Metabolic engineering of Escherichia coli for the production of hydrocarbons substituting gasoline = 대장균을 이용한 가솔린 대체연료 생산에 관한 연구

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Recent global issues associated with the rapid increase in the oil price and exhaustion of fossil fuels revealed the urgent necessity of alternative fuels and chemicals from renewable carbon sources. With this rising concerns, metabolic engineering has become a promising tools for the microbial production of bio-based fuels and chemicals due to its ability can modify metabolic network towards various bioproducts. The biogasoline and its substitutes, which is biopropanol, were produced by systems metabolic engineering. First, the medium chain length alkanes (MCLAs), hydrocarbon range from C5 to C14, can be used directly as transportation fuels, gasoline or jet fuel. Bio-based production of MCLAs can indeed solve the current crisis of limited fossil fuels and global environmental problems. However, production of MCLAs has not been reported. Here we report for the first time, the bio-based production of MCLAs by metabolic engineering of Escherichia coli. Final engineered strain was able to produce a mixture of hydrocarbon including nonane and (C9), tridecane (C13) and pentadecane (C15) up to 0.95 g/l from 100 g/l glucose. This result now opens a new way towards development of sustainable platform technologies for the production of transportation fuels using renewable biomass. Second, 1-propanol was produced from L-threonine-overproducing TH20 strain in which flux toward L-threonine was increased by removing feedback inhibition of aspartokinase I and III and threonine dehydratase, eliminating negative transcriptional regulation and suppressing L-threonine degradation. The L-threonine precursor pool in this strain was also increased by deleting genes for competing pathways and replacing the native promoter of the ppc gene, encoding phosphoenolpyruvate carboxylase, with the strong constitutive tac promoter. This engineered L-threonine-overproducing strain, TH20, has now been engineered for 1-propanol production from 2-ketobutyrate by rational metabolic engineering. We first eliminated feedback inhibition of L-isoleucine by replacing the ilvA gene encoding a mutated threonine dehydratase (designed to block L-threonine degradation in the TH20 strain) with another mutated ilvA gene containing changes in four nucleotides. We also replaced the native promoter of the ilvA gene with the tac promoter. The ilvI, ilvH, ilvB, and ilvN genes (encoding competing pathway enzymes) were deleted to make more precursors available for 1-propanol biosynthesis. Moreover, rpoS was deleted to relieve the RpoS-mediated general stress response and up-regulate L-threonine metabolism. Finally, using an in silico flux-response analysis, we demonstrated that fermentation from glycerol, which generates twice the amount of reducing equivalents compared to glucose fermentation, was advantageous for 1-propanol production. The final engineered strain, PRO2, harboring plasmids overexpressing atoDA, $adhE^{mut}$, thrA, thrB, thrC, ackA and cimA, produced 10.3 g $L^{-1}$ of 1-propanol from 40 g $L^{-1}$ of glycerol in aerobic fed-batch fermentation. This report describes the first successful production of 1 propanol under respiro-fermentative culture conditions through systems metabolic engineering. Moreover, the concentration obtained is the highest reported for bio-based production of 1-propanol.
Advisors
Lee, Sang Yupresearcher이상엽researcher
Description
한국과학기술원 :생명화학공학과,
Publisher
한국과학기술원
Issue Date
2012
Identifier
325007
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 생명화학공학과, 2012.8,[154 p. :]

Keywords

metabolic engineering▼abiofuel▼agasoline▼adiesel▼aescherichia coli; 대사공학▼a바이오연료▼a가솔린▼a디젤▼a대장균

URI
http://hdl.handle.net/10203/264876
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=848961&flag=dissertation
Appears in Collection
CBE-Theses_Ph.D.(박사논문)
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