SK281320B6 - Aspergillus terreus strain and fermentation of lovastatine with this strain. - Google Patents

Abstract

A new strain of Aspergillus terreus CCM 8236 was obtained by classical mutagenesis methods. A process for the aerobic fermentation of lovastatin by an Aspergillus terreus CCM 8236 strain in the form of an acid in submerged culture on a laboratory scale is described. The inoculum is prepared in low pH soil in the range of 4.5 to 4.7. A mixture of glucose and lactose in a mutual ratio of 0.5 to 0.7% by weight is used as the carbon source in the production medium. of glucose and 11 to 15 wt. lactose and cotton or soy flour is used as a complex source, the concentration of free ammonia ions being at most 0.07 g / l. Under these conditions, the strain produces more than 2000 mg / l of lovastatin in 170 hours

Description

Technical field

The invention relates to a novel strain of the microorganism Aspergillus terreus, obtained by the classical mutagenesis method (combined action of chemical and physical mutagen), which produces a high amount of lovastatin in an optimized medium with a suitable carbon and nitrogen source.

BACKGROUND OF THE INVENTION

Lovastatin (MK-803, monacolin K, mevinoline) is a secondary metabolite that competitively inhibits the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase (EC 1.1.1.34), thereby reducing endogenous cholesterol synthesis, thus providing considerable possibilities in the treatment of hyperlipemia. The inhibition of HMG CoA reductase not only reduces the level of cholesterol in mammalian cells, but also induces low density lipoprotein (LDL) cholesterol receptors in the liver, changes in ubiquinone production, protein isoprenylation, juvenile hormone synthesis in insects, plant sterols and pigments, and fungal gibberellins. Chemically, lovastatin is (1S, 3S, 7S, 8S, 8aR) -1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8- [2 - [(2R, 4R) -tetrahydro-4] hydroxy-6-oxo-2H-pyran-2-yl] ethyl] -1-naphthyl (S) -2-methylbutyrate. Its biosynthesis has been demonstrated in Aspergillus terreus ATCC 20542 using labeled acetate substrates through a polyketide pathway. Although the exact structure of the key intermediates between acetate and lovastatin is still unknown, there are several working hypotheses of biosynthesis (Wagschal et al., J. Chem. Soc., Perkin Trans. 1, 2357-2363, 1996). Lovastatin occurs in two forms: the acid form and the lactone form, the biologically active acid form. Since 1987, lovastatin has been used in the form of tablets to reduce hypercholesterolemia and prevent ischemic heart disease.

U.S. Pat. No. No. 4,231,938 and E. P. 0 022 478 describe the fermentation preparation of lovastatin and dihydroxymevinoline by the production strains of Aspergillus terreus ATCC 20541 and ATCC 20542.

U.S. Pat. No. No. 4,387,242 describes the fermentation preparation of a hypocholesterolemic and hypolipaemic agent having a structure related to lovastatin by the same strain of Aspergillus terreus ATCC 20542.

E. P. 0 556 699 relates to a process for the preparation of a production strain for lovastatin from an Aspergillus genus which does not initially produce it (e.g., Aspergillus oryzae).

U.S. Pat. No. No. 5,403,728 describes a microbial process for the preparation of both lovastatin acid and lactone by aerobic fermentation of a submerged culture of a novel Aspergillus obscurus species.

Monascus species - M. anka, M. purpurous, M. ruber, M. vitreus, M. paxii (British Patent Specifications Nos. 2,046,737 and 2,049,664) are also mentioned in the patent literature as lovastatin production strains.

Other types of microorganisms that produce small amounts of lovastatin are also reported in the literature. In particular, Phoma sp. M 4452, Doratomyces nanus IFO 9551, Gymnoascus umbrinus IFO 8450 (Endo et al .: J. Antibiot. 39, 1609-1610, 1986), Pleurotus ostreatus, Pleurotus saca and Pleu rotus sapidus (Gunde-Cimerman, FEMS Microbiology Letters 11,203-) 206.1993).

Although a number of lovastatin-producing Aspergillus terreus strains have been described in the patent literature, its yield for the patented A. terreus strain ATCC 20542 is relatively low. A drawback is also that the components of the fermentation broths disclosed in U.S. Pat. No. 4,231,938, E. P. 0 022 478 and U.S. Pat. No. No. 4,387,242 are financially demanding for industrial production and thus unavailable.

