JP2010158256A - Method for transformation of yeast - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for transformation of yeast that can simply treat a large number of strains in no need of preparation of competent cells. <P>SOLUTION: The method for producing a transformant includes transforming fission yeast with a DNA for transformation, wherein the yeast cell bodies cultured on a SD solid medium is added to the treating solution including a DNA for the transformation, a carrier DNA, polyethylene glycol and a buffer solution including a lithium salt in an amount of ≥1×10<SP>7</SP>per μg of DNA 1 for transformation. Then, the mixture is incubated for ≥30 minutes at 25-35°C and the treated solution contains 0.5 to 10 μg of the DNA for transformation per 100 μL of the treated solution and the carrier DNA of ≥20-fold mass based on the DNA for transformation. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for performing transformation by introducing transformation DNA into yeast as a host.

  A heterologous protein production system using yeast as a host and using recombinant DNA technology can be easily implemented using known microbiological methods and recombinant DNA technology, and exhibits high production capacity. Volume culture has also been implemented and has been rapidly utilized for actual production. In actual production, the high production efficiency per cell obtained in the laboratory is maintained even after scale-up.

  However, when considering lower-cost production methods often required in actual production, improvement of the bacterial cell growth efficiency itself, suppression of degradation of target heterologous proteins, efficient implementation of yeast-specific modifications, nutrient sources It is considered necessary to take measures to improve the production efficiency of heterologous proteins, such as improving the utilization efficiency of the protein. For this purpose, yeast host modifications such as destroying unnecessary genes and adding necessary genes have been proposed (see Patent Document 1). In that case, it is necessary to examine whether a heterologous protein can be produced efficiently for all of a large number of modified yeast hosts. For this purpose, it is necessary to transform each of various modified yeast hosts to obtain transformants. There is.

  As a yeast transformation method, methods such as a lithium acetate method, an electroporation method, a spheroplast method, and a glass bead method have been developed so far. The electroporation method is widely used as a simple and efficient transformation method, and many studies have been conducted on a method for preparing competent cells that have high transformation efficiency and can be stored for a long period of time. However, since these methods require the preparation of competent cells, it was considered inappropriate when dealing with many strains such as mutants at once. Thus, in budding yeast such as baker's yeast (Saccharomyces cerevisiae), a simple transformation method pursuing simplicity has been developed based on the lithium acetate method (see Non-patent Documents 1 to 3). Although the transformation efficiency of the simple transformation method varies depending on the culture conditions of the bacterial cells, the transformation efficiency of about 300 transformants / μg DNA can be obtained by using the colony on the plate directly and using the 96-well microplate. (See Non-Patent Document 3). These simple transformation methods have low transformation efficiency and are effective when high efficiency is not required. Some contrivances for increasing the transformation efficiency have been made (see Non-Patent Documents 4 to 6). However, it has not yet been devised to satisfy both the convenience of handling a large number of hosts simultaneously and the increase in transformation efficiency.

  On the other hand, among yeast, fission yeast is separated from other yeasts at an early stage in the evolution process, and as a result of achieving another evolution, it is close to animal cells, as can be seen from growing by means of division instead of budding. It is known to have properties. For this reason, by using fission yeast, particularly Schizosaccharomyces pombe (hereinafter referred to as S. pombe), as a host for expressing a heterologous protein, a gene product closer to the natural body, similar to the case of animal cells, can be obtained. Expected to be obtained. However, the simple transformation method described above has been developed for budding yeast, and the development of such a simple transformation method has hardly been studied for fission yeast. Although transformation is possible even if the simple transformation method in budding yeast is applied to fission yeast, the conversion efficiency is much lower than that of budding yeast, and it is not a practical method.

International Publication No. 02/101038 Pamphlet

Rapid colony transformation of Saccharomyces cerevisiae. Rohan Baker, Nucleic Acids Reserch, 1991, 19 (8), 1945. Simple and Efficient Procedure for Transformation of Yeast.Randolph Elble, Bio Techniques, 1992, 13 (1), 18-20. An Efficient Procedure for Multiple Transformation of Yeast in Parallel. Antonio A. Firmenich and Kevin Redding, Bio Techniques, 1993, 14 (5), 713-718. Studies on the transformation of Intact Yeast Cells by the LiAc / SS-DNA / PEG Procedure.R. Daniel Gietz, Robert H. Schiestl, Andrew R. Willems, and Robin A. Woods, Yeast, 1995, 11, 355-360. Ethanol improves the transformation efficiency of intact yeast cells. Vit Lauermann Curr. Genet. 1991, 20, 1-3. An Improved Protocol for the Preparation of Yeast Cells for Transformation by Electroporation.J. R. Thompson, E. Register, J. Curotto, M. Kurtz and R. kelly, Yeast, 1998, 14, 565-571.

