Non Ribosomal Peptide Biosynthesis and Engineering Methods and Protocols 1st Edition by Michael Burkart, Fumihiro Ishikawa ISBN 9781071632130 1071632132

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ISBN 10: 1071632132
ISBN 13: 9781071632130
Author: Michael Burkart, Fumihiro Ishikawa

This volume provides new technologies on NRPSs and related carrier protein dependent synthases, including polyketide synthases (PKS) and fatty acid synthases (FAS). Chapters detail enzymology, structural biology, proteopromics, chemical biology, natural product chemistry, and bioinformatics. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and methods, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.
Authoritative and cutting-edge, Non-Ribosomal Peptide Biosynthesis and Engineering: Methods and Protocols aims to feature methods that will be beneficial to new researchers, and those wanting to adopt new methodologies into their research

Non Ribosomal Peptide Biosynthesis and Engineering Methods and Protocols 1st Edition Table of contents:

Part I: Background and Overview
Chapter 1: The Assembly-Line Enzymology of Nonribosomal Peptide Biosynthesis
1 Nonribosomal Peptides (NRPs)
2 NRP Biosynthesis
2.1 NRPS Architecture and Its Gene
2.2 A Domains
2.3 T Domains
2.4 C Domains
2.5 Te Domains
3 Outlook
References
Chapter 2: Structural Studies of Modular Nonribosomal Peptide Synthetases
1 Introduction to Modular NRPS Enzymes
2 Catalytic Domains of Non-ribosomal Peptide Synthetases
3 Structures of Modules of NRPS Enzymes
3.1 SrfA-C
3.2 AB3403
3.3 EntF
3.4 LgrA
3.5 DhbF
3.6 FmoA3
3.7 ObiF1
3.8 PchE
3.9 BmdBC
4 Conclusions and Future Directions
References
Part II: In Vivo and In Vitro Methods
Chapter 3: Using NMR Titration Experiments to Study E. coli FAS-II- and AcpP-Mediated Protein-Protein Interactions
1 Introduction
2 Materials and Methods for Proteins
2.1 M9 Minimal Media
2.2 Lysis Buffer
2.3 Urea-PAGE (Polyacrylamide Gel Electrophoresis)
2.4 SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)
2.5 Isotopically Labeled 15N-AcpP
2.5.1 Growth and Protein Expression
2.5.2 Protein Purification
2.6 Chemoenzymatic Modification of 15N-AcpP
2.6.1 Holofication and Acylation
2.6.2 Apofication and Cryptofication
2.7 Partner Proteins
2.7.1 FPLC Purification and NMR Buffers
2.7.2 15N-AcpP
2.7.3 Partner Protein
3 Materials and Methods for NMR Analysis
3.1 NMR Sample Preparation
3.1.1 Sample Requirements
3.2 NMR Spectra Collection
3.3 NMR Data Analysis
3.3.1 Chemical Shift Perturbations
3.3.2 Line Shape Analysis
3.3.3 Computational Analysis and Applications
References
Chapter 4: Chemoproteomic Profiling of Adenylation Domain Functions in Gramicidin S-Producing Non-ribosomal Peptide Synthetases
1 Introduction
2 Materials
2.1 Synthetic Procedure of Aminoacyl-AMS-BPyne Labeling Reagents
2.2 Synthetic Procedure of Aminoacyl-AMS Compounds
2.3 Bacterial Culture
2.4 Sample Preparation for Labeling Studies
2.5 Activity-Based Protein Profiling
3 Methods
3.1 Synthetic Procedures of Aminoacyl-AMS-BPyne Labeling Reagents
3.1.1 Synthesis of 2a
3.1.2 Synthesis of 3a
3.1.3 Synthesis of 4a
3.1.4 Synthesis of L-Phe-AMS-BPyne
3.1.5 Synthesis of 2b
3.1.6 Synthesis of 3b
3.1.7 Synthesis of 4b
