JPH04311381A - Method for breeding yeast capable of producing astaxanthin by protoplast fusion - Google Patents

Abstract

PURPOSE:To provide a method for breeding a yeast composed of a method for protoplast fusion of a yeast belonging to the genus Phaffia to a yeast belonging to other genuses. CONSTITUTION:The objective method is for breeding a yeast comprising a step for carrying out protoplast fusion of a protoplast formed from the yeast belonging to the genus Phaffia to one protoplast of other genuses, e.g. Saccharomyces, Candia, Hansenula or Rhodotorula. Breeding of the yeast having expected properties can readily be performed by selecting the yeast to be used.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for breeding yeast by protoplast fusion between yeasts of different genera, and the yeast obtained thereby. In particular, the present invention provides a yeast that efficiently produces astaxanthin by selecting a yeast belonging to the genus Phaffia as one of these yeasts, and a method for breeding the same.

0002

BACKGROUND OF THE INVENTION Hitherto, known industrially useful yeast breeding methods include a spore-based mating method, a mutation induction/selection method, a method for introducing foreign genes through genetic manipulation, and a cell fusion method. Among these, cell fusion methods include, for example, Saccharomyces
Attempts have been made to breed flocculating yeasts and killer yeasts using fusion methods using protoplasts of yeasts belonging to the genus S. yces. In addition, the cell fusion method between yeast of different genera is also used in the genus Torulopsis and Pichia (P.
A fusion method using protoplasts of yeasts belonging to the genus Ichia has been proposed (see JP-A-1-137986).

[0003] However, Phaffia rhodochyma, which produces the red pigment astaxanthin, is being developed for a wide range of applications in various industrial fields.
odozyma), there are no examples in the prior art literature of successful cell fusion with yeast belonging to other genera, including their protoplast formation and cell fusion using them.

0004

[Problems to be Solved by the Invention] Since yeasts belonging to the genus Phaffia produce useful pigments as described above, useful yeasts have been bred by chemical or physical mutagenesis methods. However, since this method is difficult to introduce traits that are controlled by multiple genes, there have been expectations for the development of more efficient breeding methods to replace these methods. Therefore, an object of the present invention is to enable a cell fusion method that can introduce traits that are controlled by multiple genes to yeast belonging to the genus Phaffia, and to provide a breeding method using the cell fusion method. .

[0005]

[Means for Solving the Problems] The present inventors have previously succeeded in converting yeast belonging to the genus Phaffia into protoplasts using a specific enzyme composition, and have filed a patent application for the same (
(Refer to the specification of Japanese Patent Application No. 193105/1999). Then, when we investigated the fusion of protoplasts obtained by the above-mentioned protoplastization with protoplasts of various cells, we found that a stable fusion strain could be obtained even with yeast belonging to genera other than Phaffia, and thus completed the present invention.

[0006] Therefore, the above problem is solved by providing a method for breeding astaxanthin-producing yeast, which is characterized by protoplast fusion of yeast belonging to the genus Phaffia and yeast belonging to other genera. Solvable. Furthermore, as another aspect of the present invention, astaxanthin-producing yeast produced by the above breeding method is also provided.

The present invention will be explained in detail below. As the yeast belonging to the genus Phaffia used in the present invention, any strain belonging to the genus Phaffia can be used. More specific yeasts include Phaffia rhodozyma ATCC, which is known as an astaxanthin producing strain.
24201, 24202, 24203, 2
4228, 24229, 24230, and 24261 strains, as well as mutant strains derived therefrom. As used herein, "mutant strain induced" refers to a strain that is mutated by various physical or chemical treatments, including UV irradiation or N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment. Induced bacterial strains include, for example, strains with increased astaxanthin production ability and strains to which a certain auxotrophy as a selection marker, drug resistance, etc. are added, and further bred by the method of the present invention. The concept is used to include cell fusion strains. Therefore, the yeast belonging to the genus Phaffia according to the present invention includes astaxanthin-producing yeast derived from Phaffia rhodozyma, which may not essentially belong to the genus Phaffia taxonomically. Among these, the above-mentioned ATC strain is preferable.
C (American Type Culture Collection)
In addition to the type culture of Phaffia rhodozyma 6-10-A, which has enhanced astaxanthin production ability or has an appropriate selection marker attached.
rg (FERM P-12117), 13-Leu (FERM
FERM P-12118), 109-Met (F
ERM P-12119) and 24201-Ly
s (FERM P-12120). The strains with FERM P numbers listed above were submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology on March 22, 1991.
They are deposited by date and received by the corresponding number.

