Production of secondary metabolite derived from filamentous fungus dormant gene by utilizing genome rearrangement

Abstract

To identify a dormant gene cluster of a filamentous fungus, and search a new compound derived from the dormant gene by using the identification.SOLUTION: Provided is a genome rearrangement method of a filamentous fungus which includes a genome rearrangement step of expressing a heat-resistant restriction enzyme derived from a thermophile in a filamentous fungus cell and generating random genetic recombination in a genome inside a cell by the restriction enzyme, where the restriction enzyme is expressed under the control of a homeostatic promoter in the temperature condition of approximately 28°C to approximately 37°C. Also provided is a production method of a compound (secondary metabolite) derived from a dormant gene in a filamentous fungus by utilizing the genome rearrangement method.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for genome reorganization in filamentous fungi, and a method for identifying dormant gene clusters in filamentous fungi using said method and for producing compounds (secondary metabolites) derived from dormant genes. [Background technology]

[0002] Filamentous fungi are organisms that produce useful natural products and have been used in the development of many pharmaceuticals, including penicillin and lovastatin. There are a considerable number of "dormant genes" that are not normally expressed in the genomes of living organisms, and in the genomes of filamentous fungi, there are also a huge number of dormant genes that are biosynthetic for unexplored natural products that could become pharmaceutical seeds. If we could awaken these dormant genes in filamentous fungi, it would be possible to develop new pharmaceutical seeds.

[0003] The TAQing system is a large-scale genome reorganization technology that can rapidly improve the traits of organisms (Patent Document 1, Non-Patent Document 1). In this system, a restriction enzyme is introduced into a cell and transiently expressed under an inducible promoter, which simultaneously breaks double-stranded DNA in the genome DNA, and then recombinational repair causes various genome reorganizations such as end joining, homologous recombination, translocation, and copy number variation. The TAQing system has been applied to polyploid plants and yeast, making it possible to obtain individuals with various traits. However, previous reports (see above) have not specifically described the application of the TAQing system to haploids such as filamentous fungi. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2017-99338 [Non-patent literature]

[0005] [Non-Patent Document 1] Muramoto et al., Nat Commun. 2018 May 18;9(1):1995. doi: 10.1038 / s41467-018-04256-y. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to identify dormant gene clusters in filamentous fungi and to use them to search for novel compounds derived from dormant genes. [Means for solving the problem]

[0007] The inventors have considered using the TAQing system to identify dormant genes in filamentous fungi. In conventional methods for plants targeting yeast, genome reorganization was performed by temporarily expressing a restriction enzyme under the control of an inducible promoter at 30 to 40°C to prevent cells from becoming fragile due to excessive cleavage by the restriction enzyme. However, even when the inventors applied the TAQing system to filamentous fungi according to this method, they were unable to obtain a mutant. Therefore, after repeated trial and error with various conditions such as the expression system of the restriction enzyme, the method of introducing the vector, and the temperature, time, and timing of the heat shock, they succeeded in applying TAQing to filamentous fungi and activating dormant genes through this.

