Screening Of Carbomycin Producing Strains And Study Of Fermentation Conditions

Acetylisovaleryltylosin is a very important Tylosin derivative. It is obtained through Tylosin biotransformated by Streptomyces thermotolerans ATCC 11416, which is acetylated in the 3'-OH position of Tylosin, and isovaleryled in the 4″-OH position of Tylosin.And two enzymes are acetyltransferase and isovaleryltransferase. In this study, through mutation we screened the high yield strain from ATCC11416, so that prepare for the effective transformation from Tylosin to AIV.Through the mensuration of fermentation curve to determine the fermentation conditions, we found that in the Carbomycin fermentation of Streptomyces thermotolerans, the fermentation titer and DCW(dry cell weight) reached maximum on the fifth day, then the fermentation titer and DCW had a sharp decline on the sixth day. It was presumed that it was worked by the dual factors of antibiotic degradation autolysis emissions and the reduced pH value. In this study, we used UV combined with microwave mutation method to screen the Streptomyces thermotolerans strains of high yield. After two rounds of UV mutation the fermentation titer increased from the initial 12.34μg/ml to 97.96μg/ml, which was increased by 7 times. The tolerance to Tylosin increased from the beginning of 6μg/ml to 50μg/ml. Then after a microwave mutation, the tolerance to the Tylosin also increased to 80μg/ml, however, the fermentation titer declined.Through an optimization of fermentation conditions, the best carbon and nitrogen sources were starch and peptone. We determined the best concentration of starch and peptone were 63.5g/L and 35.55g/L in the fermention respectively through surface analysis, and its fermention titer increased 5% more than primary best concentration. Adding of CaCO3 did not increase the Carbomycin yield, but after adding Leu, the precursor of isovaleryl, its fermentation efficiency up to 174.03μg/ml, which increased by 19% more than broth titer without Leu. After optimization of fermentation, Carbomycin yield increased nearly 1 time more than before optimization.After adding with Tylosin to fermentation of Streptomyces thermotolerans and the fermentation was extracted, we used HPLC-MS to determine the conversion rate from Tylosin to AIV in the fermentation broth. At the same time, we studied the effect of conversion rate on different addition of Tylosin. When the added concentration was less than 150μg/ml, conversion rate was steadily, from 74% to rise to 75%. When the added concentration was 200μg/ml, the conversion rate has dropped to 72.2%, presumably because of exceeded bacterial resistance concentration of Tylosin 80μg/ml extortionate Tylosin concentration inhibited the acetyltransferase and isovaleryltransferase activity in vivo. Therefore, optimal Tylosin adding concentration was about 1.8 times cell tolerance.