  In view of these circumstances, as a transformation method for fission yeast, finding a highly efficient method that can simultaneously and easily transform various host cells such as gene-disrupted strains efficiently produces heterologous proteins. Therefore, it is considered that simplification and efficiency of transformation is a problem necessary for selecting transformants with high production efficiency.

  The present inventor is the following invention relating to a simple method for transforming fission yeast with high transformation efficiency, which can be carried out by a simple operation using colonies directly.

In a method for producing a transformant by transforming fission yeast with transforming DNA, the transformant comprises a buffer solution containing DNA for transformation, carrier DNA, polyethylene glycol and lithium salt. Add 5 × 10 μg or more of yeast cells cultured on SD (Synthetic Dextrose) solid medium to 1 × 10 ⁷ or more per 1 μg of transformation DNA to a treatment solution containing 5 to 10 μg of carrier DNA at least 20 times the mass of the transformation DNA. And a method of transforming fission yeast, which comprises incubating at 25 to 35 ° C. for 30 minutes or more.

  Moreover, in the said transformation method, it is preferable to transform using a microplate. Furthermore, the fission yeast is preferably Schizosaccharomyces pombe.

  This transformation method is effective when transforming a large number of strains at a time because preparation of competent cells is unnecessary and a transformant can be efficiently obtained by a simple operation. In addition, simultaneous transformation into a plurality of cells using a 96-well microplate or the like can be performed by adjusting the amount of the treatment solution. As a result, it becomes possible to easily select a strain having an increased production efficiency of the heterologous protein.

2 is a graph showing the relationship between the number of cells brought in and the number of transformed cells in Example 1. The graph which shows the relationship between the incubation time by the kind of solid culture medium in Example 3, and the number of transformed microbial cells. The graph which shows the relationship between the amount of carrier DNA in Example 4, and the number of transformed microbial cells.

  The simple transformation method of budding yeast known so far is a method developed by improving the lithium acetate method with an emphasis on simplicity, and by using colonies cultured on a solid medium directly, It is possible to transform bacterial cells. The transformation efficiency varies depending on the culture conditions. When a colony on a solid medium is used directly, the transformation efficiency is lower than that in a liquid culture (liquid culture: about 10,000 transformants / μg DNA, colony: (Transformants 960 to 4000 / μg DNA) (see Non-Patent Document 1 to Non-Patent Document 3). However, when a colony of fission yeast was used instead of budding yeast in the same manner, the number of transformants was 208 / μg DNA (see Non-patent Document 3). The present invention is a simple method for transforming fission yeast that improves the transformation efficiency by optimizing the transformation conditions and is simpler and better in transformation efficiency than these conventional methods.

  The transformation method of the present invention is a method for transforming fission yeast. The fission yeast is not particularly limited as long as it is a yeast belonging to the genus Schizosaccharomyces, but Schizosaccharomyces pombe, which has been studied and applied as a transformation host, is preferred.

  In the transformation method of the present invention, fission yeast is cultured on a solid medium, and the resulting cells on the solid medium are subjected to transformation. The solid medium is not particularly limited, and a solid medium conventionally used for fission yeast culture can be used. Specifically, for example, SD (Synthetic Dextrose) solid medium, MM (Minimal medium) solid medium, YES (Yeast Extract with supplements) solid medium, and the like are preferable. Particularly, cultured yeast having high transformation efficiency in a relatively short incubation time. An SD solid medium from which can be obtained is preferred. The composition of these media is known and is described, for example, in the following documents. In the case of a liquid medium, about 3% agar can be added thereto to obtain a corresponding solid medium.

