BG61180B1 - NEW STRAINS OF FUNGUS AND THEIR USE FOR OBTAINING ANTIBIOTICS - Google Patents

Abstract

A method of producing lovastatin which comprises fermentation of a culture medium with transformed microscopic fungi from the strain of Aspergillus, selected from the species of A. arizae, A. niger, A. adulans, A. phimgatus and A. flavus, which strain is incapable of expresses lovastatin but which is transformed so as to contain a foreign DNA encoding a set of enzymes synthesizing lovastatin and a selectable marker and recovering lovastatin from the culture medium.

Description

(54) NEW MUSHROOM STAMPS AND THE USE OF THEM TO OBTAIN ANTIBIOTICS

The invention relates to novel genetically engineered fungal strains and their use for the production of antibiotics. In particular, the invention relates to novel genetically engineered Aspergillus strains and to their use for the preparation of lovastatin and its analogues.

In its chemical nature, lovastatin is [1S- [1 oc / R *], 3α, 7β, 8β / 2S ⁺ , 4S ⁺ /, 8αβ]] -dimethylbutanoic acid 1,2,3,7,8,8a-hexahydro- 3,7-Dimethyl-8- [2- / tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl] ethyl] -1-naphthalenyl ester and has the chemical formula

This compound is an antihypercholesterolemic agent as a potential inhibitor of HMG-CoA reductase, an enzyme that controls the rate of cholesterol biosynthesis. It is a fungal metabolite produced by fermentation processes using selected fungal strains.

Antibiotics such as lovastatin are metabolites that require a complex of enzymes for their synthesis. In order to enable them to be obtained by molecular cloning of antibiotic-producing microorganisms, steps such as isolation, analysis and possibly modification of the corresponding genes for several enzymes are required. Attempts to isolate such genes from similar fungal species have been linked to the production of clones carrying or individual genes from the complex, or only an incomplete gene complex / see Maagragb1 and Hopwood, Molecular Cloning of the Whole Biosynthetic Pathway of a Streptomyces Antibiotic and its Expression in a Heterologous Host ”, Nature (1984), 309, pp 462-464./.

Endo CA 1,1291794 discloses the preparation of lovastatin by fermentation using a Monnascus ruber strain.

Patent CA 1,161,380 to Monaghan et.al describes the preparation of lovastatin by fermentation using a strain of Aspergillus terreus fungus.

In these two sources describing the prior art, lovastatin is produced in parallel with other very similar chemical constituents in significant quantities from which it is to be separated. On its preparation with the participation of Monascusruber, lovastatin is obtained concurrently with monacolin V. On preparation with the participation of Aspergillus terreus, lovastatin is obtained in parallel with hydroxy acid. As long as the hydroxy acid can easily be converted to lovastatin, dihydrolovastatin can be separated from it.

It is an object of the invention to provide novel genetically engineered mutant fungal strains suitable for use in fermentation processes to produce lovastatin.

It is an object of the invention to provide novel processes for the preparation of lovastatin by fermentation involving these strains.

According to one aspect of the invention, new mutant strains of suitable species of the genus Aspergillus are provided in order to be suitable for the expression and secretion of lovastatin. These strains contain the lovastatin-producing enzyme complex genes functionally derived from the DNA of the lovastatin-producing Aspergillus terreus strains. They are obtained by protoplast transformation of DNA from the lovastatin-producing strain of Aspergillus terreus, also containing a suitable selectable marker, with another non-producing lovastatin strain selected from the group Asp.species, comprising A. oryzae, A.fumigali ^ A. niger, A.nidulans and A. flavus. Selection of transformants was performed on the basis of selective markers, identification and isolation of lovastatin-producing transformants and their subcloning.

According to another aspect of the invention, there is provided a method for the preparation of lovastatin, which involves fermentation of the Aspergillus transformed microorganism medium containing genes derived from Aspergillus terreus and encoding a lovastatin-producing enzyme complex, and restoration of the lovastatin thus formed.

The method of the invention provides for the production of lovastatin with a remarkably small amount of contaminants of dihydrolovastatin and hydroxy acid derivatives compared to the process involving Aspergillus terreus.

