JPH06217759A - Regeneration of yeast protoplast and culture medium therefor - Google Patents

Abstract

PURPOSE:To obtain a culture medium capable of not reducing the ratio of regeneration even in the application of a sever treatment for conversion to a protoplast by making up a double layer fixed medium of a layer containing carrageenan and a polyamine and another layer containing agarose. CONSTITUTION:A double layer fixed culture medium is made up of a layer containing carrageenan and a polyamine and another layer containing agarose. The former is used as the upper layer and the later is used as the lower layer in general. As the polyamine, spermidine, spermine, putrescine, etc., are exemplified.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a yeast protoplast regeneration medium and a regeneration method, and more specifically to a yeast protoplast regeneration method and a regeneration medium used therefor as means for carrying out yeast breeding by cell fusion. .

[0002]

2. Description of the Related Art In order to apply the cell fusion method, which has become popular as a breeding means for various yeasts such as brewing yeasts, it is necessary to convert the yeasts into protoplasts and regenerate the cell wall after the protoplast fusion. is there. Basically, the method for regenerating yeast protoplasts is the conventional method (Weber, H. e.
t al., CurrentGenetics, vol.4,165-166,1981; Halfma
n, HJ et al., Acrh. Microbiol., vol.134,1-4,1983;
Broda.HG et al., Biochimica et Biophysica Acta,
vol.899,25-34,1987).

In the conventional method, yeast protoplasts were mixed in an agar medium having a high osmotic pressure (1.2 to 1.4 osmol (Osm)) in a sol state at about 50 ° C., which was gelled in a petri dish and Regenerating protoplasts in gel.
However, inactivation of yeast protoplasts is likely to occur at temperatures around 50 ° C. In addition, protoplasts placed in high osmotic pressure are prone to rupture, resulting in growth inhibition. further,
When agar is used, the regeneration rate of protoplasts is low and
Since the anaerobic level is strong near the bottom of the dish, there are various problems such as a low regeneration rate and a low growth rate.

In particular, since yeasts used for general industries such as brewing do not have a genetic marker, even if cell fusion treatment of protoplasts is performed, a physical method such as a cell sorter can be used to separate the fused protoplasts. I have to rely on it. For physical selection, it is necessary to strongly act yeast cell wall lysing enzymes to produce protoplasts isolated from each other without aggregation, but when such strong protoplast formation treatment is performed, In the medium, the regeneration rate is extremely reduced, and it is difficult to separate the target yeast.

[0005]

The yeast used in the cell fusion method must be able to be regenerated at a high frequency even when subjected to a strong protoplast treatment. However, when a strong protoplast treatment is performed, the regeneration rate is actually 5 × 10 ^-5 to 1
It was about 10 ⁻⁶ , and it was difficult to obtain the target regenerated yeast.

[0006] In consideration of the present situation, the present invention establishes a regeneration medium that improves the regeneration rate even if strong protoplast treatment is performed, and establishes a method for regenerating yeast protoplasts using the medium. The purpose is to

[0007]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to solve the above-mentioned problems, and instead of agar, a layer containing carrageenan and polyamine and a layer containing agarose were used as a support base of a solid medium. It was found that the regeneration rate of yeast protoplasts can be improved by using a solid medium composed of the two layers described above and completed the present invention.

That is, the present invention provides a medium for regenerating yeast protoplasts, which comprises a solid medium having two layers, a layer containing carrageenan and polyamine, and a layer containing agarose, and to which yeast protoplasts are added. The present invention provides a characteristic method for regenerating yeast protoplasts.

The reason for using carrageenan and polyamine in the present invention is to protect the membrane protein on the surface of yeast protoplasts. In the case of a two-layer solid medium comprising a carrageenan and polyamine-containing layer and an agarose-containing layer, the former is usually used as the upper layer and the latter is used as the lower layer. By using the layer containing agarose as the lower layer, it is effective as a support base for supporting the layer containing carrageenan and polyamine, which are physically rather fragile, and also effective in increasing the regeneration rate of yeast protoplasts.

As the polyamine, spermidine,
In addition to spermine, putrescine, cadaverine, homospermidine, norspermine and the like can be used, and these may be used alone or in combination of two or more kinds.

The addition amounts of carrageenan and polyamine may be appropriately determined according to the purpose, but usually 6 to 10 g / L of carrageenan and 0.1 to 0.5 of polyamine.
g / L is contained. Further, regarding the mixing ratio of carrageenan and polyamine, the former: the latter = 12 to 100: 1 is appropriate. The amount of agarose added may be 6 to 10 g / L. Next, the ratio of the layer containing carrageenan and polyamine to the layer containing agarose is not particularly limited, but usually the former / latter = 1/2 in terms of volume ratio.
~ 1/3.

When the yeast protoplasts are regenerated using the above-mentioned medium for regeneration, the temperature is suitable to be 30 to 35 ° C. The yeast protoplasts are sprinkled in a medium in a thin layer and cultured under aerobic conditions. During the regeneration treatment, by maintaining the osmotic pressure at 0.4 to 0.6 osmol, it is possible to prevent the rupture of the yeast protoplasts in the regeneration medium and promote the growth of the regenerated yeast. As other conditions, known conditions may be adopted, and there are no special conditions due to the use of the regeneration medium of the present invention.

In addition, the medium for regeneration and the method for regeneration of the present invention are not limited to target yeasts and can be applied to various yeasts. Although brewing yeasts are used in Examples described later, the yeasts are not limited to these.

