JP2996784B2 - Cell fusion method and fused cells obtained by the method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell fusion method in which a useful gene possessed by a fungus such as a filamentous fungus or a basidiomycete is introduced into a yeast to make it usable, and the fusion obtained by the method. For cells.

[0002]

2. Description of the Related Art In recent years, progress in genetic engineering technology has been remarkable.
Many techniques for transferring genes between homologous or heterologous cells have been developed and some of them have been put to practical use. In a typical method, the presence of a useful gene in a mother cell is searched on the chromosome, confirmed, and then the useful gene portion is cut out using an appropriate restriction enzyme. Then, it is carried out by introducing into an appropriate host cell and transforming using an appropriate vector.

However, when the ability (or function) of producing a useful substance of interest is composed of a plurality of components (for example, the cellulase component of Trichoderma filamentous fungi is largely divided, , CM
It consists of three components, a C-decomposing enzyme component and a β-glucosidase component, each of which is composed of a plurality of components, which means that the necessary cellulase information is present over a large number of gene groups. Performing the above operation on the gene of each component requires complicated operations and confirmation methods, and is not practical.

[0004] DNA having genetic information of a mother cell
Can be directly transformed using a host cell, for example, a yeast protoplast.However, since an enzyme that eliminates and degrades heterologous DNA is present in the host cell, naked DNA is transformed into the yeast cell. Is likely to be destroyed even if it is
The frequency of obtaining yeast having A incorporated into the host chromosome is extremely low.

[0005] That is, it is very difficult to purify DNA transformation without breaking very large chromosomes or DNA. Most chromosomes and DNA are physically destroyed during this step.

[0006] Even if it is purified without being destroyed, it is physically destroyed again in the step of introducing it into cells. For example, even when introduced into the cells, the chromosome or DNA into which the nuclease in the yeast cells has been introduced is recognized as a foreign substance, and will be destroyed. Even if this works, only a small part of the introduced chromosome or DNA will be integrated into the yeast nucleus. Even if the gene can be introduced without being destroyed at any step, a large number of unnecessary genes other than the necessary genes are incorporated, and the genetic stability of the hybrid is uneasy.

[0007] Although there are means for obtaining equivalent microorganisms by selection and separation from the natural world, they require enormous labor and time. Even if the target strain is obtained by this method and the productivity is to be further increased, there is no means and the search may be performed in nature.

[0008]

As described above, by the conventional gene manipulation, a very large number of Trichoderma enzyme component genes are inserted into a plasmid one by one, and the gene is expressed in each one. Incorporating this promoter would require enormous effort and time. Even if it is performed, it is not clear that yeast cells can stably hold a huge number of plasmids.

On the other hand, the present inventor has found that polyploids of high homogeneity can be obtained efficiently and efficiently by treating filamentous fungi or basidiomycetes with colchicine or a colcemide derivative. No. 50091 (Japanese Unexamined Patent Application Publication No.
No. 231080), but also a filamentous fungus or a basidiomycete, especially a Trichoderma or Aspergillus fungus, is treated with a polyploid-inducing agent such as colchicine or colcemid for a long time under non-nutritive conditions. And found that the nucleus in the cell was in a miniature nucleus state.

An object of the present invention is to provide a cell fusion method in which a function comprising a plurality of pieces of genetic information is collectively introduced into a target host cell, particularly a yeast cell, by utilizing the phenomenon of the micronucleus state. More specifically, it is an object of the present invention to obtain a yeast having, for example, a cellulase-producing ability using the present method.

[0011]

In the cell fusion method according to the first aspect of the present invention, a conidia or a mycelium of a fungus having a predetermined gene is subjected to a polyploid-inducing agent under non-nutritive conditions. After exposure to form micronuclei, the cells are treated with a cell wall lytic enzyme to form protoplasts, and yeast is treated with a cell wall lytic enzyme to form protoplasts, and the two protoplasts are fused.

[0012] In the cell fusion method according to the second aspect of the present invention, after the two protoplasts are fused, a nuclear fusion treatment for fusing nuclei derived from the two protoplasts is performed.

[0013] More specifically, it discloses that the fungus is a filamentous fungus of the genus Trichoderma and that the polyploid-inducing agent is colchicine or colcemide.

[0014] In the fused cells obtained by the cell fusion method, in the fused cells obtained by the method according to any one of claims 1 to 4, the Trichoderma reesei having the cellulase-producing gene as the test cell may be used. Wherein the yeast is Saccharomyces cerevisiae, specifically, TRSC-2, TRSC-3, or TRS.
C-4 is disclosed.