SUMMARY OF THE INVENTION

By combining the use of a physical and chemical mutagen applied to the spores of the A. terreus strain, a new mutant strain of A. terreus CCM 8236 was obtained, which is characterized by overproduction of lovastatin. Compared to the A. terreus strain, ATCC 20542 achieves at least double production. Both inoculation and sporulation soil is composed of available and inexpensive raw materials, which are commonly used in the fermentation of secondary and primary metabolites. The invention is based on the use of the strain A. terreus CCM 8236 for the production of lovastatin in the form of an acid and a lactone or a pharmaceutically acceptable salt or ester thereof.

The original strain of A. terreus with the working title GB1 was provided by dr. Vollek. Based on the relatively highest production of lovastatin, it was selected as the starting material for the production strain among several wild strains, also identified as A. terreus.

The strain was treated with chemical and physical mutagens to obtain a production strain for lovastatin.

In the first breeding phase, the starting strain was influenced by the strong mutagen N-methyl, N-nitro, N-nitrosoguanidine (NTG). Due to the increased resistance of the mutants to NTG, they were alternately exposed to ultraviolet (UV) and NTG in the second phase of breeding. The isolates obtained (designated by GB and serial number) were tested for the amount of lovastatin production. In this way, a mutant with the working designation GB2033 with a markedly increased production of lovastatin was isolated and deposited at the Czech Collection of Microorganisms in Brno under the designation CCM 8236. The soil composition - both sporulation and inoculation and production - was adapted for this new strain.

The cultivation of the microorganism used in the fermentation process to which the patent relates is carried out in three steps:

1. culture sporulation

2nd inoculation stage

3rd production stage

Rapid sporulation is important for the quality of the inoculum (within 4 to 5 days at 28 ° C the culture is completely sporulated, the spores are cinnamon-brown in color, colonies are flat, velvety appearance without white edges, about 4 cm in diameter, easily washable with saline 30% glycerol).

Cultivation of the inoculum is carried out at 28 ° C at 220 rpm for 16 to 20 hours in low pH soil (4.6), which is significantly reduced during cultivation (3.0 to 3.2). A quality inoculum is characterized by the following properties:

- small pellets, well fiberised

- sediment 20%

-orientative analytical parameters: glucose content: 40 g / l phosphorus content: 200 mg / l NH ⁴⁺ content: 0,2 g NH ₂ content 0,1 g / l.

Cultivation of the production step takes place at 28 ° C at 220 rpm for 170 to 190 hours. The production medium contains a high concentration of carbon sources (glucose and lactose) in a precisely defined ratio (1: 22) in favor of lactose. As the nitrogen source it is necessary to use an organic source, e.g. soy flour, inactivated baker's yeast and the like. The highest production was achieved on soil containing Pharmamedia cotton flour as a nitrogen source. Inorganic nitrogen in the soil in the form of NH ₄ ⁺ has a significant negative effect on the production ability of the strain. There is also a shift in the proportion of carbon sources in favor of glucose.

From a morphological point of view, the microorganism culture is pelletized, the pellets are small (about 0.2 to 0.5 mm), become compact at the end of the fermentation, and conidiophores begin to form at the edges. During optimal fermentation, the sediment rises to 45-55%, the pH drops to 6.0-5.8, the concentration of phosphorus and carbon sources drops to trace amounts.

After completion of the fermentation the pH of the fermentation broth provides 40% H ₂ SO ₄ to 4,6 to 4,8. Lovastatin is extracted from the treated sample with acetone (1: 1) by sonication. Production is determined by HPLC method in an isocratic system of 65% acetonitrile and 0.1% H ₃ PO ₄ .

In the flask production assay thus performed, the new strain of A. terreus CCM 8236 produces at least 2000 mg / l lovastatin.

The identity of the product was verified by nuclear magnetic resonance imaging.

The new strain of A. terreus CCM 8236 differs from both the parent strain GB1 and the patented strain ATCC 20542 in terms of its growth abilities on various carbon sources, colony morphology and medium pigmentation (Table 1).

