The Construction Of A Strain Producing Doramectin And The Optimization Of Fermentation Conditions

Doramectin is is the third generation of avermectin derivatives, obtained by mutation biosynthesis from the Streptomyces avermitilis of α- keto acid dehydrogenase(BCDH) blocked mutant, and its chemical properties are macrolide antibiotics. Compare with other macrolide antibiotics, it has significant effects on animal diseases which are caused by endoparasite and ectoparasite. Doramectin has higher insecticidal activity and longer efficiency time than other types of abamectin drugs, so it is considered to be the most potential ofanti-parasitic drugs of broad-spectrum.α- keto acid dehydrogenase(BCDH), encoded by bkd FGH gene cluster, can catalyze the synthesis of the precursor of avermectin. In this study, we successfully made the bkd FGH gene cluster mutants blocked by homologous double crossover method and constructed the mutant LZ-01 of blocking the abamectin precursor synthesis. The mutant was unable to synthesize avermectin. If cyclohexane carboxylate was added as a precursor in the fermentation process, the unknown component 1 can be obtained by HPLC. If it had the same peak time with the standard sample of doramectin, it can be initially identified as doramectin(CHC-B1).C5-O- methyl transferase-mediated encoded by ave D gene was involed in transforming avermectin "B" component to the "A" component. In this study, we used mutant LZ-01 and made ave D gene deletions by homologous double crossover method, then constructed mutant strain LZ-02 of ave D gene deletion. If an exogenous precursor cyclohexanone carboxylate were addedduring the fermentation process, the unknown synthesis of component 1 still could be detected by HPLC. But there was none of the productionof CHC-A component. The unknown Component 1 in the fermentation product was comfirmed as doramectin by LC / MS detection.ave C gene was important gene in the biosynthetic gene cluster of avermitilis, which involved in the transforming component doramectin "2" to component "1." In this study, we obtained the ave C* gene with 10 mutation points(including D48 E and E238D) by total gene synthesis, used LZ-02 as the starting strain and built the mutant strain LZ-03 with overexpression of ave C*. Although the percentage of production in the mutant doramectin(CHC-B1) and CHC-B2 were substantially unchanged among the fermentation products, the total production of the both had significantly increased.We optimized the fermentation conditions of the mutant strain LZ-01 and LZ-02. The results showed that the optimal fermentation time of doramectin was 311 h(approximately 13 d) and the best fermentation medium contained cottonseed meal and soybean meal as the complex nitrogen source. Meanwhile, the types of inorganic salts were studied minand the best one was Zn SO4. The concentration of phosphate was studied. When the concentration of KH2PO4 in the fermentation medium was 0.5 g/L, minthe yield of Doramectin was highest, reaching up to 12.98 μg / ml.