| Endophytic fungi are an important source of natural bioactive metabolites.These metabolites can be industrially produced by using large-scale fermentation culture.Their yields are usually low and unstable which limit their industrialization.The production of these metabolites can be effectively enhanced by the metabolic regulation.Botrallin and TMC-264 belong to dibenzo-a-pyrones,and were produced by endophytic fungus Ponipodefl2(Hyalodendriella sp.Ponipodefl 2)isolated from the hybrid 'Neva' of Populus deltoides Marsh × P.nigra L..In this study,we used several strategies to optimize the culture conditions and to enhance botrallin and TMC-264 production in mycelial liquid culture of the endophytic fungus Ponipodef12.The main results are as follows.1.Using two phase culture technique by adding macroporous adsorption resin to the liquid culture medium of endophytic fungus Ponipodefl 2,the dibenzo-a-pyrones production was enhanced.Thirteen macroporous adsorption resins were screened preliminarily,and the resin DM-301 was found to be the most effective to promote botrallin and TMC-264 production of the fungus Ponipodefl 2,and the yields of botrallin and TMC-264 were 27.98 mg/L and 33.86 mg/L,which were 1.43-fold and 8.98-fold of control,respectively.The resin DM-301 was further optimized for its concentrations,addition time and incubation time.After optimization,4.38%(i.e.,3.5 g/80 mL medium)of resin DM-301 was added to the liquid medium on day 24 of culture,and incubation for 4 d,the highest yield of botrallin reached 51.47 mg/L that was 2.29-fold of the control,by adding resin DM-301 at 4.38%(g/mL)in the culture broth on day 24 and in a period of 4 d for adsorption.While the highest yield of TMC-264 reached 47.74 mg/L that was 11.76-fold of the control in a period of 6 d for adsorption.2.After eight metal ions at their different concentrations were screened with the method of"one-factor-at-a-time(OFAT)",three most effective metal ions(Zn2+,Cu2+and Mg2+)along with their optimum concentrations were obtained.The addition time and their optimum concentrations of three metal ions(Zn2+,Cu2+ and Mg2+)were further obtained respectively for improving botrallin and TMC-264 production.The combination effects of three metal ions(Zn2+,Cu2+and Mg2+)on the production of botrallin and TMC-264 by employing statistical method based on the central composite design(CCD)and response surface methodology(RSM)were evaluated.The optimal concentrations of Zn2+,Cu2+ and Mg2+for botrallin and TMC-264 production were 0.83g/L,0.20 g/L and 0.12 g/L,respectively.Under the optimal culture condition,the yields of botrallin and TMC-264 production of endophytic fungus Ponipodefl 2 both reached highest,which were 146.51 and 36.63 mg/L,respectively.3.Seven bacteria species were screened as the biotic elicitors by "one-factor-at-a-time(OFAT)".It was found that Staphylococcus haemolyticus(St-ha)was the most effectively biotic elicitor to stimulate botrallin and TMC-264 production of endophytic fungus Ponipodef12.The effects of the extracellular and intracellular polysaccharides of autoclaved culture broth,filtering autoclaved culture broth,normal St-ha culture broth,and pure culture broth by filtering St-ha bacteria were compared.The results showed that the treatment of pure culture broth by filtering St-ha bacteria mostly enhanced the botrallin and TMC-264 production of endophytic fungus Ponipodef12,and the yields of botrallin reached highest(49.79 mg/L)with 1.25%(1 mL/80 mL)of St-ha added into the culture medium,which was 2.19-fold of control,while the yield of TMC-264 had maximum value(15.44 mg/L)when the added concentration of St-ha was 6.25%(5 mL/80 mL),which was 2.69-fold of control.4.Four factors(i.e.,pH,K2HPO4·3H2O,NaCl and CH3COONa)along with their different concentrations were screened by "one-factor-at-a-time(OFAT)" to evaluate their effects on botrallin and TMC-264 production of endophytic fungus Ponipodef12,and their optimum concentrations were obtained,respectively.The combination effects of pH,K2HPO4·3H2O,NaCl,CH3COONa as well as the Zn2+ were studied by employing statistical method based on the Box-Behnken design(BBD)and response surface methodology(RSM),and a quadratic predictive model was developed.The optimal conditions were pH as 5,K2HPO4·3H2O concentration as 1.75 g/L,NaCl concentration as 0.25 g/L,CH3COONa concentration as 0.01 g/L,and ZnSO4·7H2O concentration as 0.02 g/L.Under the optimum culture condition,the yields of botrallin and TMC-264 of endophytic fungus Ponipodef12 reached 86.95 mg/L and 15.84 mg/L,respectively.5.Combined the 5 factors(pH,K2HPO4·3H2O,NaCl,CH3COONa and ZnSO4·7H2O)with the best optimized resin DM-301 and the best optimized bacteria St-au,and eight different processing were set up,i.e.,liquid cultural medium of PDB(PDB),liquid cultural medium of PDB which contains 5 factors(PDB-5),liquid cultural medium of PDB which contains the bacteria St-au(PDB-B),liquid cultural medium of PDB which contains 5 factors and bacteria St-au(PDB-5-B),liquid cultural medium of PDB which contains resin DM-301(PDB-R),liquid cultural medium of PDB which contains 5 factors and resin DM-301(PDB-5-R),liquid cultural medium of PDB which contains bacteria St-au and resin DM-301(PDB-B-R),and liquid cultural medium of PDB which contains 5 factors,bacteria St-au and resin DM-301(PDB-5-B-R).The result showed that there were different effects among the different treatments on botrallin and TMC-264 production.And PDB-5-R had the most significant enhanced for botrallin production,and the yield of botrallin reached 80.83 mg/L,which was 3.71-fold of control.While the yield of TMC-264 had maximum value(10.84 mg/L)with PDB-5-B-R treatment,which was 2.02-fold of control.The results in this study developed some effective strategies to improve the production of botrallin and TMC-264 in fermentation culture of endophytic fungus Ponipodef12.It will provide the basis for the large-scale production of botrallin and TMC-264 by using means of biotechnology,and lay the foundation for their application in industrial scale. |
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