Characterization of the sporulation control protein SsgA using an efficient method to create and screen random mutant libraries in streptomycetes

Filamentous actinomycetes are commercially widely used as producers of natural products. However, the mycelial life-style of actinomycetes has been a major bottleneck in their commercialization, and screening is difficult due to the poor 5 growth in microtitre plates. We previously demonstrated that the enhanced expression of the cell division activator protein SsgA results in fragmented growth of streptomycetes, with enhanced growth rates and improved product formation. We here describe a novel and efficient method to create, maintain and screen mutant libraries in Streptomyces, and its application for the functional analysis of 10 S. coelicolor ssgA. The variants were amplified directly from deep-frozen biomass suspensions. Around 800 ssgA variants including single amino acid substitution mutants for over half of all SsgA residues were analysed for their ability to restore sporulation to an ssgA mutant. The essential residues were clustered in three main sections, and hardly any were in the carboxy-terminal 15 third of the protein. The majority of the crucial residues is conserved among all SsgA-like proteins (SALPs). However, the essential residues L29, D58 and S89 were conserved only in SsgA orthologues and not in other SALPs, suggesting an SsgA-specific function. AC CE PT ED on Jne 4, 2017 by gest ht://aem .sm .rg/ D ow nladed fom