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Development of a Novel Escherichia coli Host Strain for Long-chain Fatty Acid Hydroxylation

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Authors
배진형
Advisor
김병기
Major
공과대학 화학생물공학부
Issue Date
2014-02
Publisher
서울대학교 대학원
Keywords
Hydroxy long-chain fatty acidLong-chain fatty acid hydroxylationLong-chain fatty acid degradationLong-chain fatty acid transportFadDFadL
Description
학위논문 (석사)-- 서울대학교 대학원 : 화학생물공학부, 2014. 2. 김병기.
Abstract
Hydroxy long-chain fatty acids, having both a carboxyl group and a hydroxyl group, are precursors for pseudo-ceramides, polyesters, and lactones. A major problem of hydroxylation of long-chain fatty acid (LCFA) in Escherichia coli is that transport and degradation of LCFA are tightly linked by long-chain fatty acyl-CoA synthetase (FadD). In this study, an effective way to prevent degradation of fatty acid without impairing transport was proposed. It requires manipulating two endogenous proteins related with LCFA transport, i.e. FadD and FadL, and a heterologous enzyme which consumes LCFA. CYP153A from Marinobacter aquaeolei which converts palmitic acid into ω-hydroxy palmitic acid was expressed in E. coli as a model system. With CYP153A in the cell, the ability to transport LCFA was successfully maintained even when fadD was deleted. However, the lack of long-chain fatty acyl-CoA (LCFA-CoA), which is synthesized by FadD, is known to downregulate outer membrane LCFA transporter gene (fadL). This problem was solved by the overexpression of fadL from an additional vector. It relieved not only repression by FadR but also catabolite repression to allow glucose to be used as carbon source which can be rapidly catabolized. When fadD deletion and fadL overexpression were combined, 2.6 g/L of palmitic acid was converted to 2.4 g/L of ω-hydroxy palmitic acid, which was 5.5-fold increase compared to wild-type strain. This simple genetic manipulation generally can be applied to any LCFA hydroxylation using E. coli.
Language
English
URI
https://hdl.handle.net/10371/127084
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College of Engineering/Engineering Practice School (공과대학/대학원)Dept. of Chemical and Biological Engineering (화학생물공학부)Theses (Master's Degree_화학생물공학부)
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