Genetic Engineering Of Doramectin Producing Strain

Doramectin is a new kind of Avermectins with high insecticidal activity and low toxic effect.It was produced by knock-out of the key genes bkd FGH responsible for the biosynthesis of the starter unit in Avermectins,with addition of the exogenous precursor cyclohexoic acid.Due to the lack of industrial strains for efficient Doramectin production in China,the demand for Doramectin in agriculture,animal husbandry,pet medicine and other aspects depends heavily on imports.In this study,the efficient production strains of Doramectin were tried to be constructed,which laid a research foundation for the localization of Doramectin.The bkd FGH gene deletion mutant strain Streptomyces avermitilisΔbkd constructed in the previous study was used as the starting strain,and doramectin was produced by addition of sodium cyclohexane carboxylate.In the industrial production of Doramectin,addition of a large amount of sodium cyclohexane carboxylate would greatly increase the cost.In order to enable cells to become self-sufficient,six genes related to cyclohexane coenzyme A biosynthesis from the Phoslactomycin B biosynthetic gene cluster were organized into two operons,which were respectively controlled by the constitutive strong promoters P_{erm E*}and P_{azi A4}.This genetic circuit was cloned into the Streptomyces integrative vector p SET152,to give rise to the precursor biosynthetic elements expression plasmid p CHC.The plasmid was introduced into the gene deletion mutant strain S.avermitilisΔbkd by conjugation.The constructed recombinant expression strain S.avermitilisΔbkd::p CHC can produce Doramectin without the addition of exogenous precursor,suggesting the total biosynthesis of Doramectin.Even though the yield of Doramectin is significantly lower than the result of exogenous addition experiment,indicating that the biosynthetic pathway of the precursor needs further optimization.Substitution of the promoters of the cyclohexane coenzyme A biosynthetic genes with the combination of P_{erm E*}and P_{kas O*}increased the yield of Doramectin by two times.Based on the biosynthetic pathway of Avermectin and the functional information of the key biosynthetic genes,the gene ave D encoding C5-oxymethyltransferase was knocked out from the mutant strain S.avermitilisΔbkd,to obtain the double gene deletion strain S.avermitilisΔbkdΔD.Ave D can convert the"B"components into the"A"components.HR-LC-MS confirmed that the"A"components as impurities were eliminated in the ave D gene deletion mutant strain,and the"B"components as active elements were significantly increased,which increased the Doramectin production by three times.The ave C gene is crucial for determining the proportion of"1"and"2"components.The ave Cmn gene containing 10 dot mutations was introduced into S.avermitilisΔbkd,and the ave Cmn gene expression strain S.avermitilisΔbkd::p CHC-EK-Cmn was constructed.HR-LC-MS detection found that the"2"components decreased and the"1"components increased,in which the Doramectin production increased by 1.4 times.Oligomycin and Doramectin compete the same substrate pool.By knocking out the positive regulatory factors olm RI and olm RII in the oligomycin biosynthetic pathway,the triple gene deletion mutant strain S.avermitililsΔbkdΔDΔolm R were constructed.Although the Doramectin yield did not significantly increase,the toxic Oligomycin was eliminated,which improved the safety of the Doramectin product.In this study,the total biosynthesis of Doramectin was achieved by molecular biology.The production of Doramectin was gradually increased by genetic engineering,which laid the foundation for cultivation and screening of the Doramectin industrial production strains in future.