JP2006288266A - Method for producing fusant of yeast - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing a fusant by fusing cells by using a frozen protoplast of a yeast. <P>SOLUTION: The method for fusing the cells by electrical fusion of the frozen protoplast of the yeast, finished by discovering that the fusing rate is extremely increased by the electrical fusing method when fusing the frozen protoplast is provided. <P>COPYRIGHT: (C)2007,JPO&amp;INPIT

Description

  The present invention relates to a method for producing a yeast fusion, and more particularly to an efficient method for producing a yeast fusion for cell fusion of cryopreserved yeast protoplasts by electrical fusion and breeding of the yeast.

The protoplast fusion method, which is widely used as a means of breeding microorganisms, is said to be a suitable method for introducing traits involving multiple genes, and is used for breeding various industrially useful strains. .
In recent years, breeding that simultaneously fuses a large number of protoplast cells, such as a genome shuffling method (Non-patent Document 1) applying the protoplast fusion method, has attracted attention.
However, in order to perform protoplast fusion, it is necessary to prepare at least two protoplasts at the same time, and it takes a lot of labor and time to adjust the protoplast and to separate the protoplasts for each experiment. There were various obstacles to the implementation of protoplast fusion. In order to solve these problems, protoplast preservation methods have been developed, and cryopreservation of protoplasts has been attempted in several strains (Non-patent Document 2). There were many problems such as low fusion rate, and it was very difficult to use for actual breeding. This means that when a genetic marker or auxotrophic strain is fused, the fusion rate does not matter so much, but microorganisms used for general industries do not have a genetic marker. In many cases, if the fusion rate is low, selection of the fusion is extremely difficult and fatal. Thus, it is very significant if protoplasts can be stored frozen and protoplasts can be fused with high efficiency.
However, when unfrozen protoplasts are used, the fusion rate drops significantly when applied to frozen protoplasts, even though protoplast fusion, which is a general cell fusion method, has been achieved efficiently. However, frozen protoplasts of yeast could not be used for breeding by cell fusion.
Ying-Xin Zhang, Kim Perry, Victor A. Vinci, Keith Powell, Willem PC Stemmer and Stephen B. del Cardayre (2002) Nature 415, 644-646 K. Kuwano, Y. Aruga and N. Saga (1996) Plant Science 116, 117-124

  It is an object of the present invention to efficiently produce a fusion by cell fusion using frozen yeast protoplasts.

The inventors of the present invention have made extensive studies to solve the above-mentioned problems, and found that the electrical fusion method can remarkably improve the fusion rate during the fusion of frozen proplasts, thereby completing the present invention.
That is, the present invention provides a method for efficiently producing a fusion by cell fusion of frozen yeast protoplasts by electrofusion.
That is, the present invention has the following configuration.
(1) A method for producing a yeast fusion using a frozen protoplast of yeast, which comprises lysing a frozen protoplast and fusing it with cells by electric fusion. Production method.
(2) A method for producing a yeast fusion using frozen yeast protoplasts, comprising at least the following steps a) to c):
a) Process for producing yeast protoplasts
b) Freezing the protoplast of a)
c) Step of obtaining a fusion by lysing the frozen protoplast of b) and then performing cell fusion by electrofusion (3) The yeast according to (1) or (2) above, wherein the yeast is a yeast of the genus Sporobolomyces A method for producing a fusion product.
(4) The step (a) comprises the step of culturing a yeast of the genus Sporobolomyces under conditions that inhibit cell wall synthesis, and then adding two or more cell wall lytic enzymes to the solution containing the yeast. A method for producing a yeast fusion according to claim 1.
(5) A fusion produced by the production method according to any one of (1) to (4) above.
(6) A product produced by the fusion according to (5) above.

