Theophylline degradation exists in solid-state fermentation of pu-erh tea
Fungi count, caffeine and theophylline contents were determined in the natural solid-state fermentation of pu-erh tea, and results are presented in Figure 1. Fungi count (Fig. 1A) dramatically increased from day 0 to 10 and then increased slowly before day 20. After day 20, fungi count maintained a high level overt 1.0 × 105 CFU/g. Because of the metabolic activity of fungi, caffeine content (Fig. 1B) was decreased highly significantly (p < 0.01) from 36.85 ± 1.02 mg/g to 25.46 ± 1.85 mg/g during fermentation. Theophylline content (Fig. 1C) was increased highly significantly (p < 0.01) before day 20, which confirmed that caffeine-degrading fungi leaded to caffeine degradation and theophylline production. In our previous study [40], it was found that Aspergillus sydowii convert caffeine to theophylline. However, after day 20, theophylline content had a highly significant (p < 0.01) decrease from 11.18 ± 1.10 mg/g to 5.89 ± 0.65 mg/g, which shown that theophylline degradation appeared in solid-state fermentation except for caffeine degradation. Therefore, in consideration of fungal community, there existed theophylline-degrading fungi in the solid-state fermentation, which could be Aspergillus sydowii or other fungi.
Isolation and identification of theophylline-degrading fungi
Based on colony morphology, eleven filamentous fungi were initially selected and isolated from pu-erh tea. Among them, seven fungi were superior in number and coded orderly with numbers PT-1 to PT-7. Distinctive morphological features of the seven isolates were observed after cultivation at 30 °C for 5 days and documented in Table 1.
Table 1 Colony characteristics of theophylline-degrading fungi
Isolate
|
Shape
|
Surface
|
Colour
|
Exudate
|
Reference
|
PT-1
|
Circular
|
Rough
|
Black
|
None
|
[39]
|
PT-2
|
Circular
|
Rough
|
Olive green
|
Red-coloured
|
[39]
|
PT-3
|
Circular
|
Rough
|
Dark yellow colonies with white edges
|
Yellow sclerotium
|
[40]
|
PT-4
|
Irregular
|
Rough
|
Light yellow
|
Yellow sclerotium
|
[40]
|
PT-5
|
Circular
|
Rough
|
Greyish-green centre with yellow patches
|
Red pigment
|
[40]
|
PT-6
|
Circular
|
Rough
|
Iron gray bulge with milk white edges
|
None
|
Figure S1
|
PT-7
|
Irregular
|
Rough
|
Hazel green with gray back
|
None
|
Figure S2
|
The amplified sequences of ITS region were produced with sizes ranging between 502 and 546 base pairs. The amplified sequences of β-Tubulin region were produced with sizes ranging between 420 and 694 base pairs. The amplified sequences of Calmodulin region were produced with sizes ranging between 715 and 765 base pairs. Based on the DNA sequences (Table 2), seven dominating isolates were belonged to 6 Aspergillus spp. and 1 Penicillium sp., respectively. Through Neighbor-Joining analysis for both new fungi (Additional file: Figure S5), in the phylogram for Aspergillus species (Figure S5a and S5b), strain PE-6 was clustered with Aspergillus ustus and showed a 100% of identity to the tested Aspergillus ustus NRRL275; additionally, PE-7 strain was closely related to Aspergillus tamarii NRRL20818 with 99.9% of identity.
Table 2 Identification of theophylline-degrading fungi by sequence determination
Isolate
|
Primers
|
Fragments (bp)
|
Species
|
Strain number
|
identity
|
Reference
|
PT-1
|
ITS1/ITS4
|
546
|
Aspergillus niger
|
NCBT110A
|
99.8%
|
[39]
|
PT-2
|
ITS1/ITS4
|
516
|
Aspergillus sydowii
|
NRRL250
|
99.8%
|
[39]
|
PT-3
|
TS1/ITS4
|
541
|
Aspergillus pallidofulvus
|
NRRL4789
|
99.9%
|
[40]
|
Bt2a/Bt2b
|
516
|
CF1L/CF4
|
765
|
PT-4
|
TS1/ITS4
|
532
|
Aspergillus sesamicola
|
CBS137324
|
99.8%
|
[40]
|
Bt2a/Bt2b
|
515
|
CF1L/CF4
|
757
|
PT-5
|
TS1/ITS4
|
525
|
Penicillium manginii
|
CBS253.31
|
99.6%
|
[40]
|
Bt2a/Bt2b
|
420
|
PT-6
|
TS1/ITS4
|
502
|
Aspergillus ustus
|
NRRL275
|
100%
|
Figure S3
|
Bt2a/Bt2b
|
694
|
PT-7
|
TS1/ITS4
|
532
|
Aspergillus tamarii
|
NRRL20818
|
99.9%
|
Figure S4
|
Bt2a/Bt2b
|
476
|
CF1L/CF4
|
715
|
Evaluation results of theophylline-degrading fungi in solid mediums
The screening was carried out in agar solid mediums for the evaluation of the biocatalytic potential for the degradation of theophylline. All isolate tea-derived strains were inoculated into an agar solid medium with the presence of dextrose and they were also inoculated into a set of agar solid mediums with increasing theophylline concentrations. The colony diameters of potential theophylline-degrading fungi were measured and showed in Table 3.
