JP7358105B2 - Pyrithiamine-resistant mutant strain of Aspergillus filamentous fungi - Google Patents

Description

The present invention relates to a pyrithiamine-resistant mutant strain of a filamentous fungus of the genus Aspergillus, and a method for producing a genome-edited filamentous fungus of the genus Aspergillus.

Aspergillus oryzae is industrially important, and much basic research is being conducted using genetic recombination technology. In addition, it has become possible to put strains obtained through genome editing into practical use.

Many selection marker genes are known in Aspergillus oryzae. However, most of these are drug resistance genes derived from foreign organisms or auxotrophic complements to auxotrophic strains, and drug resistance genes derived from foreign organisms are treated as genetic recombination, which limits their application. Even in the case of self-cloning, which involves recombining genes from the same species, the Ministry of Health, Labor and Welfare and other competent authorities require safety reviews, making it difficult to apply them to food products. Also, auxotrophic complementation is very complicated to obtain recombinant host strains.

Therefore, an endogenous positive selection marker gene that does not have these disadvantages is the most convenient and desirable. Until now, only four endogenous positive selection marker genes that can be used in Aspergillus oryzae are known: niaD, sC, pyrG, and thiA (ptrA).

The above-mentioned niaD, sC, pyrG, and thiA (ptrA) deletion strains each contain chloric acid (or its salts), selenic acid (or its salts), 5-fluoroorotic acid (5-FOA), and pyrithiamine (PT). Selection is possible using a medium containing The selected concentrations are 0.5M (=61 g/L) for potassium chlorate, 0.1 mM (=19 mg/L) for sodium selenate, and about 2 g/L for 5-FOA. In contrast, pyrithiamine can be selected at a concentration of 0.1 mg/L, which is about 1/20 to 1/600,000, for example, in the case of Aspergillus oryzae. Since all of the above-mentioned drugs are designated as deleterious substances, poisonous substances, harmful heavy metals, or are expensive as reagents, pyrithiamine is the best in terms of environmental impact, work safety, and cost.

In addition, pyrG-deficient strains have problems in that their growth is slow due to remaining uracil requirement (see [0025] of Patent Document 1), and the reagents used for selection are expensive. In addition, niaD and sC-deficient strains are incapable of assimilating nitrate and sulfate, respectively, so they cannot be used in the culture medium, and their leaky phenotype causes background to appear ( (See Non-Patent Document 1).

The known PT resistance gene thiA is a thiamine synthase. The PT resistance mechanism by the thiA gene is unusual and functions by introducing mutations into a limited region of the 5'UTR region. This region is originally involved in an inhibitory mechanism that stops thiamine synthesis in response to intracellular thiamine (or PT), and mutations in this region cause PT resistance by continuing to synthesize thiamine even in the presence of pyrithiamine. (See Patent Document 2, Non-Patent Document 2).

Genome editing, including the CRISPR/Cas9 system, is a technology whose use has expanded rapidly in recent years. This is a technology that targets a specific sequence within the genome, specifically cuts that region, and then introduces a loss-of-function mutation or inserts another gene during the repair process. Research is being conducted using enzymes or supporting proteins such as TALEN, Cas9, Cpf1, and PPR that have the function of cleaving genes in various species such as humans and plants. There are two main methods: methods of introducing these proteins directly into proteins, and methods of directly introducing these proteins.

In genome editing, it is known that there are limits to the sequences that can be cut, and that the efficiency is significantly dependent on the sequence. In the case of thiA, mutations must be introduced only in a limited partial sequence (specifically, the promoter region), so it is unlikely to be used as a cleavage target in the first place. Furthermore, even if cleavage is possible, if the extent of the defect becomes large during the repair process, the gene itself will become functionally defective, making it impossible to acquire resistance.

Japanese Patent Application Publication No. 2015-39349 Japanese Patent Application Publication No. 2000-308491

Japan Brewery Association Journal, Volume 95, No. 7, pp. 494-502, 2000 Journal of the Japanese Society of Biotechnology, Volume 84, No. 3, pp. 89-95, 2006

An object of the present invention is to provide a pyrithiamine-resistant mutant strain of a filamentous fungus of the genus Aspergillus that can be produced by genome editing. Another object of the present invention is to provide a method for producing a genome-edited filamentous fungus of the genus Aspergillus, which enables the production of a pyrithiamine-resistant mutant strain by genome editing.

As a result of extensive research to achieve the above objective, the present inventor discovered a new pyrithiamine resistance marker gene (thiI) in Aspergillus oryzae, and targeted this gene by genome editing to functionally delete it. We obtained the knowledge that pyrithiamine-resistant mutant strains can be easily created using the following method. thiI is presumed to be a transporter, and is thought to be directly or indirectly responsible for releasing the pyrithiamine repressor of thiA.

The present invention was completed based on these findings and further studies, and provides the following pyrithiamine-resistant mutant strains of Aspergillus filamentous fungi, methods for producing genome-edited Aspergillus filamentous fungi, etc. be.

(I) Pyrithiamine-resistant mutant strain of Aspergillus filamentous fungi
(I-1) Pyrithiamine-resistant mutant strain of Aspergillus filamentous fungi that is functionally defective in the nucleic acid encoding the following protein (a) or (b):
(a) Protein consisting of the amino acid sequence described in SEQ ID NO: 1
(b) A protein containing an amino acid sequence having 90% or more identity with each of the amino acid sequences set forth in SEQ ID NOs: 2 to 4, and having transporter activity.
(I-2) A food or drink containing the pyrithiamine-resistant mutant strain described in (I-1).
(I-3) A method for producing a food or drink using the pyrithiamine-resistant mutant strain according to (I-1).