The production strain A. terreus of the present invention obtained by classical mutagenesis was deposited with the Czech Collection of Microorganisms in Brno under the number CCM 8236. It is a stable production strain with production of 2000 to 3000 mg / l of lovastatin in the form of acid and lactone, which represents an approximately 130-fold increase in production over the initial strain and at least a 2-fold increase in production over the A. terreus strain ATCC 20542. Rapid sporulation is important for the quality of the inoculum material. The inoculation stage takes place at an atypical pH. The composition of the cultivation soils fully corresponds to the requirements of industrial production in terms of both financial costs and availability of individual components.

Tab. L Morphological characteristics of strains A. terreus GB1, CCM 8236 and ATCC 20542 on Czapek-Dox agar with various carbon sources.

Carbon source A. lerrrustiSi A / errwj CCM 8236 Afrrnw ATCC 20542 glucose Inhuman air mycelium. Airy myelium Aerial mycelium velvet regular colonies » appearance, colony regular appearance. regular colonies diameter 2 J to 3 cm. $ two e shape with diameter 3 1 cooked with a diameter of 3 zones: up to 4 cm, in three zones: up to 4 cm, with two zones: 1. cenirätaa zone pale brown i. central wna pale brown 1. Central zone of pest pest 2 marginal roria white 2! rye-colored zone 3rd edge zone white will give 2nd edge zone white Soluble pigment: brown fructose Cylindrical aerial mycelium Colonies of regular shape s Vatwili · kuktnie pmiieÍGélur colonies of regular shape n diameter 3 cm. κ two zone- ivam with a diameter of 2 to cm. diameter 35 cm. with two zones- ml: in two zones: me: L «carrion zone pale brown, vo 1st Central Zone (Boar 1. central zone beige velvet look will give. 2 border zone white 2. Hela peripheral zone. Soluble pigment: yellow 2nd edge zone white Soluble pignienl: brown

Carbon source A. irfreu.tGBI A terrrus CCM 8236 A terma ATCC 29542 maltose Valvile aerial mycelium, colonies of regular shape with a diameter of 35 and 4 cm. with two zones: 1. Central zone pale brown 2nd edge zone white Airy mycelium of velvety appearance. colonies of regular shape with a diameter 35 to 4 cm. with two zones: L central zone brown 2nd edge zone Bicia Soluble ptgmcnl: yellow Valvile airy mycelium. regular colonies with a diameter of 3 to 4 cm. with two zones: 1. Central zone cinnamon brown 2nd edge zone white Soluble pigment: brown lactose Vatvlté airy mycelium. regular colonies with a diameter of 2.5 to 3 cm. with two zones: 1. Central zone pale brown 2nd edge zone white Colonies irreparable to shape> 2 to 2.5 cm in diameter, forming only a mibatrate mycelium overgrown with soil. Non-regular colonies with a diameter of 2 to 25 cm in the central zone of a cylindrical airy mycelium of the Centenary color, in the marginal zone a yellow substrate mycelium overgrown with soil. sadtarti Cylindrical aerial mycelium, colonies of regular shape with a diameter of 3 cm. and two zones: 1. Central zone pale brown 2nd edge zone white Colonies of regular shape and diameter 4 to 45 cm, strund zones: 1. central zone brown, velvety appearance 2. cotton wool beige zone 3. edge zone flat, velvet look. pest brown Vatoviié colonies of regular shape with a diameter of 15 to 2 cm. with two zones: L central zone brown. 2nd edge zone white Soluble pigment: brown glycerol Cylindrical aerial mycelium. colonies of regular others with a diameter of 3 to 35 cm. with two zones: 1st central zone yellow 2nd zone white Lobe-shaped colonies with a diameter of 1 to 15 cm. think flat. velvet look. beige coloring. Colony of lobed shape with a diameter of 1 to 1.5 cm, mycelium flat. velvety appearance, with two zones: L central zone school brown 2nd edge zone and bicia šktoh Flat aerial mycelium of velvety appearance. regular Ivani colonies with a diameter of 2.5 to 3 cm. with court zones: 1. Central zone pale brown 2. marginal zone white Aerial mycelium of velvet velvet. colonies of more or less regular shape with teardrop-shaped edges. with a diameter of 2 already 25 cm with three zones: L central zone pale brown 2. Scorpion zone 3rd edge zone white Colonies of regular shape, 2 to 3 cm in diameter. mycelium of velvety appearance, with two zones: L central zone cinnamon brown 2. marginal and white, markedly bounded

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1: Scheme of the mutation procedure for obtaining strain Aspergillus terreus CCM 8236.

