SK5593A3 - Process for preparing lovastatin - 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

Technical field

IN

The present invention relates to novel genetically engineered fungal strains and their use in the preparation of antibiotics. More specifically, it concerns new,

genetically engineered strains of Aspergillus and their use in the preparation of the medicament lovsstatin and its analogues.

State of the art; ...

Lovastatin is chemically 1,2,3, T, 8,8a-hexahydro-3,7-dimethyl 1-8-p- (tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl) ) - ethyl / ester L-naftalenylester / S / c to ^<(R) ₃ 3 o <, 7/9, 8/3 (25 ^R, 4 S ^C) 8AO / 3 // - 2-methyl and the hylbutanové chemical formula

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as it is a potent inhibitor of FllG-lOA reductase, the enzyme Pi controlling the rate of chlorolesterol biosynthesis. It is a fungal metabolite produced by the fermentation process using selected strains of fungi. i '.

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Antibiotics such as lovastatin are metabolites that require a set of several enzymes for their synthesis. Isolation is required to enable their production by molecular cloning of microorganism-V 'producing antibiotic.

Lcin, snalysis, and even a modification of the corresponding i gene;

some enzymes. Attempts have isolated such genes from the respective strains of fungi until now have provided clones, or indi vidual zeny groups, or only an unintended set of genes - β-dalpid and woodlopwood, eclecular Hloninc of of the Thole / iosynthetic Psthway of c. :

Srpression in a Heterolojzous Guest ',? ^r nture (1984), 309, p. 462-464 · í.

Canadian patent no. No. 1129794, describes the preparation of lovastatin by fermentation using a non-moss strain of Pdonascus ruber.

U.S. Pat. 116138C lionaghan et al., Describes the preparation of lovastatin by fermentation using strains of the fungus Aspergillus terreus species.

In both of these known processes, lovastatin is produced in separate amounts together with other, very chemically similar compounds, and must be separated from them. Intended use Síonsseus ruber is lovastatin produced with monacollnox J. Uses Aeoergillus terreus is accompanied by dihydrolovestatin and hydrory acid, but the hydro group can be easily lactonized to lovastatin, the dihydrolstatin must be separated from it.

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DETAILED DESCRIPTION OF THE INVENTION

The object of the present invention is to provide novel, genetically engineered siutants of strains of alder species which are capable of being used in fermentation processes for the preparation of lovastatin.

It is an object of the present invention to provide novel methods for preparing lovastatin using such strains of ori fermeration.

According to one aspect of the present invention, there are provided novel mutants of strains of the species of the genus AspcrFillus which are capable of expressing and secreting lovaatatin. These strains contain any type ^ lovastatin-producing enzyme, a funkSnom vzlahu each other, derived from PHA terreus strains Asperrillus producing lovastotír .. hoo prepared spônobom transformation protonlastov L * ?, the strain Asperyillus terreus, produkujúecho lovastatin, comprising also useful selektovetln · ¹ ' a marker, with other Aspergilnus species that do not produce lovastatin and are selected from the group consisting of A.oryzee, Ä. furr.i * atus, A.ni.ver, A.nidulans and A.flsvus, by selecting the trensformants thus formed, according to the selectable marker, identifying and isolating the lovastatin-producing transformations and their subclonates.

According to a further aspect of the invention there is provided a process for the preparation of lovastatin, which comprises fermeotizing a nutrient medium with an Asperfllue nicotransformer transformant comprising dreams derived from Asperfrillus terreus and encoding a set of enzymes that produce lovastatin and recover lovastatin thus formed.

The process of the present invention produces lovastatin with significantly less amounts of contaminating dihydrolovastrtin and hydroacid derivatives as compared to the process using Asoer-illus terreus.

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In the process of preparing the transformants of the present invention, standard techniques for preparing protoplasts from selected lovastatin-producing Aspergillus strain and standard DNA extraction techniques from lovastatin-producing Aspregillus terreus strain can be used. These techniques are well known to those skilled in the art and do not need to be discussed in greater detail here. Similarly, the protoplast transformation techniques and procedures used herein are known and standard.

