Induced effect of Ca2+ and Al3+ on chaetominine synthesis by Aspergillus fumigatus CY018 under submerged fermentation

Chaetominine (CHA), an alkaloid with a biological activity obtained from Aspergillus fumigatus CY018, has strong anticancer activity against the human leukemia cells. However, its physiological and biochemical research is limited by CHA yield in the liquid‐state fermentation, which is a problem that urgently needs effective biological solution. In this work, Ca2+ and Al3+ were found to have a strong promoting effect on CHA production after multiple metal ions screening. Then, the addition condition of Ca2+ and Al3+ was, respectively, optimized CHA production and dry cell weight. The intermediate metabolites were increased with coaddition of Ca2+ and Al3+. The activities of key enzymes of DAHPs, AroAs, and TrpCs in the CHA biosynthesis pathway were improved by 3.58‐, 3.60‐, and 3.34‐fold, respectively. Meanwhile, the transcription level of laeA, dahp, cs, and trpC was upregulated by 3.22‐, 12.65‐, 5.58‐, and 6.99‐fold, respectively, by coaddition of Ca2+ and Al3+. Additionally, the fermentation strategy was successfully scaled up to a 5‐L bioreactor, in which CHA production could attain 75.6 mg/L at 336 h. This work demonstrated that Ca2+ and Al3+ coaddition was an effective strategy for increasing CHA production, and the information obtained might be useful in the fermentation of filamentous fungi with the addition of metal ions.


INTRODUCTION
Aspergillus sp. is an endophytic fungus and has received considerable attention of researchers in recent years. 1 Aspergillus fumigatus, an endophyte found in portunus, plants, and others, has an outstanding ability of synthesizing primary and secondary bioactive metabolites. The secondary metabolites biosynthesized by A. fumigatus have shown multiple biological activities for common human diseases. Fumigaclavine C was fermented by A. fumigatus CY018 through the two-stage culture process (oscillation and rest), which was an ergot alkaloid with anti-inflammatory and antipsychotic activity. 2 The antitumor compound of Epothilone B could arrest cancer cells at the G2-M phase, the yield of which was improved with response surface methodology by A. fumigatus. 3 Chaetominine (CHA) ( Figure S1 in the Supporting Information), a quinazolinone alkaloid from the endophytic fungi of A. fumigatus CY018, was found to possess greater anticancer activity than 5-fluorouracil, which implied that CHA could be developed as an anticancer candidate in the future. [4][5][6] Moreover, it is reported that CHA was able to inhibit the germination and growth of wheat and radish at low concentrations. 7 However, the in-depth physiological and biochemical research of CHA has been seriously limited by the low yield in the submerged fermentation. Therefore, further enhancement of CHA production is urgently required considering its potential use in the field of medicine. Numerous effective biological strategies are suggested to improve the yield of secondary metabolites by a microorganism, such as medium component optimization, temperature regulation, oxygen concentration regulation, and others. [8][9][10] Microbial growth and secondary metabolite biosynthesis were significantly regulated by metal ions as suggested in the literature. Luo et al. reported an effective strategy of the metal ions (Zn 2+ , Cu 2+ , and Mg 2+ ) coaddition could enhance the production of botrallin and TMC-264 by Hyalodendriella sp. Ponipodef12. 11 Dzurendova et al. determined the effects of varying levels of metal and phosphate ions on the growth and metabolism of Mucor circinelloides, demonstrating that Mg and Zn ions were essential for the growth and metabolic activity of M. circinelloides. 12 The effects of different metal ions (Mg 2+ , Al 3+ , and Fe 3+ ) on the growth, lipid accumulation, and sedimentation of Scenedesmus sp. were explored by Kong et al., indicating that Fe 3+ could promote the microalgal lipid biosynthesis and secretion in batch culture and the addition of Al 3+ could enhance the sedimentation efficiency, whereas the addition of Al 3+ had an inhibitory effect on the biomass of Scenedesmus sp. R-16 and lipid biosynthesis. 13 It was evidently shown that the effect of metal ions would improve the biosynthesis of active metabolites by a microbe.
We also found that the addition of metal ions could change the synthesis of metabolites by A. fumigatus CY018 in the preliminary experiment. Therefore, the selection and optimization of metal ions on CHA production and biomass were investigated. Subsequently, the transcription level and enzyme activity of specific genes were analyzed by the addition of metal ions. The concentration of intermediate metabolites in the synthetic route of CHA was analyzed to demonstrate the regulatory mechanism of metal ions for CHA biosynthesis. Moreover, the additional strategy of metal ions was successfully reproduced in the 5-L bioreactor. This work would be useful in enhancing the production of target compounds in submerged fermentation.

