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Electronic Journal of Biotechnology

versão On-line ISSN 0717-3458

Electron. J. Biotechnol. vol.19 no.6 Valparaíso nov. 2016

http://dx.doi.org/10.1016/j.ejbt.2016.09.002 

RESEARCH ARTICLE

Enhancement of botrallin and TMC-264 production in liquid culture of endophytic fungus Hyalodendriella sp. Ponipodef12 after treatments with metal ions

 

Haiyu Luoa, Dan Xua, Rushan Xiea, Xuping Zhanga ,Jian Wanga, Xuejiao Donga, Daowan Laia, Ligang Zhoua*,Yang Liub

a Key Laboratory of Plant Pathology, Ministry of Agriculture/Department of Plant Pathology, College of Plant Protection, China Agricultural University, Beijing 100193, China 

b Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences/Key Laboratory of Agro-products Processing, Ministry of Agriculture, Beijing 100193, China


ABSTRACT

Background: Hyalodendriella sp. Ponipodef12, an endophytic fungus from a poplar hybrid, was a high producer of botrallin and TMC-264 with various bioactivities. In this study, the influences of eight metal ions (i.e.,Mn2+,Na+, Mg2+,Zn2+,Cu2+,Fe2+,Fe3+ and Al3+) on botrallin and TMC-264 production in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 were investigated.

Results: Three most effective metal ions (Zn2+,Cu2+ and Mg2+) along with their optimum concentrations were screened. The optimum addition time and concentrations of Zn2+,Cu2+ and Mg2+ were further obtained respectively for improving botrallin and TMC-264 production. The combination effects of 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, and two quadratic predictive models were developed for botrallin and TMC-264 production. The yields of botrallin and TMC-264, which were predicted as 144.12 mg/L and 36.04 mg/L respectively, were validated to be 146.51 mg/L and 36.63 mg/L accordingly with the optimum concentrations of Zn2+ at 0.81 mmol/L, Cu2+ at 0.20 mmol/L, and Mg2+ at 0.13 mmol/L in medium.

Conclusion: The results indicated that the enhancement of botrallin and TMC-264 accumulation in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 by the metal ions and their combination should be an effective strategy.

Keywords: Combination effects, Production, Microbial biotechnology, Process biotechnology, Bioactivities, Central composite design, Response surface methodology


 

1. Introduction

Endophytic fungi are eukaryotic organisms that inhabit intra- and/or inter-cellular healthy tissues of the plants without causing discernible symptoms of plant disease. These endophytes have proved to be a promising source of natural products with novel structures and/or strong bioactivities to show their applications in pharmaceutical, agricultural and food industry.

Hyalodendriella sp. Ponipodef12 was an endophytic fungus derived from the healthy stems of poplar hybrid 'Neva' of Populus deltoides Marsh x P. nigra L.. In our previous studies, four dibenzo-a-pyrones namely botrallin, TMC-264, palmariol B, and alternariol 9-methyl ether were obtained from Hyalodendriella sp. Ponipodef12. Among them, botrallin and TMC-264 (Fig. 1) were found to be the predominant bioactive components. Botrallin has been isolated from the fungi Botrytis alliiMicrosphaeropsis olivacea and Hyalodendriella sp., and exhibited antimicrobial, antinematodal and acetylcholinesterase inhibitory activities. TMC-264 has been isolated from the fungi Phoma sp. and Hyalodendriella sp.. It also showed antimicrobial and antinematodal activities. In addition, TMC-264 selectively inhibited interleukin-4 (IL-4) signaling by interfering with phosphorylation of the signal transducer and activator of transcription 6 (STAT6), as well as binding of the phosphorylated STAT6 to the recognition sequence, so it might be an inhibitor of IL-4 signaling for treatment of allergic diseases.

