Chapter
3.2. Genome-Guided Natural Products Discovery From Cyanobacteria
3.3. Dereplication of Analytical Information
3.4. Molecular Structure Determination
4. Culturing Filamentous Cyanobacteria
4.1. Identification of Cultures by Microscopy and Morphology
4.2. Conditions for Growth of Cyanobacteria
4.2.3. Liquid Media Preparation Procedure
4.2.4. Solid Media Preparation Procedure
4.3. Isolation of Monocultures of Cyanobacterial Strains From Field Collections
4.3.1. Materials and Media
4.3.2. Isolation Procedure
4.3.4. Growth in Solid Media
4.3.5. Morphology Considerations: Mat vs Solitary Filaments
4.3.6. Culture Maintenance: Seed Cultures and Stepwise Scale-Up
4.3.7. Culture Room Setup
4.3.8. Glassware Maintenance
4.4. Biomass Measurement Techniques
4.4.1. Measuring Cell Growth by Length
4.4.2. Dry Cell Weight and Storage
4.5. Disposal of Biohazard Waste
5. Extraction of gDNA From Cryopreserved and Fresh Cyanobacterial Samples
5.1. gDNA Extraction Considerations
5.2. Biomass Handling and gDNA Extraction
5.2.3. RNAlater Sample and Freezer Storage
5.2.4. Biomass Rinsing, Pulverization, and gDNA Extraction Procedure
5.2.5. Alternative DNA Extraction Procedure
5.2.6. DNA Quality Assessment
6. Genome Mining Natural Product Rich Marine Cyanobacteria
6.1. Cyanobacterial Genomics Overview
6.2. Obtaining High-Quality Draft Genomes From Complex Cyanobacterial Samples
6.2.1. Procedure for Cyanobacterial Genome Assembly
6.3. Obtaining Complete Genomes With Additional Long-Read Sequencing
6.4. Detection of BGC’s From Genomic Assemblies
6.5. Genome Mining Strategies for Novel Gene Cluster Families
6.5.1. Gene Cluster Networking Workflow
7. Biosynthetic Pathway Validation
7.1. Stable Isotope-Labeled Feeding to Verify Incorporation of Predicted Backbone
7.1.3. Stable Isotope Feeding Procedure
7.2. Recombinant Protein Expression
7.3. Heterologous Expression and Molecular Genetics
Chapter Two: PKS-NRPS Enzymology and Structural Biology: Considerations in Protein Production
2.1. Amino Acid Sequence Selection
2.2. Translation Start Sites
3. Selection of Expression Plasmid and Bacterial Strain
4. Affinity Handles and Fusion Partners
5.2. Chaperone Assistance
5.3. Simultaneous tRNA Augmentation and Chaperone Assistance
5.4. Coexpression of NRPS A Domains With MtbH-Like Proteins
7. Production of ACPs in Defined State
8. Production of Multidomain Proteins
11. Refinements for Crystallization
Chapter Three: Terminal Alkyne Biosynthesis in Marine Microbes
2. Enzyme Expression, Purification, and in vitro Assays
2.1. Heterologous Expression and Purification of Proteins
2.1.1. Protein Expression
2.1.2. Protein Purification
2.1.2.1. Soluble Protein Purification
2.1.2.2. Membrane-Bound Desaturase Purification
2.1.2.2.1. Detergent-Dependent Purification
2.1.2.2.1.1. Membrane Preparations
2.1.2.2.1.2. Membrane Protein Solubilization and Purification
2.1.2.2.2. Detergent-Independent Purification
2.2. In Vitro Activity Assays
2.2.1. ATP-[32P]Pyrophosphate (PPi) Exchange Assays for Fatty Acyl-ACP Ligases
2.2.2. ACP-Loading Assays for Fatty Acyl-ACP Ligases
3. An in vivo Reporting System for the Functional Reconstitution of Membrane-Bound Desaturase/Acetylenase Activities
3.1. Type III PKS Selection
3.3. Plasmid Vector Selection
3.4.1. Seed Culture Conditions
3.4.2. TB-Based Culture Conditions and Extraction of Small-Molecule Reporter
3.4.3. F1 Minimal Medium-Based Culture Condition
4. Strategies for Discovering Additional Terminal Alkyne Biosynthetic Proteins
4.1. Bioinformatics Analysis
4.2. Experimental Validation
5. Click Chemistry to Identify and Quantify Terminal Alkynes
Chapter Four: The Biochemistry and Structural Biology of Cyanobactin Pathways: Enabling Combinatorial Biosynthesis
