Preface
Contents
Contributors
Part I: General Overview
Chapter 1: Current Trends and Perspectives in Microbial Bioconversions of Steroids
1 Introduction
2 Microbial Bioconversions of Phytosterol
2.1 Phytosterols
2.2 Androstenedione (AD) and Androstadienedione (ADD)
2.3 9Ξ±-Hydroxy Androstenedione (9-OH-AD)
2.4 9,21-Dihydroxy-20-methylpregna-4-en-3-one (9-OH-4-HP)
2.5 Hydroxycholesterol
2.6 Sitolactone (HIL) and HIP
2.7 Testosterone and Boldenone
3 Microbial Enzymes for Steroid Production
3.1 Cholesterol Oxidase
3.2 3-Ketosteroid-Ξ1-dehydrogenase (KstD)
3.3 Laccases
4 Heterologous Expression of Steroidogenesis Systems and Generation of Recombinant Strains for Oxyfunctionalization of Steroids
4.1 Bacterial Steroid Hydroxylases
4.2 Mammalian Steroidogenesis System
4.3 Fungal CYPs
5 Conclusions
References
Part II: Microbial Screening and Genetic Manipulation
Chapter 2: Isolation of Environmental Bacteria Able to Degrade Sterols and/or Bile Acids: Determination of Cholesterol Oxidase...
1 Introduction
2 Materials
2.1 Preparation of Steroids as Carbon Sources for Culture Media
2.2 Isolation of Environmental Strains Able to Degrade Cholesterol and/or Cholic Acid
2.3 Identification of Strains Showing Extracellular Cholesterol Oxidase Activities
2.4 Measurement of Extracellular Cholesterol Oxidase Activity in Culture Supernatants
2.5 Measurement of 3Ξ±-Hydroxysteroid Dehydrogenase Activity in Cell Extracts from Cells Grown with Cholic Acid as Sole Carbon ...
2.6 Measurement of 17Ξ²-Hydroxysteroid Dehydrogenase Activity in Cell Extracts
2.7 UHPLC Analyses of Selected Steroids
3 Methods
3.1 Preparation of Steroids as Carbon Sources for Culture Media
3.1.1 Preparation of Cholesterol, Bile Acids, or Testosterone Emulsified with Methyl-Ξ²-cyclodextrins
3.1.2 Preparation of Cholesterol or Testosterone Emulsified in Tyloxapol
3.2 Isolation of Environmental Strains Able to Degrade Cholesterol and/or Cholate
3.3 Isolation of Environmental Strains with Cholesterol Oxidase Activity
3.4 In Vitro Determination of Cholesterol Oxidase Activity
3.5 Determination of 3Ξ±-Hydroxysteroid Dehydrogenase Activity in Cell Extracts
3.6 17Ξ²-Hydroxysteroid Dehydrogenase Activity in Cell Extracts
3.7 Analysis of Substrates and Products of In Vitro Reactions by UHPLC
4 Notes
References
Chapter 3: Selection of Biodegrading Phytosterol Strains
1 Introduction
2 Materials
2.1 Substrate
2.2 Bacterial Strains
2.3 Bacterial Growth Media
2.4 Equipment and Solvents
3 Methods
3.1 Selection of Strains Tolerant to High Concentrations of Ξ²-Sitosterol
3.2 Ξ²-Sitosterol Biotransformation
3.3 Extraction of Biotransformation Products
3.4 Analysis of Biotransformation Products
3.4.1 Analysis by TLC
3.4.2 Steroid Quantification by GLC-Mass Spectrometry Analysis
4 Notes
References
Chapter 4: Identification and Characterization of Some Genes, Enzymes, and Metabolic Intermediates Belonging to the Bile Acid ...
