JP2016158599A - Recombinant filamentous fungi and methods for producing hollow multimers - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods for accumulating hollow multimers in the culture media utilizing bacteria.SOLUTION: The invention provides a filamentous fungus carrying a heterologous expression unit comprising a polynucleotide encoding a protein that forms a hollow multimer, and a method for producing the hollow multimer comprising culturing the filamentous fungus in a culture medium to accumulate the hollow multimer in the culture medium.SELECTED DRAWING: None

Description

  The present invention relates to a genetically modified filamentous fungus and a method for producing a multimer having a lumen using the same.

  There are two methods for producing functional proteins using microorganisms: a method of accumulating the target protein in the cell and a method of secreting the target protein outside the cell. Examples of production methods for secreting heterologous proteins include bacterium belonging to the genus Bacillus (Non-patent Document 1), methanol-utilizing yeasts such as Pichia pastoris (Non-patent Document 2), Corynebacterium glutamicum, etc. Techniques using coryneform bacteria (Patent Documents 1 to 6) and filamentous fungi (Non-Patent Documents 3 and 4) have been reported. Trichoderma reesei and Acremonium cellulolyticus, which are classified as filamentous fungi, are known to secrete various cellulases in nature, and their functions are applied to cellulase production. (Patent Documents 7 and 8).

  Ferritin forms a cage supermolecular structure having an outer diameter of 12 nm having a lumen having a diameter of about 7 nm by forming a 24-mer composed of monomer units having a molecular weight of about 20 kDa. In this lumen, iron molecules can be stored as iron oxide nanoparticles. Ferritin-like proteins exist universally from animals and plants to microorganisms and are deeply involved in homeostasis of iron elements in living organisms or cells. These ferritins include oxides of metals such as beryllium, gallium, manganese, phosphorus, uranium, lead, cobalt, nickel, and chromium, as well as cadmium selenide, zinc sulfide, iron sulfide, and cadmium sulfide. It has been shown that nanoparticles such as semiconductors and magnetic materials can be artificially stored, and applied research in the field of semiconductor material engineering and medical fields has been actively conducted (Non-Patent Document 5).

U.S. Pat. No. 4,965,197 JP-T 6-502548 Japanese Patent No. 4320769 JP-A-11-169182 Japanese Patent No. 4362651 Japanese Patent No. 4730302 JP 2003-135052 A JP 2008-271927 A

M.M. Simonen and I. Palva, Microbiol. Rev. , 57, 109-137 (1993) James M.M. Cregg et al. Nat. Biotechnol. 11,905-910 (1993) Tove Christensen et al. Nat. Biotechnol. 6, 1419-1422 (1988). Nigel S. Dunn-Coleman et al. Nat. Biotechnol. , 9, 976-981 (1991) I. Yamashita et al. , Biochem Biophys. Acta, 1800, 844-857 (2010)

In protein production systems using Escherichia coli, yeast, insect cells, animal cells or extracts thereof generally used for protein production, other than the target protein contained in the cells used for the synthesis of the target protein. It was necessary to separate and purify the target protein from contaminants (eg, protein, DNA).
Moreover, the target protein accumulated excessively in the cells forms inclusion bodies and the like, and it was difficult to recover the target protein from the inclusion body.
Furthermore, it is also known that an excessively accumulated target protein reduces the ability of a host cell to increase and protein production ability and inhibits the accumulation of the target protein.
In order to avoid such problems, protein secretory production systems using bacteria such as Bacillus bacteria and coryneform bacteria are known. By using such bacteria, multimers having lumens ( It was difficult to accumulate a huge protein complex) in the medium (secretory production of multimers having lumens).

  As far as the present inventors have grasped, it has not been reported that a multimer having a lumen is expressed in a filamentous fungus by a gene recombination technique.

  As a result of intensive studies, the present inventors have succeeded in efficiently accumulating multimers having a lumen (giant protein complex) in a medium by using filamentous fungi as host cells. It came to be completed.

That is, the present invention is as follows.
[1] A filamentous fungus comprising a heterologous expression unit comprising a polynucleotide encoding a protein capable of forming a multimer having a lumen.
[2] The filamentous fungus according to [1], wherein the protein is heterologous to the filamentous fungus.
[3] The filamentous fungus according to [1] or [2], wherein the protein is ferritin.
[4] The filamentous fungus according to any one of [1] to [3], wherein the filamentous fungus belongs to the genus Acremonium.
[5] The filamentous fungus according to [4], wherein the filamentous fungus belongs to Acremonium cellulolyticus.
[6] A method for producing a multimer having a lumen, comprising culturing the filamentous fungus of any one of [1] to [5] in a medium and accumulating the multimer having the lumen in the medium.
[7] The method of [6], comprising recovering a multimer having a lumen from a medium.

  According to the present invention, a multimer having a lumen (a huge protein complex) can be efficiently accumulated in a medium. Therefore, separation and purification of the target multimer from contaminants (eg, protein, DNA) other than the target multimer contained in the cells used for the synthesis of the target multimer can be avoided. In addition, excessive accumulation of the target multimer in the cell, and hence formation of inclusion bodies in the cell can be avoided, so that the target multimer can be recovered more easily. Furthermore, since it is possible to avoid a decrease in host cell's potentiating ability and a decrease in protein production ability due to excessive intracellular accumulation of the target multimer, the production efficiency of the target multimer can be improved.

