Application Of A Double-Reporter System And A Transposition System In Streptomyces Avermitilis

Avermectin, a series of macrocyclic polyketide produced by Streptomtces avermitilis, is acknowledged as a powerful antiparasite,acaricidal, insecticidal drug. It is widely used in agricultural, veterinary and medical fields for its particular activity and property of high efficiency, low toxic and no residual. Though as an industrially important microorganism, only a few useful tools were developed for genetic manipulation in Streptomtces avermitilis, which restricts the research on regulation of biosynthesis of avermectin.A double-reporter system harboring two markers, neo and actlI-4/actâ–³actâ…¡-4, was established in this study, with the attempt to indicate biosynthesis of avermectin by it. neo and actâ…¡-4, without their original promoters, were inserted into the gene cluster for avermectin biosynthesis, while actâ–³actll-4 was integrated into the genome. The expression of the double-reporter system was relied on the expression of the gene cluster for avermectin biosynthesis. Thus, the double-reporter marked strain Streptomtces avermitilis PX1 was endued with resistance to kanamycin for neo, and the potential to produce actinorhodin for actâ…¡-4/actâ–³actâ…¡-4. However, when this system was tested in detail, neo was successfully expressed while actâ…¡-4/actâ–³actâ…¡-4 was not. Then the double-reporter system was employed to screen for high producers of avermectin by mutagenizing PX1 strain with NTG. By selecting with kanamycin,20.00% mutants were obtained with higher avermectin titres, while 5.43% were bald and deep blue with production of avermectin almost abolished.Transposon, which was a useful tool in investigating into function of genes, was acclaimed. In this study, a series of vectors for transposition were constructed according to the genetic characteristics of Streptomtces avermitilis. Besides the suicide vector pHL734, which could hardly transformed into Streptomtces avermitilis by conjugation, vectors carrying oriSCP2* or oripIJ10l were constructed. oriSCP2* and oripIJ101 are unstable replication origins in streptomycetes, with average loss frequencies of 76.00% and 99.96%, respectively. Two markers, codA and I-Scel, were introduced into the transposition system individually to enhance the efficiency for selection of mutants. A negative selection system based on codA, which may perform well in Streptomyces coelicolor, was retarded in Streptomtces avermitilis due to its vigoroso resistance to 5-fluorocytosine.I-Scel, however, may be a potent marker used in the transposition system. The strategy of high-throughput screening for mutants by transposition in Streptomtces avermitilis was challenged with continuous difficulties, thus a lot of work has to be done to modify the transposition system till an efficient one is finally erected.In addition, the l-Scel marker was also applied to construct a vector for gene disruption in Streptomyces. It has a comparative advantage over traditional vectors for no marker is left in the genome after the target gene was disrupted. melC2-melC1, which is one of the gene clusters responsible for biosynthesis of melanin in Streptomtces avermitilis, was knocked out by using this vector, resulting in a frequency of 9.09% of desired mutants.