GB2182673A - Transformation method for the actinomycete Streptomyces wadayamensis - Google Patents

Abstract

The invention relates to a transformation method for the actinomycete Streptomyces wadayamensis which comprises the formation and isolation of S. wadayamensis protoplasts, the incubation of protoplasts with plasmid DNA e.g. PIJ 365 in the presence of polyethylene glycol and Ca<++> to introduce the DNA into the protoplasts; culturing the protoplasts in a medium to regenerate them to normal cells and recovering a strain with phenotype derived from the donor DNA.

Description

SPECIFICATION Transformation Method for the Actinomycete Streptomyces Wadayamensis Summary of the Invention The present invention relates to a transformation method for the actinomycete Streptomyces wadayamensis.

Several processes are known to bring about changes in the genetic complement of an organism.

Transormation generally comprises the introduction of plasmid DNA into a cell in the presence of Ca++ ions and polyethylene glycol.

The method of the present invention comprises the formation and isolation of S. wadayamensis protoplasts, the incubation of protoplasts with plasmid DNA in the presence of polyethylene glycol and Ca++ to introduce the DNA into the protoplasts; cuituring the protoplasts in a medium to regenerate them to normal cells and recovering a strain with phenotype derived from the donor DNA.

Description ofthe Invention The method of this invention includes the formation of protoplasts from mycelium, their transformation and the selection of transformants.

Typically, the mycelium is grown rapidly, reaching the stationary phase 12 to 18 hours, for example about 15 hours, after inoculation of a seed culture into a culture medium at a volume ratio of say 1:20 to 1:75, for example about 1:50, and at a thermostatically controlled growth temperature, preferably of about 37"C. If the microorganism is not grown at a sufficiently rapid rate, the subsequent formation of protoplasts will be prevented, even where the stationary phase optical density has been reached.

Conveniently, protoplasts are formed from mycelium by the digestive action of the lytic enzyme lysozyme, at a concentration of 510 mg/ml, preferably & 0 mg/ml, with a typical enzyme concentration of 30 to 50, for example about 40, units per mg. It has been determined that this lysozyme concentration is higher than that which is required for protoplastformation from the mycelium of other Streptomyces.

Preferably, transformation occurs in a medium containing 5--200 mM Ca2+, more preferably about 100 mM Ca2+.

Preferably, transformation occurs in a medium initially containing 20 to 50% polyethylene glycol, more preferably 30 to 40% PEG, and subsequently diluted for instance to 10 to 20% PEG. The PEG is suitably of mean molecular weight around 1000.

In an embodiment of the invention protoplasts are regenerated for 4 to 7 days on a medium containing maltose. Preferably the maltose is at a concentration of not less than 2%.

In a particular embodiment of the present invention, the microorganism is grown in a nutrient medium (for example S1 medium) which is used as a preinoculum of another medium (for example S4 medium). S1 medium consists of 20 g soluble starch, 10 g peptone, 3 g malt extract and 0.5 g K2HPO4 per litre of distilled water (pH 7.0). S4 medium consists of 20 g maltose, 4 g yeast extract and 10 g malt extract per litre of distilled water (pH 7.4). The culture medium preferably contains a subinhibitory concentration of an inhibitory agent such as glycine that inhibits the cell wall synthesis.

In this way the action of the Iytic action is more effective.

For example, the microorganisms cultivated in S4 medium with 0.6% glycine at 37"C, with shaking, until the stationary phase is reached. Mycelium is recovered by centrifugation and washed twice with 10.3 m sucrose. Washed cells are resuspended in a hypertonic buffer such as P3, which is composed of 70 mM NaCI, 5 mM MgCI2, 5 mM CaCI2, 0.5 M sucrose and 2.5 mM TES (N-tris(hydroxymethyl)- methyl-2-aminoethanesulphonic acid) buffer, pH 7.2. Lysozyme, 5 to 10 mg/ml final concentration, is added. This mixture is incubated for 3 for 60 minutes at 37"C, and the formation of protoplasts is followed by optical microscopy.

Protoplasts are separated from mycelial debris by filtration through cotton. After filtration, protoplasts are washed with a hypertonic buffer such as PWP, consisting of 70 mMM NaCI, 10 mM MgCl2, 20 mM CaCI2, 0.5 M sucrose and 2.5 mM TES buffer, pH 7.2.

Washed protoplasts are then gently resuspended in PWP medium.

