Breeding High Avilamycin-Producers By Streptomyces Viridochromogenes

As a kind of orthosomycin antibiotics, avilamycin is widely used as a new type of digestive enhancer and metabolic regulator in feedstock. It is also authorized by EU for its unique structure, high safety, low-residue and toxicity. This study aimed to breed high-yield producers of avilamycin by Streptomyces viridoehrongenes. By combining the mutagenesis treatment with genome shuffling, the recombinants were adapted and regenerated on rifamycin plates. The rpoB gene sequence and metabolic fermentation of recombinants were characterized further. A HPLC method was modified to detect the avilamycin in the fermentation media.After four rounds of mutagenesis plus two rounds of protoplast fusion, two high-yielding strains, G2-10and G2-31, were obtained. Their avilamycin titers reached2242.64mg/L and2471.41mg/L which were3.16-and3.48-fold to the parental strain11-10, respectively. By five continuous sub-cultivations, both of them behaved stably and showed an industrial potential.Rifamycin resistance plate was used to screen preliminarily recombinants. The strains G2-31performed a high resistance at800mg/L compared to the original strain. Then RpoB proteins from recombinants with various resistance levels were distinguished by sequencing rpoB genes. The results showed that there were multi-site replaces in RpoB protein in the drug resistant recombinants such as G61D, S76N, and C104A. The mutation S76N was reported to confer to rifamycin resistance previously, while how to function by the other two on drug resistance needed further tests.In order to investigate the metabolic differences between the original strain and the recombinants, six strains were cultivated in the submerged. Compared to original strain, the recombinants showed a long lag phase for3d. Avilamycin synthesis in all recombinants tested had a second accumulation course in the later growth phase, and this course was not uneven. Strain G2-31consumed glucose rapidly to support growth; meanwhile G1-22consumed slowly which leaded to much low biomass and then avilamycin titer. It implied that more biomasss would promote higher avilamycin accumulation.The components of the fermentation media are complicated, so we established a new HPLC method to detect the avilamycin and this method was confirmed by LC-MS. The determined chromatographic conditions were, C18(230mm X4.6mm,5μm) column, column temperature30℃, flow rate1mL/min, wavelength220nm, injection volume20μL, and adjusting the ratio of mobile phases which were solution A (methanol) and solution B (water) by the gradient method. Under these chromatographic conditions, the components of the sample separated well and the shapes of chromatographic peaks were symmetrical. By LC-MS, the avilamycin components were confirmed to be avilamycin A and avilamycin B with the peak at33min and22.9min. and for the premix was29.004min and24min. The molecular weight of the fermentation condition was23more than that of the premix, which can explain the higher water solubility.