RU2596928C1 - STRAIN Paraphoma sp. PRODUCER OF FEOSFERID A - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, particularly, to production of biologically active substances and can be used for producing of feosferid A. Stain of mushroom Paraphoma sp., deposited in FGBNU VIZR at registration number VIZR 1.46, is proposed as a producer of a feosferid A.

EFFECT: invention allows to raise output of feosferid A.

1 cl, 2 ex

Description

The invention relates to biotechnology, in particular to the production of biologically active substances, and can be used to produce pheosperide A for various purposes, both for agricultural production and for medical purposes.

Many micromycetes are capable of forming compounds with antitumor activity (Tsuruo et al., 1985 [1]; Strobel, 2003 [2]; Rai et al., 2009 [3]; Kharwar et al., 2011 [4]; Evidente et al. , 2013 [5]; Kornienko et al., 2015 [6]). One of them is Feosferide A, which has antitumor activity, selectively inhibiting the activity of STAT3 protein at a concentration of IC ₅₀ = 6.1 × 10 ^-4 M (Bromberg et al., 1999 [7]; Maloney et al., 2006 [8] ) The properties and mechanism of action of this substance are currently being studied.

An unidentified endophytic fungus strain FA 39 is known as the producer of pheospheride A, having 97% homology with the species Phaeosphaeria avenaria (Maloney et al., 2006 [8]), Phoma sp. No. 19 (Poluektova, Berestetskiy, 2014 [9]), Paraphaeosphaeria neglecta FT462 (Li et al., 2015 [10]). For the prototype adopted strain strain FA 39.

When cultivating FA 39 on a medium containing corn, rice cereal, wheat flakes Fiber One (General Mills Sales, Inc.) and 0.1% yeast extract, the yield of pheosperide A was 1.01 g / kg (Maloney et al., 2006 [8]) . Flasks with the medium were inoculated with agar blocks of a 3-day culture of the fungus FA 39. The fungus was cultured for seven days at 25 ° C. At the end of cultivation, the resulting material (32.4 g) was extracted with methanol and re-extracted (yield 15.61 g / kg of substrate). Fractionation of the extract by chromatographic methods on silica gel made it possible to obtain a fraction containing pheosperide A as the main component. Further purification of this fraction by high performance liquid chromatography yielded 32.8 mg of pure pheospheride A with a yield of 1.01 g / kg of substrate (Maloney et al., 2006 [8]).

The disadvantage of this producer strain of the pheosperide A is the insufficiently high level of its formation.

The objective of the invention is to obtain a new producer strain that is superior to strain FA 39 in productivity of the formation of pheospheride A. The problem was solved by selecting the most productive strains.

The starting material for the selection of highly productive strains was the natural isolate Phoma sp. No. 19, isolated from the spotted leaves of Cirsium arvense, collected in Vostochnoye, Khabarovsk District, Khabarovsk Territory, 08.24.2006. Based on the study of morphological and cultural properties, the fungus was previously identified as Phoma sanguinolenta (Tode) Desm. according to the determinant of Boerema (Boerema, 2004 [11]). The extract from the mycelium of this fungus demonstrated phytotoxic activity (Berestetskii, Kurlenya, 2014 [12]).

Strain cf Paraphoma sp. 1.46 (VIZR) was selected as the most productive of the various morphological variants of Phoma sp. No. 19. For selection on the basis of the formation of pheosperid A, colonies of various morphological variants of Phoma sp. No. 19 at the KGA. The content of pheospheride A in fungal microextracts obtained by the method (Smedsgaard J., 1997 [13]) was determined by HPLC. Strain 1.46 was selected as the most productive and deposited in the State collection of microorganisms FSBIU VIZR.

