| Plant endophytic fungi can produce a large number of novel bioactive secondary metabolites,and have become an important resource of novel bioactive natural products with great potential applications in agriculture,medicine and food industry.It is of great significance for the development and utilization of secondary metabolites by improving the the production of bioactive compounds from endophytic fungi.This research mainly includes the effects of metal ions,macroporous resins and two-liquid-phase culture system on the spirobisnaphthalenes production in the liquid culture of endophytic fungus Dzf12 from Dioscorea zingiberensis C.H.Wright.The isolation and identification of spirobisnaphthalenes along with their biological activity evaluation were also studied.Meanwhile,in order to study the biosynthesis pathway of spirobisnaphthalenes,the genome sequence of endophytic fungus Dzf12 were sequenced,and the genes for secondary metabolites biosynthesis were predicted.The main results are as follows.1.In order to study the effect of metal ions on the spirobisnaphthalenes production in liquid culture of endophytic fungus Dzf12,the extraction and quantitative analysis procedures of palmarumycins C12 and C13 were optimized.Then,with one-way ANOVA method,eight metal ions(Na+?Ca2+?Ag+?Co2+?Cu2+?Al3+?Zn2+and Mn4+)were employed,and Ca2+,Cu2+,Al3+were found to be beneficial to the production of palmarumycins C12 and C13.Ca2+,Cu2+,Al3+concentration along with their addition time also had important influences on mycelia growth and palmarumycins C12 and C13 production in liquid culture of the endophytic fungus Dzf12.When Ca2+was applied to the medium at 5.0-10.0 mmol/L on day 3,Cu2+to the medium at 1.0-2.0 mmol/L on day 3,Al3+to the medium at 1.5-2.5 mmol/L on day 6,the maximal yields of palmarumycins C12 and C13 were obtained,respectively.The combination effects of Ca2+,Cu2+and Al3+ on mycelia biomass and palmarumycin C13 production were further studied.The statistical method based on the central composite design(CCD)and response surface methodology(RSM)was employed to optimize the tested variables and establish the second-order polynomial regression equation between the mycelia biomass or palmarumycin C13 production and the tested variables.When the concentrations of Ca2+,Cu2+and Al3+in medium were determined as 12.38,1.62 and 1.50 mmol/L respectively,the maximal mycelia biomass was 8.95 g dw/L,which was 1.40-fold in comparison with that(6.39 g dw/L)of the control.When the concentrations of Ca2+,Cu2+and Al3+in medium were determined as 7.58,1.36 and 2.05 mmol/L respectively,the maximal yield of palmarumycin C13 was 203.85 mg/L,which was 6.00-fold in comparison with that(33.98 mg/L)of the control.2.In order to study the effect of solid(macroporous resins)-liquid two-phase system on the spirobisnaphthalenes production in liquid culture of endophytic fungus Dzf12,ten macroporous adsorption resins(D-101,D1300,HPD-100,X-5,AB-8,DM130,ADS-17,DA-201,NKA-9 and S-8)were employed.Among them,the mycelial growth of endophytic fungus Dzf12 were slightly stimulated by resin X-5,AB-8 and ADS-17,respectively.The palmarumycins C12 and C13 yields were increased with the addition of resin HPD100,AB-8 and DA-201,especially with the addition of resin DA-201.The addditon amounts of resin DA-201 on mycelia biomass,palmarumycins C12 and C13 production were further studied.The total yields of palmarumycins C12 and C13 reached the highest levels(144.64 mg/L and 48.30 mg/L)which were 66.34-fold and1.59-fold in comparison with those(2.18 and 30.37 mg/L)of the control,respectively,when resin DA-201 was added in the medium at 4.17%on day 9.With the optimal addditon amounts of resin DA-201,the effects of resin DA-201 addition and incubation period on mycelial growth and production of palmarumycins C12 and C13 were conducted.When the 11-day-old cultures were treated with resin DA-201 at 4.17%,the total palmarumycins C12 and C13 yields reached the highest values(149.86 and 55.78 mg/L),which were 70.69-fold and 1.82-fold of control yields(2.12 and 30.70 mg/L),respectively.And the percentages of palmarumycins C12 and C13 adsorbed in resin DA-201 were 95.81%and 87.20%,respectively.3.In order to study the effect of two-liquid-phase culture system on the spirobisnaphthalenes production in liquid culture of endophytic fungus Dzfl2,seven biocompatible water-immiscible organic solvents(n-dodecane,n-hexadecane,1-hexadecene,liquid paraffin,dibutyl phthalate,butyl oleate and oleic acid)screened from ten solvents were found to improve palmarumycins C12 and C13 production in the liquid cultures of endophytic fungus Dzf12.The combination effects of addition amounts and addition time of seven organic compounds on mycelia biomass,palmarumycins C12 and C13 production were futher studied.n-Hexadecane,1-hexadecene and liquid paraffin stimulated the mycelial growth.Among them,1-hexadecene was found to be the most efficient.When 1-hexadecene was applied to the medium at 5%(v/v)on day 6,the maximal mycelia bimass was 9.60 g dw/L,which was 1.55-fold in comparison with that(6.20 g dw/L)of the control.The addition of dibutyl phthalate,butyl oleate and oleic acid to cultures of the endophytic fungus Dzfl2 significantly enhanced palmarumycin C12 yield by adsorbing palmarumycin C12 into the organic phase,respectively.With a final concentration of 5%(v/v)oleic acid fed at the beginning of fermentation(day 0),the highest palmarumycin Q2 yield(184.74 mg/L)was obtained,about 45-fold increase in comparison with the control.While the other four liquid hydrocarbons couldn't absorb any palmarumycins,n-dodecane,1-hexadecene and liquid paraffin had great influences on the production of palmarumycin C13.Liquid paraffin at 5-10%(v/v)added at the beginning of fermentation(day 0),10%(v/v)added on day 3 or 5%(v/v)added on day 6 of fermentation,the palmarumycin C13 yield reached a maximum value(about 130 mg/L),which was 4.22-fold that of the control.Two spirobisnaphthalenes namely palmarumycins C2 and C3 were first isolated from the cultures of endophytic fungus Dzf12 after treatment with 1-hexadecene.Palmarumycins C2 and C3 exhibited stronger antimicrobial activities(relatively weak antifungal activity on spore germination of M.oryzae in comparison with their antibacterial activity)and weak antioxidant activities.Meanwhile,the optimum addition strategy of 1-hexadecene for the maximal yields of palmarumycins C2 and C3 was proceeded.Addition of 1-hexadecene in medium at 10%on day 6 of culture,the maximal yields of palmarumycins C2 and C3 were obtained as 0.40 g/L and 1.19 g/L,which were 40.00 fold and 59.50 fold in comparison with those(0.01 g/L and 0.02 g/L)of the control,respectively.1-Hexadecene was first used as the second phase in two-liquid-phase culture system for producing secondary metabolites.4.Gene prediction was performed according to genomic data.In endophytic fungus Dzf12 genome,35 secondary metabolite synthesis genes were predicted,including 19 PKS genes,1 PKS-like gene,7 NRPS genes,6 NRPS-like genes and 2 terpenoids synthase genes.Ultimately g5855 gene was considered as the candidate gene for spirobisnaphthalenes biosynthesis.The production of spirobisnaphthalenes from endophytic fungus Dzf12 was improved effectively in this study.The results provided theory basis for clarifying the biosynthetic pathway of spirobisnaphthalenes and large-scale industrial producing spirobisnaphthalenes in liquid culture of endophytic fungus Dzf12. |
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