The Research Of Adenosine High-yield Strains Breeding And Fermentation Condition Optimization

Adenosine can be directly used as clinical drug for the treatment of the cardiovascular and circulatory system disease, and intermediates for the synthesis of antiviral drugs. It is mainly produced by fermentation method using sugar as raw material in industry. There are a very large development space to improvement the fermentation production and and cut costs through breeding and improving fermentation conditions. At the same time, a problem need to be solved that the production traits may be instability in the production. After a long term mutation breeding work, an adenosine producing strain Bacillus subtilis DI3-243 was screened out from an inosine producing strain, which could accumulate adenosine to 7-9 g/L. In this research, B. subtilis DI3-243 were used as starting strain to bred high yield adenosine strains through single factor and composite mutagenesis such as ion beam injection and diethyl sulfate(DES), and the fermentation condition were optimized too.1. B. subtilis DI3-243 as experimental strain is conducted the preliminary research on the fermentation conditions factors such as purine bases, glucose, organic nitrogen source etc., the DI3-243 strain could produce 14.46 g/L adenosine in shake flask fermentation with optimizing the culture medium formula by simple factor design of experiment or orthogonal experiment, which was increased by 48.2% comparing with what it used to be. The optimize culture medium formula be as follows: 250 mg/L guanine, 9 g/L glucose, corn starch 60 ml/L, yeast powder 12 g/L, yeast extract 12 g/L.2. B. subtilis DI3-243 was used as original strain which possessed the genetic markers of Xan-,Thiand SGr, higher yield strain DI4-24 was screened after composite mutagenesis treating with ion beam injection and DES, and the adenosine yield were inproved up to 16.37 g/L, which It was increased by 67.73% than the original strain. The genetic phenotypes of DI4-24 were of Xan-, Thi-, SGr and Hisauxotroph. His- was newly added genetic mark compared with the starting strain.3. In the process of experiment, a phenomenon were found that DI4-24 strains degradated easily in the process of preservation, continuous passage and fermentation production, although it was higher yield strain with the genetic markers of His- auxotroph. The degradation phenomenon showed as fermentation yield decreasing obviously, red lawn, abnormal morphology of colony and bacteria. Two kinds of abnormal colonies were separated on selective culture medium. One is of red, its genetic markers were identified and we found that His- mark was lost, which was recorded as DI4-24 R strains, adenosine production declined to 6 g/L lower. The other clone was of white and larger, the change of genetic markers were not found and adenosine yield was lower than 6 g/L too, which was recorded as DI4-24 W strains. The reason for strains degradation is caused by spontaneous mutations appearly. DI4-24 R mutation colonies ware far more than that of DI4-24 W. The former should be recovery mutation of His- auxotroph, and the recovery mutation rate was of 2.4 x 10-5. Recombinational restoration test were taken and the results showed that the repair ability of DI4-24 W DI4-24 R and DI3-243 strains were normal and even higher than that of the wild strain B.subtilis BS168, these revealed that recovery mutation may be caused for the higher repair ability.4. The influence factors to recovery mutation were analized in the process of 4 ℃ storage by measuring OD743 value of DI4-24 cell suspension. The results show that the quantity of mutant cells increased with the extension of time, and the degradation rate was more quick especially during the period of 10-20 days. Low temperature storage can reduce the degradation speed. There were better effect for reducing degradation speed when it was storaged at-80 ℃. With increasing of subculture times, strain degradations would be accelerated. Especially after 5 times subculture, the degradation speed would be more quick, and the yield reduced to 5 g/L lower.5. The influence factors to recovery mutation were investigated in the seed cultivation process using the same method mentioned above. The amount of recovery mutation cells of His- auxotroph would increased with time when the seed culture time was more than 8 h, and the increasing would be more rapid during the period of 10 to 24 h. The higher of the culture temperature, the more of back mutation occured. So the temperature of seed cultivation should be to reduce aptly to 33-35 ℃. When initial pH of seed medium rised from 7.0 to alkaline region, the amount of recovery mutation increased, and reduced to partial acid condition the amount decreased. Added substances such as carotene and folic acid in the seed culture medium could significantly reduce the recovery mutation rate, and added substances such as the tea polyphenols, cysteine and sodium selenite could increase the mutation rate. Culture condition was optimized as following: added folic acid and carotene in seed culture medium, contolled initial pH to 6.5 and cultured at 35 ℃ for 8 h. Adenosine production could be increased by 9% inoculating seed prepared as above method.6. B. subtilis DI4-24 was used as original strain, and ion beam injection as mutagen to screened strains that His- auxotroph was recovered. Then higher yield and more stable strain were selected from these auxotroph strains, among them DI4-24R-37 strain could produce glycosides 15.84 g/L its His- genetic trait restoration and mutation rate declined obviously. Enlarged fermentation experiment were taked in 500 L fermentation tank, and the glycoside production was of 28.9 g/L after 72 h culture.