1.1. What Are Cyanobactins?
1.2. How Are Cyanobactins Made?
1.3. What Are the Applications of Cyanobactins?
1.4. What Do Cyanobactins Do in Nature?
2. Biosynthetic Diversity of the Cyanobactin Family
2.1. General Biosynthetic Pathway
2.2. RSs in Precursor Peptides
2.3. Proteins of Unknown Function
2.4. Two Basic Biosynthetic Subdivisions
2.5. Cassette Duplication in Precursor Peptides
2.7. Unusual Cyanobactin Pathways
3.1. Discovery and Initial Characterization
3.2. Substrates and Products
3.3. Enzyme Structure and Function
4.1. Discovery and Initial Characterization
4.2. Substrates and Products
4.3. Enzyme Structure and Function
5.1. Discovery and Initial Characterization
5.2. Substrates and Products
5.2.1. Threonine-Serine Prenylation
5.2.2. Tyrosine Prenylation
5.2.3. Tryptophan Prenylation
5.2.4. N-Terminal Prenylation
5.2.5. Potentially Nonprenylating PatF Homologues
5.3. Enzyme Structure and Function
6.1. Discovery and Initial Characterization
6.2. Substrates and Products
6.3. Enzyme Structure and Function
7. In Vitro Construction of Artificial Cyanobactins
Chapter Five: The Enzymology of Prochlorosin Biosynthesis
1. Introduction: Lanthipeptides in Marine Cyanobacteria Populations
2. Expression and Purification of ProcA Peptide Substrates
2.2. Protocol for Heterologous Expression and Purification of ProcA Peptides
2.2.2. Materials, Buffers, and Solvents
2.2.3. Peptide Expression Procedure
2.2.4. Expressed Peptide Purification Procedure
2.3. Protocol for Preparation of Deuterium-Labeled LanA Analogues
2.3.2. Materials and Solvents
2.3.3. Procedure to Prepare Deuterium-Labeled ProcA Core Peptides
2.3.4. Procedure to Prepare Deuterium-Labeled ProcA Peptide Substrates by EPL
2.3.6. Copper-Catalyzed Alkyne-Azide Ligation Procedure
3. Expression, Purification, and Activity Assays of ProcM
3.2. Protocol for Expression and Purification of ProcM
3.2.1. Materials and Buffers
3.3. Characterization of the Enzyme
3.3.1. Mutagenesis of the ProcM Zinc-Binding Residues and Determination of Zinc Content
3.3.2. Procedure for Zinc Content Determination
3.3.3. Separation of the Cyclase Domain of ProcM
3.4. In Vitro Assay Conditions
3.4.1. Standard Procedure for ProcM Activity Assays
3.4.2. Procedure for Testing Cyclase Activity Decoupled From Dehydration
4. Product and Reaction Characterization Methods
4.1. Leader Peptide Removal Strategies
4.2. Methods to Determine Cyclization State
4.2.1. Procedure for Cys Alkylation
4.3. Methods for Investigating Dehydration and Cyclization Reactions
4.3.1. Procedure for Determining the Directionality of Dehydration
4.3.2. Procedure for Determining the Directionality of Cyclization
4.3.3. Procedure for Testing the Reversibility of Cyclization
4.4. Mass Spectrometry-Based Kinetic Assay for LanM Synthetases
4.4.2. Sample Preparation for Kinetic Studies
4.4.4. Liquid Chromatography and Electrospray Ionization Settings for Kinetic Studies
4.4.6. Data Analysis and Information Gained From Kinetic Studies
4.5. Use of Nuclear Magnetic Resonance Spectroscopy for Ring Topology Assignment
