Studies Of Genomic Transformation System And Functional Genes Of Strain Strepomyces Ahygroscopicus Var. Wuyiensis CK-15

Wuyiencin, produced by Streptomyces ahygroscopicus var. wuyiensis, is a broad-spectrum andhigh effective antibiotic with low toxicity. In the early days, the way which to achieve new strains withhigh yield mainly are traditional ways, such as induced by physical and chemical methods. Nowadaysthose approaches cannot meet the manufacture. The researches about molecular biology of antibioticgrow fast in recent years. For changing the strain that produced wuyiencin in molecular level, we did aresearch on construction of genetic transformation of S. ahygroscopicus var. wuyiensis CK-15firstly.Then, based on gene sequence that already got, we screened the genomic DNA library to get completebiosynthetic gene cluster. The mean results in this study are as follows:1. Tests showed that there was no plasmid in S. ahygroscopicus var. wuyiensis CK-15for only thegenome exists. This result shows that the whole biosynthetic gene cluster of S. ahygroscopicus var.wuyiensis CK-15exists on the genomic gene.2. Construct a genomic transformation system that is suitable for S. ahygroscopicus var. wuyiensisCK-15by conjugal transfer: heat the spores of S. ahygroscopicus var. wuyiensis CK-15for10min under50℃, cultured for2.5h under37℃, mixed with ET12567(with PUZ8002), cultured for16~18h,covered with l mL aqueous solutions of nalidixic acid and antibiotics, cultured for5~7d under30℃.And the construction of genomic transformation can lay the foundation for verification of gene function.3. Partial gene of wuyiencin biosynthetic cluster was cloned. After bioinformatic analysis, wegot14independent orf: one gene (orf1)that is related with the formation of nucleoid, threetranscription regulators (orf2, orf3, orf4),5PKS gene (orf6~9, orf14), one thioesterase gene(orf5),one glycosyltransferase gene (orf10), one aminotransferase gene (orf11), two modifierand signal transduction gene (orf12, orf13)The construction of genomic transformation of S. ahygroscopicus var. wuyiensis CK-15is afundamental work for gene operation of the biocontro agent. Based on this, the synthesized functionalgenes may be transformed into CK-15to obtaine its mutants and to verify the antagonistic effects inthese transformants. Consequently, S. ahygroscopicus strains with significantly higher wujiencinproductivity may be obtained through engineering breeding.