EP1047768A1

EP1047768A1 - A method of fermentation preparation of lovastatin by an (aspergillus terreus) strain and the strain for performing the method

Info

Publication number: EP1047768A1
Application number: EP98948055A
Authority: EP
Inventors: Gabriela Borosová; Jana Gajdosiková; Adela Vajciková; Valter Vollek
Original assignee: Biotika AS
Current assignee: Biotika AS
Priority date: 1997-10-13
Filing date: 1998-10-12
Publication date: 2000-11-02
Also published as: TR200000996T2; SK281320B6; WO1999019458A1; SK139597A3

Abstract

The new strain Aspergillus terreus CCM 8236 was obtained by means of classic mutagenesis procedures. The process of aerobic fermentation of lovastatin by the strain of Aspergillus terreus CCM 8236 in the form of acid in a submersion culture in a laboratory scale is described. The inoculum was prepared in a broth having low pH value. The mixture of glucose and lactose (at the ratio of 1:22) at the high concentration and Pharmamedia is used as the carbon and nitrogen source, respectively. The strain produced more than 2000 mg/l of lovastatin during 170 hours in the above-mentioned broth.

Description

A Method of Fermentation Preparation of Lovastatin by an Aspergillus terreus Strain and the Strain for performing the method.

Technical field

This invention relates to a micro-organism strain of an Aspergillus terreus type, obtained by the classic mutagenesis method comprising the combined action of both the physical and chemical mutagen and to a method of fermentation preparation of lovastatin by means of the strain.

Background of the invention

Lovastatin (MK-803, monacoiin, K. mevinolin) represents the secondary metabolite that inhibits competitively enzyme 3 - hydroxy - 3 metylglutaryl - coenzyme A (HMG CoA) reductase (EC 1.1.1.34) and thus decreases endogenous synthesis of cholesterol and provides wide opportunities in hyperlipaemia therapy. Inhibition of HMG CoA reductase results not only in a decrease of cholesterol level in mammal cells but also in induction of cholesterol receptors LDL, e.i. low density lipoprotein, in liver, change in ubiquinone production, protein isoprenylation, juvenile hormone synthesis in insects, plant sterols and pigments and fungal gibberellins. From the chemical point of view, 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 was proved for Aspergillus terreus ATCC 20542 by means of the labelled substrates from acetate by polyketide pathway. In spite of the fact that the precise structure of the key intermediates between acetate and lovastatin has not been known yet, some working hypotheses of biosynthesis have been already available, as reads in Wagschal et al, J.Chem.Soc, Perkin

Trans. 1 , 2357-2363, 1996. Lovastatin occurs in two forms, namely, in the form of acid and in the form of lactone while the biological active form is the former one. Lovastatin in the tablet form has been used for hyperchoiesterolaemia decrease and prevention of ischaemic heart disease since 1987. The fermentation preparation of lovastatin and dihydroxymevinolin by the production Aspergillus terreus strains of ATCC 20541 and ATCC 20542 types resp. is described in papers US 4.231.938 and EP 0 022 478.

The fermentation preparation of hypocholesterolemic and hypolipemic substanfce featuring with the structure related to lovastatin by the same strain of Aspergillus terreus ATCC 20542 is described in a paper US 4.387.242.

Process of preparation of a strain producing lovastatin using this species of Aspergillus genus that does not originally produce the above mentioned substance, e.g. Aspergillus oryzae, is described in EP 0 556 699. The method of preparation of such strain includes the transformation of A. oryzae protoplast by DNA isolated from the production strain of A. terreus.

The microbial process of preparation of lovastatin in the form of both the acid and lactone by aerobic fermentation of submersion culture of the new species Aspergillus obscurus is described in US 5.403.728.

Species of Monacus genus - M.anka, M.purpureus, M.ruber, M. vitreus, M. paxii are also mentioned as the strains producing lovastatin in the patent papers UK 2.046.737 and UK 2.049.664.

Other microorganism species during fermentation of which small lovastatin amounts are obtained are also mentioned in specialized literature. It concerns particularly to the following species: Phoma sp. M 4452, Doratomyces nanus IFO 9551 , Gymnoascus umbrinus IFO 8450 as described in the article of Endo et ai: J.Antibiot. 39, 1609-1610, 1986, Pleurotus ostreatus, Pleurotus saca and Pleurotus sapidus as described in Gunde - Cimerman, FEMS Microbiology Letters 11 , 203-206, 1993.