On the other hand, yeasts belonging to genera other than Phaffia include Saccharomyces.
myces), Hansenula
, Candida and Rhodotorula. Similar to yeasts belonging to the genus Phaffia, such yeasts are also used with the concept of including mutant strains derived from each species. Further, the method of the present invention can be applied to Aspergillus (Aspergillus).
gillus), Neurospora (Neurospora)
a) It is also applicable to the fusion of fungi commonly referred to as molds such as Rhizopus and Rhizopus.

[0009] Cell fusion between yeasts of different genera is carried out by converting each yeast into a protoplast, and then fusing the obtained protoplasts by a method known per se. Although not limited to this, the protoplastization of the present invention can be carried out as follows. For example, in the case of yeast belonging to the genus Phaffia using a general yeast growth medium, preferably a medium with a reduced amount of carbon source,
℃, and in the case of yeast other than Phaffia genus, culture is performed at 0 to 40℃. The yeast to be subjected to protoplast formation may be cultured to any of the lag phase, logarithmic growth phase, and stationary phase, but it is preferable to use yeast in the logarithmic growth phase. Bacteria were collected from the culture solution using a conventional method, and the washed cells were incubated at 0 to 37°C in a pH 5 to 8 buffer containing 30 to 200 mM 2-mercaptoethanol.
After pretreatment for 0 to 16 hours at a temperature in the range of 0 to 16 hours, centrifugation is followed by washing with an osmotic agent. As the osmotic pressure regulator, inorganic salts, monosaccharides, disaccharides, sugar alcohols, etc. are used. More specifically, inorganic salts include potassium chloride, monosaccharides include glucose, mannose, xylose, fructose, arabinose, etc., and disaccharides include sucrose, maltose, isomaltose, etc. Examples of sugar alcohols include sorbitol, mannitol, and xylitol. The concentration of the osmotic pressure regulator is 0.5-
1.2M is preferred. Next, it is brought into contact with a cell wall lytic enzyme in a buffer solution (pH 5 to 8) with controlled osmotic pressure. Examples of cell wall lytic enzymes include Novozyme 234, zymolyase, helicase, snail enzyme, and lysozyme. These may be used alone or in combination. The contact treatment with the cell wall lytic enzyme is carried out at a temperature of 0 to 37°C for 10 minutes to 24 hours with gentle shaking. The protoplast conversion rate is approximately 95% or more.

[0010] As the protoplast fusion treatment used in the present invention, any conventional method can be used, such as a method using polyethylene glycol (PEG) or an electric fusion method using a commercially available cell fusion device. Can be mentioned. For example, in the case of the electrofusion method, fusion is performed by washing the protoplasts with an osmotic pressure regulator, mixing both protoplasts, applying a high-frequency electric field, and then applying a high-voltage pulse. The conditions for applying a high-frequency electric field are that the frequency is 0.25 to 2.0 MHz;
The voltage is 20 to 40 V, and the time for applying the high frequency is the time required for protoplasts to form pearl chains, which is generally 5 to 90 seconds. Then, a high voltage pulse is applied, the field strength of which is 0.5 to 30 kV.
/cm, the pulse width is 5 to 500 μs, the number of times the high voltage pulse is applied is 1 to 5 times, and after applying the high voltage pulse, the fusion strain is stabilized by leaving it for 10 minutes or more. It is preferable.

In the present invention, the fused protoplasts are regenerated in a regeneration medium containing an osmotic pressure regulator. Specifically, for example, it is preferable to first embed protoplast fused cells using low melting point agar containing a nutrient source. Thereafter, a regenerated strain can be obtained by keeping the plate at a temperature of 0 to 40°C. The resulting fusion strain can be selected and isolated based on the differences in auxotrophy between the parent strains and the fusion strain used, as well as phenotypes such as drug resistance and temperature sensitivity.

[0012] The astaxanthin-producing yeast according to the present invention can be endowed with various characteristics by selecting the parent strain. for example,
The growth temperature limit for yeast belonging to the genus Phaffia is 27°C.
Generally, it is cultured at 20-22℃, but as a heterogeneous yeast, Saccharomyces cerevisiae (Saccharomyces cerevisiae)
By selecting a yeast that can easily grow even under higher temperature conditions of around 30° C., such as S. ces cerevisiae), it becomes possible to obtain a new yeast that grows even under high temperature conditions. Further, a new yeast with a high astaxanthin content and excellent growth ability can be created by selecting a Phaffia genus yeast with a high astaxanthin content and a yeast other than Phaffia genus with an excellent growth ability as parent strains.