Various strategies have been developed to increase metabolite yield in microorganism or plant cultures, which include optimization of medium, utilization of two-phase culture systems, addition of precursors and metal ions, as well as application of elicitation by using polysaccharides and oligosaccharides. Many metal ions (i.e.,K+,Na+,Mg2+,Ca2+,Mn2+,Fe2+,Fe3+,Co2+, Ni2+,Cu2+,Zn2+,Al3+ and Mo5+), which played important roles in cell growth and metabolism, were essential for microorganisms. In order to speed up application of botrallin and TMC-264, one of the most important approaches is to increase the yields of botrallin and TMC-264 in fermentation culture of Hyalodendriella sp. Ponipodef12. In our previous studies, obvious enhancement of botrallin and TMC-264 production in the liquid culture of Hyalodendriella sp. Ponipodef12 was achieved by in situ resin adsorption. Inthisstudy, the effects of eight metal ions on the production of botrallin and TMC-264 in liquid culture of Hyalodendriella sp. Ponipodef12 were investigated. Firstly, the single metal ion at its different concentrations was added in medium to screen its enhancing effect by "one-factor-at-a-time (OFAT)", and three most effective metal ions (Zn2+,Cu2+ and Mg2+) with their optimum concentrations were screened. Secondly, three effective metal ions (Zn2+,Cu2+ and Mg2+) along with their addition time were studied to obtain the appropriate combination of addition time and concentration for each ion for improving botrallin and TMC-264 production. Lastly, the combination effects of Zn2+,Cu2+ and Mg2+ on botrallin and TMC-264 production in liquid culture of Hyalodendriella sp. Ponipodef12 were studied by employing statistical method based on the central composite design (CCD) and response surface methodology (RSM) analysis to develop the quadratic predictive models and obtain the maximal yields of botrallin and TMC-264. To the best of our knowledge, the effects of metal ions on botrallin and TMC-264 production of Hyalodendriella sp. Ponipodef12 have not yet been reported. The purpose was to investigate the enhancing effects of the metal ions for botrallin and TMC-264 biosynthesis in liquid culture of Hyalodendriella sp. Ponipodef12, as well as to provide data supporting dibenzo-a-pyrone production on a large scale.

2. Materials and methods

2.1. Endophytic fungus and culture conditions

Endophytic fungus Hyalodendriella sp. Ponipodef12 (GenBank accession number HQ731647) was isolated from the healthy stems of the 'Neva' hybrid of P. deltoides Marsh x P. nigra L in our previous study. It was stored both on PDA (potato 200 g/L, dextrose 20 g/L and agar 20 g/L) slants at 4°C and in 40% glycerol at — 70°C in the Herbarium of the College of Plant Protection, China Agricultural University (Beijing, China). The fungus was cultured on PDA medium in Petri dishes at 25°C for 10 d. For seed culture, four plugs of agar medium (0.4 x 0.4 cm) with fungal cultures were inoculated in each 250-mL Erlenmeyer flask containing 100 mL potato dextrose broth (PDB) medium, and incubated on a rotary shaker at 150 rpm and 25°C for 5 d. For fermentation culture, about 14 mycelia pellets were inoculated in each 250-mL Erlenmeyer flask containing 80 mL PDB medium, and incubated on a rotary shaker at 150 rpm and 25°C.

2.2. Preparation of the media containingmetal ions

The cultures were supplemented with MnSO4 x H2O, MgSO4 x 7H2O, Na2SO4, FeSO4 x7H2O, ZnSO4 x 7H2O, CuSO4 x 5H2O, FeCl3 x 6H2O, AlCl3 x 6H2O and Al (NO3)3 x 9H2O, for Mn2+,Mg2+,Na+,Fe2+,Zn2+, Cu2+,Fe3+ and Al3+ on day 10 of culture, respectively. Each inorganic salt was dissolved with sterile water, filtrated through a microfilter (pore size, 0.22 µm). The levels considered for the metal ions Mn2+, Mg2+,Na+,Fe2+,Zn2+ and Cu2+ were set at 0.1, 0.5,1.0, 2.0,4.0, 8.0, and 16.0 mmol/L and the levels considered for Fe3+ and Al3+ were set at 0.1, 0.5, 1.0, 2.0, and 4.0 mmol/L based on the gradient dilution method. Each experiment had three replicates including the control cultures without addition of metal ions. As Zn2+,Cu2+ and Mg2+ were found to be the most effective metal ions (Tables S1, S2, and S3), they were applied in the next experiments at different concentrations (0.25, 0.50, 0.75,1.0 and 1.25 mmol/L for Zn2+; 0.125, 0.25, 0.50, 0.75 and 1.0 mmol/L for Cu2+; 0.0625, 0.125, 0.25, 0.50,1.0 and 2.0 mmol/L for Mg2+) on days 0, 5, 10, 15 and 20 of culture, respectively. For investigating the combination effects of Zn2+,Cu2+ and Mg2+ on the botrallin and TMC-264 production, the final concentrations of the metal ions were set by the software of Design Expert 8.0 (Stat-Ease, USA).

