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The background Pleuromutilin 1951 by kavanagh et al found that the bacteria produced by large fungal burden tricyclic two antibiotics, molecular formula: C22H34O5. The role of antibiotics in the bacterial 50S ribosomal large subunit amino acid transferase, to inhibit bacterial growth by inhibition of protein synthesis. Over the years, many pleuromutilin derivatives antibacterial activity has been studied, three of which have been developed for the listed drugs, including 1974 and 1999 from Switzerland sandoz veterinary drugs the really wonderful Zhongshengmycin and Woni Miao Lin, and GSK company listed in 2007 by Swiss drug him Palin. In recent years, the research and development of the water-soluble derivative of pleuromutilin soon, which will become pleuromutilin class of antibiotics in the future development direction. Industrial truncated Pleuromutilin production improved mainly: the traditional strains mutagenesis, fermentation medium optimization, soybean oil and nitrogen source regulation, the establishment of the fermentation process mathematical model. Improved research on cell from the molecular level has also been reported, but did not make substantive progress. At the same time the biosynthetic pathway Pleuromutilin mainly supposedly based on isotope feeding method, is not clear from molecular biology, which largely hinder people to study for pleuromutilin biological synthesis. Since, from the level of molecular biology on the pleuromutilin the biological biosynthesis difficult to achieve substantial progress in the short term, the study from the fermentation or other level is particularly important. The purpose of looking for methods that can effectively regulate the biosynthesis of pleuromutilins from the angle of metabolic regulation and optimization of fermentation. 1 investigated the fungal growth inhibitor hydrochloric Laid than terbinafine (TH) is by partially blocking the ergosterol biosynthesis can regulate metabolic flow to improve antibiotic biosynthesis ability. 2 investigated P4 fermentative production pleuromutilin process, whether the glucose feeding strategy affect pleuromutilin biosynthesis. Method 1, the metabolic regulation in shake flask fermentation level were the TH concentration gradient experiments and different time add experimental TH. Wherein, TH concentration (μg / mL), respectively: 0,4,8,12,16,40,400, add the time (h), respectively: 0,24,48,72,96,120,144 fermentation 192h detection pleuromutilin The prime unit biomass and ergosterol alcohol content changes. 2, in the optimization of fermentation experiments, the first in the shake-flask level were investigated 2%, 3% and 4% of the initial glucose P4 Growth Screening optimal initial glucose concentration. Then, in a 7L fermenter, to optimize the concentration of glucose batch fermentation to determine the glucose added time. Added time based on the optimal initial glucose concentration and the glucose fermentation 240h 7L fermenter, five kinds of glucose were investigated feeding strategy impact pleuromutilin fermentation, these five strategies were as follows: Batch fermentation (the control group) , constant speed streams plus exponential feeding pH-stat substrate concentration control glucose feeding strategy. Group above five strategies optimal method for 50L zoom verification. Results 1,12 μg / mL of TH 48h Add to shake the bottle will be most conducive to promote the pleuromutilin the biosynthesis. At this time, the yield of the pleuromutilin, bacterial biomass and hydrochloric Laid terbinafine content respectively to 3.05mg / m, 37.13g / L and 226.56mg / L, respectively, compared with the control group increased by 31%, -0.97%, and - 0.89%. It can be deduced: TH is able to effectively promote the biosynthesis of pleuromutilin. The experiment from the side corroboration pleuromutilin biosynthesis imaginary ways to provide a reference for the further study of metabolic regulation. 7L fermentor of these optimal conditions the zoom validation experiments 192h pleuromutilin yield and yield rate of 5.8mg/mL and 16.6% respectively. 2, the control group, P (Yield) = 3.2mg/mL glucose conversion rate = 26.7 mg / g; constant speed flow plus the experimental group, P = 3.6mg/mL glucose conversion rate = 32.3 mg / g; exponential feeding experimental group P = 4.42mg/mL, the rate of conversion of glucose = 28.7 mg / g; pH-stat experiment group P = 2.77mg/mL, the rate of conversion of glucose = 27.9 mg / g; glucose concentration control experimental group, P = 4.79mg/mL glucose conversion rate = 37.3 mg / g. Which are the highest P value and the rate of conversion of glucose fermentation of glucose substrate concentration control feeding strategy increased 49% and 39%, respectively, compared with the control group. Control the fermentation of glucose substrate concentration feeding strategy in 50L fermenter enlarge verify experimental results: the 192h pleuromutilin unit 10.15mg/mL. Conclusion 1, the level of the shake flask, the TH 12μg/mL 48h can effectively regulate the pleuromutilin biosynthesis, and the method is more stable in a 7L fermenter amplification process. 2, glucose substrate concentration optimal feeding strategy effects and verify the stability of amplification, can provide a reference for the industrial production of pleuromutilin.
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