Metabolic engineering of Saccharomyces cerevisiae for formatotrophic growth:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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Potsdam
[2019]
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| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | 97 Seiten Illustrationen 30 cm |
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| 245 | 1 | 0 | |a Metabolic engineering of Saccharomyces cerevisiae for formatotrophic growth |c von Jorge Gonzalez de la Cruz |
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Datensatz im Suchindex
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| adam_text | TABLE OF CONTENTS
1 INTRODUCTION.1
1.1 YEAST- A METABOLIC POWERHOUSE.1
1.2 FORMATE- A PROMISING FEEDSTOCK.2
1.3 FORMATE ASSIMILATION PATHWAYS.4
1.4 METABOLIC ENGINEERING OF YEAST FOR FORMATOTROPHIC GROWTH.11
2 AIM OF THE PROJECT.14
3 MATERIALS AND METHODS.15
3.1 REAGENTS.15
3.2 STRAINS, MEDIA AND CULTIVATION.15
3.2.1 YEAST STRAINS.15
3.2.2 ESCHERICHIA COLI STRAINS.15
3.3 MEDIA.16
3.3.1 YEAST MEDIA.16
3.3.2 MEDIA.18
3.4 PLASMID AND GENOMIC DNA EXTRACTION FROM YEAST AND E. COLI..18
3.5 GROWTH CONDITIONS AND DETERMINATION OF GROWTH RATE.18
3.6 YEAST TRANSFORMA TION.19
3.7 YEAST GENE DELETIONS VIA CRISPR CAS9 OR MARKER DISRUPTION.19
3.8 PLASMID CONSTRUCTION.20
3.9 CARBON LABELING BY LC-MS ANALYSIS..21
4 RESULTS.25
4.1 PART 1. FORMATE ASSIMILATION FOR GLYCINE BIOSYNTHESIS.25
4.1.1 DETERMINING THE FATE OF ASSIMILATED FORMATE USING 13(HABELING.33
4.1.2 EVOLUTION EXPERIMENT: ADAPTING FOR GLYCINE BIOSYNTHESIS FROM
FORMATE.38
4.2 PART 2. TOWARDS SERINE AND GLYCINE BIOSYNTHESIS FROM FORMATE.39
4.3 PART 3. FROM SERINE TO BIOMASS.49
4.3.1 THE REDUCTIVE GLYCINE PATHWAY PUT TO THE TEST.51
4.4 PART 4. AN ALTERNATIVE PATHWAY VIA GLYOXYLATE THE REDUCTIVE
GLYCINE-GLYOXYLATE
PATHWAY.52
5 DISCUSSION.55
5.1 GENERAL DISCUSSION.55
5.2 METABOLIC ENGINEERING REVISITED.58
5.3 OVERVIEW OF OUR RESULTS- FIRST MODULE.60
5.4 OVERVIEW OF OUR RESULTS- SECOND MODULE.61
5.5 OVERVIEW OF OUR RESULTS- THIRD MODULE AND THE ENTIRE PATHWAY.65
5.6 AN ALTERNATIVE PATHWAY VIA GLYOXYLATE.67
6 OUTLOOK.71
7 REFERENCES.73
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/1205568476
8 APPENDICES 79
LIST OF FIGURES
FIGURE 1 THE FORMATE BIO-ECONOMY CONCEPT.
4
FIGURE 2 METABOLIC PATHWAYS SUPPORTING GROWTH ON FORMATE IN NATURAL
FORMATOTROPHIC MICROBES.6
FIGURE 3 THE REDUCTIVE GLYCINE PATHWAY.
7
FIGURE 4 THE GLYCINE CLEAVAGE SYSTEM.10
FIGURE 5 THE CL METABOLISM IN YEAST.12
FIGURE 6 PLASMIDS CONSTRUCTS FOR OVEREXPRESSION OF THE DIFFERENT ENZYMES
OF THE REDUCTIVE GLYCINE PATHWAY..22
FIGURE 7 LC-MS RESULTS FOR GLYCINE IN TWO REPRESENTATIVE SAMPLES.23
FIGURE 8 ESTABLISHMENT OF THE THREE DIFFERENT STEPS OF THE REDUCTIVE
GLYCINE PATHWAY.26
FIGURE 9. SCHEME OF THE GLYCINE AUXOTROPH STRAIN BLOCKED IN ALL OF KNOW
GLYCINE BIOSYNTHESIS PATHWAYS.
