Abstract
Caffeine (1, 3, 7-trimethylxanthine) and theobromine (3, 7-dimethylxanthine) are the major purine alkaloids in plants, e.g., tea (Camellia sinensis) and coffee (Coffea arabica). Caffeine is a major component of coffee and is used widely in food and beverage industries. Most of the enzymes involved in the caffeine biosynthetic pathway have been reported previously. Here, we demonstrated the biosynthesis of caffeine (0.38 mg/L) by co-expression of Coffea arabica xanthosine methyltransferase (CaXMT) and Camellia sinensis caffeine synthase (TCS) in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this production platform for making other purine-based alkaloids. To increase the catalytic activity of TCS in an effort to increase theobromine production, we identified four amino acid residues based on structural analyses of 3D-model of TCS. Two TCS1 mutants (Val317Met and Phe217Trp) slightly increased in theobromine accumulation and simultaneously decreased in caffeine production. The application and further optimization of this biosynthetic platform are discussed.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Biosynthetic Pathways
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Caffeine / genetics
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Caffeine / metabolism*
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Camellia sinensis / enzymology
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Coffea / enzymology
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Industrial Microbiology / methods
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Metabolic Engineering / methods*
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Methyltransferases / genetics
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Methyltransferases / metabolism
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Mutation
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Saccharomyces cerevisiae / genetics*
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Saccharomyces cerevisiae / metabolism*
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Theobromine / genetics
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Theobromine / metabolism*
Substances
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Caffeine
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7-methylxanthosine synthase
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Methyltransferases
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caffeine synthase
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Theobromine
Grants and funding
This work was supported by grants from the Natural Science Foundation of China (NSFC No. 31170649), the Program for Changjiang Scholars, and the Innovative Research Team in University (No. IRT1101). This work was also supported in part by grants from US National Science Foundation (NSF) (MCB-0923779) and US Department of Energy (DOE) (DE-SC0001295) to O. Yu. Funding for the 4000QTRAP mass spectrometer was provided through an NSF-MRI grant (DBI-0521250). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.