Microorganism
A. fumigatus CY018 as a filamentous fungus was kindly provided by the East China University of Science and Technology. The conidia of A. fumigatus CY018 were stored in 40% glycerin at -80 • C in a refrigerator. The mycelia were activated on potato dextrose agar (PDA) medium and stored at 4 • C in a refrigerator. A. fumigatus CY018 was transferred to a fresh medium every month.

Fermentation conditions
The mycelia of A. fumigatus CY018 stored at 4 • C in a refrigerator were first transferred and cultured on PDA at 28 • C for 7 days in a biochemical incubator. An ager block (size of 1 cm 2 ) was cut from the PDA and transferred to a 500-mL shake flask with 200 mL potato dextrose broth, , which was cultured as seed medium in a shaking incubator at 180 rpm, 28 • C for 72 h. Subsequently, 7 mL of seed medium was transferred to a 250-mL Erlenmeyer flask with 50 mL fermentation medium, which was cultured in the shaking incubator at 180 rpm, 28 • C for 384 h. The composition of the fermentation medium was as follows: 100 g/L sucrose, 5 g/L ammonium acetate, 2 g/L sodium tartrate, 2.4 g/L sodium glutamate, 1.

Selection and optimization of metal ions
The biomass and CHA production was detected with the addition of 0.5 and 1 mM of metal ions (AlCl 3 , CaCl 2 , CuCl 2 , MnCl 2 , and ZnCl 2 ) at 0 h of fermentation. These solutions of metal ions were sterilized in an autoclave at 121 • C for 25 min. Whereafter, the addition concentration and addition time of CaCl 2 and AlCl 3 were further optimized biomass and CHA production.

Metabolite analysis
After fermentation, the growth of A. fumigatus CY018 was measured by dry cell weight (DCW) with the total mycelium in the 250-mL Erlenmeyer flask. The fermented hypha was harvested by the suction filter device with the weighted filter paper. The filtered mycelium was dried until constant weight was obtained, and it was weighed with an analytical balance. The determination of residual sugar in the fermentation broth was utilized by the revised anthrone sulfuric acid method. The sucrose standard curve was prepared by the experimental process of the phenol sulfuric acid method, which was used for the determination of residual sugar content in the fermentation broth. One milliliter of broth from the shake flask was taken to a mixed broth and centrifuged at 13,000 rpm and 4 • C, in which the supernatant was diluted 100 times. Then, the diluent was measured at 485 nm by a spectrophotometer, and the absorbance value was used with the standard curve of the sucrose concentration to calculate the amount of residual sugar in the fermentation broth.
The concentration of CHA was analyzed by HPLC with a C18 column (4.6 mm × 250 mm, 5 μm, Agilent ZOR-BAX Eclipse XDB-C18) under 226 nm, and the method is described in detail by Liu et al. 14 The HPLC condition was followed: the mobile phase was acetonitrile/water (40:60, v/v); the flow rate was 1 mL/min; column temperature was 25 • C; sample injection volume was 20 μL. The concentration of CHA was measured in comparison with the standard, which was isolated and purified in the laboratory.
The detection of organic acids (pyruvate, DAHP, chorismate, and tryptophan) during the CHA biosynthetic pathway was utilized by HPLC with the Agilent Hi-plex ligand exchange column (7.7 mm × 3000 mm, 8 μm) under 338 nm, in which the detector was a UV detector. The HPLC condition for organic acids was followed: the mobile phase was 0.01% trifluoroacetate; the flow rate was 1 mL/min; column temperature was 25 • C; sample injection volume was 20 μL. The concentration of organic acids was measured in comparison with the standard.

