for transformation is a sequence homologous to a transposon sequence of the host filamentous fungus present in multiple copies at different positions on the genome.

[33] The DNA construct according to

[31] or

[32] , wherein the transposon sequence used for transformation is a retrotransposon sequence.

[34] The DNA construct according to any of

[31] to

[33] , wherein the filamentous fungus is selected from filamentous fungi belonging to the genera Aspergillus, Neurospora, Penicillium, Fusarium, Trichoderma, Talaromyces, Acremonium, Mucor, or Rhizopus.

[35] The DNA construct according to any one of

[31] to

[34] , wherein the filamentous fungus is selected from filamentous fungi belonging to the genus Aspergillus.

[36] The DNA construct according to any one of

[31] to

[35] , wherein the filamentous fungus is at least one species selected from the group consisting of Aspergillus sojae, Aspergillus oryzae, Aspergillus niger, Aspergillus tamarii, Aspergillus ruuchiuensis, Aspergillus usamii, Aspergillus kawachii, and Aspergillus saitoi.

[37] The DNA construct according to any one of

[31] to

[36] , wherein the filamentous fungus is any one species selected from Aspergillus sojae and Aspergillus oryzae.

[38] The DNA construct according to any one of

[31] to

[37] , wherein the filamentous fungus is Aspergillus sojae.

[39] The DNA construct according to any one of

[31] to

[38] , wherein the modification of the promoter of the selectable marker gene is a deletion of a portion of the promoter.

[40] The DNA construct according to any one of

[31] to

[39] , wherein the selectable marker gene is the pyrG gene, pyrF gene, adeA gene, adeB gene, argB gene, argC / E gene, argA gene, niaD gene, sC gene, amdS gene, trpC gene, leu2 gene, bioDA gene, ptrA gene, hph gene, or benA gene.

[41] The DNA construct according to any one of

[31] to

[40] , wherein the selectable marker gene is the pyrG gene.

[42] The DNA construct according to any one of

[31] to

[41] , wherein 40% or more, 60% or more, or 80% or more of the full-length promoter sequence of the selectable marker gene is deleted.

[43] The DNA construct according to any one of

[31] to

[42] , wherein the length of the promoter of the selectable marker gene is 250 bp, 200 bp, 150 bp, 100 bp, 80 bp, or 60 bp or less.

[0078]

[44] A kit for transforming a host filamentous fungus that is deficient in a selection marker gene or that contains a selection marker gene whose encoded protein is functionally defective, the kit comprising the DNA construct according to any one of

[31] to

[43] .

[45] The kit according to

[44] , further comprising a host filamentous fungus that is deficient in a selection marker gene or that contains a selection marker gene whose encoded protein is functionally defective.

[0079]

[46] A filamentous fungal transformant comprising a DNA construct integrated into the chromosome of a host filamentous fungus lacking a selectable marker gene or containing a selectable marker gene whose encoded protein is functionally defective, wherein the DNA construct comprises an operably linked coding region of a target gene, an expression cassette comprising a promoter and a terminator, a transposon sequence, and the selectable marker gene with a modified promoter.

[47] The filamentous fungal transformant according to

[46] , wherein the transposon sequence is a sequence homologous to a transposon sequence of the host filamentous fungus present in multiple copies at different positions on the genome.

[48] The filamentous fungal transformant according to

[46] or

[47] , wherein the transposon sequence is a retrotransposon sequence.

[49] The filamentous fungal transformant according to any of

[46] to

[48] , wherein the filamentous fungus is selected from filamentous fungi belonging to the genus Aspergillus, Neurospora, Penicillium, Fusarium, Trichoderma, Talaromyces, Acremonium, Mucor, or Rhizopus.

[50] The filamentous fungus transformant according to any of

[46] to

[49] , wherein the filamentous fungus is selected from filamentous fungi belonging to the genus Aspergillus.

[51] The filamentous fungus transformant according to any of

[46] to

[50] , wherein the filamentous fungus is at least one species selected from the group consisting of Aspergillus sojae, Aspergillus oryzae, Aspergillus niger, Aspergillus tamarii, Aspergillus ruuchiuensis, Aspergillus usamii, Aspergillus kawachii, and Aspergillus saitoi.

[52] The filamentous fungal transformant according to any of

[46] to

[51] , wherein the filamentous fungus is any one species selected from Aspergillus sojae and Aspergillus oryzae.

