SI9300068A - Novel fungicidal strains and use thereof in antibiotic production - Google Patents

Abstract

Lovastatin is produced by a process of fermentation using a fungal transformant produced by introducing into a non-lovastatin expressing Aspergillus strain such as a strain of Aspergillus oryzae the DNA of a lovastatin-expressing strain of Aspergillus terreus.

Description

New fungal soybeans and their use in antibiotic production

The present invention relates to novel, genetically engineered fungal soybeans and their use in the preparation of antibiotics. Specifically, it refers to new, genetically engineered Aspergillus soybeans and their use in the preparation of lovastatin and its analogues.

Lovastatin is chemically 1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8- [2- (tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl) -ethyl] [1S- [1a (R-), 3a, 7 / 3,8 / 3 (2S *, 4S '), 8a / 3]] - 2-methylbutanoic acid -l-naphthalenyl ester and having the chemical formula

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

Antibiotics, such as lovastatin, are metabolites that require a series of different enzymes for their synthesis. To enable their production by molecular cloning of antibiotic-producing microorganisms, isolation, analysis, and possibly modification of the corresponding genes for multiple enzymes is required. Attempts to isolate such genes from such fungal species have thus far yielded clones that carried either single gene series or only incomplete gene series - see Malpartida and Hopwood, Molecular Cloning of the Whole Biosynthetic Pathway of a Streptomyces Antibiotic and its Expression in a Heterologous Host, Nature (1984), 309 p. 462-464.

CA-PS 1,129,794 (Endo) describes the preparation of lovastatin by fermentation using a strain of the fungal species Monascus ruber.

CA-PS 1,161,380 (Monaghan et al.) Describes the preparation of lovastatin by fermentation using strains of the fungal species Aspergillus terreus.

Both of these known processes produce lovastatin, together with other, very similar chemical compounds, in significant amounts to be separated. When produced using Monascus ruber, lovastatin appears together with monacolin J. When prepared using Aspergillus terreus, lovastatin appears with dihydrolovastatin and hydroxy acid. While hydroxy acid can be readily lactonized into lovastatin, dihydrolovastatin must be separated from it.

It is an object of the present invention to provide novel, genetically engineered mutant fungal soybeans capable of use in fermentation processes for the preparation of lovastatin.

It is a further object of the invention to provide novel processes for the preparation of lovastatin by fermentation using such strains.

According to one aspect of the present invention, it is a novel mutant fungal strain of the relevant species of the genus Aspergillus, which is capable of expressing and secreting lovastatin. These strains contain the lovastatin-producing enzyme gene in a functional relationship derived from the DNA of lovastatin-producing Aspergillus terreus strains. They are produced by a process of protoplast transformation of DNA from lovastatin-producing Aspergillus terreus strain, which also contains a suitable selectable marker, with another species of lovastatin-free Aspergillus selected from A oryzae, A fumigatus, A niger, A nidulans and A flavus, the selection of the transformants thus formed on the basis of selectable brandy, the identification and isolation of lovastatin-producing transformants and their subcloning.

According to another aspect of the invention, there is provided a process for preparing lovastatin, comprising fermenting the nutrient medium with the Aspergillus microorganism containing the genes derived from Aspergillus terreus and coding for a series of lovastatin producing enzymes and recovering the resulting lovastatin.

The process of the present invention produces lovastatin with significantly smaller amounts of dihydrolovastatin impurities and hydroxy acid derivatives compared to processes using Aspergillus terreus.

In the process for preparing the transformants of the present invention, standard protoplast preparation techniques of the selected lovastatin non-producing Aspergillus strain and standard DNA extraction techniques of the lovastatin producing Aspergillus terreus strain can be used. These techniques are well known to those skilled in the art and do not need detailed discussion here. Similarly, the techniques and processes of protoplastic transformation used herein are known and standard.

The preferred choice of non-lovastatin-producing Aspergillus strain is the Aspergillus oryzae strain, but this is not critical to the success of the invention. Other species of Aspergillus may be used, namely A niger, A nidulans, A fumigatus and A flavus. But oryzae is chosen as the preferred species because of its inertness, which makes it easy and safe to handle in large scale laboratory fermentations and fermentations.

