BG97421A - New fungal strains and use thereof in antibiotic production - Google Patents

Abstract

A method of producing lovastatin, which comprises fermentation of a culture medium with transformed microscopic fungi from the aspergillus strain, selected from the species a.orizae, a.niger, a.nidulans, a.fiMi gaTus and a.flavus, which strain is unable to express lovastatin but which has been transformed so as to contain a foreign DNA encoding a set of enzymes synthesizing lovastatin and selective marker, and recovery of lovastatin from the culture medium.

Description

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 strains of A ^ e ^ Uus and their use for the preparation of lovastatin and its analogues.

By its chemical nature, lovastatin is N is-Cia / r / _g 3a _g 7c _g 8b / 2S, 4S 8aD 3 7-dimethylbutanoic acid 1,2, 3 _g 7,8,8a-hexahydroO-3,7 'Dimethyl -8- - (tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl) -ethyl 3-naphthalenyl ester and having a chemical

This compound is an anti-Percholesterolemia agent, as a potential inhibitor of HMG-CoA reductase, an enzyme that controls the rate of cholesterol biosynthesis.

Antibiotics, such as lovastatin, are metabolites that require a complex of enzymes for their synthesis. Steps such as isolation, analysis, and possibly modification of corresponding genes for several enzymes are required to obtain them by molecular cloning of antibiotic-releasing microorganisms. Attempts to isolate such genes from similar fungal species are associated with the production of.

or clones carrying any individual genes of the complex or only an incomplete gene complex / see. 1 /.

Canadian Patent 1,1'291794 Endo describes the preparation of lovastatin by fermentation using a strain of Monassic < ¹ &^gt;

Canadian Patent 1,161,380 Monacan et al. Describes the preparation of lovastatin by fermentation using strains of the species.

In these two sources describing the state of the art, lovastatin is produced in parallel with many other chemical constituents in significant quantities to be separated. When prepared with the participation of Mopa giveg, lovastatin is obtained in parallel with monacolin I. Upon its preparation with the participation of Aureg.sub.3 (lb and b * Lggeiy, lovastatin is prepared in parallel with hydroxy acid. To the extent that hydroxy acid can readily be converted to lovastatin, dihydrolovastatin can be separated from it.

Object of the present: and so bretyunie'ze to: provide new genetically * · ··· - »·· · · · ··· · w ·· ···· · · · engineered mutant fungal strains', podhodshtshti" for use in fermentation processes to produce lovastatin.

Further, it is an object of the invention to provide new processes for the preparation of lovastatin by fermentation involving these strains.

According to one aspect of the present invention, new mutant strains of the appropriate As ^ ei ^ ittusca species provided in order to be suitable for the expression and secretion of lovastat1 ' these strains contain the functionally derived lovastatin-producing enzyme complex genes from the lovastatin-producing strains of Ae ^ et ^ ttus Herrens _r Τθ strains were obtained by the pathway in the protoplast transformation of the DNA from the lovastatin-producing strain of As> pei ^ i (tus геeggei} also containing a suitable selective marker, with another non-producing strain , selection from the group of Asp, specie including A ^ ori2ae

Selection of transformants is performed on the basis of selective markers, identification and isolation of lovastatin-producing transformants and their sub-cloning.

According to another aspect of the invention, there is provided a method for the preparation of lovastatin, which comprises fermenting the culture medium with a transformed microorganism

obtained from Aspei ' s ierrens and encoding a complex of lovastat producing enzymes and recovering lovastatin thus formed.

The method of the present invention provides for the preparation of lovastatin with a remarkably small amount of contaminants from respiratory rolovastatin and hydroxy acid derivatives compared to the process involving terreus

In the process of preparing the transformants according to the invention, standard techniques for the selection of the lovastatin-free AsyeiQ \ ttu5 strains as well as for the extra k can be used

In the same way, 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., aristae, but it is not the only one to obtain a successful result. Drums are used.A.oriZae was chosen as the preferred species, given its inertia, which contributes to the ease and safety of laboratory work and fermentation in commercial scale.

In order to select the transformed microorganisms from the untransformed, following the process of protoplast transformation, DNA from A. should contain the 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 in obtaining up to a bar result of the invention Antibiotic resistance markers such as, for example, antibiotics. ampicillin resistance, rifamycin new resistance, streptomycin resistance, etc. can be used and the resulting mixture of transformed untransformed microorganisms may be cultured in media containing a suitable antibiotic, so that only transformants containing the selective marker can survive for isolation.