[0015]

In the present invention, test cells of conidia or mycelium of a fungus having a predetermined gene are exposed to a polyploid inducing agent under non-nutritive conditions to form micronuclei, and then the cells are treated with a cell wall lytic enzyme. The protoplasts are treated, and the yeast is treated with a cell wall lysing enzyme to produce protoplasts.Because the two protoplasts are fused, a micronucleus containing a gene of a useful product consisting of a plurality of gene groups is formed, Other unnecessary gene groups can be prevented from significantly participating in hybrid formation, the frequency of hybrid acquisition in interspecies protoplast fusion can be improved, and the genetic stability of the obtained intergeneric hybrid can be improved.

That is, in the interspecies protoplast fusion using a normal nucleus, both cells are "separated" during regeneration of the fused protoplasts, resulting in fragmentation. Even if the fusion forms a colony without "segregation", it occurs only very infrequently and this fusion "segregates" frequently during subculturing. All the above problems can be overcome by performing heterogeneous fusion with the introduction side as a micronucleus. This means that genetically stable intergeneric hybrids are frequently obtained.

Further, for example, a chromosome containing a predetermined gene such as a gene of a useful product is introduced into yeast by a single operation as a micronucleus and integrated into the yeast nucleus, whereby the useful product of the species is easily and alternately obtained. Can be produced.

Further, when a fusion process for fusing nuclei derived from two protoplasts is performed after fusing the two protoplasts, the frequency of obtaining stable stable fused cells is improved. The nuclear fusion treatment is specifically an ultraviolet irradiation, a camphor treatment or the like. In this case, too, one of them is a micronucleus, so that the nuclear fusion treatment can be performed relatively easily.

More specifically, to obtain a micronucleus-retaining microbial cell of a filamentous fungus or a basidiomycete, particularly a Trichoderma or Aspergillus filamentous fungus, having the target useful gene information, the mycelium or the bacillus is required. It is obtained by exposing viable offspring to a polyploid inducer, preferably colchicine or colcemid. At this time, if a nutrient component is present in the aqueous colchicine solution to be treated, polyploid polymorphs are produced, and therefore it is necessary to perform the treatment under non-nutritive conditions such as a distilled aqueous solution. Further, it is desirable that this polyploid inducer treatment be performed for a relatively long time, for example, 10 hours to several days.

Next, using the micronucleus-retaining microbial cells as protoplasts, a fused cell is produced with the protoplasts of the host yeast to be introduced. Each protoplast is formed by a conventional method, that is, treating the target micronucleus-retaining microbial cell and the host yeast with an appropriate cell wall lysing enzyme.

Next, the fusion cells with the protoplasts of the micronucleus-retaining microbial cells and the protoplasts of the host yeast are performed by a conventional method. That is, for example, a fused cell can be obtained by treating in a polyethylene glycol aqueous solution in the presence of calcium chloride.

In order to obtain a desired fused cell containing a useful gene, the above-mentioned fused cell is cultured and regenerated in an appropriate medium, and then it is confirmed whether or not it has a desired function. This is achieved by collecting a fused cell having the function of At this time, if the fusion cells obtained by the fusion operation are further subjected to a nuclear fusion treatment such as ultraviolet irradiation, camphor treatment, etc., the probability of obtaining the desired stable fused cells is improved.

As the fused cells specifically obtained by the above operation, those which disclose TRSC-2, TRSC-3, or TRSC-4 obtained by fusing Trichoderma reesei having a cellulase-producing gene with Saccharomyces cerevisiae are disclosed. It is. Among them, TRSC-4 has been deposited as Microtechnical Laboratory No. 12446. This fused cell is a cellulase-producing yeast, has a cellulose assimilation ability, and enables alcohol direct fermentation from cellulose. Therefore, a path to the effective use of the unused resources and the waste resources is opened, which is promising socially and economically.

[0024]

【Example】

Example 1 (Colchicine treatment) Trichoderma reesei QM9
414 (ATCC 13631 strain) conidia in a 50 ml spitgrass 5% colchicine aqueous solution (pH
9.0), and reciprocal shaking at room temperature for 24 hours forms micronuclei in conidia. Similarly, micronuclei are formed in conidia-derived microbial cells that retain the micronuclei. This small micronucleus is considered to have been caused by abnormal chromosome division or chromosome deconcentration.

Example 2 (Protoplastization and Fusion Cells) Next, the obtained micronucleus protoplasts were fused with yeast protoplasts. Protoplasts were obtained from the colchicine-treated conidia-derived conidia obtained in Example 1 by 3.5.
% Cell wall lysing enzyme "Funcelase"
Treated with Yakult Honsha Co., Ltd. at 40 ° C. for 3 hours to prepare micronucleated protoplasts. Yeast cells cultured for 20 hours were subjected to the same treatment to prepare yeast protoplasts.