In spite of the fact that multiple strains of Aspergillus terreus species producing lovastatin were described in patent literature, the amount of lovastatin produced by the patented strain Aspergillus terreus ATCC 20542. is considerably low. Another disadvantage is the fact that the components of fermentation broth published in the patents US 4.231.938, EP 0 022 478 and US4.387.242 are expensive and thus unsuitable for industrial applications. Disclosure and object of the invention

A. terreus CCM 8236, the new mutant strain featuring lovastatin hyperproduction, was obtained by the combined usage of both the physical and chemical mutagens that were applied to spores of A. terreus strain. When compared with the strain A terreus ATCC 20542, the new strain at least doubles the production of lovastatin. Both the inoculation and production media contain row materials that are easily available and cheap and are normally used for fermentation of both the secondary and primary metabolites. It is a subject matter of the invention to use the strain A. terreus CCM 8236 for production of lovastatin in the form of acid and lactone or its pharmaceutically utilizable salts or esters.

The original strain A. terreus, with a temporary name of GB1 , because of the relatively highest production of lovastatin, was selected as a production strain starting material from a number of wild strains identified as A. terreus.

The above strain was subjected to action of chemical and physical mutagens in order to obtain a strain suitable for a production of lovastatin.

During the first phase of its culturing, the starting strain was affected by the strong N-methyl, N-nitro, N-nitrozoguanidine (NTG) mutagen. Because of the increased resistance of mutants to NTG, they were by turns subjected to action of ultraviolet radiation (UV) and NTG. The isolates obtained, each of them marked with letters GB and a serial number, were tested for the lovastatin production yield. The procedure resulted in isolation of a mutant marked GB2033, which features a remarkably increased lovastatin production. It was archived in the Czech collection of microorganisms in Brno, Czech Republic, under the marking CCM 8236. Broth compositions, the sporulation, inoculation and production ones, were matched to the above mentioned new strain.

Microorganism culturing used for the fermentation process according the invention consists of three steps:

1. Culture sporulation

2. Inoculation

3. Production A fast sporulation is essential for inoculum quality, therefore the culture is sporulated completely during 4-5 days at the temperature of 28 °C. Spores have brown colour; colonies are flat, have velvet-like appearance without white edges, its diameter being app. 4 cm and can be easily washed with saline solution containing 30% of glycerole.

Inoculum is cultivated at the temperature of 28 °C and 220 rpm for 16 up to 20 hours in broth having low pH value of 4.6 that further shows a remarkable decrease during culturing, going down to 3.2 or even 3.0. The high-quality inoculum has the following features:

- Pellets are small and well shredded to fibres;

- 20% sediment

- Informative analytical characteristics: glucose content: 40 g/l phosphorus content: 200 mg/l NH₄ ⁺- content: 2 g/l

NH₂ ^"- content: 1 g/l

The production step is cultured at the temperature of 28 °C at 220 rpm for 170 - 190 hours. The production broth contains high concentration of carbon sources, namely glucose and lactose, at the precisely defined ratio of 1:22 in favour of lactose. An organic source, soya flour, inactivated baker's yeast, etc., for example, shall be used as nitrogen source. The higher production yield was reached with the broth containing cotton flour Pharmamedia as nitrogen source. Inorganic nitrogen in the broth, in the form of NH₄ ⁺, shows remarkably negative effect to the production capability of the strain. The same effect is shown also by a change of a ratio of carbon sources in favour of glucose.

The microorganism culture is of the pellet-type from the morphological point of view; the pellets are small, having a diameter of app. 0.2 - 0.5 mm. They become compact towards the end of fermentation and conidiofores start to form on their edges. Sediment value increases up to 45% - 55%, pH value decreases to 6.0 - 5.8, concentration of phosphorus and carbon sources decreases to trace amounts during an optimal fermentation process. pH value of the fermentation broth is treated with 40% H₂SO₄ to the value of 4.6 - 4.8 after the fermentation process is completed. Lovastatin is extracted from the treated sample by means of acetone in a ratio of 1 :1 , while applying an ultrasound mixing. The production yield is determined by HPLC method in the isocratic system consisting of 65% of acetonitrile and 0.1% of H₃PO₄.

The new strain A. tereus CCM 8236 produces at least 2000 mg/l of lovastatin during the above-mentioned flask production test.

Product identity was verified by a nuclear magnetic resonance method.

The new strain A. tereus CCM 8236 differs from the starting strain GB1 and also from the patented strain ATCC 20542 also by its capability of growing on various carbon sources, morphology of colonies and medium pigmentation as presented in Tab. 1.