Preferably, lovastatin is a non-producing strain of Aspergillus strain Aspergillus oryzae, but is not a prerequisite for a practical successful practice of the invention. Other Aspergillus species may be used, in particular A.niger, A.nidulans, A.fumigatus, f.

A. flavus. A.oryzae is chosen to be preferred over its

Inertia, which allows easy and safe handling;

l. ním with it and fermentation on a commercial scale.

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In order to isolate transformants of microorganisms not transformed after protoplaw transformation,. and

DNA from A.terreus had contained a selectable marker, There is a wide selection of selectable markers, available &

and known to those skilled in the art, and refining the choice is not essential to a successful embodiment of the invention. Antibiotic resistance markers such as ampicillin resistance, rifampicin resistance, streptomycin resistance 1, etc., and the resulting mixture of transformants and not transformants may be used.

A - * ´ can be cultivated in a medium containing suitable antibiotic;

tic so that only transformants that contain a selectable marker will survive isolation. ·

In particular, resistance to cycloheximide is suitable as a selectable marker for Lahkosl. Cycloheximide is an inhibitor of ^one of protein synthesis, cycloheximide-making reaction in the presence cezis- * tentného strain or transformant in a culture medium, is readily detectable. f.

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For sc-selection of transformants based on a selectable marker, these are restricted to those that will express and secrete lovastatin. Only a relatively small number of total transformants produced by the protoplast transformation process have this ability. They are recognized by separate culture in a standard culture medium and analysis of the resulting medium, e.g. by RPLC, on the presence of icvestatin. Those found to be positive for lovestatin are subcultured and grow and harbor colonies of new, lovastatin-producing transformants of a fungal microorganism of the genus Aepergillus and preferably of Aspergillus species.

The invention is described for purposes of illustration in the following experimental section.

Attached questions;

Fig. 1 is a graphical representation of the HPLC analysis of the crude fungal extract produced by the experiments of Example 1 below.

Pbr.1 'is the Ηί-λΟΉ spectrum of the lovastatin compound 2 purified from a hybrid strain.

Fig. 3 is the mass spectrum of the same compound.

Fig. 4 is a representation of the morphological characteristics of the novel trsnsformant? 10At / JTBJ-V.

DETAILED DESCRIPTION OF THE INVENTION.

Example 1

Materials and methods

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Linear culture - isolates of A.-perrillus terreus and .oryzae used in this experiment were isolated from formulas of 5dy orchids from the Arriculture Csnada collection, SS Stetion, Jeaverlodge, Alberta, Canada.

Selection of cyclohexemide resistant mutants

After cultivation of both Aspereilli isolates on Czspekovos dox raáôiu containing cyclohexamide (equ. 3.1-5 drawbars) for 2 days or 2? For example, spores (10) of each A. terreu and A.oryzae isolate were dispensed in 10 µl cyclohersmid-fried dox flask = 5 ml) for 30 minutes, centrifuged at 13 QDQ for 10 minutes & then resuspended in sterile distilled water. ^L L ± o and I washed three times by stirring the resedimeatovaná odstredení®. The spores were screed suspended in sterile 1 # (v / v) solution of Tri-ton? -P? and their number was determined in terms of haemocytoneter. At a suitable dilution, they were resolved in 10 ml of cycloheximide® and elution medium and resistant mutants were isolated after 13 days incubation at? 8 + 2 ° C. For the next transformation study, the resistant terostat was produced for the cyclohexaraid.

'' rotovs orotoolastu e p?: A

The isolates of Asoerrilius oryzae and of the cycloheximide resistant lovastetin producing A.terreus (referred to herein as JA0-4703) were cultured separately in 50 ml of baked soil. rultúry were incubated for 58 hours at 28+ 2 ° C on a rotary shaker (New Srunswick ScientiTic Inc.) at 2 30 rpm, min ^"1 ·, array oreayté collected and sterile distilled water by repeated cdstredenía.

