Improvement of chaetominine production by tryptophan feeding and medium optimization in submerged fermentation of Aspergillus fumigatus CY018
© The Author(s) 2016
Received: 25 May 2016
Accepted: 27 August 2016
Published: 15 September 2016
Chaetominine (CHA) is a novel alkaloid with excellent medicinal activities produced by Aspergillus fumigatus CY018. However, its further application has been severely restricted by the low production yield. In this work, the fermentation titer of CHA was investigated by medium composition optimization and amino-acid addition strategies.
Under the optimized conditions of sucrose 115.03 g/L, ammonium acetate 3.98 g/L, d-tryptophan 3.84 g/L, KH2PO4 1.5 g/L, FeSO4·7H2O 0.02 g/L, MgSO4·7H2O 0.7 g/L, sodium glutamate 3 g/L, sodium tartrate 1.5 g/L, and CaCl2 0.045 g/L), a CHA production yield of 55.92 mg/L was obtained, which increased significantly (3.99-fold) as compared with the unoptimized basal medium. Scale-up fermentation was carried out in a 5-L bioreactor based on the shake-flask fermentation results, maximum CHA yield of 48.53 mg/L was obtained at an air flow rate of 2.0 ± 0.1 VVM and an agitation rate of 400 rpm.
These results demonstrated that medium composition optimization and amino-acid addition were useful strategies for improving CHA production via biotechnological process. The methods in this work would be useful for the biotechnological production of CHA from A. fumigatus.
KeywordsChaetominine Aspergillus fumigatus Medium optimization d-Tryptophan
Many endophytic microbes have the potential to produce bioactive natural products that may directly or indirectly be used as therapeutic agents for the treatment of various diseases (Kusari et al. 2014). Chaetominine (CHA) is a bioactive alkaloidal metabolite isolated from the endophytic filamentous fungus Aspergillus fumigatus CY018 (Yao et al. 2016). The compound exhibited strong cytotoxic activity against the human leukemia K562 and colon cancer SW1116 cell lines, which suggested that this bioactive metabolite might serve as a promising candidate for anti-cancer treatment (Kusari et al. 2014; Yao et al. 2016).
CHA was first reported to be a compound with new framework synthesized by endophytic Chaetomium sp. IFB-E015 in 2006 (Yao et al. 2016). Due to the unprecedented skeleton and its potential biological properties in treating cancer diseases, a number of efforts have been directed to the total synthesis of CHA in recent years (Snider and Wu 2007; Toumi et al. 2008; Malgesini et al. 2009; Peng et al. 2014). More recently, there are some reports indicating that endophytic Aspergillus species are also shown to produce CHA. For example, CHA is obtained from endophyte A. terreus isolated from stem of rice and A. fumigatus CY018 isolated from Cynodon dactylon (Shen et al. 2015; Liu et al. 2016). In the previous report, it was speculated that CHA might be biosynthesized from l-alanine, anthranilic acid, and d-tryptophan (Jiao et al. 2007), and the biosynthetic proposal has been practically verified by the biomimetic synthesis method (Xu et al. 2015). However, no research has focused on the bioproduction of CHA by submerged fungus cultivation, and little is known about the fermentation conditions for the improvement of its production.
The aim of this study was to develop a suitable culture medium for CHA production in the submerged fermentation of A. fumigatus CY018 and to evaluate its potential use for scaling-up of CHA sustainable production. The carbon and nitrogen source appropriate for fungus growth and CHA accumulation were first selected based on the conventional single-factor method. Secondary, the effects of amino acids, including l-alanine and d-tryptophan, which were proposed as speculated precursor units of CHA (Jiao et al. 2007), on microbial growth and CHA biosynthesis were investigated systematically. In addition, the fermentation medium was further developed and optimized by RSM for improving CHA production and scaling-up fermentation. The established medium constituents were supposed to be efficient nutritional components for the CHA production performance and would provide important support for the scaling-up production of the bioactive metabolite.
Chemical and reagents
Reagents for microorganism cultivation and product extraction, including glucose, sucrose, ammonium acetate, d-tryptophan, sodium nitrate, KH2PO4, FeSO4·7H2O, MgSO4·7H2O, sodium glutamate, sodium tartrate, methanol, and ethyl acetate were purchased from Sinopharm Chemical Reagent Company, China. Acetonitrile of HPLC grade was purchased from TEDIA Company, USA. All other chemicals used in this study were of AR grade unless indicated otherwise. Authentic CHA was provided by Prof. RX Tan (Nanjing University, China) and confirmed by the NMR and LC–MS techniques.
