ES2228281A1 - Gibberella Fujikuroi wild strains fermentation comprises production of Gibberellins GA1 and GA3 by inoculation and solvent extraction - Google Patents

Abstract

Method for the production of gibberellins GA3, by means of fermentations with the wild strain IMI58289 of the fungus Gibberella Fujikuroi. The object of this invention is a method for the production of gibberellins with the wild strain of Gibberella fujikuroi IMI58289, without the need for prior genetic manipulation of the fungus. First, the IMI58289 strain is inoculated for 6 days in a medium rich in nitrogen, conditions in which gibberellin production is inhibited. After six days, the cultures are filtered and the mycelium is washed with distilled water and resuspended in a glucose solution with microelements. Subsequent incubation under these conditions results in a gibberellin mixture with an unusually high proportion of GA3.

Description

Method of production of gibberellins GA3 by fermentation with the wild strain IMI58289 of the fungus Gibberella fujikuroi .

Object

The object of this invention is a method of producing gibberellins GA3, using the wild strain IMI58289 of Gibberella fujikuroi , without the need for prior genetic manipulation of the fungus.

Gibberellins (GAs) are natural phytohormones that regulate the growth and flowering of plants among other physiological activities. These phytohormones are at the level of traces in plants but their exogenous application produces spectacular results so they have a great interest in intensive agriculture and the beer industry. More than 100 gibberellins have been described, among which GA3 (1), also known as gibberellic acid, has traditionally been considered the most active gibberellin in plants (MacMillan, J, Natural Product Report , 1997 , 221). However, recent studies have shown that GA 1 (2) has an important biological activity, even higher than GA 3 in plants such as rice (Kobayashi M, MacMillan J, Phinney B, Gaskin P, Spray CR, Hedden P , Phytochemistry , 2000 , 55 , 317), tomato (Grünzwig JM, Rabinowitch HD, Katan J, Wodner M, Ben-Tal Y, Phytochemistry 1997 , 46 , 811), cherry tree (Huanpu M, Blake PS, Browning G; Taylor JM, Phytochemistry, 2001 , 56 , 67) or the pea (Yaxley JR, Ross JJ, Sherriff LJ, Reid JB, Plant Physiol, 2001 , 125 , 627).

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Background of the invention

The filamentous fungus Gibberella fujikuroi is an organism capable of producing gibberellins in sufficient quantities for its industrial exploitation.

The production of gibberellins by different wild or mutant strains of Gibberella fujikuroi in various culture media has been described several times. Specifically, the production of GA_ {3} (1) has been covered under different patents (GB844341, GB850018, GB886551, US2918413) as well as the production of the active mixture of gibberellins GA_ {4} + GA_ {(US6287800, CN1222575 ). However, the production of GA 3 (2) or mixtures of GA 1 + GA 3 at levels that may be useful in the agrochemical industry has not been previously described, and precisely the invention presented in this Patent application consists of a method of producing gibberellins GA3.

Description of the invention

This patent application describes a method for the production of gibberellins with the wild strain of Gibberella fujikuroi IMI58289 without the need for prior genetic manipulation of the fungus.

First, the IMI58289 line is incubated for 6 days amid nitrogen rich conditions in that the production of gibberellins is inhibited. At six days, the cultures are filtered and the mycelium is washed with distilled water and resuspended in a glucose solution with microelements. The subsequent incubation under these conditions results in a mixture of gibberellins approximately 0.1 g / l of cultivated medium, with a unusually high proportion of GA3.

The extracts of each fermentation were analyzed by 1 H NMR spectroscopy and the technique Combined Gas Chromatography-Spectrometry of Masses (CG-EM) [Column: HP-1 (Crosslinked Methyl Siloxane) 25 m x 0.2 mm x 0.33 µm; gas carrier: helium with a flow of 0.6 ml / min; temperature of injector: 260 ° C; detector temperature: 280 ° C; temperature Initial: 120 ° C, increased to 5 ° C / min to 220 ° C, increased subsequently at 3 ° C / min up to 280 ° C, where it is maintained for 10 minutes].

Description of a preferred embodiment

The fermentation was carried out in two stages. In a first stage, the nitrogen-rich culture medium was distributed in Erlenmeyer flasks, which were inoculated with a spore suspension of the wild strain IMI58289 of Gibberella fujikuroi . The flasks, covered with fatty cotton to facilitate aeration, were incubated at 30 ° C with an orbital shaking of approximately 200 rpm. After 6 days the fermentation was stopped and the mycelium was removed from the culture medium by filtration. It was then resuspended in a glucose solution with microelements and incubated for 6 days under the conditions mentioned above. Finally, the culture broth was filtered, acidified to pH 1.5-3 with HCl, and extracted with ethyl acetate. Evaporation of the solvent under vacuum and subsequent degreasing of the extract generated a residue consisting mostly of gibberellins. The majority gibberellin in this mixture was GA3 (in a proportion greater than 50%).

Claims (2)

1. Method of production of GA 3 gibberellins by fermentation with wild strains of the Gibberella Fujikuroi fungus characterized in that the wild strain of Gibbeberella fujikuroi used is IMI58289 and because it comprises the following stages: a) Inoculation by spore suspension in a culture medium rich in nitrogen. b) Incubation and fermentation of the culture with orbital agitation at a temperature between 22 ° C-32 ° C and for a period of 6 days. c) Separation of the mycelium by filtration and suspension thereof in a glucose solution with microelements d) Incubation of the new broth in the same conditions of temperature and orbital agitation and by a 6 day period. e) Filtration, acidification and extraction with organic solvents of the culture broth. f) Evaporation of the solvent from the broth of culture. 2. Residue obtained after carrying out the gibberellin production method according to claim 1, characterized in that it contains a mixture of gibberellins in an amount around 0.1 g / l of cultured medium and with a GA 3 richness greater than 50 %.