3-Methylxanthine production through biodegradation of theobromine by Aspergillus sydowii PT-2

Background Methylxanthines, including caffeine, theobromine and theophylline, are natural and synthetic compounds in tea, which could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several microbial isolates from Pu-erh tea could degrade and convert caffeine and theophylline. We speculated that these candidate isolates also could degrade and convert theobromine through N-demethylation and oxidation. In this study, seven tea-derived fungal strains were inoculated into various theobromine agar medias and theobromine liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture to investigate their potential application in the production of 3-methylxanthine. Results Based on theobromine utilization capacity, Aspergillus niger PT-1, Aspergillus sydowii PT-2, Aspergillus ustus PT-6 and Aspergillus tamarii PT-7 have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii PT-2 and A. tamarii PT-7 could degrade theobromine significantly (p < 0.05) in all given liquid mediums. 3,7-Dimethyluric acid, 3-methylxanthine, 7-methylxanthine, 3-methyluric acid, xanthine, and uric acid were detected in A. sydowii PT-2 and A. tamarii PT-7 culture, respectively, which confirmed the existence of N-demethylation and oxidation in theobromine catabolism. 3-Methylxanthine was common and main demethylated metabolite of theobromine in the liquid culture. 3-Methylxanthine in A. sydowii PT-2 culture showed a linear relation with initial theobromine concentrations that 177.12 ± 14.06 mg/L 3-methylxanthine was accumulated in TLM-S with 300 mg/L theobromine. Additionally, pH at 5 and metal ion of Fe2+ promoted 3-methylxanthine production significantly (p < 0.05). Conclusions This study is the first to confirm that A. sydowii PT-2 and A. tamarii PT-7 degrade theobromine through N-demethylation and oxidation, respectively. A. sydowii PT-2 showed the potential application in 3-methylxanthine production with theobromine as feedstock through the N-demethylation at N-7 position.


Background
Methylxanthines are natural and synthetic compounds found in many foods, drinks, pharmaceuticals, and cosmetics [1]. Caffeine (1,3,7-trimethylxanthine), theobromine (3,7-dimethylxanthine) and theophylline (1,3dimethylxanthine) are most popular and well-known methylxanthines in tea [2]. Both theobromine and theophylline have a close connection with caffeine metabolism in the physiology of tea plant (Camellia sinensis), and the former is precursor of caffeine biosynthesis and the latter is a transient metabolite of caffeine biodegradation [3,4]. Caffeine level remains stable in the processing of general teas (green tea, black tea, oolong tea and white tea) [5,6]. However, the participation of various microorganisms induced the change of caffeine content in the processing of Pu-erh tea and other dark teas [7,8].
Methylxanthines are extensively metabolized in the liver by the cytochrome P450 (CYP450) oxidase enzyme system, mainly through related N-demethylation and oxidation [21]. Although caffeine and other methylxanthines are toxic to most bacteria and invertebrates [22], several bacteria and fungi, including Pseudomonas sp. [23,24], Pseudomonas putida [25,26], Serratia marcescens, Fusarium solani [27,28], Stemphyllium sp., A. tamarii and Penicillium commune [29], have evolved the ability to metabolize caffeine. Two possible pathways of caffeine catabolism, such as N-demethylation and oxidation, are found in microorganisms, which are similar to that in animals and humans [30]. More than one Ndemethylases and oxidases, such as caffeine oxidase, xanthine oxidase and theobromine oxidase, participate into the N-demethylation and oxidation [31][32][33]. Genes (ndmA, ndmB, ndmC, ndmD and ndmE) isolated from P. putida are responsible for the entire demethylation pathway [34,35]. Additionally, genes cdhABC and tmuDHM identified in Pseudomonas sp. strain CBB1 are associated with the oxidation of caffeine and trimethyluric acid, respectively [36,37]. Therefore, we speculated that those seven microbial isolates from Pu-erh tea also could degrade and convert theobromine through Ndemethylation and oxidation.
As the second most common methylxanthine in tea, theobromine dilates blood vessels, especially coronary arteries, lowers blood pressure and increases heart rate [38]. Until now, a bacterial strain P. putida isolated from tea garden soil was demonstrated to degrade theobromine [26]. Additionally, A. niger, Talaromyces marneffei and Talaromyces verruculosus isolated from cocoa pod husks were demonstrated to degrade theobromine [39]. In this work, seven tea-derived fungal strains isolated from Pu-erh tea were used to investigate their capacity and characterization in theobromine degradation. It is confirmed that Aspergillus sydowii PT-2 and Aspergillus tamarii PT-7 could degrade theobromine in the liquid culture. Analysis of theobromine degradation metabolites and pathways revealed that 3-methylxanthine was main degradation product of theobromine in A. sydowii PT-2 culture through the N-demethylation at N-7 position. The results showed the application of A. sydowii PT-2 in the production of 3-methylxanthine with theobromine as feedstock.