For the selection of microorganisms of transformants from non-transformants by protoplast transformation, DNA from A terreus must contain a selectable marker. There is a wide selection of selectable markers that are accessible and selectable by the skilled worker, and precise selection is not critical to the success of the invention. Brands of antibiotic resistance, such as ampicillin resistance, rifampicin resistance, streptomycin resistance, etc., can be used, and the resulting mixture of transformants and non-transformants can be grown in a medium containing an appropriate antibiotic so that only transformants containing a selectable marker will survive for isolation.

Particularly preferred as a selectable marker is cycloheximide resistance for convenience. Cycloheximide is an inhibitor of protein synthesis, so that the presence of a cycloheximide-resistant strain or transformant in the culture broth is easily established.

Following the selection of transformants based on selectable brandy, they are screened for those that will express and secrete lovastatin. Only a relatively small number of all transformants produced in the protoplastic transformation process have this capability. They are identified by separate cultivation in a standard culture broth and analysis of the resulting medium, e.g. by HPLC, given the presence of lovastatin. Those tested positive for the presence of lovastatin are subcultured and cultured to obtain colonies of new transformative fungal microorganisms of the genus Aspergillus producing lovastatin, and preferably Aspergillus oryzae.

The invention is further illustrated in the following experimental reports.

In the accompanying drawings, FIG. 1 is a graphical representation of HPLC analysis of crude fungal extracts produced following the experiments of Example 1 described below;

FIG. 2 ^X H-NMR spectrum of lovastatin compounds obtained and purified from hybrid strain; FIG. 3 is the mass spectrum of the same compound;

FIG. 4 is an overview of the morphological characteristics of the new AoAt / NBJ-V transformant.

EXAMPLES

MATERIALS AND METHODS

Fungal culture - Aspregillus terreus and A. oryzae fungal isolates used in the present study were isolated from soil samples for orchids collected from Agriculture Canada, Research Station, Beaverlodge, Alberta, Canada.

SELECTION OF CYCLOHEXYMIDE RESISTANT MUTANTS

After growing both Aspergillus isolates on Czapek dox media containing cycloheximide (conc. 0, 1-5 mM) for 6 days at 28 ± 2 ° C, the spores (10 ⁸ ) of each A. terreus and A. oryzae isolate were distributed at 10 mM cycloheximide Czapek dox broth (50 ml) and incubated for 30 minutes, centrifuged at 10000 g for 10 minutes and then resuspended in sterile distilled water. They are washed three times with stirring and sedimented again by centrifugation. The spores were then suspended in sterile 1% (v / v) Triton rit-100 solution and their numbers were determined in an aliquot on a hemocytometer. Applied dilution was distributed to 10 mM cycloheximide selection medium and resistant mutants were isolated after incubation for 10 days at 28 ± 2 ° C. Isolates are screened for lovastatin production. For further transformation study, the cycloheximide resistant isolate of A. terreus producing lovastatin is selected.

PREPARATION OF PROTOPLASTS AND DNA

Aspergillus oryzae and the cycloheximide-resistant isolate A. terreus (hereinafter designated JAG-4703) producing lovastatin were grown separately in 50 ml of Czapek's dox broth. 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 sterile distilled water by repeated centrifugation.

Protoplasts from cultures of both species were obtained using Novozyma 234 (Novo-Nordisk, Novo Alle, DK 2880, Bagsvaerd, Denmark) to remove cell walls during digestion for 10 to 12 hours, generally by the method described in Dickinson & Isenberg, J. Gen. Microbiol. 128, p. 651-654 (1982). Aliquots of protoplasts of each species regenerate single cell wall viable spores at 28 ° C after 24 hours in regenerated medium (R) containing 0.1 g of agar (Difco), 5 g of sorbose and 0.35 g of EDTA in 50 ml of distilled water . When they emerge, these conflicts develop all the characteristics of their parents' culture.