“Brotoolssty from cultures of both species have been obtained using the termozyme 254 (ile-iordisk, levo ille, 288C, sverd, Denmark) to remove cell walls during p? .1.2 cleavage, generally by Ĺe.u.ej oickinsonoa and T sen bernom, ^r . Cer.ľ icrobiol. 1? °, marked 'Ja · -' · 10 Mon.

-7str. 651-Γ54 (ΐ982 \ ^{n n} protoplast fragments from each species regenerate viable, single cell spores with stems at 28 ° C for 24 hours in a regeneration agar (β) containing ,1 g agar {TJifco}, 5 in sorbose and 0.35 PDTA in 50 ml of dilute water, after germination, these spores have all the characteristics of a parental culture.

Total OKA was isolated from Anoergillue terreus protoplasts according to the procedure of Schlief &apos; s Vensinka, ^nT &apos; ractical Methods in Moleculer Biology, 33, p. 21-29 ^ lppl ^. protoolasts were suspended in a solution containing 10 mg / ml SOS, 0.1 2 N NeCl and 3.1 mtri-HCl, pH 9.0.

An equal volume of phenol saturated with tris-HCl buffer was added. The mixture was centrifuged at 1? 00? g l / min in a ppendorf centrifuge tube (Orinkmann rnatrumer, Canadc Ltd., liexdale, Onfcerio). The upper phase, containing the OKA removed, zmieSsná ethar.olom 95%, stored at-20 ^D C for 60 minutes, then centrifuged as before for 10 min. Pellet was resuspended in pH 7.0 buffer and incubated with 8 H Naase Oigina, St. Louis, KO. The phenol treated LUA was precipitated from the aqueous layer. The isolated ONA was purified by adsorption and washing on 3? A3-cellulose ose (-53-52, Whatman), pre-soaked in 10 µM tris-H? Buffer, pH 7.5 and 0.3 µl KaCl and placed. in the paeteur ointment O ΏΪ A A eluted at 13 m @ -1. tris-HCl buffer, pH 7.5, containing.

1.5M KaCl. This solution was diluted to 3.2M KaCl with DKA precipitated with two volumes of ethanol. purity? 5KA was evaluated from 2CG-32O nm spectra by determining the oomeru 2βθ ^ ^ ^{Α 2βΟ} absorbance. Only BIA with ratios of x50 of 1.50 s2.0C were used in the transformation. Seal samples, each 0.1 mg of isolated PIJA, were incubated in a regenerative dressing to ensure the absence of viable protoplasts, and none of them developed any colonies.

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-8? Rotopiact-DNA incubation

Approximately 5.2 x 10 ⁴ A.oryzao protoplasts, evaluated by a haeniocytometer computer, were incubated with 10-103 ng A.terreus DNA in 5 ml regeneration medium, the composition of which is described above. To study the possible chemical effects of DNA on lovastatin expression, 1C to 100 µg calf thymus (Sigma Chemical Co., St. Louis, IL) and DNA (3ethesda Research Laboratories) were incubated with similar amounts of A.oryza protoplasts. , Gaithaberg, 2ZP, USA) and homologous DMA A.oryzae. By including the DNA in the incubations, the protoplanet recovery was reduced by less than 10X and a comparable number of protoplasts thus treated (10µ) had no resistance to cyclohevimide.

Production of lcvaatt.tin and ätúdis

A suspension of spores developed from A.oryzae protoplasts incubated with A. terreus DNA was inoculated, ml in a third plate, not Ozapek dox agar medium containing 1? m, ^J cycloheximide. After 48 hours incubation at? 8 8 2 ° 0, the cultures reached a diameter of — 8 mm. Each evolved separately from an individual spore under these nodimimok. Those growing on cyclohexircide medium were each separately inoculated onto Czapek dox broth (50 ml in a 500 ml Erlenmeyer flask), incubated on a rotary shaker (200 rpm) at 28 ♦ 2 ° C for 12 days. Each growth medium was evaluated for lovastatin production by the method described below and analyzed by HPLC.