27
FIGURE 10. POSSIBLE COMBINATION OF PROMOTERS IN THE PLASMID LIBRARY AND
INDIVIDUAL CONSTRUCTS FOR OVEREXPRESSION OF
GENES GCV1, GCV2, GCV3, MIS1 AND LPD1.28
FIGURE 11. GROWTH EXPERIMENT OF LIBRARY OVEREXPRESSION WITH OR WITHOUT
THE LPD1 GENE, IN GLYCINE AUXOTROPH STRAIN
YCG-F..30
FIGURE 12. GROWTH EXPERIMENT OF GLYCINE AUXOTROPH STRAIN YCG-F
OVEREXPRESSING PLASMIDS PJGC3 (GCV1-3 MIS1) OR
PJGC4(GCVL-3 MIS1-LPD1).31
FIGURE 13. GROWTH EXPERIMENT WITH DIFFERENT FORMATE CONCENTRATIONS OF
GLYCINE AUXOTROPH STRAIN YCG-F,
OVEREXPRESSING PLASMID PJGC3 (GCV1-3-MIS1) WITH STRONG PROMOTERS.32
FIGURE 14 AMINO ACID LABELING IN STRAIN YCG-F WITH PLASMID LIBRARY
OVEREXPRESSION, FED WITH13C-LABELED FORMATE. 34
FIGURE 15 AMINO ACID LABELING IN STRAIN YCG-F OVEREXPRESSING PLASMIDS
PJGC3 OR PJGC4 WITH STRONG PROMOTERS, FED
WITH 13C-LABELED FORMATE.
35
FIGURE 16 AMINO ACID LABELING IN GLYCINE AUXOTROPH STRAIN YCG-F AND IN
GLYCINE AUXOTROPH STRAIN YCG-F
OVEREXPRESSING PLASMID PJGC3, WITH13 C-LABELED FORMATE OR 13C-LABELED CO
2
.
37
FIGURE 17 AMINO ACID LABELING IN THE STRAIN BY4741 AND GLYCINE AUXOTROPH
GROWING IN DIFFERENT CARBON SOURCES, FED
WITH13 C-LABELED FORMATE.
37
FIGURE 18 GROWTH CURVE OF YCG-F STRAIN EVOLUTION EXPERIMENT TO INDUCE
YEAST TO SYNTHESIZE GLYCINE FROM FORMATE.
.38
FIGURE 19 AMINO ACID LABELING IN EVOLVED YCG-F STRAIN.39
FIGURE 20 SERINE AUXOTROPH STRAIN GROWING IN SM MEDIA SUPPLEMENTED WITH
250 MM OF FORMATE.40
FIGURE 21 LABELING IN GLYCINE AND SERINE AMINO ACIDS IN STRAIN YJGCL AND
WILD TYPE BY4741, FED WITH LABELED
FORMATE PLUS NON-LABELED GLUCOSE.42
FIGURE 22 LABELING IN GLYCINE AND SERINE AMINO ACIDS IN STRAIN YJGCL AND
WILD TYPE BY4741, FED WITH GLUCOSE LABELED
IN THE FIRST CARBON PLUS NON-LABELED FORMATE.43
FIGURE 23 LABELING IN GLYCINE AND SERINE AMINO ACIDS IN STRAIN YJGCL AND
WILD TYPE BY4741, FED WITH GLUCOSE LABELED
IN THE SECOND CARBON PLUS NON-LABELED FORMATE.
44
FIGURE 24 LABELING IN GLYCINE AND SERINE AMINO ACIDS IN STRAIN YJGCL AND
WILD TYPE BY4741, FED WITH GLUCOSE LABELED
IN THE THIRD CARBON PLUS NON-LABELED FORMATE.
45
FIGURE 25 LABELING IN DIFFERENT AMINO ACIDS IN STRAIN YJGC4, FED WITH
LABELED THREONINE.
