서강대학교

초록

Chapter 1. Production of long-chain free fatty acids from metabolically engineered Rhodobacter sphaeroides heterologously producing periplasmic phospholipase A2 in dodecane-overlaid two-phase culture : Long-chain free fatty acids (FFAs) were produced from metabolically engineered R. sphaeroides heterologously producing phospholipase A2 (PLA2) in the periplasm. The FFA productivity was greatly increased by two-phase high-cell-density culture with dodecane. This approach provides the highly competitive productivity of long-chain FFAs by R. sphaeroides compared with other bacteria. The FFA production by PLA2 in periplasm can be further applied to FFA production by other photosynthetic bacteria with similar differentiated membrane systems. Thylakoid membranes (TMs) from Synechocystis sp. PCC6803 have been extensively studied to improve the sustainability of in vitro NADPH generation. The sustainability of NADPH generation of TM can be increased via silica encapsulation. Further removal of singlet-state oxygen by keeping the electron transport chain of TM working together with ROS removal by catalase highlighted the sustainable NADPH generation activity under illuminated conditions. Thus, if ROS formation by TM under light is properly suppressed or removed, it can be used to provide reducing power for in vitro biochemical reactions. Chapter 2. Sustainability of in vitro light-dependent NADPH generation by the thylakoid membrane of Synechocystis sp. PCC6803 : Background: NADPH is used as a reductant in various biosynthetic reactions. Cell-free bio-systems have gained considerable attention owing to their high energy utilization and time efficiency. Efforts have been made to continuously supply reducing power to the reaction mixture in a cyclical manner. The thylakoid membrane (TM) is a promising molecular energy generator, producing NADPH under light. Thus, TM sustainability is of major relevance for its in vitro utilization. Results: Over 70% of TMs prepared from Synechocystis sp. PCC6803 existed in a sealed vesicular structure, with the F1 complex of ATP synthase facing outward (right-side-out), producing NADPH and ATP under light. The NADPH generation activity of TM increased approximately two-fold with the addition of carbonyl cyanide-p-(trifluoromethoxy) phenylhydrazone (FCCP) or removal of the F1 complex using EDTA. Thus, the uncoupling of proton translocation from the electron transport chain or proton leakage through the Focomplex resulted in greater NADPH generation. Biosilicified TM retained more than 80% of its NADPH generation activity after a week at 30°C in the dark. However, activity declined sharply to below 30% after two days in light. The introduction of engineered water-forming NADPH oxidase (Noxm) to keep the electron transport chain of TM working resulted in the improved sustainability of NADPH generation activity in a ratio (Noxm to TM)-dependent manner, which correlated with the decrease of singlet oxygen generation. Removal of reactive oxygen species (ROS) by catalase further highlighted the sustainable NADPH generation activity of up to 80% in two days under light.