qRT-PCR for key genes
One milliliter of broth from the shake flask was taken from a mixed broth and centrifuged by 13,000 rpm at 4 • C, in which the supernatant was carefully removed. The fungus was ground and broken by a mortar with liquid nitrogen in the asepsis room. Then, 200 μL of double distilled water was utilized to dissolve the extract, which was used to analyze the bioactivity and transcription level of a key gene at taken points. The TriZol solution was utilized to extract the total RNA of A. fumigatus CY018 at a sampling point of fermentation. The extracted solution was used for the RNA concentration using a spectrophotometer at A260/280. The method of Li et al. 15 was utilized to detect the transcription level of laeA, dahp, cs, trpC, and actin with coaddition of Ca 2+ and Al 3+ . The extracted RNA and the Premix Script TM Reagent Kit (Takara) were used for reverse transcription. The Premix Ex Taq TM II (Takara) was utilized to determine transcriptional levels of key genes according to the manufacturer's procedure. The sequences of primer pairs for PCR amplification and qRT-PCR assay are displayed in Tables 1 and 2. The CFX96 real-time PCR detection system was used for the qRT-PCR experiment, and the test kit was SYBR Premix Ex Taq II Kit. The transcriptional level of the measured genes was corrected by the conserved gene Actin as a normalized internal standard. The process of qRT-PCR followed as first predenatured at 95 • C for 5 min, and amplification occurred in two steps: 5 s at 95 • C for denaturing, 30 s at 55 • C for annealing and 60 s at 72 • C for extension for 40 cycles. The CFX Manager software was used to calculate standard curves, CT values, and detect the transcriptional level of samples.

Biochemical analysis of key enzymes
One milliliter of broth from the shake flask was taken to a mixed broth and centrifuged at 13,000 rpm and 4 • C, in which the supernatant was carefully removed. The precipitated thallus was broken by a mortar with liquid nitrogen in the asepsis room. Then, 200 μL of double distilled water was utilized to dissolve the extract, which was used for analysis of bioactivity of a key enzyme in the shikimate pathway. The bioactivity of DAHP synthase (DAHPs) was analyzed as described by Liu et al. 16 A substrate solution and 100 μL of treated crude enzyme solution were incubated at 37 • C for 10 min. Subsequently, the mixture was added with 0.2 mL of 10% trichloroacetic acid and then centrifuged at 13,000 rpm for 5 min. The 0.25-mL mixed solution was added with HIO 4 , NaAsO 4 , thiobarbital for the reaction, and the final solution was centrifuged fast and determined with a spectrophotometer at 549 nm by measuring the decrease of PEP.
The AroA synthase (AroAs) was measured at 28 • C in 50-μL reaction volume mixtures (50 mM of Hepes buffer (pH 7.0), 1 mM of shikimate-3-phosphate, 1 mM of PEP, and 0.4 μg of the purified cell disruption). The detection  17 TrpC synthase (TrpCs) was measured through the reaction of indole and l-serine with purified cell disruption, following the modified method as described by Zhao et al. 18

Scale-up experiments
The experiments of lab-scale fermentation process were performed in a 5-L stirred bioreactor as mentioned by Liu et al., 19 in which was coadded with Ca 2+ and Al 3+ as the optimized concentration for 120 h. Samples were taken at an interval of 48 h.

Statistical analysis
All data obtained in the shake flask and 5-L bioreactor are the mean of experiments in triplicate. The statistical significance of differences in optimization of metal ions and detection of parameters (intermediate metabolites, enzyme activity, and transcription level of key enzymes) was evaluated using a one-way analysis of variance (ANOVA) and Duncan's multiple range tests in SPSS version 16.0. A value of p < 0.05 was considered statistically significant.

Effects of multiple metal ions for biomass and CHA production
The strategy of metal ions addition was usually beneficial to the growth of microbe and the biosynthesis of active metabolites. In the present work, the different content (0.5 and 1 mM) of five metal ions (Al 3+ , Ca 2+ , Cu 2+ , Mn 2+ , and Zn 2+ ) were individually tested for selecting the appropriate metal ion for CHA production and biomass of A. fumigatus in Figure 1. The effect of 0.5 mM Al 3+ could effectively enhance the CHA production (52.3 mg/L), which was 1.5 times that of the control. The higher concentration (1 mM) of Al 3+ -promoted CHA synthesis was less than the low concentration. The production of CHA was achieved, respectively, 55.31 and 40.18 mg/L by the addition of 0.5 and 1 mM Ca 2+ . It was found that the addition of Ca 2+ and Al 3+ could increase CHA production, but it had no obvious effect on the biomass of A. fumigatus. The effect of Cu 2+ on CHA production was decreased with the increase in the concentration, meanwhile the effect of Cu 2+ on biomass was increased. A similar result was shown by the effect of Mn 2+ , in which the inhibition effect was stronger than that of Cu 2+ . The effect of Zn 2+ completely inhibited CHA biosynthesis and decreased the biomass of A. fumigatus. In comparison with the effects of five metal ions, the low concentration of Ca 2+ and Al 3+ could significantly improve the CHA production, which indicated that Ca 2+ and Al 3+ were dominant metal ions for the CHA biosynthesis.

