Engineering Natural Product Biosynthesis: Methods and Protocols (Methods in Molecular Biology, 2489)

✍ Scribed by Elizabeth Skellam (editor)

• Publisher: Humana
• Year: 2022
• Tongue: English
• Leaves: 475
• Category: Library

✦ Synopsis

This volume highlights natural products, molecular methods for identifying, and current trends in designing non-natural natural products. Chapters guide readers through protocols on heterologous expression techniques, gene disruption, modified pathway regulators, and in-vitro studies. 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 reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.

Authoritative and cutting-edge, Engineering Natural Product Biosynthesis: Methods and Protocols aims to be a useful and practical guide to new researchers and experts looking to expand their knowledge.

Chapter 13 is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: A Bioinformatics Workflow for Investigating Fungal Biosynthetic Gene Clusters
1 Introduction
2 Materials
2.1 Basic Requirements
2.2 Conda
2.3 fungiSMASH
2.4 BiG-SCAPE
2.5 Other Software
3 Methods
3.1 Prediction of Biosynthetic Regions with fungiSMASH
3.2 Similarity Network Analysis
3.3 Build a Fasta File with Amino Acid Sequences from Core Enzymes
3.4 Phylogenetic Analysis of Core Enzymes
4 Notes
References
Chapter 2: Investigating Fungal Biosynthetic Pathways Using Heterologous Gene Expression: Aspergillus oryzae as a Heterologous...
1 Introduction
2 Materials
2.1 General Laboratory Equipment
2.2 Plasmid Design and Construction via Yeast Recombination
2.3 Plasmid Rescue in E. coli and Screening for Correct Construction
2.4 A. oryzae Transformation
2.5 Chemical Analysis
2.5.1 Small-Scale Extraction from Plates
2.5.2 Liquid Cultures for Large-Scale Fermentation and Purification
3 Methods
3.1 Plasmid Design and Construction via Yeast Recombination
3.1.1 Plasmid and Primer Design
3.1.2 Yeast Recombination
3.1.3 Plasmid Screening and Rescue in E. coli
3.2 A. oryzae Transformation
3.3 Chemical Analysis
3.3.1 Small-Scale Extraction from Plates
3.3.2 Liquid Cultures for Large-Scale Fermentation and Purification
4 Notes
References
Chapter 3: Investigating Fungal Biosynthetic Pathways Using Heterologous Gene Expression: Aspergillus nidulans as a Heterologo...
1 Introduction
2 Materials
2.1 Strains
2.2 Plasmids
2.3 Reagents for Cloning
2.4 Media and Solutions for A. nidulans Transformation and Culturing
2.5 Other Materials for A. nidulans Transformation, Culturing, and Analysis
3 Methods
3.1 Cloning of Plasmids for A. nidulans Expression
3.2 Procedure for Transformation of Plasmids into A. nidulans
3.2.1 Germlings
3.2.2 Digestion
3.2.3 Harvesting Cells
3.2.4 Transformation
3.3 Procedure for Production of Compounds and Biotransformation
3.3.1 Production of Compounds
3.3.2 Biotransformation
3.4 Procedure for RT-PCR (Reverse Transcription-Polymerase Chain Reaction) to Verify Gene Expression
4 Notes
References
Chapter 4: Investigating Fungal Biosynthetic Pathways Using Heterologous Gene Expression: Fusarium sp. as a Heterologous Host
1 Introduction
2 Materials
2.1 Equipment
2.2 PCR Reagents and Primers
2.3 Preparation of Fusarium Spores
2.4 Vector Assembly by TAR Cloning in Yeast
2.5 Recovery and Validation of Cluster-Carrying Plasmids
2.6 Protoplast-Mediated Transformation
3 Methods
3.1 Production of Fusarium Spores
