| Widely used as a new type of digestive enhancer and metabolic regulator in feedstock, avilamycin is a kind of orthosomycin antibiotics. The development of the product has the good economic and social performance for its unique structure, high safety, toxicity, and low-residue. The main purpose of this study is to improve production of avilamycin by genome shuffling based on ribosomes engineering. And avilamycin-resistance was designed to be evolution pressure to eliminate the father strains. We also characterized the derived strains on their morphological and genetic differentiation.After six rounds of protoplast fusion, three high-yielding strains were obtained, of which fusant All-3produced avilamycin up to2514.3mg/L, fusant A11-10at4006.7mg/L, and fusant A11-13at1318.7mg/L respectively. Avilamycin yields were of17.5,27.88, and9.18times to the original strain1-17. The avilamycin productivity of the strain is stable after five passage strains. It was suggested that these genome shuffled strains would be indudtrial potential.Using electron microscopy and fluorescence microscopy, the different characteristics of colonies, spores and hyphae were studied. On the solid culture, the shuffled strains showed great variations from strain1-17:light gray colonies, serrated edge; stout aerial hyphae, lots of smooth round or spherical spores, while strain1-17was of white colonies, smooth egde, linear-type spore’s silk, and hard to break, cylindrical spores.Studies in mycelium morphological differentiation in submerged cultivation showed that the original strain1-17has dense mycelium pellets with large diameter, and at48h to52h, the pellet diameter growth arrested. The shuffled strains have more spores, hard to form pellets, and their mycelium pellets loose obviously with small diameter. At24h fusant A11-13still has no obvious mycelium pellets, required a long developing course for hyphae clustering and pellets fonnation. The stage of pellet arrest of fusant All-3were extended, from48h to60h, while that of fusant A11-10delayed obviously.The fermentation growth curve of strains in500mL-triangle flasks showed that:the mycelium biomass of three fustants are obviously higher than that of the original strains1-17. Associated with morphological differentiation at the rapid growth stage, the mycelium biomass sharply increased, but almost no avilamycin was synthesized. At growth arrest phase the cell biomass kept a dynamic balance, while aviliamycin production began to accelerate significantly. Then the cell biomass reduced significanly, and secondary metabolites continued to accumulate. At72h the avilamycin production of fusants reached a peak and then decreased, later than that of strain1-17, and the production rate of fusant All-10was higher than the others. It concluded that there is a relationship between morphological differentiation characters and secondary production in fermentiation process. Accumulation of avilamycin significantly covers the arrest and the continued stages after that. Therefore a hypothesis was supposed that a relationship between mycelium morphology and its development with secondary metabolites synthesis and output of the mutant be important to avilamycin synthesis during the growth process.In order to investigated the genetic characteristics of fusants by RAPD,6primers were screened out from the34random primers. Polymorphic36bands were obtained, polymorphism detection rate at86%. NTSYS (UPGMA) cluster analysis showed that10strains have a high homology. The smallest genetic similarity coefficient was of0.56, and9isolates of which were divided into two categories at0.68: high-yielding strains class and lower-yielding strains class. However, fusant All-9with low-yielding were exception. We supposed that there be somewhat key DNA sequence in the high production strains which coferred to avilamycin production and mutant in the genome shuffling process then keep down. Some DNA sequence which smiliar to that also keep although it doesn’t matter. It still need further research to prove that. |
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