Evaluation results of tea-derived fungi in theobromine utilization
To assess theobromine utilization capacity of tea-derived fungi, each microbial isolate was inoculated into different theobromine agar medias (TAM) and theobromine liquid mediums (TLM), respectively. Colony diameters and theobromine concentrations were determined after cultivation at 30°C for 5 days. Colony diameters and sporulation time on TAM are recorded in Table 1, and theobromine concentrations in TLM are presented in Fig. 1. As shown in Table 1, apart from Aspergillus pallidofulvus PT-3 and Penicillium mangini PT-5, other microbial isolates had relatively high theobromine utilization capacity, such as Aspergillus niger PT-1, A. sydowii PT-2, Aspergillus sesamicola PT-4, Aspergillus ustus PT-6 and A. tamarii PT-7. Comparison of colony diameters on different TAM showed that dextrose or sucrose as carbon source could promote theobromine utilization partly. TAM-S with the maximal colony diameter was most suitable for theobromine utilization by candidate fungal strains.
In this study, TLM-S, TLM-D, TLM-N, TLM-SN were prepared to select potential theobromine-degrading fungi and optimal medium in the liquid culture. As shown in Fig. 1, due to the difference in cultivation modes, A. pallidofulvus PT-3, A. sesamicola PT-4 and P. mangini PT-5 could not utilize theobromine completely in all given TLM. A. niger PT-1 just used the theobromine in TLM-S slightly. Only A. sydowii PT-2, A. ustus PT-6 and A. tamarii PT-7 could utilize theobromine largely in the liquid culture. The additional carbon source promoted theobromine utilization capacity of A. sydowii PT-2 and A. tamarii PT-7 through enhancement of cell density in the liquid culture [19]. Particularly, the highest theobromine removal rate was found in TLM-S for the potential theobromine-degrading fungi, including A. niger PT-1, A. sydowii PT-2, A. ustus PT-6 and A. tamarii PT-7. The composition of TLM-S was therefore chosen as the optimal medium to investigate theobromine degradation metabolites in the liquid culture.

Theobromine degradation characterization in liquid culture
A.niger PT-1, A. sydowii PT-2, A. ustus PT-6 and A. tamarii PT-7 were inoculated into TLM-S with an increasing theobromine concentration (100, 200 and 300 mg/L, respectively), and Tissue-culture bottles were incubated in an orbital shaker (130 rpm, 30°C), respectively. The inoculated bottles were took every 24 h for the determination of theobromine and related metabolites by using high-performance liquid chromatography (HPLC). Theobromine concentrations (Additional file 1: Table S1) are presented in Fig. 2. Significant difference (p < 0.05) was found in theobromine concentrations TAM-D theobromine agar media with dextrose as carbon source, TAM-N theobromine agar media with ammonium sulphate as nitrogen source, TAM-S theobromine agar media with sucrose as carbon source, TAM-T theobromine agar media with theobromine as sole carbon and nitrogen source Theobromine catabolic intermediates were identified by HPLC using internal standard method (Table 2). 3,7-Dimethyluric acid, 3-methylxanthine, 7-methylxanthine, 3-methyluric acid, xanthine and uric acid were detected consecutively in the liquid culture. The detected metabolites showed that both N-demethylation and oxidation were found in theobromine catabolism. Quantitative analysis indicated that 3-methylxanthine was common and main demethylated metabolite through Ndemethylation at the N-7 position of theobromine in A.
The non-linear relationship between theorbromine degradation and 3-methylxanthine accumulation in A. tamarii PT-7 culture indicated that as the main intermediate of theobromine degradation, 3-methylxanthine might be degraded by A. tamarii PT-7 and other candidate isolates in the liquid culture. To investigate 3mehylxanthine metabolism, four candidate isolates were inoculated into a linearly increasing concentration of 3methylxanthine from 100 mg/L to 300 mg/L, 3methylxanthine and related metabolites were determined by HPLC (Fig. 4). Compared with other isolates, A. sydowii PT-2 and A. tamarii PT-7 could reduce 3methylxanthine significantly (p < 0.05) in all given concentrations. Particularly, A. tamarii PT-7 degrade almost all 3-methylxanthine in a low substrate concentration (100 mg/L 3-methylxanthine), and maintained a relatively high removal rate about 34.97% in 300 mg/L Fig. 3 Comparison of A. sydowii PT-2 (a) and A. tamarii PT-7 (b) on 3-menthylxanthine accumulation in the liquid culture. All data were present by mean value ± SD of three biological replications. The significance level was assessed by using the independent t-test of SPSS 20.0 compared with that in 100 mg/L of theobromine . * p < 0.05; ** p < 0.01; *** p < 0.001 Fig. 4 Effect of candidate isolates on 3-methylxanthine metabolism. Biocidal treatment without inoculation was defined as the control. All data were present by mean value ± SD of three biological replications. The lowercase letters indicated a significant difference at p < 0.05 level by using one-way ANOVA of SPSS 20.0 between different candidate isolates at same substrate concentration. The different letters show significant differences substrate concentration after cultivation for 5 days. Through the analysis of related metabolites with 3methylxanthine degradation (Additional file 1: Table S3), 3-methyluric acid, xanthine and uric acid were detected in the liquid culture, respectively. Associated with the metabolites detected in theobromine degradation, xanthine was demethylated product from 3methylxanthine through N-3 demethylation. Alternatively, 3-methyluric acid and uric acid were direct oxidative products from 3-methylxanthine and xanthine at the C-8 position, respectively.

