Breeding Of The Novel Antibiotic AGPM High Producing Strain And Studies On The Proteomics

Today, antitumor antibiotics have received much attention in the drug research and development. Recently, a new strain of Streptomyces producing a novel antibiotic, AGPM, was isolated from soil in our laboratory. It has been found that the biological activity of AGPM at 10-11M was 100～1000 times higher than that of Taxol and Taxotere against the non-small cell lung cancer and rat leucocythemia. Therefore, it is very valuable to investigate and development AGPM further as a new antitumor antibiotics or a precursor compound.However, the yield of the new antibiotic from the original strain was very low(7.8mg/L), so it is imperative to improve the microbial strain for a better production of the new antitumor antibiotics.According to the strain breeding theory ,the selection and breeding of antibiotics AGPM producing mutant and the fermentation technology of AGPM were systematically studied in this thesis. The main research contents and results are as follows:At first, Streptomyces 099 as a starting strain was stepwise treated with Diethyl sulfate(DES) and ultraviolet lighe(UV). A mutant strain TD0551 with a higher yield of AGPM was selectively bred. Under the fermentation condition without being optimized, the production of AGPM was 17.29mg/L, which was 1.22 times higher than the parental strain Streptomyces 099. At the same time, genetic character of strain TD0551 was very stable.The batch fermentation condition of strain TD0551 in shake flask was studied in this dissertation. The optimal seed and fermentation medium and culture conditions for AGPM production were developed by orthogonal design theory. Under the optimum conditions, strain TD0551 could produce AGPM 23.16mg/L by flask-shaking batch fermentation. The production of AGPM was increased by 33.9% than the initial conditions.By 30L bioreactor, the scale-up of fermentation for antibiotics AGPM was studied and the strain could produce AGPM 27.18mg/L in the optimized operating conditions in the 30L bioreactor. The AGPM morphologically structured model was constructed based on the experiment data from 30L bioreactor batch fermentation. The model simulations for mycelium growth and antibiotic formation were in good agreement with the experiment data for AGPM fermentation process.Fed batch fermentation was studied with TD0551 according to the optimum conditions of batch fermentation. Under the optimum initial glucose concentration and the in intermittent feeding ,the AGPM production could reach 30.94mg/L , improved by 33.6% than that of batch fermentation.The effect of adding oxygen-vector or emulsified oxygen-vector to the medium were investigated by flask and 2L bioreactor respectively. And the results showed that adding oxygen-vector or emulsified oxygen-vector to the medium could improve the transfer of oxygen, and then , both the production of AGPM and the growth of the strain were enhanced. The yield of AGPM finally increased by 27.3% than that of control. In addition, the relationship between production of AGPM and the mycelium morphology was investigated.The effect of adding resins on the strain growth and AGPM production was studied, and the optimum conditions were determined. In the optimum two-phase culture condition, the production AGPM was greatly increased by 55%,much higher than the task of Project of Chinese National Programs for Science and Technology Development (30mg/L).The protein patterns from the strain 099 and TD0551, and protein patterns from the strain TD0551 at different culture period were analyzed for the first time by using two-dimensional polyacrylamide, respectively, and some difference proteins concerned with AGPM production were found, and more their construction and function were determined preliminary with the help of Streptomyces 2-D database. The results demonstrated that some of the special proteins were directly related with the antibiotic AGPM biosynthesis.