Rheinisch-Westfaelische Technische Hochschule Aachen

Lehrstuhl für Biotechnologie

(This partner has entered the project on 1/5/2009)

Contact details

Leading scientist: Prof. Dr. Ulrich Schwaneberg

Address: Templergraben 55, 52056 Aachen, Germany

Phone: +49 241 80 24176

Fax: +49 241 80 22387

Web address: www.biotec.rwth-aachen.de

Qualifications and experience

The partner at the RWTH Aachen has its core expertise in biocatalyst engineering with a focus on method development for directed protein evolution. Development of novel methods comprise methods for generating diversity on the gene level (SeSaM: Sequence Saturation Mutagenesis) and novel high-throughput screening systems for oxygenases. The experimental part (Prof. Schwaneberg’s group) is complemented by the computational expertise of Dr. Roccatano’s group at the Jacobs University Bremen which was the previous host of the Schwaneberg group. Dr. Roccatano develops bioinformatics tools for benchmarking mutagenesis methods and study, using computer simulation methods, structural and dynamic properties of monooxygenases. A fruitful collaboration between the JUB/RWTH-application team is documented by six jointly published manuscripts, five of them on P450 BM-3 which will be used in WPs as model system for throughput technology development (WP3, WP4).

With the Sequence Saturation Mutagenesis (SeSaM) method we achieved a breakthrough in random mutagenesis by eliminating mutagenic hot spots, incorporating single nucleotide analogs over the whole gene, and achieving for the first time subsequent mutations (16 %) for a polymerase-based mutagenesis method. We further developed a statistical method called MAP (Mutagenesis Assistant Program) to provide protein engineers with a tool to benchmark current random mutagenesis methods. MAP investigates the consequences of mutational bias on the protein level and can guide development of random mutagenesis methods.  

Facilities

The lab infrastructure at the RWTH Aachen is dedicated to directed protein evolution and comprises three plate readers, two liquid-handling machines, a flow cytometer with sorting function, specialized screening equipment such as solid dispenser for microtiter plates, Deutz tool, and unpublished in-house pre-screening systems on agar plates and evaporation-based screening systems. For protein production and characterization six fermenters (50 L, 16 L, 3x 5 L), a French-press, and standard purification and analytical tools (Äkta, GC, HPLC) are available.

Role in the project

Workpackage 2, 3 and 4.

Key publications/patents

• Tee KL, Schwaneberg U (2006) A screening system for directed evolution of epoxygenases: validation reveals importance of position 184 in P450 BM3 for stereoselective styrene epoxydation, Angewandte Chemie, 45, 5380
• Wong TS, Roccatano D, Zacharias M, Schwaneberg U (2006) A statistical analysis of current random mutagenesis methods for directed protein evolution, J. Mol. Biol., 355, 858 (cover page)
• Nazor J, Schwaneberg U (2006) Laboratory evolution of P450 BM-3 for mediated electron transfer ChemBioChem, 7, 638
• Wong TS, Zhurina D, and Schwaneberg U (2006) The diversity challenge in directed protein evolution, Comb. Chem. High Throughput Screen., 9, 271
• Wong TS, Tee KL, Schwaneberg U (2004) Sequence Saturation Mutagenesis (SeSaM): A novel method for directed evolution, Nucleic Acids Res. 32, e26. (filed as patent WO 2005/035757).