Metabolic engineering of escherichia coli for the production of fumaric and L-malic acid = 대장균에서의 푸마르산과 L-말산 생산을 위한 대사공학적 연구

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Fumaric and L-malic acid are the naturally occurring organic acids which were key intermediates of the tricar-boxylic acid cycle. In this study, Escherichia coli was metabolically engineered for the production of fumaric and L-malic acid. For the aerobic production of fumaric acid, the iclR gene was deleted to redirect the carbon flux through the glyoxylate shunt. In addition, the fumA, fumB and fumC genes were also deleted to enhance fumaric acid formation. The resulting strain was able to produce 1.45 g/L of fumaric acid from 15 g/L of glucose in flask culture. Based on in-silico flux response analysis, this base strain was further engineered by plasmid-born (pTac15k) overexpression of the native ppc gene encoding phosphoenolpyruvate carboxylase (PPC) under the strong tac promoter, which resulted in the production of 4.09 g/L of fumaric acid. Additionally, the arcA and ptsG genes were deleted to reinforce the oxidative TCA cycle flux, and the aspA gene was deleted to block the conver-sion of fumaric acid into L-aspartic acid. Since it is desirable to avoid the use of inducer, the lacI gene was also deleted. To increase glucose uptake rate and fumaric acid productivity, the native promoter of galP gene was replaced with the strong trc promoter. The final strain (CWF812) was able to produce 28.2 g/L fumaric acid in 63h by fed-batch fermentation with the overall yield and productivity of 0.389 g/goglucose and 0.448 g/L/h. Similar approach was employed to produce L-malic acid using the iclR gene deleted strain under aerobic condi-tion. Then, we further deleted two known malate dehydrogenases (mdh and mqo) to accumulate L-malic acid under aerobic condition. However, there was no accumulation of L-malic acid in this strain. Thus, we further de-leted malic enzymes (maeB and sfcA) which could convert L-malic acid into pyruvate by gluconeogenetic activi-ties. As a result, we could obtain 0.53 g/L of L-malic acid in this strain. By over-expression of the ppc gene, we could significantly improve the L-malic acid titer up-to 3.13 g/L from 15 g/L of glucose in a flask culture. This study demonstrates the possibility for the industrial production of fumaric and L-malic acid in E. coli.
Advisors
Lee, Sang Yupresearcher이상엽researcher
Description
한국과학기술원 :생명화학공학과,
Publisher
한국과학기술원
Issue Date
2013
Identifier
325007
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생명화학공학과, 2013.2,[v, 43 p. :]

Keywords

Fumaric acid▼aL-malic acid▼aescherichia coli▼ametabolic engineering; 푸마르산▼aL-말산▼a대장균▼a대사공학

URI
http://hdl.handle.net/10203/266345
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=848982&flag=dissertation
Appears in Collection
CBE-Theses_Master(석사논문)
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