Note: The method (UV-ultraviolet light, NTG-N-methyl, N-nitro, N-nitrosoguanidine) and time of treatment (in minutes or seconds), working isolation of test isolates (GB1 to GB2050) and lovastatin production are reported. on screening medium (mg / l acid form and mg / l lactone form).

DETAILED DESCRIPTION OF THE INVENTION

Example 1 Mutagenesis of A. terreus strain GB1

Preparation of spore suspension: a continuous increase in mycelia sporulated on sporulated soil with tomato paste (2%) and oatmeal (2%) was washed with 10 ml saline and 30% glycerin (spores number 3.5 x 10 ⁷ / ml).

Influence of NTG ml spore suspension with 10 ⁶ / ml spores and 1 ml NTG stock solution (equ. 5 mg / ml) with minimal dimethylformamide (added for low solubility of NTG in water) was added to 8 ml saline. NTG concentration in this solution was therefore 0.5 mg / ml and the spore density of the order of 10 ⁵ spores / ml. Influencing of biological material took place in the dark, stirring constantly and at room temperature. Every 10 min. 1 ml of sample was taken. The sample was centrifuged rapidly and the treatment solution was thoroughly removed. The spores were suspended in 1.5 ml of saline and centrifuged again. Spore sediment was suspended in 1 ml saline and diluted 10 and 100-fold. After 200 μΐ of such suspension was

Sown on petri dishes with sporula soil. Petri dishes were cultured for 8 days at 28 ° C.

Influence by UV radiation

A 100 µl spore suspension with a number of spores of the order of 10 ⁵ and 10 ⁴ / ml was seeded onto a petri dish with sporula soil. After drying the dishes at 28 ° C for 30 min. were exposed to UV light for 20, 30, 40, 60 and 120 seconds. The source of UV radiation was a Philips 30W fluorescent lamp. Its distance from the soil surface was 20 cm. After treatment, the plates were immediately placed in the dark for 1 hour. (Avoid photoreactivation). They were cultured for 8 days at 28 ° C.

Monocolons with a diameter of about 4 cm were washed with 2 ml of saline with 30% glycerin and tested directly for lovastatin production. The primary criteria for selecting the mutants for the production assay was the quality of the sporulation and the mycelial growth rate. The selected colonies were used in a screening production assay.

Example 2

Screening production test

Selected monoisolates were tested for production ability on soil with the following composition:

lactose 100 g / l soya flour 20 g / l nanoclay 3 g / l KH ₂ PO ₄ g / 1 KC1 0.5 g / l MgSO ₄ .7H ₂ O 0.5 g / l FeSO ₄ .7H ₂ O 0.01 g / l PH 6.8 The screening production broth was inoculated with 1 ml spore suspension with a spore count of the order of 10 ⁷ spores / ml. Production was determined after 120 hours of culture. at 28 ° C and 220 rpm n HPLC. production A. terreus strain (acid + lactone) [Mg / 1] GB1 0.6 + 3.1 ATCC 20542 200.1 +4.8 CCM 8236 402.2 + 11.8

Example 3: Laboratory scale production test

1. Sporulácia

Composition of sporula soil:

tomato paste 20 g / l oat flour 20 g / l agar 20 g / l PH 6.5

Visual and microscopic evaluation of sporulation.

- 65th hour: White mycelium continuously growing over the entire surface, the culture starts to sporulate and becomes yellowish. Visible septum mycelium with growing conidiophores visible under the microscope.

- 96th hour: Culture is already completely sporulated, cinnamon mycelium. Well developed conidiophores with easily released spores.

The cultured culture was washed with saline with 30% glycerol. The spore suspension thus prepared can be stored at 4 ° C for 30 days without decreasing production capacity.

2. Inoculation stage

The presence of ions is required for good growth of the inoculum

Fe ²⁺ , M ²⁺ , Zn ²⁺ , K ⁺ , Mg ²⁺ . Ca ²⁺ . An excess of carbon source, soluble phosphorus and nitrogen in the soil is also needed. Neither of these factors should be limiting.

ml of seed broth is seeded with a 10% spore suspension and cultured at 28 ° C and 220 rpm for 20 hours.

