Overexpression Of AcyB And Immobilization Of Streptomyces Thermotolerans On Polyurethane Foam To Improve Production Of Acetylisovaleryltylosin

3’-acetyl-4"- isovalery tylosin(AIV), a kind of animal antibiotic, is widely used at livestock farming and effective at treating the disease, and has less possibility of developing drug resistance. As the method of transforming the tylosin is inefficient to produce AIV, it is necessary to cultivate a strain that has a high yield of AIV and to seek for the semi-continuous fermentation process for industrialized production.At first, this research cloned the gene acyB1-B2 that has low activity in the original strains. Then, two recombinant plasmids containing structural gene acyB1-B2 based on plasmids p IB139 and p UWL201 were transferred, separately, in Streptomyces thermotolerans(SP.11416, start strain) through intergeneric conjugal transfer. Screened from a variety of recombinant strains, the recombinant strain SP.11432 that satisfies the relevant requirements was obtained, producing the amount of AIV that is twice the amount of AIV produced by SP.11416. The tolerations of SP.11416 and SP.11432 on tylosin, AIV and Apr were investigated, separately. The results showed that the tolerations of SP.11432 was greatly improved.Second, based on the requirement-the mass of immobilized cell, the best carrier, polyurethane foam(PUF), was selected from different carriers that immobilized SP.11432. According to the structural characteristics of membrane of Streptomyces thermotolerans and the electronegativity on the surface of Streptomyces thermotolerans, some kinds of modified PUF were designed and created. Considering the maximum mass of immobilized cell and the AIV concentration, the positive-crosslinking polyurethane foam(PCPUF) was the best carrier for immobilizing S. thermotolerans. The criteria of PCPUF to immobilize cel s were also investigated.Finally, the outcome of single batch fermentation over 80 hours showed that, through the fermentation of SP.11432 immobilized by PCPUF, the peak concentration of AIV could be 33.54mg/L, more than 20.34 mg/L got from the fermentation of free cells. Compared with free cells, the immobilized cells spend less time obtaining the peak concentration. In addition, the time of single batch fermentation was 48 hours. In the process of semi-continuous fermentation, the optimal number of times of repeated batch fermentation is 6, and the best percent conversion is 86.30%.