Optimization And Metabolic Pathway Study Of 1403C, A Novel Anti-tumor Anthraquinone Produced By Mangrove Endophytic Fungus 1403

A novel anthraquinone 1403C was found and isolated from the fermentation broth of mangrove endophytic fungus 1403# (Halorosellinia sp.).1403C and its acetylate showed significant anti-tumor activity against cancer cell lines in vitro and in vivo with milder effect on human normal cells.1403R, the reduzate of 1403C, showed low anticancer activity.The prelimilary optimization of 1403C production showed that the favorable fermentation condition was:the concentration of artifitial sea water in the production medium 20%; seed culture time 60 h; seed volume 5% and initial pH 7.0. Dissolved oxygen experiments showed that Halorosellinia sp. (No.1403) could produce high 1403C at a higher level of dissolved oxygen.The effect of seed culture methods on the mycelial morphology and production of a novel anti-tumor anthraquinone 1403C by mangrove endophytic fungus Halorosellinia sp. (No.1403) was invested. M1, M2, M3 and Control were four different methods for seed cultivation. By using these methods, inoculums of different morphologies were harvested for fermentation. In the subsequent fermentation, mycelial clump was the dominant morphology in the fermentation broth M1, M2 while large pellets and mycelial clumps co-existed in M3 and Control. In all cases, production of 1403C was dependent on the morphology. The fermentations using M1, M2 and M3 increased 1403C production by 243.5%,194.8% and 70.2% than Control. Organic acid analysis showed that the pyruvate level in the fermentations M1, M2, M3 were higher than in Control during the rapid production phase of 1403C (after 36 h). Fermentation performed in the baffled Erlenmeyer flask attained a maximum 1403C production of 0.77±0.02 g/L which was 4.8-fold of the production in the unbaffled Erlenmeyer flask, both using Control as seed culture method. The present work showed that seed culture methods could significantly affect mycelial morphologies in the fermentation and thus 1403C production. The mycelial clump was more favorable than the pellet because the mycelial clump had more active growth centers which could produce 1403C and higher dissolved oxygen levels could be maintained.The effects of feeding of pathway specific inhibitors and biosynthetic precursors on 1403C production were studied. Feeding of anthranilic acid and trisodium citrate, specific inhibitors of mevalonate pathway, did not inhibite 1403C yield on biomass (YP/X (1403C)). Addition of low concentration of trimethylamine had no effect on YP/X (1403C) while high concentration (12 mM) of trimethylamine led to lower YP/X (1403C) compared to the control experiment. Feeding of shikimate, a key intermediate of shikimate pathway, did not enhance YP/X (1403C). Addition of iodoacetamide which was the specific inhibitor of polyketide synthase and fatty acids synthase strongly inhibited YP/X (1403C) with the highest inhibition rate of 94.2%. Feeding of malonic increased YP/X (1403C) by 59.2% compared to the control. Combined feeding of sodium acetate and malonic with a ratio of 1:4 increased YP/X (1403C) by 102.7%. These results suggested that the biosynthetic pathway of 1403C involves with polyketide pathway.A certain correlation between 1403C and 1403R was found in the course of fermentation. By analyzing time profiles of 1403C and 1403R production, hypothesis could be made that 1403R was transformed to 1403C from 60 h in the fermenation...