2.3. Analytical procedures

Themycelia of theendophyticfungus Hyalodendriella sp. Ponipodef12 were separated from the fermentation broth by filtration under vacuum through a pre-weighed filter paper. It was then rinsed three times with distilled water, dried in an oven at 60°C to a constant dry weight (dw), and then the mycelia dry weight was obtained.

Botrallin and TMC-264 extraction and quantification in the samples were based on the methods as described previously. For botrallin and TMC-264 analysis in mycelia, 100 mg of dry mycelia powder was deposited into a vial with 10 mL of ethyl acetate, and then subjected to ultrasonic treatment (three times, 60 min each). After removal of the solid by filtration, the filtrate was evaporated to dryness and redissolved in 1 mL of methanol. For analysis of botrallin and TMC-264 yield in medium, 5 mL of the culture broth was evaporated to dryness and extracted with 5 mL of ethyl acetate, and the liquid extraction was then evaporated to dryness and redissolved in 1 mL of methanol.

The content of botrallin and TMC-264 was analyzed by a high performance liquid chromatography (HPLC) system (Shimadzu, Kyoto, Japan), which consisted of two LC-20AT solvent delivery units, an SIL-20A autosampler, a SPD-M20A photodiode array detector, and CBM-20Alite system controller. A reversed-phase Ultimate TM XB C18 column (250 mm x 4.6 mm, 5 µm, Welch Materials, Inc., Ellicott, MD, USA) was used for separation by using a mobile phase of methanol-H2O (60:40, v/v) at a flow rate of 1 mL/min. The temperature was maintained at 40°C, and UV detection at 234 nm. The sample injection volume was 10 µL. The LC-solution multi-PDA workstation was employed to acquire and process chromatographic data. Botrallin and TMC-264 were detected and quantified with the standards prepared according to the previous method. The linear equation of botrallin by HPLC analysis was = 5.71711 9 106 X — 85,792.5 (R2 = 0.9997), and that of TMC-264 was = 1.62393 9 107 X — 137,986 (R2 = 0.9981), where was the peak area, was quality (µg) of the sample injected for each time, and was the correlation coefficient.

2.4. Methodology and design of experiments

The optimal concentrations of Zn2+,Cu2+ and Mg2+ for the enhancement of botrallin and TMC-264 production by Hyalodendriella sp. Ponipodef12 were determined by means of CCD and RSM using Design Expert 8.0 (Stat-Ease, USA). 100 µL of metal ion solutions was added to 80 mL medium in the experiments with different concentrations of ZnSO4, CuSO4 and MgSO4 on day 15, respectively. The three factors were designated as X1X2X3, and each independent variable (final metal ion concentration) in the CCD experiments was studied at five coded levels (—1.682, — 1, 0, + 1, + 1.682) (Table 1) and a set of 20 experiments were carried out (Table 2). The factors were coded according to the following equation:

where xi was the coded value of the variable Xi, while X0 was the value of Xi at the center point, and ∆was the step change of an independent variable.

The response variable (botrallin and TMC-264 production) was explained by the following second-order polynomial equation:

where was the predicted response value; a0 was the intercept term; x1x2 and x3 were coded independent variables; a1a2 and a3 were linear coefficients; a12, a13 and a23 were interaction coefficients; and a11, a22 and a33 were the quadratic term coefficients. All of the coefficients of the second polynomial model and the responses obtained from the experimental design were subjected to multiple nonlinear regression analyses.

