JP4674343B2 - How to make filamentous fungi that produce spores with low viability - Google Patents

Description

  The present invention relates to the production of filamentous fungi that have reduced the viability of spores (conidia), and are particularly suitable for biohazard countermeasures. More specifically, the present invention uses genetic engineering techniques. The present invention relates to a filamentous fungus that produces conidia with reduced viability, a method for producing the transformed filamentous fungus, and a method for reducing the viability of the filamentous fungus conidia.

  Filamentous fungi have a high protein secretion ability and are used in the production of various substances by fermentation. In recent years, transformants using these filamentous fungi as a host have been created, and further useful strains have been created. In general, the safety of transformants remains unconfirmed, but within the designated control area. It is strictly controlled.

However, conidia of filamentous fungi are likely to scatter in the air, and a great deal of labor is required to keep them in a closed environment. In the unlikely event that they enter the environment, the transformants of the filamentous fungi can easily proliferate, and these transformed filamentous fungi are thought to expand the breeding range by forming further spores. Hazard countermeasures are an urgent issue in the industry (for example, see Non-Patent Document 1).
Biotechnology Encyclopedia, CMC Corporation, p. 822-823 (October 9, 1986)

  The present invention was made in order to develop a technique for reducing and preventing biohazard as much as possible, and as a result of intensive studies from various directions, it is only necessary to kill filamentous fungal spores scattered outside the management area. Therefore, we focused on the point that it was necessary to reduce the stress resistance of the spores of the filamentous fungi that became the host for transformation.

  Therefore, the problem to be solved by the present invention is to make it easier to kill filamentous fungal spores scattered outside the management area by reducing the stress resistance of the spores of the filamentous fungi serving as the transformation host.

  The present invention has been made for the purpose of solving the above-mentioned problems. In the process of analyzing transcriptional regulatory factor genes (Atf gene group), the atfB gene of Aspergillus oryzae was deleted. It was found for the first time that the viability of E. coli is reduced, and furthermore, it was confirmed for the first time that this atfB gene-deficient strain could easily be killed when subjected to stress such as ultraviolet rays, and the results of further research based on these useful new findings It is finally completed.

  That is, the present invention is the first successful development of a technique for reducing the spore viability by deleting or mutating the atfB gene in filamentous fungi. It provides a method for easily killing in abundant outdoors.

  The present inventors have extensively studied and analyzed the transcriptional regulatory factor gene. Although the atfB gene encoding the transcriptional regulatory factor protein is present in filamentous fungi other than Neisseria gonorrhoeae, Its function is unknown. As a result of energetic analysis using a microarray or the like, the present inventors have positively transcribed a series of stress resistance genes such as the Catalase A gene (catA) and the trehalose synthase-like gene into the conidia. I found out that I was in control. However, it has not yet been elucidated that the atfB gene is involved in spore viability, and further, it has not been elucidated at all that an atfB gene deletion strain can be easily killed by ultraviolet rays or the like. It ’s the destination.

  This invention is made | formed based on such useful new knowledge, Comprising: As the embodiment, the following aspects are illustrated.

(Aspect 1)
A method for reducing the viability of spores of a filamentous fungus, characterized by completely or partially deleting or mutating the DNA of the atfB gene encoding a transcriptional regulator protein possessed by the filamentous fungus.
(Aspect 2)
The method according to aspect 1, wherein the DNA of the atfB gene is a DNA of a gene encoding the following protein (A) or (B).
(A) Protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (B) One or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and have gene expression control activity Protein (Aspect 3)
The gene according to aspect 2, wherein the gene encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 comprises the following DNA (a) or (b):
(A) DNA consisting of the base sequence shown in SEQ ID NO: 2
(B) a DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 2 under a stringent condition and encodes a protein having gene expression control activity