The present invention is a method of efficiently fusing a frozen protoplast of yeast, which has been thought to be unable to fuse cells at a high fusion rate, and according to the present invention, a fusion using a frozen protoplast is obtained. Can be obtained efficiently.
In particular, it can also be applied to yeast that does not have a genetic selection marker or yeast that does not have auxotrophy, and breeding of such yeast by cell fusion can be facilitated.
Furthermore, since the present method enables efficient production of a fusion using frozen protoplasts, it is practically possible to use a protoplast stored by a practical cryopreservation method as a protoplast storage method. It was.

(Protoplast production)
Any yeast protoplast may be used. For example, a yeast protoplast of the genus Sporobolomyces is prepared as follows. Sporobolomyces genus yeast was first cultured in YPD medium at 28 ° C for 24 hours, and then 500 μL of this culture was inoculated into 50 mL of protoplasting medium, and 20 hours later, 500 μL of 2-D-deoxyglucose 6.5% solution was inoculated. Add and incubate for 48-96 hours.
Next, the culture solution thus obtained is centrifuged, and the cells are washed with a 100 mM phosphate buffer solution (adjusted to pH 7), and further washed twice with a protoplastization buffer.
After washing, use a cell wall enzyme treatment solution containing Westase (0.5%, manufactured by Takara) and Cellulase ONOZUKA R-10 (2.5%, manufactured by Yakult Yakuhin) in the protoplastization buffer so that the cell concentration is about OD4. Suspend the cells and gently immerse them at 30 ° C for 3-5 hours. By these treatments, a protoplast solution having a protoplast conversion rate of 99% or more can be obtained.

  Further, for example, in yeast belonging to the genus Saccharomyces, protoplasts can be obtained by lysing the cell wall using endo-β-1,3-glucanase, phosphomannase, protease, etc. derived from Arthrobacter (Kitamura, K. Arch. Biochem. Biophys., 153, 403-406 (1972)).

The protoplastization rate shown here was confirmed by the following method. That is, it was confirmed that the cell wall was dissolved and removed with a microscope, the number of cells with low osmotic pressure sensitivity was determined by the osmotic shock method, and the protoplastization rate was calculated by the following formula.
More specifically, in the present application, an isotonic solution represents a buffer solution that is made isotonic with an intracellular solution by adding an osmotic pressure adjusting agent, and a hypotonic solution represents distilled water. The same number of cells were put in the same amount of isotonic solution and hypotonic solution, the number of cells in each fluid after 1 minute was counted, and the protoplastization rate was calculated according to the following formula (1).

Protoplastification rate (%) = (number of cells in isotonic solution−number of cells in hypotonic solution) / number of cells in isotonic solution × 100 (1)

  Although these protoplasts can be cryopreserved as they are, it is preferable to cryopreserve them after substituting them with a buffer to which a cryoprotectant (such as glycerol or trehalose) is added before cryopreservation.

  The yeast belonging to the genus Sporobolomyces used in the present invention may be any genus as long as it belongs to the genus Sporobolomyces, but bacteria having the ability to produce useful substances such as coenzyme Q10, carotenoids, or L-carnitine with high efficiency are desirable. Specifically, Sporobolomyces sp. NY205 strain (received as the receipt number FERM AP-20491 on April 7, 2005 at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary).

(Protoplast electrofusion)
Frozen protoplasts are thawed, washed several times with a buffer for electrofusion, and then mixed in equal amounts with both protoplasts. A buffer for electrofusion can be selected as appropriate, and for example, a buffer composed of 1.4 M Sorbitol, 0.1 mM CaCl ₂ , and 0.5 mM MgCl ₂ is exemplified. The protoplast mixed solution can be fused using a cell fusion device by applying a high frequency electric field and then applying a high voltage pulse. The conditions for applying the high-frequency electric field are a frequency of 0.25 to 2.0 MHz, an AC voltage of 10 to 40 V, and the time of applying the high frequency is the time until the protoplast forms a pearl chain, generally 5 to 90 seconds. Next, a high voltage pulse is applied, but the electric field strength is 0.1 to 20 kV / cm, and the pulse width is 5 to 500 μs. At this time, it is preferable to perform postfusion for about 1 to 10 seconds after applying a high voltage pulse. Moreover, it is preferable that the fusion strain is stabilized by allowing it to stand for 10 minutes or more after the electrofusion treatment.
The melting is desirably natural melting, and the electrofusion is desirably performed immediately after melting by mixing two liquids to be fused.

