Directed Evolution of Selective Enzymes

Manfred T. Reetz is the current Hans-Meerwein-Research-Professor at the University of Marburg (Germany) and former Director and Professor Emeritus of the Max-Planck-Institut für Kohlenforschung in Mülheim (Germany). He started his academic career in the US and returned to Germany for his doctorate, which he received in 1969 from the University of Göttingen. He was Professor at several universities in Germany and the USA and received numerous awards, including the Chirality Medal (2014), Otto Hahn Prize (2011), Tetrahedron Prize for Creativity in Organic Chemistry (2011), Arthur C. Cope Award (2009), Karl Ziegler Prize (2005) and the Leibniz Award of the German Research Council (1989). From 2007 to 2011, Reetz was Senator of the German Academy of Sciences Leopoldina. He has authored more than 550 publications, several book chapters and one book about organotitanium reagents in organic synthesis.

• Preface IX1 Introduction to Directed Evolution 11.1 General Definition and Purpose of Directed Evolution of Enzymes 11.2 Brief Account of the History of Directed Evolution 41.3 Applications of Directed Evolution of Enzymes 16References 172 Selection versus Screening in Directed Evolution 272.1 Selection Systems 272.2 Screening Systems 442.3 Conclusions and Perspectives 52References 533 Gene Mutagenesis Methods 593.1 Introductory Remarks 593.2 Error-Prone Polymerase Chain Reaction (epPCR) and Other Whole-Gene Mutagenesis Techniques 603.3 Saturation Mutagenesis: Away from Blind Directed Evolution 703.4 Recombinant Gene Mutagenesis Methods 853.5 Circular Permutation and Other Domain Swapping Techniques 913.6 Solid-Phase Combinatorial Gene Synthesis for Library Creation 923.7 Computational Tools 96References 1014 Strategies for Applying Gene Mutagenesis Methods 1154.1 General Guidelines 1154.2 Rare Cases of Comparative Studies 1184.3 Choosing the Best Strategy when Applying Saturation Mutagenesis 1304.3.1 General Guidelines 1304.3.2 Choosing Optimal Pathways in Iterative Saturation Mutagenesis (ISM) 1354.3.3 Systematization of Saturation Mutagenesis 1424.3.4 Single Code Saturation Mutagenesis (SCSM): Use of a Single Amino Acid as Building Block 1494.3.5 Triple Code Saturation Mutagenesis (TCSM): A Viable Compromise when Choosing the Optimal Reduced Amino Acid Alphabet 1514.4 Techno-Economical Analyses of Saturation Mutagenesis Strategies 1544.5 Combinatorial Solid-Phase Gene Synthesis: An Alternative for the Future? 159References 1605 Selected Examples of Directed Evolution of Enzymes with Emphasis on Stereo- and Regioselectivity, Substrate Scope, and/or Activity 1675.1 Explanatory Remarks 1675.2 Collection of Selected Examples from the Literature 2010 up to 2016 189References 1896 Directed Evolution of Enzyme Robustness 2056.1 Introduction 2056.2 Application of epPCR and DNA Shuffling 2076.3 B-FIT Approach 2116.4 Iterative Saturation Mutagenesis (ISM) at Protein–Protein Interfacial Sites for Multimeric Enzymes 2156.5 Ancestral and Consensus Approaches and their Structure-Guided Extensions 2166.6 Computationally Guided Methods 2196.6.1 SCHEMA Approach 2196.6.2 FRESCO Approach 2216.6.3 FireProt Approach 2236.6.4 Constrained Network Analysis (CNA) Approach 2246.6.5 Alternative Approaches 226References 2277 Directed Evolution of Promiscuity: Artificial Enzymes as Catalysts in Organic Chemistry 2377.1 Introductory Background Information 2377.2 Tuning the Catalytic Profile of Promiscuous Enzymes by Directed Evolution 2457.3 Conclusions and Perspectives 259References 2608 Learning from Directed Evolution 2678.1 Background Information 2678.2 Case Studies Featuring Mechanistic, Structural, and/or Computational Analyses of the Source of Evolved Stereo- and/or Regioselectivity 2698.2.1 Epoxide Hydrolase 2698.2.2 Ene-Reductase of the Old Yellow Enzyme (OYE) 2738.2.3 Esterase 2798.2.4 Cytochrome P450 Monooxygenase 2828.3 Additive versus Non-additive Mutational Effects in Fitness Landscapes 287References 296Index 303