Engineering highly efficient NADP-dependent formate dehydrogenases using a NADPH biosensor Escherichia coli strain:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Potsdam
Juli 2020
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | 115 Seiten Illustrationen, Diagramme 30 cm |
Internformat
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| 100 | 1 | |a Calzadíaz Ramírez, Liliana |e Verfasser |0 (DE-588)1216904898 |4 aut | |
| 245 | 1 | 0 | |a Engineering highly efficient NADP-dependent formate dehydrogenases using a NADPH biosensor Escherichia coli strain |c Liliana Calzadíaz Ramírez |
| 264 | 1 | |a Potsdam |c Juli 2020 | |
| 300 | |a 115 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
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| 502 | |b Dissertation |c Universität Potsdam |d 2020 | ||
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Datensatz im Suchindex
| _version_ | 1804182716310093824 |
|---|---|
| adam_text | TABLE
OF
CONTENTS
1.
ZUSAMMENFASSUNG
...........................................................................................................................
13
2.
SUMMARY
...........................................................................................................................................
15
3.
INTRODUCTION
......................................................................................................................................
16
3.1.
NADPH
PRODUCTION
IN
ESCHERICHIA
COLI
....................................................................................
16
3.2.
APPLICATIONS
OF
NADP-DEPENDENT
ENZYMES
FOR
NADPH
PRODUCTION
IN
METABOLIC
ENGINEERING
18
3.3.
MOLECULAR
ENGINEERING
STRATEGIES
...........................................................................................
20
3.4.
FORMATE
DEHYDROGENASE
.........................................................................................................
22
4.
AIM
OF
THE
PROJECT
...........................................................................................................................
24
5.
MATERIALS
AND
METHODS
...................................................................................................................
25
5.1.
CHEMICALS
.................................................................................................................................
25
5.2.
MEDIA
........................................................................................................................................
25
5.3.
STRAINS
AND
GENE
DELETIONS
......................................................................................................
25
5.4.
PLASMID
CONSTRUCTIONS
..............................................................................................................
26
5.4.1.
DIHYDROLIPOAMIDE
DEHYDROGENASE
..................................................................................
26
5.4.2.
MALIC
ENZYME
...................................................................................................................
27
5.4.3.
FORMATE
DEHYDROGENASE
.................................................................................................
28
5.5.
EVOLUTION
EXPERIMENT
OF
THE
NADPH
AUXOTROPH
STRAIN
.........................................................
29
5.6.
PROTEIN
ENGINEERING
.................................................................................................................
31
5.6.1.
MUTANT
CONSTRUCTION
.......................................................................................................
31
5.6.2.
LIBRARY
CONSTRUCTION
AND
SCREENING
.................................................................................
31
5.6.3.
GROWTH
EXPERIMENTS
.......................................................................................................
32
5.6.4.
TRANSCRIPTIONAL
ANALYSIS
...................................................................................................
33
5.6.5.
PROTEIN
PURIFICATION
..........................................................................................................
34
5.6.6.
ENZYMATIC
ASSAYS
..............................................................................................................
35
6.
RESULTS
6.1.
CHARACTERIZATION
OF
A
NADPH
AUXOTROPH
STRAIN
......................................................................
36
6.2.
NADPHAUX
STRAIN
ENABLES
DIRECT
SELECTION
FOR
NADPH
PRODUCTION
.......................................
41
6.2.1.
GROWTH
EXPERIMENTS
OF
DIHYDROLIPOAMIDE
DEHYDROGENASE
VARIANTS
............................
41
6.2.2.
ASSAYING
FOR
FORMATE-DEHYDROGENASE-DEPENDENT
GROWTH
............................................
43
6.3.
EVOLUTION
EXPERIMENT
OF
THE
NADPHAUX
STRAIN
......................................................................
47
6.3.1.
GROWTH
EXPERIMENTS
OF
MAEA-EVOLVED
VARIANTS
IN
NADPHAUX
AMAEA
........................
50
6.4.
COENZYME
ENGINEERING
OF
PSEFDH
VIA
STRUCTURE-GUIDED
DIRECTED
EVOLUTION
........................
54
6.4.1.
GROWTH
EXPERIMENT
OF
PSEFDH_WT
AND
MVAFDH
4M
.....................................................
55
6.4.2.
SWITCHING
COFACTOR
SPECIFICITY
OF
PSEFDH
........................................................................
56
6.4.3.
LIBRARY
DESIGN,
CONSTRUCTION
AND
QUALITY
CONTROL
............................................................
60
6.4.4.
SCREENING
AND
GROWTH
EXPERIMENTS
OF
PSEFDH
LIBRARY
...................................................
65
6.4.5.
ENZYME
KINETICS
OF
THE
BEST
PSEFDH
VARIANTS
.................................................................
