The life cycle of Streptomyces

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Table of contents

Literature review
I. Microbial natural product discovery in the genomics era
I.1. Diversity of microbial specialized metabolites
I.2. From genes to molecules
I.3. Streptomyces, a prolific metabolite producer
I.3.a. The life cycle of Streptomyces
I.3.b. Regulation of specialized metabolite biosynthesis in Streptomyces
I.3.b.i Pathway-specific regulators
I.3.b.ii Pleiotropic regulators
II. Ribosomally synthesized and post-translationally modified peptides
II.1. Diversity
II.2. Lanthipeptides
II.2.a. Structure
II.2.b. Biosynthesis
II.2.c. Classification
II.2.d. Biological activities
II.3. Thiopeptides
II.3.a. Structure
II.3.b. Biosynthesis
II.3.c. Classification
II.3.d. Biological activities
II.4. Lasso peptides
II.4.a. Diversity
II.4.b. Discovery
II.4.c. Biosynthesis
II.4.c.i Gene cluster organization
II.4.c.ii Maturation process
II.4.c.iii Additional modifications
II.4.d. Biological activities
II.4.e. Bioengineering using lasso peptides
III. Ecological role of RiPPs
IV. Objectives of the thesis
Materials and methods
I. Chemicals and biological materials
II. General DNA and microbiology methods
II.1. Purification of plasmids and cosmids
II.2. Isolation of genomic DNA
II.3. General Polymerase Chain Reaction methods (PCR)
II.4. Agarose gel electrophoresis
II.5. General cloning procedure
II.6. Preparation and transformation of competent E. coli cells
II.6.a. Electrocompetent cells
II.6.b. Chemical competent cells
II.7. Site-directed mutagenesis
II.8. Modification of cosmids using PCR targeting
II.9. Conjugation methods for Streptomyces strains
III. RNA methods
III.1. RNA extraction
III.2. Reverse transcription PCR (RT-PCR)
III.3. Real-time quantitative PCR (qPCR)
IV. Heterologous expression of lasso peptide gene clusters
IV.1. Generation of lasso peptide expression system
IV.1.a. Cosmid libraries
IV.1.b. Plasmid vectors
IV.2. Lasso peptide production in heterologous host
IV.3. Lasso peptide production and extraction
V. Liquid chromatography mass spectrometry (LC-MS)
VI. Generation of deletion mutants in S. sviceus-
VI.1. Inactivation using pKGLP2-based suicide plasmids
VI.2. Inactivation using modified cosmids
VI.2.a. Generation of P4H8, a P4H7 based suicide cosmid
VI.2.b. Modification of P4H8 by PCR Targeting
VI.2.c. Inactivation using P4H8-based cosmids
VI.2.d. Gene complementation in the deletion mutants of S. sviceus
VII. Gene reporter assays
VIII. Oxidative sensibility assay
IX. Morphology characterization
IX.1. Optic microscopy
IX.2. Scanning Electronic microscopy (SEM)
X. Autoinduction of sviceucin
X.1. Gene reporter assay
X.2. qPCR
Chapter I: Heterologous expression of lasso peptides from Actinobacteria
I. Introduction
I.1. Common strategies to trigger and improve the expression of cryptic or silent BGCs
I.1.a. Optimization of cultivation conditions
I.1.b. Heterologous expression
I.1.c. Metabolic engineering
I.1.d. Synthetic biology
I.2. Heterologous expression of lasso peptides
II. Results and discussion
II.1. Genome mining
II.1.a. Stackebrandtia nassauensis cluster
II.1.b. Nocardiopsis alba cluster
II.1.c. Actinoalloteichus sp. cluster
II.1.d. Streptomyces noursei cluster
II.1.e. Streptomyces venezuelae cluster
II.2. Heterologous production based on native gene clusters
II.2.a. Cosmid-based method
II.2.b. Co-expression with the pathway-specific regulator
II.3. Heterologous expression using genetically-engineered clusters
II.3.a. Promoter exchange strategy
II.3.b. Orthogonal two-plasmid expression system
III. Conclusion and perspectives
Chapter II: Regulation mechanism and ecological role of sviceucin in Streptomyces sviceus
I. Introduction
I.1. Sviceucin as a good model to study LP regulation and ecological roles
I.2. Overview of growth and development in Streptomyces
II. Results and discussion
II.1. Determining the operon structure in the sviceucin gene cluster
II.2. Growth dependence of sviceucin production
II.3. Probing the regulation mechanism of sviceucin biosynthesis
II.3.a. Is sviceucin biosynthesis controlled by SviR1 and SviR2 ?
II.3.b. Attempts to determine the SviR1 and SviR2 regulon in the svi cluster
II.3.c. Effects of deleting other genes on sviceucin production
II.3.d. Is sviceucin an autoinducing peptide?
II.4. Morphological characterization of S. sviceus mutants
II.5. Sensitivity to oxidative stress of S. sviceus mutants
III. Conclusions and perspectives
Conclusion and perspectives
I. Heterologous expression of lasso peptides from Actinobacteria
II. Regulation mechanism and physiological role of sviceucin