[0013]

EXAMPLES The present invention will be explained in more detail with the following examples. Example 11) Preparation of auxotrophic mutant Phaffia rhodozyma
a) ATCC 24201 strain was cultured at 20°C for 48 hours using YM medium (Note 1, described below), and after harvesting, it was washed twice with distilled water and then incubated with 0.02M phosphate buffer (pH 7.5).
) and suspended in 5 mL.

[0014] NTG was added to this suspension as a mutagen.
The solution was added at a concentration of μg/mL, and the suspension was shaken at 20° C. for 1 hour. After collecting the bacteria, the cells were washed twice with distilled water. After the mutation treatment, dilute the suspension approximately 1000 times with distilled water, spread 0.1 mL of the diluted suspension onto YM agar medium (Note 2, described later), and culture at 20°C for 72 hours to obtain colonies. Ta. Mutant strains were detected by the replica method using this colony plate as a master plate. That is, using sterilized velvet fabric, colonies on the master plate were replicated onto a minimal medium (Note 3, described below) and cultured at 20° C. for 72 hours. A strain that cannot grow on minimal medium was harvested from the master plate as an auxotrophic mutant strain, and a lysine auxotrophic mutant strain 24201-Ly was obtained.
s (FERM P-12120) was obtained. Also, Saccharomyces cerevisiae (Saccharomyces
cerevisiae) is treated in a similar manner,
A mutant strain L-3C with auxotrophy for leucine and uracil was obtained. This mutant strain was sent to the Microbial Technology Research Institute in 1991.
Deposited on March 22, 2015, accession number 12116 (
FERM P-12116). 2
) Phaffia rhodochyma 24201- Protoplast fusion of Lys and Saccharomyces cerevisiae L-3C Phaffia rhodochyma 24201- Lys with 0.5% glucose, 0.3% yeast extract, 0 malt extract
．． The cells were inoculated into 50 ml of a medium containing 3% peptone and 0.5% peptone, and cultured with shaking at 20°C for 2 days. On the other hand, Saccharomyces cerevisiae L-3C was cultured with shaking at 30° C. for 1 day in YM medium. Both strains were collected, washed once with sterile water, and then washed with 0.1M 2-mercaptoethanol and 0.02
M EDTA, 0.02 containing 1.2M sorbitol
The suspension was suspended in 5 mL of M Tris-HCl buffer (pH 7.8). Phaffia rhodochyma 24201-Lys for 1 hour, Saccharomyces cerevisiae L-3C for 30 minutes,
Pretreatment was performed at 20°C with gentle shaking. After pretreatment, it was washed once with 1.2M sorbitol.

