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Enhanced production of isobutanol in engineered Saccharomyces cerevisiae by blocking competitive pathways

DC Field Value Language
dc.contributor.advisor서진호-
dc.contributor.author이경묵-
dc.date.accessioned2017-07-14T06:48:10Z-
dc.date.available2017-07-14T06:48:10Z-
dc.date.issued2017-02-
dc.identifier.other000000140639-
dc.identifier.urihttps://hdl.handle.net/10371/125981-
dc.description학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2017. 2. 서진호.-
dc.description.abstractAs the global warming and climate change caused by petroleum-based fuels are intensifying, the research has actively conducted to develop bio-based fuels. Among the various candidates for ideal biofuels, isobutanol has valuable properties as a fuel such as high energy density, high octane number, low hygroscopicity and low vapor pressure. Also, isobutanol is an important platform chemical used as precursors of various chemicals.
In this study, Saccharomyces cerevisiae, which has various advantages for industrial production, was used as a host strain to produce isobutanol. Isobutanol is synthesized via the L-valine biosynthesis pathway and Ehrlich pathway in S. cerevisiae. In previous studies, engineered S. cerevisiae, which expresses the enzymes involved in isobutanol biosynthesis in the cytosol, was constructed. But an isobutanol yield of the engineered S. cerevisiae was still low. Also, there are several competitive pathways for isobutanol production in S. cerevisiae		-
dc.description.abstractbiosynthetic pathways for L-valine, L-isoleucine, L-leucine, isobutyrate and ethanol biosynthesis. In this study, the competitive pathways were eliminated by the CRISPR-Cas9 gene editing method and by overexpressing acetolactate synthase (ALS) from Bacillus subtilis, ketolacid reductoisomerase (KARI) from Escherichia coli, dihydroxyacid dehydratase (DHAD) and ketoacid decarboxylase (KDC) from Lacococcus lactis, an isobutanol yield based on glucose was improved a 8.6-fold higher than the corresponding value of the control strain. In batch cultivation in a bioreactor with gas trapping, this strain produced 662 mg/L isobutanol with 6.7 mg/g of yield.
This study indicates that competitive pathways are one of the causes for the low production of isobutanol in S. cerevisiae. By totally blocking the ethanol synthesis and screening another KDC with higher enzyme activity, it would be possible to improve the production of isobutanol in S. cerevisiae.		-
dc.description.tableofcontentsI. INTRODUCTION 1
1. Isobutanol-promising biofuel & biochemical 1
2. Production of isobutanol in microorganism 5
3. Pathway for isobutanol biosynthesis in S. cerevisiae 9
4. Competitive pathways for isobutanol production 12
5. CRISPR-Cas9 system for genome editing 15
6. Gas trapping system 16
7. Research Objectives 17
II. MATERIALS AND METHODS 18
1. Reagents 18
2. Strains and plasmids 19
2.1. Strains 19
2.2. Plasmids 22
3. DNA manipulation and transformation 28
3.1. Enzymes 28
3.2. Polymerase chain reaction (PCR) 28
3.3. Preparation of bacterial genomic DNA 29
3.4. Isolation of DNA fragments and DNA sequencing 29
3.5. Transformation of E. coli 30
3.6. Preparation of plasmid DNA 31
3.7. Yeast transformation 31
4. Genome editing by CRISPR-Cas9 system 32
5. Media and culture conditions 33
5.1. Media 33
5.2. Cultivations in flasks 34
5.3. Cultivations in bioreactor with gas trapping 35
6. Analysis 34
6.1 Dry cell weight 36
6.2 Metabolite detection 36
6.3 Isobutanol detection 37
III. RESULTS AND DISCUSSIONS 38
1. Blocking the competitive pathways in mitochondria 38
1.1. Elimination of L-valine biosynthesis pathway 38
1.2. Elimination of L-isoleucine biosynthesis pathway 42
2. Blocking the competitive pathways in cytosol 46
2.1. Elimination of isobutyrate and L-leucine biosynthesis pathway 46
2.2. Elimination of ethanol biosynthesis pathway by using pyruvate decarboxylase (Pdc)-deficient S. cerevisiae 51
2.3 Elimination of ethanol biosynthesis pathway by disrupting alcohol dehydrogenase gene 55
3. Isobutanol production in bioreactor with gas trapping 59
IV. CONCLUSIONS 62
V. REFERENCES 63
국 문 초 록 71		-
dc.formatapplication/pdf-
dc.format.extent1456469 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoko-
dc.publisher서울대학교 대학원-
dc.subjectBiofuels-
dc.subjectIsobutanol-
dc.subjectSaccharomyces cerevisiae-
dc.subjectBranched-chain amino acid biosynthesis-
dc.subjectAlcohol dehydrogenase-
dc.subjectCRISPR-Cas9-
dc.subjectGas trapping-
dc.subject.ddc630-
dc.titleEnhanced production of isobutanol in engineered Saccharomyces cerevisiae by blocking competitive pathways-
dc.typeThesis-
dc.description.degreeMaster-
dc.citation.pagesviii,73-
dc.contributor.affiliation농업생명과학대학 농생명공학부-
dc.date.awarded2017-02-
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