1 Introduction
2 Materials
2.1 Isolation of P. putida DOC21 Mutants Unable to Catabolize BA by Tn5 Transposon Mutagenesis
2.2 Identification of the Target Tn5 Insertion Point and Surrounding Sequences in the Genome of P. putida Strain DOC21
2.3 Genome Editing Through Deletion of Specific Genes Involved in Bile Acid Catabolism in P. putida DOC21
2.4 Identification of 4-Androstene-3,17-dione and 1,4-Androstadiene-3,17-dione as Metabolic Intermediates Using Specific Mutan...
2.5 Identification of StdA1 and StdA2 from P. putida DOC21 as ATP-Dependent Acyl-CoA Synthetases Involved in the Catabolism of...
2.6 Identification of ATP-Dependent Acyl-CoA Synthetase StdA3 from P. putida DOC21 Involved in the Degradation of the C and D ...
3 Methods
3.1 Isolation of Mutants of P. putida DOC21 Unable to Catabolize BAs by Tn5 Transposon Mutagenesis
3.2 Identification of the Tn5 Insertion Point in the Genome of P. putida Strain DOC21 Mutants and Location of the Genomic Sequ...
3.2.1 Construction of pJQ-Tn5 (Fig. 4)
3.2.2 Transferring of pJQ-Tn5 into the P. putida DOC21 Tn5 Mutant by Triparental Mating (Fig. 5)
3.2.3 Cloning of the DNA Fragments in Which Tn5 Has Been Inserted (Fig. 5)
3.3 Genome Editing Through Deletion of Specific Genes Involved in Bile Acid Catabolism in P. putida DOC21
3.4 Identification of 4-Androstene-3,17-dione and 1,4-Androstadiene-3,17-dione as Metabolic Intermediates Using Specific Mutan...
3.5 Identification of StdA1 and StdA2 from P. putida DOC21 as ATP-Dependent Acyl-CoA Synthetases Involved in the Catabolism of...
3.5.1 Preparation of the Substrates for StdA1 and StdA2 Assays
3.5.2 StdA1 and StdA2 ATP-Dependent Acyl-CoA Synthetase Assays
3.6 Identification of ATP-Dependent Acyl-CoA Synthetase StdA3 from P. putida DOC21 Involved in the Degradation of the C and D ...
3.6.1 Obtaining of the Substrates for StdA3 Assay
3.6.2 StdA3 ATP-Dependent Acyl-CoA Synthetase Assay
4 Notes
References
Chapter 5: Targeted Mutagenesis of Mycobacterium Strains by Homologous Recombination
1 Introduction
2 Materials
2.1 Microbial Strains
2.2 Genetic Manipulation Material
2.3 Agarose Gel
2.4 Media and Other Materials
2.5 Equipment
3 Methods
3.1 Primer Design
3.2 Vector Construction
3.3 Amplification of the Upstream and Downstream Homology Arms of Targeted Gene
3.4 Construction of Knockout Plasmids
3.5 Preparation of Mycobacterial Competent Cells
3.6 Electroporation of Knockout Plasmids into Mycobacterial Competent Cells and Twice Recombination
4 Notes
References
Part III: Genetic and Biochemical Analyses
Chapter 6: RNA Preparation and RNA-Seq Bioinformatics for Comparative Transcriptomics
1 Introduction
2 Materials
2.1 Buffers, Solutions, and Reagents
2.2 Laboratory Equipment for RNA Extraction
2.3 Bioinformatics Hardware
2.4 Software
2.5 Input Files
3 Methods
