Project Title: Directed Evolution of heme-dependent monooxygenases for the production and formulation in kit applications

Supervisor: Dr. David Schönauer, SeSaM-Biotech, Aachen, Germany

Objectives: Monooxygenases represent a highly valuable enzyme class with numerous applications in the chemical, flavor and fragrances and especially pharmaceutical industry in drug discovery. Over the course of the PhD project, the Early-Stage-Researcher 6 (ESR) will perform Directed Evolution of selected heme-dependent monooxygenases and become familiar with their production as well as whole-cell based catalysis to measure monooxygenase performance. The second half of the project will focus on stability studies of improved monooxygenase mutants with regard to formulation in kit applications. The project has strong industrial focus and throughout the 3-year program, the ESR will study several months at the premises of project partners to complement the scientific training.

Methodology: The ESR will learn and apply a broad range of molecular biology methods for the generation of monooxygenase mutant libraries by the patented SeSaM technology and the cloning and transformation in E. coli or P. pastoris expression systems. Cell culture for optimized monooxygenase production in shake flasks and optionally fermenters will be an essential part of the project as well as whole-cell biocatalysis to assess relevant monooxygenase parameters like activity, stability and substrate promiscuity. Eventually, the ESR will become familiar with analytical tools as TLC, GC or HPLC for product measurement and gain expertise in formulation of monooxygenases for kit applications. Additionally, the ESR will receive basic training in computational biology through a secondmend at partner institutions.

This scientific training will be complemented by soft-skill courses and events organized throughout the 3-year program within the network of the partner institutions, including entrepreneurship, scientific writing, preparation and presentation of posters and communication of research results to the public.

Expected Results: Development of a hydroxylation kit employing monooxygenases for commercialization.

The doctorate degree for the ESR6 will be awarded by RWTH Aachen University, one of nine elite universities of Germany.