(II) Method for producing genome-edited Aspergillus filamentous fungi
(II-1) A step of genome editing a filamentous fungus of the genus Aspergillus targeting a first nucleic acid encoding the protein of (a) or (b) below and a second nucleic acid other than the nucleic acid, and pyrithiamine resistance A method for producing a genome-edited Aspergillus filamentous fungus, including the step of selecting a pyrithiamine-resistant mutant strain using as an indicator:
(a) Protein consisting of the amino acid sequence described in SEQ ID NO: 1
(b) A protein containing an amino acid sequence having 90% or more identity with each of the amino acid sequences set forth in SEQ ID NOs: 2 to 4, and having transporter activity.
(II-2) A method for producing a food or drink using the genome-edited Aspergillus filamentous fungus obtained by the method described in (II-1).

(III) Method of use as a pyrithiamine resistance marker gene
(III-1) Method of using a nucleic acid encoding the following protein (a) or (b) as a pyrithiamine resistance marker gene for Aspergillus filamentous fungi:
(a) Protein consisting of the amino acid sequence described in SEQ ID NO: 1
(b) A protein containing an amino acid sequence having 90% or more identity with each of the amino acid sequences set forth in SEQ ID NOs: 2 to 4, and having transporter activity.

The pyrithiamine resistance marker gene (thiI) newly discovered in the present invention can acquire PT resistance even if there is a mutation in the thiI sequence or the promoter region, so unlike thiA, genome editing is required to acquire PT resistance. Available.

Furthermore, unlike niaD, sC, and pyrG, deletion of thiI does not show auxotrophy, does not make it impossible to use nitrate and sulfate in the culture medium, and allows for positive selection at low concentrations, making it safe and effective. Environmentally Friendly. In addition, strains exhibiting chlorate resistance may be false positives that do not have a mutation in the nitrate utilization pathway in the first place, or strains that have mutations in other nitrate utilization pathway genes (e.g., nirA). However, it takes a lot of effort to obtain the desired niaD-deficient strain. The same is known for sC-deficient strains that exhibit resistance to selenate. On the other hand, pyrG requires extremely large amounts of expensive drugs and requires constant supplementation of expensive nutrient sources such as uridine/uracil in low-nutrient media.

As described above, thiI is a useful selection marker gene that can be used for genome editing, allows for safer, easier and cheaper selection, and does not require requirements due to deletion.

Therefore, according to the present invention, it is possible to create a pyrithiamine-resistant mutant strain of a filamentous fungus of the genus Aspergillus using genome editing.

3 is a photograph showing the results of culturing WT, a thiA constitutively expressing strain, and a thiI-deficient strain in Test Example 2 in each medium. 3 is a photograph showing the results of genome editing for two genes in Test Example 3.

Embodiments of the present invention will be described in detail below.

Note that in this specification, the term "comprise" includes the meanings of "essentially consist of" and "consist of".

In the present invention, the term "gene" includes double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof, unless otherwise specified. Furthermore, in the present invention, the term "gene" refers to regulatory regions, coding regions, exons, and introns without distinction, unless otherwise specified.

In this specification, each URL is searched on the day before the filing date, and each law refers to the law that is in effect in Japan on the day before the filing date.

Furthermore, in the present invention, "nucleic acid", "nucleotide" and "polynucleotide" are synonymous, and include both DNA and RNA, and may be double-stranded or single-stranded.

The filamentous fungi of the genus Aspergillus targeted by the present invention are not particularly limited, and include Aspergillus oryzae, Aspergillus niger, Aspergillus kawachii, Aspergillus luchuensis, Old name: Aspergillus awamori), Aspergillus saitoi, Aspergillus sojae, Aspergillus tamarii, Aspergillus glaucus, Aspergillus fumigatus, Aspergillus flavus ( Aspergillus flavus), Aspergillus terreus, Aspergillus nidulans (Emericella nidulans), and the like. Among them, Aspergillus lutuensis, Aspergillus sojae, and Aspergillus are safe microorganisms that have been used in brewed foods such as sake, soy sauce, miso, and mirin for many years, and are frequently used hosts in Japanese industries. - Aspergillus oryzae, the model microorganism Aspergillus nidulans, and the protein production host Aspergillus niger are preferred, and the koji mold Aspergillus oryzae is more preferred.

Furthermore, it is desirable that the host Aspergillus filamentous fungus in the present invention has only its own thiI. For example, when Aspergillus oryzae is used as a host, it is desirable to exclude those in which the thiI gene of Aspergillus oryzae has been replaced with that of Aspergillus niger or has been separately introduced. The Aspergillus filamentous fungus that is the host in the present invention is preferably a host for which whole genome analysis results (base sequences) have been published, or even if not, a host that is extremely similar to the host.

(1) Pyrithiamine resistance marker gene The pyrithiamine resistance marker gene of the present invention contains a nucleic acid encoding the following protein (a), (b), or (c).
(a) Protein consisting of the amino acid sequence described in SEQ ID NO: 1
(b) A protein containing an amino acid sequence having 90% or more identity with each of the amino acid sequences set forth in SEQ ID NOs: 2 to 4 and having transporter activity.
(c) A protein having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 and having transporter activity.

The pyrithiamine resistance marker gene (thiI) derived from Aspergillus oryzae has the nucleotide sequence shown in SEQ ID NO: 5 (without intron) and SEQ ID NO: 6 (with intron), and the nucleotide sequence is shown in SEQ ID NO: 1. It encodes a protein consisting of an amino acid sequence. thiI is a gene presumed to be a transporter, and its deletion is thought to confer some form of pyrithiamine resistance to the host strain.

