S-Space College of Engineering/Engineering Practice School (공과대학/대학원) Program in Bioengineering (협동과정-바이오엔지니어링전공) Theses (Ph.D. / Sc.D._협동과정-바이오엔지니어링전공)
Metabolic engineering for overproduction of fatty acids from glucose, and its application to biosynthesis of ω-hydroxy palmitic acid in Escherichia coli
- 공과대학 협동과정 바이오엔지니어링전공
- Issue Date
- 서울대학교 대학원
- Free fatty acid; hydroxy palmitic acid; metabolic engineering; P450 monooxygenase; cofactor optimization
- 학위논문 (박사)-- 서울대학교 대학원 : 공과대학 협동과정 바이오엔지니어링전공, 2016. 2. 김병기.
- Metabolic engineering of fatty acid biosynthesis pathway combined with cofactor regeneration system was performed to overproduce palmitic acid (PA) and stearic acid (SA) for bioenergy and biomaterials such as bio-diesel and bio-degradable plastics. To produce fatty acids, thioesterase from Lactobacillus reuteri, and acyl-CoA dehydratase from Escherichia coli, the two key enzymes for fatty acid biosynthesis, were over-expressed to enhance PA biosynthesis. Additionally, acyl-CoA transferase was deleted to block fatty acid β-oxidation pathway. To direct metabolic flux into fatty acid synthesis, and control energy consumption, succinyl-CoA synthetase of TCA cycle, and transaldolase A of pentose phosphate pathway were also deleted. The engineered E. coli FFA4 strain without a P450 system could produce 503.0 mg/L of PA (C16) and 508.4 mg/L of SA (C18), of which the amounts are ca. 1.6 and 2.3 fold higher than those of the wild-type.
To hydroxylate palmitic acid (HPA) produced, CYP153A monooxygenase from Marinobacter aqueolei and redox partners CamA/B from Pseudomonas putida were over-expressed. However, the production yield of hydroxyl palmitic acid did not increase as much as free fatty acid production did, suggesting that the hydroxylation was a rate determining step (RDS) of HPA production. For the maximum production of HPA, NADH, i.e. an essential cofactor for P450 reaction, was overproduced by the integration of NAD+ dependent formate dehydrogenase (FDH) from Candida boidinii into E.coli chromosome, and the deletion of alcohol dehydrogenase (ADH). Ultimately, the E.coli strain with NADH-level optimization produced 610 mg/L of HPA, which was almost a three-fold increase in its yield compared to the same strain without NADH overproduction.