CS273373B1 - Method of ergot alkaloids' fermentation production with modification of cellular lipids - Google Patents

Abstract

The invention concerns a method of fermentation of ergot alkaloids while modifying cellular lipids, leading to an increase in the biosynthesis of ergot alkaloids and changes in their spectrum. The main purpose is a significant increase in the production of ergot alkaloids by ca 10 to 30 % with positive effect on the effectiveness of fermentation and, subsequently, on extraction, isolation and possible semi-synthetic procedures, used for production of final medical substances. The given effect is achieved by addition of mono- and dichlorophenoxyalkanoic acids and/or their derivatives, causing modification of cellular lipids, in a quantity of 5 to 300 mg/l in the production phase of the fermentation process. The active substances can be added at the start or in the course of cultivation and one specific substance or combination of substances can be used. The invention can be used in fermentation and the pharmaceutical industry.

Description

The invention relates to a method for fermentative production of ergot alkaloids with modification of cell lipids in production strains of fungi of the genus Claviceps used for the production of clavide alkaloids, lysergic acid and paspalic acid, simple lysergic acid derivatives and peptide alkaloids, which modifies cellular lipids.

To date, a number of fermentation processes for ergot alkaloids / H have been described. Kobel and JJ Sanglier, Ing; Biotechnology, Vol. 4, VCH Verlagsgesellschaft, Weinheim, 1986, p. ¹ Author's Certificate no. 199 986, 202 613 and 224 532. These processes utilize the properties of the production strains, given the possibility of expression of genetic information for the biosynthesis of ergot alkaloid under specific culture conditions, or these culture conditions favoring biosynthesis such prekurzoraci or enzyme induction. It follows that the increase in production by the above-mentioned optimization of the culture conditions is limited. Further increase of production is then dependent on breeding of new production strains, which is still based on nonspecific mutagenesis in ergot alkaloid producers resulting in uncontrolled mutations. Given the randomness of the positive mutation intervention and its low frequency, this method of further production increase is very demanding. in terms of time and material.

The aforementioned disadvantages of the methods of increasing production are overcome by a method of fermentative production of ergot alkaloids with modification of cell lipids, which is based on the fact that in the production and / or inoculum phase of the fermentation process the Clnviceps production strain is treated with mono- or dichlorophenoxyalkonic acids or their derivatives. formula I.>.

O - / li, / - COOR.

O

Y ^R 4 ° ^Η 3 where R 1 is /-CH.-/ or / -CH- / or / -C- / or /-CH.-CH.-/ 12,2,2,2 ch ₃ ch ₃ or / - / CH _2/3 ~ /

R ₂ is -H or -CgH ^ or -NH ₂

R ₃ is -Cl or -H provided that R 6 is Cl a

R 3 is -Cl or -H provided that R ₃ is Cl, either alone or in admixture at a concentration of 5 to 300 mg / L of nutrient medium.

The active ingredient is added to the nutrient medium at the beginning and / or during the production and / or inoculation phase of the fermentation process.

In the production phase of the fermentation process, the production organism is treated with substances which increase the proportion of mono and dinunsaturated fatty acids and / or reduce the total phospholipid content and / or reduce the proportion of phosphatidylethanolamine and / or reduce the membrane-functional quasiplanar demethylsterol / D content. Berg et. al., Mykosen 29, 221 (1986) and / or lowering the viscosity of the cytoplasmic membrane. Modification of the cell lipids is achieved by adding the active ingredient at a concentration of 5 to 300 mg / l of nutrient medium in the production phase of the fermentation process and the active ingredient is added to the

CS 273 373 B1 at the beginning and / or during the cultivation of the production phase. Modification of cellular lipids is achieved by adding one or more kinds of active ingredients in any ratio at a total concentration of all active ingredients of 5 to 300 mg / L.