(Aspect 4)
comprising the steps of constructing a plasmid for disrupting the atfB gene comprising DNA from which at least a part of the atfB gene has been deleted or mutated, and transforming a filamentous fungus with the obtained plasmid. A method for producing a filamentous fungus with reduced conidia viability.
(Aspect 5)
The method according to aspect 4, wherein the atfB gene disruption plasmid comprises atfB5 ′ region, marker gene, and atfB3 ′ region DNA.
(Aspect 6)
The method according to aspect 5, wherein the marker gene is an sC gene.
(Aspect 7)
The method according to any one of aspects 4 to 6, wherein the DNA in the atfB5 ′ region is the following DNA (c) or (d) or a DNA containing the DNA:
(C) DNA consisting of the base sequence shown in SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence shown in SEQ ID NO: 3
(Aspect 8)
The method according to any one of aspects 4 to 7, wherein the DNA of the atfB3 ′ region is the following DNA (e) or (f) or a DNA containing the same:
(E) DNA consisting of the base sequence shown in SEQ ID NO: 4
(F) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence shown in SEQ ID NO: 4
(Aspect 9)
When the atfB gene disruption plasmid is linearized, it is pUSCΔatfB obtained by introducing ΔatfB DNA in which the atfB5 ′ region, the sC gene, and the atfB3 ′ region are sequentially arranged into the vector pUSC. The method according to any one of Embodiments 6 to 8.

(Aspect 10)
A DNA for constructing a plasmid for disrupting an atfB gene, wherein the DNA of ΔatfB contains the following DNA (g) or (h):
(G) DNA consisting of the base sequence shown in SEQ ID NO: 5
(H) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence shown in SEQ ID NO: 5
(Aspect 11)
AtfB gene disruption plasmid pUSCΔatfB represented by DNA consisting of the base sequence shown in SEQ ID NO: 5;

  According to the present invention, filamentous fungi that produce spores with low viability can be produced, and the produced filamentous fungi have the property of being easily killed when exposed to ultraviolet rays or the like. Therefore, even if various transformants using a filamentous fungus as a host are scattered outside the management area, these transformants according to the present invention die in a short time, and biohazard is prevented in advance. Effective.

  Therefore, various gene manipulation techniques for filamentous fungi can be applied widely, and at that time, research and production are possible without excessive concern about biohazards. Usually, the enhancement of the survival of microorganisms is evaluated as an effect, but the present invention is extremely characteristic in that the opposite effect, that is, a negative effect is evaluated as a significant effect. In the field of gene manipulation technology, it has a unique and unique effect.

  In order to carry out the present invention, the atfB gene may be deleted or mutated from the filamentous fungus. The deletion of the atfB gene may be completely deleted, a part thereof may be deleted, or a region including the atfB gene may be extensively deleted. The base sequence of the atfB gene is shown in SEQ ID NO: 2 (FIG. 9) in the sequence listing, and the corresponding amino acid sequence is shown in SEQ ID NO: 1 (FIG. 8). This gene is contained in Neisseria gonorrhoeae, and is also contained in, for example, Aspergillus oryzae NS4 strain (described later), so that it can also be obtained by cutting it out.

  In order to carry out the present invention, a plasmid for disrupting the atfB gene may be constructed, and filamentous fungi may be transformed therewith. The plasmid for disrupting the atfB gene can be constructed by introducing the atfB5 'region and the atfB3' region into a vector (for example, a chromosomal integration vector pUSC). Usually, in addition to the above two regions, it is preferable to introduce a sC gene or other marker gene.

  The base sequence of the atfB5 ′ region is represented by SEQ ID NO: 3 (FIG. 10) and roughly corresponds to the promoter region, and the base sequence of the atfB3 ′ region is represented by SEQ ID NO: 4 (FIG. 11). It roughly corresponds to the area. Since these nucleotide sequences have been clarified, these regions may be excised from the gonococcal gene, or synthesized by PCR using a gonococcal genomic DNA as a template and a promoter. There are no difficulties.