The traction fermented ice (53 mL) was acidified to pH 4.0 with 17N HCl and then partitioned with ethyl acetate (133 mL, 3x). The combined ethyl acetate fractions were dried under vacuum at rt

-930 ° C, the residue was collected in acetonitrile (5 mL) and then analyzed by HPLC.

HPLC analysis

An HPLC device (Beckman Model 420) was supplied by Beckman Instruments, Toronto, Canada and contained

Altex pump (model 110 A) and injection valve. The LC-UV detector was set to 237 nm. A column of hypersil C-18 ODS silica (25 x 0.46 cm, ID) and solvent saturated acetonitrile and water (55:45 v / v) was used)

-1 'at a flow rate of 1.0 ml.min · Lovastatin production was compared and quantified from the standard lovastatin curve.

Example 1 - Results

Seventy-nine of cycloheximide resistant isolates, including four lovastatin producing isolates, were obtained from 5.2.times.10@6 A.oryzae protoplasts incubated with DNA from a cycloheximide resistant A.terreus mutant A.terreus. Transformation experiments were performed six times.

The results are shown in Table 1 below.

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Ibbulka 1 Frotoplasts to cycloheximide resistant and lovastatin expressing

Aspergillus oryzae incubated with DNA from a mutant Aspergillus terreus resistant to cycloheximide and producing lovastatin

One of the AO-IX transformation cultures retains its orginally transformed characteristics for six months. Fig. 1 illustrates 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 in Table 2 below.

Table 2

Production of lovastatin by transformed Aspergillus oryzae isolates in Czapek's dox soil (pH 6.8 µ isolate)

AO-I

Λ0-ΙΙ

AO-III

AO-IV lovastatin extract (mg / 1)

15.36 + 1.78 26.89 + 3.76 13.46 + 4.56

9.67 + 0.75

Isolate no. AO-I was subcultured five times to provide the ooAt - NBJ / 5 transformant deposited on February 25, 1992 as its viable, permanent culture in the American Type Culture Collection under the designation ATCC 74I35.

Lovastatin is produced together with its hydroxyacid analog, which is readily convertible to lovastatin e.g. by refluxing in toluene to effect lactonization and thus the lovastatin yield is increased. Lactonization was not performed in these experiments.

Lovastatin produced by the parental culture of A. terreus in Czapek's dox soil was at a concentration of 166.72 ± 5.92 mg / L and the parental A.oryzae produced no lovastatin.

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Fig.? of the accompanying drawings represents the spectrum of the lovastatin compound purified with H 2 OC LC from a hybrid strain. When compared to the known standard lovastatin spectrum, this spectrum confirms the identity of the product.

Fig. 3 of the accompanying figures represents the top spectrum of the same product and shows that the lactic acid wheels are pure 99. <\ ·

A ΌΧΑ concentration ranging from 5 to 2 µg per ml of medium does not affect either the regenerative capacity of A.oryzae protoplasts or the acquisition of resistance to cycloheximide and lovaštetiny producing isolates from incubations and D1-JA A. terreu. These values indicate that these concentrations are not a critical factor in the production of lovastatin. Similar to the calf thymus, DNA and homologous DfA of A.oryzae at concentrations from 5 ng to 5 µg per ml medium did not affect the recovery of A.oryzae protoplasts compared to untreated controls. AvSek TKA at concentrations of 10 to 100 µg per ml medium reduces regeneration to a maximum of 1%. These OKA treatments do not elicit lovastatin expression.

The regeneration medium provides a number of protoplasts comparable to that obtained by Achs et al., J. GWn. L'Icrobiol., 45, 515-523 (1966), 8 using a more complex mineral medium. Conidia and freshly congested conidia produce better viable protoplast and DNA results than mycelia. The formation of cellular aggregates during protoplast regeneration described by Acha and epol. (1966) was not observed in this system. Apparent transfer of factors responsible for the expression of slimylcycloheximide and expression of lovastatin from A.terreus to A.oryzae induces ONA trans-species transformation.

-ľ? & 310? clustering of the hairs involved in the expression of these characteristics.