In the process of preparing the transformants according to the invention, standard techniques for the selection of lovastatin-free Aspergillus strains as well as for the extraction of DNA from the lovastatin-producing strain of Asp.terreus can be used. These techniques are well known to those skilled in the art and do not need to be described in detail. Likewise, the techniques and processes of protoplastic transformation useful for the purpose of the invention are well known and standard.

The preferred choice of non-producing lovastatin strain Asp. is A. oryzae, but it is not the only one used to obtain a successful result. Other species such as A. niger, A.nidulans, A, fumigaUsH A. flavus may also be used. A.oryzae was selected as the preferred species given its inertia, which facilitates work and contributes to the safety of laboratory work and commercial fermentation.

For selection of transformed microorganisms from untransformed, after the process of protoplastic transformation, DNA from A. should contain a selection marker. There is a wide / wide / selection of selection markers suitable or / and selected by one of skill in the art, and precise selection is not particularly important for obtaining a good result according to the invention. Antibiotic resistance markers, such as ampicillin resistance, rifamycin resistance, streptomycin resistance and the like, can be used and the resulting mixture of transformed and non-transformed microorganisms can be cultured in a medium containing a suitable antibiotic so that it contains only selective marker, survive for isolation.

Cycloheximide resistance is more conveniently used as a preferred selective marker. Cycloheximide is an inhibitor of protein synthesis so that the presence of a cycloheximide resistant strain or transformant in the culture fluid is readily detectable.

After selection of the transformants based on the selection marker, they are examined to detect those that will express and secrete lovastatin. Only a relatively small amount of the total number of transformants obtained by protoplast transformation have this ability. They are recognized by separate cultivation in standard culture fluid and analysis of the resultant medium, i. by HPLC for the presence of lovastatin. Those who tested positive were subcultured and allowed to sprout to produce colonies of novel lovastatin-producing transformants of fungal microorganisms of the genus Aspergillus and preferably of the species Aspergillus oryzae.

The invention is illustrated by the following experimental examples, accompanied by figures, of which:

Figure 1 is a graphical representation of the HPLC analysis of the crude fungal extract produced according to the example described below;

Figure 2 shows the NMR spectrum of the lovastatin compound prepared and purified from the hybrid strain;

Figure 3 is the spectral mass of the same compound;

Figure 4 is an image of the morphological characteristics of the new AoAt / NBJ-V transformant.

Example 1.

Materials and methods

Fungal culture - The fungal isolates of Aspergillus terreus and A. oryzae used in this study were isolated from soil samples in which orchids collected from Agriculture Canada, Research Station, BeaVeridge, Alberta, Canada.

Selection of cycloheximide resistant mutants

The germinated Aspergillus isolates on Czapek medium containing cycloheximide (concentration 0.1-5 mM) for 6 days at 28 ± 2 ° C, spores / 10 ⁸ / from each isolate of A.terreus and A.oryzae , were dispersed in Chapek medium (50 ml) and incubated for 30 min, centrifuged at 10,000 g for 10 min, and then resuspended in sterile distilled water.

They are washed three times with stirring and again precipitated by centrifugation. The spores are then suspended in sterile Triton X-100 solution - 1% (v / V) and their number is determined in a hemocytometer. A suitable solution was dispersed in 10 mM cycloheximide in a selective medium and the resistant mutants were isolated after 10 days at an incubation temperature of 28 ° C ± 2 ° C. The isolates were tested for the production of lovastatin. Cycloheximide resistant lovastatin producing an isolate of A.terreus was selected for further transformation studies.

Production of protoplasts and DNA

A. oryzae and the cycloheximidresistant lovastatin-producing isolate of A.terreus (designated JAG-4703) were cultured separately in 50 ml of Capek culture fluid. The cultures were incubated for 58 hours at 28 ± 2 ° C on a rotary shaker / New Brunswick Scientific Inc./ at 200 rpm, then harvested and washed with distilled water by repeated centrifugation.