Total DNA was isolated from Aspergillus terreus protoplasts according to the procedure described in Schlief & Wensink, Practical Methods and Molecular Biology, 33, 21-29 (1981). The protoplasts were suspended in a solution containing 10 mg / ml SDS, 0.1 M NaCl and 0.1 M tris-HCl, pH 9.0. Add the same volume of phenol saturated with tris-HCl buffer. The mixture was centrifuged for 10 min. at 12,000 g in an Eppendorf centrifuge tube (Brinkmann Instruments, Canada Ltd., Rexdale, Ontario). The upper DNA-containing phase was removed, mixed with 95% ethanol, stored for 60 min. at -20 ° C, then centrifuged as before for 10 minutes. The pellet was resuspended in buffer pH 7.0 and incubated with RNase (Sigma, St. Louis, MO, USA), treated with phenol, and DNA precipitated from the aqueous layer. The isolated DNA was purified by adsorption and washing on DEAE-cellulose (DE-52, Whatman) pre-soaked in 10 mM tris-HCl buffer, pH 7.5, and 0.3 M NaCl contained in a Pasteui pipette. DNA was eluted with 10 mM tris-HCl buffer, pH 7.5 containing 1.5 M NaCl. Dilute this solution to 0.2 M NaCl and precipitate the DNA with two volumes of ethanol. The purity of DNA is determined from 200 to 320 nm spectra by determining the absorbance deflection Α ^ θ / Α ^. Only DNAs with ratios between 1.50 and 2.00 are used in the transformations. Six samples, each 0.1 mg of isolated DNA, are incubated in regenerated medium to ensure the absence of viable protoplasts, and none of them develops a colony.

Incubation of protoplasts - DNA

Approximately 5.2 χ 10 ⁴ protoplasts A oryzae, determined by hemocytometer counts, were incubated with 10-100 ng A terreus DNA in 5 ml of regenerable medium, with the composition as described above, and regenerated as described above. To study the possible chemical effects of DNA on lovastatin expression, 10-100 μξ of calf thymus DNA (Sigma Chemical Co., St. Louis, MO., USA), DNA (Bethesda Research Laboratories, Gaitherberg, MD, USA) and homologous A. oryzae were incubated DNA with similar proportions of A. oryzae protoplasts. When DNA is incubated in incubations, protoplast regeneration is reduced to less than 10% and cycloheximide resistance was not achieved by comparative numbers of treated protoplasts (10 ⁵ ).

LOVASTATIN PRODUCTION AND TEST

A suspension of spores developed from A oryzae protoplasts incubated with A terreus DNA was inoculated, 1 ml per petri dish, onto Czapek's dox agami medium containing 10 mM cycloheximide. After incubation for 48 hours at 28 ± 2 ° C, the cultures reach 6-8 mm in diameter. Each develops separately from the individual dispute under these conditions. Those grown on cycloheximide medium were each inoculated separately on a slurry of Czapek dox media (50 ml in a 500 ml conical flask) and incubated on a rotary shaker (200 rpm) at 28 ± 2 ° C for 12 hours. Each growth medium was examined for lovastatin production by the procedure described below and by HPLC analysis.

EXTRACTION

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

HPLC ANALYSIS

HPLC equipment (Beckman Model 420) was purchased from Beckman Instruments, Toronto, Canada, and is available from the Altex pump (Model 110 A) and injection valve. The LC-UV detector was set at 237 nm. Use a Hypersil C-18 ODS silica column (25 x 0.46 cm in diameter) and a solvent system of acetonitrile and water (55:45, v / v) at a flow rate of 1.0 ml / min. The lovastatin produced is compared and quantified from the constructed standard lovastatin curve.

EXAMPLE 1 - RESULTS

Ninety-seven cycloheximide-resistant isolates, including 4 lovastatin-producing isolates, were obtained from 5.2 χ 10 ⁵ A. oryzae protoplasts incubated with DNA from the cycloheximide-resistant mutant A. terreus-producing mutant. Transformation experiments were performed 6 times.

The results are given in the following Table 1.