Table 3 Growth of tea-derived fungi in agar solid medium (2 % w/v) with dextrose (2 % w/v) (control culture) or presence of theophylline instead of dextrose (30 °C, 5 d, pH 7.0).
Isolate fungi
|
Colony diameter (cm)
|
Control culture
|
600mg/L theophylline
|
1200 mg/L theophylline
|
1800 mg/L theophylline
|
A. niger
|
3.5 ± 0.5
|
0.5 ± 0.2
|
No growth
|
1.0 ± 0.5
|
A. sydowii
|
2.5 ± 1.0
|
0.5 ± 0.1
|
1.0 ± 0.3
|
0.5 ± 0.3
|
A. pallidofulvus
|
3.0 ± 0.5
|
No growth
|
No growth
|
0.5 ± 0.1
|
A. sesamicola
|
3.0 ± 0.5
|
No growth
|
No growth
|
0.5 ± 0.1
|
P. mangini
|
3.0 ± 1.0
|
No growth
|
No growth
|
No growth
|
A. ustus
|
2.5 ± 0.5
|
1.0 ± 0.3
|
1.5 ± 0.4
|
1.5 ± 0.4
|
A. tamarii
|
3.0 ± 0.5
|
2.0 ± 0.5
|
2.5 ± 0.5
|
3.5 ± 1.0
|
Six tea-derived strains could survive in the agar solid mediums (2% w/v) with theophylline alone. Aspergillus spp. showed a better growth in higher evaluated concentrations. Particularly, A. niger, A. sydowii, A. ustus and A. tamarii had growth in low theophylline concentration, which showed that these strains had a high utilization ratio of theophylline. A higher growth in lower concentration indicated a higher utilization of fungi direct or indirect [46]. Therefore, A. niger, A. sydowii, A. ustus and A. tamarii were considered as the potential theophylline-degrading fungi.
Selection of theophylline-degrading fungi and optimal medium in liquid culture
For theophylline biodegradation in liquid culture, with biocidal treatment as the control, seven isolates were inoculated into the basic mediums with the presence of theophylline and sucrose or dextrose as carbon source, or ammonium sulphate as nitrogen source, respectively. Theophylline concentration was determined after cultivation at 30 °C for 5 days by HPLC and results are showed in Fig. 2. Through comparisons of each isolate, A. ustus and A. tamarii could decrease theophylline concentration high significantly (p < 0.01) by about 65.15% and 95.98% in TLM-S medium, respectively. In addition, under the effect of A. niger and A. sydowii, theophylline concentrations were decreased slightly by about 1.03% and 5.19% in TLM-S medium, respectively, which showed that the theophylline utilizations of A. niger and A. sydowii were restricted. A. pallidofulvus, A. sesamicola and P. mangini had no significant (p > 0.05) impact on theophylline degradation. Hence, A. ustus, A. tamarii, A. niger and A. sydowii were selected as the potential theophylline-degrading fungi for theophylline degradation in liquid culture.
Theophylline could be used as the sole carbon and nitrogen source. The presence of other carbon or nitrogen sources had a significant impact on theophylline degradation and pathway. The optimum liquid medium was chose by comparing theophylline degradation capacity in different mediums. In contrast with other mediums (TLM-D, TLM-N and TLM-SN), theophylline degradation level had a highly significant (p < 0.01) improvement in TLM-S medium, which showed that carbon source particularly sucrose promoted theophyline degradation and nitrogen source restrained theophylline degradation to a certain extent. Therefore, TLM-S medium was chose to analyze the characterization of theophylline degradation in liquid culture.
Characterization of theophylline degradation by theophylline-degrading fungi
A. ustus, A. tamarii, A. niger and A. sydowii were inoculated into TLM-S mediums with increasing theophylline concentrations (100, 200 and 300 mg/L, respectively), and the Tissue-culture bottles were incubated in an orbital shaker (130 rpm, 30 °C). The inoculation bottles were took every 24 h for the determination of theophylline and related metabolites by HPLC, and results are presented in Fig. 3. Under the effect of A. ustus and A. tamarii, theophylline content was decreased highly significantly (p < 0.01) in all substrate concentrations. However, the concentration of theophylline inoculated by A. niger and A. sydowii was slightly decreased (p > 0.05) in all substrate concentrations. Therefore, A. ustus and A. tamarii had advantage in theophylline degradation than A. niger and A. sydowii. Both A. ustus and A. tamarii could degrade theophylline completely in low concentration (100 mg/L theophylline). However, A. ustus only degrade 79.00% theophylline in high concentration (300 mg/L theophylline), while A. tamarii could degrade theophylline almost completely in all concentrations, which showed that A. tamarii had a higher theophylline degradation capacity.