SD medium: Burke D, Dawson D, and Stearns T. Methods in yeast genetics: A Cold Spring Harbor Laboratory Course Manual. Plainview: Cold Spring Harbor Laboratory Press, 2000. pp172.
MM medium: Alfa C, Fantes P, Hyams J, McLeod M, and Warbrick E. Experiments with fission yeast: A laboratory course manual. Plainview: Cold Spring Harbor Laboratory Press, 1993.pp 134.
YES medium: Moreno S, Klar A, and Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. In: Guthrie C, Fink GR eds. Methods in Enzymology. Volume 194: Guide to yeast genetics and molecular biology. San Diego: Academic Press, Inc., 1991: 795-823.pp801.
The culture time on the solid medium is not particularly limited, but is preferably about 1 day to 10 days. In order to increase the culture efficiency, it is preferable to culture for 1 day to 5 days and use for transformation. Moreover, a nutrient source can be added to the solid medium as described above containing only a minimum amount of nutritional components if necessary. If there are too many additional nutrients, the conversion efficiency tends to decrease. It is sometimes said that the conversion efficiency is higher as the starved bacteria, but if there are too few nutrient sources, the growth of the bacteria may be insufficient. As the solid medium in the present invention, it is preferable to use the SD solid medium as described above to which 5% or less of sugar such as glucose is added.

  The transformation treatment solution used in the transformation method of the present invention comprises a transformation DNA containing a gene to be introduced, carrier DNA, a polyethylene glycol and a buffer containing a lithium salt.

  The transformation DNA may be any DNA as long as it contains the gene to be introduced, which is the purpose of transformation. Usually, plasmid DNA containing the gene, such as an expression vector, PCR for gene disruption or gene replacement Amplified fragments are used. The DNA for transformation may be circular DNA (plasmid DNA or the like) or linear DNA (PCR amplified fragment or the like). Expression vectors usually include gene regions such as a promoter gene, marker gene, and replication origin in addition to the gene. The expression vector may be constructed by introducing a target gene into a multicloning vector. The size of these DNAs for transformation is not particularly limited, but usually about 1000 to 20 Kbp is used.

  The amount of DNA for transformation in the treatment solution is 0.5 to 10 μg per 100 μL. The greater the amount of transforming DNA, the greater the number of transformed cells, but the conversion efficiency per transforming DNA is not so high and is not economical, so this amount is considered appropriate. About 5 to 2 μg is optimal.

  Carrier DNA is a component necessary for improving transformation efficiency, and Single Strand DNA having a size of about 200 to 5000 bp is usually used. For example, salmon sperm DNA, calf thymus DNA, carp sperm DNA, and the like can be used. The amount of carrier DNA used is 20 times or more the amount of DNA for transformation. As the amount of carrier DNA increases, the transformation efficiency increases. However, if the amount used is excessive, the economic efficiency is impaired, and the upper limit is suitably about 10,000 times the mass. A more preferable amount of carrier DNA is 50 to 500 times the mass of DNA for transformation.

  Polyethylene glycol (PEG) is a compound widely used in transformation methods, and its molecular weight is about 500 to 20,000, and polyethylene glycol having a molecular weight of about 1000 to 8000 is particularly preferable. Lithium salts are also widely used in transformation methods, and include mineral acid salts such as hydrochlorides and organic acid salts such as carboxylates and sulfonates. As the lithium salt, a carboxylate is particularly preferable, and a commonly used acetate is most preferable. The amount of polyethylene glycol and lithium salt in the treatment solution is a commonly used amount. For example, polyethylene glycol is preferably about 10 to 70% (w / v), and lithium salt is 10 to 500 mmol per 100 μL. The degree is preferred.

  The treatment solution is an aqueous solution having a buffering action, and usually contains Tris-HCl or EDTA in addition to the above components. These amounts are those commonly used in transformation methods. The pH is preferably adjusted to about 4.5 to 5.5. If the pH is outside this range, the transformation efficiency tends to decrease.

The cells cultured on the solid medium are added to the treatment solution and incubated to obtain transformants. The amount of bacterial cells is required to be 1 × 10 ⁷ or more per 1 μg of DNA for transformation. If the number of cells is small, a sufficient number of transformants cannot be obtained. A more preferable number of cells is 3 × 10 ⁷ or more. Moreover, when there are too many microbial cells added to a process solution, there exists a tendency for conversion efficiency to fall. Therefore, the upper limit of the number of cells is preferably about 5 × 10 ⁸ cells. A more preferable upper limit is about 1.5 × 10 ⁸ pieces. Incubation time is 30 minutes or more. Usually, the longer the incubation time, the more transformed cells, but it is not efficient to incubate for a long time, and long incubation may lead to the death of the bacteria. Therefore, the upper limit of the incubation time is suitably about 3 days. In addition, in order to perform efficient transformation, which is the object of the present invention, the incubation time should be short, and therefore, it is more preferably 48 hours or less. Most preferably, an incubation of 30 minutes to 24 hours is employed. In particular, in order to perform efficient transformation, incubation for 30 minutes to 12 hours is preferable. The incubation temperature is not particularly limited, but 25 to 35 ° C. is appropriate. After the incubation with the treatment solution is completed, the transformed cells are obtained by culturing the cells in a selective medium.