TABLE 1 Cycloheximide-resistant and lovastatin-expressing protoplasts of Aspergillus oryzae, incubated with DNA from cycloheximide-resistant mutant Aspergillus terreus producing lovastatin

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One of the transformed cultures of AO-II retained its original transformed characteristics for 6 months. FIG. 1 shows HPLC analysis of extracts obtained from recipient and donor cultures of A. oryzae and those treated with A. terreus DNA. The production of lovastatin by transformed isolates is shown below in Table 2.

TABLE 2 Production of lovastatin with transformed isolates

Aspergillus oryzae in Czapek's dox broth (pH 6.8)

Isolate no. lovastatin yield (mg / 1) AO-I 15.36 ± 1.78 AO-II 26.89 ± 3.76 AO-III 13.46 ± 4.56 AO-IV 9.67 ± 0.75

Isolate no. We subcultured AO-I 5 times to obtain the AoAt - NBJ / 5 transformer, which was deposited on 25 February. 1992 as a viable permanent culture at American Type Culture Collection under ATCC Reference 74135.

Lovastatin is produced in conjunction with its hydroxy acid analogue, which can be easily converted to lovastatin e.g. by refluxing in toluene to perform lactonization while increasing lovastatin yield. Lactonization was not performed in these experiments.

Lovastatin produced by the parental culture of A. terreus in Czapek's dox broth was 166.72 ± 5.92 mg / 1 and parental A. oryzae did not produce lovastatin.

FIG. 2 of the accompanying drawings is the ^ -NMR spectrum of lovastatin compound purified by HPLC from a hybrid strain. Compared to the known, standard lovastatin spectrum, this spectrum confirms the identity of the product.

FIG. 3 of the accompanying drawings are the mass spectrum of the same product and show that lovastatin was obtained at a purity of 99%.

DNA concentrations in the range of 5-20 ng / ml medium did not affect either the regeneration capacity of A. oryzae protoplasts or the recovery of cycloheximide-resistant and lovastatin-producing isolates from incubations with A. terreus DNA. The data indicate that these concentrations were not a limiting factor in the production of lovastatin. Similarly, calf thymus DNA, DNA, and homologous A. oryzae DNA at concentrations of 5 ng to 5 / tg per ml of medium did not affect the regeneration of A. oryzae protoplasts compared to untreated controls. However, the DNA is at 10 oz. 100 / tg per ml of medium reduced the regeneration to a maximum of 26% or. 1%. These DNA treatments did not induce lovastatin expression.

Regenerimi medium gave protoplast numbers comparable to those obtained by Acha et al. J. Gen. Microbiol., 45, 515-523 (1966) using a more complex mineral medium. Conidia and freshly sprouted conidia produced better yields of viable protoplasts and DNA than mycelium. The formation of cell aggregates during protoplast regeneration was not observed in this system, as described in Acha et al. (1966). The apparent transfer of factors responsible for expression of cycloheximide resistance and lovastatin expression from A. terreus to A. oryzae suggests interspecies DNA transformation. The co-transformation of cycloheximide resistance and lovastatin production was unexpectedly satisfactory, suggesting the physical proximity and possible accumulation of genes involved in the expression of these characteristics.

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

AoAt-NBJ / 5 MORPHOLOGY

Heads of conidia colonial, light brown. Conidiophores smooth, colorless. Vesicles semicircular, covered up to one half or two thirds with phialides arranged in two layers. Conidia spherical or ellipsoidal, with smooth colonies on Czapek agai growing very rapidly, either hairy or velvety cinnamon appearances due to conidia production. Orange exudate, change yellow to qavo.

These experiments indicate that, in principle, genetically determined antibiotic production properties of one fungal species can be expressed in another.

EXAMPLE 2 - PREPARATION PROCEDURE

Fungal culture: Transformed culture of Aspergillus oryzae (ATCC No. 74135) AoAt / NBJ-V is used to produce lovastatin.