[53] The filamentous fungal transformant according to any of

[46] to

[52] , wherein the filamentous fungus is Aspergillus sojae.

[54] The filamentous fungal transformant according to any of

[46] to

[53] , wherein the modification of the promoter of the selectable marker gene is a deletion of a portion of the promoter.

[55] The filamentous fungal transformant according to any one of

[46] to

[54] , wherein the selectable marker gene is the pyrG gene, pyrF gene, adeA gene, adeB gene, argB gene, argC / E gene, argA gene, niaD gene, sC gene, amdS gene, trpC gene, leu2 gene, bioDA gene, ptrA gene, hph gene, or benA gene.

[56] The filamentous fungal transformant according to any one of

[46] to

[55] , wherein the selectable marker gene is the pyrG gene.

[57] The filamentous fungal transformant according to any one of

[46] to

[56] , wherein 40% or more, 60% or more, or 80% or more of the full-length promoter sequence of the selectable marker gene is deleted.

[58] The filamentous fungal transformant according to any one of

[46] to

[57] , wherein the length of the promoter of the selectable marker gene is 250 bp, 200 bp, 150 bp, 100 bp, 80 bp, or 60 bp or less.

[0080]

[59] Use of the DNA construct or kit according to any one of

[31] to

[45] in producing a transformant of a host filamentous fungus containing a selectable marker gene that is deficient in the selectable marker gene or in which the function of the protein that it encodes is deficient.

[0081]

[60] Use of the DNA construct or kit according to any one of

[31] to

[45] for transforming a host filamentous fungus that lacks a selectable marker gene or contains a selectable marker gene whose encoded protein is functionally defective.

[0082] [Sequence Information List] The following table summarizes the sequence information described herein.

[0083]

[0084] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples in any way.

[0085] <Method for Measuring GDH Activity> In the following examples, glucose dehydrogenase (GDH) activity was measured as follows.

[0086] 1.025 mL of 100 mM phosphate buffer (pH 7.0), 0.3 mL of 1 M D-glucose solution, and 0.075 mL of 2 mM DCIP (2,6-dichloroindophenol) solution were mixed and incubated at 37°C for 5 minutes. Next, 0.05 mL of 15 mM PMS (phenazine methosulfate) solution and 0.05 mL of enzyme sample solution were added to start the reaction. The absorbance was measured at the start of the reaction and over time, and the decrease in absorbance at 600 nm per minute (ΔA600) associated with the progress of the enzyme reaction was determined, and GDH activity was calculated according to the following formula:

[0087]

[0088] Here, 1 U of GDH activity is defined as the amount of enzyme that reduces 1 μmol of DCIP in 1 minute in the presence of 200 mM D-glucose at 37° C.

[0089] In the formula, "1.5" is the liquid volume (mL) of the reaction reagent + enzyme reagent, and "16.3" is the millimolar extinction coefficient (cm ２ / μmol), "0.05" is the volume of the enzyme solution (mL), "1.0" is the optical path length of the cell (cm), and "ΔA600 ｂｌａｎｋ " represents the decrease in absorbance at 600 nm per minute when the reaction was initiated by adding the buffer solution used to dilute the enzyme instead of the enzyme sample solution.

[0090] Example 1: Examination of a method for introducing multiple copies of the GDH gene using a pyrG-disrupted strain as a parent strain (1) Preparation of a strain into which multiple copies of the glucose dehydrogenase (GDH) gene have been introduced. p19-GDH-pyrG3 (Figure 1: a plasmid in which Ptef-GDH-Talp-pyrG3 has been inserted into the multicloning site of pUC19), which has an expression cassette of pyrG3 (SEQ ID NO: 1) with a promoter length shortened to 56 bp and the GDH gene as a reporter gene, as described in JP 2018-68292 A, and A pyrG-deficient strain of Aspergillus sojae NBRC4239 (48 bp upstream, 896 bp coding region, and 240 bp downstream of the pyrG gene deleted) was transformed with p19-RT-GDH-pyrG3 (Figure 2: a plasmid in which RTup (SEQ ID NO: 2)-Ptef-GDH-Talp-pyrG3-RTdown (SEQ ID NO: 3)) in which retrotransposon-like sequences derived from the NBRC4239 strain (present at eight locations on the chromosome of Aspergillus sojae) were ligated upstream of Ptef and downstream of pyrG3, by the protoplast PEG method. Transformation with p19-GDH-pyrG3 yielded 31 As-pyrG3 strains, and transformation with p19-RT-GDH-pyrG3 yielded 19 As-RT-pyrG3 strains. These strain groups were designated the As-pyrG3 group and the As-RT-pyrG3 group, respectively.