Inoculation soil composition:

glucose 50 g / l CSL 10 g / l (NH ₄ ) ₂ SO ₄ g / 1 kh ₂ Mon ₄ g / 1 Ca (NO ₃ ) ₂ .4H ₂ O 2 g / l MgSO ₄ .7H ₂ O 0.25 g / l KC1 0.25 g / l FeSO ₄ .7H ₂ O 0.01 g / l MnSO ₄ .7H ₂ O 0.01 g / l ZnSO ₄ .7H ₂ O 0.002 g / l PPG 2000 0.1 ml / 50 ml pH untreated (4,6) sterilization 40 min. 115 ° C

3. Production stage.

The production broth is inoculated with 10% inoculum. The fermentation is carried out for 170 hours at 28 ° C and 220 rpm. The production of lovastatin is determined by HPLC.

Tab. 2 Effect of the lactose / glucose ratio in the production soil on lovastatin production.

tribe Carbon source ratio [glucose: lactose] 22: 1 1: 22 PH I'm sitting. [%] prod. (mg / l) acid + lact pH I'm sitting. [%] prod. mg / l] acid + lact ATCC 20542 6.5 45 23 + 4 3.4 45 43 + 14 CCM 8236 6.3 48 55 + 8 2.4 46 114 + 8

Note: Soy flour and (NH ₄ ) ₂ SO ₄ were used as the nitrogen source.

Tab. 3a Effect of nitrogen source in the production soil on the production of lovastatin.

tribe Nitrogen source soya flour + (NH ₄ ) ₂ SO ₄ soya flour pH I'm sitting. [%] prod. [mg / l] acid + lact pH I'm sitting. [%] prod. (mg / l) acid + lact. ATCC 20542 3.4 45 43 + 14 6.0 45 1005 + 0 CCM 8236 2.4 46 114 + 8 5.9 55 1986 + 0

Tab. 3b Effect of nitrogen source in the production soil on lovastatin production

tribe Nitrogen source cotton flour + (NH ₄ ) ₂ SO ₄ cotton flour PH I sit. [%] prod. [mg / l] acid + lact pH I sit. [%] prod. (mg / l) acid + lact ATCC 20542 2.9 46 105 + 15 6.0 45 1347 + 0 CCM 8236 3.4 45 202+ 16 5.8 48 2248 + 0

Note. A 1: 22 ratio of glucose and lactose was used as the carbon source.

SK 281320 Β6

The results of the experiments in the table show that the presence of ammonium sulphate in the soil at the beginning of the production stage has a significant negative impact on the production capacity. A shift in the ratio of carbon sources to glucose has the same effect. Under such conditions, the production of lovastatin is even lower than that in the screening soil. The results of the experiments also show that the choice of an organic nitrogen source is important. In all soils tested, the new strain of A. terreus CCM 8236 produced approximately 100% more lovastatin than the strain of A. terreus ATCC 20542.

Based on the presented experiments the composition of the production soil was optimized.

Optimized production land has the following composition:

glucose 5 g / l lactose 110 g / l CSL 50% 5 g / l cotton flour 21 g / l KH ₂ PO ₄ 1 g / l CaCO ₃ 5 g / l PPG 2000 2 ml / l drinking water to 11 PH 7.0

On soil with such a composition, the average production of lovastatin was 2665 mg / l (n = 7), the maximum production reached 3042 mg / l lovastatin.

Industrial usability

The new high-production strain of Aspegillus terreus CCM 8236 can be used in the industrial production of lovastatin, its salts and esters.

Claims (2)

PATENT CLAIMS Aspergillus terreus strain GB2033, producing a high amount of lovastatin obtained by classical mutagenesis and deposited at the Czech Collection of Microorganisms in Brno under the designation Aspergillus terreus CCM 8236. 2. Fermentation preparation of lovastatin in the form of an acid or lactone, or a pharmaceutically acceptable salt or ester thereof, characterized in that the strain of Aspergillus terreus CCM 8236 is cultivated in seed broth with a pH in the range of 4.5 to 4.7 and in production broth, in using a combination of glucose and lactose in a ratio of 0.5 to 0.7% glucose and 11 to 15% lactose as a carbon source, and cotton or soy flour as a complex source, with a free ammonium ion concentration of max. 0.07 g / 1st