The production strain A. terreus that is the subject of this invention and was obtained by means of the classic mutagenesis has been archived in the Czech collection of microorganisms in Brno under the number CCM 8236. It represents the stable production strain featuring the yield of 2000 - 3000 mg/l lovastatin in the forms of acid and lactone. This production rate represents approximately 130 multiple of the production rate achieved by the starting strain and at least double increase in production in comparison with the strain A. terreus ATCC 20542. Fast sporulation is essential for high quality of the inoculation material. The inoculation step is carried out at the atypical pH value. The composition of the culturing broth fully complies with industrial production requirements concerning financial expenses and availability of the individual components. Table 1 :

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

00

Description of preferred procedures

The invention shall be further described with respect to presented examples and accompanying tables and the Fig. 1 showing a scheme of the mutation procedure used to obtain the Aspergillus terreus CCM 8236 strain. In Fig. 1 there are presented abbreviations UV and NTG for ultraviolet radiation and N-methyl, N-nitro, N-nitrozoguanidine methods respectively, affection periods in minutes or seconds, temporary markings of the isolates tested from GB1 up to GB2050 and quantities of lovastatin production on the screening broth in mg/l of acid form and mg/l of lactone form.

Example 1 :

Mutagenesis of the strain of A. terreus GB1.

Preparation of spore suspension: The confluent growth of mycelium sporulated on a sporulation broth comprising 2 % of tomato puree and 2 % of oat flour, rinsed with 10 ml of saline solution and 30% glycerine and showing spore number 3.5 x 10⁷/ml.

Influencing by NTG

1 ml of spore suspension showing the spore number of 10⁶/ml and 1.0 ml of NTG stock solution having a concentration of 5 mg/ml and containing a minimal amount of dimethyl formamide, which had to be applied because of the low solubility of NTG in water, was added to 8.0 ml of saline solution. Therefore, NTG concentration in the specified solution was 0.5 mg/ml and spore density of the order of 10⁵ spores/ml.

The biological material, while continuously stirred was influenced in the dark and at a room temperature. A 1 ml sample was sampled every 10 minutes. The sample was centrifuged quickly and the influenced solution removed thoroughly and carefully. Spores were suspended in 1.5 ml of saline solution and repeatedly centrifuged. Spore sediment was suspended in 1 ml of saline solution and diluted 10-times and 100-times. Per 200 μl of the above suspension were inoculated in Petri dishes containing sporulation broth. Petri dishes were cultured at the temperature of 28 °C for 8 days. Influencing by UV radiation

100 μl of spore suspension with spore number of the order 10⁵ and 10 /ml was inoculated in Petri dishes containing sporulating broth. The dishes were dried at the temperature of 28 °C for 30 minutes and then subjected to UV radiation for 20, 30, 40, 60 and 120 seconds. A fluorescent lamp, namely 30 W Philips one, was used as a source of UV radiation. It was located at the distance of 20 cm from the broth surface. The influenced dishes were then placed immediately to the dark place for 1 hour in order to prevent photoreactivation. The dishes were cultured at the temperature of 28 °C for 8 days.

The monocolonies having the diameter of approximately 4 cm were rinsed with 2 ml of saline solution containing 30% of glycerol and tested directly for lovastatin production. The primary criteria for selection of mutants for the production test were the sporulation quality and mycelium growth rate. The colonies selected were used for the screening production test.

Example 2

Screening production test

The monoisolates selected were tested for a production capability on broth having the following composition:

Lactose 100 g / l Kcl 0.50 g/l

Soya flour 20 g / 1 MgSO₄ .7 H₂O 0.50 g/l

NaNO₃ 3 g / 1 FeSO₄ . 7 H₂O 0.01 g/l

KH₂PO₄ 1 g / 1 pH 6.8

The screening production broth was inoculated with 1 ml of spore suspension with a spore number of the order of 10⁷ spores/ml.

The sample was cultured at the temperature of 28 °C and at 220 rpm for 120 hours and the production was then determined by means of HPLC method. Production

Strain A. terreus (acid + lactone)

[mg/l]

GB1 0.6 + 3.1

ATCC 20542 200.1 + 4.8

CCM 8236 402.2 + 11.8

Example 3

Production test in a laboratory scale.

1. Sporulation.

Composition of the sporulation broth:

Tomato puree 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 confluently grown over the whole surface, the culture starts to sporulate and its colour turns yellowish. Septed mycelium with growing conidiofores is visible under a microscope.

