JPS6156995B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for regenerating protoplasts prepared from cells of microorganisms belonging to actinomycetes, that is, a method for regenerating protoplasts into normal cells having a cell wall by re-forming the lost cell wall. It is characterized by the ability to regenerate protoplasts with high regeneration efficiency by culturing protoplasts using a protoplast regeneration medium that has a multilayered structure. In genetic engineering, genetic recombination consists of creating protoplasts from certain microbial cells, incorporating foreign plasmids or complex plasmids, and then allowing the protoplasts to reform their cell walls to obtain transformed cells. The technique is conventional. Actinomycetes are a group of industrially important microorganisms that produce antibiotics, physiologically active substances, and special enzymes, and various studies are being conducted in the field of gene recombination technology of actinomycetes. Escherichia coli can take in plasmids in the presence of calcium chloride without turning them into protoplasts, but actinomycetes do not easily take up externally given plasmids like E. coli. Therefore, in order to use actinomycetes as host cells into which plasmids are taken up, the cell wall of the actinomycetes is first treated with a cell wall lytic enzyme to make it naked, that is, to form a protoplast, and then the plasmid is transferred. It is necessary to incorporate it. Thereafter, a transformed strain of actinomycetes is usually obtained by regenerating protoplasts, that is, producing cells with a normal morphology by reforming the cell wall. However, when conventionally used regeneration media are used to regenerate actinomycete protoplasts, the regeneration rate of protoplasts is extremely low (depending on the bacterial species, the regeneration rate is approximately 40%, but usually (with a reproduction rate of 2-3%), therefore,
The yield of transformed cells of actinomycetes is extremely poor. This has been a major obstacle to the development of genetic recombination technology for actinomycetes. The present inventor has conducted various studies to establish a method for regenerating actinomycete protoplasts that provides a high regeneration rate. Instead, a protoplast regeneration medium supplemented with sucrose and containing magnesium salts (preferably magnesium chloride) and calcium salts (preferably calcium chloride) at concentrations higher than those conventionally used, and containing in the regeneration medium. When actinomycete protoplasts are cultured using a new type of agar or agarose, which can also be called a multilayer culture method, the regeneration rate is more than five times that of the conventional method, reaching a maximum value of more than 90%. I found out that it happens. The present invention was completed based on this knowledge. That is, the gist of the present invention is to treat protoplasts obtained by treating the cells of a microorganism belonging to actinomycetes with a cell wall lytic enzyme, by treating them with an alkali metal succinate or sucrose and relatively high concentrations of magnesium and calcium salts. and a solidified plate of an agar medium for protoplast regeneration containing agar or agarose and other commonly used regeneration medium components as the lower layer, and an alkali metal succinate or sucrose, a relatively high concentration of magnesium salts and calcium salts, and soft agar. or from actinomycete protoplasts, which are cultured in the upper layer of a multilayered medium having an agar for protoplast regeneration or an agarose medium as an upper layer containing agarose and other commonly used regeneration medium components, and/or at the boundary between the upper and lower layers. in cell regeneration methods. The actinomycete protoplasts to be regenerated by the method of the invention can be obtained by treating cells of any strain of actinomycetes, particularly those belonging to the genus Streptomyces or Stoptoverticillium, with cell wall lytic enzymes, e.g. lysozyme. Actinomycete protoplasts can be prepared by any conventionally known method. For example, the following preparation method can be used. That is, an agar slant culture of a certain strain of actinomycetes was placed on a glucose-polypeptone-yeast medium).
(hereinafter referred to as GPY medium) [1.0% glucose, 0.4
% polypeptone, 0.4% yeast extract, 0.4%
Aqueous solution of NaC, 0.04% MgSO ₄ 7H ₂ O, 0.1% K ₂ HPO ₄ , pH 7.2] was inoculated and cultured with shaking at 28°C for 2 days to prepare a seed mother. Extract-glycine medium (hereinafter
(referred to as GPYG medium) [2-4 g of glycerin,
1g polypeptone, 4g yeast extract, 1
~15g glycine, 0.5g _MgSO44・_7H2O ,2
1 g of KH ₂ PO ₄ , 8 g of Na ₂ HPO ₄ .12H ₂ O dissolved in 1 part of deionized water] and grown at 28°C.
