Kooperativer Bibliotheksverbund

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Format: 1 online resource (xviii, 439 pages) : , illustrations.

ISBN: 0-12-815149-8 , 0-12-815148-X

Series Statement: Methods in enzymology ; Volume 608

Content: Volume 608 of the series Methods in Enzymology covers key aspects of enzyme discovery, engineering tools and platforms, and examples of applications in the enzymology of synthetic biology.Detailed methods for laboratory use of enzymes in synthetic biology applications Informative case history examples illustrating how enzyme and metabolic engineering are used to generate new products Emphasises latest developments in laboratory automation for the engineering of biology Covers many aspects of the design, build, test, learn cycle used in synthetic biology

Note: Front Cover -- Enzymes in Synthetic Biology -- Copyright -- Contents -- Contributors -- Preface -- Section I: Enzyme Discovery -- Chapter One: Enzyme Discovery: Enzyme Selection and Pathway Design -- 1. Introduction -- 2. Enzyme Selection -- 2.1. Introduction to Enzyme Selection -- 2.2. Protocol Description (Selenzyme) -- 2.2.1. Preparation Steps -- 2.2.1.1. Pathway Representation and Use of Generalized Transformations -- 2.2.2. Computing Reaction Similarity -- 2.2.2.1. Description of the General Workflow -- 2.2.2.2. Reaction Directionality -- 2.2.3. Screening, Ranking, and Selection -- 2.2.3.1. Database Screening -- 2.2.3.2. Properties Calculation -- 2.2.3.3. Ranking of Sequence Candidates -- 2.2.4. Selenzyme: Online Enzyme Selection Tool -- 2.2.4.1. Integration With RetroPath2.0 and Other Workflows -- 2.2.5. Other Applications of the Protocol -- 2.2.6. Improvements of the Protocol -- 3. Pathway Design -- 3.1. Introduction to Retrosynthesis -- 3.2. Protocol Description (RetroPath2.0) -- 3.2.1. Preparation Steps -- 3.2.1.1. Sink and Source Definition -- 3.2.1.2. Reaction Rules -- 3.2.2. Building a Retrosynthesis Network -- 3.2.2.1. General Workflow Presentation -- 3.2.2.2. Remark: Rule Scoring by Enzyme Sequence Consistency -- 3.2.3. Pathway Enumeration Between Two Pools of Compounds -- 3.2.3.1. Computing the Scope -- 3.2.3.2. Enumerating Pathways -- 3.3. Use Case: 1,4-Butanediol Pathways Prediction Using RetroPath2.0 -- 3.3.1.1. Materials and Methods -- 3.4. Other Applications of the Protocol -- 4. Summary and Conclusion -- Acknowledgments -- References -- Section II: Enzyme Engineering Tools and Platforms -- Chapter Two: Cell-Free Synthetic Biology for Pathway Prototyping -- 1. Introduction -- 1.1. The State of Metabolic Engineering -- 1.2. Emerging Cell-Free Biotechnology -- 1.2.1. Purified Enzyme Systems -- 1.2.2. Crude Cell Lysate Systems. , 2. The Cell-Free Metabolic Engineering Framework -- 3. S12 Lysate Preparation for Cell-Free Metabolic Engineering -- 3.1. Materials -- 3.1.1. Equipment -- 3.1.2. Media -- 3.1.3. Media Supplements -- 3.1.4. Bacterial Strains and Plasmids (See Table 1) -- 3.1.5. Buffers and Reagents -- 3.2. Procedure -- 3.2.1. Cell Preparation and Expression -- 3.2.2. Extract Preparation -- 3.2.3. Extract Quantification of Total Protein by Bradford Assay -- 3.2.4. Overexpressed Protein Quantification by Densitometry -- 4. Mix-and-Match Cell-Free Metabolic Engineering -- 4.1. Materials -- 4.1.1. Equipment -- 4.1.2. Buffers and Reagents -- 4.2. Procedure -- 4.2.1. Mix-and-Match Biosynthesis Reactions -- 