The aim of the doctoral thesis was to develop new methods for the detection and increase of the concentration of heterologous extracellular proteins in the bacterium Streptomyces rimosus, and to test several different approaches and evaluate their usefulness. In the initial part, we simplified the existing methods of detecting extracellular proteins by using various fluorescent proteins. Through exoproteome analysis, we obtained new information about the most abundant extracellular proteins, which we then used in the design of expression cassettes. By adding a reporter system, we optimized the CRISPR-Cas9 molecular tool and the procedure for performing deletions with this tool in the bacterium S. rimosus. The developed Cas9_SD_GusA system is also useful for engineering other bacteria where the CRISPR tool does not work well. We also designed and tested a directed evolution system based on erythromycin esterase EreB, which enables the increase of the target protein amount. With the optimized CRISPR system, we performed deletions of larger genome segments of S. rimosus and created a metabolically purified strain of S. rimosus ATCC Δotc Δ145kb, which does not produce any of the main metabolites of this bacterium (rimocidins, oxytetracycline) and is therefore theoretically more suitable for the industrial production of heterologous proteins and other products. In the constructed strain, we then tested the functionality of versions of the fusion protein mCherry-kaminizin and evaluated the performance of this reporter system for the production of the industrially relevant protease Kaminizin. The results of the work performed demonstrate the advantages and limitations of each of the tested approaches and provide a basis for further development of the host organism S. rimosus for the production of heterologous proteins and other products.
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