The fitness of the second-order polynomial model equation was evaluated by the coefficient (R2) of determination. The analysis of variance (ANOVA) and test of significance for regression coefficients were conducted by F-test. In order to visualize the relationship between the response values and test independent variables, the fitted polynomial equation was separately expressed as 3D response surfaces and 2D contour plots by the software of Design Expert.

2.5. Statistical analysis

All tests were carried out in triplicate, and the results were represented by their mean values and the standard deviations (SD).

Table 1
Coded values ( x) and uncoded values ( X) of variables in the CCD experiments.

Table 2
Central composite design matrix for the experimental design and predicted responses for botrallin and TMC-264 production.

The data were submitted to analysis of variance (one-way ANOVA) to detect significant differences by PROC ANOVA of SAS version 9.1. The term significant has been used to denote the differences for which ≤0.05.

3. Results

3.1. Effects of metal ions on botrallin and TMC-264 production

Eight metal ions (i.e.,Mn2+,Na+,Mg2+,Zn2+,Cu2+,Fe2+,Fe3+ and Al3+) were separately added in medium on day 10 of culture. The effects of the metal ions on mycelia growth and production of botrallin and TMC-264 in liquid culture of Hyalodendriella sp. Ponipodef12 were presented in Tables S1, S2, S3, S4, S5, S6, S7, S8, and S9. The enhanced capacity ofthe metal ions for botrallin and TMC-264 production was in order of Zn2+ N Cu2+ N Mg2+ N Al3+ (in the form of AlCl3) N Na+ N Fe3+ N Fe2+ N Mn2+ respectively at their appropriate concentrations. Three metal ions, Zn2+,Cu2+ and Mg2+ were the most effective to enhance production of botrallin and TMC-264. They were selected for further enhancing experiments for botrallin and TMC-264 production in liquid culture of endophytic fungus Hyalodendriella sp. Ponipodef12.

Addition time of the metal ions has been considered as a main factor to affect biosynthesis of fungal metabolites. Zn2+,Cu2+ and Mg2+ were found to be effective to improve botrallin and TMC-264 biosynthesis in liquid culture of Hyalodendriella sp. Ponipodef12 in this study. Hence, their concentrations in combination with addition time were further optimized. As the ten-day-old cultures treated with 0.5-1.0 mmol/L of Zn2+ reached ideal botrallin and TMC-264 production, the highest concentration of Zn2+ in subsequent studies was limited at 1.25 mmol/L. Similarly, the highest concentrations of Cu2+ and Mg2+ were set at 1.00 mmol/L and 2.00 mmol/L, respectively. Fig. 2 showed the effects of Zn2+,Cu2+ and Mg2+ on botrallin and TMC-264 production in liquid culture of Hyalodendriella sp. Ponipodef12, which were dependent on both their concentrations and addition time (added on days 0, 5, 10, 15 and 20).

The effects of Zn2+ and its addition time on botrallin and TMC-264 production in liquid culture of Hyalodendriella sp. Ponipodef12 were shown in Fig. 2a and Table S10. When the cultures were fed with 0.25-1.25 mmol/L of Zn2+ on days 10 and 15, the production of botrallin and TMC-264 was improved obviously. With 1.00 mmol/L of Zn2+ added on day 15, the highest yields of botrallin (133.54 mg/L) and TMC-264 (32.83 mg/L) were obtained. The total yield (botrallin plus TMC-264) was improved to reach 166.37 mg/L, which was about 5.90-fold of the control yield (28.22 mg/L).

 

Fig. 2b and Table S11 showed the effects of Cu2+ on botrallin and TMC-264 production in Hyalodendriella sp. Ponipodef12 liquid cultures, which were dependent on both Cu2+ concentrations (0.125 to 1.00 mmol/L) and its addition time (added on days 5,10,15 and 20). As shown in Fig. 2b, with 0.25 mmol/L of Cu2+ fed on day 15, the highest yields of botrallin (80.20 mg/L) and TMC-264 (31.29 mg/L) were obtained. The total yield of botrallin plus TMC-264 was improved to reach 111.50 mg/L, which was about 3.95-fold of the control yield (28.22 mg/L).