Protoplasts from cultivation of both species were obtained using Novozym 234 / Novo-Nordisk, Novo Alle, DK 2880, Bagsvaerd, Denmark / - for removal of cell walls during 10-12 h degradation, following the method described by Dickinson & Isenberg, J.Gen. Microbiol. 128, p.651-654 (1982). Equal quantities of protoplasts of all types of regenerated, viable single cell wall spores were obtained after regeneration in regeneration medium at 28 ° C for 24 h containing 0.1 g arap / Difco / 5 g sorbose and 0.35 g EDTA in 50 ml of distilled water. As they develop, these disputes display all the cultural characteristics of their parents.

The total amount of DNA was isolated from A. terreus protoplasts according to the procedure described by Schlief & Wensink, “Practical Methods in Molecular Biology”, 33, 21-29 (1981). The protoplasts were suspended in a solution consisting of 10 mg / ml SDS, 0.1 M NaCl and 0.1 Μ Tris-HCl, pH 9.0. An equal volume of phenol diluted with Tris-HCl buffer was added to this solution and the mixture was centrifuged at 12,000 g for 10 min in an Eppendorf centrifuge tube (Brinkmann Instrument, Canada, Ltd., Rexdale, Ontario). The upper phase containing DNA was removed, mixed with 95% ethanol, left for 60 min at -20 ° C, then centrifuged as before for 10 min. The pellets were resuspended in buffer pH 7.0 and incubated with RNa3a / Sigma, St. Louis, MO, USA /, was treated with phenol and DNA was precipitated from the liquid layer. The isolated DNA was purified by adsorption and washed on DEAE cellulose (DE-52, Whatman), re-wetted with Tris-HCl buffer, pH 7.5 and 0.3M NaCl and placed in a paste pipette. The DNA was eluted with 10 mM Tris-HCl buffer, pH 7.5, containing 1.5M NaCl. This solution was diluted to 0.2 M with NaCl and the DNA precipitated with two volumes of ethanol. The purity of the DNA was tested on the 200-320 nm spectrum by determining the A ₂₆₀ / A ₂₈₀ absorption rate. Only those DNAs with velocities between 1.50 and 2.00 can be used for transformations. Six samples, each 0.1 mg of isolated DNA, were incubated in regeneration media to detect the absence of viable protoplasts, and none of them developed as well as colony.

Protoplastic DNA incubation

Approximately 5.2x10 ⁴ A.oryzae protoplasts tested for hemocytometer counting were incubated with 10-100 ng A.terreus DNA in 5 ml regeneration medium, the composition of which was as described above, as well as the regeneration J. To investigate the possible chemical effect of DNA on lovastatin expression, 10-100 pg of calf thymus DNA / Sigma Chemical Co., St. Louis, MO, USA /, DNA / Bethesda Researcn Lab., Caithberg, MD, USA / and homologous DNA from A. oryzae were incubated with similar portions of A.orilae protoplasts. When incorporated into DNA incubation, protoplast regeneration is reduced to less than 10% as an analogous number of treated protoplasts fail to sprout and show any cycloheximide resistance.

Lovastatin production and testing

A suspension of spores developed from A. oryzae protoplasts incubated with A.terreus DNA was inoculated into 1 ml per petri dish on a Chapek agar medium containing 10 mM cycloheximide. After incubation for 48 hours at 28 ± 2 ° C, the cultures reach a diameter of 6-8 mm. Each of them develops from a single dispute under these conditions. Those grown on cycloheximide medium were inoculated individually on Chapek medium in 500 ml Erlenmeyer flasks and incubated on a rotary shaker (200 g) at 28 ± 2 ° C for 12 days. Each culture medium was tested for 5 lovastatin production as described above and HPLC analysis.

Extraction

The fermentation medium (50 ml) was acidified to pH 4.0 with 17 N HCl and then fractionated against ethyl acetate (100 ml, 3 times). The ethyl acetate fractions were dried in vacuo at 30 ° C, the residue collected in acetonitrile (5 ml) and subjected to HPLC analysis.

