SPP 1170:  Directed Evolution to Optimise and Understand Molecular Biocatalysts

• Subject Area: Biology
• Term: from 2004 to 2010
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5472117

All cellular reactions are catalysed by proteins (enzymes) or RNAs (ribozymes). Life cannot be understood without understanding how these molecular biocatalysts function and how they are stabilised. Within the Priority Programme the novel approach of directed evolution will be used to generate novel molecular biocatalysts with tailored properties. This will provide new insights into the relationship between sequence, structure and function of enzymes and ribozymes, and help better understanding their natural evolution. Moreover, directed evolution will be used to generate enzymes with useful properties for industrial applications.
Directed evolution imitates millions of years of natural evolution on the short time scale of laboratory experiments and allows to manipulate biocatalysts in a completely new fashion. In a directed evolution experiment, first large gene libraries are generated by random mutagenesis. From these libraries novel enzymes or ribozymes are isolated by elaborate screening or selection techniques, followed by their in-depth characterisation by state-of-the- art tools of protein and nucleic acid chemistry, enzymology, structural biology and theoretical chemistry. In contrast to the more traditional rational design, directed evolution does not require a detailed a priori knowledge of the structure or mechanism of a biocatalyst. It is, moreover, particularly instructive because it can provide unexpected solutions that go beyond the original hypothesis.
Specifically, the Priority Programme aims to answer the following questions:
-- What are the possibilities and where are the limits for optimising enzymes and ribozymes?
-- Are there common principles among enzyme- and ribozyme-mediated catalysis?
-- How much conformational stability and flexibility is required for optimal catalysis?
-- What is the structural basis of substrate- and stereo-selectivity of molecular biocatalysts?

• DFG Programme: Priority Programmes

Projects

• A general approach for in vivo ultra-high-throughput screening of engineered hydrolases (Applicant Kolmar, Harald )
• Active site recruitment in an ancestral, bifunctional ProFAR/PRA isomerase (Applicant Wilmanns, Ph.D., Matthias )
• Computational insights into directed evolution of substrate specificity and structure based combinatorial library design (Applicant Bocola, Marco )
• Coordination of the SPP 1170 (Applicant Sterner, Reinhard )
• Directed Evolution as a Means to Optimize and Understand Molecular Biocatalysts (Applicant Reetz, Manfred T. )
• Directed evolution of DNA methyltransferases (Applicant Jeltsch, Albert )
• Directed evolution of limonene epoxide hydrolase to engineer its substrate- and enantioselectivity (Applicant Arand, Michael )
• Directed Evolution of the Dnak/ClpB chaperone system - towards substrate specific optimization and complementation with orthologous systems (Applicant Reinstein, Joachim )
• Directed evolution of the S1 subsite specificity of trypsin (Applicants Bordusa, Frank ;  Stubbs, Milton T. )
• Directed evolution of transaldolases for novel specificities (Applicants Fessner, Wolf-Dieter ;  Sprenger, Georg )
• Directed evolution of transaldolases for novel specificities (Applicant Sprenger, Georg )
• Directed evolution to understand enzyme function of almost protein independent bacterial RNase P RNA versus largely protein-dependent archaeal RNase P RNA (Applicant Hartmann, Roland K. )
• (Focused) Directed Evolution to Unterstand Relationships in alpha/beta-Hydrolase-Fold Enzymes (Applicant Bornscheuer, Uwe T. )
• Generation of error-prone polymerase variants using directed evolution: Molecular basis of replication fidelity (Applicant Brakmann, Susanne )
• Generation of homing endonucleases of pre-defined specificity by directed evolution (Applicant Wende, Wolfgang )
• How general are the determinants of stability and activity? An evolutionary study with perturbed beta-lactamases (Applicant Müller, Kristian )
• Improving the catalytic activity of a thermostable anthranilate phosphoribosyl transferase by directed evolution (Applicants Seidel, Claus ;  Sterner, Reinhard )
• Improving the catalytic activity of a thermostable anthranilate phosphoribosyl transferase by directed evolution (Applicant Seidel, Claus )
• In vitro recapitulation of the evolution of tRNA nucleotidyltransferases (CCA-adding enzymes) (Applicant Mörl, Mario )
• In vitro selection of enzymes by mRNA-Display (Applicant Famulok, Michael )
• In vitro selection of thermostable ribozymes (Applicant Jäschke, Andres )
• Lipase function and folding analyzed by in vivo high throughput screening of combinatorial enzyme libraries (Applicants Jaeger, Karl Erich ;  Kolmar, Harald )
• Origins of differential response to DNA lesions by DNA polymerases: Insights throuth directed enzyme evolution (Applicant Marx, Andreas )
• Sequence diversity and antibiotic resistance - a molecular model of short- and long-range effects of mutations in serine lactamases (Applicant Pleiss, Jürgen )
• Stabilization of enzymes by Proside (Applicant Schmid, Franz-Xaver )
• The interplay between specificity and stability in lactamases: molecular modeling of flexibility and dynamics (Applicant Pleiss, Jürgen )
• Understanding mediated/direct electron transfer and solvent resistance by iterative cycles of directed monooxygenase evolution and refinement of computational models (Applicant Schwaneberg, Ulrich )

• Spokesperson: Professor Dr. Reinhard Sterner