Light Regulation On Anticancer Compound 1403C Production By Marine Fungus Halorosellinia Sp.1403

1403C (also called SZ-685C), a structurally novel polyketide with striking anticancer activities, was isolated from marine mangrove endophytic fungus Halorosellinia sp.1403 by Sun Yat-sen University. It was proven to induce apoptosis of breast cancer cell by suppression of the Akt/FOXO pathway, and of great research value as potential Akt inhibitor and anticancer drug candidate. It was shown in previous studies that marine fungus Halorosellinia sp.1403 made responses to light, and in present work the effects of different irradiation conditions were investigated on cell growth and 1403C biosynthesis by analyzing fermentation parameters and cell metabolic behaviors, so as to design an effective light regulation strategy for 1403C biosynthesis.Firstly, extraction and determination methods of intracellular enzymes and intracellular/extracellular organic acids were established for real-time measure of cell metabolism. During the culture of Halorosellinia sp.1403, red pigments (mainly 1403C) gradually accumulated on the surface of mycelia with the increase of broth pH, which affected the assay of enzymes activities. After a series of experiments, the optimized conditions were confirmed as follows. Fresh mycelia were first washed by pre-cooled weak acid solution (pH 5.0) for 3 min to remove the pigments and washed to neutral later. Then mycelia were cell-disrupted by MiniBeadbeater-16 for 15 min to release cytosolic/mitochondrial enzymes. Enzymes activities were determined by corresponding biochemical/immunological kits. Meanwhile, intracellular organic acids were extracted with hot water, and intracellular/extracellular organic acids were quantified with high performance liquid chromatography.Based on these methods above, the light-responded alternations of growth, metabolic behaviors and polyketides biosynthesis were studied in this work. The colony with long mycelia and much red pigments was obtained in agar plate exposed under white light. In shake flask culture, white light showed a positive regulation on 1403R and 1403C biosynthesis and a negative regulation on griseofulvin biosynthesis, and the optimized light intensity was 350±20 lux. Besides, a suitable light pulse strategy could further improve 1403C production. Similarly, white light promoted 1403C biosynthesis in 5-L bioreactor culture. The maximal 1403C production was 1.35 g/L, which was 41% higher than 0.96 g/L in dark. Through the analysis on fermentation parameters and cell metabolism in the fermentation process, it was found that white light could regulate a series of metabolic behaviors to provide sufficient carbon skeleton and reducing power for accumulation of biomass,1403R and 1403C prior to 60 h, while white light caused synchronous descent of production and biomass after 60 h. Therefore a light-dark shift strategy in bioreactor fermentation was presented, and Halorosellinia sp.1403 was grown under white light during 0-60 h, then shifted to dark after 60 h. The severe decrement of product and biomass accumulations during later stage of fermentation were markedly ameliorated by this strategy and the maximal 1403C production (1.67 g/L) was 24% and 74% higher than those obtained under constant white light and dark conditions, respectively.As mentioned above, because white light was favorable for the accumulation of several organic acids involving in glucose metabolism in the early stage of fermentation, the regulations of specific organic acids under white light and dark conditions were investigated. It was found that no obvious improvement of 1403C production was obtained by feeding organic acids when Halorosellinia sp.1403 was grown in dark, and positive effect on 1403C accumulation was observed on the premise of culture under white light. When 3 mmol/L pyruvate was fed by single-point feeding strategy (at 36 h or 48 h) and two-points feeding strategy (at 36 h and 48 h), there was a striking promotion of 1403C production.In L-malate feeding experiment,1403C productions were significantly improved by two-points feeding strategies (at 36 h and 48 h) at a dose of 3 mmol/L,6 mmol/L and 9 mmol/L, respectively, as well as two-points feeding strategy (at 24 h and 36 h) at a dose of 6 mmol/L.The regulation of fermentation by various monochromatic lights split from white light was studied subsequently. In shake flask culture, green/blue light had similar effect as white light and inspired metabolic flux into the biosynthesis of octaketone (directing to 1403C), not heptaketone (directing to griseofulvin). Therein, the highest 1403C production was achieved under green light, while the lowest one was obtained under red light. In 5-L bioreactor culture, green light strengthened the glucose metabolism and the biosynthesis of 1403R and 1403C in the early stage of fermentation, and the enhancement was more significant than white light. The maximal 1403C production (1.38 g/L) was achieved at 72 h under green light, with a slight increase in production and 12 h reduction in fermentation time comparing with white light condition. Blue light also strengthened the glucose metabolism, and the enhancement was also more significant than white light. However, it led to higher biomass but lower 1403C production than white light. In addition, the lowest 1403C production was still obtained under red light among all of the monochromatic lights.