0.016M citrate phosphate buffer (pH
Phaffia rhodozyma 2420 into 5 mL of protoplastization solution consisting of 5.8) and 1.2 M sorbitol.
In the case of 1-Lys, 1.6 mg/mL Novozyme 234 (manufactured by Novo Nordisk) was added to Saccharomyces spp.
In the case of S. cerevisiae L-3C, 1 mg/mL of zymolyase (manufactured by Seikagaku Corporation) was dissolved, and then the pretreated bacterial cells were suspended. With gentle shaking, Phaffia rhodochyma 24201-Lys was kept warm at 20°C for 2 hours, and Saccharomyces cerevisiae L-3C was kept at 20°C for 1 hour. At this time, it was observed under a microscope that the cells had become spherical protoplasts. The rate of protoplast formation by this treatment was 99% or more for both strains. After washing once with 1.2 M sorbitol, both protoplast suspensions were mixed at a ratio of 1:1, and the densities in 1.2 M sorbitol were adjusted to 5 x 107/mL. Using this suspension, protoplast fusion was performed at room temperature using a cell fusion device SSH-2 (manufactured by Shimadzu Corporation). When a high frequency electric field was applied for 20 seconds at a frequency of 1 MHz and a voltage of 40 V, the formation of pearl chains was observed. Then the electric field strength is 7kV
The fusion operation was completed by applying two high voltage pulses of 60 μs/cm and a pulse width of 60 μs. Thereafter, the chamber was allowed to stand for about 10 minutes, and then the treated protoplast suspension was mixed with 1.2M sorbitol, 0.
67% Yeast Nitrogen Base (Yes
t Nitrogen Base W/O Amino
acids (manufactured by Sigma), spread on a minimal medium containing 2% glucose, 3% agar, and further a medium consisting of 1.2 M sorbitol, 0.67% yeast nitrogen base, 2% glucose, and 1% low melting point agar. were layered and the protoplasts were buried. This was cultured at 20°C, and after 10 days it was possible to obtain a fusion strain that grew and regenerated on a minimal medium. It has also been confirmed that each parent strain does not grow on minimal medium, and the regenerated strain is considered to be a fusion strain. At this time, Fafia Rodochima 24201-
The regeneration rate of Lys was 2%, and the regeneration rate of Saccharomyces cerevisiae L-3C was 0.5%. The fusion frequency of both protoplasts was 5 x 10-5. One of the fusion strains thus obtained, PS-F1, was grown in YM medium for 2 hours.
After shaking culture at 0° C. for 6 days, astaxanthin content and bacterial cell yield were measured. Astaxanthin content 0.2
0 (mg/g-dry bacterial cells), and the dry bacterial weight per medium was 4.52 (g/L). The astaxanthin content of Phaffia rhodochyma 24201-Lys is 0.
15 (mg/g-dry cell mass), and the dry cell weight was 4.
．． 80 (g/L), the fusion strain PS-F1 had almost the same astaxanthin productivity as the parent strain. Example 2 Phaffia rhodochyma 24201-Lys (F
ERM P-12120) and Saccharomyces cerevisiae L-3C (FERM P-12116) were converted into protoplasts in the same manner as described above, and protoplast fusion was performed. The settings were the same as in Example 1, except that the voltage intensity, pulse width, and high voltage pulse application used were 5 kV/cm, 100 μs, and 3 times, respectively. The fusion-treated protoplast suspension was spread on the same minimal medium as in Example 1, layered, and then cultured at 20°C. After two weeks of culturing, a particularly red colony was selected as the fusion strain PS-F2 from among the colonies that grew and regenerated in the minimal medium. After culturing the PS-F2 strain in YM medium at 20°C for 6 days with shaking, the astaxanthin content and bacterial cell yield were measured, and the astaxanthin content was 0.65 (mg/g-dry bacterial cell), and the dry bacterial weight was 4. .95 (g/L), and the parent strain 24201-Lys (astaxanthin content 0).
．． Astaxanthin content was significantly increased compared to 15 mg/g (dried bacterial cells).

The above PS-F2 strain was deposited with the Microbial Technology Research Institute on March 22, 1991, and has accession number 1.
No. 2121 (FERM P-12121). Example 3 Phaffia rhodochyma 24201-Lys (
FERM P-12120) and Saccharomyces cerevisiae IFO 1136 strain, which does not have an amino acid requirement, were converted into protoplasts according to Example 1, and then protoplast fusion was performed. At this time, Saccharomyces cerevisiae I
The protoplastization rate of strain FO 1136 was 99.9%, and the regeneration rate was 0.15%. Among the colonies grown on minimal medium, orange colonies that seem to be producing astaxanthin were selected as the fusion strain PS.
-Selected as F3. PS-F3 strain was cultured with shaking in YM medium at 20°C for 6 days and the astaxanthin content was measured. As a result, the astaxanthin content was 0.18 (m
g/g-dry bacterial body), dry bacterial weight was 4.71 (g/g-dry bacterial body).
L) and had astaxanthin productivity equivalent to that of the parent strain.

[0017] The following media are shown in footnotes in the above examples. Note 1: YM medium...YM Br
oth (manufactured by Difco) Note 2: YM agar medium...YM Agar (manufactured by Difco) Note 3: Minimal medium...Difco-Yeast Nit
rogen Base W/O
Amino acids (Difco
company) 6.7g/L
glucose

20g/L agar
20g/L

[0018]

[Effects of the Invention] The present invention provides a method for protoplast fusion of yeast belonging to the genus Phaffia and yeast belonging to other genera. This method is suitable for breeding astaxanthin-producing yeast that have good properties for traits that are controlled by multiple genes, which is difficult to do using conventional mutation induction and selection methods.

According to the present invention, for example, a strain with high astaxanthin production ability and excellent growth ability can be obtained, and by selecting a strain to be a fusion partner, a strain that can grow at various culture temperatures can be provided. Bacterial strains with these excellent traits have great advantages in the industrial fermentation production of astaxanthin.

Claims (2)

[Claims] 1. A method for breeding astaxanthin-producing yeast, which comprises protoplast fusion of yeast belonging to the Phaffia genus and yeast belonging to other genera. 2. An astaxanthin-producing yeast produced by the method according to claim 1.