The complex regulation of the lipopetpide synthesis

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Table of contents

GENERAL INTRODUCTION
CHAPTER 1: LITERATURE REVIEW
1. BACILLUS SUBTILIS: IN A NUTSHELL
1.1 Bacillus subtilis and its genome
1.2 The complex regulation of the lipopetpide synthesis
1.3 Industrial applications
2. INSIGHTS INTO NON-RIBOSOMAL PEPTIDE SYNTHESIS (NRPS), UNIQUE MACHINERY
2.1 Introduction
2.2 Primary domains
2.3 Secondary domains
2.4 Classification
2.5 Precursors of the NRPS mechanism and their regulation mechanisms
2.6 Interest of the NRPS peptides produced by B. subtilis
3. SURFACTIN: EXPLORING FROM GENE TO PRODUCT
3.1 Discovery
3.2 Structure
3.3 Properties
3.4 Biosynthesis
3.5 Regulators
3.6 Structure and activity relationship
3.7 Analysis
3.8 Application
4. OVERPRODUCTION OF SURFACTIN
4.1 Present scenario
4.2 Role of various factors
4.3 Direct approach
4.4 Linkage between Direct and Indirect approach
4.5 Indirect approach
CHAPTER 2: MATERIALS AND METHODS
MATERIALS
1 Strains
2 Plasmids
3 Genotype of plasmids and E.coli strains
4 Culture Media
METHODS
5 DNA Manipulation
6 Formalization of reaction network
7 Mutant construction
8 Transformation
9 Surfactin analysis
10 LC-MS-MS analysis of surfactin isomer
11 Extracellular metabolome
CHAPTER 3: RESULTS AND DISCUSSION
1. Is intracellular pool of leucine a limiting factor for surfactin biosynthesis?
2. How to enhance leucine production within the intracellular pool ?
2.1 Formalization of the model of BCAA metabolic pathway
2.2 Knockout prediction
2.3 Mutant constructed
2.4 Growth and surfactin analysis
2.5 Publication and Valorization of these results
3. FACTORS INFLUENCING SURFACTIN PRODUCTION
3.1 Role of nutrient composition
3.2 Impact of oxygen transfer
3.3 Other factors
3.4 Publication and Valorization of these results
4. ROLE OF PYRUVATE FLUX
4.1 Mutant constructed
4.2 Growth and surfactin analysis
4.3 Carbon metabolism and surfactin production
5. IMPACT OF DELETION ON SURFACTIN VARIANTS
5.1 Isoform analysis
5.2 Extracellular metabolite analysis
5.3 Publication and Valorization of these results
6. PERMEASE
6.1 Mutant constructed
6.2 Growth and surfactin analysis with native promoter and constitutive promoter
6.3 Publication and Valorization of these results
CHAPTER 4: CONCLUSION AND PERSPECTIVES
APPENDIX I VECTORS AND PLASMIDS
pGEM-T Easy
pDG1661
pBC16
pUC57
pBG124
pBG127
Synthesized sequence (PrepU – ermC- p srfAA)
APPENDIX II MEDIA COMPOSITIONS
LB MEDIUM
COMPETENCE MEDIUM
LANDY MEDIUM
TSS SUPPLEMENTED WITH 16 AMINO ACIDS
AMBER MEDIUM
APPENDIX III
APPENDIX IV