FIG. 1 is a diagram showing an SDS-PAGE electrophoresis image of a horse-derived ferritin protein contained in a culture supernatant of a recombinant filamentous fungus. FIG. 2 is a diagram showing an SDS-PAGE electrophoresis image of Pyrococcus furiosus-derived ferritin protein contained in the culture supernatant of the recombinant filamentous fungus. FIG. 3 is a view showing a transmission electron microscope image of a horse-derived ferritin protein contained in the culture supernatant of the recombinant filamentous fungus. FIG. 4 is a view showing a transmission electron microscope image of a Pyrococcus furiosus-derived ferritin protein contained in the culture supernatant of the genetically modified filamentous fungus.

  The present invention provides a filamentous fungus comprising a heterologous expression unit comprising a polynucleotide encoding a protein capable of forming a multimer having a lumen.

  The term “expression unit” refers to a predetermined polynucleotide to be expressed as a protein (in the present invention, a polynucleotide encoding a protein capable of forming a multimer having a lumen) and a promoter operably linked thereto. The smallest unit that enables transcription of the polynucleotide, and thus production of the protein encoded by the polynucleotide. The expression unit may further contain elements such as a terminator that functions in filamentous fungi, a ribosome binding site, and a drug resistance gene. The expression unit may be DNA or RNA, but is preferably DNA.

  The term “heterologous expression unit” means that the expression unit is heterologous to the host cell (filamentous fungus in the present invention). Therefore, in the present invention, at least one element constituting the expression unit is heterologous to the filamentous fungus. Examples of the elements constituting the expression unit that are different from those of filamentous fungi include the elements described above. Preferably, one or both of a polynucleotide encoding a protein capable of forming a multimer having a lumen and constituting a heterologous expression unit, or both are heterologous to a filamentous fungus. Accordingly, in the present invention, one or both of a polynucleotide encoding a protein capable of forming a multimer having a lumen, or a promoter, is not a filamentous fungus (eg, prokaryotic and eukaryotic organisms, or microorganisms, Insects, plants, and animals such as mammals) or viruses, or artificially synthesized. Alternatively, the polynucleotide encoding a protein capable of forming a multimer having a lumen may be heterologous to the filamentous fungus. Preferably, the protein capable of forming a multimer having a lumen is heterologous to the filamentous fungus.

  The term “protein capable of forming a multimer having a lumen” refers to a protein having the ability to form a multimer having a space inside by protein association. Some proteins are known as such proteins. For example, such proteins include proteins that can form a 24-mer with a lumen (eg, ferritin), and proteins that can form a multimer with a lumen (eg, 12-mer). Examples of the protein capable of forming a multimer having a lumen include Dps (DNA-binding protein from starved cells) capable of forming a 12-mer having a lumen. Dps forms a cage structure consisting of an outer diameter of 9 nm and a lumen having a diameter of about 5 nm by forming a 12-mer composed of monomer units having a molecular weight of about 18 kDa. Molecules can be stored as iron oxide nanoparticles. Furthermore, in addition to iron, ferritin includes oxides of metals such as beryllium, gallium, manganese, phosphorus, uranium, lead, cobalt, nickel, and chromium, as well as cadmium selenide, zinc sulfide, iron sulfide, and cadmium sulfide. It has been shown that nanoparticles such as semiconductors and magnetic materials can be artificially stored, and application research in the field of semiconductor material engineering and medical fields has been actively conducted (I. Yamashita et al., Biochem Biophys). Acta, 2010, vol. 1800, p.846.). Dps may be referred to as NapA, bacterioferritin, Dlp or MrgA depending on the type of bacteria from which it is derived, and Dps has known subtypes such as DpsA, DpsB, Dps1, and Dps2. (See T. Haikarainen and A. C. Pagepagerion, Cell. Mol. Life Sci., 2010 vol. 67, p. 341). Accordingly, in the present invention, the term “Dps” is intended to include proteins referred to by these aliases. A protein capable of forming a multimer having a lumen may have its N-terminal part and / or C-terminal part exposed on the surface of the multimer.

  Proteins that can form multimers with lumens may be derived from prokaryotes or eukaryotes. Proteins that can form multimers with lumens may also be derived from animals such as microorganisms, insects, plants, and mammals.

In a preferred embodiment, the protein capable of forming a multimer having a lumen may be a protein derived from a microorganism that produces a protein capable of forming a multimer having a lumen. Examples of such microorganisms include Listeria genus, Staphylococcus genus, Bacillus genus, Streptococcus genus, Vibrio genus, Escherichia buru, and Escherichia buru. ), Borrelia, Mycobacterium, Campylobacter, Thermosychococcus, and Deinococcus, Pyrococcus, Pyrococcus, and Pyrococcus (Corynebacterium) genus That bacteria and the like.
Examples of bacteria belonging to the genus Listeria include Listeria innocua and Listeria monocytogenes. Examples of bacteria belonging to the genus Staphylococcus include Staphylococcus aureus. Examples of bacteria belonging to the genus Bacillus include Bacillus subtilis. Examples of bacteria belonging to the genus Streptococcus include Streptococcus pyogenes and Streptococcus suis. Examples of bacteria belonging to the genus Vibrio include Vibrio cholerae. Examples of bacteria belonging to the genus Escherichia include Escherichia coli. Examples of the bacterium belonging to the genus Brucella include Brucella Melitensis. Examples of bacteria belonging to the genus Borrelia include Borrelia burgdorferi. Examples of the bacterium belonging to the genus Mycobacterium include Mycobacterium smegmatis (Mycobacterium smegmatis). Examples of bacteria belonging to the genus Campylobacter include Campylobacter jejuni. Examples of the bacterium belonging to the genus Thermocinecococcus include Thermocinecococcus elongatas. Examples of the bacteria belonging to the genus Deinococcus include Deinococcus Radiodurans. Examples of bacteria belonging to the genus Pyrococcus include Pyrococcus furiosus. Examples of bacteria belonging to the genus Corynebacterium include Corynebacterium glutamicum.

