Guanine deaminase provides evidence of the increased caffeine content during the piling process of pu'erh tea

Abstract

Wet piling is a key process for producing pu'erh tea because various components change under the action of microorganisms. Among these components, caffeine content is increased. Evidence has indicated a salvage pathway for caffeine biosynthesis in microbes, in which xanthine is methylated in the order of N-3 → N-1 → N-7. In addition, guanine can be used to synthesize xanthine through guanine deaminase (EC: 3.5.4.3). In this study, we investigated the variation in caffeine content during piling fermentation with supplementary guanine, ¹⁵N-labeled guanine and xanthine. We cloned the guanine deaminase gene (GUD1) from Saccharomyces cerevisiae (one dominant strain in piling fermentation). The results revealed that [¹⁵N]xanthine could be synthesized from [¹⁵N]guanine, and [¹⁵N]caffeine was also detected during piling with supplementary [¹⁵N]xanthine. Furthermore, ScGUD1 could catalyze the conversion of guanine to xanthine, which is likely to be methylated for caffeine synthesis under microorganism action. The obtained results revealed the mechanism underlying the increased caffeine content during piling of pu'erh tea.

Fig. 1. Variation in caffeine content during piling fermentation with multiple concentrations of exogenous guanine. The significances of differences were compared between the groups of fermentation time on different guanine concentrations (*p < 0.05). Data represent the mean ± standard deviation of three independent experiments.

Fig. 2. Mass spectra of [¹⁵N]xanthine in samples of imitated pu'erh tea for piling with exogenous [¹⁵N]guanine. (A) LC-MS chromatograms of purine standards and piling fermented tea samples; (B) identification of [¹⁵N]guanine (m/z: 157.0422) and [¹⁵N]xanthine (m/z: 157.0279) in imitated pu'erh tea samples. G, guanine; X, xanthine; 1-MX, 1-methylxanthine; 3-MX, 3-methylxanthine; 7-MX, 7-methylxanthine; Tb, theobromine; Tp, theophylline; Cf, caffeine.

Fig. 3. Mass spectra of [¹⁵N]caffeine in samples of imitated pu'erh tea samples for piling with exogenous [¹⁵N]xanthine. (A) LC-MS chromatograms of purine standards and piling fermented tea samples; (B) MS-MS spectra identification of [¹⁵N]xanthine (m/z: 155.2000) and [¹⁵N]caffeine (m/z: 197.1000). G, guanine; X, xanthine; 1-MX, 1-methylxanthine; 3-MX, 3-methylxanthine; 7-MX, 7-methylxanthine; Tb, theobromine; Tp, theophylline; Cf, caffeine.

Fig. 4. Possible caffeine biosynthetic pathways in tea plants (shown as green arrows) and in the piling process of pu'erh tea under microorganism action (blue arrows).

Fig. 5. Cloning of ScGUD1, construction of recombinant of pMAL-ScGUD1, and enzyme assay in E. coli. (A) Agarose gel electrophoresis of PCR-amplified ScGUD1. M, DNA marker; (1) PCR product of ScGUD1. (B) SDS-PAGE electrophoresis of recombinant pMAL-ScGUD1. M, Protein marker; (1) total crude protein from the induced cells was transformed using the pMAL-c5X vector alone; (2) suspension of total crude protein from the induced cells using the pMAL-c5X vector by 16 °C; (3) suspension of total crude protein from the induced cells using the pMAL-c5X vector by 30 °C; (4) total crude protein from the induced cells was transformed using the pMAL-ScGUD1; (5) suspension of total crude protein from the induced cells using the pMAL-ScGUD1 by 16 °C; (6) suspension of total crude protein from the induced cells using the pMAL-ScGUD1 by 30 °C. (C) Purification of recombinant pMAL-ScGUD1. M, Protein marker; (1) purified MBP from suspension of total crude protein; (2) purified pMAL-ScGUD1 from suspension of total crude protein. (D) Enzyme assay of recombinant of pMAL-ScGUD1 in vitro. The reactions of recombinant pMAL-ScGUD1 and the control strain were measured through HPLC. (E) In vivo enzyme activity determination of the recombinant pMAL-ScGUD1. The reactions of recombinant pMAL-ScGUD1 and the control strain were measured with and without exogenous guanine through HPLC.

Fig. 6. In vivo activity determination of recombinant pRSF-ScGUD1 (A) and pZ8-ScGUD1 (B) with or without exogenous guanine through HPLC.