3.1 Samples Harvest and RNA Stabilization
3.2 RNA Extraction
3.3 Removal of Contaminant DNA
3.4 Alignment of the Sequencing Reads to the Genome Sequence
3.5 Differential Expression Analysis with splineTimeR
4 Notes
References
Chapter 7: Bidimensional Analyses of the Intra- and Extracellular Proteomes of Steroid Producer Mycobacteria
1 Introduction
2 Materials
2.1 Bacterial Growth Media
2.2 Bacterial Strain
2.3 Biological and Chemical Materials
2.4 Buffers and Solutions
2.5 Acrylamide/Bis-acrylamide Gels
2.6 Equipment
3 Methods
3.1 Protein Collection: Secretome
3.2 Protein Collection: Intracellular Proteome
3.3 Protein Quantitation by Bradford Method
3.4 Sample Integrity Validation
3.5 2D Clean-Up
3.6 2D-DIGE Analysis
4 Notes
References
Chapter 8: Fungal Sterol Analyses by Gas Chromatography-Mass Spectrometry Using Different Derivatives
1 Introduction
2 Materials
2.1 Reagents
2.1.1 Fungal Strains
2.1.2 Media
2.1.3 Saponification
2.1.4 Acetylation
2.1.5 Trimethylsilylation
2.1.6 GC-MS
2.2 Common Equipments
2.2.1 Inoculation
2.2.2 Biomass Obtention
2.2.3 Saponification
2.3 Lab Equipment
2.3.1 Extraction and Derivation
2.3.2 GC-MS
3 Methods
3.1 Cell Culture
3.1.1 On Solid Medium
3.1.2 Aspergillus fumigatus in Liquid Medium
3.1.3 Candida spp. in Liquid Medium
3.2 Saponification
3.3 Derivation
3.3.1 Acetylation
3.3.2 Trimethylsilylation
3.4 GC-MS Analyses
3.4.1 General Parameters
3.4.2 Free and Acetylated Sterols
3.4.3 TMSi Ether Derivatives
4 Interpretation Keys
5 Notes
References
Chapter 9: Cholesterol Assay Based on Recombinant Cholesterol Oxidase, ABTS, and Horseradish Peroxidase
1 Introduction
2 Materials
2.1 Strains and Culture Media
2.2 Molecular Cloning
2.3 Recombinant COXNs Purification
2.4 Recombinant COX Activity Measurements
3 Methods
3.1 Cultivation of N. simplex
3.2 Cloning of Cholesterol Oxidase Gene from N. simplex
3.3 Cultivation of Recombinant E. coli BL21(DE3)
3.4 Purification of Recombinant Cholesterol Oxidase
3.5 Spectrophotometric Measurement of COX Activity and Cholesterol Concentration Assay
3.5.1 COX Activity Detection in MCD-Based Buffers
3.5.2 COX Activity Detection in Cholate Assay Buffer
4 Notes
References
Chapter 10: Measuring the Interaction of Sterols and Steroids with Proteins by Microscale Thermophoresis
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents and Supplies
3 Methods
3.1 Solubilization of Cholesterol Sulfate
3.2 Solubilization of Pregnenolone Sulfate
3.3 Fluorescent Labeling of the His-Tagged Protein
3.4 Pretest of the Fluorescent-Labeled Protein
3.5 Ligand-Binding Check
3.6 Determining the Affinity of Ligand Binding
4 Notes
References
Part IV: Fermentation and Biocatalysis for Steroids Production
Chapter 11: Cultivation of Mycolicibacterium spp. Mutants in Miniaturized and High-Throughput Format to Characterize Their Gro...