HPLC Analysis

HPLC equipment / Beckman 420 model / is provided by Beckman Instruments, Toronto, Canada and consists of an Altex pump / Model

110 A / and injection valve. The LC-UV detector was set at 237 nm. A C-18 ODS silica column (25 x 0.46 cm, i.d./ dissolution system consisting of acetonitrile and water / 55: 45, V / V) was used at a slow rate of 1.0 ml / min. The lovastatin production was compared and quantified from the constructed standard lovastatin curve.

Example 1. Results

Seventy-nine cycloheximide-resistant isolates, including four lovastatin-producing isolates, were obtained from A.oryzea protoplasts incubated with the DNA of the cycloheximide-resistant lovastatinpro15 mutant of A.terreus. Transformation experiments are performed six times.

The results are given in the following Table 1.

Table 1

Cycloheximide-resistant and lovastatin-expressing protoplasts of A.oryzae, incubated with DNA from the cycloheximide-resistant lovastatin-producing mutant of A.terreus.

No Protoplasts ml Regen number. protoplasts after prot. fusion, No. / ml % Regeneration DNA / ng / Number of lovastatin producing isolates 1. 7.4 x 10 * 4.1 x 10 ⁵ 56 100 0 2. 5.2 x 10 ⁵ 3.1 x 10 ⁵ 60 75 2 3. 5.2 x 10 ⁵ 3.7 x 10 ⁵ 72 50 2 4. 4.0 x 10 ⁵ 2.7 x 10 ^s 70 25 0 5. 2.6 x 10 ⁵ 1.5 x 10 ⁵ 65 10 0 6. 1.6 x 10 ⁵ 1.1 x 10 * 70 0 0

One of the transformed AOII crops has received its original transformation characteristics in six months. Figure 1 illustrates HPLC analysis of extracts obtained from the recipient and donor cultures of A.oryzae and those treated with DNA obtained from A.terreus. The production of lovastatin using transformed isolates is further shown in Table 2.

Table 2

Preparation of lovastatin, expressed in mg / Ι, from transformed isolates of Aspergillus oryzae in Chapek food broth / pH 6.8 /

Isolate no Lovastatin Productivity / mg / 1 / AO - I 15.36 ± 1.78 AO - 11 26.89 ± 3.76 AO - III 13.46 ± 4.56 AO - IV 9.67 ± 0.75

Isolate No. AO-Ι was subcultured five times to obtain AoAt NBJ / 5 transformant, which was deposited on February 25, 1992 as a viable, permanent culture in ATCC No. 74135.

Lovastatin is produced in association with a hydroxy acid analog of it, which is readily transformed into lovastatin, for example by fusion into toluene to effect lactonization and, therefore, to increase the productivity of lovastatin. Lactonization is a must in these experiments.

Lovastatin produced by A.terreus parental culture in the Chapek food broth is 166.72 ± 5.92 mg / l, and A. oryzae parental culture does not produce any amount of lovastatin.

Figure 2 represents the NM-NMR spectrum of the lovastatin compound purified by HPLC from the hybrid strain. Compared to the standard known lovastatin spectrum, this spectrum represents the identity of the product.

Figure 3 - shows the spectral mass of the same product and shows that lovastatin is obtained with a purity of 99%.

DNA concentrations are 5 to 29 ng per ml of medium, without affecting either the regenerative capacity of A.oryzae protoplasts or the recovery of cycloheximide resistance and lovastatin-producing isolates from DNA incubation. The data indicate that these concentrations are not a limiting factor in the production of lovastatin. Similarly, calf thymus, DNA, and homologous DNA from A.oryzae at concentrations of 5 ng to 5 pg per ml of medium and did not affect the regeneration of A.oryzae protoplasts compared to untreated controls. However, at 10 and 100 μg per ml of medium, DNA reduces regeneration to a maximum of 26% and 1% respectively. These DNA treatments do not express lovastatin expression.

The regeneration medium results in an amount of protoplasts comparable to those obtained by Acha et al. J.Cen.Microbiol., 45, 515523 (1966), using a mineral-rich medium. Conidia and freshly ripened conidia produce more than viable protoplasts and DNA from the mycelium. The formation of cell aggregates during protoplast regeneration described by Acha et.al. (1966) is not observed in this system. The apparent transfer of factors responsible for the expression of cycloheximide resistance and lovastatin expression from A.terreus to A.oryzae indicates interspecific transformation of DNA. The co-transformation of cycloheximide resistance and lovastatin productivity are unexpectedly satisfactory and suggest physical proximity and likely accumulation of genes involved in the expression of these characteristics.