47
FIGURE 26 GROWTH EXPERIMENT OF SERINE AUXOTROPH STRAIN YJGC3 THAT
OVEREXPRESSES PLASMID PJGC14 OR PJGC6 IN SM
MEDIA WITH FORMATE 250 MM OR WITHOUT FORMATE.48
FIGURE 27 LABELING IN DIFFERENT AMINO ACIDS IN STRAIN YJGC3 WITH PLASMID
OVEREXPRESSION PJGC14 ORPJGC6, FED WITH
LABELED FORMATE.49
FIGURE 28 GROWTH EXPERIMENT OF WILD TYPE YEAST YPH500 OVEREXPRESSING
PLASMID PJGC038 TO TEST GROWTH ON SERINE
AS SOLE CARBON SOURCE.50
FIGURE 29 REDUCTIVE GLYCINE-GLYOXYLATE PATHWAY VARIANT IN YEAST STRAIN
YJGC5, DELETED IN ICL1 GENE.
52
FIGURE 30 GROWTH CURVES FOR STRAIN YJGC5 OVEREXPRESSING THE GCV SYSTEM
GENES WITH THE ADDITION OF AGX1 TO TEST
GROWTH ON FORMATE AND ACETATE.53
FIGURE 31 FORMATE ASSIMILATION PATHWAYS. 56
FIGURE 32 BIOMASS YIELD ANALYSIS OF PATHWAYS SUPPORTING FORMATOTROPHIC
GROWTH IN SILICO.
57
FIGURE 33 SUPERIMPOSITION OF ESHMTAND ETA ACTIVE SITE ENTRANCE.64
FIGURE 34 REDUCTIVE GLYCINE-GLYOXYLATE PATHWAY VARIANT.68
|
| adam_txt |
TABLE OF CONTENTS
1 INTRODUCTION.1
1.1 YEAST- A METABOLIC POWERHOUSE.1
1.2 FORMATE- A PROMISING FEEDSTOCK.2
1.3 FORMATE ASSIMILATION PATHWAYS.4
1.4 METABOLIC ENGINEERING OF YEAST FOR FORMATOTROPHIC GROWTH.11
2 AIM OF THE PROJECT.14
3 MATERIALS AND METHODS.15
3.1 REAGENTS.15
3.2 STRAINS, MEDIA AND CULTIVATION.15
3.2.1 YEAST STRAINS.15
3.2.2 ESCHERICHIA COLI STRAINS.15
3.3 MEDIA.16
3.3.1 YEAST MEDIA.16
3.3.2 MEDIA.18
3.4 PLASMID AND GENOMIC DNA EXTRACTION FROM YEAST AND E. COLI.18
3.5 GROWTH CONDITIONS AND DETERMINATION OF GROWTH RATE.18
3.6 YEAST TRANSFORMA TION.19
3.7 YEAST GENE DELETIONS VIA CRISPR CAS9 OR MARKER DISRUPTION.19
3.8 PLASMID CONSTRUCTION.20
3.9 CARBON LABELING BY LC-MS ANALYSIS.21
4 RESULTS.25
4.1 PART 1. FORMATE ASSIMILATION FOR GLYCINE BIOSYNTHESIS.25
4.1.1 DETERMINING THE FATE OF ASSIMILATED FORMATE USING 13(HABELING.33
4.1.2 EVOLUTION EXPERIMENT: ADAPTING FOR GLYCINE BIOSYNTHESIS FROM
FORMATE.38
4.2 PART 2. TOWARDS SERINE AND GLYCINE BIOSYNTHESIS FROM FORMATE.39
4.3 PART 3. FROM SERINE TO BIOMASS.49
4.3.1 THE REDUCTIVE GLYCINE PATHWAY PUT TO THE TEST.51
4.4 PART 4. AN ALTERNATIVE PATHWAY VIA GLYOXYLATE THE REDUCTIVE
GLYCINE-GLYOXYLATE
PATHWAY.52
5 DISCUSSION.55
5.1 GENERAL DISCUSSION.55
5.2 METABOLIC ENGINEERING REVISITED.58
5.3 OVERVIEW OF OUR RESULTS- FIRST MODULE.60
5.4 OVERVIEW OF OUR RESULTS- SECOND MODULE.61
5.5 OVERVIEW OF OUR RESULTS- THIRD MODULE AND THE ENTIRE PATHWAY.65
5.6 AN ALTERNATIVE PATHWAY VIA GLYOXYLATE.67
6 OUTLOOK.71
7 REFERENCES.73
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/1205568476
8 APPENDICES 79
LIST OF FIGURES
FIGURE 1 THE FORMATE BIO-ECONOMY CONCEPT.