3.1.8 Synthesis of L-Pro-AMS-BPyne
3.1.9 Synthesis of 2c
3.1.10 Synthesis of 3c
3.1.11 Synthesis of 4c
3.1.12 Synthesis of L-Val-AMS-BPyne
3.1.13 Synthesis of 2d
3.1.14 Synthesis of 3d
3.1.15 Synthesis of 4d
3.1.16 Synthesis of L-Orn-AMS-BPyne
3.1.17 Synthesis of 2e
3.1.18 Synthesis of 3e
3.1.19 Synthesis of 4e
3.1.20 Synthesis of L-Leu-AMS-BPyne
3.2 Synthetic Procedures of Aminoayl-AMS Compounds
3.2.1 Synthesis of 7a
3.2.2 Synthesis of L-Phe-AMS 8a
3.2.3 Synthesis of 7b
3.2.4 Synthesis of L-Pro-AMS 8b
3.2.5 Synthesis of 7c
3.2.6 Synthesis of L-Orn-AMS 8c
3.2.7 Synthesis of 7d
3.2.8 Synthesis of Gly-AMS 8d
3.2.9 Synthesis of 7e
3.2.10 Synthesis of L-Ala-AMS 8e
3.2.11 Synthesis of 7f
3.2.12 Synthesis of L-Val-AMS 8f
3.2.13 Synthesis of 7g
3.2.14 Synthesis of L-Leu-AMS 8g
3.2.15 Synthesis of 7h
3.2.16 Synthesis of L-Ile-AMS 8h
3.2.17 Synthesis of 7i
3.2.18 Synthesis of L-Asn-AMS 8i
3.2.19 Synthesis of 7j
3.2.20 Synthesis of L-Gln-AMS 8j
3.2.21 Synthesis of 7k
3.2.22 Synthesis of L-Ser-AMS 8k
3.2.23 Synthesis of 7l
3.2.24 Synthesis of L-Thr-AMS 8l
3.2.25 Synthesis of 7m
3.2.26 Synthesis of L-Met-AMS 8m
3.2.27 Synthesis of 7n
3.2.28 Synthesis of L-Tyr-AMS 8n
3.2.29 Synthesis of 7o
3.2.30 Synthesis of L-Trp-AMS 8o
3.2.31 Synthesis of 7p
3.2.32 Synthesis of L-Asp-AMS 8p
3.2.33 Synthesis of 7q
3.2.34 Synthesis of L-Glu-AMS 8q
3.2.35 Synthesis of 7r
3.2.36 Synthesis of L-Lys-AMS 8r
3.2.37 Synthesis of 7s
3.2.38 Synthesis of L-Arg-AMS 8s
3.2.39 Synthesis of 7t
3.2.40 Synthesis of L-His-AMS 8t
3.3 Bacterial Culture
3.3.1 Bacterial Propagation Procedure
3.3.2 Bacterial Culture Procedure
3.4 Sample Preparation for Labeling Studies
3.5 Activity-Based Protein Profiling
3.6 Competitive Activity-Based Protein Profiling
4 Notes
References
Chapter 5: In Vitro Biochemical Characterization of Excised Macrocyclizing Thioesterase Domains from Non-ribosomal Peptide Syn…
1 Introduction
2 Materials
2.1 Thioesterase (TE) Expression
2.2 Peptide Synthesis
2.3 Biochemical Assays
3 Methods
3.1 TE Cloning and Expression
3.1.1 TE Cloning
3.1.2 Recombinant TE Expression
3.1.3 Recombinant TE Purification
3.1.4 Recombinant TE Concentration and Ultrafiltration
3.2 Peptide Synthesis
3.2.1 Resin Loading
3.2.2 Substitution Level Estimation
3.2.3 Peptide Synthesis
3.2.4 Peptide Cleavage from Resin
3.2.5 Thiol Synthesis, Thioester Synthesis, and Peptide Deprotection
3.3 Biochemical Assays
3.3.1 Assay Preparation and Incubation
3.3.2 LCMS Analysis of TE Product Distribution
3.3.3 Kinetic Characterization of TE
4 Notes
References
Chapter 6: Chemo-Enzymatic Synthesis of Non-ribosomal Macrolactams by a Penicillin-Binding Protein-Type Thioesterase
1 Introduction
2 Materials
2.1 Synthesis of Seco-Surugamide B-SNAC
2.1.1 Solid-Phase Peptide Synthesis
2.1.2 Thioesterification, Deprotection, and Precipitation
2.1.3 HPLC Purification
2.2 Synthesis of Surugamide B
2.2.1 Solid-Phase Peptide Synthesis
2.2.2 Macrocyclization, Deprotection, and Precipitation
2.2.3 HPLC Purification
2.3 Preparation of a Recombinant SurE
2.3.1 Cloning of surE Gene
2.3.2 Protein Purification
2.4 In Vitro Cyclization Mediated by SurE
3 Methods
3.1 Synthesis of Seco-Surugamide B-SNAC (Fig. 3)
3.1.1 Solid-Phase Peptide Synthesis (SPPS)