The further optimized concentrations of Mg2+ were set as 0.0625, 0.125, 0.25, 0.50,1.00 and 2.00 mmol/L, and were added on days 0, 5, 10, 15 and 20, respectively. The effects of Mg2+ on mycelia growth and production of botrallin and TMC-264 were showed in Fig. 2c and Table S12. When the cultures were fed with 0.0625-2.00 mmol/L of Mg2+ on days 10 and 15, the production of botrallin and TMC-264 was improved obviously. The highest yields of botrallin (63.85 mg/L) and TMC-264 (32.21 mg/L) were obtained when Mg2+ was added on day 15 at 0.125 mmol/L and 0.50 mmol/L, respectively. The total yield of botrallin plus TMC-264 was improved to reach 90.34 mg/L, which was about 3.20-fold of the control yield (28.22 mg/L).

3.2. Combination effects ofcopper, zinc and magnesium ions on botrallin and TMC-264 production

According to the above single-factor experiments, Zn2+,Cu2+ and Mg2+ all showed their obvious effects on botrallin and TMC-264 production in liquid culture of Hyalodendriella sp. Ponipodef12 separately at their most suitable concentrations and addition time which were 1.00 mmol/L of Zn2+ added on day 15, 0.25 mmol/L of Cu2+ added on day 15, and 0.125 mmol/L of Mg2+ added on day 15. In order to study the combination effects of Zn2+,Cu2+ and Mg2+, the suitable concentrations of Zn2+,Cu2+ and Mg2+ in medium for botrallin and TMC-264 production were determined using CCD experiments and RSM analysis. Three ions were all added on day 15, and five levels of each variable (metal ion concentration, mmol/L) were set by the software of Design Expert 8.0 (Stat-Ease, USA), which are presented in Table 1. Subsequently, 20 trials of CCD were carried out to optimize the production of botrallin and TMC-264. The values of response Y1 (botrallin yield) and Y2 (TMC-264 yield) under the different ions combinations were presented in Table 2. There was a considerable variation of botrallin and TMC-264 yields depending upon the different ions combination. Botrallin and TMC-264 yields ranged from 31.80-140.00 mg/L, and 7.70-36.00 mg/L, respectively.

The empirical relationships between botrallin and TMC-264 yields (Y1 and Y2) and the tested variables (metal ion concentrations) were obtained by application of RSM. By employing multiple regression analysis on the experimental data, the response variables (Y1 and Y2) and the tested variables were related by the following second-order polynomial equations [Equation 3 and Equation 4]:

Statistical testing of the model was performed with Fisher's F-test to obtain the mathematical relationship between response and process variables. In order to ensure a good model, the test for significance of regression model was performed and applied with the ANOVA. Table 3 showed the results of ANOVA for production of botrallin and TMC-264. Values of prob. (p) N  less than 0.05 indicated model terms are significant for production of botrallin and TMC-264. The non-significant lack-of-fit (more than 0.05) showed that each quadratic model was valid for present study. Non-significant lack-of-fit was good for data fitness in the model of this study. The predicted R2 of 0.9973 and adjusted R2 of 0.9984 for botrallin, and predicted R2 of 0.9815 and determination coefficients adjusted R2 of 0.9928 for TMC-264 were reasonable agreement with their values of R2 (0.9992 and 0.9962), which were closer to 1.0, indicated the better fitness of model in the experimental data. In addition, low variation coefficient (CV) of models for botrallin and TMC-264 indicated that the quadratic multinomial regression models for botrallin and TMC-264 had high reliability.