Practically, a preferred selective marker, for convenience, uses the cycloheximide resistance. Cycloheximide is an inhibitor of protein synthesis so that the presence of a cycloheximide-resistant strain or transformant in the culture fluid is readily detected.

After selection of the transformants based on the selection marker, they are examined for the detection of those that will express and secrete lovastatin. "Only a relative small amount of the total number of transformants, per 5 u? Gn ggh." · · · · · · · · · · · · · · Possess this ability .T * is c * is pa3fio * 6? Fe.BaV h £ ez separately cultured in standard culture fluid, and analysis of the resultant environment,

ie, by HPLC, for the presence of lovastatin. Those who showed a positive test result are sub-cultured and allowed to sprout to produce colonies of new, lovastatin-producing fungal microorganisms and preferably of the species As ^ ex ^ CCus orlZae,

The invention is further illustrated by the following experimental cases, accompanied by drawings.

FIGURE 1 is a graphical representation of the crude mushroom extract produced by Example 1, described below, by HPLC.

FIGURE 2 is the H-CMK spectrum of the lovastatin compound obtained and purified from the hybrid strain ΐ

FIGURE 3 θ spectral mass of the same compound>

FIGURE 4 is an image of the morphological characteristic of the new AoAt / LTBI-V transformant,

EXAMPLE 1

MATERIALS AND METHODS

MUSHROOM CULTURE - mushroom isolates: of As ^ e-ujittus 1egegei and A, oriZae used in the present study were isolated from soil samples. in which orchids collected by A ^ r1si {tig Canada, ^ ebeared grow

Siaii σπ, -BeaV erld ^ e, AlBerla, Canada ^

SELECTION OF HA MUTANTS RESISTANT TO CYCLOHEXIMIDE

The sprouted asperidas of Asper ^ i / lus on a medium of Crdre> <

containing cycloheximide-Hconcentrate. 0.1-5 mM / b for 6 days at 28 ± 2 ° C spores (10 ⁸ ) of each isolate of A, 4g, and A. ori ^ ae, were dispersed in medium of ds (50 ml) and incubated for 30 min, the centrifuge is quenched at 10,000 rpm for 10 min, and then resuspended in sterile distilled beaker .. 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 .. ⁱⁿ 10 cyclohexia. ^ ^is ^ ^ §Y PE ^ and ⁿ ^ 1VD priate solution and resistant mutants were isolated after 10 days at incubation temperature 28 ° C and 2 ° C. ^ The isolates are tested for the production of lovastatin. Cycloheximinid-resistant lovastatin producing isolate from A 'tag is selected for further transformation studies.

Obtaining Protoplasts and DNA

A. orlZae and the cyclohexamide-resistant lovastatin-producing isolate of A. ierreus / here designated <1AC - 4703 $ were cultured separately in 50 ml of Capek culture fluid. The cultures were incubated for 58 hours at 28 + 2 ° C. of> rotary shaker / Nex) Btumx / ici, 9 cl entitle Inc. / at 200 g, are then collected and washed with distilled water by repeated centrifugation.

Protoplasts from the cultivation of the two species were obtained using IOL> r- (t 234 / Novo- ^ orJisi ^ ovo Attest to remove the cell walls during 10-12 h degradation, following the method described by D1sk1št> opG 1 $ spouse <^, .θβπ.

Mlcr otioC 128, -p. 651-654 / 1982 / Amounts of protoplasts of all types of regenerated, viable, single cell wall spores are obtained after regeneration in a regeneration medium at

28 ° C for 24 hours containing 0.1 g of agar / D1 / co / 5 g of sorbose and 0.35 g

WA in 50 ml of distilled water. In the development of these thesis spores exhibit all the cultural characteristics of their parents ..

-7 The total amount of &: ioyi] Zaio: from: protoplasts of A,

are suspended in a solution consisting of 10 mg / ml $ D $, 0.1 M NaCl and 0.1 M Tris-HCl, pH 9.0 .. The same volume of phenol diluted with Tris-HCl buffer is added to this solution and mixture was centrifuged at 120C

harvesting DNA was removed, mixed with 95% ethanol, left for 60 mS at -20 ° C, then centrifuged as before 10 min.

