A1 Production of recombinant proteins with Bacillus megaterium and Aspergillus niger
The production of recombinant proteins is an integral part of industrial biotechnology. Dependent on the properties and the desired purity of a given protein, the downstream processing represents a time-consuming and cost intensive production step. Thus, a production host capable of protein secretion into the culture broth is desirable to reduce production expenses. Both organisms Bacillus megaterium and Aspergillus niger, used in this “Sonderforschungsbereich” possess this feature of interest. Therefore, they are used as models to systematically study the production and export of recombinant proteins. In this context, the project A1 focuses on the development of genetic tools for both organisms which allow for an efficient production and secretion of proteins of interest. Diverse prokaryotic and eukaryotic glycosyltransferases like mannosyl-, fucosyl- and sialyltransferases are used as model enzymes.
Electron microscopic image of (A) Bacillus megaterium and (B) Aspergillus niger. Manfred Rohde, HZI, Braunschweig, 2007.
The aim of project A1 is to systematically analyse and optimise the whole protein production process at the level of transcription, translation, production and secretion. On transcriptional level, strong inducible promoters will be identified and their potency in regulated recombinant gene expression will be analysed. Moreover, the effect of genetic elements known to enhance mRNA stability will be analysed with respect to the yields of recombinant proteins. To increase the translational efficiency, rare tRNAs are identified and coproduced during recombinant protein production. Finally, various export leader-peptides of host proteins known to be strongly secreted will be applied to enhance the translocation of a given protein.
Altogether, all research subjects require a tight cooperation with other projects within the “Sonderforschungsbereich”: Data of the genome of B. megaterium (derived from project Z) will be needed e.g. for the identification of rare tRNA genes while results of the proteome analysis of A. niger (generated in project B4) are of central importance for the identification of alternative export leader-peptides to enhance protein secretion. The identification of possible bottlenecks within the production process of recombinant proteins with the help of systems biological modelling (in cooperation with projects B3 and B4 for A. niger and B8, B9 and B10 for B. megaterium, respectively) will be used for a rational optimisation of both model organisms. Therefore, our project additionally focuses on the development of suitable genetic systems enabling a directed, stable inactivation of chromosomal genes.