4
FIGURE 2 METABOLIC PATHWAYS SUPPORTING GROWTH ON FORMATE IN NATURAL
FORMATOTROPHIC MICROBES.6
FIGURE 3 THE REDUCTIVE GLYCINE PATHWAY.
7
FIGURE 4 THE GLYCINE CLEAVAGE SYSTEM.10
FIGURE 5 THE CL METABOLISM IN YEAST.12
FIGURE 6 PLASMIDS CONSTRUCTS FOR OVEREXPRESSION OF THE DIFFERENT ENZYMES
OF THE REDUCTIVE GLYCINE PATHWAY.22
FIGURE 7 LC-MS RESULTS FOR GLYCINE IN TWO REPRESENTATIVE SAMPLES.23
FIGURE 8 ESTABLISHMENT OF THE THREE DIFFERENT STEPS OF THE REDUCTIVE
GLYCINE PATHWAY.26
FIGURE 9. SCHEME OF THE GLYCINE AUXOTROPH STRAIN BLOCKED IN ALL OF KNOW
GLYCINE BIOSYNTHESIS PATHWAYS.
27
FIGURE 10. POSSIBLE COMBINATION OF PROMOTERS IN THE PLASMID LIBRARY AND
INDIVIDUAL CONSTRUCTS FOR OVEREXPRESSION OF
GENES GCV1, GCV2, GCV3, MIS1 AND LPD1.28
FIGURE 11. GROWTH EXPERIMENT OF LIBRARY OVEREXPRESSION WITH OR WITHOUT
THE LPD1 GENE, IN GLYCINE AUXOTROPH STRAIN
YCG-F.30
FIGURE 12. GROWTH EXPERIMENT OF GLYCINE AUXOTROPH STRAIN YCG-F
OVEREXPRESSING PLASMIDS PJGC3 (GCV1-3 MIS1) OR
PJGC4(GCVL-3 MIS1-LPD1).31
FIGURE 13. GROWTH EXPERIMENT WITH DIFFERENT FORMATE CONCENTRATIONS OF
GLYCINE AUXOTROPH STRAIN YCG-F,
OVEREXPRESSING PLASMID PJGC3 (GCV1-3-MIS1) WITH STRONG PROMOTERS.32
FIGURE 14 AMINO ACID LABELING IN STRAIN YCG-F WITH PLASMID LIBRARY
OVEREXPRESSION, FED WITH13C-LABELED FORMATE. 34
FIGURE 15 AMINO ACID LABELING IN STRAIN YCG-F OVEREXPRESSING PLASMIDS
PJGC3 OR PJGC4 WITH STRONG PROMOTERS, FED
WITH 13C-LABELED FORMATE.
35
FIGURE 16 AMINO ACID LABELING IN GLYCINE AUXOTROPH STRAIN YCG-F AND IN
GLYCINE AUXOTROPH STRAIN YCG-F
OVEREXPRESSING PLASMID PJGC3, WITH13 C-LABELED FORMATE OR 13C-LABELED CO
2
.
37
FIGURE 17 AMINO ACID LABELING IN THE STRAIN BY4741 AND GLYCINE AUXOTROPH
GROWING IN DIFFERENT CARBON SOURCES, FED
WITH13 C-LABELED FORMATE.
37
FIGURE 18 GROWTH CURVE OF YCG-F STRAIN EVOLUTION EXPERIMENT TO INDUCE
YEAST TO SYNTHESIZE GLYCINE FROM FORMATE.
.38
FIGURE 19 AMINO ACID LABELING IN EVOLVED YCG-F STRAIN.39
FIGURE 20 SERINE AUXOTROPH STRAIN GROWING IN SM MEDIA SUPPLEMENTED WITH
250 MM OF FORMATE.40
FIGURE 21 LABELING IN GLYCINE AND SERINE AMINO ACIDS IN STRAIN YJGCL AND
WILD TYPE BY4741, FED WITH LABELED
FORMATE PLUS NON-LABELED GLUCOSE.42
FIGURE 22 LABELING IN GLYCINE AND SERINE AMINO ACIDS IN STRAIN YJGCL AND
WILD TYPE BY4741, FED WITH GLUCOSE LABELED
IN THE FIRST CARBON PLUS NON-LABELED FORMATE.43
FIGURE 23 LABELING IN GLYCINE AND SERINE AMINO ACIDS IN STRAIN YJGCL AND
WILD TYPE BY4741, FED WITH GLUCOSE LABELED
IN THE SECOND CARBON PLUS NON-LABELED FORMATE.