The nucleic acid encoding the protein (a) (hereinafter referred to as "the nucleic acid (a)") can be cloned from Aspergillus oryzae by, for example, shotgun cloning. In addition, the nucleic acid (a) can also be obtained by performing PCR using primers designed based on the base sequences of SEQ ID NOs: 5 and 6 and an Aspergillus aspergillus genome library as a template. It can also be obtained by screening an Aspergillus aspergillus genome library by hybridization using probes designed based on the base sequences of SEQ ID NOs: 5 and 6. It can also be obtained by chemical synthesis.

The nucleic acid encoding the protein (b) includes corresponding nucleic acids of species other than Aspergillus oryzae, natural or non-natural allelic variants, and the like. In addition, as a protein that contains an amino acid sequence having 90% or more identity with each of the amino acid sequences set forth in SEQ ID NOs: 2 to 4 and has transporter activity, proteins with 91% or more identity in the amino acid sequence, 92 % or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. As shown in the Examples below, among the amino acid sequences set forth in SEQ ID NO: 1, the amino acid sequences set forth in SEQ ID NOs: 2 to 4 are regions that are highly conserved in Aspergillus filamentous fungi. Furthermore, it is preferable that the protein has an identity of 75% or more with the amino acid sequence set forth in SEQ ID NO: 1, and the identity in the amino acid sequence is, for example, 76% or more, 77% or more, 78% or more, 79% or more. % or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, and 85% or more.

Nucleic acids encoding the protein (c) include corresponding nucleic acids of species other than Aspergillus oryzae, natural or non-natural allelic variants, and the like. In addition, proteins that have 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 and have transporter activity include proteins that have 91% or more, 92% or more, or 93% or more identity in the amino acid sequence. , 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

In addition to the amino acid sequence described in SEQ ID NO: 1, any of the amino acid sequences described in SEQ ID NO: 7, 8, and 9 can be added to the above (a) and (c).

Amino acid sequence identity (%) can be determined using programs commonly used in the art (eg, BLAST, FASTA, etc.) with default settings. Here, the transporter activity more specifically refers to the phenotype of pyrithiamine resistance when the gene encoding the protein is deleted. Such a modified pyrithiamine resistance marker gene can be produced according to a conventional method.

A pyrithiamine-resistant mutant strain can be created by functionally deleting the above-mentioned pyrithiamine-resistant marker gene. The functional defect is not particularly limited, and includes, for example, a defect in a gene region encoding a protein, a defect in a promoter region or a transcription start point (including the region immediately before it), or destabilizing mature mRNA. (For example, deletion of the polyA addition site) can result in a reduction in transporter activity and impart pyrithiamine resistance to the host. Preferably, the deletion is in a gene region encoding a protein.

The pyrithiamine resistance marker gene (thiI) of the present invention can acquire PT resistance even if there is a mutation in the thiI sequence or the promoter region, so unlike thiA, genome editing can be used to acquire PT resistance. There is an advantage. In addition, unlike other positive selection marker genes, niaD, sC, and pyrG, deletion of thiI does not show auxotrophy, does not make it impossible to use nitrate and sulfate in the medium, and does not cause It has the advantage of being able to perform positive selection based on concentration, being safe, inexpensive, and environmentally friendly. Furthermore, the phenotype is clear and selection is easy.

Thus, thiI is a useful selection marker gene that can be used for genome editing, allows safer selection, and does not require requirements due to deletion.

(2) Pyrithiamine-resistant mutant strain of Aspergillus filamentous fungus The pyrithiamine-resistant mutant strain of Aspergillus filamentous fungus of the present invention has a functional defect in the nucleic acid encoding the protein (a), (b), or (c) above. It is characterized by the presence of

In the case of plate culture, this mutant can grow, for example, even if the Czapek Dox medium contains pyrithiamine in an amount of usually 0.1 mg/L or more.

Here, the site in which the function of the nucleic acid is rendered defective may be either in the sequence or in the promoter region, and is not particularly limited as long as it is capable of deleting the function (transporter activity) of the protein encoded by the nucleic acid. Functional deficiency here includes not only complete loss of protein function, but also a reduction in protein function to such an extent that resistance to pyrithiamine is obtained. Furthermore, functional deficiency includes not only complete failure to express the protein encoded by the nucleic acid, but also a decrease in protein expression to the extent that resistance to pyrithiamine is obtained.

There are no restrictions on the coding region of the protein (a), (b) or (c) above on the chromosome that is the target of genome editing, but the region from the start codon to the stop codon is preferred, and among these, the region that is not spliced is preferred. A coding region other than the region is more preferred. Furthermore, when the distance from the start codon to the stop codon is 100% (2261 bp), the region of 80% (1809 bp) from the start codon (e.g. 1-1809 bp), preferably 60% (1357 bp), 40% (904 bp), 20% (452 bp), 0% (1 bp including the start codon), or a region between them (20-80%, etc.). However, bp expressed in % is rounded off.

Examples of methods for functionally deficient nucleic acids include genome editing and homologous recombination, with genome editing being preferred. More specific methods for deleting a gene include inserting a stop codon into the coding region, causing a frame shift to cause translation into a different protein, or inserting a stop codon as a result. Homologous recombination can be performed according to conventional methods, and genome editing can be performed according to the method described below. A pyrithiamine-resistant mutant strain can be isolated by selecting a pyrithiamine-resistant mutant strain using pyrithiamine resistance as an index after making the nucleic acid functionally defective.

Furthermore, the pyrithiamine-resistant mutant strain of the filamentous fungus of the genus Aspergillus of the present invention may be one in which the nucleic acid (thiI gene) encoding a protein originally possessed by the strain is deficient in function.

