FR2499589A1 - Regulation of fermentative ergot alkaloid prodn. - by adding precursor or stimulant for isoleucine biosynthesis - Google Patents

Abstract

A process is claimed for regulating the amt. and proportions of alkaloids obtained by culturing mutant strains of Claviceps purpurea which produce principally ergocornine (Ia), alpha- ergocryptine (Ib) and beta-ergocryptine (Ic). The process comprises using a cpd. which acts as a precursor for isoleucine biosynthesis, or which stimulates isoleucine formation by biochemical control, as a regulator during conventional fermentation. The regulator can be methyloxalacetic acid, alpha-ketobutyric acid, threonine, homoserine, homocysteine and/or methionine. It can be used in an amt. of 0.01-10 (pref. 0.05-5.0) g/l. The proportions of (Ic) can be increased to the level required for prodn. of dihydroergotoxine, which is used as the (m)ethanesulphonate for treatment of peripheral and cerebral circulatory disorders.

Description

 The present invention relates to a process for regulating the quantity and the proportions of the alkaloids produced under saprophytic conditions, by mutant strains of Claviçeps purpurea which produce mainly ergocornine, I'cret 8-ergocryptine.

 It is known that methanesulfonate and dihydroergotoxin ethanesulfonate, respectively, are used in medicine as regulators of central metabolism as well as central and peripheral circulation. The preparation increases the synthesis of central nervous system proteins and inhibits catecholamine-stimulated adenylcyclase. This product also has a weakly sedative action, inhibits reflex tachycardia, improves the blood supply to the brain and lowers blood pressure. In medicine, the product is mainly used for the treatment of diseases which are due to peripheral or cerebral circulatory disorders.

 Dihydroergotoxin is prepared by hydrogenation of ergotoxin. The product which forms is a mixture of dihydroergocristine, dihydroergocornine and dihydroergocryptine (cf. Hungarian patent nO 129,061).

Dihydroergocryptine comes in the form of two variants, namely the forms a- and ss- (Experientia23, 991 19677) and according to the most recent works, the pharmacological action of the two forms is not entirely identical - . The form a- is more active in a contraceptive test on rats, while the form ss- exerts a stronger vasopressor action on brainless cats (Experientia 33, 1552 / i9727). This is the reason why in the prescriptions of preparations containing dihydroergotoxin, the proportion of a- and B-dihydroergocryptine is well determined. The proportion of the form a- to the form ss- considered as ideal is 2 : 1 and the dihydroergotoxin with the optimal composition contains the components dihydroergocristine, dihydroergocornine, a-dihydroergocryptine and ss-dihydroergocryptine in a 3: 3: 2: 1 ratio. The tolerances allowed in the relationship between form a- and form S- are different in the various countries. Thus, in the United States and in several European countries, the admissible limit values for the content of ss-dihydroergocryptine are from 26.7 to 44% of the total content of dihydroergocryptine, while. than in other European countries and in JAPAN, the tolerance is 28.6-40 t.

 The initial stage in the preparation of methanesulfonate or dihydroergotoxin ethane sulfonate is the preparation of non-hydrogenated ergot alkaloids (ergocristine, ergocornine, a- and B-ergocryptine).

 These alkaloids are prepared biologically.

A possibility of preparation is represented by the parasitic biosynthesis in which one infects rye and one obtains the alkaloid produced by extraction from the plant. This method is mainly used for the preparation of ergocristine (strains used: the strain deposited in the American Type Culture Collection under the number ATCC 20103 and described in the Patent
British No 1,192,912 from Societa Farmaceutici Italia; the strain deposited by Richter Gedeon Vegyészeti Gyar RT under the number MNG 00163 in the National Collection Hon groins4.

Furthermore, biosynthesis can take place by the saprophytic route by isolating the alkaloids from the fermentation broth. The best known strains for implementation by this route are the ATCC 20102 strain which produces ergocryptine and ergotamine (Patent
British 1,158,380), strain ATCC 20106 which produces ltergocornine and ergosine (British Patent I 184,039), strain ATCC 20019 which produces ergocryptin (US Patent No. 3,485,772), strain of Società Farmaceutici Italia which produces ss-ergocryptine and ergosine (which is deposited in the Personal Collection of this Company under the nO FI 7374 and which is described in the Belgian Patent nO 824 987).