44
FIGURE 24 LABELING IN GLYCINE AND SERINE AMINO ACIDS IN STRAIN YJGCL AND
WILD TYPE BY4741, FED WITH GLUCOSE LABELED
IN THE THIRD CARBON PLUS NON-LABELED FORMATE.
45
FIGURE 25 LABELING IN DIFFERENT AMINO ACIDS IN STRAIN YJGC4, FED WITH
LABELED THREONINE.
47
FIGURE 26 GROWTH EXPERIMENT OF SERINE AUXOTROPH STRAIN YJGC3 THAT
OVEREXPRESSES PLASMID PJGC14 OR PJGC6 IN SM
MEDIA WITH FORMATE 250 MM OR WITHOUT FORMATE.48
FIGURE 27 LABELING IN DIFFERENT AMINO ACIDS IN STRAIN YJGC3 WITH PLASMID
OVEREXPRESSION PJGC14 ORPJGC6, FED WITH
LABELED FORMATE.49
FIGURE 28 GROWTH EXPERIMENT OF WILD TYPE YEAST YPH500 OVEREXPRESSING
PLASMID PJGC038 TO TEST GROWTH ON SERINE
AS SOLE CARBON SOURCE.50
FIGURE 29 REDUCTIVE GLYCINE-GLYOXYLATE PATHWAY VARIANT IN YEAST STRAIN
YJGC5, DELETED IN ICL1 GENE.
52
FIGURE 30 GROWTH CURVES FOR STRAIN YJGC5 OVEREXPRESSING THE GCV SYSTEM
GENES WITH THE ADDITION OF AGX1 TO TEST
GROWTH ON FORMATE AND ACETATE.53
FIGURE 31 FORMATE ASSIMILATION PATHWAYS. 56
FIGURE 32 BIOMASS YIELD ANALYSIS OF PATHWAYS SUPPORTING FORMATOTROPHIC
GROWTH IN SILICO.
57
FIGURE 33 SUPERIMPOSITION OF ESHMTAND ETA ACTIVE SITE ENTRANCE.64
FIGURE 34 REDUCTIVE GLYCINE-GLYOXYLATE PATHWAY VARIANT.68 |
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| author | Gonzalez de la Cruz, Jorge |
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| spelling | Gonzalez de la Cruz, Jorge Verfasser (DE-588)1205039430 aut Metabolic engineering of Saccharomyces cerevisiae for formatotrophic growth von Jorge Gonzalez de la Cruz Potsdam [2019] 97 Seiten Illustrationen 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Universität Potsdam 2019 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf https://d-nb.info/1205568476/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032737385&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Gonzalez de la Cruz, Jorge Metabolic engineering of Saccharomyces cerevisiae for formatotrophic growth |
| subject_GND | (DE-588)4113937-9 |
| title | Metabolic engineering of Saccharomyces cerevisiae for formatotrophic growth |
| title_auth | Metabolic engineering of Saccharomyces cerevisiae for formatotrophic growth |
| title_exact_search | Metabolic engineering of Saccharomyces cerevisiae for formatotrophic growth |
| title_exact_search_txtP | Metabolic engineering of Saccharomyces cerevisiae for formatotrophic growth |
| title_full | Metabolic engineering of Saccharomyces cerevisiae for formatotrophic growth von Jorge Gonzalez de la Cruz |
| title_fullStr | Metabolic engineering of Saccharomyces cerevisiae for formatotrophic growth von Jorge Gonzalez de la Cruz |
| title_full_unstemmed | Metabolic engineering of Saccharomyces cerevisiae for formatotrophic growth von Jorge Gonzalez de la Cruz |
| title_short | Metabolic engineering of Saccharomyces cerevisiae for formatotrophic growth |
| title_sort | metabolic engineering of saccharomyces cerevisiae for formatotrophic growth |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1205568476/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032737385&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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