The culture was cultured with shaking for 24 to 26 hours, and the culture was centrifuged to collect bacterial cells, washed with a 0.5 M sucrose aqueous solution, and further centrifuged. NaC, 5mM
MgC ₂ , 5mM CaC ₂ , 25mM TES buffer (PH7.4) (NEGood, Arch, Biochem.
Biophys., 96, 653-61 (1962); CA57,
1269d (1962)), suspend in an aqueous solution containing 0.5M sucrose], add the required amount of lysozyme (a type of cell wall lytic enzyme), and culture with gentle shaking at 28°C. Continue the shaking culture until at least 95% of the cells have lost their cell walls, that is, become protoplasts when observed using a phase contrast microscope. When the mixture is then centrifuged at 1500 rpm for 10 minutes at 0°C, intact cells with undissolved cell walls precipitate, and the protoplast fraction is collected as a supernatant. When this supernatant is centrifuged at 3500 rpm for 10 minutes at 0°C, protoplasts collect as a precipitate, hence the common name.
PWP medium [70mM NaC, 10mM MgC ₂ .
20mM CaC ₂ , 25mM TES buffer (PH
7.4), an aqueous solution containing 0.5M sucrose], and centrifuged at 3500 rpm for 10 minutes at 0°C again, and the precipitated protoplasts were
Suspend in PWP medium and store as washed protoplasts. The alkali metal succinate salt contained in the upper and lower layers of the multilayer medium used in the method of the present invention can be a sodium salt or a potassium salt, and its concentration is 10 to 18% (Wt/V, i.e., weight/ (capacity) range. Further, the magnesium salt can be magnesium chloride salt (MgC ₂ .6H ₂ O) or magnesium sulfate (MgSO ₄ .7H ₂ O), and its concentration is preferably 0.4 to 1.0% (Wt/V). Also, the calcium salt can be calcium chloride, the concentration of which is 0.2-1.0% (Wt/
V) is better. These concentrations are higher than the magnesium salt concentration in conventionally used general media, which is 0.05.
~0.1% (Wt/V) and calcium salt concentration
It is preferably higher than 0.001 to 0.05% (Wt/V). In addition to the above-mentioned components, the upper and lower layers of the multilayer culture medium of the present invention contain 0.5 to 2.0% of components commonly used in actinomycete protoplast regeneration media.
(Wt/V) glucose, 0.2-1.0% (Wt/V)
of polypeptone, 0.2-1.0% (Wt/V) yeast extract and 0.02-0.2% (Wt/V) as metal salts.
V) KC or K ₂ SO ₄ , 0.01-0.05% (Wt/
V) K ₂ HPO ₄ can be blended. Also,
The medium PH is adjusted to 6.5-7.2, for this
0.25M TES buffer or HEPES buffer [NE
Good, Arch.Biochem.Biophys., 96, 653-61
(1962): CA, 57, 1269d (1962)]. The agar contained in the lower layer of the multilayer medium may be ordinary agar, but the agar or agarose contained in the upper layer must have a gelling (solidification) temperature lower than that of ordinary agar and must contain sodium succinate. It must be of a soft quality that can maintain a non-solid state at around 38℃ or below.
Point Agarose” (Bethesda Research La−
boratories, Inc.) or “LGT Agarose”
(manufactured by Miles Laboratories Inc., USA). In particular, the amount of soft agar or agarose in the upper layer is preferably in the range of 0.3 to 0.7% (Wt/V). The succinate contained in the upper and lower layers of the multilayer culture medium of the present invention can be replaced with 10 to 18% (Wt/V) sucrose. However, it is best to avoid mixing succinate and sucrose. Culture media suitable as the multilayer culture medium of the present invention include R ₃
There is a medium which has the following composition.