Identification of the species of fungus was carried out on the basis of a combination of morphological and cultural characters. Additional identification was carried out by molecular methods. According to the results of multilocus analysis of the ITS region and the LR5 and TEF genes, the strain was named cf Paraphoma sp. 1.46 (VIZR). The nucleotide sequences of internal transcribed spacers (ITS) of ribosomal genes of 575 pairs of nucleotides, 28S rRNA and TEF 1-alpha genes of 854 and 459 genes, respectively, are deposited in the GenBank database (Banklt 1840305).

The fungus has moderate growth on standard agarized nutrient media (potato glucose agar (KHA), maltose agar (MA), oat agar (OA) and апapek agar with vitamins (CHA) in the dark at 24 ° C. The fungus forms slightly on the KGA colony raised, olive-gray, velvety with smooth gray-blue edges. The average colony diameter is 57.75 mm. On OA medium, the colonies are open, cobweb-velvety light gray with smooth edges. Red pigment was observed along the edge. The average colony diameter is 56.75 mm. Colonies on MA olive gray col eta, slightly raised, with fluffy mycelium. The edges of the colony are smooth, dark olive in color. The average diameter of the colony is 43.5 mm. On the agarized Chapek medium, slightly raised velvety colonies of gray color with even edges of light gray color were observed. The reverse is olive gray with dark olive edges. The average diameter of the colony is 54.5 mm. No sporulation of the fungus was detected on any nutrient medium. When cultivated under artificial lighting conditions, the fungus releases red pigment into the agar medium.

The strain is stored in test tubes on mowed potato-glucose agar in a domestic refrigerator at a temperature of + 5-8 ° C.

Example 1. Obtaining the extract from the culture cf Paraphoma sp. 1.46 pearl barley

Pearl barley was used as a substrate for solid-phase cultivation of the fungus. 150 g cereals and 100 ml water were added to 25 1-liter conical flasks. The grain substrate was sterilized by autoclaving for 30 minutes at 121 ° C. The relative humidity of the substrate is 40 ± 5%. The substrate was inoculated with two blocks (5 mm in diameter) cut from the edge of the seed culture cf Paraphoma sp. 1.46. The fungus was cultivated for 30 days in the dark at a temperature of 24 ° C. The flasks were shaken every 2 days to prevent adhesion of the substrate. The substrate colonized with the fungus was dried with a stream of sterile air. The combined dry material (3.75 kg) was extracted with 50% aqueous acetone (10 L). After evaporation of the acetone, the aqueous extract was extracted with hexane (5 L), discarding the organic layer. Then, the aqueous phase was extracted with ethyl acetate (2.5 L × 3). The combined extract was dehydrated by filtration through anhydrous sodium sulfate and evaporated on a rotary evaporator at a temperature not exceeding 40 ° C. The yield of extract was 2.84 g / kg of substrate.

Example 2. Obtaining pheosperide A from the mycelium extract cf Paraphoma sp. 1.46

Fractionation of the extract was carried out on Chromabond C-18 concentrating cartridges (Macherey-Nagel, Germany) with a sorbent mass of 10,000 mg. The dry residue dissolved in acetonitrile was added in batches of 1 g to a cartridge preconditioned with 20 ml of acetonitrile and 30 ml of 0.1% formic acid. Fractionation of the extract was carried out with a solvent system of acetonitrile - 0.1% formic acid in a ratio of 0: 100; 25:75; 50:50; 100: 0, with a volume of 70 ml. The fraction containing pheosperide A was obtained in the acetonitrile - 0.1% formic acid system in a ratio of 50:50. Further fraction separation was carried out using semi-preparative HPLC on a BUCHI chromatograph on a PuriFlash-25 SiHC cartridge (Interchim, France) with a sorbent mass of 40 g using a prepatron with an extract preliminarily sorbed on silica gel. The extract was separated in a hexane-ethyl acetate system (in a gradient from 0 to 80% ethyl acetate) with a flow rate of 50 ml / min; phytotoxin was detected at a wavelength of 260 nm. As a result of the separation, the pheosperide A was obtained with a purity of more than 96%. The yield of pheosperide A was 1.9 g / kg of substrate.