  The plasmid for disrupting the atfB gene may be prepared by introducing a promoter region and a terminator region into the vector (FIG. 1). For example, by introducing the atfB5 ′ region and the atfB3 ′ region into the vector pUSC, An atfB gene disruption plasmid pUSCΔatfB containing the sC gene and lacking the atfB gene can be constructed. The sequence of (atfB5 ′ region) − (sC gene) − (atfB3 ′ region) is named ΔatfB sequence, its base sequence is shown in SEQ ID NO: 5 (FIGS. 12 to 16), and the outline of gene disruption is shown in FIG. Shown in In the nucleotide sequence of SEQ ID NO: 5, nucleotide numbers 1 to 8 are NotI restriction enzyme sites, 9 to 2049 are atfB5 'sequences, 4249 to 7501 are SC sequences, and 7525 to 9524 are atfB3' sequences.

The characteristics of this plasmid are as follows.
(1) When it is “linear”,
(2) “Promoter region”, “sC gene”, “terminator region” are arranged in order,
(3) “Promoter region” and “terminator region” are arranged with directionality. further,
(4) The directionality of sC may be arbitrary. Also,
(5) Sequences other than “promoter region”, “sC gene”, and “terminator region” may or may not be present.
(6) The “promoter region” and “terminator region” may be longer or shorter than this. However, if it is short, the efficiency of genetic recombination may decrease.

(7) The sC gene may be changed according to the strain to be introduced, or may be a gene that complements other auxotrophy. The sC gene is not essential in that it may be a drug resistance gene or another marker gene, and other marker genes can be used as appropriate.

  In this plasmid, the sC region functions as a so-called marker gene and is included for the purpose of efficiently obtaining a transformant. The sC gene is a gene involved in sulfate (sulfur source) assimilation. When this gene is introduced into the NS4 strain, which is deficient in sulfate utilization, the mutation is complemented so that a single transformant on the auxotrophic medium is obtained. Can be easily separated. If anything, Amp (ampicillin resistance gene) is less necessary in the transformant of Neisseria gonorrhoeae, and Amp is useful when a plasmid is amplified in E. coli.

(8) The DNAs of these genes are introduced into pUSC and other appropriate vectors to construct an atfB gene disruption plasmid (FIG. 1). pUSC is described in the following literature, and is also distributed by the Research Institute for Liquors, and there is no difficulty in obtaining it. O. Yamada, B .; R. Lee, K.M. Gomi: Transformation System for Aspergillus oryzae with Double Auxotropic Mutations, niaD and sC. Bioscience, Biotechnology, and Biochemistry 61 (8), 1367-1369, 1997

(9) AtfB gene disruption plasmid when using atfB5 ′ region (SEQ ID NO: 3 FIG. 10) as a promoter region, using atfB3 ′ region (SEQ ID NO: 4 FIG. 11) as a terminator region, and using pUSC as a vector The construction of (pUSCΔatfB: its entire base sequence is shown in SEQ ID NO: 5) is shown in FIG. In addition, (atfB5 'region)-(sC gene)-(atfB3' region) is named ΔatfB sequence, and its base sequence is shown in SEQ ID NO: 5 (FIGS. 12 to 16).

(10) This plasmid is a genus of Alpergillus (Aspergillus et al., Aspergillus et al., Aspergillus oryzae), Aspergillus usami, Aspergillus kawachii, etc. In addition to various filamentous fungi such as Penicillium spp. And Rhizopus spp., They can generally be used in various organisms.

  Hereinafter, although the Example of this invention is described, this invention is not limited only to these Examples.

(Example 1: Creation of Aspergillus oryzae that forms conidia having low resistance to stress such as ultraviolet rays by deleting the atfB gene present in Aspergillus oryzae)
Isolation and confirmation of the atfB gene-disrupted strain was performed as follows.