Optimization of addition time and concentration by dominant metal ions
For further enhancement of CHA production, the addition concentration and time of Ca 2+ and Al 3+ were, respectively, investigated, which was based on selection results of metal ions. The different concentrations (0, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7 mM) of Ca 2+ were added and analyzed about CHA production and biomass of A. fumigatus in the Figure 2A. The tendency of CHA production was gradually rose and then slowly declined with the increasing concentration of Ca 2+ . When the content of Ca 2+ was 0.5 mM, the CHA production reached the maximum value (58.8 mg/L), which was ascertained as the optimized concentration. However, the effect of the Ca 2+ concentration was not obvious on the biomass of A. fumigatus, in which biomass of 0.5 and 0.55 mM Ca 2+ was relatively lower than others. After the addition concentration of Ca 2+ was confirmed, the addition time (0, 72, 120, 168, 216, 264, 312 h) of Ca 2+ was subsequently studied and the results are shown in Figure 2B. The CHA production was sharply increased and reached the peak (62.74 mg/L, at 120 h) during 0-120 h, then became constant. The biomass of A. fumigatus had an analogous tendency at optimization of the concentration; which was that the tendency of biomass was not correspond to that of CHA production. At this point, the optimized addition concentration and time of Ca 2+ , respectively, were determined as 0.5 mM and 120 h.  Figure 3A. The concentration of Al 3+ was increased slowly from 0 to 0.45 mM, then remained constant from 0.45 to 0.6 mM, and decreased slowly from 0.6 to 0.7 mM. When the content of Al 3+ was 0.5 and 0.55 mM, the CHA production was, respectively, 49.45 and 49.15 mg/L. The effect of the addition concentration of Al 3+ on biomass was weak, the trend of which was relatively constant. The appropriate addition concentration of Al 3+ was defined as 0.5 mM, then the addition time of Al 3+ was determined. The addition time of Al 3+ was set at 0, 72, 120, 168, 216, 264, 312 h, and the results are shown in Figure 3B. The effect of addition time of Al 3+ was increased quickly and then decreased slowly, in which the maximum value of the CHA production was 50.43 mg/L at 120 h. Therefore, the optimized conditions of Ca 2+ and Al 3+ addition were determined, which was added with 0.5 mM Ca 2+ and Al 3+ at 120 h.

Intermediate metabolites response to metal ions addition
The addition condition of Ca 2+ and Al 3+ was already optimized (0.5 mM, 120 h), then the change in the trend of intermediate metabolites in the synthesis pathway of CHA was studied by exploring the mechanism of metal ions addition. Pyruvate was rapidly formed at the early stage of fermentation (24-96 h) and slowly consumed at the late stage of fermentation, in which the concentration of pyruvate after coaddition of Ca 2+ and Al 3+ was lower than the control as shown in Figure 4A. Before 120 h of fermentation, the change and concentration of DAHP were similar to both the experimental group and control group. The production of DAHP with the addition of metal ions was higher than the control from 168 to 312 h ( Figure 4B). The tendency of the chorismate biosynthesis was similar to that of DAHP, in which the biosynthetic capacity of chorismate was also higher than the control after coaddition of Ca 2+ and Al 3+ ( Figure 4C). The concentration of tryptophan with coaddition of Ca 2+ and Al 3+ was fast increased from 0 to 216 h and then gradually decreased from 216 to 360 h, in which the produced tryptophan was far more than thr control during 120-384 h. Clearly, the addition of Ca 2+ and Al 3+ was beneficial to the biosynthesis of intermediate metabolites (DAHP, chorismite, and tryptophan) in the CHA biosynthetic pathway.

