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Batch type feed-batch fermentation method for streptomyces roseosporus to produce Daptomycin efficiently

A technology of Streptomyces roseospora and daptomycin, applied in the field of fed-batch fed-batch fermentation for efficient production of daptomycin by Streptomyces roseospora, can solve the problem of low yield of daptomycin, high production cost, no Fermentation optimization and other issues to achieve good industrial application and promotion value, increased yield, and easy control of the fermentation process

Active Publication Date: 2012-07-04
NANTONG YIKAI MEDICAL DEVICES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are domestic strains producing daptomycin to initially realize pilot production, but without fermentation optimization, the yield of daptomycin is low and the production cost is high. Therefore, the fermentation process of daptomycin is optimized to further improve The yield of daptomycin is of great significance

Method used

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  • Batch type feed-batch fermentation method for streptomyces roseosporus to produce Daptomycin efficiently
  • Batch type feed-batch fermentation method for streptomyces roseosporus to produce Daptomycin efficiently
  • Batch type feed-batch fermentation method for streptomyces roseosporus to produce Daptomycin efficiently

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Take spores of Streptomyces reseosporus at room temperature and make spore suspension with sterile water. Then get 0.5 milliliter of spore suspension and inoculate in 250 milliliter shake flasks that 30 milliliters of seed culture medium are housed, 30 ℃, in 180~220rpm shaker, cultivate 48 hours. Then inoculate into 7.5L automatic fermenter with 1% inoculum amount. Among them, the fermentation medium components (glucose 1.0g / L, dextrin 3g / L, casein 1.0g / L, peanut cake powder 0.6g / L, L-asparagine 0.12g / L, K 2 SO 4 0.6g / L, initial pH7.0. ) and then fermented for 144 hours at 30°C with the ventilation rate controlled at 0.8v / v / m. The daptomycin concentration in the fermentation broth was monitored by HPLC. Daptomycin fermentation unit increased to 41±0.6mg / L; if figure 1 shown.

Embodiment 2

[0025] Take spores of Streptomyces reseosporus at room temperature and make spore suspension with sterile water. Then get 0.5 milliliter of spore suspension and inoculate in 250 milliliter shake flasks that 30 milliliters of seed culture medium are housed, 30 ℃, in 180~220rpm shaker, cultivate 48 hours. Then inoculate into 7.5L automatic fermenter with 4% inoculum amount. Among them, the fermentation medium components (glucose 1.0g / L, dextrin 3g / L, casein 1.0g / L, peanut cake powder 0.6g / L, L-asparagine 0.12g / L, K 2 SO 4 0.6g / L, initial pH7.0. ) Then, at 32°C, at 0-30h, the pH is controlled at 6.5, and the dissolved oxygen is 45%; at 30h-144h, the pH is controlled at 7.0, the ventilation rate is controlled at 0.8v / v / m, and fermentation is carried out for 144 hours. The daptomycin concentration in the fermentation broth was monitored by HPLC. The fermentation unit of daptomycin is 85 ± 0.8mg / L, and the output ratio of Example 1 has been improved by 2 times. figure 2 shown...

Embodiment 3

[0027] Take spores of Streptomyces reseosporus at room temperature and make spore suspension with sterile water. Then get 0.5 milliliter of spore suspension and inoculate in 250 milliliter shake flasks that 30 milliliters of seed culture medium are housed, 30 ℃, in 180~220rpm shaker, cultivate 48 hours. Then inoculate into 7.5L automatic fermenter with 3% inoculum amount. Among them, the fermentation medium components (glucose 1.0g / L, dextrin 3g / L, casein 1.0g / L, peanut cake powder 0.6g / L, L-asparagine 0.12g / L, K 2 SO 4 0.6g / L, initial pH7.0. ) and then, at 28°C, at 0-30h, control pH6.5, dissolved oxygen 45%,; 30h-144h, control pH7.0, ventilation volume control 0.8v / v / m, 30h at a speed of 0.10mL / h Start feeding 1:1 capric acid and methyl oleate solution to ferment for 144 hours. The daptomycin concentration in the fermentation broth was monitored by HPLC. The daptomycin fermentation unit is 110 ± 0.8mg / L ratio and has improved to embodiment 1 output and improved 2.68 tim...

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Abstract

The invention relates to a batch type feed-batch fermentation method for streptomyces roseosporus to produce Daptomycin efficiently, comprising the following steps: taking streptomyces roseosporus slant spore at normal temperature to prepare spore suspension by sterile water; inoculating 0.5ml of the spore suspension in a 250ml shake flask filled with 30ml of seed culture medium, and culturing for 48h in a shaker when the temperature is 28-32 DEG C and the revolution speed is 180-220 rpm; adding 1% of inoculum size in an automatic 7.5 LDE fermentation tank loaded with fermentation medium; adding one or combination of cofactor ferroheme or leucovorin or pyridoxal phosphate VB6 with the concentration of 0.5-30mg / L; and controlling the throughput to be 0.8-1.2v / v / m, the pH value to be 6.5-7.5 and fermenting for 90-144h at the temperature of 28-32 DEG C. By adopting the method in the invention to regulate biological enzyme activity to increase intracellular metabolism flux introduced in Daptomycin synthesis, the Daptomycin yield is improved; the method has simple operation and low cost; and in the streptomyces roseosporus fermented and cultured by the invention, the fermentation unit of the Daptomycin can be improved by 0.24-4.6 times compared with a contrast sample.

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation, in particular to a batch-type fed-batch fermentation method for efficient production of daptomycin by Streptomyces roseospora. Background technique [0002] "Streptomyces roseosporus" (Latin name: "Streptomyces roseosporus") is an actinomycete of the genus Streptomyces, and its synthetic daptomycin (English name "daptomycin") is N -decanoyl-L-tryptophanyl-L-asparaginyl-L-aspartyl-L-threonylglycyl-L-ornithyl-L-D-aspartyl-D-alanine Acyl-L-aspartylglycyl-D-seryl-threo-3-methyl-L-glutamyl-3-alanine ε1-lactone is composed of a decane side chain with A cyclic β-amino acid peptide chain N-terminal tryptophan connected, is a new macrolide antibiotics. [0003] Daptomycin is a calcium ion-dependent antibiotic. In the presence of calcium ions, daptomycin will bind to cell membrane proteins in the form of non-covalent bonds, and the daptomycin-binding protein ( DBPs) as its target site, da...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02C12R1/465
Inventor 闻建平宇光海贾晓强
Owner NANTONG YIKAI MEDICAL DEVICES CO LTD
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