Directed development of Bacillus megaterium for applications in recombinant protein production

The work presented in this thesis focused on the directed genetic optimisation of the Gram-positive bacterium Bacillus megaterium for recombinant protein production. For this purpose gene expression was systematically analysed and improved on transcriptional and translational level. A novel gene expression system was developed based on the highly processive RNA polymerase of the bacteriophage T7. Using this system, the green fluorescent protein (GFP) was successfully produced intracellularly to significant higher amounts (49.7 mg l-1) compared to the reference using the well-established xylose-inducible promoter system (7.2 mg l-1). Interestingly, the application of the T7 RNA polymerase dependent gene expression system for the secretory production of the exoenzyme levansucrase resulted in significantly lower protein yields possibly due to an overload of the secretion machinery and the subsequent induction of stress responses. Furthermore, a versatile codon test system was developed and individual codons were experimentally identified which are limiting the efficiency of the translational process in B. megaterium. The subsequent identification of a rare tRNA gene and its coexpression significantly elevated the rate of translation of corresponding codons into the respective polypeptide chain as estimated by the formation of the model protein GFP. First insights into the molecular basis for the development of natural competence in B. megaterium were obtained. Overproduction of the putative central transcription factor ComK for competence development resulted in partial cell lysis. Nevertheless, for the first time the ability of B. megaterium to develop a functional machinery for DNA-uptake and -integration was demonstrated. As a proof of concept two eukaryotic proteins of industrial importance, a human keratin-binding domain and a fungal hydrophobin, were produced with B. megaterium in cooperation with the BASF SE. Multiple parameters like codon usage optimisation, the nature of the signal peptide or the cultivation conditions were analysed in order to improve the yield in secretory production of the respective proteins. It was shown that intracellular aggregation and degradation severely hampered quantitative production of the keratin-binding domain. The fungal hydrophobin on the other hand could successfully be produced, secreted and purified to homogeneity and its surface active properties were demonstrated.