4.5.1. Procedure for Determination of Ring Topology by NMR Spectroscopy
Section II: Biosynthesis by Bacterial Symbionts of Marine Invertebrate Animals
Chapter Six: Chemoenzymatic Dissection of Polyketide β-Branching in the Bryostatin Pathway
2. Previous Studies and Identification of Cryptic Components of Bryostatin Biosynthesis
3. Initial Biochemical Analysis of Novel Bry Gene Products
3.1. BryU, β-Branch Donor ACP
3.2. BryT, Enoyl-CoA Hydratase
3.3. BryA, Carboxyl O-Methyltransferase
4. Chemoenzymatic Regiochemistry Determination
4.1. Analysis of the BryT Dehydration Product
4.1.1. Materials and Equipment
4.1.2. In Situ γ-Deuterated HMG-CoA Synthesis and LC/MS Analysis
4.1.3. Large-Scale Product Isolation and NMR Analysis
4.2. Analysis of Bry β-Branching O-Methylation
4.2.1. Materials and Equipment
4.2.2. Carrier Protein Methylation and LC/MS Analysis
5. Summary and Conclusions
Chapter Seven: Radical S-Adenosylmethionine Peptide Epimerases: Detection of Activity and Characterization of d-Amino Aci ...
2. Equipment, Consumables, and Reagents
3. Detection of Epimerase Activity From rSAM Epimerases
4. Orthogonal D2O-Based Induction System
Chapter Eight: Cobalamin-Dependent C-Methyltransferases From Marine Microbes: Accessibility via Rhizobia Expression
2. Heterologous Host and Vector Considerations
2.2.2. Buffers, Solutions, and Reagents
2.2.3. Procedure for Creating a New MCS in pLMB51
3. Rhizobia Transformation
3.2. Buffers, Solutions, and Reagents
4. Rhizobia Expression and Protein Purification
4.2. Buffers, Solutions, and Reagents
4.3. Protein Expression Procedure
4.4. Protein Purification Procedure (See Note 2 in Section 4.5)
5. Data Acquisition and Analysis of Peptide and Protein Targets
5.2. Buffers, Solutions, and Reagents
5.3. Procedure for Proteinase K Digestion, Sample Preparation, and LC-ESI-MS/MS Analysis
5.4. MaxQuant Identification of Methylated Peptides (See Note 8 in Section 5.5)
6. Summary and Conclusions
Chapter Nine: Biosynthetic Insights of Calyculin- and Misakinolide-Type Compounds in ``Candidatus Entotheonella sp.”
1.1. Calyculin-Type Compounds
1.2. Misakinolide-Type Compounds
1.3. Polyketide and Nonribosomal Peptide Biosynthesis
2. Targeting Biosynthetic Pathways
2.1. Key Biosynthesis Gene Detection
2.2. Biosynthetic Gene Cluster Cloning
2.2.1. Complex Metagenomic Library Construction and Screening
2.2.2. Bioinformatic Analysis
2.3. Compound Producer Identification
3. Functional Studies of Key Pathway Components
3.1. General Concept for Protein Activity Assays
3.2. Activity Assay for PS
3.2.1. Instrument, Buffers, and Reagents
3.2.2. Procedure (Pöplau et al., 2013; Ueoka et al., 2015)
3.3. Activity Assays of Phosphotransferase
3.3.1. Instrument, Buffers, and Reagents
3.3.2. Procedures (Wakimoto et al., 2014)
4. Summary and Conclusion
Section III: Natural Product Biosynthetic Enzymology of Marine Bacteria and Fungi
Chapter Ten: Chemoenzymatic Synthesis of Starting Materials and Characterization of Halogenases Requiring Acyl Carrier Pr ...