[0008] The present invention is based on the above findings and provides the following [1] to [7]. [1] A method for genome reorganization in filamentous fungi, comprising the steps of: A genome reorganization step of expressing a thermostable restriction enzyme derived from a thermophile in a filamentous fungal cell and causing random genetic recombination in the genome in the cell by the restriction enzyme, The method, characterized in that the restriction enzyme is expressed under the control of a homeostatic promoter at a temperature condition of 28°C to 37°C. [2] The method according to [1], wherein the thermostable restriction enzyme derived from a thermophilic bacterium is one or more selected from BclI, Sse9i, BstUI, TfiI, TseI, Tsp45I, TaqI and PhoI. [3] The method according to [1] or [2], wherein the thermostable restriction enzyme derived from a thermophilic bacterium is TaqI. [4] The method according to any one of [1] to [3], wherein the genome rearrangement step is carried out for 1 week to 12 weeks. [5] The method according to any of [1] to [4], wherein the constitutive promoter is any one selected from the group consisting of the TAA gene promoter (amyB), the GLA gene promoter (glaA), the enolase gene promoter (enoA), the glyceraldehyde-3-phosphate dehydrogenase gene promoter (gpdA), the TEF1 gene promoter (TEF1), and the α-glucosidase gene promoter (agdA). [6] Filamentous fungi include Aspergillus sp., Acremonium sp., Alternaria sp., Arthrinium sp., Aureobasidium sp., Beauveria sp., Bipolaris sp., Bjerkandera sp., Ceriporiopsis sp., Chrysosporium sp. Chaetomium, Coprinus, Coriolus, Cordyceps, Colletotricum, Cryptococcus, Eupenicillium, Emericella, Filibasidium, Fusarium, Humicola, Magna The method according to any one of [1] to [5], wherein the bacterium is any one selected from the group consisting of the genus Humicola, the genus Isaria, the genus Madurella, the genus Magnaporthe, the genus Metarhizium, the genus Mucor, the genus Myceliophthora, the genus Neocallimastix, the genus Neurospora, the genus Paecilomyces, the genus Penicillium, the genus Pestalotiopsis, the genus Phanerochaete, the genus Phlebia, the genus Piromyces, the genus Pleurotus, the genus Purepureocillium, the genus Schizophyllum, the genus Stachybotrys, the genus Salaromyces, the genus Talaromyces, the genus Thermoascus, the genus Thielavia, the genus Tolypocladium, the genus Trametes, and the genus Trichoderma. [7] A method for producing a compound derived from a dormant gene, comprising the steps of: 1) a genome recombination step of inducing random genetic recombination in a genome in a filamentous fungal cell by a method according to any one of [1] to [6]; 2) a step of comparatively analyzing the metabolic products of the mutant strain obtained in the genome reorganization step and the wild-type strain, and selecting a mutant strain that produces a compound that is not produced in the wild-type strain; and 3) culturing the mutant strain to obtain a compound derived from a dormant gene; A method comprising: Effect of the Invention

[0009] According to the present invention, a novel method for large-scale genome reorganization of filamentous fungi is provided. By utilizing the genome reorganization method of the present invention, it is possible to identify dormant gene clusters of filamentous fungi and obtain compounds derived from dormant genes. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows an outline of the experiment in Example 1. [Diagram 2] Figure 2 shows the schematic of fungal expression plasmid construction. PamyB: A. oryzae amyB promoter (inducible with starch or maltose), TamyB: A. oryzae amyB terminator, Ampr: ampicillin resistance gene, ptrA: pyrithiamine resistance gene. [Diagram 3] Figure 3 shows the sequence of the NLS-taqI gene amplified by PCR. Shading: 6 x His, SV40 NLS, and taq1 sequence, respectively. [Figure 4] Figure 4 shows transformed strains that emerged after subculture of A. niger transformants. The top panel shows transformed strains that emerged from transformants using a vector in which taqI is linked downstream of the amyB promoter. The bottom panel shows transformed strains that emerged from transformants using a vector in which taqI is linked downstream of the enoA promoter. [Diagram 5] FIG. 5 shows the results of genomic PCR (electrophoresis) of amyB#1-14. [Figure 6] FIG. 6 shows the results of metabolite analysis (HPLC) of the transformed A. niger strain. [Figure 7A-B] 7A-B show the results of antibacterial activity evaluation of A. niger transformed strain culture extracts. [Figure 8] FIG. 8 shows the results of genome extraction (electrophoresis) for sequencing of A. niger transformed strains. [Figure 9] FIG. 9 shows gene deletions and inversions observed in the transformed filamentous fungal strains (enoA 1-6 strain: gene deletion, amyB 1-8 strain: gene translocation). [Figure 10]FIG. 10 shows the SNPs observed in filamentous fungal transformants. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] 1. Genome rearrangements in filamentous fungi The present invention relates to a method for genome reorganization of a filamentous fungus using a thermostable restriction enzyme derived from a thermophile. The genome reorganization method of the present invention includes a genome reorganization step of expressing a thermostable restriction enzyme derived from a thermophile in a filamentous fungal cell and causing random genetic recombination in the genome in the cell by the restriction enzyme, and is characterized in that the restriction enzyme is expressed under the control of a homeostatic promoter at a temperature condition of about 28°C to about 37°C.

[0012] The "filamentous fungi" to which the present invention is applied are not particularly limited, and examples thereof include the genus Aspergillus, the genus Acremonium, the genus Alternaria, the genus Arthrinium, the genus Aureobasidium, the genus Beauveria, the genus Bipolaris, the genus Bjerkandera, the genus Ceriporiopsis, the genus Chaetomium, the genus Chrysosporium, the genus Coprinus, the genus Coriolus, the genus Cordyceps, the genus Colletotricum, the genus Cryptococcus, the genus Eupenicillium, the genus Emericella, the genus Filibasidium, the genus Fusarium, the genus Humicola, the genus Magna Humicola, Isaria, Madurella, Magnaporthe, Metarhizium, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Pestalotiopsis, Phanerochaete, Phle Examples include the genus Bia, Phoma, Piromyces, Pleurotus, Purepureocillium, Schizophyllum, Talaromyces, Salaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma.