FERMENTATION

Preparation of the inoculum: The fungal culture (lyophilized vial) is transferred aseptically to oblique potato dextrose agar and allowed to grow for 4 to 5 days at 25 ° C. At the end of the incubation, a suspension of conidia was prepared by adding 10 ml of triton x-100 solution (0.01%), stirring vigorously and transferring the conidia suspension to sterile rice (50 g) contained in a conical flask (250 ml capacity). Make up the flask with 10 ml of sterile water, stir vigorously and incubate for 5 to 7 days at 28 ° C. At the end of the incubation time, sterile water (50 ml) was added.

The conidia suspension was passed through a sterile muslin cloth and the filtrate containing 1 x 10 ⁸ conidia / ml) was transferred to an inoculation fermenter (capacity 50 1) with 30 1 inoculation media. The composition of the inoculation media used is as follows:

D-glucose 20 g / l malt extract 20 g / l neo-peptone 3g / l tap water 11 pH 6.8 (adjusted with 10% NaOH).

The media were sterilized for 30 minutes at 121 ° C (1.05 bar). The inoculum was allowed to grow for 17 to 20 hours at 28 ° C (aeration rate of 0.3-0.5 vvm) with 80-100% dissolved oxygen (200 rpm).

LOVASTATIN PRODUCTION

Transfer the inoculum (30 1) to a production fermenter (working volume 10001) containing the production media. The composition of the production media is as follows:

Lactose ardamine pH g / l 10 g / l

soy protein 2 g / l KC1 2 g / l KHgPO, 0.8 g / l MnSO ^ HgO 0.03 g / l betaine 0.6 g / l p ^r 2000 2 ml tap water 11

6.8 (before sterilization, adjusted with 10% NaOH / H ₂ SO ₄ , sterilization at 121 ° C for 1 hour at 1.05 to 1.41 bar.

After inoculation, the culture was allowed to grow for 7 to 8 days at 28 ° C with pH control at 6.0 to 6.2 (with 10% HgSOJ.

The ventilation rate is maintained between 0.7 and 1.0 vvm (% dissolved oxygen 80 to 100%).

2. PRODUCTION STAGE Transfer the aged fermented broth (501) aseptically into an inoculation fermenter (working volume 2000 1) containing 1500 l inoculation media (composition given above). The inoculum was allowed to grow at 28 ° C (200 rpm) for 24 hours, dissolved. oxygen 80 to 100%). Transfer Brozgo (2001) aseptically into a production fermenter (capacity 30,000 1 with a working volume of 20,000 1) containing 18,000 1 production media. The culture was allowed to grow for another 7 to 9 days at 28 ° C with pH control at 6.0 to 6.2. After 60 hours of fermentation, the pH is not controlled. The fermented broth is supplemented with nutrient media (speed 101 / h) for 5 days. The composition of the nutrient media is as follows:

D-glucose 285.7 g / 1 ardamine ph 71.4 g / 1 soy protein 14.3 g / 1 tap water 11 ph 6.8 (before sterilization) - sterilized for 30 to 45 minutes at 121 ° C. At the end of the fermentation, the broth was acidified to pH 4.0 with 10N HCl and centrifuged. The fungal cake is dried at 55 ° C and subjected to extraction.

EXTRACTION AND CLEANING

The dried fungal cake (20 kg) was homogenized with cold ethyl acetate (10 to 15 min) and the homogenized material was refluxed for 4 to 6 hours at 80 to 85 ° C, allowed to cool and filtered. The filtrate was dried in vacuo to a black tar material (2.5 kg to 3 kg). The black tar material was mixed with silica gel (2.5 kg) and CH ₂ C1 ₂ (5 1). The ClfC ^ was evaporated in vacuo and the crude tar material mixed with silica was poured onto a silica gel column (length 100 cm x diameter 22.5 cm) developed with EtOAc: hexane (1: 2 vv). Samples were collected (2000 ml, 12x). After 24 hours, the elution solvent was changed to EtOAc: Hexane (1: 1, v / v) and allowed to run for another 24 hours. Samples containing the product were collected, dried (280 g) and crystallized with methanol.