  In another preferred embodiment, the protein capable of forming a multimer having a lumen may be a protein derived from an animal such as a mammal that produces a protein capable of forming a multimer having a lumen. Mammals include, for example, humans, mice, rats, guinea pigs, rabbits, cows, horses, goats, and pigs.

  It is also preferable to use a heat-resistant protein as a protein capable of forming a multimer having a lumen. Examples of such heat-resistant proteins include proteins derived from thermophilic microorganisms such as Pyrococcus bacteria such as Pyrococcus furiosus. By using a thermostable protein, recovery of a multimer having a lumen from a medium is facilitated.

A protein capable of forming a multimer with a lumen may further be a naturally occurring protein, or a variant thereof capable of forming a multimer with a lumen. Examples of such mutants include substitution, deletion, insertion and / or 1 or several (eg, 1 to 20, 1 to 15, 1 to 10 or 1 to 5) amino acid residues. Or a variant with an addition. Those skilled in the art can easily produce such mutants based on the disclosure in the present specification and the common general technical knowledge in the field.
For example, a protein capable of forming a multimer having a lumen may be one in which a peptide moiety capable of binding to a target substance is added to its N-terminus and / or C-terminus. The term “peptide moiety capable of binding to a target substance” refers to a moiety having a peptide having affinity for an arbitrary target substance and capable of binding to the target substance. Since various peptides having affinity for a target substance are known, in the present invention, a portion having such a peptide can be used as the peptide portion. The peptide moiety capable of binding to the target substance may have only one peptide having affinity for any target substance, or the same or different species having affinity for any target substance. May have a plurality of peptides. Examples of the target substance include inorganic materials and organic materials, or conductor materials, semiconductor materials, and magnetic materials. Specifically, examples of such target substances include metal materials (eg, metals such as titanium and niobium, and metal compounds such as metal oxides), silicon materials (eg, silicon compounds such as silicon and silicon oxide). ), Carbon materials (eg, carbon nanomaterials such as carbon nanotubes, carbon nanohorns), low molecular compounds (eg, biological substances such as porphyrins, radioactive substances, fluorescent substances, dyes, drugs), polymers (eg, hydrophobic organic polymers) Or conductive polymer), protein (eg, oligopeptide or polypeptide), nucleic acid, carbohydrate, and lipid.
A protein capable of forming a multimer having a lumen may also have a secretion signal at the N-terminus of the protein capable of forming a multimer having a lumen. The secretion signal is not particularly limited as long as it can act in a filamentous fungus, for example, but a signal sequence of a gene derived from a filamentous fungus is preferable. Examples of such signal sequences include signal sequences derived from genes such as cellobiohydrolase genes (eg, cbhI, cbhII), endoglucanase genes, β-glucosidase genes, and amylase genes. When a protein capable of forming a multimer having a lumen has a secretion signal, the protein capable of forming a multimer having a lumen, to which a secretion signal is added, is expressed in a filamentous fungus. Can be cleaved, so that a multimer having a lumen without a secretion signal can be accumulated in the medium.

  The promoter constituting the heterologous expression unit is not particularly limited as long as the protein encoded by the polynucleotide linked downstream thereof can be expressed in filamentous fungi. For example, the promoter may be the same or different from the filamentous fungus. For example, a configuration or an inducible promoter that is widely used for production of recombinant proteins can be used. Examples of such promoters include PhoA promoter, PhoC promoter, T7 promoter, T5 promoter, T3 promoter, lac promoter, trp promoter, trc promoter, tac promoter, PR promoter, PL promoter, SP6 promoter, arabinose inducible promoter, cold A shock promoter and a tetracycline inducible promoter are mentioned. Preferably, a promoter having a strong transcription activity in filamentous fungi can be used. Examples of the promoter having a strong transcription activity in filamentous fungi include a promoter of a gene highly expressed in the filamentous fungus and a promoter derived from a virus. Examples of promoters of genes that are highly expressed in filamentous fungi include promoters of genes such as cellobiohydrolase genes (eg, cbhI, cbhII), endoglucanase genes, β-glucosidase genes, and amylase genes. Examples of virus-derived promoters include SV40 promoter and CMV promoter, which are known to be highly expressed in filamentous fungi.

  Filamentous fungi are microorganisms that are active in a mycelial state, and are generally called “mold”. Filamentous fungi are used in the production of fermented foods (alcohol, soy sauce) and pharmaceuticals such as antibiotics. The filamentous fungus used in the present invention is not particularly limited as long as it can secrete a multimer having a lumen (a complex of a protein encoded by a polynucleotide in a heterologous expression unit). Examples of such filamentous fungi include, for example, Acremonium / Talaromyces, Trichoderma, Aspergillus, Neurospora, Fusarium, Bacteria belonging to the genus Chrysosporium, Humicola, Emericella, and Hypocrea are preferable, and Acremonium bacteria such as Acremonium cellulolyticus are preferred. The filamentous fungus may be an original strain or a strain derived from the original strain (eg, a strain having an exogenous gene introduced, a creA mutation (eg, disruption), etc.). .

  The heterologous expression unit is contained in the filamentous fungus in a form incorporated in the genomic DNA of the filamentous fungus or in a form not incorporated in the genomic DNA of the filamentous fungus (eg, in the state of an expression vector). A filamentous fungus containing a heterologous expression unit can be obtained by transforming a filamentous fungus with an expression vector according to a conventionally known method (eg, protoplast method). If the expression vector is an integrative vector that produces homologous recombination with the filamentous fungal genomic DNA, the heterologous expression unit can be incorporated into the genomic DNA of the filamentous fungus by transformation. On the other hand, when the expression vector is a non-integrated vector that does not cause homologous recombination with the genomic DNA of the filamentous fungus, the heterologous expression unit is not incorporated into the genomic DNA of the filamentous fungus by transformation. It can exist independently from genomic DNA.