The morphology of the new AoAt-JBJ / 5 transformants is shown in Fig. ‘4 and πη £ β is characterized by the following:

AoAt-tBJ / 5 morphology

Conidellar heads columnar, light brown. Eonidiophores smooth, colorless. Hemispherical vesicles, coated up to one-half or two-thirds with phislides arranged in two layers. The conidia are spherical or elliptical, a smooth colony on Czapekovo agar growing very fast, possibly clusters or swept Cinnamon brown during conidia formation. Orange oriate, changing from yellow to brown.

These experiments demonstrate that, essentially, genetically determined production of antibiotics, characteristically produced by one species of fungi, can be expressed in another species.

Example 2

Method of production

Mold culture: Ea production of lovastatin was used with transformed cultures of Aspergillus oryzae (ATCC® 7413 $) AoAt / KBJ-V.

Permentácia;

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preparation of a vaccine culture; The pliean culture (lyophilized flask) was aseptically transferred to Potato-: Dextrose Oblique Agor & left at 25 ° 0 for 4-5 days. At the end of the incubation, the cidial suspension was prepared by adding 10 ml of a solution of three tons of X-100 (0.01 5E) vigorously stirred and the conidial suspension was transferred to

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8 g (30 g) in a su lsuaeyer beaker ( ^Ί 5 µl volume). 3.Care with 13 ml of sterile water, vigorously stirred and incubated at 28 ° C. in hold. Sterile water (50 ml) was added at the end of the incubation period. The gentidial suepension for 81a with sterile muslin was added and the filter containing 1 x 10 6 conidia / ml was transferred to a seed feraentor (50 L capacity) with 30 L seed. The inoculation of the vaccine medium was as follows:

D-glucose 20 g / l malt extract 20 g / l neo-peptone 3 g / l tap water 1 1

Ph? S (adjusted with 10% NaOH). (Védium was sterilized in the 1? 1 ° C * 'Q min (15 psi). The seed culture was nonechaná Rašla 17 and? 20 hours at 28 ° ^N (rýchloet The air Anie 0.3 - 0.5 vvm) and 80, and ? 100% dissolved oxygen (OCO rpm ^"1).

Lovastatin production

Inoculation culture of the production fermenter of the production medium, as follows:

lactose 60 ardamine pH 10 soy protein 2

KCL 2 ^{τ _ΏΪ?} -0 ₄ 0.8! ₄ .4 & _? 0 0,03 belfiin 0,6

Ί p tap water 1 µl prior to sterilization, treated with 105 N ^& lt ^; OR ^& gt ^; / 3, SO4, sterilization at 121 ° 7 for 1 hour ori 15 and * 20 psi.

(30 L) was transferred to a vessel (10 00 L working volume), containing the production medium was g / l

6/1

6/1 g / 1 g / 1 g / 1 g / 1 ml f

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After inoculation, the culture was allowed to grow for 7 to 8 days at 28 ° C with pH control between 6.0 and 6.7 (10% H 2 SO 4).

The aeration rate was maintained between 0.7 1.0 vvm dissolved oxygen (80-100%).

The second production stage of the day-old fermented broth (50 L) was transferred aseptically to a seed fermenter (2000 L working oil) containing 1500 L seed medium (composition above). The seed culture was allowed to Rašla 24 hours at 28 ° in the (200 rpm ¹⁾ rozp.kyslík 80 to 100%. administered (200 L) was aseptically transferred to a production fermenter (30,000 L) of capacity 3 with a working volume of 20,000 L containing 18,000 L of production medium. The culture was allowed to grow for an additional 7-9 days at 28 ° C with a controlled pH of 6.0 to 6.2. After 60 hours of fermentation, the pH was not controlled. Fermentation was supplemented with nutrient medium (10 l / h) after 5 µm.

The composition of the nutrient medium was as follows;

L-glucose ardamine pH soy protein tap water

285.7 g / 71.4 g / 11.4 g / l

1 pH 6.8 sterilized at 121 ° C for 30 to 45 min before sterilization. At the end of the fermentation, the soil was acidified to pH 4.0 with 10 N HCl and centrifuged, the mold cake was dried at 55 ° C and extracted.

