Bioprocess Optimization And Metabolic Regulation Of Fibrinolysis Active FGFC1 Production From Marine Fungus

FGFC1, a structurally novel compound isolated from cultures of a marine-derived fungus Stachybotrys longispora FG216, was proven to hold fibrinolytic activity and it has potential value for devolpment of antithrombotic agents. In this work, we aim to enhance FGFC1 production by bioprocess optimization and metabolic regulation so as to provide FGFC1 product enough for pharmaceutical research.In our work, statistical methodologies including one-factor-at-a-time design, Plackett-Burman design and response surface methodology were used to facilitate FGFC1 production. FGFC1 production reached 9.13 g/L under optimized conditions, which increased 10.1 times compared to that under original conditions.Furthermore, metabolic pathway analysis and regulation were performed to improve FGFC1 production. Metabolic inhibition and precursor feeding experiments indicated that FGFC1 was synthesized through shikimic acid pathway and mevalonate pathway. FGFC1 production in the optimized fermentation medium was not increasd by feeding precursors of shikimic acid pathway and mevalonate pathway, which indicated that these precursors were not limited factors for FGFC1 synthesis in the optimized fermentation medium. On the other hand, L-ornithine, as a key part of FGFC1 chemical structure, was found to be a precursor for FGFC1 production. Adding different concentration of L-ornithine resulted in an excited discovery of a new key intermediate of FGFC1, which was named as FGFC3. Therefore, three key compounds were identified in FGFC1 biosynthesis, which were L-ornithine, FGFC2 and FGFC3. After time course analysis of these compounds during fermentation process, FGFC1 production bioprocess was divided into four phases, and then an effective metabolic regulation strategy was proposed to increase FGFC1 production based on its metabolic features of different phases. Finally, the FGFC1 production reached 9.92 g/L, which increased 16.3% compared to the control group.Fermentation of FGFC 1 was scaled up. Based on fermentation results in shaking flask and bioreactor cultures, the scale-up criterion was proposed to maintain dissolved oxygen tension at 30～40% throughout the fermentation process and to keep similar impeller linear velocity, fungal morphology and pH. Accordingly, FGFC1 production reached 7.66 g/L and 6.57 g/L in the fermentation in 50 L and 1000 L bioreactors, respectively. The vigorous growth and morphology changes caused by intense shear stress in larger bioreactors could be the main reason for production decrease. This work established an effective process for large scale fermentation of FGFC1, which greatly improved pharmaceutical research.