[Table] Adjust the above ingredients to 1 with deionized water. Furthermore, in another composition example of the multilayer medium, asparagine, proline, and ammonium succinate are used in place of polypeptone and yeast extract in the _R3 medium. When performing protoplast regeneration operations,
First, an agar plate that will be the lower layer of the multilayer culture medium of the present invention is solidified and prepared, and the protoplasts to be regenerated are sown on it, and then a soft agar or agarose medium that is not solidified and kept at a temperature of 38°C or less is prepared. Place as the upper layer. Mix the upper layer medium well with the protoplasts in an unsolidified state, and then mix the upper layer medium in a solidified state, generally at a temperature of 25 to 30°C, preferably 28°C.
Culture the protoplasts for 3-15 days at a temperature of °C. At this time, it was observed that when the surface of the upper layer was air-dried or dried with a desiccant, the regeneration rate of protoplasts was improved compared to when no drying was performed. On the 3rd to 4th day of culture, colonies derived from intact cells that had been mixed in the protoplasts appeared, and then on the 8th to 12th day of culture, colonies of cells obtained by regeneration of protoplasts (regenerated) appeared. colonies) appear. This regenerated colony is collected using a conventional method. The regeneration method shown in the present invention can also be applied to cell fusion, which is one type of genetic manipulation. That is, the method of the present invention can be used as a basic technique when two types of cells are respectively used as protoplasts and cell fusion is performed and then regenerated. In addition, as an application to transformation operations, it can be used not only as a regeneration method after transformation, but also by applying the regeneration operation according to the present invention to actinomycetes containing plasmids, to host cells that do not contain plasmids or that selectively have plasmids. You can also make For example, protoplasts are prepared from the naturally isolated strain 18a of Streptomyces kasugaensis MB-273 by the method shown in Examples (a) and (b) below,
The mycological properties of the regenerated colonies obtained by culturing using the multilayer method of the present invention were intermediate between those of the MB273 strain and the 18A strain. When we investigated their plasmid composition, they were roughly divided into two groups, one of which contained large amounts of plasmid pSK3 and pSK2, but no pSK1 was found. In the other group, plasmids pSK1, pSK2, and 3 were not detected at all. A strain that does not contain this plasmid (designated strain M518) is suitable as a host cell for transformation. Furthermore, actinomycetes obtained by the regeneration method of the present invention, such as Streptomyces kasugaensis
The present inventors have confirmed that the M518 strain derived from MB-273-18a (a strain that does not contain a plasmid) exhibits safety as it is free of pathogenicity and parasitism when used as a host cell. . That is,
Immunosuppressants were administered to ICF mice (20 mice in SPF1 group).
Cortisone was administered and 1×10 ⁷ CFU of the M518 strain was injected, and the animals were compared with a non-cortisone-administered group, but there were no deaths in either group, and no significant difference was observed in changes in body weight. Wistar type rat (SPF)
1 × 10 ⁹ CFU of M518 strain was orally administered, but after administration
No viable bacteria were detected in the feces until the 14th day, suggesting that the viable bacteria die rapidly within the rat's digestive tract. In addition, no pathogenicity or parasiticity was detected in experiments using 15 cynomolgus monkeys, indicating that the M518 strain created by this regeneration method has sufficient safety as a host cell. Next, the method of the present invention will be explained with reference to examples. Example 1 (a) Preparation of protoplasts Streptomyces kasugaensis MB273
- The mutant strain MB273-18a obtained by natural isolation from the C4 strain (isolated from a soil sample collected at Yoshinoyama, Nara Prefecture) (Feikoken Bibori No. 5938) was
It was inoculated into GPY medium and cultured with shaking at 28°C for 2 days to obtain a seed mother. Next, this was subcultured into the above-mentioned GPYG medium and cultured with shaking at 28°C for 26 hours. 8 ml of the culture was taken and centrifuged at 8000 rpm for 15 minutes to collect bacterial cells (approximately 0.2 g).
