Rational engineering of the methylerythritol 4-phosphate (MEP) pathway for terpenoid production through metabolic control analysis:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Aachen
Apprimus Verlag
2018
|
| Ausgabe: | 1. Auflage |
| Schriftenreihe: | Applied microbiology
Volume 9 |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | ix, 173 Seiten Illustrationen, Diagramme 21 cm |
| ISBN: | 9783863596361 |
Internformat
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| 016 | 7 | |a 1163346268 |2 DE-101 | |
| 020 | |a 9783863596361 |c Broschur |9 978-3-86359-636-1 | ||
| 035 | |a (OCoLC)1047395356 | ||
| 035 | |a (DE-599)DNB1163346268 | ||
| 040 | |a DE-604 |b ger |e rda | ||
| 041 | 0 | |a eng | |
| 044 | |a gw |c XA-DE | ||
| 049 | |a DE-29T |a DE-83 | ||
| 100 | 1 | |a Volke, Daniel |e Verfasser |0 (DE-588)1163407003 |4 aut | |
| 245 | 1 | 0 | |a Rational engineering of the methylerythritol 4-phosphate (MEP) pathway for terpenoid production through metabolic control analysis |c vorgelegt von Master of Science Daniel Volke |
| 250 | |a 1. Auflage | ||
| 264 | 1 | |a Aachen |b Apprimus Verlag |c 2018 | |
| 300 | |a ix, 173 Seiten |b Illustrationen, Diagramme |c 21 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 490 | 1 | |a Applied microbiology |v Volume 9 | |
| 502 | |b Dissertation |c RWTH Aachen University |d 2017 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 830 | 0 | |a Applied microbiology |v Volume 9 |w (DE-604)BV042644516 |9 9 | |
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| 999 | |a oai:aleph.bib-bvb.de:BVB01-030496904 | ||
Datensatz im Suchindex
| _version_ | 1804178743672963072 |
|---|---|
| adam_text | TABLE OF CONTENTS
TABLE OF
CONTENTS..................................................................................................................................................
I
SU M M AR
Y....................................................................................................................................................................V
ZUSAMMENFASSUNG..............................................................................................................................................
VI
ABBREVIATIONS.......................................................................................................................................................VII
I.
INTRODUCTION............................................................................................................................................
1
1.1
TERPENOIDS........................................................................................................................................................................1
1.2 MVA AND MEP PATHWAY- BIOSYNTHESIS OF DMAPP AND IPP THROUGH TWO
ANALOGOUS PATHWAYS
.......................
3
1.3 THE MEP PATHWAY: ENZYMATIC STEPS AND
REGULATION...............................................................................................
6
1.3.1 THE MEP PATHWAY IN E. CO
LI................................................................................................................................
9
1.4 BRANCHES OF THE MEP PATHWAY IN E.
COLI...................................................................................................................14
1.5 STOICHIOMETRY OF THE MEP AND MVA PATHW
AY.......................................................................................................
15
1.6 THE VALUE OF TERPENOIDS FOR HUMAN
SOCIETY............................................................................................................16
1.7 TERPENOID PRODUCTION IN RECOMBINANT ORGANISMS USING THE MEP PATHW
AY.....................................................17
1.7.1 ISOPRENE AND ISOPRENE
SYNTHASE.....................................................................................................................
19
1.8 METABOLIC CONTROL
ANALYSIS..........................................................................................................................................21
1.9 OBJECTIVES OF THE RESEARCH AND BACKGROUND TO THE RESEARCH PROBLEM
.............................................................24
II. MATERIAL AND
METHODS.....................................................................................................................27
11.1 MATERIAL AND
CHEMICALS.........................................................................................................................................27
11.1.1
INSTRUMENTS..........................................................................................................................................................27
11.1.2
SOFTWARE................................................................................................................................................................28
11.1.3
CHEMICALS.............................................................................................................................................................
28
11.1.4
STRAINS....................................................................................................................................................................
29
11.1.5
NUCLEOTIDES..........................................................................................................................................................29
11.2 MICROBIOLOGICAL
METHODS......................................................................................................................................35
11.2.1 CULTURE M
EDIA......................................................................................................................................................
