Rational engineering of the methylerythritol 4-phosphate (MEP)

Name: Rational engineering of the methylerythritol 4-phosphate (MEP) | pathway for terpenoid production through metabolic control analysis
Brand: Apprimus Verlag
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pathway for terpenoid production through metabolic control analysis

Daniel Volke(Autor*in)

Apprimus Verlag

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Erschienen am 25. Juli 2018

186 Seiten

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978-3-86359-650-7 (ISBN)

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Inhalt

Intro
Table of Contents
I. Introduction
I.1 Terpenoids
I.2 MVA and MEP pathway- Biosynthesis of DMAPP and IPP through two analogous pathways
I.3 The MEP pathway: Enzymatic steps and regulation
I.3.1 The MEP pathway in E. coli
I.4 Branches of the MEP pathway in E. coli
I.5 Stoichiometry of the MEP and MVA pathway
I.6 The value of terpenoids for human society
I.7 Terpenoid production in recombinant organisms using the MEPpathway
I.7.1 Isoprene and Isoprene synthase
I.8 Metabolic control analysis
I.9 Objectives of the research and background to the research problem
II. Material and methods
II.1 Material and chemicals
II.1.1 Instruments
II.1.2 Software
II.1.3 Chemicals
II.1.4 Strains
II.1.5 Nucleotides
II.1.6 Oligonucleotides
II.2 Microbiological methods
II.2.1 Culture media
II.2.2 Determination of optical density of E. coli cultures
II.2.3 Culture conditions
II.2.4 Heterologous expression und homologous overexpression in E. coli BL21
II.2.5 Long-term storage of bacterial cultures
II.2.6 Preparation of E. coli lysate
II.2.7 Preparation of inverted membranes from E. coli BL21
II.2.8 Transformation of E. coli
II.2.8.1 Heat shock
II.2.8.2 Electroporation
II.3 Biochemical methods
II.3.1 Agarose gel electrophoresis
II.3.2 Protein concentration determination
II.3.3 DNA sequencing
II.3.4 PCR (polymerase chain reaction)
II.3.5 Colony PCR for screening for genomic alterations and engineered plasmids
II.3.6 Plasmid preparation
II.3.7 PCR and gel clean-up of DNA fragments
II.3.8 Splicing by overlap extension PCR
II.3.9 Gibson assembly (sequence-independent cloning)
II.3.10 Recombineering
II.3.11 DXP synthesis
II.3.12 Indirect substrate transport test of proton-dependent transporter in inverted membranes
II.3.13 SDS-PAGE
II.3.13.1 Coomassie staining
II.3.14 Western blot
II.3.15 Deoxyxylulose 5-phosphate synthase purification from A. thaliana
II.3.15.1 Cloning
II.3.15.2 Overexpression and purification of AtDxs
II.3.15.3 In vitro enzyme assay for Dxs
II.4 Chromatography
II.4.1 LC-MS/MS
II.4.1.1 Targeted Metabolomics
II.4.1.1.1 Sample preparation for targeted metabolomics
II.4.1.2 Kinetic 13C incorporation analysis into the MEP pathway intermediates
II.4.1.2.1 Calculation of label incorporation
II.4.1.2.2 Additional Information for targeted metabolomics
II.4.1.3 Targeted Proteomics
II.4.1.3.1 Sample preparation
II.4.1.3.2 In Silico prediction of peptide mass and fragment size
II.4.1.3.3 Synthetic internal standard and calibration curve
II.4.1.3.4 Separation and detection of peptides
II.4.2 GC-MS/MS
II.4.2.1 Isoprene detection and quantification
III. Statistical analysis
IV. Results
IV.1 Establishment and optimization of targeted proteomics for the enzymes of the MEP pathway
IV.2 Establishment of targeted metabolomics for the intermediates of the MEP pathway
IV.3 Establishing isotopically non-stationary 13C metabolic flux analysis of the MEP pathway
IV.3.1 Mathematical description of the label incorporation
IV.4 Enhanced isoprene production in E. coli through overexpression of native MEP pathway genes
IV.4.1 Construction of vectors for isoprene production in E. coli
IV.4.2 Isoprene production of engineered E. coli strains
IV.4.3 Protein expression of engineered strains
IV.4.4 Metabolite concentration in engineered strains
IV.4.5 Expression of additional genes of the MEP pathway
IV.5 Establishment and optimization of recombineering for the generation of mutation libraries
IV.6 Metabolic control analysis of the MEP pathway
IV.6.1 Construction of RBS libraries through recombineering
IV.6.2 Influence of RBS sequence on enzyme concentration
IV.6.3 MEP pathway intermediate concentrations in dxs expression library
IV.6.4 Export of MEP pathway intermediates
IV.6.5 Isoprene production of the expression mutants
IV.6.6 Flux through the MEP pathway
V. Discussion
V.1 Establishment of targeted metabolomics for the intermediates of the MEP pathway
V.2 Establishment of isotopically non-stationary 13C metabolic flux analysis of the MEP pathway
V.3 Establishment and optimization of recombineering in E. coli BL21
V.4 MEP pathway enzyme concentration in E. coli and implications
V.5 Isoprene production in E. coli
V.6 Metabolic control analysis of the MEP pathway in E. coli
V.6.1 Perturbation of the MEP pathway
V.6.2 Metabolite concentration in dxs expression library
V.6.3 Concentration control coefficients of Dxs
V.6.4 Export of intermediates of the MEP pathway
V.6.5 Flux through the MEP pathway and flux control coefficients of Dxs
V.6.6 Isoprene production in dxs expression library
V.7 Conclusion from this research and objectives for future research
VI. References
VII. Appendix
VII.1 Characterization of Deoxyxylulose 4-phosphate synthase from A. thaliana
VII.1.1 Purification
VII.1.2 Optimization of reaction conditions
VII.1.3 Kinetic characterization of AtDxs
VII.1.4 Inhibition of AtDxs by ß-cyclocitral
VII.1.5 Discussion of the results of the characterization of the AtDxs
VII.2 Nucleotide sequence of the codon optimized isoprene synthase from P. alba for E. coli
VII.3 MEcPP concentration in the expression strength library
VII.4 Calculations of the terpenoid yield achieved and theoretical maximum yield
VII.5 Alternative targeted proteomics method
VII.6 Hydrophilic interaction chromatography for targeted metabolomics
VII.7 Calibration curve targeted proteomics
VII.8 Calibration curve targeted metabolomics
VII.9 Additional data for isotopically non-stationary 13C metabolicflux analysis
VII.9.1 Determination of needed initial glucose concentration
VII.9.2 Quenching by fast centrifugation
VII.9.3 Quenching by fast reduction of broth temperature
VII.9.4 Data from isotopically non-stationary 13C metabolic flux analysis
VII.10 Expression of the isc operon
VII.11 Analysis of the fosmidomycin resistance protein
VII.12 Capturing isoprene in an aerobic fermentation process
VII.13 Purification of Dxs from P. alba through glutathione sepharose affinity chromatography
VII.14 References for appendix
VIII. List of figures
IX. List of tables

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