Cytochemical studies / Voříšek et al., In: Ergot alkaloids and their derivatives, Academia, Praha, 1983, p.75 / localized the formation of ergot alkaloids by fungi of the genus Claviceps into membranes of endoplasmic reticulum, membranes of homologous vacuoles and lipoprotein filling vacuoles. For example, peptide alkaloids (ergopeptides) are deposited in lipid droplets (Neumann et. al., Biochem. Physiol. Pflanzen 174, 504 (1979)]. Most ergot alkaloid synthesis enzymes in Claviceps fungi, except tryptophan dimethylallyltransferase / Heinstein et. al., Biochem. Biophys. Res. Commun. 44, 1244, 1971] and DMAT-N-methyltransferases (Otsuka et. al., Planta Medica 40, 109 (1980)] is located on membranes predominantly in the endoplosmic reticulum. Their activity is mostly associated with the microsomal fraction of cells. The activity of these enzymes is influenced by conformational interactions and the quality of the membranes, respectively their lipid composition. According to theoretical assumptions, the manipulation of lipid metabolism, respectively by influencing the cell membrane composition in the desired direction, can regulate the ergot alkaloid biosynthesis in terms of increasing their production or altering the composition of the produced alkaloid mixture.

There is an increase in ergot alkaloid production, a change in the composition of the produced alkaloids, stabilization of their production and modification in the composition of cell lipids. For example, 4-chlorophenoxy acids, 2,4-chlorophenoxy acids and their derivatives, e.g. DL-2- (4-chlorophenoxy) propionic acid or

2,4-dichlorophenoxyacetate. These substances are known for their auxin effects on plants. For example, 2,4-dichlorophenoxyacetic acid is used as a herbicide and some of them have hypolipaemic effects on higher animals, for example 2- (4-chlorophenoxy) 2-methylpropionic acid ethyl ester (Klofibrate). The effects of these substances on microorganisms are relatively low - p-chlorophenoxypropionate (0.1-2 mg / L) increases glutamic acid yields by 30% in Lactobacillus arabinosus and Leuconostoc mesenteroides cultures and can substitute the biotin required by the cultures to grow (Kinoshita K. et al., Nippon Nogei Kagahu Koishi 36, 760-763 (1962). p-Chlorophenoxyacetate (0.2 mM) increases the production of chlortetracycline in a low-producing strain. Streptomyces aureofaciens from 38 to 150 mg / L / Vanek Z .: Folia Biol. 4100-106 (1958)]. The acids are added in the form of salt solutions, preferably sodium, which are sterilized by filtration. In thermal sterilization, the effect of the substances is somewhat reduced, probably by hydrolysis of the chlorine atoms on the aromatic system. This disadvantage can be overcome by increasing the concentration of the substance or by using a dichloro derivative, preferably 2- (2,4-dichlorophenoxy) propionate. Sterilization can also be accomplished by dissolving chlorophenoxy acids in 2 N NaOH, which is autosterile. The solution is then neutralized under sterile conditions with conc. HCl to about pH 8 and optionally diluted with sterile water. Thus, negative heat sterilization and demanding filter sterilization can be avoided. When cultivated in and / or on media containing substances modifying the composition of cellular lipids, the proportion of mono- and di-unsaturated acids, especially oleic and linoleic acids, increases, and palmitic acid content decreases. Subsequently, the fatty acid unsaturation index increases. The total phospholipid content (as determined by TLC-FID) is reduced to about 50% of the control, which is related to an overall decrease in the lipid content of the mycelium. The proportion of phosphatidylethanolatine decreases sharply and the phosphatidylcholine index of phosphatidylethanolamine significantly increases.

´ The change in the lipid composition of cell membranes during ergot alkaloid fermentation will also have an impact on the production of ergot alkaloids, both on the total content of the sum of alkaloids by increasing it and on the proportion of individual alkaloids (spectrum) in the total sum. Optimal concentrations of lipid modifying substances that maximize the production of the sum of alkaloids are strain specific and should be determined for the particular production strain used and the particular culture conditions.

CS 273 373 Bl

The main advantage of the process according to the invention is the effective possibility to substantially increase (up to 100%) the production of ergot alkaloids in the fermentation technologies used, where the possibilities of further increasing the production by the known methods are exhausted. A further advantage is the possibility of using existing bred production strains while eliminating the need for difficult further breeding without the certainty of achieving similarly increased production.