35
11.2.2 DETERMINATION OF OPTICAL DENSITY OF E. COLI CULTURES
...................................................................................
38
11.2.3 CULTURE
CONDITIONS..............................................................................................................................................
38
11.2.4 HETEROLOGOUS EXPRESSION UND HOMOLOGOUS OVEREXPRESSION IN E. COLI
BL21..........................................38
11.2.5 LONG-TERM STORAGE OF BACTERIAL
CULTURES........................................................................................................
38
11.2.6 PREPARATION OF E. COLI
LYSATE..............................................................................................................................
39
11.2.7 PREPARATION OF INVERTED MEMBRANES FROM E. COLI
BL21..............................................................................39
11.2.8 TRANSFORMATION OF F.
COLI...................................................................................................................................40
11.3 BIOCHEMICAL M
ETHODS............................................................................................................................................
40
11.3.1 AGAROSE GEL
ELECTROPHORESIS..............................................................................................................................40
11.3.2 PROTEIN CONCENTRATION DETERM
INATION............................................................................................................
40
11.3.3 DNA
SEQUENCING..................................................................................................................................................
41
11.3.4 PCR (POLYMERASE CHAIN
REACTION)....................................................................................................................
41
11.3.5 COLONY PCR FOR SCREENING FOR GENOMIC ALTERATIONS AND ENGINEERED
PLASMIDS
......................................
42
11.3.6 PLASMID
PREPARATION...........................................................................................................................................
42
11.3.7 PCR AND GEL CLEAN-UP OF DNA FRAGM
ENTS.......................................................................................................
43
11.3.8 SPLICING BY OVERLAP EXTENSION
PCR...................................................................................................................
43
11.3.9 GIBSON ASSEMBLY (SEQUENCE-INDEPENDENT
CLONING)............................................................................43
11.3.10
RECOMBINEERING..............................................................................................................................................44
11.3.11 DXP
SYNTHESIS..................................................................................................................................................
44
11.3.12 INDIRECT SUBSTRATE TRANSPORT TEST OF PROTON-DEPENDENT
TRANSPORTER IN INVERTED MEMBRANES.... 45
11.3.13
SDS-PAGE.........................................................................................................................................................
45
11.3.14 WESTERN B LO
T....................................................................................................................................................46
11.3.15 DEOXYXYLULOSE 5-PHOSPHATE SYNTHASE PURIFICATION FROM A.
THALIANA.................................................46
II. 4
CHROMATOGRAPHY.................................................................................................................................................48
11.4.1
LC-MS/MS..............................................................................................................................................................
48
11.4.2 GC-MS/M
S.............................................................................................................................................................58
III. STATISTICAL
ANALYSIS........................................................................................................................................................
59
IV.
RESULTS.......................................................................................................................................................61
IV. 1 ESTABLISHMENT AND OPTIMIZATION OF TARGETED PROTEOMICS FOR THE
ENZYMES OF THE MEP PATHW AY
........
61
IV.2 ESTABLISHMENT OF TARGETED METABOLOMICS FOR THE INTERMEDIATES OF THE
MEP P ATHW AY
...........................
63
IV.3 ESTABLISHING ISOTOPICALLY NON-STATIONARY 13C METABOLIC FLUX
ANALYSIS OF THE MEP PATHWAY.......................65
IV.3.1 MATHEMATICAL DESCRIPTION OF THE LABEL
INCORPORATION.............................................................................66
IV.4 ENHANCED ISOPRENE PRODUCTION IN COLI THROUGH OVEREXPRESSION OF
NATIVE MEP PATHWAY GENES
......
68
IV.4.1 CONSTRUCTION OF VECTORS FOR ISOPRENE PRODUCTION IN
COLI...................................................................
68
IV.4.2 ISOPRENE PRODUCTION OF ENGINEERED COLI STRAINS
..................................................................................
70
IV.4.3 PROTEIN EXPRESSION OF ENGINEERED
STRAINS..................................................................................................71
IV.4.4 METABOLITE CONCENTRATION IN ENGINEERED
STRAINS.....................................................................................72
IV.4.5 EXPRESSION OF ADDITIONAL GENES OF THE MEP
PATHWAY..............................................................................73
IV.5 ESTABLISHMENT AND OPTIMIZATION OF RECOMBINEERING FOR THE GENERATION
OF MUTATION LIBRARIES
.............