3.1.2 Thioesterification, Deprotection, and Precipitation
3.1.3 HPLC Purification
3.2 Synthesis of Surugamide B (Fig. 4)
3.2.1 Solid-Phase Peptide Synthesis (SPPS)
3.2.2 Macrocyclization, Deprotection, and Precipitation
3.2.3 HPLC Purification
3.3 Preparation of Recombinant SurE
3.3.1 Cloning of the surE Gene
3.3.2 Preparation of Recombinant SurE
3.4 In Vitro Cyclization Mediated by SurE
3.4.1 In Vitro Reaction of SurE
3.4.2 Analysis of the Reaction Mixture
4 Notes
References
Chapter 7: Directed Evolution of the BpsA Carrier Protein Domain for Recognition by Non-cognate 4′-Phosphopantetheinyl Transfe…
1 Introduction
2 Materials
2.1 Library Preparation
2.2 Library Screening
2.3 Protein Expression and Purification
2.4 Kinetic Measurement of PPTase Activity
3 Methods
3.1 PCP Domain Template Preparation and Error-Prone PCR
3.2 Vector Preparation and Quality Assessment
3.3 Library Construction and First-Tier Screening
3.4 Second Tier Liquid Screening
3.5 Expression and Purification of BpsA Variants
3.6 Expression and Purification of PPTases
3.7 In Vitro Quantification of Indigoidine Synthesis Rate
4 Notes
Bibliography
Chapter 8: Unraveling Structural Information of Multi-Domain Nonribosomal Peptide Synthetases by Using Photo-Cross-Linking Ana…
1 Introduction
2 Materials
2.1 Equipment
2.2 Chemicals and Solutions
2.3 Material for MS Analysis
3 Methods
3.1 Design of the Expression Plasmid for the NRPS Protein with the Unnatural Photo-Cross-Linking Amino Acid
3.2 Production of NRPS Protein with p-benzoyl-L-phenylalanine (BpF) Incorporated by Using Nonsense Suppression
3.3 Cell Lysis and Protein Purification
3.4 Photo-Cross-Linking Assay
3.5 Analysis of Photo-Cross-Linked Proteins by Coomassie-Stained SDS-PAGE and by Western Blotting
3.6 In-Gel Tryptic Digest of Photo-Cross-Linked Proteins
3.7 MS-MS Analysis
3.8 Data Processing
4 Notes
References
Chapter 9: A Chemoenzymatic Approach to Investigate Cytochrome P450 Cross-Linking in Glycopeptide Antibiotic Biosynthesis
1 Introduction
2 Materials
2.1 Synthesis of Protected Amino Acids
2.2 Solid-Phase Peptide Synthesis (SPPS)
2.3 Peptidyl Hydrazide Displacement with CoA
2.4 Protein Expression and Purification
2.5 Loading of Peptidyl-CoA onto apo PCP-X Didomain
2.6 CYP450-Catalyzed Cross-Linking Reaction
2.7 Equipment
3 Methods
3.1 Peptidyl-CoA Synthesis
3.1.1 Amino Acid Synthesis
3.1.2 Solid-Phase Peptide Synthesis
3.1.3 Peptidyl Hydrazide Displacement with CoA
3.2 Protein Expression and Purification
3.2.1 PCP-X Didomain Expression and Purification
3.2.2 Cytochrome P450 Oxy Expression and Purification
3.2.3 Sfp Mutant R4-4 Expression and Purification
3.2.4 PuR/PuxB Expression and Purification
4 Enzymatic Cyclization
4.1 Loading of Peptidyl-CoA onto an Apo PCP-X Didomain
4.2 CYP450-Catalyzed Cross-Linking Reaction on Type I-IV GPAs
5 Notes
References
Chapter 10: Cross-Linking of the Nonribosomal Peptide Synthetase Adenylation Domain with a Carrier Protein Using a Pantetheine…
1 Introduction
2 Materials
2.1 Synthesis of the Bromoacetamide Pantetheine Probe
2.2 Recombinant Proteins
2.2.1 A-Domain Recombinant Protein
2.2.2 CP Recombinant Protein
2.2.3 CoaA, CoaD, and CoaE Recombinant Proteins
2.2.4 Sfp Recombinant Protein
2.3 Cross-Linking Reaction
