Date of Award


Degree Name

Master of Science


Biological Sciences

First Advisor

Dr. Todd J. Barkman

Second Advisor

Dr. Yan Lu

Third Advisor

Dr. Andre Venter


Theobroma, Cacao, Evolution, Caffeine, Biochemistry

Access Setting

Masters Thesis-Open Access


Caffeine biosynthesis is widely distributed in flowering plants and requires three consecutive methylation steps of xanthine alkaloids. Genes that have previously been reported to participate in the multi-step pathway in Coffea sp. (coffee) and Camellia sinensis (tea) encode members of the SABATH family of methyltransferases. Two genes highly expressed in fruits of Theobroma cacao (cacao) are orthologous to the caffeine genes in tea and appear to have diversified following gene duplication. Biochemical characterization of the enzymes (XMTs) encoded by these genes strongly suggest an unprecedented major pathway to theobromine, a precursor to caffeine. These findings imply that caffeine biosynthesis evolved convergently in plants and raise two major questions about the evolution of the caffeine pathway in cocoa: 1) What ancestral conditions led to duplication, evolution, and maintenance of paralogous XMTs in the evolution of theobromine accumulation in cacao fruits? and 2) How has gene duplication played a role in the independent evolution of flux through the caffeine pathway? To answer these questions, two ca. 50-million-year-old enzyme ancestors of the cocoa XMTs were resurrected in vitro and biochemically characterized with xanthine alkaloids as substrates.