3.2 Target Associated Recombination (TAR)-Mediated Cloning of Targeted Gene Cluster
3.2.1 PCR-Amplification of Cluster Comprising Fragments A1-A(X)
3.2.2 Plasmid Backbone Preparation
3.2.3 Yeast Transformation
3.3 Recovery and Validation of TAR Constructs
3.3.1 Extraction of Plasmid DNA from Yeast Transformants
3.3.2 Recovery and Proliferation of Constructs in E. coli
3.3.3 Validation of TAR Constructs
3.4 Transformation of the Targeted Gene Cluster into F. graminearum
3.4.1 Preparation of TAR Construct Carrying the Targeted Gene Cluster DNA
3.4.2 Protoplast-Mediated Transformation
3.4.3 Selecting Mutants
3.4.4 Screening Fungal Transformants with Colony PCR
3.5 Mutant Genome Validation
3.6 Identification of Target Compounds Produced by Targeted Gene Cluster
4 Notes
References
Chapter 5: Heterologous Expression of Fungal Biosynthetic Pathways in Aspergillus nidulans Using Episomal Vectors
1 Introduction
2 Materials
2.1 Cloning of Biosynthetic Genes on AMA1 Vectors
2.2 Aspergillus nidulans Strain Construction and Analysis
3 Methods
3.1 Cloning of Biosynthetic Genes on AMA1 Vectors
3.2 Preparation of Aspergillus nidulans Protoplasts
3.3 Transformation of Aspergillus nidulans
3.4 Small-Scale Culture and Metabolic Profile Analysis
4 Notes
References
Chapter 6: Targeted Genetic Engineering via Agrobacterium-Mediated Transformation in Fusarium solani
1 Introduction
2 Materials
2.1 Instruments
2.2 Sterile Equipment
2.3 Strains
2.4 Plasmids
2.5 Biological Kits
2.6 Enzymes
2.7 Primers
2.7.1 Primers for Construction of the Overexpression Vector pSHUT4::geneX
2.7.2 Primers for Validation of the pSHUT4::geneX TAR Construct
2.7.3 Primers for Construction of the Gene Deletion Vector pKO-geneY
2.7.4 Primers for Validation of the pKO-geneY TAR Construct (See Fig. 2c)
2.7.5 Primers for Colony PCR Screening of F. solani OE::geneX Transformants (See Fig. 3a)
2.7.6 Primers for Colony PCR Screening of F. solani ΔgeneY Transformants (See Fig. 3b)
2.8 Solutions
2.9 Antibiotics Stocks
2.10 Media
3 Methods
3.1 Purification of Fungal DNA
3.2 Preparation of Fragments for Construction of Overexpression Vector (See Fig. 1)
3.3 Preparation of Fragments for Construction of Deletion Vector (See Fig. 2)
3.4 Assembly of Vector Constructs
3.5 Isolation of TAR Constructs
3.6 Validation of Vector Constructs
3.7 Transforming Validated Vectors into A. tumefaciens
3.7.1 Preparation of Competent Agrobacterium tumefaciens Cells
3.7.2 Electroporation of A. tumefaciens
3.8 Production of F. solani Spores
3.9 Targeted Genetic Engineering via Agrobacterium-Mediated Transformation
3.9.1 Day 1
3.9.2 Day 2
3.9.3 Day 3
3.9.4 Day 6
3.9.5 Day 10-14
3.10 Isolation of Transformants
3.11 Screening Fungal Transformants with Colony PCR
3.11.1 Preparation of Colony PCR Template DNA
3.11.2 Screening OE::geneX Overexpression Mutants (See Fig. 3a)
3.11.3 Screening ΔgeneY Knockout Mutants (See Fig. 3b)
3.12 Preparation of OE::geneX ΔgeneY Double Mutants
4 Notes
References
Chapter 7: Investigating Fungal Biosynthetic Pathways Using Pichia pastoris as a Heterologous Host
1 Introduction
2 Materials
2.1 Plasmid Construction
2.2 P. pastoris Transformation
2.3 Extraction and Detection of Products
2.4 Transcriptional Level Analysis
2.5 Protein Expression Level Analysis
3 Methods
3.1 Pathway Assembly by Big Plasmid Carrying Multiple Biosynthetic Genes
3.1.1 Construction of Expression Plasmids
3.1.2 P. pastoris Transformation