By using the pyrithiamine-resistant mutant strain of the filamentous fungus of the genus Aspergillus of the present invention, food and drink products, particularly fermented food and drink products, can be produced. Such foods and drinks include, for example, sake (including sake lees as a by-product), doburoku, fermented products made from grains such as five-grain rice and millet (including the form of koji and even alcoholic fermentation), Amazake, food and drinks containing soy sauce, miso, mirin, vinegar, shochu, awamori (or the grain koji that is the raw material for these (for example, miso koji and wheat koji for soy sauce and miso)), rice koji, etc. (including confectionery and sweets), pickles containing (salt) koji, etc., dried bonito flakes, aged meat (including not only livestock meat but also fish meat), and other forms of food containing cultured koji mold (supplements, etc.) (including nutritional supplements). Examples of the food and drink products of the present invention include those containing the pyrithiamine-resistant mutant strain of the Aspergillus filamentous fungus of the present invention. Such food and drink products can be produced according to conventional methods.

(3) Method for producing genome-edited Aspergillus filamentous fungi In the present invention, genome editing is not subject to any restrictions as long as it is a technology that (cuts and) edits a specific genomic DNA region; Refers to methods of deletion, substitution, and insertion of genes in a host that do not fall under the category of "genetically modified organisms" as defined in the "Act on Securing Biological Diversity through Regulation of the Use of Modified Organisms, etc."; Any genetic modification that does not include this is sufficient. In addition, items that can or have the potential to meet certain conditions, such as ``proving that the edited organism does not contain DNA/RNA synthesized outside cells'' (e.g. If it can be easily proven through whole genome analysis that it is not included in the target organism), it can be defined as a genome-edited organism that does not fall under the category of "genetically modified organism" created by genome editing. In addition, "self-cloning," which is genetic recombination using only the nucleic acids of organisms belonging to the same taxonomic species as the host (Enforcement of the Act on the Conservation of Biological Diversity through Regulations on the Use of Genetically Modified Organisms, etc.) Those resulting from natural mutations (natural occurrences), including chemical treatments, UV treatments, etc., are excluded. For a simple definition of genetically modified organisms, refer to the website of the Ministry of Education, Culture, Sports, Science and Technology (https://www.lifescience.mext.go.jp/bioethics/anzen_faq/#1-4), for example. Methods of deleting genes, that is, not only the TALEN, ZFN, and CRISPR/Cas methods described below, but also modified or new methods of these methods, are "methods of modifying the genes of organisms that do not fall under genetically modified organisms." If so, it is included in the genome editing of the present invention, and there are no restrictions on its use. For example, something like CasX (Nature 566, 218-223 (2019)) may be used. Although there are no restrictions on the deletion, substitution, and insertion of host genes through genome editing, deletion is preferred.

The method for producing a genome-edited Aspergillus filamentous fungus of the present invention includes a first nucleic acid encoding the protein (a), (b), or (c) above, and a second nucleic acid other than the nucleic acid. The method is characterized by including the steps of editing the genome of a filamentous fungus of the genus Aspergillus as a target, and selecting a pyrithiamine-resistant mutant strain using pyrithiamine resistance as an index.

Genome editing can be performed on Aspergillus filamentous fungi using a nuclease that acts on any site in the genome. The nucleases include TALEN protein, ZFN protein, a complex of Cas9 protein and guide RNA, a complex of nickase-modified Cas9 protein and each of a pair of guide RNAs, a complex of Cpf1 protein and guide RNA, and a complex with deaminase. A complex of a modified nickase or null mutant Cas9 protein and guide RNA, a PPR-DNA cleavage domain fusion protein, and a site (or enzyme) with nuclease activity and its nuclease to any genome sequence (base sequence). It is sufficient that the composition consists of a site (hereinafter referred to as an "auxiliary site") that assists the action (such as binding to a base sequence), and may be integrated or separated. (hereinafter referred to as "genome editing composition"), genome editing can be performed.

Genome editing consists of, for example, the following steps 1 to 4. 1) Determining the target genome editing region and designing and manufacturing guide RNA as an auxiliary site, 2) Introducing guide RNA and a site with nuclease activity into target cells, 3) Drugs Selection process based on resistance, etc.; 4) Confirmation process of genome editing by phenotypic or sequence analysis.

In step 1), for example, it can be designed using a known algorithm/design assistance software (CRISPR design tool, CRISPRdirect, CHOPCHOP, GGGenome, etc.) or manually, depending on the characteristics of the nuclease. can. To take CRISPRdirect as an example, if you set the target host as the "Aspergillus oryzae RIB40 genome" and enter the target gene (for example, SEQ ID NO: 5), it will present multiple genes with no off-targets. In addition, sgRNA can be designed by removing the portion corresponding to NGG from the target DNA and then attaching an accompanying sequence "GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU" (SEQ ID NO: 10) as shown in Cloning vector pCnCas9 (GenBank: MH220818). . That is, as long as the host and target gene sequences (particularly the ORF region in this application) are known, an optimal sgRNA can be automatically designed.

In the step 2), for example, a method of introducing an expression cassette of a gene encoding an enzyme and an auxiliary site into a target cell in the form of a plasmid or a virus, a method of directly introducing mRNA into a target cell, a method of introducing an expression cassette of a gene encoding an enzyme and an auxiliary site into a target cell, There are methods such as forming a complex and then introducing it into target cells. Considering off-target effects and side effects of cleaving similar sequences, it is preferable to introduce the complex by a method that reduces off-target effects, such as by introducing the above-mentioned complex.

Step 3) may include a step of performing single colony isolation using a medium containing a drug.

In step 4), for example, it is possible to just confirm the phenotype such as color and drug resistance, but it is also possible to include the step of confirming the deletion using PCR method and the step of specifically identifying the deleted sequence using a sequencer. May contain.

