Engineering a novel biosynthetic pathway in <i>Escherichia coli</i> for the production of caffeine

Li, Mengmeng; Sun, Ying; Pan, Si-an; Deng, Wei‐Wei; Yu, Oliver; Zhang, Zhengzhu

doi:10.1039/c7ra10986e

Cited by 11 publications

(17 citation statements)

References 37 publications

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“…Xanthine alkaloids in the extracts were measured using an ACQUITY UPLC H-Class instrument (Waters, the United States) and separated on a ACQUITY UPLC BEH C18 Column (2.1 × 100 mm, 1.7 μm in particle size and 130 Å in pore size) at a flow rate of 0.3 ml/min at 35°C with 1 μl sample volume, the mobile phase was selected as 0.2% (v) acetic acid in water (A), and methanol (B), and the gradient elution was as follows: B 6% from 0 to 2 min, to 8% at 3.5 min, to 10% at 4.5 min, to 18% at 7.5 min, to 26% at 10.5 min, to 80% at 13.5 min and kept at 80% for 1 min, to 6% at 16 min and kept at 6% for 1 min, the detection wavelength was set to 278 nm ( Li et al, 2017 ; Pan et al, 2019 ). Xanthine alkaloids were identified by using a high resolution quadrupole time-of-flight mass spectrometry (QTOF MS) on the positive-ion mode, the Agilent 1290 UPLC was interfaced with an Agilent 6545 Q-TOF LC/MS system equipped with electrospray ionization source (ESI).…”

Section: Methodsmentioning

confidence: 99%

Metabolite and Transcriptome Profiling on Xanthine Alkaloids-Fed Tea Plant (Camellia sinensis) Shoot Tips and Roots Reveal the Complex Metabolic Network for Caffeine Biosynthesis and Degradation

Deng

Cheng

et al. 2020

Front. Plant Sci.

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While caffeine is one of the most important bioactive metabolites for tea as the most consumed non-alcohol beverage, its biosynthesis and catabolism in tea plants are still not fully understood. Here, we integrated purine alkaloid profiling and transcriptome analysis on shoot tips and roots fed with caffeine, theophylline, or theobromine to gain further understanding of caffeine biosynthesis and degradation. Shoot tips and roots easily took up and accumulated high concentrations of alkaloids, but roots showed much faster caffeine and theophylline degradation rates than shoot tips, which only degraded theophylline significantly but almost did not degrade caffeine. Clearly feedback inhibition on caffeine synthesis or inter-conversion between caffeine, theophylline, and theobromine, and 3-methylxanthine had been observed in alkaloids-fed shoot tips and roots, and these were also evidenced by significant repression of TCS and MXMT genes critical for caffeine biosynthesis. Among these responsively repressed genes, two highly expressed genes TCS-4 and TCS-8 were characterized for their enzyme activity. While we failed to detect TCS-4 activity, TCS-8 displayed N -methyltransferase activities towards multiple substrates, supporting the complex metabolic network in caffeine biosynthesis in tea plants since at least 13 TCS-like N -methyltransferase genes may function redundantly. This study provides new insight into complex metabolic networks of purine alkaloids in tea plants.

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Section: Methodsmentioning

confidence: 99%

Metabolite and Transcriptome Profiling on Xanthine Alkaloids-Fed Tea Plant (Camellia sinensis) Shoot Tips and Roots Reveal the Complex Metabolic Network for Caffeine Biosynthesis and Degradation

Deng

Cheng

et al. 2020

Front. Plant Sci.

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“…Finally, the supernatants were ltered through 0.22 µm lter membranes to glass vials. Caffeine content was quanti ed by ultra-performance liquid chromatography (UPLC) system (Waters Corporation, USA) equipped with a reverse-phase C18 column and tunable ultraviolet detector at a ow rate of 0.3 mL min − 1 , according to the method described by Li et al [57]. The RNAs were also extracted from those two parts by using Fruit-mate™ for RNA Puri cation (Takara, Beijing, China) for the gene expression analysis.…”

Section: Pathogenicity Test Histopathological Observation and Fungalmentioning

confidence: 99%

Inhibition Mechanism of Caffeine in Tea Pathogenic Fungi Botryosphaeria Dothidea and Colletotrichum Gloeosporioides

Thangaraj

Deng

Cheng

et al. 2020

Preprint

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Background Caffeine acts as an antifungal agent; however, its underlying inhibition mechanism remains unclear. To investigate variations in caffeine antifungal activity, this study was evaluate the inhibition mechanisms of caffeine on phytopathogens Botryosphaeria dothidea and Colletotrichum gloeosporioides through transcriptomic analysis, which causes leaf necrosis and anthracnose on tea leaves, respectively.Results Our in vitro results shows that the antifungal activity of caffeine quite different between B. dothidea and C. gloeosporioides. Furthermore, microscopic observation and bioactivity assay results proved that caffeine destroyed the fungal protective layers (cell wall and cell membrane) and subcellular organelles. The transcriptomes of these two fungi exposed to different caffeine concentrations were analyzed by high-throughput sequencing and the results showed that caffeine repressed gene expression involved in the pathways of mRNA surveillance, ribosome biogenesis in eukaryotes, aminoacyl-tRNA biosynthesis, etc. Particularly, caffeine affected gene expression in melanin biosynthesis in C. gloeosporioides, resulting in the disturbances of melanin production and fungal pathogenicity. Manually infected tea leaves showed melanin pigment was important to appressorium development and fungal infection by histopathological observation and fungal biomass quantification. Besides, C. gloeosporioides successfully enters into the host tissue even plant could build multilayer defenses against the fungus, this might be due to the variation in pathogen targets and quantities of secondary metabolites by the host cells.Conclusions Overall, our findings indicate that caffeine in the environment can reduce the fungal growth and physiological activities. However, the genome-wide expression showed different transcriptional profiles between these fungal isolates. Thus, we believe that target of the caffeine varied among isolates and melanin may play a major role in fungus caffeine tolerance. Further in-depth research required to conclude this hypothesis.

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“…The standards and sample extracts were analyzed using a Waters HPLC system (Waters, Milford, MA, USA) through a reversed-phase column (Waters; 100 A, 5 mm, 4.6 mm Â 250 mm), and the detection method was described previously. 14 wavelength was set at 274 nm. The samples were eluted at a ow rate of 0.3 mL min À1 , with an injection volume of 5 mL for the ltrate.…”

Section: Caffeine Content Variation During Piling Fermentation With Smentioning

confidence: 99%

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

Pan

Sun

et al. 2019

RSC Adv.

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Engineering a novel biosynthetic pathway in Escherichia coli for the production of caffeine

Abstract: This work demonstrated a novel biosynthetic pathway to produce caffeine in Escherichia coli.

Cited by 11 publications

References 37 publications

Metabolite and Transcriptome Profiling on Xanthine Alkaloids-Fed Tea Plant (Camellia sinensis) Shoot Tips and Roots Reveal the Complex Metabolic Network for Caffeine Biosynthesis and Degradation

Metabolite and Transcriptome Profiling on Xanthine Alkaloids-Fed Tea Plant (Camellia sinensis) Shoot Tips and Roots Reveal the Complex Metabolic Network for Caffeine Biosynthesis and Degradation

Inhibition Mechanism of Caffeine in Tea Pathogenic Fungi Botryosphaeria Dothidea and Colletotrichum Gloeosporioides

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

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