Therewereno significant differences between predicted and actual values in 20 trials of CCD for botrallin and TMC-264 yields shown in Table 2, which indicated that the models had high degree of fit. Furthermore, we also analyzed for the significance of each secondary coefficient of the quadratic multinomial regression model with the results shown in Table 4. Statistical testing of the model was performed with Fisher's F-test. Greater F-value and smaller p-value indicated that second item had more significant effect on values. Table 4 showed that most regression coefficients, especially all quadratic term coefficients of the two models were very significant, and demonstrated the research variables (i.e.,Zn2+,Cu2+,Mg2+ concentrations) and their interactions had significant roles in the formation of botrallin and TMC-264.

The three-dimensional (3D) response surface and two-dimensional (2D) contour plots were the graphical representation of the regression equation used to determine the optimum values of the variables within the ranges considered. The entire relationships between

Table 3
ANOVA for the second-order polynomial models.

 

Table 4
Regression results of the central composite design.

reaction factors and responses could be better understood by examining the planned series of response surface plots generated from the predicted models [Equation 3 and Equation 4] by using the Design Expert software 8.0. The interactive effects among the three independent variables being at fixed level on the production of botrallin and TMC-264 were shown in 3D surface and 2D contour plots (Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8), respectively. The maximum predicted value was identified by the surface confined in the smallest ellipse in the contour diagram. Elliptical contours could be obtained when there was a perfect interaction between the independent variables.

An elliptical response surface in the entire region was found from the second order quadratic equation for the botrallin production with the interaction of Zn2+ and Cu2+ concentrations (Fig. 3). The results showed that botrallin production was considerably affected by varying the concentrations of Zn2+ and Cu2+. The maximum production of botrallin was predicted at the given ranges of both Zn2+ and Cu2+ concentrations. The production decreased at the maximum and minimum values of ranges considered in both parameters.

Fig. 4 shows the response surface plots for variation in the botrallin production, as a function of Cu2+ and Mg2+ concentration by keeping the concentration of Zn2+ at 1.0 mmol/L. The production of botrallin was affected by the concentrations ofCu2+ and Mg2+. The production of botrallin increased with increasing of Cu2+ concentration at a certain level. The effect of Mg2+ on botrallin production was similarly with Cu2+.

Fig. 5 also showed the elliptical response surface plots of botrallin production as a function of Zn2+ and Mg2+ concentration. The predicted botrallin production decreased at the higher and lower values of ranges for both Zn2+and Mg2+ concentrations. Maximum production was obtained near the center points ofthe response surface.

Among Fig. 6Fig. 7 and Fig. 8, all were found an elliptical response surface in the entire region from the second order quadratic equation for interactions cyclically on the yields of TMC-264 between two variables among three variables. The TMC-264 production was considerably affected by varying the concentrations of Zn2+,Cu2+ and Mg2+. The maximum production of TMC-264 was predicted at given ranges of Zn2+,Cu2+ and Mg2 + concentration. The TMC-264 production decreased at the maximum and minimum values ofranges considered among three parameters.

The highest yields of botrallin and TMC-264 were predicted as 144.12 mg/L and 36.04 mg/L by solving the quadratic regression equations [Equation 3 and Equation 4] between botrallin and TMC-265 yields and three metal ions at the optimal concentrations of Zn2+ at 0.81 mmol/L; Cu2+ at 0.20 mmol/L; Mg2+ at 0.13 mmol/L in medium. In order to verify the optimization results as well as to validate the model developed, a set of experiment with five replications was performed according to the media constituent presented in Table 5. Under the determined conditions, the mean yield values of botrallin (146.51 mg/L) and TMC-264 (36.63 mg/L) were obtained from the actual experiments, which were individually 6.84-fold and 6.44-fold of those (21.41 mg/L and 5.69 mg/L) in the original basal medium. Based on the Student t-test, the above model was satisfactory and adequate for reflecting the expected optimization as no significant differences were observed between the predicted maximum yields of botrallin and TMC-264 and the experimental ones.