ZFN (zinc-finger nuclease) is an artificial nuclease in which a DNA cutting domain derived from a nuclease domain of a restriction enzyme is linked to a zinc finger repeat that specifically recognizes and binds to a target sequence. This is an example of a genome editing protein molecule itself recognizing a target sequence on the genome. The ZFN-based genome editing technology itself is widely known as a first-generation genome editing method. It is common to connect about three zinc finger repeats of one unit that recognize 3 bases, and design it so that it recognizes a target sequence of around 18 bases. Most commonly, nuclease domains are used. Typically, the C-terminus of the zinc finger domain and the N-terminus of the DNA cleavage domain are linked.

When a DNA double-strand break occurs in the genome of a filamentous fungus of the Aspergillus genus, the double-strand break site is repaired by non-homologous end joining (NHEJ), but during this process, one or more bases are frequently deleted. As a result of this addition, mutations such as frame shifts and generation of stop codons occur, resulting in genome modifications such as destruction of target genes. By allowing a double-stranded DNA fragment to coexist at the DNA double-strand break, it is also possible to knock the DNA fragment into the break site.

TALEN (Transcription activator-like effector nuclease) is a second-generation genome editing tool that uses the TAL effector protein of the plant pathogenic bacterium Xanthomonas genus as a DNA-binding domain and connects it to a nuclease domain derived from the restriction enzyme FokI etc. . TAL effector proteins have a repeating sequence with 34 amino acids as one unit, and each unit of sequence recognizes one base. The number of repeating units corresponds to the chain length of the target sequence. The DNA-binding domain of TALENs is generally designed to recognize a target sequence of about 18 bases. Methods for designing TALEN DNA-binding domains are well known, and design tools are also known.

The complex of Cas9 protein and guide RNA is a complex of a protein molecule with DNA cleaving activity and a guide nucleic acid molecule that recruits the protein molecule to a target sequence part on the genome. Generates DNA double-strand breaks. Genome editing technology that uses Cas9 protein and guide RNA is a third-generation genome editing technology known as the CRISPR/Cas system. This technology itself is well known, and various kits, design tools, etc. are known.

Cas (CRISPR-Associated Proteins) proteins derived from any bacteria may be used. Cas9 derived from the genus Streptococcus is commonly used in the known CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-Associated Proteins) system, including S. pyogenes, S. solfataricus, and S. solfataricus. Cas9 from the genus solfataricus is preferred. Cas9 protein can be obtained by expressing and recovering from various known Cas9 expression vectors using appropriate host cultured cells, or commercially available Cas9 proteins can also be used.

The guide RNA has a structure in which a chimeric sequence of short CRISPR RNA (crRNA) and transactivation crRNA (tracrRNA) is linked to the 3' side of the same sequence as the target sequence on the genome (T is replaced by U). single-stranded RNA can be used. Such single-stranded RNA can be prepared by conventional chemical synthesis. As the chimeric sequence, those used in known CRISPR/Cas systems can be used.

The target sequence to be adopted as the guide RNA is approximately 17 to 25 bases, for example, 19 to 25 bases adjacent to the upstream side of an appropriate PAM (protospacer adjacent motif) sequence, from the genome sequence of the Aspergillus filamentous fungus to be genome edited. A sequence of about 22 bases, especially about 19 to 20 bases, may be used. The PAM sequence varies depending on the species and type of bacteria from which the Cas9 used is derived; in the case of Cas9 derived from Streptococcus pyogenes type II, which is commonly used in known CRISPR/Cas systems, the PAM sequence is 5'-NGG ( N is A, T, G or C).

In the genome of Aspergillus filamentous fungal cells into which a complex of Cas9 protein and guide RNA is directly introduced, a double-strand break in the target sequence occurs between the third and fourth bases upstream of the PAM sequence. To set the target sequence of the guide RNA, first search for an appropriate PAM sequence in the sequence of the target gene or target region to be edited, select its upstream adjacent sequence as a candidate target sequence, and then select the Aspergillus filamentous sequence to be edited. A sequence with few similar sequences in the bacterial genome sequence may be selected as the final target sequence.

The complexes between the nickase-modified Cas9 protein and each of a pair of guide RNAs are two types (pairs) of complexes in which one of the two types of guide RNAs and the nickase-modified Cas9 protein are associated. The nickase-modified Cas9 protein is a modified protein in which one of the two original cleavage domains of the Cas9 protein has been inactivated, and it cleaves only one of the DNA double strands at the target site. Therefore, in order to cleave both strands of DNA, two types of guide RNAs (a pair) that recognize the target sequence on one strand of DNA and another target sequence on the other strand are used. Must be used in combination with nickase-modified Cas9. The double nicking method using nickase-modified Cas9 protein is expected to reduce off-target cleavage.

Nickase-modified Cas9 itself is known, and general nickase-modified Cas9 includes a Cas9 mutant with a D10A mutation (aspartic acid No. 10 replaced with alanine) in the RuvC1 nuclease domain, and a H840A mutation (in the HNH nuclease domain). A Cas9 mutant is known in which histidine No. 840 is replaced with alanine). Any nickase-modified Cas9 can be used in the present invention.

A complex of a nickase-modified or null mutant Cas9 protein linked to deaminase and a guide RNA consists of a protein molecule with base conversion activity and a guide nucleic acid molecule that recruits the protein molecule to a target sequence portion on the genome. This is an example of a complex. A null mutant Cas9 protein is a mutant in which both of the two cleavage domains of the wild-type Cas9 protein are inactivated, and can be prepared, for example, by introducing both the D10A mutation and H840A mutation described above.