[0013] Preferably, the filamentous fungus is of the genus Aspergillus or Penicillium, more preferably of the genus Aspergillus.

[0014] Aspergillus genus includes Aspergillus aculeatus, Aspergillus aculetinus, Aspergillus awamori, Aspergillus brasiliensis, Aspergillus candidus, Aspergillus clavatus, Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus luchuensis, Aspergillus sojae, Aspergillus ustus, Aspergillus wentii, Aspergillus longivesica, Aspergillus bisporus, Aspergillus ostianus, Aspergillus melleus, Aspergillus luteo-niger, Aspergillus elegans, Aspergillus carneus, Aspergillus sclerotiorum, Aspergillus asperescens, Aspergillus Examples of such fungi include Aspergillus kawachi, Aspergillus udagawae, Aspergillus nidulans, Aspergillus niger, and Aspergillus oryzae.

[0015] The Penicillium genera include Penicillium albocoremium, Penicillium aurantiogriseum, Penicillium bilaiae, Penicillium camemberti, Penicillium candidum, Penicillium chrysogenum, Penicillium claviforme, Penicillium commune, Penicillium crustosum, Penicillium digitatum, Penicillium echinulatum, Penicillium expansum, Penicillium glabrum, Penicillium imranianum, Penicillium italicum, Penicillium lacussarmientei, Penicillium lusitanum, Penicillium purpurogenum, Penicillium roqueforti, Penicillium stoloniferum, Penicillium ulaiense, Penicillium verrucosum, Penicillium viridicatum, Penicillium paxilli, Penicillium oxalicum, Penicillium griseofulvum, Penicillium brevicompactum, Penicillium albiadum, Penicillium aculeatum, Penicillium adametzii, Penicillium multicolor, Penicillium Purpurescens, Penicillium nalgiovense, Penicillium chermesinum, Penicillium nalgiovense、Penicillium oxalicum、Penicillium verrucosum、Penicillium eapansum、Penicillium italicum etc.

[0016] The "thermophilic bacterium-derived heat-resistant restriction enzyme" used in the present invention is not particularly limited, but is a heat-resistant restriction enzyme having an optimum temperature of about 75°C or less, preferably about 50°C to about 75°C, and more preferably about 50°C to about 65°C.

[0017] Examples of thermostable restriction enzymes derived from thermophilic bacteria with an optimum temperature of 75° C. or lower include Apol, Bell, BfuAI, BspQI, BSSHI, BtsCI, Nt.BspQI, and Sfil (optimum temperature: about 50° C.); BsiWI, BslI, BsmAI, BsmBI, BtsI, Fatl, Faul, NtBstNBI, Smll, and Sse9I (optimum temperature: about 55° C.); BsaBI, BsaJI, BsaWI, BsiEI, BssKI, BstAPI, and BstEII. , BstNI, BstUI, BstYI, BtgZI, and Mwol (optimal temperature about 60°C); BsiHKAI, BsmFI, BsmI, BsrDI, BsrI, BstBI, Nb.BsmI, Nb.BsrDI, PI-PspI, T Examples include aqI, Tfil, Tsel, Tsp45I, Tsp509I, TspRI, Tth111I, AccIII (optimal temperature about 65°C); ApeKI, Phol, PspGI, TspMI (optimal temperature about 75°C).

[0018] From the viewpoint of cleavage efficiency, it is preferable that the recognition site of the thermostable restriction enzyme derived from a thermophile is 4 to 6 bases long.

[0019] In terms of optimal temperature and recognition site, preferred thermostable restriction enzymes derived from thermophilic bacteria are ApeKI, BsrI, BssKI, BstNI, BstUI, BtsCI, FatI, FauI, PhoI, PspGI, SmlI, Sse9I, TaqI, TfuI, TseI, Tsp45I, and TspRI. In addition, in consideration of the relationship between the optimum temperature of a restriction enzyme and the growth temperature of filamentous fungal cells, preferred heat-stable restriction enzymes derived from thermophilic bacteria include ApeKI, BsaBI, BsaJI, BsaWI, BsIEI, BslI, BsmBI, BsmI, BspQI, BsrDI, BsrI, BssKI, BstAPI, BstBI, BstNI, BstUI, BstYI, FatI, FauI, MwoI, Nb.BsmI, Nb.BsrDI, PspGI, SfiI, SmlI, TaqI, TfiI, TliI, TseI, Tsp45I, Tsp509I, TspMI, TspRI, Tth111I, and the like.