The isolated DNA is purified by adsorption and washed on DEAE - cellulose (DE-52, ^ patmap), again wetted with Tris-HCl buffer, pH 7.5 and 0.3 ^ PaCl and placed in a _{pipette. The} DNA was eluted with 10 mM Tris-HCl buffer, pH 7.5 containing 1.5 N NaCl. This solution was diluted to 0.2 M with -blaCI and the DNA was precipitated with two volumes of ethanol. tested the 200-320 nm spectrum by determining the ^ 60 ^ 280 absorption rate.

Only those DNAs with velocities between 1.50 and 2.00 can be used for transformations, Six samples each of 0.1 mg of isolated DNA are incubated in regeneration media to detect viable protoplasts and none did not open any colony.

by hemocytometer counting, incubated with 10-100 ng A, tegive · · · · · · · · · · · · · · · · · · · · · · · ·

DNA in 5 ml regeneration with ^ &lt; + &gt; ED &lt; + &gt; &apos; s &quot; n &apos; which is as described by πσ-up &apos; and also its regeneration. To investigate the possible chemical effect of DNA on lovastatin expression &quot; 10 -100 io Veal Thymus DNA / ^ CAemicAi Co /, DNA / Beiiiesda Pesearcn Tav ,, Sapegwego »NUC and homolog

The DNA of Aβ1 / ae is incubated with similar portions of Aβ2a protoplasts. When included in the DNA incubation, the protoplastic radium regeneration is reduced to less than 10 ^ as an analogous number of treated protoplasts fail sprout and show any cycloheximide resistance.

LOVASTATIN PRODUCTION AND TESTING

A suspension of spores developed from A, Og1 / ae protoplasts incubated with A terrens DNA was inoculated into 1 ml per petri dish on a Chapek agar medium containing 10 cycloheximide. After incubation for 48 hours at a temperature of 28 ± 2 ° C, the cultures reach a diameter of 6-8 mm. Each ^C develops from a single spore under these conditions. Those that grow on cycloheximide medium are inoculated individually on a Chapek-liquid 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 examined for lovastatin projection in the manner described above and by HPIC 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, 3x). The ethyl acetate fractions were dried in vacuo at 3 (/ ⁾ C _{> the} residue was collected in acetonitrile (5 ml) and subjected to HP1C analysis

• β · · · · · ·

HP1C ANALYSIS

HPIC equipment (Model Besmap 420) is provided by Neskmap InsirnMeiiT (Toronto, Canada) and consists of an Altex pump (Model 1ΊΌ A) and an injection valve ^ C-detector set to 237 nm. A C-18 0D ^> silica column / 25x0.46 cm, Ι.οΙ dissolution system consisting of acetonitrile and water / 55 * 45, V / was used at a slow rate of 1.0 ml / min. The lovastatin produced 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 protoplasts of A. orlZae .incubated with DNA from cycloheximide-resistant lovastatin-producing mutants of A. teg.

The results are given in the following Table 1.

TABLE ι - Cycloheximide-resistant and lovastatin-expressing protoplasts of A.orl'Zae incubated with cycloheximide-resistant · tenovastatin-producing mutant of A. terrens

No. Protoplasts Number of reg ..% Regene- DNA ^{/ n} ? ^ζ Number of lovastatin pro, isolating isolates. ml protoplasts after prot. / Ml walkie talkie 1 »7.4 χ 10 ⁵ 4, 1 χ S ⁵ 56 100 0 2. 5.2 χ 10 ⁵ 3.1 x 10 ⁵ 60 75 2 3. 5.2 χ 10 ⁵ 3.7 χ 10 ⁵ 72 50 2 4. 4.0 χ 10 ⁵ 2.7 χ 10 ⁵ 70 25 0 5. 2.6 x 10, / * * 1 C. -V * Ί \ \ · 1.5 X 10 ⁵ 1 ν ν ί H5 65 g? n 10 L. 0 l

• · · ·

One of the transformed AO-AND cultures has obtained its original transformation characteristics for six months. Figure 1 illustrates the HPIC analysis of extracts obtained from recipient and donor cultures of A., oriZae and those treated with DNA derived from A. ierren ^. The production of lovastatin using transformed isolates is shown below in Table 2.

