title: Metabolic engineering of Saccharomyces cerevisiae for caffeine and theobromine production.
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.
authors.affiliation: Key Laboratory of Tea Biochemistry and Biotechnology, Ministry of Education, Anhui Agricultural University, Hefei, PR China.
authors.forename: Lu
authors.initials: L
authors.lastname: Jin
authors.affiliation: Conagen Inc., St. Louis, Missouri, United States of America.
authors.forename: Mohammad Wadud
authors.initials: MW
authors.lastname: Bhuiya
authors.affiliation: Key Laboratory of Tea Biochemistry and Biotechnology, Ministry of Education, Anhui Agricultural University, Hefei, PR China.
authors.forename: Mengmeng
authors.initials: M
authors.lastname: Li
authors.affiliation: Key Laboratory of Tea Biochemistry and Biotechnology, Ministry of Education, Anhui Agricultural University, Hefei, PR China.
authors.forename: XiangQi
authors.initials: X
authors.lastname: Liu
authors.affiliation: Conagen Inc., St. Louis, Missouri, United States of America.
authors.forename: Jixiang
authors.initials: J
authors.lastname: Han
authors.affiliation: Key Laboratory of Tea Biochemistry and Biotechnology, Ministry of Education, Anhui Agricultural University, Hefei, PR China.
authors.forename: WeiWei
authors.initials: W
authors.lastname: Deng
authors.affiliation: Key Laboratory of Tea Biochemistry and Biotechnology, Ministry of Education, Anhui Agricultural University, Hefei, PR China.
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.affiliation: Donald Danforth Plant Science Center, St. Louis, Missouri, United States of America.
authors.forename: Oliver
authors.initials: O
authors.lastname: Yu
authors.affiliation: Key Laboratory of Tea Biochemistry and Biotechnology, Ministry of Education, Anhui Agricultural University, Hefei, PR China.
authors.forename: Zhengzhu
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D053898:Biosynthetic Pathways; D002110:Caffeine / Q000235:genetics / Q000378:metabolism*; D028241:Camellia sinensis / Q000201:enzymology; D040503:Coffea / Q000201:enzymology; D007218:Industrial Microbiology / Q000379:methods; D060847:Metabolic Engineering / Q000379:methods*; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D013805:Theobromine / Q000235:genetics / Q000378:metabolism*
chemical_list: D002110:Caffeine; C477002:7-methylxanthosine synthase; D008780:Methyltransferases; C120479:caffeine synthase; D013805:Theobromine