75
IV.6 METABOLIC CONTROL ANALYSIS OF THE MEP PATHW
AY................................................................................................78
IV.6.1 CONSTRUCTION OF RBS LIBRARIES THROUGH
RECOMBINEERING.........................................................................78
IV.6.2 INFLUENCE OF RBS SEQUENCE ON ENZYME CONCENTRATION
...........................................................................
80
IV.6.3 MEP PATHWAY INTERMEDIATE CONCENTRATIONS IN DXS EXPRESSION LIBRARY
..............................................
82
IV.6.4 EXPORT OF MEP PATHWAY
INTERMEDIATES......................................................................................................
85
IV.6.5 ISOPRENE PRODUCTION OF THE EXPRESSION M UTANTS
....................................................................................
87
IV.6.6 FLUX THROUGH THE MEP P ATHW
AY...................................................................................................................88
TABLE OF CONTENTS III
V.
DISCUSSION...............................................................................................................................................
91
V.1 ESTABLISHMENT OF TARGETED METABOLOMICS FOR THE INTERMEDIATES OF THE
MEP P ATHW AY...........................91
V.2 ESTABLISHMENT OF ISOTOPICALLY NON-STATIONARY 13C METABOLIC FLUX
ANALYSIS OF THE MEP PATHWAY
..............
92
V.3 ESTABLISHMENT AND OPTIMIZATION OF RECOMBINEERING IN 6. COLI BL21
.............................................................
93
V.4 MEP PATHWAY ENZYME CONCENTRATION IN . COLI AND
IMPLICATIONS..................................................................
94
V.5 ISOPRENE PRODUCTION IN .
COLI..............................................................................................................................
98
V.6 METABOLIC CONTROL ANALYSIS OF THE MEP PATHWAY IN .
COLI.............................................................................103
V.6.1 PERTURBATION OF THE MEP PATHW
AY................................................................................................................
103
V.6.2 M ETABOLITE CONCENTRATION IN DXS EXPRESSION LIBRARY
..................................................................................
106
V.6.3 CONCENTRATION CONTROL COEFFICIENTS OF
DXS....................................................................................................107
V.6.4 EXPORT OF INTERMEDIATES OF THE MEP
PATHWAY.............................................................................................110
V.6.5 FLUX THROUGH THE MEP PATHWAY AND FLUX CONTROL COEFFICIENTS OF DXS
....................................................
I L L
V.6.6 ISOPRENE PRODUCTION IN DXS EXPRESSION
LIBRARY............................................................................................113
V.7 CONCLUSION FROM THIS RESEARCH AND OBJECTIVES FOR FUTURE RESEARCH
............................................................
115
VI.
REFERENCES.............................................................................................................................................123
V II.
APPENDIX.................................................................................................................................................141
VII.1 CHARACTERIZATION OF DEOXYXYLULOSE 4-PHOSPHATE SYNTHASE FROM A
THALIANA
............................................
141
VII.1.1
PURIFICATION......................................................................................................................................................141
VII.1.2 OPTIMIZATION OF REACTION
CONDITIONS........................................................................................................
142
VII.1.3 KINETIC CHARACTERIZATION OF
ATDXS..............................................................................................................
142
VII.1.4 INHIBITION OF ATDXS BY
SS-CYCLOCITRAL...........................................................................................................143
VII.1.5 DISCUSSION OF THE RESULTS OF THE CHARACTERIZATION OF THE ATDXS
..........................................................
144
VII.2 NUCLEOTIDE SEQUENCE OF THE CODON OPTIMIZED ISOPRENE SYNTHASE FROM
P. ALBA FOR . COLI
.....................
146
VII.3 MECPP CONCENTRATION IN THE EXPRESSION STRENGTH
LIBRARY..............................................................................147
VII.4 CALCULATIONS OF THE TERPENOID YIELD ACHIEVED AND THEORETICAL
MAXIMUM Y IE LD ........................................148
VII.5 ALTERNATIVE TARGETED PROTEOMICS
METHOD..........................................................................................................148
VII.6 HYDROPHILIC INTERACTION CHROMATOGRAPHY FOR TARGETED
METABOLOMICS....................................................... 149
VII.7 CALIBRATION CURVE TARGETED
PROTEOMICS..............................................................................................................