  The expression vector used in the present invention may further contain elements such as a terminator that functions in filamentous fungi, a ribosome binding site, and a drug resistance gene in addition to the above-mentioned minimum unit as a heterologous expression unit. Examples of drug resistance genes include resistance genes for drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.

  The expression vector may further comprise a region allowing homologous recombination with the filamentous fungal genome for homologous recombination with the filamentous fungal genomic DNA. For example, an expression vector may be designed such that the heterologous expression unit contained therein is located between a pair of homologous regions (eg, homology arms homologous to a specific sequence in the filamentous fungal genome, loxP, FRT). Good. The genome region (target of homologous region) of the filamentous fungus into which the heterologous expression unit is to be introduced is not particularly limited, but may be a locus of a gene having a high expression level in the filamentous fungus. Examples of such genes include cellobiohydrolase genes (eg, cbhI, cbhII), endoglucanase genes, β-glucosidase genes, and amylase genes.

  The expression vector may be a plasmid, viral vector, phage, integrative vector, or artificial chromosome. An integrative vector may be a type of vector that is integrated entirely into the genome of the host cell. Alternatively, the integrative vector may be a vector in which only a part (eg, a heterologous expression unit) is integrated into the genome of the host cell. The expression vector may further be a DNA vector or an RNA vector (eg, retrovirus). As the expression vector, a commonly used expression vector may be used. Examples of such expression vectors include pUC (eg, pUC19, pUC18), pSTV, pBR (eg, pBR322), pHSG (eg, pHSG299, pHSG298, pHSG399, pHSG398), RSF (eg, RSF1010), pACYC ( Examples include pACYC177, pACYC184), pMW (eg, pMW119, pMW118, pMW219, pMW218), pQE (eg, pQE30), and derivatives thereof.

  The present invention also provides a method for producing a multimer having a lumen. The method of the present invention comprises culturing the filamentous fungus of the present invention in a medium and accumulating a multimer having a lumen in the medium.

  The culture can be performed in any medium, but a liquid medium is preferred. The culture temperature is, for example, 15 to 42 ° C, preferably 20 to 37 ° C. The culture of the filamentous fungus can be performed, for example, at pH 5.5 to 8.5, preferably pH 6-8. Under such conditions, the filamentous fungus grows sufficiently, so that a large number of multimers having lumens can be obtained. The culture can be performed, for example, for about 1 day to 1 week.

  As the liquid medium, any liquid medium used for culturing filamentous fungi can be used. For example, examples of the liquid medium include those containing carbohydrates such as glucose, fructose, glycerol and starch as a carbon source. The liquid medium may also contain an inorganic or organic nitrogen source (eg, ammonium sulfate, ammonium chloride, casein hydrolyzate, yeast extract, polypeptone, bactotryptone, etc.). The liquid medium may further contain other nutrient sources such as inorganic salts (eg, sodium or potassium diphosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride), vitamins (eg, vitamin B1). ), May contain antibiotics.

  The method of the present invention may further comprise recovering a multimer having a lumen from the culture medium. For example, such recovery involves removing the filamentous fungus of the present invention from the culture medium by centrifuging the medium containing the filamentous fungus of the present invention and the multimer having the lumen, and then including the multimer having the lumen. It can be performed in a medium. When a heat-resistant protein is used as a protein that can form a multimer having an inner lumen, a protein other than the heat-resistant protein contained in the medium can be denatured by heating the medium. Therefore, in such a case, by using a denatured protein separation method (eg, centrifugation), a multimer having a lumen and composed of a heat-resistant protein can be easily purified. A multimer having a lumen can be highly purified by appropriately combining known methods such as salting-out and various types of chromatography (eg, ion exchange chromatography, gel filtration chromatography).

  A multimer having a lumen is useful, for example, in the development of devices and materials excellent in photocatalytic activity or electrical characteristics. Specifically, the multimer having a lumen is useful as a material or a component in the production of a photoelectric conversion element (eg, a solar cell such as a dye-sensitized solar cell), a hydrogen generation element, or a semiconductor memory element. The filamentous fungus of the present invention and the method of the present invention using the same can improve the productivity of multimers having lumens. Therefore, the filamentous fungus and method of the present invention are useful for the development of devices and materials having excellent photocatalytic activity or electrical characteristics, for example.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

Example 1: Construction of a template plasmid for chromosome recombination of filamentous fungi Plasmid plasmid for recombination template of chromosomal DNA of Acremonium cellulolyticus (also known as Talaromyces cellulolyticus), which is a filamentous fungus As A., which is a filamentous fungus. a pyrF (ura5) gene encoding orotate phosphoribosyl transferase derived from cellulolyticus and A plasmid (pUC-pyrF-cbhI) loaded with a promoter, extracellular secretion signal and terminator of the cbhI gene encoding cellulolyticus-derived cellobiohydrolase I was constructed.