A series of experiments was conducted to find out theophylline degradation pathway through the identification of catabolic intermediates by HPLC using internal standard method (Table 4). Related degradation metabolites were not found in the liquid culture inoculated by A. niger and A. sydowii. 1,3-Dimethyluric acid, 3-methylxanthine, 3-methyluric acid, xanthine and uric acid were detected consecutively in the liquid culture. 3-Methylxanthine was common and main metabolite through N-demethylation at the position N-1 of theophylline in A. ustus and A. tamarii. Xanthine was the further demethylated metabolite in theophylline degradation found in A. ustus and A. tamarii through N-demethylation at the position N-3 of 3-methylxanthine. In contrast to A. ustus isolate that additional metabolites including 1,3-dimethyluric acid and 3-methyluric acid were identified in the culture through the oxidation of theophylline and 3-methylxanthine, respectively [19]. Only uric acid was identified in A. tamarii culture as the oxidation product of xanthine, which showed the differences in degradation metabolites and pathways between A. ustus and A. tamarii.
Table 4 Theophylline degradation metabolites detected in the liquid media inoculated by Aspergillus fungi
Metabolite
|
Fungal isolates
|
A. ustus
|
A. tamarii
|
A. niger
|
A. sydowii
|
1,3-dimethyluric acid
|
+
|
-
|
-
|
-
|
1-methylxanthine
|
-
|
-
|
-
|
-
|
3-methylxanthine
|
+
|
+
|
-
|
-
|
1-methyluric acid
|
-
|
-
|
-
|
-
|
3-methyluric acid
|
+
|
-
|
-
|
-
|
xanthine
|
+
|
+
|
-
|
-
|
uric acid
|
-
|
+
|
-
|
-
|
TLM-S media were analyzed by HPLC for 1,3-dimethyluric acid, 1-methylxanthine, 3-methylxanthine, 1-methyluric acid, 3-methyluric acid, xanthine and uric acid.
Production of 3-methylxanthine or xanthine through theophylline degradation
Several xanthine derivatives including 3-methylxanthine have been synthesized chemically for use in medical industry [47]. Except for engineering a microbial platform for de novo biosynthesis of diverse methylxanthins [48], bioconversion from cheaper feedstocks such as caffeine, theophylline and theobromine was an effective pathway to produce high value methylxanthines via metabolically engineered microorganisms [21]. In this study, A. ustus and A. tamarii could degrade theophylline, and 3-methylxanthine and xanthine were detected as degradation metabolites. The microbial utilization of 3-methylxanthine and xanthine were investigated as substrate through liquid culture of isolates. The concentrations of 3-methylxanthine and xanthine were determined by HPLC after cultivation for 5 days. As shown in Fig. 4, A. ustus and A. tamarii had a significant (p < 0.05) or a highly significant (p < 0.01) impact on 3-methylxanthine degradation with a removal ratio of about 27.05% and 84.29%, respectively. Associated with the metabolites in theophylline degradation, 3-methyluric acid and xanthine were 3-methylxanthine degradation metabolites through oxidation and N-demethylation, respectively.
Although A. ustus and A. tamarii could utilize 3-methylxanthine and xanthine significantly, 3-mthylxanthine and xanthine concentrations were accumulated largely in theophylline degradation. To investigate the application in the production of 3-methylxanthine and xanthine using theophylline-degrading fungi with theophylline as feedstock, the quantitative determination of 3-methylxanthine and xanthine were carried out in all theophylline concentrations inoculated by A. ustus and A. tamarii, respectively. The concentrations of 3-methylxanthine in A. ustus and A. tamarii cultures are presented in Fig. 5. We monitored the accumulation of 3-methylxanthine over the course of inoculated culture by A. ustus and A. tamarii. 3-Methylxanthine was detected in the culture medium after 24 h for the first time, and increased significantly with cultivation. Over a 7-day period cultivation of A. ustus (Fig. 5a), 49.68 ± 2.97 mg/L, 83.82 ± 3.35 mg/L and 129.48 ± 5.81 mg/L of 3-methylxanthine were accumulated and increased significantly with increasing initial theophylline concentrations, respectively. Due to high degradation capacity of 3-methylxanthine in A. tamarii culture, 3-methylxanthine concentration (Fig. 5b) stayed at a low level that only 56.72 ± 5.81 mg/L of 3-methylxanthine was accumulated in 300 mg/L of theophylline after a 7-day period cultivation. Hence, strain A. ustus exhibited a continuing accumulation of 3-methylxanthine over the course of liquid culture, and increasing initial theophylline concentrations could improve the production of 3-methylxanthine.
It is clear from the data presented in Fig. 6 that a reaction containing theophylline in A. tamarii culture provided linear conversion of theophylline to xanthine. Over a 7-day period cultivation of A. tamarii, 35.88 ± 6.65 mg/L, 103.95 ± 4.82 mg/L and 159.11 ± 10.8 mg/L of xanthine were accumulated and increased significantly with increasing initial theophylline concentrations through N-demethylation at the position N-3 of 3-methylxanthine, respectively. Therefore, xanthine was main metabolite in theophylline degradation over the course of A. tamarii liquid culture, which showed that A. tamarii could be used for the production of xanthine with theophylline as feedstock through the microbial conversion.