- 96th hour: The culture has been already sporulated, mycelium has cinnamon colour. Well-grown conidiofores with easily releasing spores.

The sporulated culture was rinsed with saline solution containing 30% of glycerol. The spore suspension stored at the temperature of 4 °C for 30 days showed no decrease of production capability and therefore can be stored at this condition.

2. Inoculation step

To ensure good inoculum growth a presence of Fe²⁺, Mn²⁺, Zn²⁺, K⁺, Mg²⁺ and Ca²⁺ ions is inevitable. The excessive amount of carbon source, soluble phosphorus and nitrogen in a broth is also necessary. None of the above factors shall be a limiting one. 50 ml of the inoculation broth was inoculated with 10% of spore suspension and cultured at the temperature of 28 °C and at 220 rpm for 20 hours.

Composition of the inoculation broth:

Glucose 50 g/l FeSO₄ . 7 H₂O 0.01 g/l

CSL 10 g/l MnSO₄ . 7 H₂O 0.01 g/

(NH₄)₂SO₄ 1 g/l ZnSO₄ . 7 H₂O 0.002 g/l

KH₂P0₄ 1 g/l KCI 0.25 g/l

Ca(NO₃)₂ • 4 H₂O 2 g/l PPG 2000 0.1 ml/50 ml

MgSO₄ . 7 H₂O 0.25 g/l pH without any treatment 4.6

Sterilisation 40 min at 115 °C

3. Production step

10% of inoculum was inoculated on the production broth. Fermentation was carried out at the 28 °C and at 220 rpm for 170 hours. Lovastatin production was determined by HPLC method.

Table 2.

Influence of lactose and glucose ratio in the production broth on lovastatin production

Table 3a.

Influence of nitrogeneous source in the production broth to lovastatin production

Strain Nitrogen source soya flour + (NH₄)₂SO₄ soya flour

/ pH sediment prod, [mg/l] pH sediment prod, [mg/l] [%] acid + lactone [%] acid + lactone

ATCC 20542 3.4 45 43+ 14 6.0 45 1005 + 0

CCM 8236 2.4 46 114 + 8 5.9 55 1986 + 0

Table 3b.

Strain Nitrogen source cotton flour + (NH )₂SO cotton flour pH sediment prod, [mg/l] pH sediment prod, [mg/l] [%] acid + lactone [%] acid + lactone

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: As a carbon source there were used glucose and lactose at the ratio of 1 :22.

From the results presented in Tab. 3a and Tab. 3b it is evident that the presence of ammonium sulfate in the broth at the beginning of the production step has remarkably negative influence to production capability. The same effect is also achieved by a change of a carbon source ratio in favour of glucose. Lovastatin production is under such conditions even lower than its production in the screening broth. Test results have also proved that selection of nitrogen as the organic source has its importance. The new strain of A. terreus CCM 8236 produced lovastatin at the amount which is approximately by 100% higher in comparison with results achived with the strain of A. terreus ATCC 20542 in all kinds of the broth tested.

The content of the production broth was then optimized on the basis of the experiments presented as follows:

Glucose 5 g/l

Lactose 110 g/l

CSL 50% 5 g/l

Cotton flour 21 g/l

KH₂PO₄ 1 g/l

CaCO₃ 5 g/l

PPG 2000 2 ml/l

Potable water up to 1 I pH 7.0

The average lovastatin production reached in the broth of the above mentioned composition was 2665 mg/l (n = 7); the maximum lovastatin production reached was 3042 mg/l.

Industrial application

This new high-production microorganism strain Aspergillus terreus CCM 8236 can be used for industrial production of lovastatin, its salts and esters.

Claims

1. The strain of Aspergillus terreus with the temporary name GB 2033 obtained by methods of the classic mutagenesis and archived in the Czech Republic collection of microorganisms in Brno under the name Aspergillus terreus CCM 8236.

2. A method of fermentation preparation of lovastatin in the form of acid or lactone or its pharmaceutically utilizable salts or esters comprising a culturing of the strain of Aspergillus terreus CCM 8236 in an inoculum broth having pH value in a range of 4.5 - 4.7 and an application of a carbon source at a form of a combination of glucose and lactose, the combination featuring 0.5 - 0.7%o of glucose and 11 - 15% of lactose, and an application of a cotton flower or soya flower as a complex source in the production step, and at the same time the concentration of the ammonia ions is in a range 0 - 0.07 g/l.