  A. Cell bodies were collected from a colony of cellulolyticus TN strain (FERM BP-685, Japanese Patent Publication No. 61-162167) with a toothpick and suspended in 50 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The solution heat-treated at 95 ° C. for 10 minutes and the primers (SEQ ID NOs: 5 and 6) were mixed and subjected to PCR to amplify the region including 1 kb above and below the pyrF gene. Amplification was performed using KOD FX (TOYOBO, Japan) as a PCR enzyme. The obtained PCR product was ligated according to a standard protocol using pGEM T-easy vector (Promega, USA). In the DNA solution obtained, E. E. coli JM109 was transformed and cultured in an LB agar medium (containing 100 mg / L ampicillin) at 37 ° C. overnight to form colonies. Plasmid extraction was performed from the transformant that formed the colonies using QIAprep Spin Miniprep Kit (QIAGEN, USA), and the base sequence of the DNA mounted on pGEM T-easy vector was decoded. A plasmid whose base sequence was confirmed to be the target was cleaved with EcoRI and DraI, and a pyrF fragment (2.9 kbp) was obtained by agarose gel excision purification using QIAEXII Gel Extraction Kit (QIAGEN, USA). The pyrF fragment has EcoRI cleavage sites at both ends, and the pyrF fragment can be excised from the vector by digesting the pGEM T-easy vector carrying the pyrF fragment with EcoRI. However, the size of the pyrF fragment obtained by digesting the vector with EcoRI and the size of the pGEM T-easy vector fragment were equivalent, and it was expected that the two would be difficult to distinguish by electrophoresis for agarose gel excision purification. DraI was added to fragment only the sequence.

  Subsequently, the A. cerevisiae extracted using MO Bio Ultra Clean Sol DNA kit (MO BIO Laboratories, USA). The region (4.6 kbp) from the promoter to the terminator of the cbhI gene was amplified by PCR using the genomic DNA extraction of cellulolyticus TN strain as a template and primers (SEQ ID NOs: 7 and 8). As the PCR enzyme, KOD FX (TOYOBO, Japan) was used. This PCR product was ligated to pUC19 (Takara Bio Inc., Japan) using an In-Fusion Advantage PCR cloning kit (Takara Bio Inc., Japan) according to a standard protocol by cloning enhancer treatment. In the ligated DNA solution, E. coli was used. E. coli JM109 was transformed and cultured in an LB agar medium (containing 100 mg / L ampicillin) at 37 ° C. overnight to form colonies. Plasmid extraction was performed from the transformant that formed the colony using QIAprep Spin Miniprep Kit (QIAGEN, USA). The plasmid was linearized by cutting with the restriction enzyme EcoRI and dephosphorylated using Alkaline Phosphatase (Calf intestine) (Takara Bio Inc., Japan). Like the DNA fragment, pUC19-cbhI (7.3 kbp) linearized with EcoRI and the pyrF fragment (2.9 kbp) were mixed at a molar ratio of 1: 3, and Ligation High (Takara Bio Inc., Japan) Ligation was performed according to standard protocols. E. coli with the DNA solution. E. coli JM109 was transformed and cultured in an LB agar medium (containing 100 mg / L ampicillin) at 37 ° C. overnight to obtain a transformant. Using the QIAprep Spin Miniprep Kit (QIAGEN, USA), an expression plasmid (pUC-pyrF-cbhI) carrying the pyrF gene, the cbh1 gene and its regulatory promoter was extracted from the transformant. In addition, those in which the ORFs of the pyrF gene and the cbhI gene are reversed were selected. Construction of a template plasmid for recombination of each target gene (ferritin gene to which the secretory signal sequence of the cbhI gene was added) by mounting the target gene in the downstream region of the secretory signal of the cbhI gene of this pUC-pyrF-cbhI did.

Example 2: DNA preparation for the construction of genetically modified filamentous fungi for ferritin expression DNA encoding horse-derived ferritin (SEQ ID NO: 1, 2) or archaeon Pyrococcus furiosus-derived ferritin (SEQ ID NO: 3, 4) In order to construct a filamentous fungus, Acremonium cellulolyticus, inserted into the chromosome, plasmids for gene recombination were constructed by the following procedures.

  First, a template plasmid for gene recombination (pUC-fer-0-pyrF) carrying a fer-0 gene (SEQ ID NO: 1) encoding a ferritin protein derived from a horse was constructed. PCR product amplified using pUC-pyrF-cbhI as a template and primers (SEQ ID NOs: 9, 10) and pMK2-fer-0 (K. Iwahori, K. Yoshizawa, M. Muroka, I. Yamashita, Inorg. Chem) 2005, 44, 6393.) as a template and PCR products amplified with primers (SEQ ID NOs: 11 and 12), respectively, were purified by Wizard SV Gel and PCR Clean-Up System (Promega, USA). In order to fuse the purified PCR product using In-Fusion HD Cloning Kit (Takara Bio Inc., Japan), a PCR product using pUC-pyrF-cbhI as a template DNA and pMK2-fer-0 as a template DNA In-Fusion enzyme mix was added to the solution in which the PCR products were mixed at 3: 2, and the mixture was allowed to stand at 50 ° C. for 15 minutes. JM109 was transformed with the DNA solution, and colonies were formed by culturing overnight at 37 ° C. in an LB agar medium (containing 100 mg / L ampicillin). PUC-fer-0-pyrF was recovered from the bacteria that formed the colonies using Wizard Plus Minipreps System (Promega, USA).

  P.P. A template plasmid for gene recombination (pUC-PfFn-pyrF) carrying a gene encoding a ferriotin protein derived from furiosus (SEQ ID NO: 3, hereinafter referred to as PfFn gene) was also constructed. PCR product amplified using pUC-pyrF-cbhI as a template (SEQ ID NOs: 9, 10) and a primer (SEQ ID NO: 13, 14) using PfFn gene DNA fully synthesized by GenScript (USA) as a template The PCR products amplified in (1) were purified by Wizard SV Gel and PCR Clean-Up System (Promega, USA). In order to fuse the purified PCR product using In-Fusion HD Cloning Kit (Takara Bio Inc., Japan), a PCR product using pUC-pyrF-cbhI as a template and a PCR product using PfFn as a template 9: In-Fusion enzyme mix was added to the solution mixed in 5 and allowed to stand at 50 ° C. for 15 minutes. JM109 was transformed with the DNA solution, and colonies were formed by culturing overnight at 37 ° C. in an LB agar medium (containing 100 mg / L ampicillin). PUC-PfFn-pyrF was recovered from the bacteria that formed the colony using Wizard Plus Minipreps System (Promega, USA).