The morphology of the new AoAt transformer - NBJ / 5 is shown in Figure 4 and can be characterized as follows.

AoAt morphology - NBJ / 5

Conidial heads, elongated and light brown. Conidiophores smooth and colorless. Germ bags - hemispherical, covered up to half or up to one third of the phialids (the last branch of the conidiocarrier or conidiocarrier itself), arranged in two layers. Conidia are spherical or elliptical, smooth colonies of Chapek agar medium, growing too rapidly, both fluffy and velvety in brownish-brown color, to form conidia. Orange exudate, turning from yellow to brown.

These experiments show that the genetically determined antibiotic production characteristics of one fungal species can be expressed in another.

Example 2. Method of Preparation

The fungal culture is a transformed culture from Aspergillus oryzae / ATCC No. 74135 / AoAt - NBJ / V and used to prepare lovastatin.

Fermentation

Preparation of seeds. The fungal culture (lyophilized) was aseptically transferred onto potato-dextrose agar and allowed to sprout at 25 ° C for 4-5 days. Towards the end of incubation, the conidial suspension was prepared by adding 10 ml of Triton X-100 (0.01%) solution, stirred and the conidial suspension was transferred to sterile rice (50 g), placed in Erlenmeyer flasks (250 ml capacity). . Fill the flask with another 10 ml of sterile water, shake gently and incubate at 28 ° C for 5-7 days. At the end of the incubation period, sterile water (50 ml) was added.

The conidial suspension was passed through a sterile gauze and the filtrate containing 1x10 'conidia / ml was transferred to a fermenter with a capacity of 501 with 30 l of culture medium. The composition of the culture medium contains in g / l the following ingredients: E - glucose 20; malt extract 20; neoppton 3 and tap water 1 1.

pH is 6.8, adjusted with 10% -NaOH. / The medium was sterilized at 121 ° C for 30 min (15 psi). The seeds were allowed to germinate for 17-20 h at 28 ° C / aeration rate of 0.3 - 0.5 VVM6 with 80-100% soluble oxygen. (200 rpm).

Production of lovastatin

The seeds (30 l) are transferred to a fermenter (1000 l, working volume) containing the fermentation medium. The composition of the medium is as follows:

Lactose 60 g / l Ardamin pH 10 g / l Soy protein 2 g / l KS1 2 g / l KN ₂ PO ₄ 0.8 / 1 MnS0 ₄ H ₂ 0 0.03 g / l

Betaine 0.6 g / l

P200 2 ml

Tap water 1 L pH was 6.8 (before sterilization, brought with 105 NaOH / H ₂ 0 ₄ ), sterilization at 121 ° C for 1 hour at 15-20 psi.

After inoculation, the culture was allowed to grow for 7-8 days at 28 ° C with pH control at 6.0-6.2 (with 10% H ₂ SO ₄ ).

The aeration rate is maintained between 0.7 - 1.0 VVM /% dissolved oxygen 80100% /.

2ND Production stage

A seven-day fermentation liquid (50 l) was aseptically transferred to a fermenter (2,000 l working volume) containing 1500 l of the nutrient medium (composition above). The crop was allowed to sprout for 24 hours at 28 ° C / 200 rpm / DO. 80-100%. The fermentation fluid (200 l) was aseptically transferred to a fermenter with a capacity of 30,000 l and a working volume of 20,000 l containing 18,000 l of the culture medium. The culture was allowed to sprout for 7-9 days at 28 ° C with pH control at 6.0 - 6.2. After 60 hours of fermentation, the pH is no longer controlled. The fermentation liquid is supplemented with a feed medium / speed of 10 Ι / h / for 4 days. The nutrient medium contains the following ingredients: D-glucose 285.7 g / l; Ardamin pH

71.4 g / l Soy protein 14.3 g / l and tap water 1 1.

pH 6.8 (before sterilization) - sterilization at 121 ° C for 30-45 min. At the end of the fermentation, the fermentation liquid was acidified to pH 4.0 with 10 N NC1 and centrifuged. The fungal residue was dried at 55 ° C and extracted.

