Fermentation Optimization Of HDN-1 From The Antarctic Fungus Oidiodendron Truncatum GW3-13

HDN-1 was a secondary metabolite isolated from Antarctic Oidiodendron truncatum GW3-13, which is a epipolythiodioxopiperazine (ETP) compound containing a novel three sulfur bridge structure. Cytotoxity assay using five different cancer cell lines showed that this compound has high bioactivity. Especially, HDN-1 can induce the M2-HL60 leukemia cell differentiation and apoptosis in 20 nm concentrations, and has no obvious effect on the normal white blood cell vitality and body weight in mice; Molecular mechanism research suggested that this novel natural product is a kind of inhibitors role in HSP90-C. At present, HDN-1 has been targeted as a new anti-tumor drug for new drug research and development.The main aim of this study is to improving production of HDN-1 through optimize the composition of culture medium and fermentation conditions, scale-up in 5L fermenter, and develope a complete and efficient preparation of HDN-1, laying a foundation for toxicological studies and animal experiments.Firstly, we investigated the effect of culture temperature, initial pH, and fluid volume and rotate speed to the growth of strain and yield of HDN-1 Shake flask experiment suggests that the optimal fermentation temperature is 25℃. The optimal initial pH of growth strains GW3-13 is pH 5.0. In 500 ml triangle liquid containing 100 ml bottle is advantageous to the strains to produce HDN-1, the speed of 180 r/min is more suitable for growth. Secondly, the optimum carbon source(glucose 、soluble starch、maltose)and nitrogen source(yeast extract、peptone、corn flour)were determined by using single factor test. Then the ratio between the compositions of culture medium was determined by employing ten levels with eight factors of uniform design experiment, and we got an available model and a medium composition. On the basis of uniform design, three significant factors(glucose. yeast extract、MgSO4·7H2O)were screened by Plackett-Burman design (PBD). At last, Box-Behnken design experiment was used that we successfully optimized the ratio between the three factors, and developed a high yield culture medium composition. The maximum production of HDN-1 got to 54.7 mg/L in flask,9.6 times higher than before.According to dissolved oxygen, simulated the shear stress and defoaming agent experiments in shaking flask fermentation, the strain is not sensitive to the shear stress,2% defoamer did not affect the production. Combined with the above results, we studied the different ventilation, feeding, intermittent control pH factors and different blade combination in 5 L fermentation tank. The results showed that ventilation 1 VVM, free pH in the entire fermentation process is good for the strain to produce HDN-1, and in this condition the maximum yield is 39.4 mg/L. The production of HDN-1 has no obvious promoting by adding medium at culturing; Compared to shaking flask, mycelial morphology in fermenter is loose, and has no significant difference compared with shaking flask. Experiments of different stirring blade combinations showed that it is no effect on the cell growth and the production of HDN-1 under the condition of upper six-flat turbine impeller and lower four-pitched turbine impeller. It is seemed that a loose sparse state of strain is advantageous to the biomass accumulation and synthesis, and the maximum yield of HDN-1 is 42.1 mg/L. Different from six flat blade, the rate of oxygen consumption increased, and this may be increased by gas-liquid mixing degree.And we have developed a complete and efficient preparation of HDN-1 separation process, improveing the efficiency of the compound purification.