 It appears from the above that these selected strains produce not only the alkaloids important for the preparation of ergotoxin, but also other alkaloids of ergot rye, that is to say that their fermentation is not selectively oriented towards the production of ergotoxin.

Hungarian researchers have selected for the first time strains of Claviceps purpurea which produce mainly a- and ss-ergocryptine (Hungarian patent nO 152 238; nO deposit of the strain
MNG 0022; Hungarian patent nO 164 816, nO of deposit of the strain: MNG 0088). However, even in the case of these strains, the adjustment of the proportions of the alkaloids relative to each other during fermentation is not carried out. The proportions of the three alkaloids produced relative to each other can only be adjusted subsequently by decomposing the mixture of alkaloids obtained, into its components, using the complicated methods available, and again mixing together the separate components, in appropriate ratios.

On the other hand, a process is known in which two strains are fermented simultaneously (Patent
Switzerland No. 577 556), one of which produces ergocornine, ergocryptine and their isomers, while the other produces ergocristine. The alkaloids indicated in a report which is not mentioned more precisely are obtained from the common culture juice of the two strains. Adjustment of these proportions is also not obtained with this process. The fact that fermentation, which involves two strains simultaneously, can be much more difficult to master than a process in which only the optimum culture of a single strain should be taken into account is also a problem.

H. KOBEL established (in the conference he gave in 1977 to BALE at the meeting of the Federation of Societies
Microbiological EuropeenneX, that the biosynthesis of peptide alkaloids may be influenced by certain amino acids. The biosynthesis of ergocornine containing valine in its peptide part, can be promoted by adding valine, the biosynthesis of a-ergocryptine which contains leucine, may be promoted by the addition of leucine. In contrast, the biosynthesis of S-ergocryptine which contains isoleucine cannot be influenced by the addition of isoleucine. H. KOBEL indicated that the probable cause of this remarkable phenomenon would be represented by the fact that the growth of the strains capable of producing ss-ergocryptine would be inhibited by ltisoleucine.

 As in the culture juices of known strains which produce ergocryptine or ergocornine and ltergocryptine, the proportion of $ -ergocryptine is also lower than that required, the appropriate proportion cannot be adjusted either.

 The present invention therefore aims to influence the fermentation of mutants of the claviceps purpurea strain in a favorable direction.

 The Applicant has now been able to surprisingly demonstrate that the fraction of B-ergocrptin present in the fermentation juice increases when the production of alkaloids of the mutant strains of Claviceps purpurea is influenced, using instead of isoleucine, a compound that behaves as a precursor in the biosynthesis of isoleucine. Therefore, the precursors of isoleucine do not inhibit the growth of the strains.

 The Applicant has also been able to demonstrate that the production of alkaloldes can also be regulated by using compounds which stimulate the formation of isoleucine, as a result of biochemical regulation. The precursors and the regulatory compounds are keto-acids, hydroxy-acids, amino-acids containing from 4 to 6 carbon atoms, whose biosynthesis starts from aspartic acid or phosphate of this acid.

To influence the fermentation in a favorable direction, the Applicant has also selected new mutant strains. During these tests, she selected a new mutant strain of Claviceps purpurea, which was deposited on May 9, 1979 under the no.
MNG 00186 in the Hungarian National Collection. This strain has a morphology similar to that of a yeast and produces mainly ergocornine and 6-ergocryptine, so that it is capable of increasing the content of B-ergocryptine.

The Applicant has finally been able to demonstrate that the proportions of the alkaloids produced practically do not change, and that the total amount of alkaloids in the fermentation juice increases, however, when the new strain is used for the production of alkaloids and adding to the fermentation juice a compound which behaves as a precursor in the biosynthesis of isoleucine or a compound which stimulates
the formation of isoleucine as a result of biochemical regulation.