(Regeneration of fusion protoplasts)
A fusion can be obtained by regenerating protoplasts fused according to the present invention using a regeneration medium containing an osmotic pressure regulator. More specifically, for example, the fused protoplast is first embedded using an agar containing a nutrient source while keeping the temperature as low as possible, and then the plate is cultured in an incubator for 3 to 20 days to obtain a fusion. be able to. The obtained fusion can be selected based on differences in auxotrophy and drug resistance from the parent strain, differences in the composition and content of the product, and the like. Various amino acids can be used when auxotrophy is used as a guide, and amino acid analogs (β-2-thienylalanine, ethionine, 5-methyltryptophan, etc.) and nucleic acid analogs (6-mercaptopurine, 2,6-diaminopurine, 5-fluorouracil) can be used.
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to an Example at all.

(Freezing preservation method)
Sporobolomyces sp. NY205 strain (FERM AP-20491) was inoculated into YPD medium and cultured at 28 ° C. for 24 hours. Next, 500 μL of this culture solution was inoculated into 50 mL of the following protoplast-forming medium, and 20 hours later, 500 μL of a 2-D-deoxyglucose 6.5% solution was added and cultured for 48 to 96 hours.
The cells obtained by centrifuging the culture solution were washed with a 100 mM phosphate buffer solution (adjusted to pH 7), and further washed twice with a protoplastization buffer. After washing, in the cell wall enzyme treatment solution containing Westase (0.5%) and Cellulase ONOZUKA R-10 (2.5%) in the protoplastization buffer, suspend the cells so that the cell concentration is about OD4, Soaked gently at 30 ° C for 3-5 hours. Protoplast conversion rate by this treatment was over 99%. Subsequently, the protoplast is suspended in a cryopreservation buffer and gradually frozen in a -80 ° C freezer to stably store the protoplast. After protoplast thawing, the protoplast suspension was diluted to an appropriate concentration with a protoplastization buffer, and the number of protoplast cells (A) in 100 μL of the diluted solution was counted using a hemocytometer. In addition, 100 μL of the same diluted solution is placed in a YPD upper layer agar kept at 41 to 43 ° C., embedded by pouring on the lower layer agar, and incubated at 28 ° C. for 4 to 8 days (B) Counted. Using the values of (A) and (B), the regeneration rate was calculated by the following equation (2), and a high regeneration rate of 20% was shown.

            Regeneration rate (%) = (B) / (A) x 100 (2)

Unless otherwise specified, the following media and buffers were used in the examples and comparative examples.
YPD medium 1% yeast extract, 2% polypeptone, 2% glucose
Protoplast medium Glucose 1%
Yeast extract 0.15%
DL-methionine 0.02%
DL-homocysteine 0.025%
L-cysteine 0.15%
KH ₂ PO ₄ 0.35%
Na ₂ HPO ₄ 12H ₂ O 0.25%
NH ₄ Cl 0.15%
MgSO ₄ 7H ₂ O 0.02%
CaCl ₂ 2H ₂ O 0.001%
FeCl ₃ 6H ₂ O 0.0008%
ZnSO ₄ 7H ₂ O 0.00001%
Protoplast cryopreservation buffer
0.037M citric acid, 0.126M Na ₂ HPO ₄ 12H ₂ O, 0.5M tartrate, 15 wt% glycerol protoplastization buffer
0.037M citric acid, 0.126M Na ₂ HPO ₄ 12H ₂ O, 0.5M tartrate
YPD Agar Yeast Extract 1%, Polypeptone 2%, Glucose 2%, 0.6M Sorbitol, 2% Agar
YPD Upper Agar Yeast Extract 1%, Polypeptone 2%, Glucose 2%, 0.6M Sorbitol, 1.5% Agar
YPD lower layer agar yeast extract 1%, polypeptone 2%, glucose 2%, 0.6M Sorbitol, 2% Agar
SD medium
0.67% Yeast nitrogen base without amino acids (Difco, 0919-15-3), 2% glucose, 2% agar