Extraction and purification

The dried mold cake (20 kg) is homogenized with stale ethyl acetate (10-15 min) and the homogenized material is refluxed at 80-85 ° C for 4-6 hours, allowed to cool and filtered. The filtrate is dried under vacuum to a black tarry material of 2.5 kg to 3 kg. the black breath-16 material was mixed with silicepel (2.5 µl ^of CH ₂ Cl ₂ (5 liters). CH ₂ Cl ₂ ^was washed in vacuo and the silica mixed with the silica was poured onto a silica gel glass column (100 cm). length x 22.5 cm diameter) by developing with EtOAC / hexane (1: 3 v / v) Samples (2000 µl, 12x) are collected, after 24 hours the solvent elution mixture is replaced with EtOAc * hexane (1: 1 v / v). The samples containing the product were combined, 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 and 40 g. 60 minutes at 60 to 65 ° 0. The hot methanol mixture is filtered and the filtrate is incubated at 4 ° C for 4-6 hours. The crystalline needles are separated from the mother liquor and rinsed with cold methanol. The crystals are dried under vacuum, weighed (190 g) and stored in a desiccator at 4 ° C.

The process of the present invention can provide lovastatin yields that are quite high due to cell wall rupture of new Aspergillus oryzae transformants, so that in this process the degree of cell lysis prior to obtaining lovastatin can be eliminated. This substantially simplifies co-current recovery and purification. As described above, lovastatin according to the present soosobum can be obtained by solvent extraction, without the need to form esters, salts, etc., during the recovery. Solvent extraction is much simpler and more economical to obtain and purify than chromatography.

The invention has been described in detail in the specific examples, but is not limited thereto. Its scope is defined in the appended claims.

Claims (12)

PA 'f E λ T L O N T E N G L ISSES ¹ 'Λ * A process for the preparation of lovastatin, characterized in that the transformed nutrient medium is regenerated. fungal microorganism of the Aapergillus strain selected from the group of A.oryzae, A.niger, A.nidulars _p . A. figus and A.flavus, which strain is unable to express g lovastatin but has been transformed so that the content is a DNA coding for a set of enzymes of the schoonic lovastatin synthesis, and a selectable marker, and lovastatin is recovered from; nutrient medium. r. • K p * pT ' 2. The method according to claim 1, wherein the transformed microorganism comprises a foreign substance. DNA obtained from lovaatatin producing Aaoergillus f terreus species. 3. A method according to claim 2, wherein the Aspergillus strain is a strain of the genus roryzae. r V • 5 i · 4. A method according to any of the preceding claims, characterized in that the foreign SNA introduced into the transformant contains genes for resistance to the cyclo-heyi.mi.dti to a selectable marker. Ť 'c. ΰ 5. the person of any of the preceding claims; It is characterized by the fact that it is a transfurmant? is AoAt-tcJ / 5 as described and defined. í i 6. A sosob according to any one of the preceding claims, characterized in that it comprises a further step of lactonization of the hydroxyacid analogue resulting from the fermentative preparation of lovastatin. . -187. The method according to any one of the preceding claims, characterized in that the recovery of lovastatin is carried out by solvent extraction. 8. New fungal transformants capable of producing enzymes capable of synthesizing lovastatin, wherein said transformants are strains of Aspergillus sp. selected from A.oryzae, A.nidulans, A.fumigatus, and A.flavus, which are naturally incapable of expressing lovastatin, but containing foreign DNA containing genes encoding the production of enzymes capable of synthesizing lovastatin. The fungal transformant of claim 9, wherein Aspergillus sp. is selected from the group consisting of A.oryzae, A. niger, A.nidulans and A.fumigatus. 10. A fungal transformant according to claim 10, wherein Aspergillus sp. is A.oryzae. The fungal transformant of claim 11, wherein the foreign ONA is derived from a lovastatin expressing strain of Aspergillus terreus. A fungal transformant according to claim 12 and comprising a protoplast transformation product of total DNA from said A.terreus strain into said oryzae strain. 13. AoAt-NEJ / 5 fungal transformants as described and defined herein.