67
6.4.6.
DECONVOLUTION
OF
KEY
MUTATIONS
OF
VARIANT
V9
...............................................................
69
6.4.7.
EXPLORING
NON-ADDITIVE
EFFECTS
IN
KEY
RESIDUES
OF
PSEFDH
V9
.........................................
72
7.
DISCUSSION,
CONCLUSIONS
AND
OUTLOOK
................................................................................................
75
8.
REFERENCES
........................................................................................................................................
81
9.
SUPPLEMENTARY
INFORMATION
.............................................................................................................
87
9.1.
OLIGONUCLEOTIDES
USED
FOR
NADPHAUX
CONSTRUCTION
...............................................................
88
9.2.
OLIGONUCLEOTIDES
PRIMERS
USED
................................................................................................
89
9.3. OLIGOS
DESIGNED
FOR
PSEFDH
MUTANTS
....................................................................................90
9.4.
DESIGNED
OLIGOS
BY
DNAWORKS
FOR
ADO
....................................................................................
91
9.5.
INITIAL
PSEFDH
MUTANTS
SCREENED
AND
SEQUENCES
...................................................................
92
9.6.
QUIKCHANGE
PROTOCOL
................................................................................................................
93
9.7.
PROTOCOL
FOR
ADO
FRAGMENT
SYNTHESIS
......................................................................................
94
9.8.
PROTEIN
AGAROSE
GEL
OF
MAEA
VARIANTS
......................................................................................
96
9.9.
STEADY-STATE
PARAMETERS
OF
MALATE
DEHYDROGENASE
(MAEA)
WT
AND
VARIANTS
......................
97
9.10.
PROTEIN
AGAROSE
GEL
OF
PSEFDH
VARIANTS
...................................................................................
99
9.11.
STEADY-STATE
PARAMETERS
OF
PSEFDH
(WT)
AND
VARIANTS
.......................................................
100
6
|
| adam_txt |
TABLE
OF
CONTENTS
1.
ZUSAMMENFASSUNG
.
13
2.
SUMMARY
.
15
3.
INTRODUCTION
.
16
3.1.
NADPH
PRODUCTION
IN
ESCHERICHIA
COLI
.
16
3.2.
APPLICATIONS
OF
NADP-DEPENDENT
ENZYMES
FOR
NADPH
PRODUCTION
IN
METABOLIC
ENGINEERING
18
3.3.
MOLECULAR
ENGINEERING
STRATEGIES
.
20
3.4.
FORMATE
DEHYDROGENASE
.
22
4.
AIM
OF
THE
PROJECT
.
24
5.
MATERIALS
AND
METHODS
.
25
5.1.
CHEMICALS
.
25
5.2.
MEDIA
.
25
5.3.
STRAINS
AND
GENE
DELETIONS
.
25
5.4.
PLASMID
CONSTRUCTIONS
.
26
5.4.1.
DIHYDROLIPOAMIDE
DEHYDROGENASE
.
26
5.4.2.
MALIC
ENZYME
.
27
5.4.3.
FORMATE
DEHYDROGENASE
.
28
5.5.
EVOLUTION
EXPERIMENT
OF
THE
NADPH
AUXOTROPH
STRAIN
.
29
5.6.
PROTEIN
ENGINEERING
.
31
5.6.1.
MUTANT
CONSTRUCTION
.
31
5.6.2.
LIBRARY
CONSTRUCTION
AND
SCREENING
.
31
5.6.3.
GROWTH
EXPERIMENTS
.
32
5.6.4.
TRANSCRIPTIONAL
ANALYSIS
.
33
5.6.5.
PROTEIN
PURIFICATION
.
34
5.6.6.
ENZYMATIC
ASSAYS
.
35
6.
RESULTS
6.1.
CHARACTERIZATION
OF
A
NADPH
AUXOTROPH
STRAIN
.
36
6.2.
NADPHAUX
STRAIN
ENABLES
DIRECT
SELECTION
FOR
NADPH
PRODUCTION
.
41
6.2.1.
GROWTH
EXPERIMENTS
OF
DIHYDROLIPOAMIDE
DEHYDROGENASE
VARIANTS
.
41
6.2.2.
ASSAYING
FOR
FORMATE-DEHYDROGENASE-DEPENDENT
GROWTH
.
43
6.3.
EVOLUTION
EXPERIMENT
OF
THE
NADPHAUX
STRAIN
.
47
6.3.1.
GROWTH
EXPERIMENTS
OF
MAEA-EVOLVED
VARIANTS
IN
NADPHAUX
AMAEA
.
50
6.4.
COENZYME
ENGINEERING
OF
PSEFDH
VIA
STRUCTURE-GUIDED
DIRECTED
EVOLUTION
.
54
6.4.1.