Extraction and purification

The dried fungal residue (20 kg) was subjected to homogenization with cold ethyl acetate (10-15 min) and the homogenized material returned to 80-85 ° C for 4-6 hours, allowed to cool and filtered. The filtrate was dried in vacuo to a black material with a gummy consistency (2.5 kg - 3 kg). The material was mixed with silica gel (2.5 kg) and CH ₂ Cl ₂ (5 1). The CH ₂ Cl ₂ was evaporated in vacuo and poured into the silica gel glass column (100 cm long x 22.5 cm in diameter) with EtOAc: Hexane (1: 2 VV). Samples (2,000 11.12 x) were collected.

After 24 hours, the elution mixture was changed to EtOAc: Hexane (1: 1 V.V) and left for another 24 hours. Samples containing the product were poured, dried (280 g) and crystallized with methanol.

Crystallization

The yellow dried product (280 g) was dissolved in 2.8 L of methanol and boiled for 40-60 min at 60-65 ° C. The hot methanol mixture was filtered and the filtrate was incubated for 4-6 hours at 4 ° C. The white crystalline needles were separated from the mother's fluid and rinsed with cold methanol. The crystals were dried under vacuum, weighed (190 g) and kept in a desiccator at 4 ° C.

The method according to the invention can result in the production of lovastatin, which is sufficient to cause the cell walls of the new A.oryzae transformants to rupture, so that this stage of cell lysis can be eliminated before the recovery of lovastatin. This greatly simplifies the on-line recovery and purification of the product. As described above, lovastatin according to the present process can be recovered by soluble extraction without the need to form esters, salts, etc. during the recovery period. Soluble extraction is much simpler and is a much more economical recovery and purification process than chromatography.

To the extent that the invention is described in detail by the specific experiments described above, it should be understood that it is not limited thereto. Its scope is defined by the claims formulated.

Claims (13)

Claims 1. A method for the preparation of lovastatin, which comprises fermentation of a culture medium with transformed microscopic fungi of an Aspergillus strain selected from A.oryzae, A.niger, A.nidulans, A. fumigalis and A. flavus strains, which is incapable of strain expresses lovastatin but which has been transformed to contain foreign DNA encoding a complex of lovastatin syntheses and a selectable marker and recovery of lovastatin from the culture medium. The method of claim 1, wherein the transformed microorganism contains foreign DNA derived from the lovastatin-producing species of Aspergillus terreus. The method of claim 2, wherein said strain of the microorganism is a strain of A.oryzae. A method according to any one of the preceding claims, wherein the foreign DNA introduced into the transformant contains cycloheximide-resistant genes as a selective marker. A method according to any of the preceding claims, wherein the transformant is AoAt - NBJ / 5, registered in ATCC under No. 74135. A method according to any one of the preceding claims, comprising an additional lactonization step of a hydroxy acid analogue formed in the fermentation process to lovastatin. A method according to any one of the preceding claims, wherein the recovery of lovastatin is effected by soluble extraction. 8. New fungal transformants carrying a complex of enzymes capable of synthesizing lovastatin, these transformants being selected as A.species strains by A.oryzae, A.niger, A.nidulans, A.fumigalis and A. .flavus and are naturally unable to express lovastatin, but containing foreign DNA containing genes encoding a complex of enzymes become capable of synthesizing lovastatin. The fungal transformants according to claim 9, wherein Aspergillus is selected from A.oryzae, A.niger, A.nidulans and A.fumigalis. A fungal transformant according to claim 10, wherein A.species is A.oryzae. The fungal transformants of claim 11, wherein the foreign DNA is derived from a lovastatin-expressing strain of A. terreus. A fungal transformant according to claim 12 and comprising the product of protoplastic transformation of the total DNA from A.terreus strain into A.oryzae strain. 13. AoAt - NBJ / 5 fungal transformants registered in ATCC under No. 74135.