[0020] In particular, thermostable restriction enzymes derived from thermophilic bacteria used in the present invention are preferably BclI, Sse9i, BstUI, TfiI, TseI, Tsp45I, TaqI and PhoI, and more preferably TaqI.

[0021] The thermostable restriction enzyme derived from thermophilic bacteria is expressed in a filamentous fungal cell under the control of a homeostatic promoter, preferably under the control of a homeostatic high expression promoter. The homeostatic promoter is not particularly limited as long as it functions in a filamentous fungal cell. Examples of the homeostatic promoter include the TAA gene promoter (amyB), GLA gene promoter (glaA), enolase gene promoter (enoA), glyceraldehyde-3-phosphate dehydrogenase gene promoter (gpdA), TEF1 gene promoter (TEF1), and α-glucosidase gene promoter (agdA), which are induced by starch or maltose. All of these are homeostatic high expression promoters. Preferred promoters are amyB, amyB, enoA, and gpdA, and particularly preferred homeostatic promoters are amyB and enoA.

[0022] A filamentous fungal cell expressing a thermostable restriction enzyme derived from a thermophile under the control of a constitutive promoter can be prepared according to a known method, that is, an expression vector containing a gene encoding the thermostable restriction enzyme derived from a thermophile operably linked to the above-mentioned constitutive promoter is prepared and introduced into a filamentous fungal cell.

[0023] The expression vector to be used is not particularly limited as long as it functions in filamentous fungi, and examples thereof include pUPTR2, pUPTRA2_PenoA, pAUR316 DNA, and pPTRII DNA.

[0024] The expression vector may incorporate an enhancer, a terminator, a selection marker, etc., as necessary. The gene encoding the thermostable restriction enzyme derived from a thermophilic bacterium may be optimized in terms of codons according to the host, as necessary.

[0025] The expression vector can be introduced into the filamentous fungal cells by known methods such as the PEG method, electroporation method, particle gun method, etc. In the present invention, it is particularly preferable to introduce the expression vector by the PEG method.

[0026] Expression of a thermostable restriction enzyme derived from a thermophile in a filamentous fungal cell is carried out at a temperature of about 28° C. to about 37° C., preferably about 28° C. to about 32° C., and more preferably about 30° C. In previous reports (such as Muramoto et al. cited above), a temporary heat shock was applied to yeast in the expression induction step to improve the expression efficiency of the thermostable restriction enzyme, but in the present invention, no such heat shock is applied.

[0027] As used herein, the term "about" refers to a value that varies by plus or minus 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%, respectively, from the reference value. Preferably, the term "about" refers to a range of plus or minus 10%, 5%, or 1%, respectively, from the reference value.

[0028] Under the above temperature conditions, the filamentous fungal cells constitutively express the restriction enzyme under the expression conditions of the constitutive promoter.

[0029] The medium to be used is not particularly limited as long as it is suitable for culturing the filamentous fungus to be used, and a solid medium is preferable, and CD + KCl + pyrithiamine agar medium (Czapeck-Dox Broth (Difco) 3.5%, KCl (0.6 M), pyrithiamine (0.1 μg / mL), agarose (1.5%)) is more preferable.

[0030] The expressed thermostable restriction enzymes from thermophiles randomly cleave the genome within filamentous fungal cells, and the resulting double-strand breaks (DSBs) are repaired by recombinational repair to result in various genome rearrangements, including end joining, homologous recombination, translocations, copy number variation, and multiple single-base mutations.

[0031] The above-mentioned genome reorganization step by expression of restriction enzymes is carried out for preferably about 1 week to about 12 weeks, more preferably about 2 weeks to about 8 weeks, and even more preferably about 3 weeks to about 4 weeks.

[0032] 2. Method for producing compounds (secondary metabolites) derived from dormant genes When the change in trait obtained by awakening the dormant genes results in the production of a compound in the mutant strain that is not produced in the wild type, it becomes possible to produce a new compound by utilizing the identified dormant gene cluster. The present invention also provides a method for producing such a compound derived from a dormant gene.

