Study On The Fermentation Process Of Dalesconols A And B By A Marine-Derived Fungus Daldinia Eschscholzii

Dalesconols A(DA)and B(DB)are two skeletally unprecedented polyketides isolated from the marine-derived fungus Daldinia eschscholzii IFB-TL01.They are of significant immunosuppressive activity comparable to that of cyclosporine A(CsA),which is a clinically used immune-compromising compound,suggesting that DA and DB may serve as the promising candidates for immunosuppressive treatments.Furthermore,DA and DB have better selective index values than that of CsA.However,their further pharmaceutical researches have been severely restricted by the low production from submerged fermentation.Allowing for the urgent need in looking for new immunosuppressant,it is quite necessary to improve dalesconols(DA and DB)production from the fermentation process.In this work,efforts will be made to improve the production of dalesconols through the optimization of medium,seed culture,fermentation condition,and fermentation mode and addition of metal ion and precusor.Based on the results,the enzyme acitivity and gene transcriptional level are analyzed to find out the effects of the optimization strategy on the fermentation process.Then,the fermentation process will scale up to a 500-L bioreactor to meet the demand of pharmaceutical research.The effects of different media(M1,M2,and M3)on fungus growth,dalesconols biosynthesis and metabolites were firstly tested and compared.M2 was more favored for dalesconols production and 18.87 mg/L dalesconols production was obtained.Detection of four organic acids in three implied that acetyl-CoA supply was not the root cause of differences on DA and DB production between M1 and M2.The highest DHN accumulation and the lowest dalesconols production in M1 might be caused by the lack of laccase to catalyze the polymerization of DHN.The laccase activity of 3.30 U/mL in M2 was the 9-folds higher than that in M1.Intermediates and enzyme analysis indicated that laccase had the major contribution to dalesconols biosynthesis.The key role of laccase on dalesconols biosynthesis was experimentally confirmed.Fermentation condition and medium play a vitol roles in the fermentation of secondary metabolites.The condition in the submerged fermentation of Daldinia eschscholzii is optimized.Origninal pH is set at 7.0.Termentation temperature was controlled at 28? at first,and then changed to 34 ? at 72h.7 mL seed broth is inoculated into 50 mL fermentation medium after 144h cultivation.Statistical methodologies including Plackett-Burman and response surface designs were used to develop new medium to facilitate dalesconols production.Under the proposed optimized medium,the dalesconols production reached 36.66 mg/L.Based on the biosynthetic pathway of dalesconols,dalesconols production was supposed to be elevated by the regulation of secondary metabolism in D.eschscholzii.Addition of polyketide pathway precursor acetate or malonate is unable to improve dalesconols production,neither the addition of mevalonate pathway inhibitor citrate or lovastatin.Ca2+induction was employed to up-regulate of laccase activity and further enhanced dalesconols production(76.90 mg/L),which was 122.8%higher than that in the control.To further probe the mechanism underlying the enhanced dalesonols production by calcium addition,Ca2+ channel and calmodulin inhibitors were applied to investigate the involvement of calcium/calmodulin signaling in regulating dalesconols production.After supplementation with 5 mM Ca2+ at 24 h,dalesconols production reached 84.33 mg/L,which resulted in a 1.57-fold enhancement compared to the control.The key role of calcium/calmodulin signaling in dalesconols biosynthesis was confirmed by treatment with Ca2+ channel and calmodulin inhibitors.The transcriptional levels of dalesconols biosynthetic genes were up-regulated after CaCl2 addition and down-regulated after inhibitors were added.The results demonstrated that Ca2+ addition induces dalesconols biosynthesis through up-regulation of dalesconols biosynthesis genes via regulation of calcium/calmodulin signaling.The fermentation process in shake-flask scale was not sufficient for large-scale production of dalesconols.Efforts devoted into the reproduction of the process bioreactor system are required considering its further application.After optimizing the agitation speed and air flow,dalesconols production reached 94.83 mg/L in the fed-batch fermentation.By using the scale-up strategy,similar impeller linear velocity together with similar tendency of dissolved oxygen,the fermentation process was successfully reproduced in pilot scale bioreactor systems.In addition,dalesconols production reached 79.41 mg/L in a 500-L bioreactor.The information obtained in this work would be helpful to the large-scale production of dalesconols and other useful conidia associated secondary metabolites.