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6-geranyl geranyl diphosphate (GGPP) synthase, 8-sesquiterpene synthase and pleuromutilin high-yield strains, and application of strains

A 1.6-GGPP and pleuromutilin technology, applied in the field of microbial fermentation, can solve the problems of unsuitability for industrial production, less fermentation conditions for pleuromutilin, and increased costs

Active Publication Date: 2012-10-03
山东胜利生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]However, from the discovery of pleuromutilin to now, there are relatively few studies on the fermentation conditions of pleuromutilin, and the yield of the product reported in the literature is only about 2.2mg / ml , low output, not suitable for industrial production
From the perspective of industrialization, although the production capacity of pleuromutilin in my country is large, the titer level of the strain is low, which increases the cost of production

Method used

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  • 6-geranyl geranyl diphosphate (GGPP) synthase, 8-sesquiterpene synthase and pleuromutilin high-yield strains, and application of strains
  • 6-geranyl geranyl diphosphate (GGPP) synthase, 8-sesquiterpene synthase and pleuromutilin high-yield strains, and application of strains
  • 6-geranyl geranyl diphosphate (GGPP) synthase, 8-sesquiterpene synthase and pleuromutilin high-yield strains, and application of strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] The strain Clitopilus prunulus TM110519 CGMCC NO. 6094 is used to produce pleuromutilin, and the process is as follows:

[0115] 1. Medium used

[0116] Incline medium (g / L): Potato 200, glucose 20, agar 20, water balance, pH 1.0.

[0117] Seed medium (g / L): glucose 50, yeast extract 15, MgSO 4 0.5, Ca(NO 3 ) 2 0.05, KH 2 PO 4 1. Water balance, pH 1.0.

[0118] Fermentation Basic Medium (g / L): Glucose 50, Corn Steep Steep Powder 20, MgSO 4 0.4, CaCO 3 1. Soybean oil 4. The balance of water, pH 1.0.

[0119] 2. Method

[0120] The strains were inoculated on the slant medium, and cultured at 20°C for 10 days to activate the strains; the activated strains were inoculated into the seed medium, and the primary seed culture was carried out at 20°C and 500 r / min. Shake culture for 10 days, then carry out secondary seed culture with 15% inoculum amount in the same seed culture medium and under the same conditions as secondary seed, and cultivate for 5 days to obta...

Embodiment 2

[0122] Clitopilus prunulus TM110518 CGMCC No. 6093 was used as the strain to produce pleuromutilin by fermentation using the medium and method of Example 1. The fermentation liquid is detected by HPLC method, and the detection result shows that the biological potency of pleuromutilin in the fermentation liquid is 11 mg / ml.

Embodiment 3

[0124] The strain Clitopilus prunulus TM110519 CGMCC NO. 6094 is used to produce pleuromutilin, and the process is as follows:

[0125] 1. Medium used

[0126] Incline medium (g / L): Potato 180, glucose 25, agar 15, water balance, pH 7.0.

[0127] Seed medium (g / L): glucose 45, yeast extract 20, MgSO 4 0.3, Ca(NO 3 ) 2 0.1, KH 2 PO 4 0.5, water balance, pH 5.0.

[0128] Fermentation Basic Medium (g / L): Glucose 45, Corn Steep Steep Powder 25, MgSO 4 0.2, CaCO 3 2. Soybean oil 3. The balance of water, pH 1.0.

[0129] 2. Method

[0130] The strains were inoculated on the slant medium, cultured at 35°C for 5 days, and the strains were activated; the activated strains were inoculated into the seed medium, and the primary seed culture was carried out at 35°C and 100 r / min. Shake culture for 5 days, then carry out secondary seed culture with 5% inoculum amount in the same seed medium and under the same conditions as secondary seed liquid, and cultivate for 3 days to ob...

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Abstract

The invention discloses 6-geranyl geranyl diphosphate (GGPP) synthase, 8-sesquiterpene synthase and pleuromutilin high-yield strains, and application of the strains. Two high-yield strains of the pleuromutilin are obtained by screening and mutagenesis, gene sequences of the GGPP synthase and the sesquiterpene synthase which have significant influence on the yield of the pleuromutilin are provided, and the reason of the yield improvement of the pleuromutilin produced by the strains is determined, so that a new path is provided for improving the yield of the pleuromutilin further. Compared with strains in the prior art, the two strains have the advantage that the capacity of producing the pleuromutilin is high; and the yield of the pleuromutilin is improved obviously by selecting and optimizing the fermentation condition further, the requirement of industrial mass production is met, and the cost is reduced.

Description

technical field [0001] The present invention relates to a pleuromutilin synthase gene, a pleuromutilin-producing strain and applications thereof, in particular to GGPP synthetase, a sesquiterpene synthase, a high-yield strain of pleuromutilin, a pleuromutilin synthesis pathway and applications , belongs to the technical field of microbial fermentation. Background technique [0002] Pleuromutilin is the precursor of chemically synthesized Tiamulin, and its molecular formula is C 22 h 34 o 5 , with a molecular weight of 378.51, belongs to diterpenoids and is a secondary metabolite of the higher fungus Basidiomycetes Pleurotus mutilus or other fungi, and is a natural diterpenoid with a wide range of antibacterial activities. The antibacterial mechanism of pleuromutilin is to inhibit the synthesis of competent bacterial protein, that is, it acts on the ribosome in the bacterial body, mainly showing antibacterial, and showing bactericidal effect at high concentrations. As a c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/60C12N1/14C12P15/00C12R1/645
Inventor 马景武赵硕珍王炜王莉李平作马丽丽
Owner 山东胜利生物工程有限公司
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