  Finally, A. of the gene encoding each ferritin. A DNA fragment used for integration into cellulolyticus chromosomal DNA was prepared. Specifically, PCR was carried out using pUC-fer-0-pyrF or pUC-PfFn-pyrF as a template and PCR using M13 primers (SEQ ID NOs: 15 and 16). The PCR products were purified by agarose electrophoresis and Wizard SV Gel and PCR Clean-Up System (Promega, USA), respectively, thereby obtaining 10 mg of purified DNA.

Example 3: Construction of a parent strain of a recombinant filamentous fungus for expressing a heterologous protein A parent strain F09ΔcreA of a strain expressing a heterologous protein such as ferritin was constructed by the following procedure.

A. cellulolyticus strain S6-25 (NITE BP-01685, hereinafter referred to as S6-25 strain) was converted to 5 g / L Solkafloc (International Fiber Corp, North Tonawanda, NY, USA), 24 g / L KH ₂ PO. ₄ , 5 g / L (NH ₄ ) ₂ SO ₄ , 2 g / L Urea, 1.2 g / L MgSO ₄ .7H ₂ O, 0.01 g / L ZnSO ₄ .7H ₂ O, 0.01 g / L MnSO _4. A medium containing 5H ₂ O, 0.01 g / L CuSO ₄ .5H ₂ O, and 20 g / L Bacto Agar was inoculated and cultured at 30 ° C. One agar disk obtained by punching the vicinity of the end of the colony formed on the agar medium with a straw was used for 40 g / L solka floc, 10 g / L Bacto petone, 6 g / L KNO ₃ , 2 g / L Urea, 1. A medium (pH 4.0) containing 6 g / L KCl, 1.2 g / L MgSO ₄ .7H ₂ O, 12 g / L KH ₂ PO ₄ was inoculated, and swirl culture was performed at 30 ° C. and 220 rpm for 10 to 11 days. . Next, 5 mL of the culture solution was filtered with a glass filter (pore size: 40 to 100 μm) to remove the remaining solka floc and long mycelia. The obtained filtrate is centrifuged (at 5000 rpm for 3 minutes) to collect mycelia, and the obtained mycelium contains 0.1% Tween 80, 0.05% MgSO ₄ .7H ₂ O, 0.5% NaCl. The washing operation of suspending in the solution and centrifuging the suspension (at 5000 rpm for 3 minutes) was performed twice. The cell suspension after washing is appropriately diluted so that the turbidity (OD 660 nm) is 1.0, 1 mL is dispensed into a petri dish (bottom diameter: about 35 mm), and ultraviolet light is used using a 15 W germicidal lamp. Was irradiated. The cell suspension after irradiation is appropriately diluted, and a minimal medium (10 g / L Glucose, 10 mM NH ₄ Cl, 10 mM KH ₂ containing 1.2 g / L 5-fluoroorotic acid, 1 g / L Uracil, 1 g / L Uridine is used. _{PO 4, 7mM KCl, 2mM MgSO} 4, 0.06mg / L H 3 BO 3, 0.26mg / L (NH 4) 6 Mo 7 O 24 · 4H 2 O, 1mg / L FeCl 3 · 6H 2 O, 0 ₄ mg / L CuSO ₄ .5H ₂ O, 0.08 mg / L MnCl ₂ , 2 mg / L ZnCl ₂ , 20 g / L Bacto Agar), and cultured at 30 ° C. Here, 5-fluoroic acid is an analog of the intermediate of the uracil biosynthesis pathway, and is toxic to strains in which the uracil biosynthesis pathway functions normally. Therefore, by selecting with a medium containing 5-fluoroorotic acid, it is possible to obtain a strain that has entered the uracil biosynthetic pathway and has stopped functioning. A mutant grown in a medium containing 5-fluoroorotic acid is transferred to a minimal medium containing 1 g / L Uracil, 1 g / L Uridine, and a minimal medium not containing these, and the minimal containing 1 g / L Uracil, 1 g / L Uridine. The strain that grew only on the medium was designated F09 strain. In general, it has been known that in strains exhibiting 5-fluoroorotic acid resistance, it is highly possible that a mutation has occurred in the pyrF gene or the pyrG gene encoding orotidine 5′-phosphate decarboxylase and has no function. Therefore, the nucleotide sequence of these regions on the chromosome of F09 strain was determined. As a result, in the F09 strain, the 629th base of the pyrF gene (SEQ ID NO: 17) was changed from A to G, and it was presumed that the pyrF gene product did not function due to this mutation.

