Screening High-Yielding Streptomyces Avermitinlis And Resconstructing Avermectin Polyketide Synthases

In this study, the wild-type avermectin-producing strain Streptomyces avermitilis K310 was used as the start strain. The essential fermentation parameters about this strain were determined to prepare for the next work. S.avermectilis K310 was cyclically mutated by NTG and HNO2 consecutively. Uniform design was introduced to optimize the fermentation medium prescriptions. A high-yielding mutant S. avermitilis SY311 was screened out. The total titer in 40ml shake flask fermentation was increased from 100μg/ml to 4500μg/ml and the titer of effective composition B1a in 20ml shake flask fermentation was 1500μg/ml, which have reached the anticipated object.The enoylreductase (ER) PCR primers were designed from the conservative sequences in the ER genes. With the primers, the gene fragment milDH2-ER2 which may contain milbemycin PKS modular2 DH2-ER2 was gained from the plasmid (22) g-8-E-10. MilDH2-ER2 was sequenced and analysed with the Frameplot and the BLASTp and with the methods on bioinformations the complete milbemycin PKS modular2 DH2-ER2 was made sure. It have successfully submitted to the GenBank and the accession number is AY396043.Streptomyces avermitilis polyketide synthase genes were reconstructed applying the principle of combinatorial biosynthesis. The heterozygosis PKS modular2 gene was attained and through gene recombination the engineering strain could directly produce ivermectin, which is the drug of first choice for the treatment of lymphatic filariasis and river blindness in...