Another advantage is the possibility to achieve a substantial increase in ergot alkaloid production without any modification of the technical production equipment. A further advantage of the process according to the invention is the possibility of substantially shortening the cultivation process to an optimum time in terms of economy of ergot alkaloid production. Another advantage resulting from the increased amount of ergot alkaloids in the final fermentation product is the intensification of the extraction and isolation processes, which results in a further reduction in the cost of producing the final product. Another advantage is the possibility of utilizing changes in the alkaloid spectrum to obtain the necessary amount of those alkaloids that are produced by the existing production strains only in minor quantities, or utilizing these changes to optimize the spectrum of alkaloids in terms of the final product (s). Another advantage is the stabilizing effect of the addition of cell lipid modifying agents on non-standard fermentation yields (e.g., low viability or suboptimal amounts of inoculum and the like).

The method of fermentative production of ergot alkaloids with modification of cell lipids is exemplified by the following examples without limiting the subject matter of the invention in any way.

Example 1

The production strain Claviceps purpurea 59 (MSC No. 252 603) is inoculated into inoculation medium containing 100 g of sucrose, 10 g of asparagine in 1 liter,

0.1 g L-cysteine, 0.1 g of yeast extract, 0.25 g KHgPO ^, ^ 0.03 g MgS0 7H ₂ 0, 0.02 g ZnS0 ^ 7H ₂ 0, 1 g of Ca (N0. (j) ₂ .4H ₂ O, dissolved in water, pH 5 15, 60 ml in 300 ml Erlenmeyer flasks. Inoculate each flask with 5x10? conidia of said strain. The flasks were incubated for 10 days. at 26 ° C on a rotary shaker (4 rpm, 55 mm excetr). Sodium 2- (2,4-dichlorophenoxy) propionate solution is prepared by dissolving the free acid in an equivalent amount of 2N NaOH and adjusting the pH of the solution to 8.0 with HCl. This solution is sterilized by filtration through a sterile sinter filter S5 and added in an amount of 20 mg of active ingredient per liter to the production medium containing 100 g of sucrose in 1 liter, 16.8 g of citric acid, 10 g of (NH4gSO4). 25 g KHgPO ^, 0.25 g of magnesium sulfate. 7H ₂ O, 0.15 g ZnS0 ₄ 7H ₂ 0, 0.02 g FeS0 ₄ 7H ₂ 0, 0.12 g KC1, 1.2 g CaCl ₂ pH 5.5 The medium thus prepared, dispensed 60 ml in 300 ml Erlenmeyer flasks, is seeded with 8% by volume submersible inoculum and cultured under the same conditions as the inoculum for 21 days. Sodium 2,4-dichlorophenoxy) propionate proceeds in parallel cultivation as a control, changing the composition of cellular lipids during cultivation (as determined by V. Kren et al., FEMS Microbiol. Lett. 22, 359, 1985) and there is a significant increase in the production of clavin alkaloids. and changes in their spectrum compared to control. The results are shown in Table 1.

Example 2

The Claviceps purpurea 59 production strain was cultured as in Example 1 except that DL 2- (4-chlorophenoxy) propionate sodium was added to the medium at a concentration of 100 mg / L. The resulting changes in cell lipid composition and alkaloid production are shown in Tab. 2.

Example 3

The production strain Claviceps purpurea EK 10 was cultured under the conditions of Example 1, except that 2- (2,4-dichlorophenoxy) propionate sodium was added to the production medium to a final concentration of 25 mg / L. Compared to the control, the amount of alkaloids increased from 2,240 to 3,218 mg / l after 16 days of cultivation and after 20 days of cultivation

CS 2373 373 B1 from 3,218 to 3900 mg / L and to change the ratio of agroclavin to elymoclavin from 19:81 to 30: 68.

1 ··

Example 4

The production strain Claviceps purpurea EK 10 was cultured under the conditions of Example 1 except that 2- (2,4-dlchlorophenoxy) propionate sodium was added to the production medium to a final concentration of 100 mg / L. Compared to the control, after 16 days of cultivation, the sum of alkaloids increased from 2,240 to 2,902 mg / l, after 20 days cultivation increased from 3,218 to 3,401 mg / l without changing the percentage of elymoclavin.