The identification of phytotoxin was carried out by spectrometric methods. The ultraviolet spectra of phytotoxin were recorded in acetonitrile on a Beckman Coulter DU 800 instrument; mass spectra of the substance were recorded by HPLC - MS on a Thermo Scientific TSQ Quantum Access instrument (triple quadrupole mass spectrometer) equipped with a diode array and mass detector. Ionization of the sample was carried out by electrospray. The ¹ H and ¹³ C-NMR spectra of phytotoxin were recorded on a Bruker AVANCE III 400 Ultrashield Plus spectrometer at a frequency of 400.1 and 100.6 MHz (Bruker, Germany), respectively. The sample was dissolved in deuterated dimethyl sulfoxide. The following spectra were recorded: proton spectrum ¹ H; carbon spectrum, ¹³ C; carbon spectrum ¹³ With DEPT; two-dimensional proton-proton spectrum ¹ H - ¹ H COSY; two-dimensional proton-proton spectrum ¹ H - ¹ H ROESY; two-dimensional carbon-proton spectrum ¹ H- ¹³ C HMQC; two-dimensional carbon-proton spectrum ¹ Н- ¹³ С НМВС. Complete information on the molecular structure of pheospheride A was obtained from x-ray diffraction analysis. It was found that the pheosperide A in the crystal exists as an enantiomeric pair (Abzianidze et al., 2015 [14]).

Thus, the strain of the fungus cf Paraphoma sp. 1.46 is interesting as a highly productive producer of pheospheride A. The productivity of the strain allows you to get pheospheride A in the amount of 1.9 g / kg, which is 47% higher than the productivity of strain FA 39.

Literature

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G.J., Irinyi L. Phoma Saccardo: Distribution, secondary metabolite production and biotechnological applications. Critical Reviews in Microbiology. 2009. V. 35 (3). P. 182-196.3. Rai M., Deshmukh P., Gade A., Ingle A.,

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7. Bromberg J.F., Wrzeszczynska M.H., Devgan G., Zhao Y., Pestell R.G., Albanese C., Darnell J.E. STAT3 as an Oncogene. Cell. 1999. V. 98. P. 295-303.

8. Maloney K.N., Hao W., Xu J., Gibbons J., Hucul J., Roll D., Brady S.F., Schroeder F.C, Clardy J. Phaeosphaeride A, an inhibitor of STAT3-dependent signaling isolated from an endophytic fungus. Org Lett. 2006. P. 4067-4070.

9. Poluektova E.V., Berestetskiy A.O. Isolation and characterization of phytotoxins produced by phoma sp. 19. book of proceedings 7th world congress on allelopathy. Complex interactions in a changing climate. Congress held in Vigo, Spain. July, 28 - August, 1, 2014. P. 114.

10. Li CS, Ding Y., Yang BJ, Miklossy G., Yin HQ, Walker LA, Turkson J., Cao S. A new metabolite with a unique 4-pyranone-γ-lactam-1,4-thiazine moiety from a hawaiian-plant associated fungus. Org Lett. 2015.

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12. Berestetsky A.O., Kurlenya A.S. Antimicrobial properties of phytopathogenic micromycetes. Mycology and Phytopathology. 2014.48 (2). S. 123-134.

13. Smedsgaard J. Micro-scale extraction procedure for standardized screening of fungal metabolite production in cultures. J Chromatogr A. 1997. V. 760 (2). P. 264-270.

14. Abzianidze V.V., Poluektova E.V., Bolshakova K.P., Panikorovskii T.L., Bogachenkov A.S. and Berestetskiy A.O. Crystal structure of natural phaeosphaeride A. Acta Cryst. 2015. V. E71. P. 625-626.

Claims (1)

The strain of the fungus Paraphoma sp., Deposited in FGBNU VIZR under registration number VIZR 1.46 - producer of theospherid A.