| Streptomyces produce various secondary metabolites, which are widely used in medicine, agriculture and veterinary medicine. Streptomyces coelicolor is the model organism for study of bacterial secondary metabolism. In this study the Streptomyces transposition mutagenesis system was optimized, developed into a functional genomics research tool and applied to genome-wide functional screening in S. coelicolor.Firstly, the Tn5 transposase gene was engineered to improve its product’s transposition activity by site-directed mutagenesis, and to increase its expression by codon optimization and utilization of an ermE* strong promoter. Using this engineered Tn5 transposase gene, as well as the mosaic ends, several mini-Tn5 transposon vectors were constructed. Among them, pHL724 and pHL734 mediated transposition efficiently in S. coelicolor, with which the transposition frequency could reach as high as 10-6/cfu. represented about 300 mutants per plate for a typical conjugation. The only difference between that two vectors is the location of the E. coli plasmid origin of replication (ori), which is located within the mini-Tn5 in pHL734, but outside of the mini-Tn5 in pHL724. Randomly selected mutants were showed to contain only one copy of Tn5 insertion in their chromosomes by Southern blot. The inserted loci of some mutants were rescued and sequenced to demonstrated the presence of the 19 bp transposon end sequences at both ends of the mini-Tn5 and 9 bp directed repeat of target sequence, suggesting the occurrence of typical Tn5 transposition.To screen for functional genes, a transposon mutagenesis library of 60,000 mutants of S. coelicolor was constructed with pHL724.127 mutants with significant phenotype changes were selected. Insertion sites of 38 mutants were located by rescuing and sequence, showing that 4 genes were inserted more than once in 13 of these mutants, and same phenotype was produced by mutations in each gene.The pHL724-derivated mutants were not ideal for rescuing cloning due to the lack of an ori site within the mini-Tn5. For this reason, a transposon mutagenesis library of 50,000 mutants was constructed using pHL734, which has an internal ori site. A total of 1478 mutants with phenotype change were selected, with their insertion loci rescued, and their inserted sites sequenced. It was showed that the Tn5 transposition insertion has a slight preference at 19 bp sequences around the target site. In total,240 genes were inserted more than once in 725 of these mutants.184 mutants were white (whi),392 were bald (bld),440 overproduced actinorhodin (ACT),579 mutants produced fewer or no ACT.Thirty eight ACT-overproducing mutants were chose to perform multi-parental genome shuffling (GS) experiment to study the feasibility of GS. Protoplasts of 38 mutants were made and fused by PEG-mediated fusion experiment, the fusion mixture was diluted and spread on R5 medium for regeneration. The regenerated clones were subjected to two studies. In the first study,96 single colonies were subjected to PCR to trace the specific transposon-tags of each parent. It was showed that 1 colony contained 6 tags,1 contained 5 tags,4 colonies contained 4 tags,15 colonies contained 3 tags, implying that these colonies arised from fused cell. Twenty colonies from each of these 21 fusion strains were subjected to PCR to trace the tags. However, no one contained two or more tags, suggesting that despite of cell fusion, recombination between tags had not occurred. In the second study, eight colonies which produced even more ACT than all parents were selected from the multi-parents fusion plates, and diagnosed by PCR. It was showed that all 8 strains had only one tag, suggesting that the ACT over-production was not resulted from GS. |
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