TABLE 2

Preparation of lovastatin, expressed in mg / l, from transformed isolates of Ας, ρειψΓΖυε, oriZae in Chapek food broth / pH 6.8 /

Isolate No Lovastatin Productivity / mg / l / ΑΟ - I 15,36 + 1.78 ΑΟ - II 26,89 i 3.76 ΑΟ - III 13,46 i 4,56 ΑΟ - π 9.67 + 0.75

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

Lovastatin is produced in association with a hydroxy acidic analogue thereof, which is readily transformed into lovastatin e.g. by fusion into toluene to effect lactonization and therefore to increase the productivity of lovastatin. Lactonization is not mandatory in these experiments,

The lovastatin produced by the A ^ egegian parent culture in Chapek's food broth is 166.72 ± 5.92 mg / l and the parental culture of A .. oriZae does not produce any amount

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Figure 2 of the accompanying drawings is the l-spectrum of the lovastatin compound purified _{by the} hybrid strain HPIC. By comparison with the standard, known lovastatin spectrum, this spectrum represents the identity of the product.

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

The concentrations of the JK range from 5 to 2U pg per ml of the medium without affecting either the regeneration lids ^^^ jj ^ DM ^? / ^ ¹ nor the recovery of cycloheximide resistant and lovastatin-producing isolates from incubations with DNK.Dannite show that these concentrations are not a limiting factor in the production of porous tynn. Likewise, tilth, thymus, DNA and homologous DNA from A. orlZae at concentrations of 5 ng to 5 ^ g per ml of medium and do not affect regeneration of the protoplasts from A.r'1 / ae compared to the untreated controls · However, at 10 and 1UU and per ml of medium reduced regeneration DNA 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 Acta et al. T _d SepD «1sgov1o ^., 45.515 to 523 '/ 1966 / using richer in minerals sreda.Konidiite and freshly matured conidia produce more of viable protoplasts and of JK ^from! mycelium.The formation of cell aggregates during the protoplastic regeneration described by Acta et! .. / 1966 / is not observed in this system. The apparent transfer of factors responsible for the expression of cycloheximide resistance and lovastatin expression by

A .. 1eggei <> to A „orlZae shows the interspecies

G transformation of DNA · The co-transformations 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 transformant - NBJ / 5 θ shown in Figure 4 and can be characterized as follows · AATA morphology - ΝΒΙ / 5

Cnidial heads elongated, light brown. Konidiefori _smooth; colorless. Seeding bags -hemisferichni coated to half or a third of fialidi / last node of konidienosetsa or very konidienosets / arranged in two sloya.Kaniliyat is spherical or ellipsoidal, smooth colonies on Czapek agar medium, growing too fast _d as fluffy as well as velvety brownish brown until conidial. Orange exudate turning from yellow to brown ..

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

EXAMPLE 2 - METHOD OF PREPARATION

Mushroom culture 'Transformed culture from A ^ eu ^ XLus orlZae / ATCC No. 74135 / AoAt / MB! -V is used to produce lovastatinFESENTATION

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. At 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

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Erlenmeyer flasks (250 ml capacity). Fill the flask with another 10 ml of sterile water, shake gently and incubate at 2E C for 5-7 days. At the end of the incubation period, sterile water (50 ml) is 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 50 l with 30 l of culture medium. The composition of the culture medium was as follows:

E - glucose 20 g / l malt extract 20 g / l neo-peptone 3r / l tap water 1l pH is 6.8, brought with 10% -PARN, / The medium is sterilized at 1 <* 1 ° C for 30 min / 15 p ^> 1 /, seeds, allowed to grow for 17-20 hours at 28 ° C / speed of aeration 0,3 - 0,5 VVM / _with 80-100% dissolved oxygen. / 200grm /.

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 KCI 2 g / l «^ 4 0.8 g / l MgNo 0.03 g / l Betaine 0.6 g / l ^P 20O0 2 ml Tap water 1 liter

pH was 6.8 / prior to sterilization, brought with 105 psi / H 2 O ^ sterilization at 121 ° C for 1 hour at 15-20 pdr

After inoculation, the culture was allowed to grow for 7-8 days at 28 ° with pH control at 6.0-6.2 / 10% ^ 2 ^ 4 *

The aeration rate is maintained between 0.7 - 1.0 VVM /% dissolved oxygen 80-100% /.

