The widely distributed phylum Actinobacteria constitutes to one of the largest and most diverse groups of microorganisms in nature. It is notable for their remarkable potential in the degradation of diverse compounds. Moreover, they are versatile producers of bioactive natural products. Consequently, actinobacteria hold considerable economic significance and utility for various applications. Due to their capability to catalyze a diverse range of enzymatic reactions, uncommon in other organisms, they become attractive candidates for biotechnological applications.

Recently, the actinobacterium Gordonia rubripertincta CWB2 and related strains G. rubripertincta CWB7 and Rhodococcus erythropolis CWB15 were isolated. Earlier studies demonstrated strain CWB2"s ability to degrade the plastic precursor styrene and related compounds. In contrast to other styrene degraders, it employs a "hybrid" pathway, resulting in the central metabolite phenylacetic acid, a compound with high industrial demand. CWB2 also showed its ability to produce ibuprofen in a co-metabolic process. Noteworthy is the involvement of glutathione and glutathione S-transferases in these catabolic processes. The same degradation pathway was found in related strains CWB7 and CWB15.

To understand the role of involved enzymes, this study aims at the generation of knockout strains and the establishment of genetic tools for actinobacterial protein production. Both strains were characterized to enable an easy operation.

Therefore, genomic, morphological, and physiological analysis was initially performed. Efficient protocols for electrocompetent Gordonia and Rhodococcus cells were established. Moreover, various protocols for knockout were tested. For protein production, expression plasmids were tested with sfGFP as reporter gene regarding their transformation efficiency and its protein yield.

Overall, this study presents not only the morphological and physiological characterization of recently found G. rubripertincta CWB7 and R. erythropolis CWB15, but also the understanding of involved enzymes. Furthermore, these strains were explored as alternative expression strains for protein production.