Enzyme expression response to metal ions addition
After investigating intermediate metabolites with coaddition of Ca 2+ and Al 3+ , the activities of key enzymes (DAHPs, AroA, and TrpC) in the CHA biosynthetic pathway were studied. The key enzymes played an important role in the CHA production with the addition of metal ions. DAHPs was a key enzyme in the shikimate pathway, which was also an important enzyme in the CHA biosynthetic pathway. The activity of DAHPs with coaddition of Ca 2+ and Al 3+ was increased first and then decreased, which was all higher than the control at every sampling time. The DAHPs activity was significantly improved 3.58-fold than control at 216 h ( Figure. 5A). AroAs induced the synthesis of 3-dehydroquinic acid, which was an important node of the CHA biosynthetic pathway. The change in the trend of AroAs activity was analogous to DAHPs as shown in Figure 5B, and the AroAs activity was improved with different degrees. TrpCs induced indole glycerol phosphate to synthesize tryptophan, which was a rate-limiting enzyme in the CHA biosynthetic pathway. The coaddition of Ca 2+ and Al 3+ did not have very obvious effect compared with the other two enzymes, but the TrpCs activity was increased 3.34-and 1.81-fold than control at 168 and 216 h, respectively ( Figure 5C). The effect of Ca 2+ and Al 3+ coaddition was useful in enhancing the activities of key enzymes in the CHA biosynthetic pathway.

Gene expression response to metal ions addition
In the previous work, the author explored the biosynthetic path of CHA and found that the key genes significantly  16,19 To further explore the underlying mechanism of coaddition of Ca 2+ and Al 3+ in CHA production, transcription levels of four genes laeA (a global regulator), and dahp, cs, and trpC (CHA biosynthetic pathway) were analyzed by qRT-PCR. The expression levels of these genes with coaddition of Ca 2+ and Al 3+ at 120, 168, 216, 264, 312, and 360 h were sampled and investigated ( Figure 6).
As shown in Figure 6A, the transcription level of laeA was obviously upregulated at 168, 216, and 264 h, which were, respectively, 2.89-, 3.22-, and 2.46-fold than the control. The expression level of dahp was increased by about 12.65-fold at 168 h and 5.51-fold at 216 h ( Figure 6B), which were affected stronger than other genes with a supplement of Ca 2+ and Al 3+ . The transcription level of the cs gene was upregulated at 216, 264, and 312 h and expressed with 5.58-, 3.32-, and 2.67-fold increase with the addition of Ca 2+ and Al 3+ ( Figure 6C). The change in the trend of the trpC gene was similar to dahp, but the effect of the trpC gene was less than dahp. The expression of trpC was increased by about 6.99-fold at 168 h and 2.12-fold at 216 h ( Figure 6D). These results indicated that the key genes of the CHA biosyn-thetic pathway were upregulated by coaddition of Ca 2+ and Al 3+ .

Scale-up CHA production to a lab-scale bioreactor
The supplement effect of Ca 2+ and Al 3+ was observed, and the scale-up experiment was carried out in the 5-L stirred bioreactor. As shown in Figure 7A, B, the time profiles of the 5-L bioreactor and shake flask with coaddition of Ca 2+ and Al 3+ were investigated. The trend of DCW was similar to both the 5-L bioreactor and shake flask, and it was increased slowly then declined slightly. However, the value of DCW in the 5-L bioreactor was little lower than that in the shake flask at the end of fermentation. The change in the trend of residual sugar was also analogous in the 5-L bioreactor and shake flask, and the consumption rate of sugar in the 5-L bioreactor was faster than that in the shake flask. The tendency of pH was consistent, which was increased fast then declined and finally stabilized at around 4 in the 5-L bioreactor and shake flask. When Ca 2+ and Al 3+ were coadded in the 5-L bioreactor, the value of pH rose and then declined quickly at 120 h. The DO continued to decrease during 0-120 h as shown in Figure 6B, then it became constant at 20% from 120 to 336 h. The trend of CHA production in the 5-L bioreactor and shake flask was both first increased then slowly decreased. The maximum CHA production in the 5-L bioreactor was 75.6 mg/L at 336 h, which was higher than that (37.6 mg/L, at 360 h) in the shake flask. Therefore, the scale-up experiment was successfully performed in the laboratory, in which the residual sugar, pH, and CHA production in the 5-L bioreactor were all better than the shake flask.