Effects of pH and metal ions on 3-methylxanthine production
The lower degradation capacity of 3-methylxanthine in liquid culture (Fig. 4) confirmed that A. sydowii PT-2 had application potential in production of 3methylxanthine with theobromine as feedstock. Metal ions and pH were principal factors influencing theobromine biodegradation and 3-methylxanthine production. Two series of experiments, such as a pH range from 3 to 7 and various metal ions, including Fe 2+ , Ca 2+ , Mg 2+ , Mn 2+ , Cu 2+ and Zn 2+ , were prepared in TLM-S to investigate the influences of pH and metal ions, respectively. A. sydowii PT-2 exhibited a high sensitivity to pH, showing the best theobromine degradation and 3methylxanthine production at pH 5 (Fig. 5a). Cu 2+ and Zn 2+ restrained theobromine degradation and 3methylxanthine production significantly (p < 0.05), only Fe 2+ promoted 3-methylxanthine production significantly (p < 0.05) compared with the control (Fig. 5b).

Discussion
Besides the traditional Traube synthesis, bioconversion offers an alternative way to produce 3-methylxanthine by using appropriate starter strain and precursor substances. Algharrawi et al. reconstructed an engineered Escherichia coli with genes ndmA and ndmD from P. putida, capable of producing 3-methylxanthine from exogenously fed theophylline [41]. Mckeague et al. engineered the eukaryotic microbial host Saccharomyces cerevisiae for the de novo biosynthesis of methylxanthines [42]. Additionally, 3-methylxanthine was main intermediate metabolite of theobromine through the N-7 demethylation by relevant fungi [31].
It was established that the isolates generally preferred TLM-S in which extra sucrose enhanced theobromine degradation efficiency. Theobromine degradation efficiency of four candidate isolates was entirely different Fig. 5 Effects of pH (a) and metal ions (b) on theobromine degradation and 3-methylxanthine production in A. sydowii PT-2 culture, respectively. The pH in the inoculated mediums was adjusted by phosphate buffer. Metal ions were added into the culture solution at a concentration of 2 mM. The culture solution without extra addition was defined as the control. The reaction (pH 6.0) was carried out at 30°C for 5 days on an incubator shaker (130 rpm). Data are presented as mean value ± SD of three biological replications. The lowercase letters indicated a significant difference at p < 0.05 level by using Tukey's multiple comparison test for one-way ANOVA. The different letters show significant differences (Fig. 2). Theobromine degradation capacity of A. niger PT-1 and A. ustus PT-6 were limited in the liquid culture, which might be related to cultivation method and medium components. For relatively high theobromine degradation efficiency, A. tamarii PT-7 and A. sydowii PT-2 were selected to investigate theobromine degradation pathway and application potential in the production of 3-methylxanthine.
We confirmed that A. sydowii mainly produced theophylline through N-demethylation at the N-7 position of caffeine, other N-demethylated metabolites, such as 1,7-dimethylxanthine, 7-methylxanthine and 3-methylxanthine, were detected during tea fermentation, which showed that A. sydowii could release related N-demethylase [43]. In this study, apart from caffeine, A. sydowii PT-2 also could remove the N-7 methyl of theobromine to formulate 3methylxanthine. Although A. ustus largely converted theophylline into 3-methylxanthine through the N-1 demethylation [19], absence of N-demethylase removing the N-7 methyl limited theobromine degradation efficiency in the liquid culture. A. tamarii PT-7 exhibited broad-spectrum capacity in methylxanthines degradation, including theobromine, theophylline and 3-methylxanthine by releasing various N-demethylases and oxidases, respectively. The high degradation of 3-methylxanthine reduced the accumulation of 3-methylxanthine in A. tamarii PT-7 culture. Therefore, A. sydowii PT-2 was best in production of 3methylxanthine with theobromine as feedstock.
Substrate concentration, pH and metal ions had profound impacts on theobromine degradation and 3methylxanthine production. A. sydowii PT-2 produced the maximum accumulation of 3-methylxanthine in the liquid culture of 300 mg/L theobromine. Comparisons showed that the optimal pH was 5 and Fe 2+ promoted the conversion of theobromine into 3-methylxanthine significantly (p < 0.05), which provided optimum condition for the growth of A. sydowii PT-2 and enzymatic reaction of relevant N-demethylase.