In the complex of deaminase-mutant Cas9 and guide RNA formed in the cell, the guide RNA hybridizes to the complementary strand of the target sequence, and base rewriting is performed by the activity of deaminase in one of the DNA duplexes. , a mismatch occurs within the DNA duplex. In the process of repairing this mismatch, the other base is changed to retain the rewritten base, the mismatch is further converted to another base, or one or more bases are deleted or inserted, resulting in Various mutations are introduced onto the genome.

It is known that with this technology, which does not generate DNA double-strand breaks, editing sites (sites where mutations occur) can be controlled by the type of Cas9 protein variant used, the size of the target sequence, etc. Examples of deaminases include activation-induced cytidine deaminase (converts cytosine to uracil), adenosine deaminase (converts adenine to hypoxanthine), and guanosine deaminase (converts guanine to xanthine).

The complex of Cpf1 protein and guide RNA is an example of a complex of a protein molecule with DNA cleaving activity and a guide nucleic acid molecule that recruits the protein molecule to a target sequence part on the genome. It causes DNA double-strand breaks at specific sites. As the guide RNA, single-stranded RNA having a structure in which a chimeric sequence of crRNA and tracrRNA is linked to the 3' side of the target sequence can be used, similar to that used in the known CRISPR/Cas system. Unlike the Cas9 protein, the cut by the Cpf1 protein results in a protruding end, but like the CRISPR/Cas system, a single Cpf1-guide RNA complex can cleave both strands of double-stranded DNA.

PPR (pentatricopeptide repeat) protein is a nucleic acid binding protein discovered in plants. PPR proteins have a structure in which more than ten PPR motifs of 35 amino acids are consecutive, and each motif corresponds to a base on a one-to-one basis. Advances in analysis of the PPR nucleic acid recognition code have made it possible to design proteins that specifically bind to desired nucleic acid sequences. A PPR-DNA cleavage domain fusion protein in which a DNA cleavage domain such as the nuclease domain of the restriction enzyme FokI is linked to a PPR protein can also be used for genome editing.

The genome editing composition can be introduced into Aspergillus filamentous fungal cells, for example, by converting the Aspergillus filamentous fungus cells into protoplasts, adding the genome editing composition to a protoplast solution, and introducing the genome editing composition into the protoplasts. This can be done by importing. The protoplast solution can be prepared by a conventional method using an Aspergillus filamentous fungus cell wall lytic enzyme such as Yatalase.

The Aspergillus filamentous fungus protoplast density and the concentration of the genome editing composition used when introducing the genome editing composition can be appropriately set depending on the Aspergillus filamentous fungus used, the type of the genome editing composition, etc. can. When using a genome editing composition applying the CRISPR/Cas system, efficient genome editing can be achieved by using approximately 10 μg or more of Cas9 protein for approximately 10 ⁶ filamentous fungal protoplasts.

In the present invention, in order to co-edit two target genes, it is desirable to use two pairs of genome editing compositions. In the present invention, since one of the target genes is a pyrithiamine resistance marker gene (first nucleic acid), positive selection of edited strains (disrupted strains) is possible. Even if the editing results for the other target gene (second nucleic acid) cannot be positively selected or the phenotype is not clear, screening using pyrithiamine resistance as an indicator will allow the second target gene to be selected. Candidate strains with edited nucleic acids can be obtained. After obtaining a candidate strain, alteration of the second nucleic acid can be confirmed by sequencing the vicinity of the editing target site of the second nucleic acid.

In the present invention, the target gene is destroyed by DNA double-strand breaks or base conversion by genome editing of the pyrithiamine resistance marker gene (first nucleic acid). It is also desirable to perform genome editing on the second nucleic acid to destroy the target gene through nucleic acid double-strand cleavage or base conversion.

In the present invention, knock-in can also be performed by genome editing. The nucleic acid fragment to be knocked in is not particularly limited. The nucleic acid fragment to be knocked in can also include a promoter sequence, if necessary. When knocking in a nucleic acid fragment downstream of a transcription start point within some gene region, the promoter sequence can be omitted. In addition, when performing knock-in genome editing in animal cells or plant cells, a 5' homologous region and a 3' homologous region having the same sequences as the upstream and downstream regions of the target site are placed at both ends of the knock-in DNA fragment, and homologous recombination is performed. It is necessary to insert a DNA fragment into the DNA double-strand break. Furthermore, since non-homologous recombination activity is high in filamentous fungal cells of the genus Aspergillus, it is not necessary to include such a homologous region in the DNA fragment.

When using plate culture, selection of pyrithiamine-resistant mutant strains obtained by genome editing can be performed using, for example, Czapek Dox medium containing pyrithiamine of 0.1 mg/L or more.

Furthermore, food and drink products can be produced using the genome-edited Aspergillus filamentous fungi obtained by the method of the present invention. Examples of such foods and drinks include the above-mentioned sake and other foods and drinks.

Examples are given below to explain the present invention in more detail. However, the present invention is not limited to these Examples in any way.

Test example 1
We destroyed thiI in A. oryzae and confirmed whether PT resistance could be obtained.

<Design of guide RNA (sgRNA)>
For the DNA sequence of the target sequence, a known production auxiliary site was used, the following target sequence was selected, and sgRNA was designed by a known method.
Target array:
5'- GGCGAAGACGAGACGGCGAGG-3' (SEQ ID NO: 11),
sgRNA:
GUACGGCGAAGACGAGACGCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 12)

<Method for disrupting host thiI gene>
・Protoplast formation RIB40 (=NBRC 100959) was used as the bacterial strain.

Manual for the protoplast-PEG method commonly used in transformation (e.g., Takara Bio, pPTR II DNA (product code 3622), URL: http://catalog.takara-bio.co.jp/com/tech_info_detail Genome editing was performed using .php?mode=2&masterid=M100003051&unitid=U100003806).

