From gene to product - Development of biotechnological processes by integrating genetic and engineering methods

The production, secretion and purification of recombinant proteins in eukaryotic as well as prokaryotic systems are major topics of industrial biotechnology. During the first application period (2001-2004) microbial expression systems for the filamentous nonpathogenic fungi Aspergillus niger as well for the Gram positive bacterium Bacillus megaterium were established. It has an efficient secretion systems which makes it applied in industrial production processes. The proteins produced by this organism possess posttranslational modifications. B. megaterium is nonpathogenic and able to metabolize numerous carbon sources. Further, it shows a great potential for protein secretion and, important for recombinant protein production, has a stable plasmid replication system. The major goal of the first funding period of SFB 578, the establishment of a system for the secretion of the recombinant proteins, which included different homologous and heterologous glycosyltransferases into the growth medium was reached. During the second funding period (2004-2008) the expression systems for both organisms were further enhanced. This included the integration of different small affinity tags for purification of the intra- as well the extracellular produced recombinant proteins. Proteins could now be easily purified and used for different activity tests. Here, new glycosyltransferases recombinantly overproduced in A. niger and B. megaterium were characterized, respectively [Homann et al., 2009, Zuccaro et al., 2008]. New promoters and leader peptides were identified by secretome analysis for both hosts. These promoters could be induced using simple cheap carbon sources. For B. megaterium a further promoter system based on a two vector system was developed. With this phage dependent system it was possible to produce recombinant proteins intracellularly.