[0033] The method of the present invention for producing a compound derived from a dormant gene in a filamentous fungus comprises the following steps: 1) a genome reorganization step of expressing a thermostable restriction enzyme derived from a thermophile in a filamentous fungal cell and causing random genetic recombination in the genome of the cell by the restriction enzyme, A genome reorganization step, characterized in that the restriction enzyme is expressed under the control of a homeostatic promoter at a temperature condition of 28 ° C to 37 ° C. 2) a step of comparatively analyzing the metabolic products of the mutant strain obtained in the genome reorganization step and the wild-type strain, and selecting a mutant strain that produces a compound that is not produced in the wild-type strain; and 3) culturing the mutant strain to obtain a compound derived from a dormant gene; A method comprising:

[0034] Step 1 is carried out according to the procedure described in the preceding paragraph 1. Step 2 involves, for example, using a selection marker such as a drug resistance gene to select a transformant, and then selecting a trait mutant from the transformant, which has traits different from those of the parent strain, using phenotypes such as growth rate, hyphal morphology, and spore formation ability as indicators. Next, the metabolic products of the trait mutant and the wild strain are compared and analyzed to select a mutant that produces a compound not produced by the wild strain. Comparative analysis of the metabolic products can be carried out, for example, by culturing the mutant and wild strain, and examining the metabolic product compositions of both using HPLC, LC-MS, GC-MS, MALDI-TOF, MS, or the like. In addition, extracts from both can also be compared by evaluating biological activities such as antibacterial activity.

[0035] Regarding step 3, if the obtained mutant strain happens to produce the compound derived from the dormant gene with high efficiency, the mutant strain can be used as is for the production of the compound derived from the dormant gene. However, the compound derived from the dormant gene resulting from genome reorganization can usually only be produced in trace amounts in the mutant strain. In such cases, it is efficient to clone all the genes (dormant gene cluster) required for the production of the compound derived from the dormant gene and produce it by recombinant production.

[0036] The dormant gene cluster can be identified by comparative analysis of the transcriptome of the mutant strain with that of the wild-type strain. Alternatively, it can be identified from the chemical structure of a compound derived from the dormant gene. The identified dormant gene cluster can be cloned from the mutant strain by a known method and introduced into an appropriate host cell to produce the compound. The host cell may be a heterologous cell or a homologous cell.

[0037] 3. Method for creating compound-producing strains derived from dormant genes The present invention also provides a method for producing a compound-producing strain derived from a dormant gene. In one embodiment, a method for producing a compound producing strain derived from a dormant gene comprises the steps of: 1) a genome reorganization step of expressing a thermostable restriction enzyme derived from a thermophile in a filamentous fungal cell and causing random genetic recombination in the genome of the cell by the restriction enzyme, A genome reorganization step, characterized in that the restriction enzyme is expressed under the control of a homeostatic promoter at a temperature condition of 28 ° C to 37 ° C. 2) A process of comparatively analyzing the metabolic products of the mutant strain obtained in the genome reorganization process and the wild-type strain, and identifying the mutant strain that produces a compound that is not produced in the wild-type strain as a strain that produces a compound derived from a dormant gene.

[0038] If a dormant gene cluster involved in the production of a compound not produced in the wild type can be identified and cloned, the dormant gene cluster or a part of it can be introduced into a homogenous or heterogenous host to obtain a strain that produces the compound derived from the dormant gene, which enables more efficient production of the compound derived from the dormant gene. EXAMPLES

[0039] Example 1: Obtaining secondary metabolites from dormant genes by TAQing in filamentous fungi 1. Introduction Gene expression was induced using the amyB promoter, which is constitutively expressed in the presence of starch or maltose, and the enoA gene, which can be constitutively expressed in the glycolytic pathway.

[0040] According to a previous report (Muramoto et al., supra), the transfected strain was cultured and grown, then heat-shocked at 42°C for 4 hours, and then inoculated onto a medium. However, no transformed strains were obtained.

[0041] Based on the conditions for yeast, the taqI gene-introduced strain was cultured under gene expression conditions, and the resulting mycelium was then protoplasted. The protoplast solution was then heat-shocked at 42°C for 1, 3, or 6 hours, and then inoculated onto a medium. However, no TAQing (indicating genome rearrangement) phenomenon, such as changes in survival rate or phenotypic changes, was observed.

[0042] This was thought to be due to differences in the efficiency of repair and reorganization after restriction enzyme digestion due to differences in ploidy between filamentous fungi (haploid) and yeast (diploid). Therefore, the taqI-introduced strain was serially subcultured (15 mL of agar medium in a 9 cm diameter petri dish) at 30°C, the lower limit of restriction enzyme activity, and the emergence of transformed strains was observed.

