Gene Mining Of Ansamycin And Aminocyclitol Antibiotics In Actinomycetes

Actinobacteria are important sources of bioactive natural products. Traditional methods for natural product screening from fermentation broths include many steps and are time-consuming, inefficient and without premilinary information of the expected compounds. The booming molecular biology and biosynthetic technology shed new lights on the isolation of particular compounds. By gene methods we had screened830strains of actinobacteria for candidates with the potential of producing ansamycins and aminocyclitols.According to the special genes in the biosynthetic pathway of ansamycins and aminocyclitols respectively, we designed degenerate primers for gene screening by polymer chain reaction from different actinobacteria’ genome. After the sequencing of PCR products, we analyzed the16S rRNA evolutionary relationship of those gene-positive ones. Based on the phylogenetic tree, strains in unique branch were chosen and the transcriptional levels of specific genes were tested. Furthermore, methods such as Southern Blot, genome scann and fermentation media optimization were used to identification and isolation the target compounds.3-amino-5-hydroxybenzoic acid (AHBA) is the precursor of the synthesis of ansamycins, and the AHBA synthase is in the final step of the reaction. In this study, we have screened16AHBA synthase gene positive actinobacteria from600stored strains in our lab by using AHBA-F/R2premiers which have been designed before. Then we designed a pair of new degenerate primers AHBA-A’F/CR based on derived AHBA synthases including mitomycin’s. Other37positive actinobacteria have been screened from the600stored ones, as well as33from230newly ones. Sequencing results of these positive strains showed plenty of polymorphisms in AHBA synhthase gene. Among them,16S rRNA genes from33strains were anlayzed by phylogenetic trees, and22of them were conducted translational level test of AHBA synthase gene. The results showed that only A00941, A00968can express AHBA gene in such condition, while A00941showed better. We then used AHBA as substrate to feed these two strains and analyzed their fermentation products by HPLC. Some peaks of corresponding products were observed appearing or growing higher after feeding. After genome scan, bioinformatic analysis of the putative ansamycin biosynthetic gene cluster revealed6AHBA-related biosynthesis genes and partial polyketide synthetic genes in one scaffold. Subsequent work of fermentation optimizing of A00941is ongoing.In the process of screening aminocyclitol antibiotics, we designed degenerate primers CYC-AF/DR according to homologous protein sequence of2,5-alcohol ketone synthase.20positive strains have been discovered and sequenced, while5of them were homologous with aminocyclitol synthease genes in Genbank.