  Using this F09 strain as a parent strain, a disrupted strain of the creA gene (SEQ ID NO: 18) was constructed by the following procedure. The creA gene is a gene encoding a transcription factor involved in catabolite repression. The creA gene is known to be involved in cellulase expression in filamentous fungi (Mol Gen Genet. 1996 Jun 24; 251 (4): 451-60, Biosci Biotechnol Biochem. 1998 Dec; 62 (12 ): 2364-70). First, A.I. A creA gene disruption DNA fragment having a sequence linked in the order of cellullyticus creA gene upstream region, hygromycin resistance gene, and creA gene downstream region was prepared according to the following procedure. A. Using the cellulolyticus genomic DNA as a template, the upstream region of the creA gene was amplified by PCR using primers (SEQ ID NOs: 19 and 20), and the downstream region of the creA gene was amplified by PCR using primers (SEQ ID NOs: 21 and 22). In addition, the hygromycin resistance gene was amplified by PCR using pcDNA3.1 / Hygro (+) (Life Technologies) carrying the hygromycin resistance gene as a template and primers (SEQ ID NOs: 23 and 24). The PCR product was purified using Wizard SV Gel and PCR Clean-Up System (Promega). The purified PCR product was incorporated into the pUC19 plasmid attached to the kit using In-Fusion HD Cloning Kit (Takara Bio Inc., Japan) and ligated. The ligation product is E. coli. E. coli JM109 was transformed and cultured in an LB agar medium (containing 100 mg / L ampicillin) at 37 ° C. overnight to form colonies. The pUC-creA :: hyg plasmid was obtained from the obtained transformant using Wizard Plus Miniprep System (Promega). A DNA fragment for creA gene disruption was amplified using pUC-creA :: hyg plasmid as a template and primers (SEQ ID NOs: 19 and 22), and concentrated and purified by ethanol precipitation.

Next, the F09 strain was inoculated into a medium containing 12 g / L Potato Dextrose Broth (Difco) and 20 g / L Bacto Agar (Difco) and cultured at 30 ° C. One agar disk in which the vicinity of the end of the colony formed on the agar medium was punched with a straw was inoculated into a medium containing 24 g / L Potato Dextroze Broth and swirled at 30 ° C. and 220 rpm for 2 days. The bacterial cells were collected by centrifugation (5000 rpm, 5 minutes), and 30 ml of a solution (pH 6.0) containing 10 g / L Yatalase (Takara Bio Inc., Japan), 10 mM KH ₂ PO ₄ , 0.8M NaCl was added and shaken. Then, the reaction was carried out at 30 ° C. for 2 hours, and the cell wall was digested and protoplasted. After removing the residue with a glass filter, the protoplasts were collected by centrifugation (2000 rpm, 10 minutes) and suspended in 1 ml with Tris-HCl buffer (pH 7.5) containing 1.2 M Sorbitol, 10 mM CaCl _2. A solution was prepared. To 200 μl of this protoplast solution, 50 μl of the previously prepared creA gene disruption DNA fragment 10 mg and 400 g / L PEG4000, 10 mM CaCl ₂ containing Tris-HCl buffer (pH 7.5) was added and left on ice for 30 minutes. Thereafter, 1 ml of Tris-HCl buffer (pH 7.5) containing 400 g / L PEG 4000 and 10 mM CaCl ₂ was added and mixed, and the mixture was left at room temperature for 15 minutes for transformation. After standing, the protoplasts recovered by centrifugation (2000 rpm, 10 minutes) are seeded in a minimal medium containing 1 g / L Uracil, 1 g / L Uridine and 1 M Sucrose, and cultured at 30 ° C. for 1 day, then 0.5 g / L. A medium containing Hygromycin B, 24 g / L Potato Dextrose Broth, 7 g / L Bacto Agar was overlaid, and further cultured at 30 ° C. for 3 days to select the target strain. The emerged colonies were inoculated into a minimal medium containing 0.5 g / L Hygromycin B and cultured at 30 ° C. for 4 days. After confirming that the creA gene region was replaced with the hygromycin resistance gene, the F09-derived creA-disrupted strain (F09 ΔcreA strain) was obtained.

Example 4: Construction of a genetically modified filamentous fungus for ferritin expression A protoplast solution of the F09 ΔcreA strain was prepared in the same manner as in Example 3, and the horse-derived ferritin constructed in Example 2 was mounted on 200 μl of the protoplast solution. 10 mg of purified DNA or P.I. Transformation was performed in the same manner as in Example 3 using 10 mg of purified DNA loaded with furiosus-derived ferritin. After the transformation reaction, protoplasts recovered by centrifugation (2000 rpm, 10 minutes) were seeded in a minimal medium containing 1M sucrose. This bacterium was cultured at 30 ° C. for 4 days, and the target strain was selected based on the ability to grow on a medium not containing uracil. Here, since the F09 ΔcreA strain has a point mutation in the pyrF gene, the pyrF gene product does not function and cannot grow in a medium not containing uracil. However, the DNA fragment used for gene recombination carries a wild type pyrF gene together with each ferritin gene, and by incorporating this DNA fragment into genomic DNA, the F09 ΔcreA strain can grow even in a medium not containing uracil. become. The emerged colony was inoculated into a minimal medium and cultured at 30 ° C. for 4 days, and then colony PCR was performed to confirm that the transformed fragment was inserted on the genomic DNA, and the equine-derived ferritin-expressing gene recombinant filamentous strain and P. A furiosus-derived ferritin-expressing gene recombinant filamentous strain was obtained. For confirmation of equine-derived ferritin-expressing gene recombinant filamentous strains, primers (SEQ ID NOs: 25 and 26), For confirmation of the furiosus-derived ferritin-expressing gene recombinant filamentous strain, colony PCR was performed with a combination of primers (SEQ ID NOs: 25 and 27). The resulting filamentous fungus (A. cellulolyticus) is equine or P. DNA encoding furiosus-derived ferritin (extracellular secretion signal of cbhI gene added to the N-terminus of ferritin) and a heterologous expression unit comprising a cbhI promoter.