3.1.3 Cultivation and Sampling of P. pastoris
3.1.4 Analysis of Production
3.2 Pathway Assembly by CRISPR-Cas9 Mediated Multiple-Gene Integration
3.2.1 Construction of Expression Vectors
3.2.2 P. pastoris Transformation
3.2.3 Cultivation and Sampling of P. pastoris
3.2.4 Analysis of Production
4 Notes
References
Chapter 8: Evolutionary Genome Mining for the Discovery and Engineering of Natural Product Biosynthesis
1 Introduction to Secondary Metabolism and Evolutionary Genome Mining
1.1 Principles of Evolutionary Genome Mining
2 Functional Annotation and Databases for NP Research
2.1 Functional Annotation of Microbial Genomes
2.1.1 myRAST Genome Annotation
2.2 Databases of Known BGCs
3 Genome Mining Programs
3.1 antiSMASH
3.1.1 General Use of antiSMASH to Identify BGCs
3.1.2 Job Submission
3.1.3 Stringency Levels
3.1.4 Extended Parameters
3.1.5 Interpreting Results
3.2 Algorithms Predicting Enzyme Substrate Specificity
3.2.1 SEARCHGTr (Glycosyltransferase Specificity)
3.2.2 NRPS and PKS Substrate Specificity Prediction
4 NP Specialty Databases
5 Evolutionary Genome Mining of NPs
5.1 EvoMining
5.1.1 Preparation of EvoMining Databases
Genome Database (G-DB)
Central Families Database (CF-DB)
Natural Products Database (NP-DB)
Internal EvoMining Databases
5.1.2 EvoMining Job Submission
A Note on Docker Images
5.2 CORASON
5.3 BiG-SCAPE
5.4 ARTS
5.5 DeepBGC
6 Concluding Remarks
References
Chapter 9: Inducing Global Expression of Actinobacterial Biosynthetic Gene Clusters
1 Introduction
2 Materials
2.1 Overexpressing a Regulatory Gene of Interest
2.2 Conjugation from Escherichia coli into Streptomyces
2.3 Media for E. coli Growth and Growth of Desired Streptomyces Exconjugants
2.4 RNA Extraction (See Note 3)
2.5 Antimicrobial Bioassays
2.5.1 Media for Bioassays
2.5.2 Possible Indicator Strains (See Note 5)
3 Methods
3.1 Genetic Manipulation
3.1.1 Moving Construct from E. coli Cloning Host into E. coli ET12567/pUZ8002
3.1.2 Conjugation from E. coli into Streptomyces and Creating a Spore Stock
3.2 PCR Check for Strain Integrity
3.3 Expression Analyses
3.3.1 RNA Extraction
3.4 Antimicrobial Bioassays (See Note 17)
3.4.1 Monitoring Antimicrobial Production Using the Pancake´´ Bioassay Technique 3.4.2 Monitoring Antimicrobial Production Using thePlug´´ Bioassay Technique
3.4.3 Monitoring Antimicrobial Production Using the ``Plug and Pour´´ Bioassay Technique
4 Notes
References
Chapter 10: Engineering Modular Polyketide Biosynthesis in Streptomyces Using CRISPR/Cas: A Practical Guide
1 Introduction
1.1 Strategic Planning and Key Points
1.2 General Considerations Prior to Adapting the Selected CRISPR/Cas Vector
1.3 Expression of the Cas9
1.4 Design and Expression of sgRNA
1.5 Design of Editing Templates for Deletion, Insertion and Mutation
1.6 Transfer of CRISPR Plasmids into the Target Streptomyces by Intergeneric Conjugation
1.7 Potential Issues with the pSG5 Replicon and Revertants
1.8 Comparison to PCR Targeting
2 Materials
2.1 General Items
2.2 Media and Buffers
2.3 Equipment
2.4 Strains and Plasmids
3 Methods
3.1 Preparation of E. coli ET12567 (pUZ8002) Conjugation Donor Strain
3.1.1 Preparation of Electroporation-Competent Cells
3.1.2 Electroporation of E. coli ET12567 (pUZ8002) with the CRISPR Plasmid
3.2 Preparation of the Streptomyces Conjugation Acceptor Strain
3.3 Intergeneric Conjugation
3.4 Genotype Screening by Colony PCR