The protoplasts obtained as described above have the ability of forming colonies when spread on an appropriate medium such as M2YE, consisting of 103 g sucrose, 20 g maltose, 4 g dextrose, 10 g malt extract, 5 g yeast extract. 0.1 g casaminoacids (Difco), 3 g L-proline, 0.25 g K2SO4, 10.12 g MgCl2.6H2O, 0.05 g KH2PO4, 2.95 g CaCl2.2H2O, 0.2 g NaOH, 5.7 g TES, 2 ml trace elements solution and 22 g agar, per litre.Trace elements solution per litre is: 40 ml ZnCl2, 200 mg FeCI3.6H2O,10 mg CuCI2.2H2O, 10 mg MnCl2.4H2O, 10 mg Na2B4O7. 1 0H2O, 10 mg (NH4)6Mo7024.4H2O.

The regeneration is carried out at about 30"C for 4 to 7 days. The number of protoplasts able to form colonies under such conditions ranges from 1% to 10% relative to the number of protoplast counted on the hemocytometer before plating. The number of cells that are not true protoplasts is calculated by plating dilutions of the sample on a hypotonic medium, where protoplasts are not able to grow, such as M22 medium, which consists of 10 g glucose, g malt extract, 4 g yeast extract and 20 g agar per litre of distilled water (pH 7.4) The ratio cells/protoplasts usually obtained is < 10-6.

Protoplasts produced by the above method may be transformed with plasmid DNA before regeneration. Introduction of DNA into protoplasts is performed by mixing protoplasts with the DNA in a suitable hypertonic buffer which contains polyethylene glycol, preferably of a mean molecular weight of 1000, and calcium ions to facilitate the uptake of DNA. The buffer may also contain sucrose or an alternative stabilising agent.

A suitable hypertonic buffer is T buffer consisting of 70 mM sucrose, 1.4 Mm K2SO4, 100 mM CaCI2, 200 ml trace elements solution and 50 mM Trismaleic acid, pH 8.0. After the PEG-Ca++ treatment, protoplasts are washed with PWP buffer, resuspended in the same buffer, and regenerated.

Transformants are selected among the regenerating protoplasts in M2YE medium. M2YE medium is the R2YE medium as described in Current Topics Microbiol Immunol 96(1982), 69, with 2% maltose substituted for the 1% glucose.

The selection is based on the phenotype derived from the donor DNA. Plasmids obtained from transformants show a total identity with the plasmids used in the transformation when analyzed by digestion with restriction endonucleases, and are able to retransform S. wadayamensis protoplasts.

The number of transformants usually obtained is 106 per microgram of DNA (10-2 per microgram of DNA and per regenerated protoplasts).

In order that the present invention may be further set forth it will be illustrated by way of the following non-limiting examples; EXAMPLE 1 Isolation of plasmid plJ365 Plasm it PIJ365 was extracted from a culture of Streptomyces lividans cells which contained this plasmid. 10109 spores of S. Lividans were used for inoculating 500 ml of YEME medium supplemented with sucrose to a final concentration of 34%, Mg Cl2 to a final concentration of 5 mM.

glycine to a final concentration of 0.5% and 2 pglml thiostrepton. YEME medium consists of 3 g yeast extract, 3 g malt extract, 5 g peptone and 10 g glucose per litre. The microorganism was grown for 48 hours at 30"C with shaking. Mycelium was recovered by centrifugation for 15 minutes at 9,280xg, at room temperature, and was washed twice with 100 ml of 10 mM Tris-HCI pH 8.0, 10 mM ethylenediaminetetraacetate (EDTA) and 0.1 M sucrose. The pellet was resuspended in 50 ml of 50 mM glucose, 10 mM EDTA, 25 mM Tris-HCI pH 8.0, and lysozyme, 5 mg/ml (41.4 Units per mg); was added. The mixture was incubated for 15 minutes at 33"C. Lysis was achieved by adding 100 ml of 1% sodium dodecysulfate (SDS) in 0.2 N NaOH.After mixing, the mixture was allowed to stand 15 minutes at room temperature. 50 ml of 5 M potassium acetate were added and the resultant solution was placed in ice for 45 minutes. After centrifugation at 15,300xg for 20 minutes at 4 C, the supernatentwas recovered and then precipitated by addition of 0.6 volumes of isopropyl alcohol. After 60 minutes at room temperature, the precipitate was recovered by centrifugation for 10 minutes at 1 5,300xg, at room temperature, washed once with 70% enthanol and dissolved in 8 ml of TE buffer (TE buffer is 10 mM Tris-HCI pH 8.0 and 1 mM EDTA).

Then, 8 g of CsCI were dissolved with gently stirring, and 1 ml of 10 mg/ml ethidium bromide solution was added. The final solution was then centrifuged at 147,000xg, for 40 hours at 20"C. After the density gradient centrifugation, the circular covalently closed DNA was visualized by irradiation with UV light and seen as a band in the lower part of the centrifuge tube. The band was extracted by making a puncture in the wall of the tube. The ethidium bromide present in the fraction was removed by extraction several times with CsCI-saturated isopropyl alcohol. Then the fraction was dialysed against 2,000 volumes of TE buffer. Usually 0.4--0.6 mg of plJ365 are obtained.