(Create mutant strain)
Sporobolomyces sp. NY205 strain is cultured in YPD medium at 28 ° C. for 24 hours, collected, washed twice with 100 mM phosphate buffer solution (adjusted to pH 7), and suspended in 20 mL of this buffer. After adding 600 μL of Ethyl methanesulfonate and stirring the suspension vigorously, the suspension was gently soaked at 28 ° C. for 1 hour. One hour after the mutation treatment, the collected cells were washed twice with 5% sodium thiosulfate, the cells were suspended in 1 mL of sterile distilled water, 100 μL of the diluted suspension was applied to the YPD plate, and 28 ° C. For 72 to 120 hours to obtain mutant colonies. When the obtained colonies were examined for amino acid requirements, leucine-requiring strains, lysine-requiring strains, methionine-requiring strains, and arginine-requiring strains were obtained. Each auxotrophic strain can grow on YPD agar but cannot grow on SD medium.
In addition, the leucine-producing ability spontaneous recovery rate of the leucine-requiring strain, the lysine-requiring strain spontaneous recovery rate, the methionine-requiring strain methionine-producing ability spontaneous recovery rate, and the arginine-requiring strain arginine-producing ability spontaneous recovery rate Both were less than 1/10 ⁵ .

(Fused protoplast fusion)
The produced NY205 mutants, leucine-requiring strain and lysine-requiring strain, were protoplasted by the method described in Example 1 and stored frozen. After thawing, mix equal amounts of each protoplast, wash twice with electrofusion buffer (1.4 M Sorbitol, 0.1 mM CaCl ₂ , 0.5 mM MgCl ₂ ), and then use this buffer to give a protoplast concentration of 10 ⁸ cells / mL. Adjusted as follows. Add the protoplast solution to the 0.5 mm cell between the electrodes, apply a 1 MHz frequency, 15 V AC voltage for 10 seconds using a cell fusion device (Neppagene, LF101), then apply DC voltage twice at 600 V (pulse width 10 μsec). (Pulse interval 0.1 sec) was applied and post-fusion was performed for 2 seconds. The electric field intensity of the high voltage pulse at this time was 12,000 V / cm. After standing for 10 minutes, dilute the solution after cell fusion to an appropriate concentration, put the diluted solution in a YPD upper layer agar kept at 41-43 ° C, and pour it on the lower layer agar for 4-10 days. The protoplasts after cell fusion were grown by incubating at 28 ° C. Colonies that grew on these plates were inoculated into SD plates where protoplasts that did not fuse cells could not grow. After culturing at 28 ° C. for 2 to 5 days, the number of colonies (X) of the fusion grown on the SD plate was counted, and the fusion rate was calculated by the following formula (3) using the total number of inoculated bacteria (Y). However, these fusion experiments showed a high fusion rate of 4% or more.

                Fusion rate (%) = (X) / (Y) x 100 (3)

Comparative Example 1

(Fusion of frozen protoplasts by conventional PEG method)
The leucine-requiring strain and arginine-requiring strain, which are NY205 mutants produced by the method described in Example 2, were protoplasted by the method described in Example 1 and stored frozen. After thawing, an equal amount of each protoplast was mixed and centrifuged, and then the supernatant was removed. A PEG4000 solution (100 mM Tris, 50 mM CaCl ₂ , 35 wt% PEG4000, pH 7.5) was added to the protoplast so that the protoplast cell concentration was 10 ⁸ cells / mL, and the mixture was allowed to stand at 30 ° C. for 20 minutes. The cell-fused solution is diluted to an appropriate concentration, and the diluted solution is put into a YPD upper layer agar kept at 41 to 43 ° C, poured onto the lower layer agar, and incubated at 28 ° C for 4 to 10 days. Grow. The fusion rate by this method was less than 0.05%.