Each of the prepared protoplasts was 0.1 M
After heating in 33% polyethylene glycol containing CaCl ₂ at 35 ° C. for 30 minutes, the mixture was centrifuged at 1000 g for 5 minutes for fusion treatment. The fusion protoplasts were washed at 0.
When applied to an agar medium containing 1% CMC-Na, the same medium is overlaid and cultured at 28 ° C. After about 1 week, regeneration and colony growth are observed, and a part of the colonies passes through the CMC-Na layer and the surface of the medium. Were selected as fused cells. In addition, even if it cultured for 7 days, the yeast original strain only formed a minute colony on the boundary surface, could not break through the upper layer, and did not appear on the surface (because the original strain has no cellulase productivity, CMC-Na Although it cannot grow in the presence, the fusion strain can grow due to cellulase productivity). Trichoderma regenerates in this medium, but only 14
It did not appear on the surface after 7 days of culture.
The fusion operation was performed three times, and one at a time was randomly selected from the fusion strains generated on the surface, and TRSC-2, TRSC-3,
It was TRSC-4.

Example 3 (Nuclear Fusion Treatment) Many of the colonies obtained in Example 2 isolated Trichoderma cells, and were presumed to be genetically unstable. In the nuclear fusion treatment, a colony was placed on a medium supplemented with 0.1% camphor (camphor) and cultured for several days, and a growing colony was defined as a colony in which the nuclear fusion treatment was completed.

Example 4 (Confirmation of Cellulase Producing Ability) Cellulase producing ability was confirmed using the obtained colonies. Table 1 below shows the results of the frequency of fusion of strains having cellulase-producing ability.

[0029]

[Table 1]

The protoplast fusion in the table was carried out to
What appeared on the medium surface after passing through the C-Na layer was defined as the number of fused cells, and the ratio to the number of initial protoplasts was determined and defined as the fusion frequency. First fusion (number of T. reesei protoplasts: number of yeast protoplasts = 6 × 10 ⁴ : 4 × 10
^{In 4} ), 9 such colonies were formed for 100,000 protoplasts. This colony was transferred to Camphor and C
After culturing on a medium containing MC-Na, an aqueous solution of Congo Red was added, and the mixture was allowed to stand for 15 minutes. After washing with saline, a pale yellow transparent area was observed immediately below and around the colony, and it was confirmed that cellulase was produced. .

In the case of this example, when cultivation was carried out in a medium containing no camphor, Trichoderma cells appeared as sectors (produced as fan-shaped heterogeneous colonies) and covered the surface of the medium. Regarding the possibility of contaminants or mycelium contamination of Trichoderma, similar results were obtained when the fused strain was once applied to a plate and colonies were isolated. Has been deleted.

As shown in the table, protoplast fusion was further performed by changing the number of each protoplast, and a large number of fusion strains were obtained. However, the acquisition frequency was almost the same, and it was confirmed that the fusion strains could be acquired at a constant frequency. Was done.

Example 5 (Cellulase-producing ability) Next, the cellulase production of the fusion strain treated by nuclear fusion was examined.
Trichoderma, a yeast strain, and a fusion strain (TRSC-2, TRSC-3, TRSC-) subjected to nuclear fusion treatment on an agar medium containing 0.1% camphor and 0.1% CMC-Na.
4) was rotated and cultured in a liquid medium containing 0.1% camphor and 0.1% CMC-Na at 28 ° C for 6 days, and then the cells were removed by centrifugation, and the supernatant was precipitated with ethanol. Was dissolved in 0.1 M acetate buffer (pH 5.0) to obtain a sample.

These samples were placed in a 7 cm long glass tube,
Disk gel electrophoresis was carried out with a 50 mm disk gel, and after completion of the electrophoresis, the gel was cut every 5 mm, and this was homogenized and suspended in 0.1 M acetate buffer (pH 5.0). CMC degradation activity as three main components of cellulase,
The presence or absence of Avicel degrading activity and Salicin degrading activity was determined by reacting each fraction with a substrate at 40 ° C. for 1 hour.

[0035]

[Table 2]

As shown in Table 2, in Trichoderma,
A plurality of components were present in each of the CMC degradation activity, Avicel degradation activity, and salicin degradation activity.

The fusion strain TRSC-2,
TRSC-3 and TRSC-4 all formed clear areas with Congo red, but fractions showing each decomposition activity were from one to three.
Individual gels were present on the gel. From this result, it was confirmed that all the components required for cellulose degradation were present in the fusion strain.