[0091] (2) Measurement of GDH activity in the obtained GDH multicopy-introduced strains All strains of the obtained As-pyrG3 group and As-RT-pyrG3 group were inoculated into Pinex medium (2% Pinex #2 (Matsutani Chemical Industry), 1% Hypolypeptone (Nihon Pharmaceutical), 0.5% yeast extract, 0.25% potassium dihydrogen phosphate, 0.25% dipotassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.4% soy sauce oil) and cultured at 30 ° C. for 4 days. After culture, the cells were homogenized using a homogenizer, disrupted using a multi-bead shocker, and the supernatant was collected after centrifugation to prepare a disrupted cell solution. The GDH activity of this disrupted cell solution was measured, and the GDH activity per mL of culture solution was calculated.

[0092] The number of copies of the inserted GDH gene and the number of strains obtained in the As-pyrG3 group and the As-RT-pyrG3 group are shown in Table 1. The copy number of the GDH gene was calculated from the relative GDH activity to a strain into which one copy of GDH was inserted, in the same manner as described in JP 2018-68292 A.

[0093]

[0094] As can be seen from the table above, linking a retrotransposon sequence to a plasmid made it easier to obtain strains with high GDH activity. Furthermore, when the GDH46 copy equivalent strain, which had the highest GDH activity among the As-RT-pyrG3 group, was examined by quantitative PCR for the number of copies of the GDH gene present on the chromosome, it was found that 66 copies of the GDH gene had actually been inserted onto the chromosome. These findings demonstrate that linking a retrotransposon sequence to a plasmid is an effective method for efficiently introducing multiple copies of any gene into Aspergillus oryzae.

[0095] [Example 2] Experiment for introducing multiple copies of GDH using a plasmid containing a homologous sequence of rDNA In many eukaryotes, including filamentous fungi, ribosomal DNA (rDNA) is conserved as a tandem repeat sequence in which 18S rDNA and 28S rDNA are alternately arranged on the chromosome.

[0096] In this example, we attempted to introduce multiple copies of any gene onto a chromosome by using a plasmid containing a homologous rDNA sequence to transform a filamentous fungus, similar to the case where a plasmid containing a retrotransposon sequence was used.

[0097] (1) Preparation of a strain containing multiple copies of the GDH gene using a plasmid containing a homologous sequence of rDNA Using the p19-RT-GDH-pyrG3 used in Example 1 as a template, PCR was performed using the following primer set 1 (Ptef-invF2 and PpyrG-invR2) to obtain a DNA fragment consisting of Ptef-GDH-Talp-pyrG3, and PCR was performed using primer set 2 (PdRTup-invR1 and PdRTdown-invF1) to obtain a DNA fragment containing the multi-cloning site of pUC19. Furthermore, PCR was performed using the chromosomal DNA of the Aspergillus sojae NBRC4239 wild-type strain as a template with the following primer set 3 (P18SrDNA_F1 and P18SrDNA_R1) to obtain a DNA fragment (SEQ ID NO: 4) consisting of a 1.8 kbp 18S rDNA sequence, and PCR was performed with primer set 4 (P28SrDNA_F1 and P28SrDNA_R1) to obtain a DNA fragment (SEQ ID NO: 5) consisting of a 1.8 kbp 28S rDNA sequence.

[0098]

[0099] The four DNA fragments were ligated by infusion using HiFi DNA Assembly Mix (NEW ENGLAND BioLabs) to obtain p19-rDNA-GDH-pyrG3, which contains 18SrDNA-Ptef-GDH-Talp-pyrG3-28SrDNA inserted into the multicloning site of pUC19 (Figure 3).

[0100] Using p19-rDNA-GDH-pyrG3, Aspergillus sojae NBRC4239 KP-del strain (deleting 48 bp upstream, 896 bp coding region, and 240 bp downstream of the pyrG gene, as well as 110 bp upstream, 2007 bp coding region of the ku70 gene) was transformed by the protoplast PEG method to obtain 24 As-rDNA-pyrG3 strains. These were designated the As-rDNA-pyrG3 group.