2. Equipments and Consumables
3. Preparation of Acyl-S-CPs
3.2.1. Preparation of Plasmid DNA
3.2.2. Expression and Purification of Recombinant Proteins
3.2.3. Chemoenzymatic Synthesis of Pyrrolyl-S-CPs
4. Halogenation of Acyl-S-CPs
Chapter Eleven: Preparation, Assay, and Application of Chlorinase SalL for the Chemoenzymatic Synthesis of S-Adenosyl-l-M ...
2. Preparation and Assay of Chlorinase SalL
2.1. Purification of Recombinant SalL From Escherichia coli
2.1.1. Cloning of the salL Gene Into an Expression Vector
2.1.2. Expression of salL in E. coli BL21(DE3) and Purification of Recombinant SalL Enzyme
2.2.2. Instrumentation and Solvents
3. Chemoenzymatic Synthesis of SAM Analogs
3.1. Overview of Chemoenzymatic Methods to Prepare SAM and SAM Analogs
3.2. SalL-Mediated Chemoenzymatic Synthesis of SAM and SAM Analogs
3.2.3. Stock Solutions and Buffers
3.2.4. Analytical Instrumentation
3.3. In Situ Chemoenzymatic Synthesis of SAM Coupled to DNA Methylation by HhaI
3.3.3. Stock Solutions and Buffers
3.4. In Situ Chemoenzymatic Synthesis of SAM Coupled to Teicoplanin Methylation by MtfA
3.4.3. Stock Solutions and Buffers
3.4.4. Expression and Purification of MtfA Methyltransferase
3.5. In Situ Chemoenzymatic Synthesis of 13C-Labeled SAM Coupled With Isotope Incorporation Into Teicoplanin via Methylat ...
3.5.3. Stock Solutions and Buffers
3.5.4. Analytical Instrumentation
3.6. In Situ Chemoenzymatic Synthesis of SAM Coupled to RGG Peptide Methylation by rPRMT1
3.6.3. Stock Solutions and Buffers
3.6.4. Procedure Peptide Methylation Using rPRMT1
4. Summary and Conclusions
Chapter Twelve: In Vitro Analysis of Cyanobacterial Nonheme Iron-Dependent Aliphatic Halogenases WelO5 and AmbO5
2. In Vitro Analysis of WelO5 and AmbO5 Halogenases
2.1. Purification of WelO5 and AmbO5 Proteins
2.2. Procurement of Substrates for WelO5 and AmbO5 Proteins
2.3. Activity Assay for WelO5 and AmbO5 Proteins
Chapter Thirteen: Characterization and Biochemical Assays of Streptomyces Vanadium-Dependent Chloroperoxidases
2. Molecular Biology of Streptomyces VCPO Genes
3. Heterologous Protein Expression and Purification
3.1. Recombinant VCPO Expression
3.2. Purification of Recombinant Streptomyces VCPO Enzymes
4. Enzymatic Assays of Heterologously Expressed Streptomyces VCPO Enzymes
4.1. Monochlorodimedone Assay
4.2.1. NapH1 Cyclization of Naphthomevalin to Napyradiomycin A1
4.2.2. NapH3 α-Hydroxyketone Rearrangement to Generate Naphthomevalin
4.2.3. Multitasking Catalytic Activities of Mcl24 to Generate Merochlorins A, B, and X
5. Conclusions and Future Outlook
Chapter Fourteen: Unusual ``Head-to-Torso” Coupling of Terpene Precursors as a New Strategy for the Structural Diversi ...
2. The Biosynthesis of the Bacterial Merochlorin Antibiotics That Feature a Uniquely Branched Sesquiterpene Moiety
4. Heterologous Production and Purification of Mcl22
5. Enzymatic and Chemical Synthesis of Isosesquilavandulyl Diphosphate
6. HPLC Analysis of Enzymatically and Chemically Produced Isosesquilavandulyl Diphosphate and Other Prenyl Diphosphates
7. Cyclolavandulyl Diphosphate Synthase (CLDS) Synthesizes Cyclolavandulyl Diphosphate (CLPP) by Catalyzing Both Terpene ...