Conclusions
This paper describes related metabolites with theobromine degradation and explores potential application of tea-derived fungi in the production of 3-methylxanthine. Among seven microbial isolates from Pu-erh tea, both A. sydowii PT-2 and A. tamarii PT-7 showed higher theobromine degradation capacity in TAM and TLM. 3,7-Dimethyluric acid, 3methylxanthine, 7-methylxanthine, 3-methyluric acid, xanthine and uric acid were detected by using HPLC in A. sydowii PT-2 and A. tamarii PT-7 culture, respectively, which confirmed the existence of N-demethylation and oxidation in theobromine catabolism. Compared with that in A. tamarii PT-7 culture, 3-methylxanthine was accumulated largely in A. sydowii PT-2 culture along with theobromine degradation and showed a linear relation with initial theobromine concentration. A. sydowii PT-2 was an appropriate starter strain most suitable for the production of 3-methylxanthine, which could produce 177.12 ± 14.06 mg/L 3-methylxanthine in TLM-S with 300 mg/L theobromine. Additionally, pH at 5 and metal ion of Fe 2+ promoted the production of 3-methylxanthine significantly (p < 0.05). This paper presents an alternative way for 3-methylxanthine production through the microbial conversion of A. sydowii PT-2 with theobromine as feedstock.

Strains and reagents
Tea-derived fungal strains (Table 3) used in this study were isolated from Pu-erh tea and identified based on colony characteristics, conidial structure and PCR amplified sequences, and stored at − 20°C in our microbiology laboratory before further processing. Theobromine, 3,7dimethyluric acid, 3-methylxanthine, 7-methylxanthine, 3-methyluric acid, 7-methyluric acid, xanthine and uric acid were purchased from Sigma-Aldrich Co., Ltd. HPLC-grade acetonitrile and ammonium formate were purchased from Thermo Fisher Scientific Co., Ltd. Other reagents, including agar, dextrose, sucrose and ammonium sulphate, were analytical grade.

Evaluation of growth of tea-derived fungi on theobromine agar medias
For each strain, spore suspension was adjusted to 1.0 × 10 7 CFU/mL for inoculation after cultivation on PDA media at 30°C for 72 h, respectively [19]. Four kinds of TAM contained 20 g/L agar and 600 mg/L theobromine were carried out to evaluate theobromine utilization, which included theobromine agar media with 2.0 g/L dextrose as carbon source (TAM-D), theobromine agar media with 1.01 g/L ammonium sulphate as nitrogen source (TAM-N), theobromine agar media with 2.0 g/L sucrose as carbon source (TAM-S) and theobromine agar media only with theobromine as sole carbon and nitrogen source (TAM-T), respectively. Plates of each TAM were inoculated with 10 uL spore suspension and incubated at 30°C. At 24-h intervals for 5 days, colony diameters were measured [44]. The isolated strains were categorized based on their total colony diameters as follows: low theobromine utilization = diameter ≤ 7.9 cm; moderate theobromine utilization = diameter 8.0-15.9 cm; high theobromine utilization = diameter ≥ 16.0 cm. Tea-derived fungi selected for further study were those that showed at least moderate theobromine utilization (diameter ≥ 8.0 cm) on agar medias.