Example 5: Production of ferritin protein using a ferritin-expressing gene recombinant filamentous strain (LA6652 p192, LA7155p86, 87)
Each ferritin-expressing gene recombinant filamentous strain was inoculated into a medium containing 12 g / L Potato Dextrose Broth (Difco) and 20 g / L Bacto Agar (Difco), and cultured at 30 ° C. One agar disk obtained by punching out the vicinity of the tip of the mycelia extended on the agar medium with a straw is 20 mL of 50 g / L Solka Floc, 24 g / L KH ₂ PO ₄ , 5 g / L (NH ₄ ) ₂ SO _4. 3 g / L Urea, 1 g / L Tween 80, 1.2 g / L MgSO ₄ .7H ₂ O, 0.01 g / L ZnSO ₄ .7H ₂ O, 0.01 g / L MnSO ₄ .5H ₂ O, 0.01 g / L Inoculated into a liquid medium containing CuSO ₄ .5H ₂ O and cultured at 30 ° C. and 220 rpm for 7 days. The obtained culture solution was centrifuged (15000 rpm for 15 minutes), and the supernatant containing the protein was collected. Contaminants contained in the supernatant were removed by centrifugation (15000 rpm, 5 minutes), and then left at 60 ° C. for 20 minutes. The solution was centrifuged (15000 rpm, 5 minutes), and the supernatant was collected. The protein contained in the soluble fraction after the heat treatment was concentrated using an ultrafiltration membrane (Amicon Ultra-0.5 mL, 10 kDa, Merk Millipore, USA), and the solvent of the protein solution was replaced with water. And the protein contained in each fraction obtained by centrifugation and heat processing was confirmed by SDS-PAGE. Further, the protein concentration in the solution was determined by the Lowry method.

  FIG. 1 shows an SDS-PAGE electrophoresis image of a culture supernatant obtained by culturing a horse-derived ferritin-expressing gene recombinant filamentous strain. Lane 1 is the culture supernatant, Lane 2 is the contaminant obtained by centrifuging the culture supernatant, Lane 3 is the culture supernatant from which the contaminant has been removed, and Lane 4 is the culture supernatant from which the contaminant has been removed. The suspension of the heat-treated sample was suspended in Laemmli sample buffer (Bio-Rad, USA) in Lane 4 and the culture supernatant from which the culture supernatant from which contaminants had been removed was suspended. After heating for a minute, electrophoresis was performed. A band could be confirmed in the vicinity of the molecular weight (20,000) of the equine-derived ferritin monomer from the SDS-PAGE electrophoresis image (arrow in FIG. 1). And it was suggested that the protein exists in the soluble fraction even after heat treatment. The concentration of protein present in the soluble fraction after heat treatment was 1.9 mg / ml.

  FIG. The SDS-PAGE electrophoresis image of the culture supernatant which cultured the furiosus origin ferritin expression gene recombinant filamentous strain is shown. Lane 1 is the culture supernatant, Lane 2 is the contaminant obtained by centrifuging the culture supernatant, Lane 3 is the culture supernatant from which the contaminant has been removed, and Lane 4 is the culture supernatant from which the contaminant has been removed. The suspension of the heat-treated sample was suspended in Laemmli sample buffer (Bio-Rad, USA) in Lane 4 and the culture supernatant from which the culture supernatant from which contaminants had been removed was suspended. After heating for a minute, electrophoresis was performed. P. A band can be confirmed in the vicinity of the molecular weight (20,300) of the monomer of furiosus-derived ferritin (arrow in FIG. 2), suggesting that the protein exists in the soluble fraction even after heat treatment. The concentration of protein present in the soluble fraction after heat treatment was 3.3 mg / ml.

Example 6: Evaluation of ferritin protein using genetically modified filamentous fungi If the protein contained in the protein solution after heat treatment is ferritin, a multimer (24-mer) having a lumen is formed, and a transmission electron microscope ( A TEM) analysis should confirm the particulate structure. Therefore, the shape of the protein contained in each protein solution in which the solvent was replaced with water was stained with 3% phosphotungstic acid (PTA) and analyzed by TEM (JEM2200-FS, 200 kV).

  As a result, equine-derived ferritin-expressing gene recombinant filamentous strain and The supernatant of the furiosus-derived ferritin-expressing gene recombinant filamentous strain contained a large number of particulate proteins having a diameter of 12 nm. In the particulate protein, the center was observed to be black, and it was also observed that the nanoparticle considered to be derived from tungsten was included. From this result, the soluble fraction after the heat treatment contains ferritin derived from horses (FIG. 3) and P. a. It was found that a large amount of furiosus-derived ferritin (FIG. 4) was contained. Further, from the TEM image, almost no structures other than the particulate protein were observed, suggesting that the amount of other contaminating proteins was small.

  From the above, this technique using a recombinant filamentous fungus is excellent in productivity of multimers having lumens, and can easily purify multimers having lumens by simple methods such as centrifugation and heat treatment. Indicated.

Claims (7)

  A filamentous fungus comprising a heterologous expression unit comprising a polynucleotide encoding a protein capable of forming a multimer having a lumen.   2. The filamentous fungus of claim 1, wherein the protein is heterologous to the filamentous fungus.   The filamentous fungus according to claim 1 or 2, wherein the protein is ferritin.   The filamentous fungus according to any one of claims 1 to 3, wherein the filamentous fungus belongs to the genus Acremonium.   The filamentous fungus according to claim 4, wherein the filamentous fungus belongs to Acremonium cellulolyticus.   A method for producing a multimer having a lumen, comprising culturing the filamentous fungus according to any one of claims 1 to 5 in a medium, and accumulating the multimer having the lumen in the medium.   7. The method of claim 6, comprising recovering the multimer having the lumen from the medium.