CRYSTALIZATION

The yellow dried product (280 g) was dissolved in 2.81 methanol for 40 to 60 minutes at 60 to 65 ° C. The hot methanol mixture was filtered and the filtrate was incubated for 4 to 6 hours at 4 ° C. White crystalline needles are removed from the mother liquor and rinsed with cold methanol. The crystals were dried in vacuo, weighed (190 g) and kept in a desiccator at 4 ° C.

The process of the present invention may result in lovastatin yields high enough to cause the cell walls of new Aspergillus oryzae transformants to rupture, so that the cell lysis rate can be eliminated prior to lovastatin recovery. This greatly simplifies the downstream recovery and cleaning process. As described above, lovastatin of the present invention can be recovered by the solvent extraction process without having to form its esters, salts, etc. during recuperation. Solvent extraction is a much simpler and more cost-effective recovery and purification process than chromatography.

While the invention has been described in detail with respect to specific experiments, it is understood that it is not limited thereto. Its scope is defined in the attached claims.

For

NOVOPHARM LIMITED:

LJUBLJANA /

PATENT OFFICE

Claims (13)

A method for the preparation of lovastatin, characterized in that it comprises fermenting the nutrient medium with the transformed fungal microorganism of the Aspergillus strain selected from the group of species A. oryzae, A. niger, A. nidulans, A. fumigatus and A. flavus, and this strain is incapable of expressing lovastatin, but has been transformed to contain foreign DNA encoding a series of enzymes capable of synthesizing lovastatin and a selectable marker, and recovering lovastatin from the nutrient medium. The method according to claim 1, characterized in that the transformed microorganism contains foreign DNA derived from the species Aspergillus terreus, which produces lovastatin. A method according to claim 2, characterized in that the Aspergillus strain is a strain of A. oryzae. The method according to any of the preceding claims, characterized in that the foreign DNA introduced into the transformant contains cycloheximide resistance genes as a selectable marker. Method according to any one of the preceding claims, characterized in that the transformer is AoAt-NBJ / 5 as described and defined herein. Process according to any one of the preceding claims, characterized in that it involves an additional lactonization step of the hydroxy acid analogue produced by the fermentation process into lovastatin. Method according to any one of the preceding claims, characterized in that lovastatin is recovered by solvent extraction. 8. New fungal transformants of a series of enzymes capable of synthesizing lovastatin, characterized in that these transformants are Aspergillus strains selected from A. oryzae, A. niger, A. nidulans, A. fumigatus and A. flavus, which is naturally incapable of expressing lovastatin, but contains genes that contain foreign DNA encoding for a series of enzymes capable of synthesizing lovastatin. 9. Fungal transformants according to claim 9, characterized in that the Aspergillus species from 15 is read from the group consisting of A. oryzae, A. niger, A. nidulans and A. fumigalis. The fungal transformants according to claim 10, characterized in that the species is Aspergillus A. oryzae. 11. Fungal transformants according to claim 11, characterized in that the foreign DNA is derived from lovastatin-expressing strain of the species Aspergillus terreus. 12. Fungal transformants according to claim 12, characterized in that they comprise the product of protoplast transformation of the entire DNA from the A. terreus strain into the A. oryzae strain. 13. AoAt-NBJ / 5 fungal transformants as described and defined herein. For NOVOPHARM LIMITED: PATENT OFFICE LJUBLJANA e SUMMARY New fungal soybeans and their use in antibiotic production Lovastatin is produced by a fermentation process using a fungal transformant produced by introducing the DNA of the lovastatin-expressing strain Aspergillus terreus into a non-lovastatin-expressing Aspergillus strain, such as the strain Aspergillus oryzae. A_A> - 1 I I 0 4 β 12 FIG. 1A ΑΛ - * —-— --- i and 1 O k 8 12 FIG · IB 0 1 1 4 8 FIG. 1C 1 12 .A _A λ_Α—, S_ 0 4 8 FIG. 1D » 12 A; extract from A. oryzae (parent) B; A. terreus extract (parent) C; standard lovastatin D; extract from transformed A. oryzae PATENT OFFICE Ljub PATENT OFFICE LJUBLJANA / co cn CM t>. m mο · Rsi