Development of versatile bacterial expression systems for use in recombinant protein production, metabolic engineering, and systems biology

Recombinant proteins and biologically active bacterial secondary metabolites are high-value industrial products. Quite often, however, their efficient production in industrial bioreactors is hampered by low levels of expression of the corresponding genes. In the current proposal it is suggested to address this problem through development of versatile vectors and optimized bacterial host strains. For efficient recombinant protein expression, a system comprising RK2-based broad- host-range vectors and the i nducible Pm/xylS promoter system will be further developed based on recent discoveries concerning the influence of promoter and 5' untranslated mRNA sequences on the level of protein expression in various hosts. The utility of the new systems will be test ed in medium-scale fermentations at SINTEF for production of medically relevant proteins. New RK2-based and actinophage-based vectors for expression of entire gene clusters for biosynthesis of secondary metabolites, as well as bacterial hosts capable of a ccommodating the vectors and expressing biosynthetic genes at high level will also be developed. The latter will be tested for efficient production of a bacterial secondary metabolite discovered in the course of our ongoing bioprospecting project in colla boration with SINTEF. Taken together, these novel systems shall provide efficient tools for biotechnological and pharmaceutical industry dealing with production of recombinant proteins and antibiotics.