| Ganoderma lucidum,a well-known traditional Chinese medicinal mushroom,produces various kinds of natural bioactive compounds,such as ganoderic acids(GAs)and polysaccharide.GAs possesses significant pharmacological activities including anti-tumor,anti-invasion,anti-metastasis and anti-HIV properties,etc.At present,low production of GAs has become a bottleneck for its widespread application.Therefore,it is necessary to improve GA production in mycelia fermentation of G.lucidum.Microparticle-enhanced cultivation(MPEC)has been applied to increase production of secondary metabolites during cultivation of filamentous microorganisms.Howerver,there is no reports on enhancement of ganoderic acids by controlling fungal morphology with microparticles talcum(Talc)addition in submerged culture of G.lucidum.In this work,effects of Talc on the mycelia morphology and the yeilds of GAs were investigated in submerged culture of G.lucidum.Our results showed that addition of Talc significantly reduced the diameter of G.lucidum pellets.In the range from 5 to 15 g/L Talc,the equivalent diameter of G.lucidum was decreasing with increasing Talc concentration.But Talc concentrations exceeding 15 g/L did not led to significant decrease of equivalent diameter compared to that with 15 g/L Talc.The equivalent diameters were 3.33±0.16?2.97±0.01?2.49±0.04?2.04±0.12 and 1.97±0.07 mm with 0?5?10?15 and 20 g/L Talc,respectively.Moreover,addition of Talc significantly increased the levels of individual and total GAs compared to those in the control(without Talc addition).Especially,the obtained maximum levels of GA-Mk,GA-T,GA-Me and the total content of GAs reached 6.02±0.29,5.08±0.14,1.71±0.09 ?g/100 mg dry cell weight(DCW)and 1.51±0.02 mg/100 mg DCW with the 15 g/L Talc in submerged culture,respectively,which are 4,1.9,1.4 and 1.6-folds higher than those in the control,respectively.Hereby,Talc addition is an effective strategy to engineer the morphology of G.lucidum into smaller diameter pellets and strongly boost production of GAs.In order to unravel the underlying mechanism of the hyperproduction of GAs,we determined the accumulations of intermediate metabolites(lanosterol and squalene)and the transcription levels of several key genes in GA biosynthetic pathway under two culture conditions.The maximum accumulated amounts of lanosterol and squalene were 3.69±0.23 and 34.86±6.41 ?g/ 100 mg DCW with the 15 g/L Talc in submerged culture of G.lucidum.These values were 2.6 and 4.2 times higher than those in control.The transcription levels of farnesyl diphosphate synthase(fps)and a CYP450 gene cyp5150l8 were up-regulated by 2.35 and 1.53-folds compared with those in the control.Our results suggested that the hypreproduction of GAs with Talc addition may be related to the higher accumulation of intermediate metabolites and the upregulated transcription levels of fps and cyp5150l8.Futhremore,we also conducted bioreactor fermentations using a 3-L bioreactor to evaluate the developed cultivation strategy.Addition of Talc in the bioreactor also led to the smaller equivalent diameter pellets and higher production of GAs,which confiremed the previous results in the shake-flask fermentation.The obtained maximum contents of GA-Mk,GA-T and GA-Me were 12.17±0.23,9.62±0.23,4.30±0.13 ?g/ 100 mg DCW respectively,which were 2.7,1.8 and 1.4 folds higher than those obtained in sumerged cultivation without Talc.These results indicated that the developed strategy is efficient for higher production of GAs in bioreactor scale.In conculsion,Talc-enhanced cultivation reduced the equivalent diameter of pellets and significantly increased the yields of GAs in submerged cultivation of G.lucidum.In addition,the presence of Talc induced the upregulation of the fps and cyp5150l8 and increased the accumulation of squalene and lansoterol in a submerged G.lucidum culture.The developed strategy is useful for developing a better GA production process in future large-scale bioreactors. |
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