3.5 Curing CRISPR Plasmids Containing a Temperature-Sensitive Replicon
3.6 Genotype Confirmation
3.6.1 Isolation of Streptomyces Genomic DNA
3.6.2 PCR Amplification of the Edited Genomic DNA
4 Notes
References
Chapter 11: CRISPR/Cas9-Based Methods for Inactivating Actinobacterial Biosynthetic Genes and Elucidating Function
1 Introduction
2 Materials
2.1 Golden Gate Assembly
2.2 Gibson Assembly
2.3 Introduction of Plasmid DNA into Actinomycetes
2.4 Validation of Mutants
2.5 Plasmid Clearance
2.6 Preparation of a Spore Stock
2.7 Metabolite Purification
3 Methods
3.1 Design of sgRNAs and HR Arms for pCm2 Retargeting
3.2 Construction of Retargeted pCRISPomyces-2
3.3 Introduction of Plasmid DNA into Actinomycetes
3.4 Validation of Mutants
3.5 Plasmid Clearance
3.6 Preparation of a Spore Stock
3.7 Metabolite Purification and LC-MS Analysis
4 Notes
References
Chapter 12: Understanding and Manipulating Assembly Line Biosynthesis by Heterologous Expression in Streptomyces
1 Introduction
2 Materials
2.1 Bioinformatic Analysis of Actinobacterial Biosynthetic Gene Clusters
2.2 Construction of Heterologous Expression Vector for Streptomyces
2.3 Transformation of Streptomyces
3 Methods
3.1 Bioinformatics Analysis of Assembly Line Enzymes
3.2 Construction of Heterologous Expression Vector for Streptomyces
3.3 Transformation of Streptomyces
4 Notes
References
Chapter 13: Heterologous Expression, Purification, and Characterization of Type II Polyketide Synthase Acyl Carrier Proteins
1 Introduction
2 Materials
2.1 General Equipment
2.2 Amplification of ACP Genes
2.3 Gibson Assembly
2.4 Preparation and Transformation of Plasmids
2.5 Expression of ACPs in E. coli
2.6 Purification and On-Column Phosphopantetheinylation of ACPs
2.7 Cyanylation of Holo-ACP Ppant Arm
2.8 Collecting and Analyzing a Vibrational Spectrum
2.9 Colorimetric ACP-KS Mechanistic Cross-Linking Assay
2.10 Tracking Ppant Sequestration Activity Using Raman Spectroscopy
2.11 Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis of ACPs
3 Methods
3.1 Amplification of ACP Genes for Assembly into Expression Vectors
3.2 Gibson Assembly and Transformation of Plasmid into Competent Cells
3.3 Preparation of Amplified Plasmid and Transformation into Expression Strain
3.4 Expression of ACPs in E. coli
3.5 Purification and On-Column Phosphopantetheinylation of ACPs
3.6 Cyanylation of Holo-ACP Ppant Arm
3.7 Collecting and Analyzing a Vibrational Spectrum of ACP-SCN
3.8 Colorimetric ACP-KS Mechanistic Cross-Linking Assay Utilizing Ellman´s Reagent (DTNB) (See Note 44)
3.8.1 Preparation of ACP-TNB-
3.8.2 Quantification of Cross-Linking Activity
3.9 Tracking Ppant Sequestration Activity Using Raman Spectroscopy
3.9.1 Loading of Alkyne Probe-Labeled Acyl Chain
3.9.2 Data Collection
3.10 Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis of ACPs
4 Notes
References
Chapter 14: Cyanobacterial Genome Sequencing, Annotation, and Bioinformatics
1 Introduction
2 Materials
2.1 DNA Extraction
2.2 Quality Check
2.3 Bioinformatics
3 Methods
3.1 Extraction of High-Molecular-Weight Genomic DNA
3.2 Hybrid Genome Sequencing
3.3 Assembly of the Cyanobacterial Reference Genome
3.4 Functional Annotation of the Cyanobacterial Genome
3.5 Genome Mining for the Discovery of Novel Cyanobacterial Natural Products
3.5.1 Overview of Computational Tools and Databases
3.5.2 Exemplary Workflow for the Mining of Cyanobacterial Genomes