Assessment of theobromine-degrading fungi in theobromine liquid mediums
Theobromine liquid medium (TLM) was prepared by using 4.0 g/L NaNO 3 , 1.3 g/L KH 2 PO 4 , 0.19 g/L Na 2 H-PO 4 ·7H 2 O, 0.26 g/L CaCl 2 ·2H 2 O, 0.15 g/L MgSO 4 , 2.0 g/ L sucrose and 300 mg/L theobromine in distilled water [45]. To investigate the influence of carbon and nitrogen source on theobromine degradation, the modifications used either 5 g/L sucrose or 10 g/L dextrose as carbon source in theobromine liquid medium with sucrose as carbon source (TLM-S) or theobromine liquid medium with dextrose as carbon source (TLM-D), and 1.01 g/L ammonium sulphate as nitrogen source in theobromine liquid medium with ammonium sulphate as nitrogen source (TLM-N), and 5 g/L sucrose and 1.01 g/L ammonium sulphate in theobromine liquid medium with sucrose and ammonium sulphate as carbon and nitrogen sources (TLM-SN), respectively. The spore suspension was adjusted to 1.0 × 10 7 CFU/mL for inoculation after eluting by using sterile saline solution with identical theobromine concentration. Both spore suspension and TLM were adjusted for pH 6.0 by phosphate buffer. For each isolate, control and experimental mediums (25 mL each) were inoculated with spore suspension with 4% inoculum size (v/v) that 1 mL spore suspension was inoculated into each medium, and biocidal treatment was defined as the control. Theobromine concentration was determined after cultivation at 30°C for 5 days on an incubator shaker (130 rpm), respectively.

Analysis of theobromine degradation metabolites in liquid culture
Through comparisons (Fig. 1), TLM-S therefore was chosen as the optimal medium to analyze theobromine degradation in the liquid culture. A series of TLM-S with different initial theobromine concentrations (100, 200 and 300 mg/L, respectively) were set up and a 6-day period cultivation of each selected isolate were carried out on an incubator shaker (130 rpm, 30°C). At intervals of up to 24 h for 6 days, an aliquot of each culture was filtered through a 0.45 um syringe filter. Theobromine  [19,31]. Standard calibration curves were prepared from solutions of theobromine, 3-methylxanthine, 7methylxanthine, xanthine, 3,7-dimethyluric acid, 3methyluric acid, 7-methyluric acid and uric acid. Internal standard method was used to aid in the identification of metabolites related to theobromine catabolism [19]. 3-Methylxanthine was quantificationally analyzed as the main intermediate metabolite in the liquid culture of A. sydowii PT-2 and A. tamarii PT-7, respectively.

Influence of potential isolates on 3-methylxanthine metabolism
3-Methylxanthine liquid mediums were prepared as above described with 5 g/L sucrose as carbon source and a linearly increasing concentration of 3-methylxanthine from 100 mg/L to 300 mg/L to explore the effect of four candidate isolates, including A. niger PT-1, A. sydowii PT-2, A. ustus PT-6 and A. tamarii PT-7, respectively. Each candidate isolate was inoculated with 4% inoculum size (v/v) and 3-methylxanthine concentration was determined by HPLC after cultivation at 30°C for 5 days on an incubator shaker (130 rpm), respectively.

Effects of pH and metal ions on theobromine degradation and 3-methylxanthine production
Effect of pH on theobromine degradation and 3methylxanthine production was investigated in TLM-S with a pH range from 3 to 7 adjusted by phosphate buffer [18,46]. In order to study the effect of metal ions, Fe 2+ , Ca 2+ , Mg 2+ , Mn 2+ , Cu 2+ and Zn 2+ were added into the culture solution in the form of salts (viz.  [32]. Theobromine and 3methylxanthine concentrations were determined after cultivation at 30°C for 5 days on an incubator shaker (130 rpm), respectively.

Statistical analysis
Three biological replications were carried out to ensure validity and repeatability. All data are presented as mean value ± standard deviation (SD). The independent t-test and Tukey's multiple comparison tests for one-way analysis of variance (ANOVA) were carried out by using SPSS 20.0 for Windows to determine significant difference level.
Additional file 1: Table S1. Comparison of theobromine concentrations detected by HPLC in liquid culture of different candidate isolates. Table S2. Production of 3-methylxanthine in TLM-S with different substrate concentrations inoculated by A. sydowii PT-2 and A. tamarii PT-7, respectively. Table S3. Related metabolites with 3-methylxanthine degradation detected in the liquid culture of different candidate isolates.