 The present invention therefore relates to a method for regulating the quantity and the proportions of the alkaloids produced under saprophytic conditions, by mutant strains which produce mainly ergocornine-, a- and e-ergocryptine. The essential characteristic of the process according to the present invention is represented by the fact that in the fermentation, which is known in itself, as a regulator, a compound which behaves as a precursor in the biosynthesis of the isoleucine or a compound that stimulates the formation of isoleucine as a result of biochemical regulation.

 In accordance with the invention, mutant strains of Claviceps purpurea are used in the process which produce mainly ergocornine, a and ss-ergocryptine. The preferred strains are the strains deposited in the Hungarian National Collection under the numbers MNG 0022, MNG 0088 and MNG 00186.

 The fermentation is carried out in submerged culture, under aerobic conditions, on a liquid nutritive medium containing carbon and nitrogen sources, mineral salts and possibly other additives, at a temperature between 20 and 260C and at a value between 5.2 and 6.8, for 4 to 8 days.

 Fermentation is carried out in the presence of an additive (precursor) regulating the proportions of alkaloids.

As a regulatory additive, a compound is used which behaves as a precursor in the biosynthesis of isoleucine (Lehninger: Biochemistry,
Worth Publ. Inc. NY, page7o4 t19757) or a compound that stimulates the biosynthesis of isoleucine by biochemical regulation.

 The precursors of isoleucine in the biosynthesis of the latter are, in the order of their formation, as follows: via homoserine and threonine or 3-methylaspartic acid (2-amino acid -3-methylsuccinic) and methyl oxalacetic acid (2-methyl-3-oxo-succinic acid), a-ketobutyric acid, then passing through a-acetyla-hydroxybutyric acid, a ss-dihydro-ss-methylvaleranic and a-keto-B-methylvaleranic acid.

 The compounds which stimulate the formation of isoleucine by biochemical regulation belong biochemically to aspartates, that is to say to compounds whose biosynthesis starts from aspartic acid or its phosphate. These are mainly keto acids, hydroxy acids and amino acids containing 4 to 6 carbon atoms. Homocysteine or methionine are preferred.

The compound which regulates the production of alkaloids is added to the fermentation juice at a rate of 0.01 to
3 3 10 kg / m, and preferably at a rate of 0.05-5.0 kg / m3.

The aqueous solution, possibly weakly acidic, of the regulating compound is added after sterilization, all at once, or divided into several fractions, or continuously during one of the periods of fermentation.

 In previous patent applications, the alkaloid content of the fermentation juice is frequently indicated by the values measured by spectrophotometry.

The total alkaloid content was also measured, in this case, by spectrophotometry; however, the results of the measurements were not found to be specific.

 The different components of the ergotoxin were determined separately by elution chromatography or by quantitative liquid chromatography.

 In addition to the foregoing provisions, the invention also comprises other provisions, which will emerge from the description which follows.

 The invention will be better understood with the aid of the additional description which follows, which refers to examples of implementation of the method which is the subject of the present invention.

 It should be understood, however, that these examples of implementation are given solely by way of illustration of the object of the invention, of which they do not in any way constitute a limitation.

EXAMPLE 1
200 ml of GK nutrient medium introduced into a 500 ml Erlenmeyer flask are inoculated with a culture of the Claviceps purpurea MNG 0022 strain cultivated on AIC agar-agar nutrient medium. The Erlenmeyer flask is incubated for three days on a shaking table, shakes at a frequency of 300 min 1. the inoculum thus obtained is itself seeded on 5 liters of a nutrient medium St in a fermenter with a volume of 10 liters . The culture is carried out for 7 days at 240 ° C. at a stirring speed of 240 min, under aeration with 0.5 liters of air per liter of fermentation juice and per minute. The fermentation juice is added at 40 °, 64th, 88th and 112th hours, as a precursor, an aqueous solution containing 10% methyloxalacetic acid (0.5 g of precursor / liter of fermentation juice). The proportions of the ergocornine and ergocrytpine alkaloids reach the desired value on the 7th day of fermentation. The total alkaloid content, which can be measured using a color reaction, is 1150 μl / ml. The assay by thin layer chromatography (J. of Chrom. 87, 433 g1973 /) reveals the presence of 300 y / ml of ergocornine, 180 y / ml of ergocryptine and 110 y / ml of their dextrorotatory epimers The ratio between a- and 8-ergocryptine is 66:34 by examination by chromatography in liquid phase under high pressure (J. Pharm.

