Metabolic Regulation And Fermentation Optimization Strategy For Pleuromutilin Biosynthesis

BackgroundPleuromutilin discovered by Kavavagh for the first time in 1951, is one of the tricyclic structural diterpenoid antibiotics with a molecular formula of C22H34O5. It selectively inhibits the synthesis of 50s subunits protein in ribosome through interacting with glutamyl transferase,and thus restrains the growth of gram-positive bacteria and mycoplasmas. In the past years, many pleuromutilin derivatives have been successfully developed, and three of which had been approved as drugs by FDA, which include tiamulin and valnemulin for veterinary, and retapamulin for human. In addition, with the advantage of high absorption, water-soluble pleuromutilin derivatives developed rapidly in recent years and will become stars in the future. At present, improved strategies for pleuromutilin production mainly includes traditional breeding species, medium optimization, regulation of carbon and nitrogen, mathematical modeling and so on. Research that bacteria improvement on the molecular level has not made substantive progress. The biosynthetic pathway of pleuromutilins which is imaginarily proposed by isotope tracing has not been clarified by molecular biology which blocks further research of pleuromutilin biosynthesis. So, it is necessary that we should focus on fermentation or other levels to explain the biosynthesis of pleuromutilin. ObjectiveIn this study, we attempted to look for effective strategy to regulate pleuromutilin biosynthetic by metabolic regulation and fermentation optimization.1,We focus on study that whether fungal growth inhibitor terbinafine hydrochloride(TH) could effectively regulates biosynthetic of pleuromutilins by partially blocking the biosynthesis of ergosterol metabolic to increase pleuromutinlins biosynthetic capacity.2,If glucose feeding strategies could affect the biosynthesis of pleuromutinlins in fermentation.Methods1,In metabolic regulation study, we investigated the effect of different concentrations of TH and various adding time of TH on pleuromutilins biosynthetic. The concentrations of TH(μg/mL)contains:0,4,8,12,16,40,400. The adding time(h)of TH contains:0,24,48,72,96,120,144. Pleuromutilin production, biomass and erogterol production was analyzed when fermentation was 192h.2,In fermentation optimization study, we investigated the effect of different concentration of Glc including 2%,3% and 4% on cell growth to get initial Glc concentration. Based on initial Glc concentration, we investigated adding time of TH in 7L bioreactor. Then, we studied effect of 5 Glc feeding strategis on 240h pleuromutilin fermentation. The 5 strategies contains:batch fermentation, constant feeding, index feeding,pH-stat feeding and Glc feedback feeding. Finally, we carried out 50L amplification verification of pleuromutilins fermentation by optimazation strategy.Results1,12g/mL of TH was the optimal concentration for the synthesis of pleuromutilin when added into flask at 48h. Under this condition, the pleuromutilin production, cell biomass, and erogterol production were 3.05mg/mL,37.13g/L and 226.56mg/L, which increased by 36%,-0.97% and -0.89%. respectively, compared with the control. As a conclusion, TH could effectively promote the biosynthesis of pleuromutilin, and it, on the other hand, proved the hypothesis of biosynthetic pathway for pleuromulitin, which can serve as reference for the research of metabolic regulation of pleuromutilin. We carried out the experiment of producing pleruomutilin in 7L bioreactor. It was found that the production of pleruomutilin was 5.8mg/mL, which was 16.6% more than that of control.2,the pleuromutilin production (mg/mL) and conversion rate of glucose (mg/g) were 3.2,26.7 for control; 3.6,32.2 for constant feeding; 4.42,28.7 for exponential feeding; 2.77,27.9 for pH-stat; 4.79,37.3 for controlled glucose concentration respectively. As one can see, the best strategy was controlled glucose concentration, which increased by 49% for pleuromutilin production and 39% for conversion rate of glucose compared with the control, respectively. Moreover,50L amplification experimental result verified that pleruomutilin production at 192h was 10.15mg/mLConclusion1,12μg/mL TH can greatly promote biosynthesis of pleruomutilin at 48h and this method is stable in 7L bioreactor fermentation.2,Controlled glucose concentration experiment was the best strategy of all and it coule be reference for industrial production.