[0014]

EXAMPLES The present invention will be described in detail below with reference to examples. Example 1 Brewery Technology Laboratory Sapporo Breweries Ltd. Preserved brewer's yeast B
SRI YB3-8 strain and BSRI YB9-12 strain were treated with cell wall lysing enzyme (thymolyase 20T) at 10 ⁸ cells / cell.
When the mg enzyme was allowed to act for 2 to 3 hours, it was possible to produce protoplasts in which there were no aggregation and individually isolated.
The protoplasts prepared under these conditions were allowed to undergo cell fusion using an electrofusion device, and then only the fused protoplasts could be separated using a physical means such as a cell sorter. By using the method of combining the electrofusion and the cell sorter, it has become relatively easy to obtain a fused yeast of brewing yeasts that does not have a genetically selective marker, which was extremely difficult in the past.

However, when the protoplasts prepared under these conditions are regenerated in a conventional medium, the regeneration rate becomes 1 to 2 × 10 ^{−5 as} shown in FIG. 1, and it is difficult to obtain the fused yeast of both strains. Became. On the other hand, using the medium for regeneration shown in Table 1, particularly, 10 ml of the lower layer medium was sprayed with 3 ml of the mixed solution of the upper layer medium and protoplast at 35 ° C. on a cooled petri dish to regenerate the protoplasts. As shown in FIG. 1, the reproduction rate was 3 to 7 × 10 ⁻⁴ , which was 15 to 70 times that of the conventional method.

[0016]

[Table 1] Table 1 Example of regeneration medium Composition of upper layer medium (1 L) Composition of lower layer medium (1 L) Glucose 20 g Glucose 20 g Bactopeptone 20 g Bactopeptone 20 g Yeast extract 10 g Yeast extract 10 g Yeast nitrogen Yeast nitrogen base 6.7 g Base 6.7g Sodium chloride 7.5g Sodium chloride 7.5g Carrageenan 8g Agarose 8g Spermidine 0.1g ───────────────────────────── Pressure 0.4-0.7 osmol Osmolality 0.4-0.7 osmol

Since the substrate of the upper layer medium is fragile, it is not possible to form a solid medium in a petri dish with only a mixed liquid of the upper layer medium and protoplasts. Therefore, when the protoplasts were regenerated using a mixed solution of 13 ml of the lower layer medium and 0.1 ml of the protoplasts, the regeneration rate was 5 to 8 × 10 ^{−5 as} shown in FIG. 1, which was 2. Although it is 5 to 8 times, the improvement of the reproduction rate is not sufficient. Therefore, as described above, it is found that using two layers of medium, and in particular, overlaying a mixed solution of the upper layer medium and protoplast on the lower layer medium is effective as a method for frequently regenerating the protoplasts.

As is clear from the above results, the method for regenerating yeast protoplasts according to the present invention is particularly effective when the fused protoplasts between yeasts that do not have a genetic selection marker, such as brewing yeasts, are physically separated. It can be seen that it is effective as a method for regenerating protoplasts at high frequency.

[0019]

INDUSTRIAL APPLICABILITY The present invention is a medium for regenerating yeast protoplasts and a method for regenerating yeast protoplasts using the medium. According to the present invention, yeast protoplasts can be regenerated at a high frequency, and particularly genetic selection. Breeding by cell fusion of yeast having no marker can be facilitated.

[Brief description of drawings]

FIG. 1 shows the relationship between the treatment time of yeast cell wall with a lytic enzyme and the regeneration rate.

[Explanation of symbols]

A Beer yeast BSRI YB in the two-layer medium of the present invention
Regeneration of strain 3-8 B Beer yeast BSRI YB in the two-layer medium of the present invention
Regeneration of 9-12 strain C Beer yeast BSRI YB3 on conventional agar medium
-8 strain regeneration D Beer yeast BSRI YB9 on conventional agar medium
-Regeneration of strain 12 E Regeneration of brewer's yeast BSRI YB3-8 strain with agarose-containing lower layer medium of the present invention F Regeneration of beer yeast BSRI YB9-12 strain with agarose-containing lower layer medium of the present invention

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location (C12N 1/16 C12R 1: 865)

Claims (16)

[Claims] 1. A medium for regenerating yeast protoplasts comprising a solid medium having two layers, a layer containing carrageenan and polyamine and a layer containing agarose. 2. The regeneration medium according to claim 1, wherein the layer containing carrageenan and polyamine is the upper layer and the layer containing agarose is the lower layer. 3. The medium for regeneration according to claim 1, which contains 6 to 10 g / L of carrageenan and 0.1 to 0.5 g / L of polyamine. 4. The regeneration medium according to claim 1, wherein the compounding ratio of carrageenan and polyamine is the former: the latter = 12 to 100: 1. 5. The regeneration medium according to claim 1, which contains 6 to 10 g / L of agarose. 6. The volume ratio of the layer containing carrageenan and polyamine to the layer containing agarose is 1 / 2-.
The medium for regeneration according to any one of claims 1 to 5, which is 1/3. 7. The regeneration medium according to claim 1, wherein the yeast is brewer's yeast. 8. A method for regenerating yeast protoplasts, which comprises adding yeast protoplasts to the medium for regeneration according to claim 1. 9. The regeneration method according to claim 8, wherein the regeneration medium has a layer containing carrageenan and polyamine as an upper layer. 10. The method for regenerating according to claim 8, wherein yeast protoplasts are added to the layer containing carrageenan and polyamine. 11. The regeneration method according to claim 8, wherein the regeneration treatment is performed at an osmotic pressure of 0.4 to 0.7 osmol. 12. The regeneration method according to claim 8, wherein the regeneration medium according to claim 3 is used. 13. The regeneration method according to claim 8, wherein the regeneration medium according to claim 4 is used. 14. The regeneration method according to claim 8, wherein the regeneration medium according to claim 5 is used. 15. The regeneration method according to claim 8, wherein the regeneration medium according to claim 6 is used. 16. The method according to claim 8, wherein the yeast is brewer's yeast.