Optimization of Ca 2+ and Al 3+ concentration and addition time
The effective factors improving the production of metabolites were studied by many researchers. The temperature, pH, agitation speed, surfactants, metal ions, and so on were optimized to enhance the yield of the target compound. [20][21][22] The metal ions play an important role in the growth and metabolite synthesis in the microbe. 23,24 The results of the present study indicated that Ca 2+ and Al 3+ increased the production of CHA, and other ions had different degrees of reduction of the production of CHA in Figure 1. Clearly, the effect of Ca 2+ and Al 3+ on CHA production was better than that of Cu 2+ , Mn 2+, and Zn 2+ . Lima et al. 25 reported the effects of different metal ions (2.5 mmol L -1 AgNO 3 , CuSO 4 , CaCl 2 , CoCl 2 , NaCl, FeSO 4 , MgSO 4 , MnSO 4 , and ZnSO 4 ) on β-mannanase activities and found different metal ions had different auxo-action on enzyme activity. Chauhan and Jha 26 found the metal ions (Na + , K + , Pb 2+ , Ca 2+ , Cu 2+ , and Co 2+ ) enhanced enzyme activity of laccase from Pseudomonas sp. S2. Therefore, the promoted effect of metal ions on the biosynthesis of active metabolites was verified by researchers.
The addition concentration and time of Ca 2+ and Al 3+ were optimized, in which the effect of different addition of optimized Ca 2+ and Al 3+ had different influence on the biosynthesis of CHA. The optimized concentration and time of Ca 2+ and Al 3+ were both 0.5 mM and 120 h, in which the production of CHA was, respectively, 62.74 and 50.43 mg/L. A similar phenomenon has been reported in other studies. Wei et al. 27  and Al 3+ were both 0.5 mM and at 120 h of fermentation time. * Indicates statistical significance (p < 0.05) compared to the control without inhibitors dalesconols A and B biosynthesis by Daldinia eschscholzii via calcium/calmodulin signaling. 28 Therefore, the optimization of addition concentration and time of Ca 2+ and Al 3+ was necessary for further clarifying the mechanism of Ca 2+ and Al 3+ and enhancement of CHA production.

Effect of Al 3+ and Ca 2+ on metabolic parameters
The purpose of metabolic parameters was to analyze the mechanism of Ca 2+ and Al 3+ on the improvement of CHA production based on previous work. The accumulation of DAHP, chorismate and tryptophan with coaddition of Ca 2+ and Al 3+ was higher than the control group ( Figure 3B-D). Meanwhile, the production of tryptophan was far higher than the control, which was an important intermediate metabolite in the CHA biosynthesis pathway and resulted in increasing CHA production. However, the amount of pyruvate with the addition of optimized metal ions was lower than the control ( Figure 3A). The carbon source might flow to the EMP pathway instead of the CHA synthesis pathway, which led to lower pyruvate production. Analogously, Zhou et al. 29 investigated the addition effect of calcium carbonate for promoting biosynthesis of l-methionine by metabolically engineered Escherichia coli W3110-BL, in which the concentration of organic acids (a-KG, succinic acid, lactic acid, formic acid, and acetic acid) had varying degrees of change with the addition of calcium carbonate.
In addition, the time course of DCW, residual sugar, pH, and CHA production are compared in Figure S2 in the Supporting Information. The trend of DCW and CHA production was improved after the addition of Ca 2+ and Al 3+ . The residual sugar was consumed more quickly than the control. The pH was briefly increased after coaddition of optimized Ca 2+ and Al 3+ , then showed the same trend of control. Chakraborty et al. 30 explored the effect of aluminum on multiple parameters (chlorophyll and carotenoids, photosynthetic efficiency, starch, and sucrose) for expounding physiological mechanisms of aluminum toxicity tolerance in Azolla microphylla Kaulf. Based on the above discussion, the coaddition of Ca 2+ and Al 3+ could promote the biosynthesis of intermediate metabolites (DAHP, chorismate, and tryptophan) and residual sugar consumption to enhance DCW of A. fumigatus CY018 and CHA production.