GROWTH
EXPERIMENT
OF
PSEFDH_WT
AND
MVAFDH
4M
.
55
6.4.2.
SWITCHING
COFACTOR
SPECIFICITY
OF
PSEFDH
.
56
6.4.3.
LIBRARY
DESIGN,
CONSTRUCTION
AND
QUALITY
CONTROL
.
60
6.4.4.
SCREENING
AND
GROWTH
EXPERIMENTS
OF
PSEFDH
LIBRARY
.
65
6.4.5.
ENZYME
KINETICS
OF
THE
BEST
PSEFDH
VARIANTS
.
67
6.4.6.
DECONVOLUTION
OF
KEY
MUTATIONS
OF
VARIANT
V9
.
69
6.4.7.
EXPLORING
NON-ADDITIVE
EFFECTS
IN
KEY
RESIDUES
OF
PSEFDH
V9
.
72
7.
DISCUSSION,
CONCLUSIONS
AND
OUTLOOK
.
75
8.
REFERENCES
.
81
9.
SUPPLEMENTARY
INFORMATION
.
87
9.1.
OLIGONUCLEOTIDES
USED
FOR
NADPHAUX
CONSTRUCTION
.
88
9.2.
OLIGONUCLEOTIDES
PRIMERS
USED
.
89
9.3. OLIGOS
DESIGNED
FOR
PSEFDH
MUTANTS
.90
9.4.
DESIGNED
OLIGOS
BY
DNAWORKS
FOR
ADO
.
91
9.5.
INITIAL
PSEFDH
MUTANTS
SCREENED
AND
SEQUENCES
.
92
9.6.
QUIKCHANGE
PROTOCOL
.
93
9.7.
PROTOCOL
FOR
ADO
FRAGMENT
SYNTHESIS
.
94
9.8.
PROTEIN
AGAROSE
GEL
OF
MAEA
VARIANTS
.
96
9.9.
STEADY-STATE
PARAMETERS
OF
MALATE
DEHYDROGENASE
(MAEA)
WT
AND
VARIANTS
.
97
9.10.
PROTEIN
AGAROSE
GEL
OF
PSEFDH
VARIANTS
.
99
9.11.
STEADY-STATE
PARAMETERS
OF
PSEFDH
(WT)
AND
VARIANTS
.
100
6 |
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| id | DE-604.BV047430894 |
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| index_date | 2024-07-03T17:58:27Z |
| indexdate | 2024-07-10T09:11:58Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-032833368 |
| oclc_num | 1196247520 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | 115 Seiten Illustrationen, Diagramme 30 cm |
| publishDate | 2020 |
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| publishDateSort | 2020 |
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| spelling | Calzadíaz Ramírez, Liliana Verfasser (DE-588)1216904898 aut Engineering highly efficient NADP-dependent formate dehydrogenases using a NADPH biosensor Escherichia coli strain Liliana Calzadíaz Ramírez Potsdam Juli 2020 115 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Universität Potsdam 2020 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf https://d-nb.info/1217353186/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032833368&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p dnb 20201008 DE-101 https://d-nb.info/provenance/plan#dnb |
| spellingShingle | Calzadíaz Ramírez, Liliana Engineering highly efficient NADP-dependent formate dehydrogenases using a NADPH biosensor Escherichia coli strain |
| subject_GND | (DE-588)4113937-9 |
| title | Engineering highly efficient NADP-dependent formate dehydrogenases using a NADPH biosensor Escherichia coli strain |
| title_auth | Engineering highly efficient NADP-dependent formate dehydrogenases using a NADPH biosensor Escherichia coli strain |
| title_exact_search | Engineering highly efficient NADP-dependent formate dehydrogenases using a NADPH biosensor Escherichia coli strain |
| title_exact_search_txtP | Engineering highly efficient NADP-dependent formate dehydrogenases using a NADPH biosensor Escherichia coli strain |
| title_full | Engineering highly efficient NADP-dependent formate dehydrogenases using a NADPH biosensor Escherichia coli strain Liliana Calzadíaz Ramírez |
| title_fullStr | Engineering highly efficient NADP-dependent formate dehydrogenases using a NADPH biosensor Escherichia coli strain Liliana Calzadíaz Ramírez |
| title_full_unstemmed | Engineering highly efficient NADP-dependent formate dehydrogenases using a NADPH biosensor Escherichia coli strain Liliana Calzadíaz Ramírez |
| title_short | Engineering highly efficient NADP-dependent formate dehydrogenases using a NADPH biosensor Escherichia coli strain |
| title_sort | engineering highly efficient nadp dependent formate dehydrogenases using a nadph biosensor escherichia coli strain |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1217353186/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032833368&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT calzadiazramirezliliana engineeringhighlyefficientnadpdependentformatedehydrogenasesusinganadphbiosensorescherichiacolistrain |
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