1 Introduction
2 Materials
2.1 Cultivation of Mycolicibacterium and Sampling
2.2 Equipment and Materials for Extraction and Mass Spectrometry Analysis
3 Methods
3.1 Cultivation of Strains
3.2 Sampling Procedures
3.3 Extraction of Phytosterols and Steroids
3.4 RapidFire-MS Analysis and Data Analysis
4 Notes
References
Chapter 12: Ξ²-Sitosterol Bioconversion in Small-Scale Devices: From Microtiter Plates to Microfluidic Reactors
1 Introduction
2 Materials
2.1 Culture Media and Equipment for Mycobacterium sp. NRRL B-3805 Growth
2.2 Microbioreactor Fabrication and Assembly
2.3 AD Extraction and HPLC Analysis
2.4 Sterilized Materials
3 Methods
3.1 Microbioreactor Fabrication
3.2 Preparation of Resting Cells of Mycobacterium sp. NRRL B-3805
3.3 Bioconversion Trials Using Resting Cells of Mycobacterium sp. NRRL B-3805 in Microtiter Plates
3.4 Bioconversion Trials Using Resting Cells of Mycobacterium sp. NRRL B-3805 in Microbioreactors
3.5 Bioconversion Runs Using Growing Cells of Mycobacterium sp. NRRL B-3805 in Microtiter Plates
3.6 Bioconversion Trials Using Growing Cells of Mycobacterium sp. NRRL B-3805 in Microbioreactors
4 Notes
References
Chapter 13: Biocatalysis of Steroids by Mycobacterium sp. in Aqueous and Organic Media
1 Introduction
2 Materials
2.1 Media Preparation
2.2 Bioconversion Assay
2.3 Bacterial Strains
2.4 HPLC
3 Methods
3.1 Bacterial Growth
3.2 Side-Chain Cleavage of Sitosterol in Aqueous Systems
3.3 Side-Chain Cleavage of Sitosterol in Organic-Aqueous Systems
3.4 Side-Chain Cleavage of Sitosterol Using Green Solvents
3.5 Determination of Side-Chain Cleavage Activity
4 Notes
References
Chapter 14: Scale-Up of Phytosterols Bioconversion into Androstenedione
1 Introduction
2 Materials
2.1 Vegetative Stages
2.1.1 First Vegetative and Second Vegetative Stages (Flask Scale)
2.1.2 Third Vegetative Stage (5 L Bioreactor)
2.2 AD Bioconversion at 5 L Scale
2.3 AD Bioconversion at 20 L Scale
2.4 AD Analysis
2.4.1 AD Analysis by HPLC
2.4.2 AD Analysis by TLC
2.5 General Equipment
3 Methods
3.1 Vegetative Stages
3.1.1 Mycolicibacterium neoaurum B-3805 First Vegetative Stage (100 mL)
3.1.2 M. neoaurum B-3805 Second Vegetative Stage (300 mL)
3.1.3 M. neoaurum B-3805 Third Vegetative Stage (5 L)
Media and Bioreactor Preparation
Incubation
3.2 AD Bioconversion at 5 L Scale
3.2.1 Media and Bioreactor Preparation
3.2.2 Bioconversion of Phytosterol to AD
3.3 AD Bioconversion at 20 L Scale
3.3.1 Media and Bioreactor Preparation
3.3.2 Bioconversion of Phytosterol to AD
3.4 Quantification of Sterols and AD
3.4.1 Analysis by HPLC
Sample Extraction
Standard Preparation
Parameter Analysis
3.4.2 Analysis by TLC
Sample Extraction
TLC Development
4 Notes
References
Chapter 15: Bioconversion of Phytosterols into Androstenedione by Mycolicibacterium
1 Introduction
1.1 Microbial Transformation of Phytosterols to Steroids
1.2 Phytosterols and Steroids Are Almost Insoluble in Water
1.3 Two-Phase Systems
1.4 Cyclodextrins
1.5 Cultivation of Mycolicibacterium
1.6 Sampling
1.7 Analysis
2 Materials
2.1 Cell Culture
2.2 Sterol and Steroid Analysis
3 Methods
3.1 Preparation of Standard Inoculum
3.2 Purity Check of Standard Inoculum
3.3 Shake Flask Cultivation
3.4 Shake Flask Bioconversion of Phytosterol to AD
3.5 Fermenter Cultivation
3.6 Fermenter Bioconversion of Phytosterol to AD
3.7 Sample Collection
3.8 Sample Extraction and Preparation
3.9 LC-MS Analysis
3.10 Calculation of Results
4 Notes
References
Chapter 16: Selective Microbial Conversion of DHEA into 7Ξ±-OH-DHEA
1 Introduction
2 Materials
2.1 Cultivation and Transformation
2.2 TLC and HPLC Analysis
3 Methods
3.1 Generation of First Inoculum
3.2 DHEA Bioconversion
3.3 TLC Analysis
3.4 HPLC Analysis
4 Notes
References
Chapter 17: Production of 11Ξ±-Hydroxysteroid Derivatives by Corynebacterium glutamicum Expressing the Rhizopus oryzae Hydroxyl...