4.3
The induced effect of Ca 2+ and Al 3+ on the biosynthesis of CHA To explore the induced effect of Ca 2+ and Al 3+ on the biosynthesis of CHA, the activities of key enzymes and transcriptional levels of key genes in the CHA biosynthesis pathway were investigated. The tendency of key enzymes (DAHPs, AroAs, and TrpCs) were first increased and then reduced after coaddition of Ca 2+ and Al 3+ following fermentation as shown in Figure 5, in which the peak appeared at 216 h. Then, the enzyme activity by coaddition of Ca 2+ and Al 3+ was decreased gradually along the CHA biosynthetic pathway after 216 h of fermentation. The activity of ligninolytic enzyme complex and the antioxidant enzyme in the white-rot fungus Trametes trogii 4 6 was improved by lignocellulose degradation after the addition of copper ions. 31 And, the change in tendency of laccase, SOD, and CAT activities with the addition of Cu 2+ were closely analogous to the present work. The phosphoglucomutase (PGM) activity from Cordyceps militaris was investigated with the addition of metal ions. 32 And, results showed Ca 2+ , Zn 2+ , Mg 2+ , Fe 2+ , and Fe 3+ could improve the activity of PGM and mRNA transcription level of the pgm gene. These results demonstrated that the activities of key enzymes helped to accumulate the target compounds with the addition of optimized metal ions.
The inner function of Ca 2+ and Al 3+ coaddition in the CHA biosynthesis was investigated at the transcription level of key genes (laeA, dahp, cs, and trpC) (Figure 6). The expression of laeA, as a global regulatory gene, was increased by coaddition of Al 3+ and Ca 2+ , which meant the effect of metal ions addition might promote the cell development and production of CHA/intermediate metabolites in the CHA biosynthesis pathway. The impact of the global secondary metabolite regulators LaeA on echinocandin B production was researched, and enhanced with the use of industrial production strain Aspergillus pachycristatus NRRL 11440. 33 Meanwhile, the transcription levels of dahp, cs, and trpC were also upregulated at different times and degrees. Clearly, the effect of time on the transcription level of dahp and trpC was reacted in advance of enzyme activities of DAHPs and TrpCs, which indicated that the effect of Ca 2+ and Al 3+ coaddition was in accordance with the the central law. A similar phenomenon was observed when elicitation (amendment) of aluminum chloride improved callus biomass growth and reserpine yield in Rauvolfia serpentina leaf callus, in which SOD, APX, and CAT activities were promoted by Al 3+ addition. 34 The enhancement of omega-3 fatty acids by Chlorella sorokiniana was obtained via Ca 2+ -induced homeoviscous adaptation, in which the relative expression profiles of accD and rbcL were upregulated by the addition of Ca 2+ . 35 Xu et al. 36 found that the induction of calcium ions could improve the production of ganoderic acid and intermediate metabolites (squalene and lanosterol), which resulted in upregulation of the transcription levels of key genes (hmgr, fps, sqs, and cyp) with the addition of Ca 2+ . Therefore, the inducing effect of Ca 2+ and Al 3+ lead to upregulate the transcription levels of key genes (laeA, dahp, cs, and trpC) and improving the activities of key enzymes (DAHPs, AroAs, and TrpCs) for enhancement of CHA production.
In conclusion, CHA could be taken as a drug candidate worthy of study owing to its attractive anticancer activity. Herein, the selected Ca 2+ and Al 3+ were optimized with the addition concentration and time as 50 mM and 120 h, in which CHA production reached 62.74 and 50.43 mg/L with the suitable condition of Ca 2+ and Al 3+ , respectively. The effect of Ca 2+ and Al 3+ coaddition improved the concentration of intermediate metabolites (DAHP, chorismate, and tryptophan) and activities of key enzymes (DAHPs, AroAs, and TrpCs). Simultaneously, the transcription levels of laeA, dahp, cs, and trpC were upregulated by 3.22-, 12.65-, 5.58-, and 6.99-fold, respectively. Furthermore, the fermentation process was successfully scaled up to the 5-L bioreactor, in which CHA production could achieve 75.6 mg/L at 336 h and higher than that in the shake flask. This work will be helpful in further research and development of CHA and active secondary metabolites produced by other filamentous fungi.

A C K N O W L E D G M E N T S
This work was supported by Funding for school-level research projects of Yancheng Institute of Technology (XJR2019066) and North Jiangsu Science and Technology Special Project-Enriching Civilization and Enhancing the County (SZ-YC202040).

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.