[0043] 2. Materials and Methods (1)Equipment and equipment used PCR reactions were performed using the oligonucleotide primers shown in Table 1 using a Thermal Cycler LifeECO (Nippon Genetics). The oligonucleotide primers used were purchased from FASMAC Co., Ltd. (Kanagawa, Japan). HPLC analysis was performed using a JASCO-1555-10 Intelligent Sampler, a JASCO PU-4180 RHPLC Pump, and a JASCO MD-4017 Photo Diode Array Detector (JASCO). The analytical column used was a COSMOSIL Packed Column 5C18-MS-II (φ4.6 mm × 150 mm, Nacalai tesque). UPLC-MS analysis was performed using an ACQUITY UPLC H-Class Plus system (Waters) connected to a PDA eλ detector (Waters) and an SQ Detector2 (Waters). The analytical column used was an ACQUITY UPLC® BEH C18 column (130A, 1.7 μm, 2.1 mm × 50 mm; Waters). GC-MS analysis was performed using an EVOQ GC-TQ (Bruker) and a DB-5MS (Agilent) analytical column. MALDI-TOF analysis was performed using an autoflex maX TOF / TOF (Bruker).

[0044] [Table 1]

[0045] (3) DNA manipulation in general PCR was performed using PrimeSTAR® Max DNA Polymerase (Takara Bio). PCR products and linear plasmids were purified using QIAEX® II Gel Extraction Kit (QIAGEN). DNA cloning was performed using E. coli DH5α (Competent Quick, Toyobo) according to the company's protocol, and recombinant plasmids were extracted from E. coli cells using GenElute™ Plasmid Midiprep Kit (Sigma-Aldrich) and Plasmid Midi Kits (Qiagen).

[0046] (4) Transformed host Escherichia coli DH5a (Competent quick, TOYOBO) was used for cloning of the plasmids, and Aspergillus niger IFM 58835 was used as a host for heterologous expression.

[0047] (5) Construction of plasmids for heterologous expression in filamentous fungi (Figure 2) The NLS-taqI (NLS: nuclear localization signal) gene was obtained from the Graduate School of Arts and Sciences, University of Tokyo. The DNA was amplified from pET15b_NLS-TaqI Plasmid provided by Kunifumi Ohta (Figure 3). The NLS-taqI gene was purified and the restriction sites (Kp The NLS-taqI gene-containing plasmid was inserted into the cytoplasmic expression plasmid. The mide was constructed by ligating the NLS-taqI gene to the pUPTRA2 vector digested with KpnI using the In-Fusion (registered trademark) HD ​​Cloning Kit (Takara Bio). The introduction of NLS-TaqI into the constructed vector was confirmed by PCR. The NLS-taqI region of the TAQ1 gene was confirmed by sequence analysis using the primers in Table 1. Sequence analysis was outsourced to FASMAC.

[0048] (6) Transformation of A. niger Transformation of A. niger was carried out using the protoplast-polyethylene glycol (PEG) method as follows: A. niger host strain was cultured on agar medium (PDA supplemented with 0.6 M KCl, 0.05% L-arginine and 0.05% adenine). Spores and mycelium of the host strain were inoculated into 60 mL of Czapek-Dox + Casamino acids medium (3.5% Czapek-Dox broth (Difco), 0.5% Casamino acids vitamin assay (Difco)). After culturing at 30°C for 20 h (reciprocal shaking), the mycelium was collected by filtration and washed with water. The mycelium was incubated with 10 mL of protoplast buffer (5 mg mL-1 Yatalase (Takara Bio), 1 mg mL-1 Cellulase Onozuka R-10 (SERVA Electrophoresis GmbH), 0.6 M KCl, 10 mM phosphate buffer (pH 6.0)). After 3 h of incubation at 30°C, undigested mycelium was removed by filtration through Miracloth (Merck) and centrifuged at 2500 rpm for 5 min to recover protoplasts. The protoplasts were washed with 0.6 M KCl and then resuspended in 0.5 ml of Sol I (0.6 M KCl, 10 mM CaCl 2 The cells were resuspended in 0.2 equivalents of Sol II (40% PEG4000, 50 mM CaCl 2, 50 mM Tris-HCl (pH 8.0)) was added with gentle mixing to bind 10-20 μg of plasmid DNA (<20 μL). After incubation on ice for 40 min, 1 mL of Sol II was added to the protoplast suspension with gentle mixing. After an additional 15 min of incubation at room temperature, 20 mL of Sol I was added and the protoplasts were harvested by centrifugation at 2000 rpm for 5 min. Protoplasts were resuspended in 600 μL of Sol I and the suspension was mixed with pre-solidified soft agar (3.5% Czapek-Dox broth, 0.6 M KCl, 0.6% agar, 0.1 μg / ml pyrithiamine). The protoplast-soft agar mixture was then layered on a CD+Ptr agar plate (3.5% Czapek-Dox broth, 0.6 M KCl, 1.5% agarose, 0.1 μg / ml pyrithiamine). After culturing at 30°C for 2-3 weeks, the resulting cells (transformants) were transferred onto a CD+Ptr agar plate and cultured for recovery.