4 Notes
References
Chapter 15: Single Crossover to Inactivate Target Gene in Cyanobacteria
1 Introduction
2 Materials
3 Methods
3.1 Construction of Plasmid for Knocking out Target Gene
3.2 Preparation of E. coli Strains
3.3 Preparation of Anabaena 7120 Culture
3.4 Conjugal Transformation of a Cargo Plasmid into Anabaena 7120
3.5 Verification of Single Crossover Knockout Mutants
3.6 Complementation Experiment
4 Notes
References
Chapter 16: Double Crossover Approach to Inactivate Target Gene in Cyanobacteria
1 Introduction
2 Materials
3 Methods
3.1 Construction of Plasmid for Knocking Out Target Gene
3.2 Construction of Plasmid for Knocking out Target Gene Via Overlap Extension PCR
3.3 Preparation of E. coli Strains
3.4 Preparation of Anabaena 7120 Culture
3.5 Conjugal Transformation of a Cargo Plasmid into Anabaena 7120
3.6 Screening for Double Crossover Mutants
3.7 Verification of Double Crossover Knockout Mutants
3.8 Complementation Experiment
4 Notes
References
Chapter 17: Expression of Cyanobacterial Biosynthetic Gene Clusters in Escherichia coli
1 Introduction
2 Materials
2.1 Equipment
2.2 Consumables
2.3 Buffers, Solutions, and Media
2.4 Biologicals
3 Methods
3.1 Transformation of BGC Construct into E. coli GB05-Red
3.2 PCR Amplification of Promoter Resistance Cassette
3.3 Recombineering of the Promoter Cassette into the BGC Vector
3.4 Screening
3.5 Fermentation
4 Notes
References
Chapter 18: Saccharomyces cerevisiae as a Heterologous Host for Natural Products
1 Introduction
2 Platform Strains
3 Design of Expression Cassettes
4 Cell Factory Engineering
5 Biosensors
6 Outlook
References
Chapter 19: Investigating Plant Biosynthetic Pathways Using Heterologous Gene Expression: Yeast as a Heterologous Host
1 Introduction
2 Materials
2.1 Ectopic Gene Expression
2.1.1 Strains and Plasmids (Table 1)
2.1.2 Reagents and Reagent Preparation
2.1.3 Equipment
2.2 Pathway Reconstruction Through Genomic Integration
2.2.1 Strains and Plasmids (Table 3)
2.2.2 Reagents and Reagent Preparation
2.3 Strain Engineering
2.3.1 Strains and Plasmids (Table 5)
2.3.2 Reagents and Reagent Preparation (Same as Subheading 2.2.2)
2.4 Metabolite Isolation and Analysis
2.4.1 Strains and Plasmids (Table 7)
2.4.2 Reagents and Reagent Preparation
2.4.3 Equipment
3 Methods
3.1 Ectopic Gene Expression
3.1.1 Construction of Yeast Expression Plasmid Using Gateway Cloning
3.1.2 Construction of Yeast Expression Plasmid Using Gibson Assembly
Gibson Assembly Followed by Gateway LR Cloning
Direct Gibson Assembly (Alternative to Above)
3.1.3 Yeast Plasmid Transformation
3.2 Pathway Reconstruction Through Genomic Integration
3.2.1 Preparation of DNA Fragments Encoding Gene Expression Cassettes
3.2.2 Yeast Transformation with the DNA Fragments
3.2.3 Screening for Positive Clones
3.2.4 Removing the Selectable Marker
3.3 Strain Engineering
3.3.1 Overexpression of Precursor Biosynthetic Pathway to Enhance Product Titer
3.3.2 Inactivation of Competing Enzymes to Redirect the Flux Toward Desired Product
3.4 Metabolite Isolation and Analysis
3.4.1 Yeast Culturing and Fermentation
3.4.2 Metabolite Extraction from Cell Culture
3.4.3 Metabolite Analysis and Characterization
3.4.4 Compound Quantification
4 Notes
References
Chapter 20: Rapid Combinatorial Coexpression of Biosynthetic Genes by Transient Expression in the Plant Host Nicotiana bentham...