(1) Preparation of plasmid for disrupting atfB gene A construct for disrupting the atfB gene was constructed by introducing about 2 kbp at the 5 ′ side and about 2 kbp at the 3 ′ side of the atfB gene into the chromosome integration vector pUSC (FIG. 2). An atfB gene-disrupted strain was obtained by transforming Neisseria gonorrhoeae NS4 using this vector (FIG. 3).

  That is, as described below, the atfB5 ′ region (SEQ ID NO: 3: FIG. 10) and the atfB3 ′ region (SEQ ID NO: 4: FIG. 11) were respectively prepared, and these regions were introduced into the vector pUSC for atfB gene disruption. Plasmid pUSCΔatfB (its construction is shown in FIG. 2 and the entire base sequence is shown in SEQ ID NO: 5 (FIGS. 12 to 16)) was prepared.

(Introduction method)
(A) Using Primer 1 and Primer 2, the promoter region was amplified from Aspergillus genomic DNA by PCR, and the resulting fragment was cleaved with restriction enzymes PstI and AatII.
(B) pUSC was cleaved with restriction enzymes PstI and AatII, and the fragment obtained in (a) was introduced into the cut site.
(C) Using primer 3 and primer 4, the terminator region was amplified from the koji mold genomic DNA by PCR, and the resulting fragment was cleaved with restriction enzyme NotI.
(D) The plasmid obtained in (b) was cleaved with PstI, smoothed, and then cleaved with NotI. PUSCΔatfB was prepared by combining this fragment with the fragment prepared in (c).

  Primer 1 shows its base sequence in SEQ ID NO: 6 (FIG. 17), Primer 2 shows its base sequence in SEQ ID NO: 7 (FIG. 18), and Primer 3 shows its base sequence in SEQ ID NO: 8 (FIG. 19). The primer 4 has the base sequence shown in SEQ ID NO: 9 (FIG. 20).

  NS4 strain is used as the host. The NS4 strain (Aspergillus oryzae NS4) is known from the above-mentioned literature and is also distributed in our laboratory, so there is no difficulty in obtaining it.

(2) Confirmation of the disrupted strain by genomic Southern As a result of performing the genomic southern using the 3′-side region of atfBORF as a probe (FIG. 4), an expected 3.5 kbp band was observed in the gene disrupted strain. Since no band was detected in the genomic Southern using the ORF portion as a probe, it was confirmed that the strain was an atfB gene disrupted strain. That is, genomic DNA was cleaved with the restriction enzyme EcoRI and subjected to electrophoresis. As a result, when the ORF part was used as a probe, a band of 8 kbp was detected in the NS4 strain, but since no ORF was present in the disrupted strain, it was confirmed that no signal was detected (left side of the drawing), and the atfB3 ′ region was detected. As a probe, a band of 8.3 kbp was detected for NS4 and a 3.5 kbp band was detected for the disrupted strain (right side of the drawing). FIG. 5 is an explanatory diagram of the genome Southern.

(2) Confirmation by Northern analysis Northern analysis was performed using RNA collected from Aspergillus in the analyzer formation stage (37 hours after bran culture) and using atfB cDNA as a probe (FIG. 6). Since the signal seen in niaD300 was not confirmed in the disrupted strain, it was confirmed that atfB was expressed at this time and that the atfB gene disruption was successful. As is clear from this result, since the atfB gene does not exist in the atfB disrupted strain, the atfB gene is not detected in the conidia formation stage (right side of the drawing). In the figure, EtBr represents ethidium bromide, and niaD is a strain lacking the ability to assimilate nitrate in Aspergillus oryzae and is distributed at this laboratory.

(4) Measurement of various stress tolerances (Fig. 7)
Heat resistance The conidia suspended in distilled water was incubated at 50 ° C. for a predetermined time and applied to an agar medium to confirm the number of surviving cells.
Oxidation resistance Conidia suspended in 50 mM or an aqueous hydrogen oxide solution were incubated at 30 ° C. for a predetermined time and applied to an agar medium to confirm the number of viable cells.
Ultraviolet resistance The number of survivors was confirmed by irradiating the conidia applied to the agar medium with an ultraviolet irradiator.