2ZD PRODUCTION STAGE

A seven-day fermentation liquid (50 l) was aseptically transferred into a fermenter (2,000 l working volume) containing 1500 l of the culture medium (composition above). The sowing culture was allowed to sprout for 24 hours at 28 ° C / 20 and gm / DO. 80-100%. The fermentation fluid (200 l) is aseptically transferred to a fermenter with a capacity of 30 0U0 lis working volume of 20,000 l containing 18,000 l of the culture medium, the culture is allowed to sprout to

7-9 days at 28 ° C is pH control at 6.0 - 6.2.After 60 hours of fermentation, the pH is no longer controlled. The fermentation liquid is supplemented with sub-

medium / speed 10 l / h / for 5 days. The nourishing environment is as follows : D-glucose 285.7 g / l Ardamin pH 71.4 g / l Soy protein 14.3 g / l / « * tap water 1 liter 6.8 / before sterilization / - sterilization at 121 ° C

30-45 min. At the end of fermentation, the fermentation liquid was acidified to pH 4.0 with 10 µl HCl and centrifuged. The mushroom residue was dried at 55 ° C and extracted.

EXTRACTION AND Purification • ·

EXTRACTION AND PURIFICATION IS '* * * * *

The dried mushroom 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 resinous consistency with silica gel / 2.5 kg / / 2.5 kg - 3 kg /. The material is mixed and CHgCI ^ / 5l /., ^C-D 2 was evaporated under vacuum and combined with the silicon raw material was poured on a silica gel glass column / 100 cm in length x 22.5 cm in diameter / a

EtOAc: Hexane / 1: 2 V _r V /. Samples (2,000 11, 12x) were collected.

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

CRYSTALIZATION

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

The method of the present invention may result in the production of lovastatin sufficient to cause the cell walls of the new transformants of A, orl / ae to rupture, so that this stage of cell lysis can be eliminated I before lovastatin is restored .This significantly 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 and the like. during the recovery period

rhyme extraction is much simpler and is a much more economical gap for recovery and purification than chromatography.

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

Claims (12)

Patent Claims A method for the preparation of lovastatin, which involves fermentation of a culture medium with transformed fungal microorganisms στ strain A s. p Ltus selected from a group of species A .. oriZae, A. p1 ^ er, A _r p10i1aP $, A. ^ im ^ a ^ and A, y'lav '^ n, which strain is unable to express lovastatin but which has been transformed to contain foreign DNA , encoding a complex of enzymes, yadidai I synthesize lovastatin and a selective marker and recover lovastatin from the culture medium. 2, The method of claim 1 wherein the transformed microorganism contains foreign _DNA> obtained from a lovastatin-producing strain As Ums »^ ^ eggei, The method of claim 2, wherein said strain of the microorganism is an A .. oryZae strain, 4, A method according to any one of the preceding claims, wherein the foreign DNA introduced into the transformant comprises, cyclohexia; -resistant genes as a selective marker, 5, according to any one of the preceding claims, wherein the transformant is AqAt-HBI / 5 as described and defined. 6, 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. 7, A method according to any of the preceding claims, wherein the recovery of lovastatin is accomplished by soluble extraction. 8, New mushroom transformants, carriers of an enzyme complex, enesoPSH-da - 17 • β · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · capable of synthesizing lovastatin , like these transformants, in quality! device to the strains of type A / ^ ⁰¹ ^ are selected from the group formed by A 'og1Hae, A.p1oeg, A..p1s1i ^ ap <; , A. and imin and A. iavus, and which are naturally incapable of expressing lovastatin, but containing foreign DNA containing genes encoding a complex of enzymes are capable of synthesizing lovastatin. 9. Mushroom transformants according to claim 9, wherein A. is selected from the group consisting of A, Og1 / ae, A.p1 ^ er, A.p1c1i / ap5 and A.y ^ Mi ^ a'kis · 10. Mushroom transformants according to claim 10, wherein s ^ G '> e, s θ & · oriZae · The fungal transformants of claim 1, wherein the date DNA is derived from lovastatin expressing a kerreMs strain. . А. alien— А. Mushroom transformants according to claim 12 and comprising the product of protoplastic transformation of the total DNA from A. ierrens strain into A. oriZae strain.