1 Introduction
2 Materials
2.1 Media and Antibiotics
2.2 Cloning
2.3 Agrobacteria Transformation
2.4 Growing Nicotiana benthamiana
2.5 Agrobacteria Infiltration
2.6 Harvesting and Metabolite Extraction
2.7 GC-MS Analysis
3 Methods
3.1 Cloning Using pEAQ-HT
3.1.1 Vector Preparation
3.1.2 Insert Preparation
3.1.3 Construct Assembly and Transformation
3.2 Cloning Using pHREAC
3.2.1 Vector Preparation
3.2.2 Insert Preparation
3.2.3 Construct Assembly and Transformation
3.3 Transformation of Agrobacteria
3.3.1 Preparation of Agrobacterium tumefaciens Electrocompetent Cells
3.3.2 Electroporation
3.4 Seeding and Potting of Nicotiana benthamiana Plants
3.4.1 Seeding
3.4.2 Potting
3.5 Agroinfiltration
3.5.1 Preparing Agrobacterium Strains for Infiltration
3.5.2 Infiltration
3.6 Harvesting and Metabolite Extraction
3.7 GC-MS Analysis
4 Notes
References
Chapter 21: Optimized Tools and Methods for Methanotroph Genome Editing
1 Introduction
2 Materials
2.1 Microbial Strains and Plasmids
2.2 Culture Medium
2.2.1 E. coli Strains
2.2.2 Methylococcus capsulatus Nitrate Mineral Salts (NMS) Cultivation and Mating Medium
2.3 Polymerase Chain Reaction and Isothermal Assembly
3 Methods
3.1 Design and Construction of CRISPR-Cas9 Plasmid Via Isothermal Assembly for M. capsulatus Gene Targeting
3.2 Optimized Methanotroph Conjugation Protocol
4 Notes
References
Chapter 22: Microarray-Based Screening of Putative HSP90 Inhibitors Predicted and Isolated from Microorganisms
1 Introduction
2 Materials
2.1 Contactless Protein Spotting
2.2 Blocking and Washing of the Membranes
2.3 Preparation of Incubation Solutions
2.4 Incubation and Scanning of the Membranes
2.5 Calculation of Binding Activity
3 Methods
3.1 Contactless Protein Spotting
3.2 Blocking and Washing of the Membranes
3.3 Preparation of Incubation Solutions
3.4 Incubation of the Membranes
3.5 Preparation of the Membranes for Scanning
3.6 Scanning of the Membranes
3.7 Evaluation of the Fluorescence Intensities
4 Notes
References
Chapter 23: Isolation of Water-Soluble Metabolites from Marine Invertebrates and Microorganisms
1 Introduction
2 Materials
2.1 Macroporous Adsorptive Resins
2.2 Macroporous Adsorptive Resin Pretreatment
2.3 Hydrophilic Extract Adsorption on Resins
2.4 Macroporous Resin Desorption and Extract Preparation
2.5 Amberlite XAD-2, XAD-4, and XAD-7 1:1:1 Resin Mixture Cleaning and Storage
2.6 Diaion HP-20 Resin Cleaning and Storage
2.7 Resin Extract Cleanup
3 Methods
3.1 Macroporous Adsorptive Resin Pretreatment
3.2 Hydrophilic Extract Adsorption on Resins
3.3 Resin Desorption and Extract Preparation
3.4 Amberlite XAD-2, XAD-4, and XAD-7 (1:1:1) Mixture Cleaning and Storage
3.5 Diaion HP-20 Resin Cleaning and Storage
3.6 Resin Extract Cleanup
4 Notes
References
Chapter 24: Natural Product Investigation in Lichens: Extraction and HPLC Analysis of Secondary Compounds in Mycobiont Cultures
1 Introduction
1.1 HPLC Analyses of Lichens and Mycobiont Cultures
2 Materials
2.1 Solvents and Reagents
3 Methods
3.1 Extraction of Secondary Compounds from the Lichen Mycobiont Culture
3.2 Filtration
3.3 HPLC Analysis
4 Notes
References
Index