Studies On Interspecies Protoplast Fusion Of S.mycarofaciens And Characters Of Its Fusion Cell

Using Midecamycin producer S. mycarofaciens var. 464 and Erythromycin producer S. erythreus as original strains, studies were carried out for researching higher producers and enhancing content of Midecamycin (MDM) AI, which could use propionate as precursor to reduce production costs.According to the literature, the utilization rate of propionic acid is thirteen times to that of acetic acid by propionate kinase in S. erythreus. But in S. mycarofaciens, there is no distinctive propionate kinase, so that the utilization rate of propionic acid is lower than that of acetic acid. For this reason, it is hoped that the higher producers would be obtained by protoplast interspecies fusion between S. mycarofaciens and S. erythreus, which would transfer propionate kinase of S. erythreus to S. mycarofaciens. Therefore, the fusion cells could use propionic acid as precursor in synthesis of MDMA₁, which would reduce the production costs.First, after investigation of two original strains' biological characteristics, we studied the main influence factors on protoplasts formation and regeneration in S. mycarofaciens and S. erythreus, and determined the best protoplasts formation and regeneration conditions of two original strains. The former shake-cultured in S' medium at 28癈, 220r.min^-1 for 24h, lysised by 3mg/ml lysozyme, keeping warm at 32 癈 for 50⁶0min, regenerated on R_5' medium, 28 癈 for 5⁶d. The latter used two-step culture, then used Img/ml lysozyme keeping warm at 37癈 for Ih; the protoplasts were plated on R5' regeneration medium at 28 癈 for 5d.Second, procedures of effective protoplasts fusion and U.V. irradiation or heat treatment protoplasts fusion were developed. Equal protoplast suspensions (containing about 108/ml protoplasts) of each strain were mixed and washed by centrifugation, then the pellets were resuspended in PEG4000 solution (35%) at 37癈 for 2min. After centrifugation and resuspension, protoplasts were dilutedand plated on regeneration medium.Last, fusion cells were screened repeatedly, and the better fusion cells, as Rhl07, Rhl09, Rh109-2, and Rh 109-15 et al were obtained. Using Rh 109-2 as original strain, we investigated the biological characteristics of fusion cells.Rh 109-2 can use G01 medium, and grow well on the medium containing 0.3% propionate. The average liter was 30% higher than original strain during normal fermentation. Using propionate as half precursor instead of 01 precursor, the fermentation titer of Rh 109-2 does not reduce, and the content of MDMA₁ increases. When using starch as only carbon source of seed medium and the improved fermentation conditions, the content of MDMAi increases to 93%.