4.2.2. Biosynthesis Analysis -- 5. Cell-Free Protein Synthesis-Driven Metabolic Engineering -- 5.1. Materials -- 5.1.1. Equipment -- 5.1.2. Buffers and Reagents -- 5.2. Procedure -- 5.2.1. CFPS-ME Reactions -- 5.2.2. Quantification of Protein Produced In Vitro via Radioactive Incorporation -- 5.2.3. Quantification of Protein Produced In Vitro via Split-GFP Construct -- 5.2.4. Metabolite Quantification -- 6. Summary and Conclusions -- Acknowledgments -- References -- Chapter Three: Fast and Flexible Synthesis of Combinatorial Libraries for Directed Evolution -- 1. Introduction -- 1.1. Mutagenesis: Introducing Sequence Variation -- 1.2. Combinatorial Libraries -- 2. Methods -- 2.1. Primer Design -- 2.1.1. Notes and Troubleshooting -- 2.2. Synthesis of Mutagenic Megaprimers by Asymmetric PCR -- 2.2.1. Equipment -- 2.2.2. Reagents and Buffers -- 2.2.3. Protocol -- 2.2.4. Notes and Troubleshooting -- 2.3. Assembly of Full-Length Gene Using Mutagenic Megaprimers -- 2.3.1. Equipment -- 2.3.2. Reagents and Buffers -- 2.3.3. Protocol -- 2.3.4. Notes and Troubleshooting -- 3. Library Ligation, Transformation, and Quality Control -- 3.1. Buffers and Reagents -- 3.2. Protocol. , 3.3. Notes and Troubleshooting -- 4. Summary and Conclusion -- Acknowledgments -- References -- Section III: Enzyme Families: Parts and Platforms for Chemical Diversity -- Chapter Four: Sesquiterpene Synthase-Catalyzed Conversion of a Farnesyl Diphosphate Analogue to a Nonnatural Terpenoid Ether -- 1. Introduction -- 2. Sesquiterpenes Production and Purification -- 2.1. Recombinant Enzyme Production and Purification -- 2.2. Determination of Optimum Catalysis Conditions -- 2.2.1. Magnesium Ion Concentration -- 2.2.2. Optimum pH -- 2.2.3. Enzyme Concentration -- 3. Enzymatic Synthesis of Cyclic Ether -- 3.1. Enzyme Activity Assay -- 3.2. Preparative Scale Incubation -- 4. Conclusion -- References -- Chapter Five: In Vivo Platforms for Terpenoid Overproduction and the Generation of Chemical Diversity -- 1. Introduction -- 2. In Vitro Reconstitution of the MVA Pathway and Targeted Engineering of Value-Added Terpenoids -- 2.1. In Vitro Reconstitution of the MVA Pathway -- 2.1.1. Protein Expression and Purification for In Vitro Reconstitution -- 2.1.2. Extraction and Detection of Farnesene -- 2.2. Targeted Engineering of Farnesene -- 2.2.1. Plasmids for the MVA Pathway and Farnesene Overexpression -- 2.2.2. Shake-Flask Fermentation and Analysis of α-Farnesene Production in Engineered Strains -- 2.3. Targeted Engineering of Lycopene -- 2.3.1. Plasmids for Lycopene Overproduction -- 2.3.2. Analysis of Lycopene Titers in Engineered Strains -- 2.4. Targeted Engineering of Astaxanthin -- 2.4.1. Plasmid Construction for Astaxanthin Overproduction -- 2.4.2. Fermentation and Quantification of Astaxanthin Produced by E. coli -- 2.5. Targeted Engineering of Taxadiene -- 2.5.1. Plasmid Construction for Taxadiene Overproduction -- 2.5.2. Overproduction and Detection of Taxadiene in E. coli. , 3. Genome Mining of Terpene Cyclases and the Generation of Chemical Diversity -- 3.1. Genome Mining of Terpene Cyclases and Exploration of the Chemical Diversity of Terpenoids -- 3.1.1. Identification of Class I Terpene Synthases -- 3.1.2. Construction of Plasmids -- 3.1.3. In Vitro Assays of Terpene Cyclases -- 3.1.4. Fermentation and Purification of Terpenoids -- 4. Conclusion -- Acknowledgments -- References -- Chapter Six: Imine Reductases, Reductive Aminases, and Amine Oxidases for the Synthesis of Chiral Amines: Discovery, Char ... -- 1. Introduction -- 1.1. Imine Reductases and Reductive Aminases -- 1.2. Amine Oxidases -- 2. Protein Engineering -- 2.1. Introduction -- 2.2. Amine Oxidase Libraries and Screening -- 2.2.1. Introduction -- 2.2.2. Library Generation -- 2.2.3. Solid-Phase Screen -- 3. Biotransformations, Intensification, and Scale-Up -- 3.1. RedAms and IREDs -- 3.1.1. Analytical-Scale Biotransformations -- 3.1.1.1. Whole Cell -- 3.1.1.2. Cell-Free Extracts and Purified Enzyme -- 3.1.2. Intensification and Scale-Up -- 3.1.2.1. Typical Procedure for Preparative-Scale Reductive Amination Exemplified With Cyclohexanone and Allylamine -- 3.2. Amine Oxidase-Catalyzed Deracemization -- 3.2.1. Analytical-Scale Deracemization Using Whole-Cell MAO-N -- 3.2.2. Analytical-Scale Deracemization Using Purified MAO-N or MAO-N Cell Lysate -- 3.2.3. Preparative-Scale Deracemization Using Whole-Cell MAO-N -- 4. In Vitro and in vivo Cascades -- 4.1. Biocatalytic Hydrogen Borrowing -- 4.2. Amine Oxidase/IRED Cascade for Enantioselective Amine Synthesis -- 4.3. Carboxylic Acid Reductase, Transaminase, and IRED Cascade Reactions -- 5. Conclusions -- References -- Further Reading -- Chapter Seven: Experimental Protocols for Generating Focused Mutant Libraries and Screening for Thermostable Proteins -- 1. Introduction -- 2. Single Mutants Generation. , 2.1. Primer Design -- 2.1.1. Equipment -- 2.1.2. Procedure -- 2.1.3. Notes -- 2.2. QuikChange Library Creation -- 2.2.1. Equipment -- 2.2.2. Buffers and Reagents -- 2.2.3. Procedure -- 2.2.4. Notes -- 2.3. Expression and Protein Purification in a 96-Well Plate -- 2.3.1. Equipment -- 2.3.2. Buffer and Reagents -- 2.3.3. Procedure -- 2.3.4. Notes -- 2.4. Melting Temperature Screening -- 2.4.1. Equipment -- 2.4.2. Buffers and Reagents -- 2.4.3. Procedure -- 2.4.4. Notes -- 3. Combining Mutations -- 3.1. Golden Gate Gene Shuffling -- 3.1.1. Equipment -- 3.1.2. Buffers and Reagents -- 3.1.3. Procedure -- 3.1.4. Notes -- 3.2. Gibson Shuffling -- 3.2.1. Equipment -- 3.2.2. Buffers and Reagents -- 3.2.3. Procedure -- 3.2.4. Notes -- 3.3. Final Stabilized Mutant -- 3.3.1. Equipment -- 3.3.2. Buffers and Reagents -- 3.3.3. Procedure -- 4. Summary and Conclusions -- Acknowledgments -- References -- Further Reading -- Chapter Eight: Characterization of Cytochrome P450 Enzymes and Their Applications in Synthetic Biology -- 1. Introduction -- 2. Expression and Purification of Microbial Cytochrome P450 Enzymes -- 2.1. Expression and Purification of P450 BM3 (CYP102A1) -- 2.1.1. Equipment -- 2.1.2. Buffers and Reagents -- 2.1.3. Procedure -- 2.1.4. Notes -- 2.2. Expression and Purification of OleT (CYP152L1) From Jeotgalicoccus sp. ATCC 8456 -- 2.2.1. Procedure -- 2.2.2. Notes -- 3. Expression and Purification of Eukaryotic P450s in E. coli -- 3.1. P450 Construct Design -- 3.2. Routes to Overexpression of Eukaryotic CYPs in E. coli -- 3.3. Expression and Purification of Human CYP2D6 -- 3.3.1. Equipment and Reagents -- 3.3.2. Procedure -- 3.3.3. Notes -- 3.4. Purification of the Native Form of a Saccharomyces cerevisiae CYP51 Enzyme -- 3.5. Examples of Purification of Cytochrome P450 Enzymes From Other Eukaryotes. , 4. Expression and Characterization of P450 Redox Partner Systems.

Language: English