Construction Of The Heterologous Expression Library And Screening For Functional Genes From Streptomyces Griseoruber AL7

Streptomyces are important antibiotic producers. S. griseoruber AL7 is isolated from soil sample collected in Shennongjia, Hubei, and showed high activity against various plant pathogenic fungi and bacteria. The type of antibiotics produced by it has not yet identified. In the research of secondary metabolism, biosynthesis gene clusters are first cloned into E. coli strains, then transferred into actinomycetes to make gene disruption or heterologous expression by another E. coli strain.There is no such strain which can finish these two tasks at the same time. So we have constructed one non-restricting and methylation deficient E. coli strains, XL1-blue MR Adcm, by successively deleting the DNA methylation genes dcm from the cosmid-based cloning host XL1-blue MR.S. griseoruber AL7 genome scanning shows that it’s 9.9 Mb, and there are 197 largecontigs. The biggest contig00171 is 464 kb. There are 16 PKS,2 NRPS and 1 terpene genes in the genome and the contig00171 contain 1 each the 3 kinds meanwhile.We constructed the high-quality cosmid library which the average size of inserts is 40 kb by multiple PFGE extraction of DNA. The host of the library is LP3, that is XL1-blue MR△dcm with pUZ8002. With the method of high-throughput conjugation of cosmid library into expression host S. lividans TK24, we get the expression library.The expression library were fermentated in MS, R5, and NA medium, with Rhodotorula, Mycobacterium smegmatis and S. coelicolor M600 as indicator bacteria for mass bioassay. The 52 clones identified from all the preliminary screened clones. Aignment the 52 cosmids terminal sequencing with the genome showed that these cosmids are not in the same contig, and there are three cosmids situated in contig00171. Verify the stability of biological activity by replacement of the expression host. Analysis the relevant genes may cause antibacterial activity based on gene annotation.In this studty, the use of the new method that high throughput heterologous expression of the high quality genome library offers an effective model for large-scale screening research, such as gene cluster and secondary metabolites.