EXAMPLE 2 Transformation of S. wadayamensis with plasmid plJ365 S wadayamensis was grown by inoculating 108 spores in 10 ml of S1 medium and incubating at 37"C for 48 hours with shaking. 0.5 ml of this culture were used to inoculate 25 ml of S4 medium, supplemented with glycine, 0.6% final cententration. The microorganism was incubated at 37"C with shaking, for 14 hours. The mycelium was recovered by centrifugation at 2,200xg for 15 minutes and washed twice with 10.3% sucrose.

Cells were resuspended in 10 ml of P3 buffer and lysozyme, 10 mg/ml final concentration, was added.

After incubation of the mixture at 37"C, with gentle shaking for 45 minutes, protoplasts were harvested and separated from micelial debris by centrifugation through cotton at 600x9 for7 minutes. After washing protoplasts once with PWP buffer, they were resuspended in 1 ml of the same buffer and counted on a hemocytometer. The number of protoplasts was 1.6x 109 per millilitre.

Transformation was carried out mixing 0.2 ml of protoplasts with 2 pg of plJ365 DNA. 0.5 ml of 35% PEG in T buffer were added as soon as possible and, after 1 minutes a new addition of 0.5 ml of 15% PEG in T buffer was made. After 3 minutes, the PEG present in the mixture was recovered by centrifugation at 600xg for7 minutes. The pellet was resuspended in 1 ml of PWP buffer and dilutions in the same buffer were made. Aliquots of 100 p were gently mixed with 2.5 ml of M2YE-0.6% agar and spread on M2YE plates (partially desiccated). Plates were incubated at 30"C.

Since plasmid plJ365 carries genes for resistance to thiostrepton and heamycin the selection of transformants was based on the growth of the microorganism in the presence of these antibiotics.

After 40 hours of incubation at 30do, thiostrepton 25 xug/ml or neomycin 10 yg/ml final concentration, were added and the plates were incubated for 7-10 days more, until transformants were able to form colonies. Under these conditions, 6x104 transformants per pg of DNA were obtained.

Protoplasts that had not been transformed were unable to grow under the antibiotic concentration cited above.

EXAMPLE 3 Isolation of plasmid plJ365 from S. wadayamensis 1 O-1 99 spores of a S. wadayamensis transformed with plasmid plJ365 as described in example 1, were used to inoculate 500 ml of S4 medium supplemented with MgCI2 to a final concentration of 5 mM, glycine to a final concentration of 0.5 and 2 pg/ml thiostrepton. The microorganism was grown for 24 hours at 37"C, with shaking. The plasmid was obtained in the way described in example 1 for the isolation of plasmid plJ365 from S. lividans cells. 120 pg of plasmid were obtained.

EXAMPLE 4 Transformation of S. wadayamensis with plasmid plJ365 isolated from S. wadayamensis cells.

The isolation of protoplasts, the transformation method and the selection of transformants were carried out as described in example 1. With plasmid obtained from S. wadayamensis cells, the frequency of transformation increases to 1 .4x 106 transformants per pg of DNA.

Claims (11)

1. A method for the transformation of the acti no mycete Streptomyces wadayamensis which comprises the formation and isolation of S.

wadayamensis protoplasts, the incubation of protoplasts with plasmid DNA in the presence of polyethylene glycol and Ca++ to introduce the DNA into the protoplasts; culturing the protoplasts in a medium to regenerate them to normal cells and recovering a strain with phenotype derived from the donor DNA.

2. A method as claimed in claim 1, wherein the mycelium is grown rapidly and reaches the stationary phase in 12 to 18 hours after inoculation of a seed culture into a culture medium.

3. A method as claimed in claim 2 wherein the culture medium contains a sub-inhibitory amount of a cell wall inhibitor.

4. A method as claimed in any of the preceding claims, wherein protoplasts are formed from mycelium by the digestive action of a lytic enzyme.

5. A method as claimed in claim 4 wherein the lytic enzyme is at a concentration of 5--10 mg/ml.

6. A method as claimed in any of the preceding claims wherein transformation occurs in a medium containing 50 to 200 mm Ca2+.

7. A method as claimed in any of the preceding claims wherein transformation occurs in a medium initially containing 20 to 50% polyethylene glycol.

8. A method as claimed in any of the preceding claims wherein protoplasts are regenerated for 4-7 days on a medium containing maltose.

9. A method as claimed in any of the preceeding claims wherein the plasmid DNA is derived from the plasmid plJ365.

10. A method as claimed in any of the preceding claims wherein the phenotype is characterised by a resistance to the antibiotics thiostrepton or noemycin.

11. Streptomyces wadayamensis transformed by the method of any preceding claim.