S-Space College of Engineering/Engineering Practice School (공과대학/대학원) Dept. of Chemical and Biological Engineering (화학생물공학부) Theses (Master's Degree_화학생물공학부)
Development of a Novel Escherichia coli Host Strain for Long-chain Fatty Acid Hydroxylation
- 공과대학 화학생물공학부
- Issue Date
- 서울대학교 대학원
- Hydroxy long-chain fatty acid; Long-chain fatty acid hydroxylation; Long-chain fatty acid degradation; Long-chain fatty acid transport; FadD; FadL
- 학위논문 (석사)-- 서울대학교 대학원 : 화학생물공학부, 2014. 2. 김병기.
- 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.