  From the above results, it was proved that the atfB gene-disrupted strains were significantly lower in the survival rate due to various stresses than the wild strains, so that they were easily killed in the outdoors in sunlight.

The plasmid for atfB gene disruption is shown. pUSCΔatfB plasmid is shown. It is a gene disruption schematic. It is an electrophoretic diagram which shows the result of the genomic Southern of a disrupted strain. (Drawing substitute photo). It is explanatory drawing of a genome Southern. It is an electrophoretic diagram which shows the result of the Northern analysis of a destruction strain (drawing substitute photograph). The resistance of Aspergillus oryzae to various stresses is shown. The amino acid sequence shown by sequence number 1 of a sequence table is shown. This shows the base sequence represented by SEQ ID NO: 2 in the sequence listing. The base sequence shown by SEQ ID NO: 3 is shown. The base sequence shown by sequence number 4 is shown. The base sequence shown by sequence number 5 is shown. The same as above. The same as above. The same as above. The same as above. Primer 1 is shown. Primer 2 is shown. Primer 3 is shown. Primer 4 is shown.

Claims (9)

To completely or partially delete or mutate the DNA of the atfB gene encoding the following transcriptional regulator protein (A) or (B) possessed by Aspergillus sp. A method for reducing stress resistance of spores of the genus Aspergillus characterized by
(A) Protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (B) One or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and have transcriptional regulatory factor activity protein The method according to claim 1, wherein the gene encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 comprises the following DNA (a) or (b):
(A) DNA consisting of the base sequence shown in SEQ ID NO: 2
(B) a DNA that hybridizes with a DNA comprising the base sequence shown in SEQ ID NO: 2 under a stringent condition and encodes a protein having a transcription factor activity A step of constructing a plasmid for atfB gene disruption comprising DNA obtained by deleting or mutating at least part of the atfB gene encoding the transcription factor protein of (A) or (B) below: A method for producing an Aspergillus genus having reduced conidia stress resistance, comprising the step of transforming an Aspergillus genus with a plasmid and destroying the atfB gene of genomic DNA .
(A) Protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (B) One or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and have transcriptional regulatory factor activity protein   The method according to claim 3, wherein the atfB gene disruption plasmid comprises DNA in which the atfB5 'region, the marker gene, and the atfB3' region are sequentially arranged when linearized. .     The method according to claim 4, wherein the marker gene is an sC gene. The method according to claim 4 or 5, wherein the DNA in the atfB5 'region comprises the following DNA (c) or (d):
(C) DNA consisting of the base sequence shown in SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence shown in SEQ ID NO: 3 The method according to any one of claims 4 to 6, wherein the DNA of the atfB3 'region contains the following DNA (e) or (f):
(E) DNA consisting of the base sequence shown in SEQ ID NO: 4
(F) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence shown in SEQ ID NO: 4   The method according to any one of claims 5 to 7, wherein the atfB gene disruption plasmid is a plasmid obtained by introducing atfB5 'region and atfB3' region DNA into the vector pUSC. 9. The DNA in which the atfB5 ′ region, the sC gene, and the atfB3 ′ region are arranged in order when linearized, comprises the following DNA (g) or (h): DNA for preparing a plasmid for disrupting the atfB gene according to 1.
(G) DNA consisting of the base sequence shown in SEQ ID NO: 5
(H) DNA that is hybridized under stringent conditions with DNA consisting of the base sequence shown in SEQ ID NO: 5 and that is DNA consisting of the base sequence shown in SEQ ID NO: 3 or DNA that consists of the base sequence shown in SEQ ID NO: 3 DNA comprising a DNA that hybridizes under stringent conditions, and a DNA that comprises the nucleotide sequence shown in SEQ ID NO: 4 or a DNA that hybridizes under stringent conditions with the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4