Abstract
Bacillus megaterium, the "big beast," is a Gram-positive bacterium with a size of 4 × 1.5 µm. During the last years, it became more and more popular in the field of biotechnology for its recombinant protein production capacity. For the purpose of intra- as well as extracellular protein synthesis several vectors were constructed and commercialized (MoBiTec GmbH, Germany). On the basis of two compatible vectors, a T7 RNA polymerase driven protein production system was established. Vectors for chromosomal integration enable the direct manipulation of the genome. The vitamin B(12) biosynthesis of B. megaterium served as a model for the systematic development of a production strain using these tools. For this purpose, the overexpression of chromosomal and plasmid encoded genes and operons, the synthesis of anti-sense RNA for gene silencing, the removal of inhibitory regulatory elements in combination with the utilization of strong promoters, directed protein design, and the recombinant production of B(12) binding proteins to overcome feedback inhibition were successfully employed. For further system biotechnology based optimization strategies the genome sequence will provide a closer look into genomic capacities of B. megaterium. DNA arrays are available. Proteome, fluxome and metabolome analyses are possible. All data can be integrated by using a novel bioinformatics platform. Finally, the size of the "big beast" B. megaterium invites for cell biology research projects. All these features provide a solid basis for challenging biotechnological approaches.