Part I: Directed Evolution
1 Continuous Evolution of Proteins In Vivo
1.1 Introduction
1.2 Challenges in Achieving In Vivo Continuous Evolution
1.3 Phage‐Assisted Continuous Evolution (PACE)
1.4 Systems That Allow In Vivo Continuous Directed Evolution
1.5 Conclusion

2 In Vivo Biosensors for Directed Protein Evolution
2.1 Introduction
2.2 Nucleic Acid‐Based In Vivo Biosensors for Directed Protein Evolution
2.3 Protein‐Based In Vivo Biosensors for Directed Protein Evolution
2.4 Characteristics of Biosensors for In Vivo Directed Protein Evolution
2.5 Conclusions and Future Perspectives

3 High‐Throughput Mass Spectrometry Complements Protein Engineering
3.1 Introduction
3.2 Procedures and Instrumentation for MS‐Based Protein Assays
3.3 Technology Advances Focusing on Throughput Improvement
3.4 Applications of MS‐Based Protein Assays: Summary
3.5 Conclusions and Perspectives

4 Recent Advances in Cell Surface Display Technologies for Directed Protein Evolution
4.1 Cell Display Methods
4.2 Selection Methods and Strategies
4.3 Modifications of Cell Surface Display Systems
4.4 Recent Advances to Expand Cell‐Display Directed Evolution Techniques
4.5 Conclusion and Outlook

5 Iterative Saturation Mutagenesis for Semi‐rational Enzyme Design
5.1 Introduction
5.2 Recent Methodology Developments in ISM‐Based Directed Evolution
5.3 B‐FIT as an ISM Method for Enhancing Protein Thermostability
5.4 Learning from CAST/ISM‐Based Directed Evolution
5.5 Conclusions and Perspectives

Part II: Rational and Semi‐Rational Design
6 Data‐driven Protein Engineering
6.1 Introduction
6.2 The Data Revolution in Biology
6.3 Statistical Representations of Protein Sequence, Structure, and Function
6.4 Learning the Sequence‐Function Mapping from Data
6.5 Applying Statistical Models to Engineer Proteins
6.6 Conclusions and Future Outlook

7 Protein Engineering by Efficient Sequence Space Exploration Through Combination of Directed Evolution and Computational Design Methodologies
7.1 Introduction
7.2 Protein Engineering Strategies
7.3 Conclusions and Future Perspectives

8 Engineering Artificial Metalloenzymes
8.1 Introduction
8.2 Rational Design
8.3 Engineering Artificial Metalloenzyme by Directed Evolution in Combination with Rational Design
8.4 Summary and Outlook

9 Engineered Cytochromes P450 for Biocatalysis
9.1 Cytochrome P450 Monooxygenases
9.2 Engineered Bacterial P450s for Biocatalytic Applications
9.3 High‐throughput Methods for Screening Engineered P450s
9.4 Engineering of Hybrid P450 Systems
9.5 Engineered P450s with Improved Thermostability and Solubility
9.6 Conclusions

Part III: Applications in Industrial Biotechnology
10 Protein Engineering Using Unnatural Amino Acids
10.1 Introduction
10.2 Methods for Unnatural Amino Acid Incorporation
10.3 Applications of Unnatural Amino Acids in Protein Engineering
10.4 Outlook
10.5 Conclusions

11 Application of Engineered Biocatalysts for the Synthesis of Active Pharmaceutical Ingredients (APIs)
11.1 Introduction
11.2 Conclusions

12 Directing Evolution of the Fungal Ligninolytic Secretome
12.1 The Fungal Ligninolytic Secretome
12.2 Functional Expression in Yeast
12.3 Yeast as a Tool‐Box in the Generation of DNA Diversity
12.4 Bringing Together Evolutionary Strategies and Computational Tools
12.5 High‐Throughput Screening (HTS) Assays for Ligninase Evolution
12.6 Conclusions and Outlook

13 Engineering Antibody‐Based Therapeutics: Progress and Opportunities
13.1 Introduction
13.2 Antibody Formats
13.3 Antibody Discovery
13.4 Therapeutic Optimization of Antibodies
13.5 Manufacturability of Antibodies
13.6 Conclusions

14 Programming Novel Cancer Therapeutics: Design Principles for Chimeric Antigen Receptors
14.1 Introduction
14.2 Metrics to Evaluate CAR‐T Cell Function
14.3 Antigen‐Recognition Domain
14.4 Extracellular Spacer
14.5 Transmembrane Domain
14.6 Signaling Domain
14.7 High‐Throughput CAR Engineering
14.8 Novel Receptor Modalities

Part IV: Applications in Medical Biotechnology
15 Development of Novel Cellular Imaging Tools Using Protein Engineering
15.1 Introduction
15.2 Cellular Imaging Tools Developed by Protein Engineering
15.3 Application in Cellular Imaging
15.4 Conclusion and Perspectives

Index