Example 6 (Genetic Stability Test) The genetic stability of the cellulase productivity of the fusion strain treated by nuclear fusion was examined by subculturing. 1 nuclear fusion-treated fusion strain
The number of generations was counted, and the number of colonies formed by applying the resultant to a medium containing CMC-Na was examined to determine what percentage of the first generation retained cellulase productivity. From the newly formed colonies on the medium containing CMC-Na, one was appropriately selected and used as the next generation, and the same operation such as counting was performed again and the fifth generation was examined.

[0039]

[Table 3]

As shown in Table 3, in the original strain, no colonies growing on this medium were obtained. However, T
When RSC-2, TRSC-3, and TRSC-4 were subcultured, even in the fifth generation, they were still grown in the presence of CMC-Na, and it was confirmed that cellulase productivity was not lost.

As described above, a stable fused strain retaining cellulase productivity was obtained by protoplast fusion with yeast using micronuclei. This micronucleus is considered to increase the possibility of obtaining stable hybrids between different species and different genera.

Further, a useful product which has conventionally been produced only with filamentous fungi and had a limit in cost can be produced at low cost by using yeast. Furthermore, the present invention is sufficiently applicable to plants, and it is considered that useful components such as pigments and alkaloids can be produced in yeast.

Further, the obtained cellulase-producing yeast enables direct alcohol fermentation from cellulose. Therefore, a path is opened to the effective use of unused resources and waste resources. Promising socially and economically.

In addition, Aspergillus sp. And Sporotrichum
It is also possible to obtain micronuclei and to introduce useful genes into yeast in the case of filamentous fungi of the genus (Sporotrichum), basidiomycetes such as shiitake mushrooms, and Kawatake mushrooms.

[0045]

EFFECTS OF THE INVENTION As described above, the present invention relates to a method in which a test cell of a conidia or a mycelium of a fungus having a predetermined gene is exposed to a polyploid-inducing agent under non-nutritive conditions to form a micronucleus. Is treated with a cell wall lytic enzyme to produce protoplasts, and yeast is treated with a cell wall lytic enzyme to form a protoplast, and the two protoplasts are fused. To improve the frequency of hybrid acquisition in interspecies protoplast fusion, and to improve the genetic stability of the interspecies hybrid obtained by integrating the gene into the micronucleus and making other unnecessary gene groups less involved in hybridization. Can be.
Also, it introduced into yeast once genes useful product as micronuclei, by incorporating the yeast nucleus, can easily be produced alternately on the kind of useful products.

Furthermore, when a fusion process for fusing nuclei derived from two protoplasts is performed after the fusion of two protoplasts, the frequency of obtaining the desired fused cells is improved. The nuclear fusion treatment is specifically ultraviolet irradiation, camphor treatment, or the like. In this case, the fusion treatment can be performed relatively easily because one of them is a micronucleus.

As a specific example of the obtained fused cells, a TRSC- fusion of Trichoderma reesei having a cellulase-producing gene and Saccharomyces cerevisiae was used.
2, TRSC-3 or TRSC-4. This fused cell is a cellulase-producing yeast, has a cellulose assimilation ability, and enables alcohol direct fermentation from cellulose. Therefore, a path to the effective use of the unused resources and the waste resources is opened, which has the effect of being promising socially and economically.

──────────────────────────────────────────────────続 き Continued on front page (51) Int.Cl. ⁷ Identification code FI C12R 1: 885) (C12N 1/19 C12R 1: 865)

Claims (5)

(57) [Claims] 1. A method comprising exposing test cells of conidia or mycelium of a fungus having a predetermined gene to a polyploid inducing agent under non-nutritive conditions to form micronuclei, and treating the cells with a cell wall lytic enzyme. A cell fusion method comprising treating a yeast with a cell wall lysing enzyme to produce a protoplast, and fusing the two protoplasts. 2. The cell fusion method according to claim 1, wherein after the two protoplasts are fused, a nuclear fusion treatment for fusing nuclei derived from the two protoplasts is performed. Wherein the fungus Trichoderma (Trichoderma)
The cell fusion method according to claim 1, wherein the cell fusion method is a genus filamentous fungus. 4. The cell fusion method according to claim 1, wherein the polyploid-inducing agent is colchicine or colcemide. 5. The fusion cell obtained by the method according to claim 1, wherein the test cell is Trichoderma reesei having a cellulase producing gene, and the yeast is Saccharomyces cerevisiae. (Saccharomyc
es cerevisiae)
C-2, TRSC-3 or TRSC-4.