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Culture and Biotransformation Media
2.3 Steroids
2.4 Bacterial Preculture
2.5 Steroid Extraction
2.6 Monitoring Steroid Biotransformation by TLC Analysis
2.7 Steroid Quantification: HPLC-DAD Chromatography-Mass Spectrometry
2.8 General Equipment
3 Methods
3.1 Shaken Flask Biotransformation
3.1.1 Preculture Preparation
3.1.2 Culture Preparation
3.1.3 Steroid Biotransformation by Resting Cells
3.2 Steroid Extraction of Shaken Flask Biotransformation
3.3 Monitoring Sterol Biotransformation by TLC Analysis
3.4 Monitoring Sterol Biotransformation by DAD-HPLC-MS Analysis
4 Notes
References
Chapter 18: Obtaining of 24-Norchol-4-ene-3,22-dione from Phytosterol with Mutants of Mycolicibacterium neoaurum
1 Introduction
1.1 Parent Bacterial Strains and Gene Target
1.2 Overview of the Gene Knockout Method
1.3 Overview of Biotransformation Process
1.4 Overview of Steroid Product Isolation and Purification
2 Materials
2.1 Bacterial Strains and Nutrient Media
2.2 Genomic DNA Isolation, Recombinant Plasmid Construction, and Gene Knockout
2.3 Phytosterol Biotransformation
2.4 Steroid Product Isolation and Purification
2.5 Steroid Analyses
3 Methods
3.1 Maintenance of M. neoaurum Stock Cultures
3.2 Genomic DNA Isolation from M. neoaurum
3.3 Recombinant Plasmid Construction
3.4 M. neoaurum Electrocompetent Cell Preparation
3.5 Electroporation and Gene Knockout
3.6 Preparation of M. neoaurum Inoculum for Biotransformation
3.7 Phytosterol Biotransformation
3.8 Separation of Biomass
3.9 Extraction of Steroids
3.10 Purification of 24-NCED and 20-HMP
3.11 HPLC Analysis of Steroids
4 Notes
References
Chapter 19: Stabilization of Enzymes by Using Thermophiles
1 Introduction
2 Materials
2.1 Bacterial Growth Media
2.2 Bacterial Strains
2.3 Biological and Chemical Materials
2.4 Equipment
3 Methods
3.1 epPCR
3.2 Mutant Library Preparation
3.3 Selection of Thermostable Mutants
4 Notes
References
Chapter 20: Stigmasterol Removal by an Aerobic Treatment System
1 Introduction
2 Materials
2.1 Equipment
2.2 Raw Materials and Solvents
3 Methods
3.1 Inoculum
3.2 Primary Treatment System of Kraft Mill Influent
3.3 Influent
3.4 Aerated Lagoon (AL) Treatment
3.5 Activated Sludge (AS) Treatment
3.6 Moving Bed Biofilm Reactors (MBBR Treatment): Start-Up and Operation
3.7 Performance of All Reactors
3.8 Extraction Techniques and CG-MS Sample Determinations
3.9 Determination of Phytosterols
4 Notes
References
Part V: Steroids Application
Chapter 21: Glucocorticoid Effect in Cancer Patients
1 Introduction
1.1 Definitions
2 Effect of Glucocorticoids (GC) on the Immune Response
3 The Role of MicroRNA in Glucocorticoid Action
4 Glucocorticoids and Apoptosis
5 Impact of Glucocorticoid Use on Cancer-Related Symptoms
6 Future Perspectives
References
Index