Study On Increasing The Yield Of Tylosin And Its Derivative Acetyl-isovalery Tylosin

Tylosin,a 16-membered macrolide antibiotic obtained from Streptomyces fradiae through fermentation,is widely used as a special antibiotic for feed additive of livestock and poultry worldwide and as a feed additive.Acetyl-isovalery tylosin,one of the tylosin derivatives,can be obtained through biotransformation of tylosin by Streptomyces thermotolerans.Its antimicrobial activity against mycoplasma is 4 times higher than that of tylosin.Since acetyl-isovalery tylosin has inhibitory effect on a variety of antibiotic-resistant bacteria,it is also widely used as veterinary drugs worldwide.The yield of tylosin was increased by the expression of crotonyl-Co A reductase(CCR)and P450 oxidase in Streptomyces fradiae QL100.Overexpression of the CCR genes orf4*,ccr2 from tylosin-producing S.fradiae,and the CCR genes SBI-01394 and SBI-02836 from milbemycin-producing Streptomyces bingchenggensis,exhibited22.48%,21.31%,26.08% and 23.03% tylosin yield increases in tylosin production,respectively.Overexpression of the homologous P450 oxidase gene tyl I increased the yield by 11.37%,while overexpression of the exogenous P450 oxidase gene ros C decreased the yield by 30%.The recombinant plasmids of acetylation gene acy A,isovalerization gene acy B1,positive regulation gene acy B2 and acy B1B2 were cloned into the multi-copy plasmid p KC1140 to improve the yield of acetyl-isovalery tylosin.The results showed that the four genes increased the production level of acetyl-isovalery tylosin by 3.30%,4.27%,5.23% and 14.69%,respectively.Subsequently,the effects of acy B1B2 on the production level of acetyl-isovalery tylosin using intergative vector was also investigated,and the recombinant plasmids p SET153-acy B1B2 were constructed.The results showed that p SET153-acy B1B2 increased the yield of acetyl-isovalery tylosin by 12.38%,which was a little less than that of p KC1140-acy B1B2,while the stability of p SET153-acy B1B2 vector was much better than that of p KC1140.The tolerance of the two expression strains to tylosin was 150 μg/m L,both of which increased by 50%,In conclusion,p SET153 plasmid was selected as the expression vector for acy B1B2,and its expression strain will be used for industrial production of acetyl-isovalery tylosin..In order to further improve the yield of acetyl-isovalery tylosin,the shaking flask fermentation conditions of Streptomyces thermotolerans / p SET153-acy B1B2 was optimized by response surface methodology.The optimal fermentation conditions were obtained: initial p H was 8.0,shaking speed was 260 r/min,fermentation temperature was 30 °C,fermentation time was 96 h,the amount of seed inoculation was 7%,media volume was 60 m L and the seed age was at 48 h.Based on the above condition,the yield of acetyl-isovalery tylosin reached 7700.61 μg/ml,which was 15.46% higher than that of original fermentation condition.The error with the predicted value was 3.21%.The results showed that it was feasible to optimize the fermentation conditions for acetyl-isovalery tylosin bio-conversion by response surface experiment.