[0049] (7) Selection of A. niger transformants Transformants were selected based on pyrithiamine resistance.

[0050] (8) Subculture of A. niger transformants and selection of transformed strains (Figure 4) A. niger transformants were transferred to fresh CD+Ptr agar plates approximately every 1-2 weeks and subcultured at 30°C. Transformants were selected based on changes in appearance.

[0051] (9) Confirmation of gene insertion in A. niger transformed strains (Figure 5) Approximately 5 to 10 transformants were obtained per transformation experiment. Therefore, gene insertion in all transformants was confirmed by PCR. Integration of the target gene into A. niger transformants was confirmed by PCR using genomic DNA template and the primers shown in Table 1. Genomic DNA of transformants was extracted as follows; a small piece of mycelium of A. niger strain grown at 30°C for several days was taken from an agar plate and incubated in TE (pH 8.0) buffer at 95°C for 10 minutes. This genomic DNA solution was diluted with water to prepare PCR template of appropriate concentration.

[0052] (10) Cultivation of A. niger transformed strains and metabolite analysis (Figure 6) All transformants that passed the gene integration check were cultured and their metabolite profiles were analyzed by reversed-phase HPLC. Each transformant was inoculated into 30 mL of PDB medium (2.4% Potato dextrose broth (Difco)) and cultured at 30 °C and 150 rpm for approximately 3 weeks (20–22 days). The culture solution (3 mL) was extracted with EtOAc (2 mL), and the organic layer was concentrated using a centrifugal evaporator. The residue was then dissolved in 150 μL of MeOH to prepare samples for reversed-phase HPLC and LC-MS analysis. The lyophilized mycelium (20 mg) was extracted with MeOH (1 mL), and the extract was concentrated using a centrifugal evaporator. The residue was then dissolved in 100 μL of MeOH to prepare samples for HPLC and LC-MS analysis. 10 μL of the sample was used for HPLC and LC-MS analysis. HPLC analysis was performed using a COSMOSIL 5C18 Packed Column (4.6 mm ID × 150 mm, Nacalai Tesque) with a flow rate of 1.0 mL / min of acetonitrile and water containing 0.01% trifluoroacetic acid (0–11.5 min: 20:80, 1.5–11.5 min: linear gradient of 20–100:0, 11.5–20 min: 100:0).

[0053] (11) Antibacterial activity of culture extracts from transformed A. niger strains (Figure 7) Antibacterial activity tests were performed on the culture extracts of the transformed strains and wild-type strains prepared above. 5 μL of a 20 mg / mL solution of each extract was used for antibacterial activity tests using the disk method. First, antibacterial activity against Escherichia Coli DH5α, Staphylococcus aureus, and Mycobacterium smegmatis was performed. Extracts that showed antibacterial activity against Staphylococcus aureus were evaluated for their antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE), carbapenem-resistant Klebsiella pneumoniae (CRE), multidrug-resistant Acinetobacter baumannii (MDRA), and multidrug-resistant Pseudomonas aeruginosa (MDRP).

[0054] (12) Genome extraction of A. niger mutant strains for whole genome sequence analysis (Figure 8) A wild-type strain and transformants of A. niger were cultured on CD agar medium (3.5% Czapek-Dox broth, 0.6 M KCl, 1.5% agarose, 0.1 μg / ml pyrithiamine). Spores and mycelia from the plates were inoculated into 60 mL of Potato dextrose medium (2.4% Potato dextrose broth). After 2-3 days of culture (rotary shaking) at 30°C, mycelia of each strain were collected by filtration, washed with water, and frozen in liquid nitrogen. The frozen mycelia were crushed with a Multi-beads Shocker (YASUI KIKAI), and genomic DNA was extracted from A. niger cells using the DNeasy® Plant Mini Kit (QIAGEN).

