Authored by one of the world's leading organic chemists, this authoritative reference provides an overview of basic strategies in directed evolution and introduces common gene mutagenesis, screening and selection methods.
Throughout the text, emphasis is placed on methodology development to maximize efficiency, reliability and speed of the experiments and to provide guidelines for efficient protein engineering. Professor Reetz highlights the application of directed evolution experiments to address limitations in the field of enzyme selectivity, substrate scope, activity and robustness. He critically reviews recent developments and case studies, takes a look at future applications in the field of organic synthesis, and concludes with lessons learned from previous experiments.

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 IX

1 Introduction to Directed Evolution 1

1.1 General Definition and Purpose of Directed Evolution of Enzymes 1

1.2 Brief Account of the History of Directed Evolution 4

1.3 Applications of Directed Evolution of Enzymes 16

References 17

2 Selection versus Screening in Directed Evolution 27

2.1 Selection Systems 27

2.2 Screening Systems 44

2.3 Conclusions and Perspectives 52

References 53

3 Gene Mutagenesis Methods 59

3.1 Introductory Remarks 59

3.2 Error-Prone Polymerase Chain Reaction (epPCR) and Other Whole-Gene Mutagenesis Techniques 60

3.3 Saturation Mutagenesis: Away from Blind Directed Evolution 70

3.4 Recombinant Gene Mutagenesis Methods 85

3.5 Circular Permutation and Other Domain Swapping Techniques 91

3.6 Solid-Phase Combinatorial Gene Synthesis for Library Creation 92

3.7 Computational Tools 96

References 101

4 Strategies for Applying Gene Mutagenesis Methods 115

4.1 General Guidelines 115

4.2 Rare Cases of Comparative Studies 118

4.3 Choosing the Best Strategy when Applying Saturation Mutagenesis 130

4.3.1 General Guidelines 130

4.3.2 Choosing Optimal Pathways in Iterative Saturation Mutagenesis (ISM) 135

4.3.3 Systematization of Saturation Mutagenesis 142

4.3.4 Single Code Saturation Mutagenesis (SCSM): Use of a Single Amino Acid as Building Block 149

4.3.5 Triple Code Saturation Mutagenesis (TCSM): A Viable Compromise when Choosing the Optimal Reduced Amino Acid Alphabet 151

4.4 Techno-Economical Analyses of Saturation Mutagenesis Strategies 154

4.5 Combinatorial Solid-Phase Gene Synthesis: An Alternative for the Future? 159

References 160

5 Selected Examples of Directed Evolution of Enzymes with Emphasis on Stereo- and Regioselectivity, Substrate Scope, and/or Activity 167

5.1 Explanatory Remarks 167

5.2 Collection of Selected Examples from the Literature 2010 up to 2016 189

References 189

6 Directed Evolution of Enzyme Robustness 205

6.1 Introduction 205

6.2 Application of epPCR and DNA Shuffling 207

6.3 B-FIT Approach 211

6.4 Iterative Saturation Mutagenesis (ISM) at Protein–Protein Interfacial Sites for Multimeric Enzymes 215

6.5 Ancestral and Consensus Approaches and their Structure-Guided Extensions 216

6.6 Computationally Guided Methods 219

6.6.1 SCHEMA Approach 219

6.6.2 FRESCO Approach 221

6.6.3 FireProt Approach 223

6.6.4 Constrained Network Analysis (CNA) Approach 224

6.6.5 Alternative Approaches 226

References 227

7 Directed Evolution of Promiscuity: Artificial Enzymes as Catalysts in Organic Chemistry 237

7.1 Introductory Background Information 237

7.2 Tuning the Catalytic Profile of Promiscuous Enzymes by Directed Evolution 245

7.3 Conclusions and Perspectives 259

References 260

8 Learning from Directed Evolution 267

8.1 Background Information 267

8.2 Case Studies Featuring Mechanistic, Structural, and/or Computational Analyses of the Source of Evolved Stereo- and/or Regioselectivity 269

8.2.1 Epoxide Hydrolase 269

8.2.2 Ene-Reductase of the Old Yellow Enzyme (OYE) 273

8.2.3 Esterase 279

8.2.4 Cytochrome P450 Monooxygenase 282

8.3 Additive versus Non-additive Mutational Effects in Fitness Landscapes 287

References 296

Index 303