3 Methods
3.1 Synthetic Procedure for the Preparation of the Bromoacetamide Pantetheine Analog
3.1.1 Synthesis of p-Methoxybenzylidene (PMB)-Protected Pantothenic Acid
3.1.2 Synthesis of 2-Azidoethylamine
3.1.3 Synthesis of PMB-Protected Pantetheine Azide
3.1.4 Synthesis of PMB-Protected Pantetheine Amine
3.1.5 Synthesis of PMB-Protected Bromoacetamide Pantetheine Analog
3.1.6 Synthesis of Bromoacetamide Pantetheine Analog
3.2 Procedure for the Cross-Linking Reaction
3.2.1 Preparation of Crypto-CP
3.2.2 Cross-Linking Reaction
4 Notes
References
11: A Practical Guideline to Engineering Nonribosomal Peptide Synthetases
1 Introduction
1.1 The eXchange Unit (XU)
1.2 Type S NRPS
2 Materials
2.1 Plasmids and Strains
2.2 Strain Cultivation and Stock Solutions
2.3 Kits and Equipment
2.4 PCR Amplification
2.5 DNA Assembly
3 Methods
3.1 Plasmid Design
3.1.1 XU Concept Fusion Site
3.1.2 SYNZIPs
SYNZIP Plasmid Construction
Fusion Sites for SYNZIP Insertion
3.2 PCR Amplification
3.3 DpnI and Gel Extraction
3.4 DNA Assembly
3.5 Competent Cells
3.6 Transformation via Electroporation
3.7 Plasmid Verification and Sequencing
3.8 Production Cultures-Heterologous Protein Expression in E. coli
4 Notes
References
Chapter 12: Probing Substrate-Loaded Carrier Proteins by Nuclear Magnetic Resonance
1 Introduction
2 Materials
2.1 Protein Expression and Purification
2.2 Stock Solutions
2.3 NMR Samples
2.4 NMR Spectrometer
3 Methods
3.1 Temperature Calibration
3.2 Control Experiments
3.3 In Situ Loading of Holo-ArCP with Salicylate
4 Notes
References
13: Ribosomal Synthesis of Peptides Bearing Noncanonical Backbone Structures via Chemical Posttranslational Modifications
1 Introduction
2 Materials
2.1 Flexizyme-Mediated Acylation to Prepare Nonproteinogenic acyl-tRNAs
2.2 Expression of Peptides Containing Noncanonical Amino Acids
2.3 Posttranslational Chemical Modification of BrvG to Backbone Azole Moieties
2.4 Posttranslational Chemical Modification of AzHyA to Hhc Units
3 Methods
3.1 Flexizyme-Mediated Acylation to Prepare Nonproteinogenic acyl-tRNAs
3.2 Expression of Peptides Containing Noncanonical Amino Acids
3.3 Posttranslational Chemical Modification of BrvG to Backbone Azole Moieties
3.4 Posttranslational Chemical Modification of AzHyA to Hhc Units
4 Notes
References
Chapter 14: Thioester Capture Strategy for the Identification of Nonribosomal Peptide and Polyketide Intermediates
1 Introduction
2 Materials
2.1 Synthesis of the Thioester Capture Agent Biotin-Cys
2.2 Capture Strategy Validation with PKS, AziB (from the Azinomycin Biosynthetic Pathway)
2.2.1 In Vitro Expression, Posttranslational Modification, and Purification of AziB
2.2.2 Protein Validation with 15% Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
2.2.3 Generation of AziB Intermediate In Vitro
2.2.4 Reaction Between the AziB Intermediates and the Thioester Capture Agent Biotin-Cys
2.2.5 Purification of Biotin-Cys-Captured AziB Intermediate
2.3 Capture Strategy Validation with NRPS (ClbN, Colibactin Biosynthesis Pathway)
2.3.1 In Vitro Expression, Posttranslational Modification, and Purification of ClbN
2.3.2 Generation ClbN Intermediate In Vitro
2.3.3 Generation of pSETAziA3ΔaziA6
