Many Actinomycetes are important producers of secondary metabolites with potent properties like antimicrobial, anti-tumor or immunosuppressive activity. However, their genetic engineering is often prohibited by mechanisms that prevent uptake and/or stable establishment of foreign DNA. CRISPR-Cas systems, for example, are known as adaptive immunity systems and recently also the involvement of CRISPR-Cas in other cellular processes including DNA-repair or cell-differentiation was described. Nevertheless, the knowledge about the function of CRISPR-Cas in Actinomycetes is still very limited.
The goal of this project is the functional analysis of CRISPR-Cas systems in antibiotic-producing actinomycetes. This includes adaptive immunity as well as functions beyond immunity, such as a potential role of CRISPR-Cas in regulation of antibiotic production. For this, the industrial aminoglycoside antibiotic producer Streptoalloteichus tenebrarius is used as model strain. It is particularly interesting, as its genome has 33 predicted biosynthetic gene clusters and three CRISPR-Cas systems (Region 1-3).
In order to perform phage infection assays, an extensive screening for phages infecting S. tenebrarius was conducted. It was shown, that S. tenebrarius is not included in the host range of the tested phages.
Investigation of the immunity function using primed assays requires the identification of the PAM sequence. Since genetic manipulation of S. tenebrarius is challenging, the CRISPR-Cas systems of S. tenebrarius were heterologously expressed in Escherichia coli. The cells were transformed with a PAM-library and the abundance of the individual PAM sequences after cas-gene induction was evaluated. No decrease in abundance was observed for all tested PAM sequences, suggesting that no PAM-recognition took place. Therefore, PAM-assays using Streptomyces coelicolor as a more closely related host are planned.
In addition, CRISPR-Cas deletion mutants in S. tenebrarius were generated. The deletion of CRISPR-Cas Region 1 led to a sporulation-deficient phenotype. This suggests an involvement of CRISPR-Cas in cell-differentiation processes in S. tenebrarius.