Sci. 67, 98 1978 /).

 The alkaloids are isolated in a manner known per se from the fermentation juice.

The nutrient media used have the following composition 11 AIC agar-agar medium
. Mannitol 40.0 g
. Citric acid 7.0 g
. Corn maceration liquor 2.0 g
. Monopotassium phosphate 1.0 g
. Magnesium sulfate 0.3 g
. Agar powder 25.0 g
. Ammonia up to pH 5.2-5.3
. Water up to 1000 ml
The desired pH value for the nutrient medium is adjusted during boiling. Then the nutrient medium is introduced in fractions of 6 ml each into test tubes and allowed to harden by placing the test tubes in an oblique position, under oblique agar-agar form.

nutrient medium GK
. Trypcasin 7.0 g
. Citric acid 4.1 g
. Monopotassium phosphate 0.3 g
. Magnesium sulfate 0.3 g
. Ammonia up to pH 5.7-5.8
. Water up to 840 ml
The nutrient medium is introduced into flasks at the rate of fractions of 80 or 168 ml each. 16 and 32 ml of a 50% glucose solution are added to each flask.

nutrient medium St
. Sucrose 100.0 g
. Succinic acid 10.0 g
. 0.25 g monopotassium phosphate
. 0.25 g magnesium sulfate
. Ammonium nitrate 1.0 g
. Calcium chloride 1.0 g
. Ammonia up to pH 5.2-5.3
. Water up to 1000 ml
The nutrient medium is sterilized in fractions of 0.1 or 5 or 100 liters.

EXAMPLE 2
200 ml of GK nutrient medium contained in a 750 ml Erlenmeyer flask are inoculated with a culture of the mutant strain of Clavicepspurpurea MNG 0088 cultivated on nutrient medium with AIC agar-agar. The culture is incubated for 3 days at 240C on a shaking table shaken at a frequency of 300 min 1. 5 liters of culture medium TC 54, in a 10-liter laboratory fermenter, are inoculated with the culture obtained. The culture is incubated for 3 days at 240 ° C. and at a shaking speed of 240 min 1 under aeration using 0.5 l of air per liter of fermentation juice and per minute.

100 liters of culture medium St is contained in a fermenter resistant to acid corrosion, equipped with a stirrer, the inoculum obtained and the culture is fermented for 6 days at 240C and at a speed d agitation of 120 min 1, under aeration with 0.3 liters of air per liter of fermentation juice and per minute. To the culture is added during the first 5 days of fermentation, a 5% aqueous solution of α-ketobutyric acid, as a precursor, at a rate of 20 ml / hour.

 After the interruption of fermentation, the total alkaloid content is 920 y / ml. Using the liquid chromatography method indicated in Example 1, we detect the presence of 260 y / ml of ergocornine, 155 y / ml of α-ergocryptine and 95 y / ml of B- ergocryptine. In addition, 80 µg / ml of ergocorninin-cryptinin can be measured in the fermentation juice.

The nutrient medium TC 54 has the following composition. Sucrose 100.0 g. Citric acid 10.0 g. Sodium chloride 10.0 g. Monopotassium phosphate 0.5 g. Magnesium sulfate 0.5 g. Ammonia up to pH 5.7-5.8. Water up to 1000 ml
The nutrient medium is sterilized in a laboratory fermenter in 5 liter fractions.

EXAMPLE 3
100 ml of GK nutrient medium contained in a 500 ml Erlenmeyer flask are inoculated with a culture of the mutant strain of Claviceps purpurea MNG 0088 cultivated on nutrient medium with St. agar. The culture is incubated for 3 days at 240C on a shaking table shaken at a frequency of 300 min 1. 100 ml of nutrient medium T 25 are inoculated with 10 ml of the culture obtained. This culture is incubated for 5 days at 20 ° C. also on a shaking table. At the 20th hour, 0.5 g of threonine is added as a precursor to the fermentation juice. On the 5th day, the total concentration of alkaloids in the juice is 1200 y / ml. Using the method indicated in Example 1, 260 y / ml of ergocornine, 140 y / ml of α-ergocryptine and 80 y / ml of B-ergocryptine are detected.