2.3.4 In Vitro Expression, Posttranslational Modification, and Purification of AziA3
3 Methods
3.1 Synthesis of the Biotin-Cys Thioester Capture Agent
3.2 Demonstration of the Capture Strategy with the PKS AziB (from the Azinomycin Biosynthetic Pathway)
3.2.1 In Vitro Expression, Posttranslational Modification, and Purification of AziB
3.2.2 Protein Analysis by 15% Sodium Dodecyl Sulfate Polyacrylamide (SDS-PAGE) Gel Electrophoresis
3.2.3 Generation of AziB Intermediate In Vitro
3.2.4 Reaction Between the AziB Intermediate and the Biotin-Cys Thioester Capture Agent
3.2.5 Purification of Biotin-Cys-Captured AziB Intermediate (See Note 4)
3.2.6 Analysis of the AziB Intermediate-Capture Product by LCMS
3.3 Demonstration of the Capture Strategy with the NRPS ClbN (Colibactin Biosynthetic Pathway)
3.3.1 In Vitro Expression, Posttranslational Modification, and Purification for ClbN
3.3.2 Generation of the ClbN Intermediate In Vitro
3.3.3 Reaction Between the ClbN Intermediate and the Biotin-Cys Thioester Capture Agent
3.3.4 Purification of the Biotin-Cys-Captured ClbN Intermediate
3.3.5 Analysis of the ClbN Intermediate-Capture Product by LCMS
3.4 Demonstration of the Capture Strategy with the NRPS AziA3 (azinomycin Biosynthetic Pathway): Elucidation of an Unknown Int…
3.4.1 Generation of the Genetic Knockout Plasmid pSETAziA3ΔaziA6
Construction of the Disruption Plasmid pKCAziA6
Generation of the S. sahachiroi ΔaziA6 Disruption Mutant
Construction of Plasmid pSET152_AziA3
Generation of S. sahachiroi pSETAziA3ΔaziA6
3.4.2 In Vitro Expression, Posttranslational Modification, and Purification of AziA3
3.4.3 Reaction Between the AziA3 Intermediate and the Thioester Capture Agent Biotin-Cys
3.4.4 Purification of Biotin-Cys-Captured AziA3 Intermediate
3.4.5 Analysis of the AziA3 Intermediate-Capture Product by LCMS
4 Notes
References
Chapter 15: Chemical Labeling of Protein 4′-Phosphopantetheinylation in Surfactin-Producing Nonribosomal Peptide Synthetases
1 Introduction
2 Materials
2.1 Synthetic Procedure of 4′-Phosphopantetheinylation Labeling Reagent
2.2 Bacterial Culture
2.3 Sample Preparation for the Labeling Studies
2.4 Chemical Labeling of Protein 4′-Phosphopantetheinylation in SrfAB-NRPS
3 Methods
3.1 Synthesis of Probe 1
3.1.1 Synthesis of 3
3.1.2 Synthesis of 4
3.1.3 Synthesis of 5
3.1.4 Synthesis of 8
3.1.5 Synthesis of 9
3.1.6 Synthesis of Probe 1
3.2 Bacterial Culture
3.2.1 Bacterial Propagation
3.2.2 Bacterial Culture
3.3 Sample Preparation for Labeling Studies
3.4 Chemical Labeling of Protein 4′-Phosphopantetheinylation in SrfAB-NRPS
4 Notes
References
Part III: Bioinformatics Methods
Chapter 16: Norine: Bioinformatics Methods and Tools for the Characterization of Newly Discovered Nonribosomal Peptides
1 Introduction
2 Materials
2.1 Norine
2.2 Smiles2Monomers
2.3 rBAN
2.4 MyNorine
3 Methods
3.1 Querying the Norine Database
3.1.1 How to Query the Database with Annotations?
3.1.2 Structure Search
3.2 Submission of a New Peptide
3.3 Study Case
3.3.1 Overview on NRPs Sharing Traits with FL6M
3.3.2 Structure Comparison with NRPs Stored in Norine
4 Notes

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Tags: Michael Burkart, Fumihiro Ishikawa, Non Ribosomal, Peptide Biosynthesis, Engineering