Activation of Silent gene clusters in Streptomyces species
Open Access
- Author:
- Ferreira, Joao
- Millennium Scholars Program:
- Biochemistry and Molecular Biology (B M B)
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisor:
- Timothy Charles Meredith, Thesis Supervisor
- Keywords:
- Streptomyces
Secondary metabilite
natural products
Silent gene clusters - Abstract:
- Streptomyces is a genus of Gram-positive bacteria commonly found in soil environments. An important attribute of Streptomyces is its ability to produce secondary metabolites with different bioactive properties, including antibacterial compounds. Around 50 percent of the antibiotics currently in use are derived from the secondary metabolites of streptomyces. These metabolites are encoded by large biosynthetic gene clusters within the bacteria’s genome, and a single species can have more than 30 gene clusters. Whole genomic sequencing has identified silent gene clusters encoding secondary metabolites of unknown biological activity. Expression of these gene clusters and characterizing their products could serve as a source of novel antibiotics. The goal of this research is to explore a method of activation of silent gene clusters, through tying secondary metabolite synthesis to primary metabolite production and thus necessary for growth and development. Through the use of CRISPR plasmids, the thyX gene was excised from S. avermitilis creating an auxotroph. The thyX gene was reinserted adjacent to silent secondary metabolite gene cluster of interest, and once removed from the thymidine/thymine supplemental media, S. avermitilis develops suppressor mutations that allow it to now express thyX along with the gene cluster of interest to be expressed. In a proof of principle study, suppressor mutants were generated for the SA20 (encoding oligomycin) and SA7 (encoding avermectin) gene clusters. Suppressor mutations were then generated for each gene cluster and the production of their compound was screened with a yeast bioassay for oligomycin and thin layer chromatography for avermectin. Sequencing of suppressor mutants identified most mutants engaged in local chromosome duplication of the thyX gene to survive genetic pressures. Another mutant possessed a mutation at the rpme1 gene locus. Future studies are needed to identify a mutant that produces an overabundance of secondary metabolites in order to widely apply this method in silent gene cluster activation.