SU1406163A1 - Strain of rhodobacter sphaeroides bacteria - producer of coenzyme of q sub ten - Google Patents

Abstract

The invention relates to the microbiological industry, in particular to the strains for the biosynthesis of ubiquinone. The aim of the invention is to obtain a new strain-producer of ubiquinone, which has biosynthetic activity. Strain 2R No. 122 is obtained after treatment of Rhodobacter sphaeroidas 2R cells with nitrosoguanidine, followed by selection of clones with a reduced growth rate under nitrogen fixation conditions. The nutate strain provides the yield of the target product in the amount of 6.9 mg per 1 g of dry cells, which is twice the productivity of the original strain. i (L

Description

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The invention relates to the microbiological industry, in particular to the strains used for the biosynthesis of ubiquinone Q, 0

The purpose of the invention is to obtain a new strain with enhanced biosynthetic activity.

To obtain such a strain, the starting wild strain of Rhodobacter sphaero-des 2R is treated with nitrosoguanidine, followed by selection of clones with a reduced growth rate under nitrogen-fixing conditions.

The strain Rhodobacter sphaeroides 2R No. 122 is deposited in the All-Union Collection of Microorganisms under the number 1593D and is characterized by the following cultural-morphological and physical-biochemical characteristics.

After 5 days of cultivation, agarized (1.2% Difco-arap) medium-free medium forms small, light pink rose colonies of a correct rounded shape with a diameter of 0.7-1.0 mm, and dry red colony on the medium with bonding. diameter 2-3 mm.

The cells are short sticks, no different from wild-type cells.

It belongs to the group of gram-negative bacteria.

Optional anaerobic.

Capable of nitrogen fixation.

Has active nitrogenase when incubated on medium containing (NH4) 2S04.

Relation to carbon sources: use Na malate, Na lactate, glucose.

Relation to nitrogen sources: well assimilates (NH4) amino acids: glutamine, alanine, proline, glutamate.

Optimal growth conditions: temperature 30-32 ° С, illumination 1500-2000 lx, pH .7.0.

In Ormedor medium with growth in the light after 72 hours of incubation, the cell concentration is 2-4 X 10 cells per ml.

For the cultivation of the Rhodobacte sphaeroides 2RN, 122 strain, Ormerod medium of the following composition is used, g / l

MgS04-7H O 0,200

CaCli 2HiO 0.075

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FeS04 7HzO0,011

EDTA0.020

Trace elements1 md

, 900

KHjPO 0.600

(NH4) jS041,250

d51-malate Na4-6

Vitamins 1 ml

The composition of trace elements, g / l:

H BOa2,860

ZnS04 7HiO .0,240

C (Koz) Mr. - ZNgO0,040.

MnS04 4H, 0 2,100

, 200,750

The composition of vitamins, µg / l:

Biotin100

Nicotinic acid 100

Thiamine100

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C r i me R 1. To obtain a strain, a three-day culture of the bacteria Rhodobacter sphaeroides 2R (2-10 cells / ml) was removed from the culture fluid and resuspended in 0.01 M K-phosphate buffer, pH 6.8. Then, nitrosoguanidine is added to the suspension to a final concentration of 100 µg / ml and held at room temperature in the dark for 60 minutes. The mutagen-washed cells are seeded in liquid medium with 0.125% ammonium sulfate and incubated in the light for 3 days. To deplete the intracellular pool of nitrogen sources, the cells are washed and kept for 1 day in a nitrogen-free medium. In order to enrich the population with mutants, the cell suspension is incubated for 20 hours with penicillin (2 mg / ml), then sown on an agar medium with 0.01% (W) 804I grown under nitrogen fixation conditions - in an N atmosphere at illumination of 1500-2000 lux. . After 8–10 days, small colonies with a diameter of 0.1–0.5 mm were isolated and these clones were tested for their ability to grow in a medium without sources of bound nitrogen and on media with 0.125% (N114) -2 SO4 from 10 mM glutamate sodium,, 10 mM glutamine, alanine, proline.

 EXAMPLE 2 To obtain a biomass containing ubiquinone Q, a 4-day culture grown on agar medium is used as a seed. Cells are grown in liquid medium at 30-32 ° C and illuminated at 1500 lux to the stationary growth stage.

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Claims (1)

Compared to the initial yield, the formula of the left product provided by strain 2R No. 122 is 6.9 mg per strain of Rhodobacter aphae1 g dry cells against 2.4 mg / g of so-called VKM B-1593D producing ubiquishly correspondingly Q.