[0101] (2) Measurement of GDH activity in strains with multiple GDH copies introduced using a plasmid containing a homologous rDNA sequence. GDH activity was measured in all strains in the As-rDNA-pyrG3 group, and the estimated copy number of the GDH gene introduced onto the chromosome was calculated, as in Example 1. As a result, the average copy number of the GDH gene in the As-rDNA-pyrG3 group was 2 copies, the maximum copy number was 4 copies, and no strains with 5 or more copies introduced were obtained.

[0102] Example 3 Experiment to introduce multiple copies of GDH using a pyrG-ku70-disrupted strain as a parent strain When attempting transformation by homologous recombination in filamentous fungi, the frequency of homologous recombination can be increased by using a strain in which the ku70 gene has been deleted in advance, and therefore a strain in which the ku70 gene, which is essential for non-homologous recombination in filamentous fungi, has been deleted was used as the parent strain in Example 2. However, it cannot be denied that the deletion of ku70 in the parent strain may be the cause of the significant decrease in the efficiency of obtaining multi-copy strains when a plasmid containing a homologous rDNA sequence was used.

[0103] Therefore, in this example, an experiment was carried out to introduce multiple copies of GDH using a plasmid containing a retrotransposon sequence, using a strain lacking both pyrG and ku70 as a parent strain.

[0104] (1) Preparation of a strain containing multiple copies of the GDH gene introduced using the KP-del strain as a parent strain Aspergillus sojae NBRC4239 KP-del strain (deleted 48 bp upstream of the pyrG gene, 896 bp of the coding region and 240 bp downstream, and 110 bp upstream of the ku70 gene and 2007 bp of the coding region) was transformed using the p19-RT-GDH-pyrG3 prepared in Example 1 by the protoplast PEG method to obtain 17 As-KPdel-RT-pyrG3 strains. These were designated the As-KPdel-RT-pyrG3 group.

[0105] (2) Measurement of GDH activity in GDH multicopy-introduced strains obtained using a KP-del strain as a parent strain and a plasmid containing a retrotransposon sequence As in Example 1, GDH activity was measured in all strains included in the As-KPdel-RT-pyrG3 group, and the estimated copy number of the GDH gene introduced onto the chromosome was calculated. The copy numbers of the inserted GDH gene and the number of strains obtained in the As-pyrG3 group, As-rDNA-pyrG3 group, and As-KPdel-RT-pyrG3 group are shown in the table below.

[0106]

[0107] In the As-pyrG3 group, no strains with 40 or more copies of GDH introduced could be obtained, whereas the maximum copy number of GDH in the As-KPdel-RT-pyrG3 group was 41. Furthermore, the efficiency of multicopy introduction was dramatically improved compared to the As-rDNA-pyrG3 group.

[0108] These results suggest that rDNA sequences, which exist in multiple copies in tandem on the chromosome, are not suitable for use as sequences ligated to plasmids to improve the efficiency of obtaining multicopy strains. It is therefore important to use retrotransposon sequences, which exist in multiple copies at different locations on the chromosome. Furthermore, it was found that when a plasmid containing a retrotransposon sequence is used, multicopy strains can be obtained efficiently, regardless of whether the host strain used has ku70 or not.

[0109] In the above examples, Aspergillus sojae was used as the host filamentous fungus, but it would be generally understood by those skilled in the art that the multicopy introduction technique shown in the above examples can be applied to filamentous fungi other than Aspergillus sojae, taking into account the close relationships determined by genome analysis, etc. For this reason, although not specifically shown in the above examples, experiments will be conducted using other host filamentous fungi (Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus flavus, Aspergillus tamarii, Aspergillus ruuchiuensis, Aspergillus usami, Aspergillus kawachi, Aspergillus saitoi, etc.) in which multiple copies of a desired gene (GDH gene) are introduced into the chromosome, with the transposon sequence and other parameters modified according to the type of host filamentous fungus, in accordance with the description in the above examples.

Claims

1. A method for transforming a filamentous fungus, comprising the steps of: providing a host filamentous fungus that is deficient in a selection marker gene or that contains a selection marker gene in which the function of the protein it encodes is defective; and transforming the host filamentous fungus with an expression cassette containing a coding region of a target gene, a promoter and a terminator, a transposon sequence, and the selection marker gene with a modified promoter.