Specifically, instead of adding plasmid DNA in the protoplast-PEG method, the following were added and tested.
・Mix a commercially available cas9 solution (derived from S. pyogenes, recombinant, with a nuclear localization signal added to the C-terminus) and the synthesized sgRNA described above and leave to stand to form a Cas9-sgRNA complex (Cas9 guide RNA complex). ) formed

After genome editing, selection was performed using pyrithiamine-containing CD selection medium as described in the manual.

<Results>
More than 50 colonies were observed. When we amplified some of the fragments by PCR and sequenced them, a mutant Aspergillus oryzae with the target gene thiI was confirmed. Part of the thiI gene was deleted, resulting in a frame shift.

Test example 2
The phenotype of one thiI-deficient strain obtained in Test Example 1 was confirmed.

<Method>
・Medium PDA medium (potato dextrose agar)
Czapek-dox agar medium (refer to the manual above for composition)
Czapek-dox agar +0.1 ppm PT
- Strain WT (RIB40), a strain constitutively expressing thiA (RIB40 with a mutation in the same region as ptrA (gene)), and a thiI-deficient strain obtained in Test Example 1 (10 ⁵ spore spot inoculation) were used. . The above-mentioned thiA constitutively expressing strain was selected from PT-resistant strains that had grown in a PT-containing medium due to natural mutation.
・Culture: Stand at 30℃ for 3 days

<Results>
The results are shown in Figure 1. The thiI-disrupted strain was no different from WT when treated with normal PDA or Cz-dox. Furthermore, PT resistance was no different from that of the thiA constitutively expressing strain.

Test example 3
Since we confirmed that pyrithiamine resistance could be conferred by thiI genome editing, we investigated whether this property could be applied to co-transformation (co-genome editing). The target gene was wA (NCBI Reference Sequence: XM_001822648/AO090102000545 similar gene) (SEQ ID NO: 13), and it was confirmed by the color of the spores.

<Method>
After designing and synthesizing sgRNA for wA genome editing (for gene deletion) and mixing it with Cas9 to form a Cas9-sgRNA complex as in Test Example 1, sgRNA for thiI genome editing as in Test Example 1 was prepared and synthesized. The same procedure as Test Example 1 was carried out except that it was added at the same time as the sgRNA complex.

<Results>
The results are shown in Figure 2. When the obtained strain was confirmed, some of the conidia were white, confirming the wA-deficient phenotype.

Test example 4
We investigated whether the genome editing of the present invention could be used for genes other than wA.

<1>
(Method) The same procedure as in Test Example 3 was conducted except that the bacterial strain and target gene were changed as follows.
Strain Aspergillus oryzae practical koji strain for sake sake OSI1013 (accession number FERM P-16528)
Target gene Glucoamylase glaB (GenBank: AB007825 similar gene) (SEQ ID NO: 14)
(Results) Among the many PT-resistant strains obtained, 12 strains were selected and sequenced. Among them, we were able to obtain a strain lacking the target gene glaB.

<2>
(Method) The same procedure as in Test Example 3 was conducted except that the bacterial strain and target gene were changed as follows.
Bacterial strain Aspergillus oryzae ferriclysin high producing strain F16
Target gene Ferricrysin synthesis transcription factor sreA (NCBI Reference Sequence: NC_036437/AO090026000719 similar gene) (SEQ ID NO: 15)
(Results) Among the PT-resistant strains, multiple high-producing strains of ferriclysin were confirmed. Yellow ferriclysin staining was observed at a frequency similar to wA.

Test example 5
We investigated whether thiI homologs exist in species other than Aspergillus oryzae and could be used as markers.

<Method>
Homologues of thiI of Aspergillus oryzae were searched for in other Aspergillus genera. Then, the gene believed to be a homolog was disrupted in the same manner as in Test Example 1.

Bacterial strain/target gene number/estimated protein number Aspergillus nidulans/NBRC 33017 strain/NW_101456 REGION: complement(745..2761) (SEQ ID NO: 16)/Protein XP_868888 (SEQ ID NO: 7) (1380th C was removed The amino acid sequence obtained by re-annotating the base sequence is shown in SEQ ID NO: 8)
・Aspergillus niger NBRC strain 33023/NT_166519 REGION: complement(2422142..2425115) (SEQ ID NO: 17)/Protein XP_001400106 (SEQ ID NO: 9)
- Aspergillus soae OSI2020 (stocked by Gekkeikansha), the same genes, proteins, and sgRNA as Aspergillus aspergillus in Example 1 were used.

<Results>
The genes of Aspergillus nidulans, Aspergillus niger, and Aspergillus soae encoding the thiI-like gene of Aspergillus oryzae were edited in the same manner as in Test Example 1, respectively. When these similar genes were disrupted, a disrupted strain could be selected on PT medium, similar to Aspergillus oryzae.

