Theophylline degradation exists in solid-state fermentation of pu-erh tea
Fungi count, caffeine and theophylline contents were determined in natural SSF 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. 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, showing that theophylline degradation appeared in SSF except for caffeine degradation. Therefore, in consideration of fungal community, there are theophylline-degrading fungi in the SSF, 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 the SSF of 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
|
Color
|
Exudates
|
Reference
|
PT-1
|
Circular
|
Rough
|
Black
|
None
|
[40]
|
PT-2
|
Circular
|
Rough
|
Olive green
|
Red-coloured
|
[40]
|
PT-3
|
Circular
|
Rough
|
Dark yellow colonies with white edges
|
Yellow sclerotium
|
[41]
|
PT-4
|
Irregular
|
Rough
|
Light yellow
|
Yellow sclerotium
|
[41]
|
PT-5
|
Circular
|
Rough
|
Greyish-green centre with yellow patches
|
Red pigment
|
[41]
|
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 sequences obtained from the pure isolate in this study were deposited in GenBank under the accession number from MT065763 to MT065769 and from MT084116 to MT064123. Based on the DNA sequences in Table 2 and additional file 1: Figure S3 and S4, seven dominating isolates were belonged to 6 Aspergillus spp. and 1 Penicillium sp., respectively. Through neighbor-joining analysis in the phylogram for Aspergillus species (Additional file: Figure S5a and S5b), strain PT-6 was clustered with Aspergillus ustus and showed a 100% of identity to the tested Aspergillus ustus NRRL275; additionally, strain PT-7 was closely related to Aspergillus tamarii NRRL20818 with 99.9% of identity. In general, those seven candidate isolates were identified as Aspergillus niger, Aspergillus sydowii, Aspergillus pallidofulvus, Aspergillus sesamicola, Penicillium manginii, Aspergillus ustus and Aspergillus tamarii based on their morphological features and amplified sequences, respectively.
Table 2 Identification of theophylline-degrading fungi by sequence determination
Isolate
|
Primers
|
Fragments (bp)
|
Accession number a
|
Species
|
Strain number
|
identity
|
PT-1
|
ITS1/ITS4
|
546
|
MT065763
|
Aspergillus niger
|
NCBT 110A
|
99.8%
|
PT-2
|
ITS1/ITS4
|
516
|
MT065764
|
Aspergillus sydowii
|
NRRL 250
|
99.8%
|
PT-3
|
ITS1/ITS4
|
541
|
MT065765
|
Aspergillus pallidofulvus
|
NRRL 4789
|
99.9%
|
Bt2a/Bt2b
|
516
|
MT084116
|
CF1L/CF4
|
765
|
MT084120
|
PT-4
|
ITS1/ITS4
|
532
|
MT065766
|
Aspergillus sesamicola
|
CBS 137324
|
99.8%
|
Bt2a/Bt2b
|
515
|
MT084117
|
CF1L/CF4
|
757
|
MT084121
|
PT-5
|
ITS1/ITS4
|
525
|
MT065767
|
Penicillium manginii
|
CBS 253.31
|
99.6%
|
Bt2a/Bt2b
|
420
|
MT084118
|
PT-6
|
ITS1/ITS4
|
502
|
MT065768
|
Aspergillus ustus
|
NRRL 275
|
100%
|
CF1L/CF4
|
694
|
MT084122
|
PT-7
|
ITS1/ITS4
|
532
|
MT065769
|
Aspergillus tamarii
|
NRRL 20818
|
99.9%
|
Bt2a/Bt2b
|
476
|
MT084119
|
CF1L/CF4
|
715
|
MT084123
|
a GenBank/EMBL/DDBJ accession number
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 in theophylline degradation. 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
|
600 mg/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 isolates could survive in the agar solid mediums (2% w/v) with theophylline. 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 as carbon source directly [40, 46]. Therefore, A. niger, A. sydowii, A. ustus and A. tamarii were considered as potential theophylline-degrading fungi.
Selection of theophylline-degrading fungi and optimal medium in liquid culture
For theophylline biodegradation in liquid culture, seven isolates were inoculated into TLM with the presence of theophylline and sucrose or dextrose as carbon source, or ammonium sulphate as nitrogen source, respectively. Theophylline concentration and fungal dry mass were determined after cultivation at 30 °C for 5 days. Results are showed in Fig. 2 and Additional file 2: Table S1, respectively. Through comparisons of each isolate, with the presence of carbon source such as sucrose or dextrose, although all isolates could survive and maintain metabolic activity in TLM, theophylline utilization efficiency was different. A. pallidofulvus, A. sesamicola and P. mangini had no ability to use theophylline. Theophylline utilization of A. niger and A. sydowii was restricted in liquid culture, theophylline removal ratios were about 1.03% and 5.19% in TLM-S, respectively. Only A. ustus and A. tamarii could utilize caffeine significantly in all given TLM. Hence, A. ustus, A. tamarii, A. niger and A. sydowii were potential theophylline-degrading fungi for theophylline degradation in liquid culture.
The presence of additional carbon or nitrogen sources had a significant impact on theophylline degradation and pathway. The optimum liquid medium was chose by comparing theophylline removal ratios 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. In addition, extra sucrose promoted theophylline degradation in TLM-S inoculated by A. ustus and A. tamarii through enhancing cell density in liquid culture. Therefore, TLM-S was selected to analyze characterization of theophylline degradation in liquid culture.
Characterization of theophylline degradation inoculated by theophylline-degrading fungi
A. ustus, tamarii, A. niger and A. sydowii were inoculated into TLM-S with increasing theophylline concentrations (100, 200 and 300 mg/L, respectively), and 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 effects of A. ustus and A. tamarii, theophylline decreased highly significantly (p < 0.01) in all substrate concentrations. However, theophylline decreased slightly (p > 0.05) in all concentrations inoculated by A. niger and A. sydowii. Therefore, A. ustus and A. tamarii had more 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 given 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). 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 a 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 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, 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 culture 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 inoculated by Aspergillus fungi 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 [22]. In this study, 3-methylxanthine and xanthine were common and main products in theophylline degradation by .A. ustus and A. tamarii. Microbial utilization of 3-methylxanthine and xanthine were investigated in liquid culture of isolates. 3-Methylxanthine and xanthine concentrations 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. Additionally, A. tamarii had a highly significant (p < 0.01) impact on xanthine degradation with a removal ratio of about 51.77%. Associated with the metabolites detected in theophylline degradation, 3-methyluric acid and xanthine were 3-methylxanthine degradation metabolites through oxidation and N-demethylation, respectively.
Despite of significant impacts on 3-mthylxanthine and xanthine degradation, 3-mthylxanthine and xanthine concentrations were accumulated largely in TLM-S inoculated by A. ustus and A. tamarii, respectively. To investigate the application in production of 3-methylxanthine and xanthine by using theophylline-degrading fungi with theophylline as feedstock, quantitative determinations of 3-methylxanthine and xanthine were carried out in all theophylline concentrations inoculated by A. ustus and A. tamarii, respectively. 3-Methylxanthine and xanthine concentrations in A. ustus and A. tamarii cultures are presented in Fig. 5. We monitored the accumulation of 3-methylxanthine and xanthine 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, 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.
Xanthine concentration over a 7-day period cultivation of A. ustus (Fig. 5c) maintained at a low level below 15.00 mg/L in all substrate concentrations. However, Fig. 5d showed 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 microbial conversion.