2. The method according to claim 1, comprising transforming the host filamentous fungus with a DNA construct in which a coding region of a target gene, an expression cassette containing a promoter and a terminator, a transposon sequence, and the selectable marker gene having a modified promoter are operably linked.

3. The method according to claim 1, wherein the transposon sequence used for transformation is a sequence homologous to a transposon sequence of the host filamentous fungus present in multiple copies at different positions on the genome.

4. The method of claim 1, wherein the transposon sequence used in the transformation is a retrotransposon sequence.

5. The method of claim 1, wherein the filamentous fungus is selected from filamentous fungi belonging to the genera Aspergillus, Neurospora, Penicillium, Fusarium, Trichoderma, Talaromyces, Acremonium, Mucor, or Rhizopus.

6. The method of claim 1, wherein the filamentous fungus is selected from the filamentous fungi belonging to the genus Aspergillus.

7. The method according to claim 1, wherein the filamentous fungus is at least one selected from the group consisting of Aspergillus sojae, Aspergillus oryzae, Aspergillus niger, Aspergillus tamarii, Aspergillus ruuchiuensis, Aspergillus usamii, Aspergillus kawachi, and Aspergillus saitoi.

8. The method according to claim 1, wherein the filamentous fungus is any one selected from Aspergillus sojae and Aspergillus oryzae.

9. The method of claim 1, wherein the filamentous fungus is Aspergillus sojae.

10. The method according to any one of claims 1 to 9, wherein the modification of the promoter of the selectable marker gene is a deletion of a portion of the promoter.

11. The method according to any one of claims 1 to 9, wherein the selectable marker gene is pyrG gene, pyrF gene, adeA gene, adeB gene, argB gene, argC / E gene, argA gene, niaD gene, sC gene, amdS gene, trpC gene, leu2 gene, bioDA gene, ptrA gene, hph gene, or benA gene.

12. The method according to any one of claims 1 to 9, wherein the selectable marker gene is the pyrG gene.

13. The method according to any one of claims 1 to 9, wherein 40% or more, 60% or more, or 80% or more of the full length sequence of the promoter of the selectable marker gene is deleted.

14. The method according to any one of claims 1 to 9, wherein the length of the promoter of the selectable marker gene is 250 bp, 200 bp, 150 bp, 100 bp, 80 bp, or 60 bp or less.

15. A method for producing a transformant of a filamentous fungus, comprising the steps of: providing a host filamentous fungus that is deficient in a selection marker gene or that contains a selection marker gene in which the function of the protein it encodes is defective; and transforming the host filamentous fungus with a DNA construct in which the coding region of a target gene, an expression cassette containing a promoter and a terminator, a transposon sequence, and the selection marker gene with a modified promoter are operably linked.

16. A method for producing a target protein, comprising culturing a transformant of a filamentous fungus obtained by the method according to claim 15 to produce a target protein encoded by the target gene.

17. A DNA construct for transforming a host filamentous fungus containing a selection marker gene that is deleted or whose encoded protein is defective in function, wherein a coding region of a target gene, an expression cassette containing a promoter and a terminator, a transposon sequence, and the selection marker gene with a modified promoter are operably linked to the DNA construct.

18. A kit for transforming a host filamentous fungus containing a selection marker gene that is missing a selection marker gene or whose encoded protein is defective in function, the kit comprising a DNA construct in which a coding region of a target gene, an expression cassette containing a promoter and a terminator, a transposon sequence, and the selection marker gene with a modified promoter are operably linked.

19. A filamentous fungal transformant, in which a DNA construct has been integrated into the chromosome of a host filamentous fungus containing a selection marker gene that is deleted or whose encoded protein is defective in function, wherein the DNA construct operably links the coding region of a target gene, an expression cassette containing a promoter and a terminator, a transposon sequence, and the selection marker gene with a modified promoter.

20. Use of a DNA construct in producing a transformant of a host filamentous fungus containing a selection marker gene that is deleted or whose encoded protein is defective in function, wherein the DNA construct is operably linked to a coding region of a target gene, an expression cassette containing a promoter and a terminator, a transposon sequence, and the selection marker gene with a modified promoter.

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