Microorganisms and preparation of inoculum
The CHA-producing strain (A. fumigates CY018) was an endophytic fungus isolated from Cynodon dactylon, which was provided by Prof. RX Tan (Liu et al. 2016). The strain was regularly maintained on potato-dextrose agar (PDA) slant and stored at 4 °C. For the seed culture, the slant was inoculated in the PDA liquid medium and cultivated at 180 rpm at 28 °C for 48 h.
The original fermentation medium consisted of 100 g/L of sucrose, 3.5 g/L of sodium nitrate, 1.5 g/L of KH2PO4, 0.02 g/L of FeSO4·7H2O, 0.7 g/L of MgSO4·7H2O, 3 g/L of sodium glutamate, and 1.5 g/L of sodium tartrate, and the initial pH of the medium was adjusted to 6.0 using 1-mol/L HCl before sterilization. Shake-flask fermentation was performed by inoculating 7-mL seed culture in a 250-mL flask containing 50 mL of fermentation medium. Then, the flasks were incubated at 28 °C on a rotary shaker (180 rpm) for 16 days.
Medium optimization by the RSM experimental design
Process variables used central composite design with actual factor levels corresponding to coded factor level
Independent variables (g/L)
The mathematical model generated during CCD implementation was validated by conducting experiment on given optimal medium setting. All data obtained in this work were the mean of triplicate experiments, and the error bars indicated the corresponding standard deviation (SD). P values were used to check the significance of the differences among cultures under different conditions. A value of P < 0.05 was considered statistically significant.
Lab-scale bioreactor system experiments
In the lab-scale bioreactor process, the experiments were performed in a 5-L stirred bioreactor which equipped with two layers of six-flat-blade disk turbine impellers. 400-mL inoculum was inoculated into the bioreactor containing 2.6-L fermentation medium. The fungus was cultured for 16 days at 28 ± 0.2 °C with the stirring speed of 200–500 rpm. The air flow was set at 1.5 ± 0.1 VVM during the whole process and 0.3 % (v/v) antifoam (mixture of organic polyether dispersions) was added before autoclaving.
Biomass accumulation was estimated using the dry cell weight (DCW) analysis (Tey et al. 2014). A sample was filtered through a 0.45-μm millipore cellulose filter that retained the hyphae. The hyphal solids were washed with sterile distilled water and dried to constant weight by air blowing thermostatic oven. Residual sugar was determined by the anthrone-sulfuric acid method (Cai et al. 2010). The broth was extracted three times with an equal volume of ethyl acetate. The upper liquor was collected and evaporated under reduced pressure. The dried crude extract was dissolved in methanol and prepared for the HPLC analysis after filtration (0.45 μm).
The HPLC system of SHIMADZU LC-10Avp plus with a PDA detector (SPD-M20A) and a C18 column (4.6 mm × 250 mm, 5 μm, Agilent ZORBAX Eclipse XDB-C18) was used to analyze the concentration of CHA. The mobile phase was acetonitrile/water (35:65). The HPLC analysis was under the following condition: flow rate, 1 mL/min; column temperature, 28 °C; UV wavelength, 226 nm; and sample injection volume, 20 μL. The quantification of CHA in samples was based on the comparison of peak areas to the external standard.
Results and discussion
The effect of carbon sources on fungus growth and CHA production
The effect of nitrogen source and concentration on fungus growth and CHA production
As shown in Fig. 2b, the DCW of fungus A. fumigatus increased with the increase concentration of ammonium acetate during the tested concentration ranges and reached maximum (17.32 g/L) at the concentration of 4.5 g/L. The effect of ammonium acetate concentration on the production performance of CHA was evaluated simultaneously; the maximum fermentation yield (31.28 mg/L) was obtained at 3.5 g/L of ammonium acetate (Fig. 2b), which was much higher than the original fermentation medium and other tested nitrogen concentration. CHA is an alkaloidal metabolite of A. fumigatus CY018 (Liu et al. 2016). Thus, there should be an intuitive connection between the nitrogen concentration and CHA accumulation. Consequently, it is not surprising that lower nitrogen concentration was negative for the CHA production. However, higher concentration of ammonium acetate (>3.5 g/L) in culture medium had a negative effect on CHA accumulation (Fig. 2b). In a previous report, inhibition of the production of secondary metabolites by high concentration of nitrogen source was also observed, which suggested the inhibition mechanism was due to the depression of enzymes in primary or secondary pathways related to the product biosynthesis (Wei et al. 2012). Among all the tested nitrogen sources and concentrations, ammonium acetate of 3.5 g/L revealed to the most optimal condition for improving CHA production (32 mg/L) and was selected for further medium optimization in the subsequent experiments.