  The bacterial cells can be pretreated before treatment with the treatment solution to increase the conversion efficiency, and the bacterial cells can be post-treated after the incubation and before culturing in the selective medium to increase the conversion efficiency. A typical post-processing is heat shock processing. For example, the transformation efficiency can be increased by suspending the cells in water after the incubation and exposing them to a temperature of about 38 to 45 ° C. for 5 to 30 minutes, followed by culturing in a selective medium. As the pretreatment, known treatments such as hypertonic solution treatment and reducing agent treatment for improving the conversion efficiency can also be carried out in the present invention to improve the transformation efficiency. As the hypertonic solution treatment, there is a treatment of contacting with a relatively high concentration aqueous solution of sugar or salt, such as a method of contacting with a 2.0 M sorbitol aqueous solution. As the reducing agent treatment, there is a treatment in contact with DTT (dithiothreitol). These pre-processing and post-processing can be performed by combining a plurality of processes.

  In the transformation method using the method of the present invention, the following method is used as a basic protocol for obtaining a transformant most efficiently (using the least amount of raw materials and obtaining the desired number of transformed cells in the shortest time). is there. Based on this method, a transformant can be obtained using the method of the present invention converted according to the purpose.

  1. The cells are cultured in an SD solid medium supplemented with 2% glucose.

  2. As the treatment solution, 10 μL of aqueous DNA solution for transformation (containing 1.0 μg of DNA for transformation), 10 μL of aqueous solution of carrier DNA (containing 100 μg of carrier DNA), 70 μL of aqueous solution of treatment raw material (50 w / v PEG4000, Use 100 mM lithium acetate, 10 mM Tris-HCl and 1 mM EDTA: pH = 4.9) and the remaining water mixed to 100 μL.

3. Above 1. About 5 × 10 ⁷ cells of the above 2. And then transforming by incubation at 30 ° C. for 1 hour, and then adding the treatment solution containing the transformed cells directly to the selective medium.

  4). 3. above. The transformation treatment is performed using a microplate.

  Since the above basic protocol adapts to a wider range of conditions, it was based on a gentle and simple procedure as much as possible, so it can be used not only for normal strains but also for mutant strains that exhibit various properties such as temperature and osmotic pressure sensitivity. It seems possible. By this method, an efficiency of about 3000 transformants / μg DNA / 1 h can be obtained.

  Since the transformation method of the present invention directly uses colonies on the plate, it is not necessary to prepare competent cells, the operation is very simple, and the amount of reaction solution is small, so a 96-well microplate was used. Application to simultaneous transformation of multiple strains is possible. Furthermore, this method can add a pretreatment of bacterial cells to improve transformation efficiency. By adding the pretreatment, the incubation time can be shortened and the transformation efficiency can be further improved.

Hereinafter, the present invention will be described in more detail with reference to specific examples. In Examples and the like, pAL known as DNA for transformation (Tanaka, K., Yanagida, T., Kawasaki, Y., Kai, M., Furuya, K., Iwasaki, M., Murakami, H., Yanagida , M. and Okayama, H. (2000) Fission yeast Eso1p is required for establishing sister chromatid cohesion during S phase. Mol. Cell. Biol. 20, 3459-3469), and using commercially available salmon sperm DNA as carrier DNA. used. As a strain, Schizosaccharomyces pombe ARC039 (S. pombe: h ⁻ , leu1-32, ura4-C190T) was used. The incubation treatment was performed using a 96-well microplate.

[Example 1]
The yeast cells were cultured on an MM solid medium for 3 days, and colonies formed there were directly treated with 100 μL of treatment solution [10 μL of pAL aqueous solution (including 1.0 μg of pAL), 10 μL of carrier DNA aqueous solution (100 μg of carrier). DNA), 70 μL of the aqueous treatment solution (50 w / v PEG 4000, 100 mM lithium acetate, 10 mM Tris-HCl and 1 mM EDTA: pH = 4.9) and the remaining water were mixed to make 100 μL. In addition to the above, the cells were incubated at 30 ° C. for 1 day, heat shock treated at 42 ° C. for 15 minutes, applied onto a selective medium, cultured, and the number of transformants was measured. FIG. 1 shows the relationship between the number of cells brought in and the number of transformed cells.