The total content of ergocorninine and ergocryptinine is 130 y / ml.

 The nutrient medium with agar-agar St differs from the liquid nutrient medium St indicated in Example 1 in that 25 g of agar-agar powder (Difco) are added to it per liter. After adding the agar, the nutrient medium is brought to the boil. Then it is introduced in fractions of 6 ml each into test tubes, it is sterilized and allowed to harden by placing the test tubes in an oblique position, to obtain oblique agaragar.

The nutrient medium T 25 has the following composition. Sucrose 300.0 g. Citric acid 15.0 g. Yeast extract 1.0 g. Monopotassium phosphate 0.5 g. Potassium sulfate 0.5 g. Ammonia up to pE 5.2-5.3. Water up to 1000 ml
The nutrient medium is introduced into flasks in 100 ml fractions and sterilized.

EXAMPLE 4
The procedure is as described in Example 3, however using 0.05 g of thomoserine as a precursor in place of threonine. On the 5th day of fermentation, the total alkaloid content is 880 μg / ml. The fermentation juice contains 220 y / ml ergocornine, 150 y / ml a-ergocryptine and 80 y / ml ss-ergocryptine. Ergocorninine and ergocryptinine together represent 180 y / ml.

EXAMPLE 5
The procedure is as described in Example 3, however using 0.05 g of homocysteine as precursor.

On the 5th day of fermentation, the total concentration of alkaloses is 700 y / ml. The fermentation juice contains 190 y / ml of ergocornine, 85 y / ml of a-ergocryptine and 90 y / ml of ss-ergocryptine. Ergocorninine and ergocryptinine together represent 150 y / ml.

EXAMPLE 6
100 ml of GK nutrient medium contained in a 500 ml Erlenmeyer flask are seeded with a culture of the strain of
Claviceps purpurea MNG 00186 cultivated on nutrient medium with agar St. This culture is incubated for 4 days at 240C on a shaking table shaken at a frequency of 300 min 1. 10 ml fractions of the culture obtained are each introduced into 8 flasks each containing 100 ml of nutrient medium St. The cultures are fermented for 7 days at 240C also on a shaking table. No precursor is added to 4 of the balloons. In the other 4 flasks, 0.02 g of methionine dissolved in 2 ml of water are added at the 24th and 48th hours of fermentation. The fermentation is interrupted on the 7th day.

 The fermentation juices grown without the addition of a precursor are combined and their alkaloid content is determined: ergocornine: 80 y / ml; a-ergocryptine 15 y / ml; B-ergocryptine: 30 y / ml.

 Fermentation juices grown with the precursor are also collected and examined. They have a total alkaloid concentration of 250 y / ml, 50 y / ml of α-ergocryptine and 100 y / ml of B-ergocryptine.

 As is apparent from the above, the invention is in no way limited to those of its modes of implementation, embodiment and application which have just been described more explicitly; on the contrary, it embraces all the variants which may come to the mind of the technician in the matter, without departing from the framework, or the scope, of the present invention.

Claims (3)

 1 - Process for regulating the quantity and the proportions of alkaloids produced under saprophytic conditions by mutant strains of Claviceps purpurea which mainly produce ergocornine, a-and ss-ergocryptine, which process is characterized in that which is used during fermentation known in itself as a regulator, a compound which behaves as a precursor in the biosynthesis of isoleucine, or a compound which stimulates the formation of isoleucine by biochemical regulation .  2 - Process according to Claim 1, characterized in that methyloxalacetic acid, α-ketobutyric acid, threonine and / or homoserine is used as the precursor of isoleucine.  3 - Method according to Claim 1 or Claim 2, characterized in that homocysteine and / or methionine is used as a compound stimulating the formation of isoleucine by the biochemical route.  40- Method according to any one of Claims 1 to 3, characterized in that the regulator is used at the rate of 0.01 to 10 kg / m3 of fermentation juice, and preferably at the rate of 0.05- 5.0 kg / m3 of fermentation juice.