Engineering of artificial enzyme complexes mediated by heterospecific coiled-coil zippers or synthetic protein-protein interaction domains in Saccharomyces cerevisiae

Cora A. Mignat*, Thomas Thomik*, Eckhard Boles, Mislav Oreb

Institute of Molecular Biosciences, Goethe-University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt am

Main, Germany, +49 69 798-29331

Mignat@bio.uni-frankfurt.de, thomik@bio.uni-frankfurt.de
In metabolic engineering approaches, yield and synthesis rate of a desired product are often compromised by diversion of precursor compounds by competing pathways. One possibility to circumvent this problem is the so called substrate channeling, whereby the metabolite is directly passed between the enzymes. An additional benefit of this strategy results from increasing the local concentration of the metabolite, which leads to improved substrate-saturation of the enzymes and, consequently, faster reaction kinetics.
In order to develop a toolbox for substrate channeling in engineered S. cerevisiae, we fused several enzymes involved in the central carbon metabolism with protein-protein interaction modules. For establishing a direct interaction between enzymes, we used heterospecific coiled-coil zippers. As an alternative approach, modular scaffold proteins, comprising serially arranged protein-protein interaction domains, can be used to mediate complex formation. This strategy has been successfully employed in E.coli with the production of mevalonate (Dueber et al. 2009). The effect of fusing protein-protein interaction modules to the enzymes on their activity and productivity of selected pathways is discussed.
Financial support by the German Federal Ministry of Food, Agriculture and Consumer Protection following a decision of the German Bundestag is gratefully acknowledged (project ECO-FERM, FKZ
22031811).

Presenting authors

*contributed equally