8. Mcl22 (ISLPPS), CLDS, and LPPS Are Structurally Related to cis-Prenyl Diphosphate Synthases
9. Summary and Conclusion
Chapter Fifteen: Identification of Enzymes Involved in Sesterterpene Biosynthesis in Marine Fungi
2. Genome Sequencing and Bioinformatics Analysis of Terpene-Related Genes in A. ustus 094102
2.1.1. Buffers and Reagents
2.2. In Silico Analysis for Terpene Synthesis Genes
2.2.1. Software and Database
3. In Vivo Identification of Gene (Clusters) Involved in Sesterterpene Ophiobolin Biosynthesis
3.1. Preparation of Fungal Protoplast
3.1.2. Buffers and Reagents
3.2. Construction of Gene-Targeting Cassette
3.2.2. Buffers and Reagents
3.3. Transformation of Gene-Editing Cassette to Fungal Protoplast
3.3.2. Buffers and Reagents
3.4. Identification of Positive Transformants
3.5. Gene Complementation
3.6. Gene Cluster Inactivation
3.7. HPLC Detection of Ophiobolins and Drimanes
3.7.2. Materials and Reagents
4. In Vitro Functional Characterization of Au8003, Au13192, and Au6298
4.1.1. Software and Database
4.2.2. Buffers and Reagents
4.2.3.1. RNA Extraction From Strain Aspergillus sp. 094102
4.2.3.2. Reverse Transcription
4.2.3.3. Amplification of Au8003 and Au6298
4.2.3.4. Amplification of Au13192
4.2.3.5. Recovery of Target DNA
4.2.3.6. Target DNA Cloning
4.2.3.7. Verification of Cloning Vectors
4.3. Protein Expression and Purification
4.3.2. Buffers and Reagents
4.3.3.1. Recovery of Target DNA Fragments and pET28a Fragment
4.3.3.2. Ligation of Target DNA and pET28a Fragments
4.3.3.3. Verification of Expressing Vectors
4.3.3.4. Expression Quality Analysis
4.3.3.5. Purification of Expressed Proteins
4.4. In Vitro Enzymatic Reaction
4.4.2. Buffers and Reagents
4.4.3.1. Au8003 in vitro Reaction
4.4.3.2. Au13192 in vitro Reaction
4.4.3.3. Au6298 in vitro Reaction
4.5.2. Buffers and Reagents
4.5.3.1. Sample Preparation of Au8003 and Au13192 in vitro Reaction Products
4.5.3.2. Sample Preparation of Au6298
4.5.3.3. Preparation for Running GC-MS
4.5.3.4. GC-MS Conditions
5. Discussion and Conclusion
Chapter Sixteen: Assaying Oxidative Coupling Activity of CYP450 Enzymes
2. Equipments and Consumables
3. Preparation of Recombinant Proteins
3.1. Plasmid DNA for Recombinant Protein Expression
3.2. Expression and Purification of Recombinant Proteins
4.1. Analytical Scale Assays
4.2. Preparative Scale Assays
Chapter Seventeen: Preparation and Characterization of the Favorskiiase Flavoprotein EncM and Its Distinctive Flavin-N5-O ...
2. Heterologous Production and Purification of EncM
3. Chemical Synthesis of Poly-β-Ketone Substrate Analogs
4. Aerobic and Anaerobic EncM Activity Assays and HPLC Analysis
5. Spectroscopic Characterization of EncM
6. Crystallization and Structural Elucidation of EncM
7. Evidence for the Flavin-N5-Oxide Cofactor of EncM
8. Strategies to Identify Novel Enzymes Employing a Flavin-N5-Oxide Cofactor
9. Summary and Conclusion
Marine enzymes and specialized metabolism - Part A
( Volume 604 )
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