[0055] 3.Results When the TaqI gene-introduced strains were subcultured at temperatures ranging from 28 to 32°C, the appearance of strains with different characteristics was observed after an average of two subcultures. Both the amyB and enoA promoters examined in this study yielded transformed strains, and there were no differences between the promoters. A total of five transformations were performed to obtain 47 taqI gene-introduced strains, which were then subcultured. As a result, the appearance of transformed strains was confirmed in about half of the strains. Of these, 20 strains were cultured in liquid culture and the secondary metabolite production profiles were investigated, and about 10 strains showed profiles different from the parent strain. Furthermore, DNA analysis showed that large-scale gene deletions and translocations were observed in two strains (Figure 9), and many strains showed numerous SNPs (Figure 10). These trends were different from the rearrangement trends seen in yeast.

[0056] 4. Discussion In mutants that show a different secondary metabolite production profile from that of the parent strain, it is believed that dormant genes are awakened, producing secondary metabolites that are not produced in the parent strain (wild type). If the dormant gene cluster required for the production of the secondary metabolites can be cloned, it will be possible to produce secondary metabolites derived from dormant genes more efficiently. [Industrial Applicability]

[0057] According to the present invention, dormant genes in filamentous fungi can be awakened, making it possible to develop new pharmaceutical seeds. [Sequence List Free Text]

[0058] SEQ ID NO: 1: primer (Taq1-IFpUKpn1-FW) SEQ ID NO: 2: primer (Taq1-IFpUKpn1-RV2) SEQ ID NO: 3: primer (Taq1-seqR3) SEQ ID NO: 4: primer (pUSA2-RV2) SEQ ID NO: 5: primer (Taq1-IFpUenoA_Kpn1-FW) SEQ ID NO:6: TaqI sequence (Fig.2)

Claims

1. A method for genome reorganization in filamentous fungi, comprising the steps of: A genome reorganization step of expressing a thermostable restriction enzyme derived from a thermophile in a filamentous fungal cell and causing random genetic recombination in the genome in the cell by the restriction enzyme, The method is characterized in that the restriction enzyme is expressed under the control of a constitutive promoter at a temperature condition of 28°C to 37°C.

2. The method according to claim 1, wherein the thermostable restriction enzyme derived from a thermophilic bacterium is one or more selected from the group consisting of BclI, Sse9i, BstUI, TfiI, TseI, Tsp45I, TaqI and PhoI.

3. The method according to claim 1, wherein the thermostable restriction enzyme derived from a thermophilic bacterium is TaqI.

4. The method according to claim 1, wherein the genome reorganization step is carried out for 1 week to 12 weeks.

5. The method of claim 1, wherein the constitutive promoter is any one selected from the group consisting of the TAA gene promoter (amyB), the GLA gene promoter (glaA), the enolase gene promoter (enoA), the glyceraldehyde-3-phosphate dehydrogenase gene promoter (gpdA), the TEF1 gene promoter (TEF1), and the α-glucosidase gene promoter (agdA).

6. Filamentous fungi include Aspergillus spp., Acremonium spp., Alternaria spp., Arthrinium spp., Aureobasidium spp., Beauveria spp., Bipolaris spp., Bjerkandera spp., Ceriporiopsis spp., Chrysosporium spp., Ch. aetomium, Coprinus, Coriolus, Cordyceps, Colletotricum, Cryptococcus, Eupenicillium, Emericella, Filibasidium, Fusarium, Humicola, Magna Humicola, Isaria, Madurella, Magnaporthe, Metarhizium, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Pestalotiopsis, Phanerochaete, Phlebia, Piromyc The method according to claim 1, which is any one selected from the group consisting of the genus Es, Pleurotus, Purepureocillium, Schizophyllum, Stachybotrys, Salaromyces, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma.

7. A method for producing a compound derived from a dormant gene, comprising the steps of: 1) A genome reorganization step of causing random genetic recombination in a genome in a filamentous fungal cell by the method according to any one of claims 1 to 6; 2) A step of comparatively analyzing the metabolic products of the mutant strain obtained in the genome reorganization step and the wild-type strain, and selecting a mutant strain that produces a compound that is not produced in the wild-type strain; and 3) culturing the mutant strain to obtain a compound derived from a dormant gene; A method comprising:

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