4. Discussion

Metal ions belong to the abiotic elicitors and play an integral role in the growth and metabolite production of microorganisms, and the addition of metal ions to enhance secondary metabolite production of fungi has been widely reported. The mycelia pellet formation and fumaric acid production were significantly affected by the trace metal ions Mg2+,Zn2+,Fe2+,and Mn2+ in fermentation culture of Rhizopus oryzae ATCC 20344. The mycelia growth and polysaccharide production were obviously enhanced by the metal ions Zn2+,Se2+ and Fe2+ in submerged culture of Ganoderma lucidum. Versicolorin production of Aspergillus parasiticus was completely dependent on Zn2+. Cu2+ stimulated dipicolinic acid synthesis in Penicillium citreoviride strain 3114, and improved the production of laccase by the white-rot fungus Pleurotus pulmonarius in solid state fermentation.

Addition of two or more metal ions in medium could synergistically enhance or inhibit metabolite production of fungi. Zinc ion (Zn2+) promoted the production of alternariol, and Mn2+ acted synergistically with Zn2+ to provide a further three fold stimulation of alternariol synthesis in Alternaria alternata. Zn2+ enhanced the production of ergot and quinoline alkaloids, and both Fe2+ and Cu2+ synergistically inhibited alkaloid biosynthesis in the cultures of Penicillium citrinum. Ca2+,Cu2+ and Al3+ synergistically enhanced palmarumycin production in liquid culture of endophytic fungus Berkleasmium sp. Dzf12. When Al3+ concentration was higher than 4 mmol/L, palmarumycin C12 production was favored, and palmarumycin C13 production was suppressed.

5. Conclusion

In this work, the metal ions were first studied for their stimulatory effects on botrallin and TMC-264 production in liquid culture of Hyalodendriella sp. Ponipodef12. Based on the one-factor-at-a-time (OFAT) experiments, three metal ions Zn2+,Cu2+ and Mg2+ at their appropriate concentrations showed the most significant enhancing effects on the production of botrallin and TMC-264, and were selected for the further study for the combination of their addition time and concentrations. When the cultures were respectively fed with 0.25-1.25 mmol/L of Zn2+, 0.125-1.00 mmol/L ofCu2+, and 0.06252.00 mmol/L of Mg2+ on days 10 and 15 of culture, the yields of botrallin and TMC-264 were improved obviously. The combination interactions of Zn2+,Cu2+ and Mg2+ for botrallin and TMC-264 production were further optimized by employing a statistical method based on CCD and RSM. The yields of botrallin and TMC-264, which were predicted as 144.12 mg/L and 36.04 mg/L respectively, were validated to be 146.51 mg/L and 36.63 mg/L accordingly with the optimum concentrations of Zn2+ at 0.81 mmol/L, Cu2+ at 0.20 mmol/L, and Mg2+ at 0.13 mmol/L in medium. There were no significant differences between the experimental and predicted values, which indicated that the models had high degree of fitting, and could guide the production of botrallin and TMC-264 in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12. The results indicated that enhancement of botrallin and TMC-264 accumulation in liquid culture of Hyalodendriella sp. Ponipodef12 by the metal ions should be an effective strategy for large-scale production of botrallin and TMC-264 in the future. As the combination effects of only three metal ions (i.e.,Zn2+,Cu2+ and Mg2+) have been studied for their enhancing effects on botrallin and TMC-264 production in this work, more metal ions in the medium, as well as other parameters like pH, temperature, oxygen supply, precursors, should be considered in the future work. Furthermore, the mechanism of action of the metal ions on botrallin and TMC-264 biosynthesis also need to be studied in detail. After a series of optimization for their biosynthesis conditions, we could obtain the final medium for maximum production of botrallin and TMC-264 by natural fermentation of the endophytic fungus Hyalodendriella sp. Ponipodef12.

Table 5
The optimal culture conditions for botrallin and TMC-264 production of Hyalodendriella sp. Ponipodef12.

Conflict of interest

The authors declare that they have no conflict of interest.

Financial support

This work was co-financed by the grants from the Hi-Tech R&D Program of China (2011AA10A202) and the National Basic Research Program of China (2013CB127805).

 

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Article history: Received 19 April 2016 Accepted 1 September 2016 Available online 14 September 2016

* Corresponding author. E-mail address: lgzhou@cau.edu.cn (L. Zhou).

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