151
VII.8 CALIBRATION CURVE TARGETED
METABOLOMICS.........................................................................................................152
VII.9 ADDITIONAL DATA FOR ISOTOPICALLY NON-STATIONARY 13C METABOLIC FLUX
ANALYSIS..............................................153
VLL.9.1 DETERMINATION OF NEEDED INITIAL GLUCOSE
CONCENTRATION......................................................................153
VLL.9.2 QUENCHING BY FAST
CENTRIFUGATION.............................................................................................................
154
VLL.9.3 QUENCHING BY FAST REDUCTION OF BROTH TEM
PERATURE.............................................................................155
VLL.9.4 DATA FROM ISOTOPICALLY NON-STATIONARY 13C METABOLIC FLUX
ANALYSIS....................................................156
VII.10 EXPRESSION O F THE ISC OPERO
N...........................................................................................................................
159
VII.11 ANALYSIS OF THE FOSMIDOMYCIN RESISTANCE P RO TEIN
......................................................................................159
VII.12 CAPTURING ISOPRENE IN AN AEROBIC FERMENTATION PROCESS
..........................................................................
162
VII.13 PURIFICATION OF DXS FROM P. ALBA THROUGH GLUTATHIONE SEPHAROSE
AFFINITY CHROMATOGRAPHY
...........
165
VII.14 REFERENCES FOR
APPENDIX.................................................................................................................................
167
V III. LIST OF
FIGURES.......................................................................................................................................
169
IX. LIST OF
TABLES..........................................................................................................................................171
ACKNOWLEDGMENTS............................................................................................................................................
173
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| isbn | 9783863596361 |
| language | English |
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| physical | ix, 173 Seiten Illustrationen, Diagramme 21 cm |
| publishDate | 2018 |
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| publisher | Apprimus Verlag |
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| series | Applied microbiology |
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| spelling | Volke, Daniel Verfasser (DE-588)1163407003 aut Rational engineering of the methylerythritol 4-phosphate (MEP) pathway for terpenoid production through metabolic control analysis vorgelegt von Master of Science Daniel Volke 1. Auflage Aachen Apprimus Verlag 2018 ix, 173 Seiten Illustrationen, Diagramme 21 cm txt rdacontent n rdamedia nc rdacarrier Applied microbiology Volume 9 Dissertation RWTH Aachen University 2017 (DE-588)4113937-9 Hochschulschrift gnd-content Applied microbiology Volume 9 (DE-604)BV042644516 9 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030496904&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Volke, Daniel Rational engineering of the methylerythritol 4-phosphate (MEP) pathway for terpenoid production through metabolic control analysis Applied microbiology |
| subject_GND | (DE-588)4113937-9 |
| title | Rational engineering of the methylerythritol 4-phosphate (MEP) pathway for terpenoid production through metabolic control analysis |
| title_auth | Rational engineering of the methylerythritol 4-phosphate (MEP) pathway for terpenoid production through metabolic control analysis |
| title_exact_search | Rational engineering of the methylerythritol 4-phosphate (MEP) pathway for terpenoid production through metabolic control analysis |
| title_full | Rational engineering of the methylerythritol 4-phosphate (MEP) pathway for terpenoid production through metabolic control analysis vorgelegt von Master of Science Daniel Volke |
| title_fullStr | Rational engineering of the methylerythritol 4-phosphate (MEP) pathway for terpenoid production through metabolic control analysis vorgelegt von Master of Science Daniel Volke |
| title_full_unstemmed | Rational engineering of the methylerythritol 4-phosphate (MEP) pathway for terpenoid production through metabolic control analysis vorgelegt von Master of Science Daniel Volke |
| title_short | Rational engineering of the methylerythritol 4-phosphate (MEP) pathway for terpenoid production through metabolic control analysis |
| title_sort | rational engineering of the methylerythritol 4 phosphate mep pathway for terpenoid production through metabolic control analysis |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030496904&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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| work_keys_str_mv | AT volkedaniel rationalengineeringofthemethylerythritol4phosphatemeppathwayforterpenoidproductionthroughmetaboliccontrolanalysis |