Example 5

An inoculum of a production strain of Claviceps fusiformis W 1 is prepared according to the procedure of Example 1. In the production phase, the production medium prepared according to Example 1, without addition of active ingredient, inoculated with 8% by volume of inoculum, is used as a control. Another variant is the production medium according to Example 1 with the addition of sodium 2- (2,4-dichlorophenoxy) propionate in amounts of 0,20,50,100,150,200 and 300 mg / l, inoculated with 3.2% by volume of inoculum, to simulate a non-standard course of cultivation with attenuated inoculum. The resulting alkaloid production after 19 days of cultivation is shown in Table 3 ·

Example 6

The C. purpurea 59 production strain is cultured under conditions identical to Example 1.

In the production phase, the production medium is used as a control without the addition of active substances that modify cell lipids. Another variant is the production medium with the addition of sodium salt of 2,4-dichlorophenoxyacetic acid at concentrations of 10, 25 and 50 mg / l and with the addition of sodium salt of 2- (2,4-dichlorophenoxy) propionic acid at concentrations of 10, 25 and 50 mg / l. . The resulting alkaloid production after 5, 11 and 20 days of culture is shown in Table 4.

Example 7

Production strain Claviceps paspali FA MS. No. 224,532 is cultured on the surface of a solidified nutrient medium containing 10 g peptone, 40 g glucose, g malt extract, 20 g agar dissolved in water for 1 day at 24 ° C per liter. The grown myoelium was wiped with <90 ml of sterile medium of the same composition except for agar, in a 500 ml boiling flask, and cultured for 4 days at 24 ° C on a rotary shaker (3.7 rpm, eccentric 70 mm). Under sterile conditions, the flask mycelium is drained by suction on a Sohleicher Schuell No. 5393 porcelain filter and washed with 100 ml of distilled water. The mycelium is suspended in 80 ml of distilled water and homogenized with an Ultra-Turrax for 5 s. 6% by volume of this suspension is seeded with a production medium containing 30 g of raanitol, 30 g of succinic acid, 30 g of propylene glycol in 1 liter. g K ₂ HP0 _4, 0.5 g MgS0 _{4 <7H 2} 0, 0.03 g Cb / N0 _3/2 .4H ₂ 0, 0.01 g MnS0 ₄ .4H ₂ 0, and 0.1 g of NnNOp pH 5.2. Following the procedure of Example 1, a solution of sodium-chloro-phonoxyacetic acid sodium salt was prepared and added to the production medium to a final concentration of 45 mg / L. The production medium is filled in 80 ml in 500 ml beakers. The production phase is cultured at 23 ° C on a rotary shaker (4 rpm, eccentric 55 min) for 16 days. Parallel culture was performed as a control in the production medium without the addition of sodium 2,4-dichlorophenoxy acetone. Compared to control an increase in the content of the isomers hydroxycthylamidu lysergic acid from 1008 to 1300 rag.l ^- '.

Example 8

Production strain Claviceps purpurea CCM F-725 / MS. author's certificate No 202 613,

Č3? No. 22 2391 / is cultivated on the surface of a solidified nutrient medium o

The composition of Example 1 was transferred to 80 ml inoculum medium in a 300 ml Erlenmeyer flask at 2.10. Inoculation medium contains in 1 liter 50 g sucrose 20 g corn extract and 1 g of KH ₂ P0 _4, dissolved in water, pH 6.2. The inoculated flasks were incubated for 48 hours at 24 ° C on a rotary shaker (4 rpm, eccentric 55 mm). An amount of 0.5% by volume of this submersible inoculum is inoculated with the production seed

CS 273 373 B1 nutrient medium containing in 1 1300 g sucrose, 4 g yeast extract, 10 g target.