Regulation of CHA production by amino acids addition
The addition of precursor during fermentation was supposed to be a useful experimental tool for improving product yield. Adding side-chain precursor during penicillin fermentation process to greatly increase its industrial production is the most successful example (Eriksen et al. 1994). Although the addition of speculated precursors at the beginning of fermentation showed less effective on the CHA production, however, the fermentation productivity was significantly changed by the variation of the addition time and concentration. As shown in Fig. 3b, the addition of l-alanine (10 mM) at different fermentation time caused various changes in the fermentation performance, and a maximum product yield of 34.96 mg/L was obtained at the addition time of 264 h. By adding d-tryptophan at 192 h (Fig. 3c), the CHA production reached maximum value of 42.79 mg/L, which was 20 % higher than the optimal l-alanine addition and much higher than the control (non-addition of amino acid). The results indicated that the addition of d-tryptophan at specific fermentation time could be considered as a useful strategy for regulation of CHA production.
Thereafter, the effect of tryptophan concentration on fungus fermentation was further evaluated for improving CHA production. At fermentation time of 192 h, adding lower concentration of tryptophan (5 mM) slightly increased the CHA production, and higher concentrations (9–17 mM) were more beneficial for the production performance (Fig. 3d). However, further improvement of the tryptophan concentration (21–29 mM) resulted in a decreased production of CHA (Fig. 3d). Similar phenomenon was also observed in the submerged fermentation of Streptomyces sp. for dinactin production, which demonstrated that the higher doses of precursors inhibited the synthesis of dinactin (Zhou et al. 2015). In this study, the addition of 17 mM tryptophan at 192 h was the most suitable feeding strategy for improving the CHA production. Under this condition, the production reached 53.93 mg/L in the shake-flask fermentation, which was 2.85 times higher than that in original medium without optimization.
Medium optimization using central composite design
Central composite design and response values
(A) Sucrose (g/L)
(B) Ammonium acetate (g/L)
(C) d-Tryptophan (g/L)
CHA production (mg/L)
53.29 ± 1.13
53.92 ± 0.73
46.23 ± 2.51
43.21 ± 0.77
44.92 ± 1.16
51.79 ± 2.05
52.82 ± 0.12
53.62 ± 1.71
43.74 ± 0.95
57.82 ± 0.21
43.23 ± 2.55
45.38 ± 1.42
47.83 ± 0.73
44.93 ± 1.34
44.94 ± 0.95
45.78 ± 2.11
39.78 ± 2.76
59.42 ± 0.22
47.94 ± 0.94
52.86 ± 1.55
ANOVA analysis for responses Y[CHA(mg/L)]
Sum of squares
Degree of freedom
Prob > F
Lack of fit
To verify the optimization results, experiments were performed under the predicted optimal condition. The experimental results (55.92 mg/L) closely agreed with the values obtained from RSM and, hence, validated the findings of response surface optimization.
Bioreactor fermentation using the optimal medium composition and tryptophan feeding strategy
In this work, the fermentation medium optimization for improving CHA production in submerged fermentation of A. fumigatus was investigated. Sucrose and ammonium acetate were selected as preferable carbon and nitrogen sources for the CHA production. In addition, adding tryptophan, a speculated precursor CHA, at fermentation time of 192 h showed great improvement in the production performance. After carbon source, nitrogen source, and tryptophan addition optimization by RSM, the CHA production could reach 55.92 mg/L in the shake-flask fermentation, which was 3.99-fold to the initial production. Based on the results in flask shaker, the fermentation process was successfully scaled-up in a lab-scale bioreactor, in which the CHA production could reach 48.53 mg/L. The information obtained demonstrated that the optimized fermentation process is an easy and effective method for improving and scale-up production of CHA.
dry cell weight
response surface methodology
central composite design
analysis of variance
YPZ was in-charge of the experiments and paper writing. RHJ offered experimental strain. LYY participated in the experiments and paper writing. YHL directed the study as the tutor. All authors read and approved the final manuscript.
This work was supported by the National High Technology Research and Development Program of China (2013AA092901) as well as the National Special Fund for State Key Laboratory of Bioreactor Engineering (2060204).
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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