When the amino acid sequences of thiI of these Aspergillus filamentous fungi were analyzed, the following underlined region of thiI of Aspergillus oryzae was highly conserved.
MAPAQQQADLLVDQDAHPIVNEEVRSQTESQDDAALLRTMGYKPVLHRTYTLFENFATTFA ALYFVGGVRVTFSTGIAAGGNLAYWTSYLVTMVFTYITAAVIAEVCSASPSAGSIYLWAAEAGGPRFGRLLGFIVAWWSTTAWTTFCASNTQAAVNYMLSELTVFNVDFPTDTSSVKFRA VQWICTEILLALAAILNFMPPKYFRYVFWFSSMVVLLDFILNIIWLPIGAHNTWGFRTAE EAFMSTYNGTGAPPGWNWCLSYLATAGILIGFDASGHVAEETKNASITAARGIFWSTVVS GIGGLATIILFLFCAPDPDTLFSFGSPQPFVPLYAVVLGRGGHIFMNVICIVALWLNTAI AIVAASRLVFAVARDGVLPFSSWVSKVHNGQPRNAVIVVWAVAALVTCTLLPSDVAFTSL VSAAGVPSAAAYGLICLARLICTPKRFPKPQWSLGKWSKPFQFIGVFWNGWVVAVLFSPYAFPVTGANLNYAPIIMAGVTIFALISYFAMPEEAWLPRNRISHFIDSKGAQATVEEVERPSGEDQGTSTPRL(配列番号１)
The underlined portions are, in order, conservation region 1 (SEQ ID NO: 2), conservation region 2 (SEQ ID NO: 3), and conservation region 3 (SEQ ID NO: 4). In addition, a homology search was conducted for amino acid sequences with high identity to SEQ ID NO: 2-4.

Amino acid sequence identity values for thiI between Aspergillus oryzae and other Aspergillus species are shown in the table below. Furthermore, the overall amino acid identity was approximately 80% or more, and it was revealed that each amino acid sequence satisfying this condition and the gene encoding it were one gene for each host.

Test example 6 (reference)
Constitutive expression of thiA is associated with deletion of RegionA of the thiA gene. And such mutations need only stay within 13bp of RegionA. On the other hand, in the CRISPR/Cas system, the target sequence (20 bp) must be adjacent to three bases (NGG) called the PAM sequence. In other words, the target design for inducing mutations within RegionA is limited to one of the following.
TCTGGATAATACCAGC GAAA AGG (AGG is a PAM sequence, Region B in the target sequence is underlined, and Region A is the rest.)

Therefore, mutations must actually occur within 4 bp. On the other hand, mutations caused by the CRISPR/Cas system frequently result in mutations of 10 bp or more in the genus Aspergillus, and the possibility of mutations occurring within 4 bp is not high. We actually investigated whether genome editing targeting the 5'UTR riboswitch region of thiA (ptrA) is possible.

<Method>
Test Example 1 was carried out in the same manner as in Test Example 1, except that the target sequence was changed as follows.
TCTGGATAATACCAGCGAAA (SEQ ID NO: 18)

<Results>
No PT-resistant strain could be obtained.

Claims (8)

A pyrithiamine-resistant mutant strain of Aspergillus filamentous fungi that is functionally defective in the nucleic acid encoding the following protein (a) or (b):
(a) Protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 and 7 to 9
(b) Contains an amino acid sequence that has 90 % or more identity with the amino acid sequence set forth in SEQ ID NOs: 1 and 7 to 9 , and the phenotype when the gene encoding the amino acid sequence is deleted A protein that confers resistance to pyrithiamine . A pyrithiamine-resistant mutant strain of Aspergillus filamentous fungi that is functionally defective in the nucleic acid encoding the protein (c) or (d) that it originally possesses:
(c) Protein consisting of the amino acid sequence described in SEQ ID NO: 1
(d) Contains all amino acid sequences that have 90% or more identity with the amino acid sequences listed in SEQ ID NOs: 2 to 4, and the phenotype when the gene encoding the amino acid sequence is deleted is pyrithiamine resistant. protein. A food or drink product comprising the pyrithiamine-resistant mutant strain according to claim 1 or 2 . A step of editing the genome of Aspergillus filamentous fungi by targeting a first nucleic acid encoding the protein of (a) or (b) below and a second nucleic acid other than the nucleic acid, and pyrithiamine resistance using pyrithiamine resistance as an indicator. A method for producing a genome-edited Aspergillus filamentous fungus, including the step of selecting a resistant mutant strain:
(a) Protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 and 7 to 9
(b) Contains an amino acid sequence that has 90 % or more identity with the amino acid sequence set forth in SEQ ID NOs: 1 and 7 to 9 , and the phenotype when the gene encoding the amino acid sequence is deleted A protein that confers resistance to pyrithiamine . The following steps include editing the genome of Aspergillus filamentous fungi by targeting a first nucleic acid that encodes the protein (c) or (d) that it originally possesses, and a second nucleic acid other than the nucleic acid, and A method for producing a genome-edited Aspergillus filamentous fungus, including the step of selecting a pyrithiamine-resistant mutant strain as an indicator:
(c) Protein consisting of the amino acid sequence described in SEQ ID NO: 1
(d) Contains all amino acid sequences that have 90% or more identity with the amino acid sequences listed in SEQ ID NOs: 2 to 4, and the phenotype when the gene encoding the amino acid sequence is deleted is pyrithiamine resistant. protein. A method for producing food and drink products using the genome-edited Aspergillus filamentous fungus obtained by the method according to claim 4 or 5 . A method of using a nucleic acid encoding the following protein (a) or (b) as a pyrithiamine resistance marker gene for Aspergillus filamentous fungi:
(a) Protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 and 7 to 9
(b) Contains an amino acid sequence that has 90 % or more identity with the amino acid sequence set forth in SEQ ID NOs: 1 and 7 to 9 , and the phenotype when the gene encoding the amino acid sequence is deleted A protein that confers resistance to pyrithiamine . A method of using the following nucleic acid encoding the protein (c) or (d) that it originally has as a pyrithiamine resistance marker gene for Aspergillus filamentous fungi:
(c) Protein consisting of the amino acid sequence described in SEQ ID NO: 1
(d) Contains all amino acid sequences that have 90% or more identity with the amino acid sequences listed in SEQ ID NOs: 2 to 4, and the phenotype when the gene encoding the amino acid sequence is deleted is pyrithiamine resistant. protein.