This bacterial cell was suspended in 8 ml of the above P3 medium (in an L-shaped tube), and 0.2 ml of 12 mg/ml lysozyme was added.
was added and gently shaken at 28°C for 30 to 40 minutes. It was confirmed using a phase contrast microscope that more than 95% of the cells had become protoplasts. The entire culture solution was cooled to 0°C and centrifuged at 1500 rpm for 10 minutes.
After 5 minutes, intact cells were precipitated. The protoplasts were collected as a supernatant, which was further centrifuged at 3500 rpm for 10 minutes at 0°C. The precipitated protoplasts were taken and
Add 8 ml of PWP medium and incubate at 0°C for 10 minutes at 3500 rpm.
A washing operation was performed by centrifugation of pm.
The precipitated protoplasts were suspended in 8 ml of PWP medium to obtain washed protoplasts. (b) Regeneration of protoplasts Take the protoplast suspension of Streptomyces kasugaensis strain MB273-18a prepared in (a) above, dilute it with PWP medium, and dilute it to 2×10 ³
Make a diluted suspension containing protoplasts/ml. Pour 0.1 ml of these onto a solidified agar plate of R ₃ medium for the lower layer, and spread on top of that agar plate with R ₃ medium for the upper layer (not solidified, but at a temperature of 38°C or lower). After pouring 4 ml of (retention) and mixing well with the protoplasts, the upper layer was also solidified at 28°C. After drying the upper surface with air or using a desiccant, culture the protoplasts at 28°C. After 3 to 4 days of culture, colonies derived from a small amount of intact cells mixed with the protoplasts appeared, and after 8 to 12 days of culture, regenerated colonies of protoplasts appeared. The number of regenerated colonies was counted on the 12th day of culture, and the regeneration rate of protoplasts was calculated using the following formula. Regeneration rate = number of regenerated colonies / number of sample protoplasts x 10
0 (c) Instead of the above MB-273-18a strain, Streptomyces fradiae KCC strain S-0579 (ISP5063), Streptomyces niveus
ISP5088 strain (NRRL 2466), Streptomyces kanamyceticus At-533 (ATCC
12853); Streptomyces fumidus
ISP5263 (ATCC12760), and Streptoberticillium machiuense ISP5221
(TCC23934), protoplasts were prepared in the same manner as in the previous section (a), and the protoplasts were regenerated and the regeneration rate was calculated in the same manner as in the previous section (b). (d) For comparison, each protoplast was regenerated using a conventional regeneration method and the regeneration rate was calculated. The conventional regeneration medium used in the comparative test is called R ₁ medium or R ₂ medium and has the following composition.

[Table] In the comparative test using the conventional method, the above R ₁ or
The _R2 medium was solidified as a monolayer, on which protoplasts were seeded and cultured at 28°C. The results of each of the above tests are summarized and presented below.

【table】

【table】

Claims (1)

[Claims] 1. Protoplasts obtained by treating cells of a microorganism belonging to actinomycetes with a cell wall lytic enzyme are treated with an alkali metal succinate or sucrose, relatively high concentrations of magnesium and calcium salts, and agar or agarose. and other commonly used regeneration medium components, as the lower layer, a solidified plate of an agar medium for protoplast regeneration, and an alkali metal succinate or sucrose, a relatively high concentration of magnesium salts and calcium salts, soft agar or agarose, and others. A method for regenerating cells from actinomycete protoplasts, which comprises culturing in the upper layer of a multilayer medium having an agar or agarose medium for protoplast regeneration as an upper layer and/or at the boundary between the upper and lower layers, which contains commonly used regeneration medium components. . 2. Prior to the start of culture, the surface of the upper layer is air-dried or dried with a desiccant. Claim 1
The method described in section.