Comparative Example 2

(Fusion of frozen protoplasts by conventional PEG method)
Fused protoplasts were fused in the same manner as in Comparative Example 1 except that PEG8000 solution, PEG6000 solution, PEG3350 solution, PEG6000S6 solution, PEG6000S3 solution, PEG6000K3 solution, PEG4000S6 solution, PEG4000S3 solution or PEG4000K3 solution were used for fusion. When the strains were grown, all these 9 methods showed a fusion rate of 0.05% or less.

The composition of each PEG solution used in Comparative Example 2 is shown below.
PEG8000 solution _{(100 mM Tris, 50mM CaCl 2} , 35 wt% PEG8000, pH 7.5)
PEG6000 solution _{(100 mM Tris, 50mM CaCl 2} , 35 wt% PEG6000, pH 7.5)
PEG3350 solution _{(100 mM Tris, 50mM CaCl 2} , 35 wt% PEG3350, pH 7.5)
PEG6000S6 solution _{(100 mM Tris, 50mM CaCl 2} , 35 wt% PEG6000,0.6M Sorbitol, pH7.5)
PEG6000S3 solution _{(100 mM Tris, 50mM CaCl 2} , 35 wt% PEG6000,0.3M Sorbitol, pH7.5)
PEG6000K3 solution _{(100 mM Tris, 50mM CaCl 2} , 35 wt% PEG6000,0.3M KCl, pH7.5)
PEG4000S6 solution _{(100 mM Tris, 50mM CaCl 2} , 35 wt% PEG4000,0.6M Sorbitol, pH7.5)
PEG4000S3 solution _{(100 mM Tris, 50mM CaCl 2} , 35 wt% PEG4000,0.3M Sorbitol, pH7.5)
PEG4000K3 solution _{(100 mM Tris, 50mM CaCl 2} , 35 wt% PEG4000,0.3M KCl, pH7.5)

Comparative Example 3

(Protoplast fusion)
The fusion strain was grown by fusing protoplasts in the same manner as in Comparative Example 1 except that the methionine-requiring strain and arginine-requiring strain, which were NY205 mutants produced by the method described in Example 2, were used. However, the fusion rate by this method was 1.04%.

  The present invention provides a cell fusion method for efficiently fusing a frozen protoplast of yeast, which has been thought to be unable to fuse cells at a high fusion rate, and in particular, a genetic selection marker. It can also be applied to yeasts that do not have them or those that do not have auxotrophy, and breeding by cell fusion of such yeasts can be facilitated.

Claims (6)

  A method for producing a yeast fusion using a frozen protoplast of yeast, which comprises lysing the frozen protoplast and fusing it with cells by electrofusion. A method for producing a yeast fusion using a frozen protoplast of yeast, comprising at least the following steps a) to c):
a) Process for producing yeast protoplasts
b) Freezing the protoplast of a)
c) Step of lysing the frozen protoplast of b) and cell fusion by electrofusion to obtain a fusion product   The method for producing a yeast fusion according to claim 1 or 2, wherein the yeast is a yeast belonging to the genus Sporobolomyces.   The yeast according to claim 3, wherein the step a) comprises the step of culturing a yeast of the genus Sporobolomyces under conditions that inhibit cell wall synthesis, and then adding two or more cell wall lytic enzymes to the solution containing the yeast. A method for producing a fusion product.   The fusion body manufactured by the manufacturing method in any one of Claims 1-4. A product produced by the fusion according to claim 5.