In the above transformation method, when the amount of cells brought in was changed as shown on the horizontal axis of FIG. 1, the number of transformants obtained (/ μg DNA) was changed as shown on the vertical axis, and the resulting transformation The number of cells reached a maximum when the amount of cells brought in was 5 × 10 ⁷ to 1 × 10 ⁸ (/ 100 μL treatment solution). Since the transformation efficiency does not reach 500 cells / μg DNA when the number of cells is less than 1 × 10 ⁷ , yeast cells cannot be efficiently transformed under the condition that 3 × 10 ⁷ or more per 1 μg of DNA for transformation is not added. It became clear.

[Example 2]
The same experiment as in Example 1 was performed, except that the number of cells brought in was 5 × 10 ⁷ (/ 100 μL treatment solution) and the amount of pAL in the treatment solution was changed. As a result, when the amount is less than 0.5 μg per 100 μL of the treatment solution, the transformation efficiency is 500 / μg DNA or less (315 μg / μg DNA in the case of 0.1 μg pAL), indicating that efficient transformation is not possible. It was.

[Example 3]
Yeast cells are cultured on SD solid medium, MM solid medium, and YES solid medium for 3 days respectively, and colonies formed there are brought in and used as 5 × 10 ⁷ cells (/ 100 μL treatment solution). The same experiment as in Example 1 was performed. FIG. 2 shows the relationship between the incubation time and the number of transformed cells depending on the type of solid medium.

  When cells grown on an SD solid medium were used, the number of transformants obtained was maximized after 1 hour incubation (600 cells / μg DNA / 1 h). When the MM solid medium was used, the number of transformants increased with the incubation time (3000 / μg DNA / 72h), and the number of transformants obtained was the highest among the three types of media used, but the long incubation time. Took time. When the YES solid medium was used, the transformation efficiency was low (about 1/10 of the MM solid medium). However, in any case, when the incubation time was set to less than 30 minutes, the transformation efficiency was 500 pieces / μg DNA or less, and it was found that efficient transformation was impossible.

[Example 4]
The same test as in Example 1 was conducted, except that the number of brought-in cells was 5 × 10 ⁷ (/ 100 μL treatment solution) and the amount of carrier DNA in the treatment solution was changed. When the carrier DNA was increased to 100 μg, an increase in transformation efficiency could be confirmed. However, when the amount of carrier DNA was less than 20 times the mass of pAL, the transformation efficiency was 500 pieces / μg DNA or less, and efficient transformation was achieved. I found it impossible. FIG. 3 shows the relationship between the amount of carrier DNA and the number of transformed cells.

  In order to construct a heterologous protein production system using recombinant DNA technology using yeast as a host, it is necessary to develop a yeast host that can produce the heterologous protein more efficiently. In order to develop a yeast host, it is necessary to evaluate whether a heterologous protein can be efficiently produced for a large number of modified yeast hosts. For this purpose, transformation is performed by transforming various modified yeast hosts. Need to gain body. In particular, the method of the present invention can be used as a convenient and efficient transformation method useful for efficiently evaluating this yeast host.

Claims (6)

In a method for producing a transformant by transforming fission yeast with transforming DNA, the transformant comprises a buffer solution containing DNA for transformation, carrier DNA, polyethylene glycol and lithium salt. 5-10 [mu] g, added carrier DNA to transformants for DNA processing solution containing 20 weight equivalents or more, SD (Synthetic Dextrose) solid medium on the culture of yeast cells transformed for DNA1μg 1 × 10 ⁷ or more per the And a method of transforming fission yeast, which comprises incubating at 25 to 35 ° C. for 30 minutes or more.   The method according to claim 1, wherein the transformation is performed using a microplate.   The method according to claim 1 or 2, wherein the fission yeast is Schizosaccharomyces pombe.   The method according to any one of claims 1 to 3, wherein the incubation time is 30 minutes to 1 hour.   The method according to any one of claims 1 to 4, wherein the yeast cells are yeast cells cultured for 1 to 10 days on the SD solid medium.   The method according to any one of claims 1 to 5, wherein the SD solid medium is an SD solid medium to which 5% or less of sugar is added.

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