citronnnu ammonium ^ 1 g MgS0 7H ₂ 0, 1.44 g Ca / noy .4HgO _2, 0.4 g of KHgPO ^, 0.2 g of L-isoleucine, 0.045 g of 2- / 2,4-dichlorophenoxy / propionate sodium and 0.06 g of sodium 2,4-dichlorophenoxyacetate dissolved in water, pK value of 6.2. The production medium was filled 40 ml into 200 ml Blacke flasks. Cultivation was carried out stationary for 2 days at 24 ° C. In the production medium without the addition of lipid modifying agents, parallel cultivation was performed as a control. The grown mycelium in the control contains, after cultivation, a sum of peptidic alkaloids of 0.86% by weight, an ergocryptine-to-cellulose ratio of 2: 1, a ratio of () / 2) -ergocryptine = 1: 1. Addition of the above amounts of sodium 2- (2,4-dichlorofunoxy) propionate and sodium 2,4-dichlorophonoxyocin enriches the sum of popic alkaloids 1.08 wt.%, The ergocryptyl glycerocornin * 2.7; 1, C ratio: </ 3 -ergocryptine = 1: 3.5 ·

Tables 1 and 2

Effect of DL 2- (4-chlorophenoxy) propionate on the composition of cell fatty acids and ergot alkaloid production by Claviceps purpurea strain 59 θ H

Variant I Fatty acids Unsaturation index Anisotropy Biomass (% total fatty acid) ointment. Membrane acid (g / l)

16: 1 18: 2 18: 1 18: 0

Checking 26.3 35.8 28.0 9.7 1,006 0.200 22,7

Addition mg / 1 active substance 22,9 44.8 20.0 7.1 1.105 0.182 15.3

Variant 2 alkaloids ⁰ total AEC CA (mg / l) -.

Checks 3 728 18 24 24 33

Addition mg / 1 .mu.C. Substance 6 500 .10 16 55 21 ^and determined as the methyl esters from the total proportion of hydrolyzable cellular lipid-measured using the fluorescent probe 1,3,6-trimethyl difenylhexatrien ^C A - agroclavine, E - elymoclavine C - chanoklavín- I, CA-chanoclavin-1-aldehyde

Table 3

Effect of sodium 2- (4-chlorophenoxy) propionate on alkaloid production in non-standard (attenuated) cultivation of C. fusioformis W1 after 19 days Variant of alkaloid production ´ (mg / 1)

Control with 8% inoculum 2 358

Control p

3-2% inoculum (attenuated) 1.685

CS 273 373 B1

Addition of active substance (rag / 1) to attenuated culture 20 May 2 no 50 2 Π5 100 ALIGN! 2 673 1 50 2 411 200 2 347 300 2 037

Table 4

Effect of sodium 2,4-dichlorophenoxyacetate (DCA) and sodium 2- (2,4-dichlorophenoxy) propionate (DCP) on ergot alkaloid production by strain C, purpurea 59.

Variant of ergot alkaloid production (mg / i)

Day 5 11th day Day 20 Control 728 2 654 7 030 DCA addition 10 mg / l 841 4 246 10 246 25 mg / l 813 4 506 9 797 50 mg / l 852 4 499 11 176 DCP addition 10 mg / l 865 4 010 9 601 25 mg / l not specified 3 946 9 715 50 mg / l 924 3 901 9 397 SUBJECT OF THE INVENTION

Claims (2)

Process for the production of ergot alkaloids with modification of cell lipids, characterized in that in the production and / or inoculation phase of the fermentation process, the nu production strain of the fungus Claviceps is treated with mono- and / or dichlorophenoxyalkanoic acids or their derivatives of the general formula I. wherein R, is / CH ₀ - / / or -CH- / or / C- / or / CH "CH _O - /, ^{1 2} H ^{22 CH} 3?. ^H 3 or / - / CHg / ^ - / R 1 is -H or -C 8 H 4 or -NH 11; CS 273 373 B1 R 1 is -Cl or -H provided that R 1 is Cl and R 1 is -Cl or -H, provided that R 1 is Cl, either alone or in admixture at a concentration of 5 to 300 mg / L of nutrient medium. Method according to claim 1, characterized in that the active substance and / or active substances are added to the nutrient medium at the beginning and / or during the production and / or inoculation phase of the fermentation process.