Caffeine Production in Yeast

The iGEM Minnesota 2012 Team is also seeking to use the tools of synthetic biology to create genetically modified yeast with the ability to synthesize caffeine. Using genes that code for caffeine biosynthesis proteins from Coffea arabica, we hope to create and subsequently insert a plasmid into Saccharomyces cerevisiae that will confer the yeast with the ability to create caffeine in the lab. Immediate goals include the creation of a caffeine-production-inducing BioBrick that we'll submit to the registry, as well as a new empty yeast plasmid backbone that future iGEM teams can use for their projects. In coffee plants, the metabolic pathway for caffeine involves the use of AMP, GMP, and IMP to produce theobromine, which is the immediate precursor to caffeine. An important intermediate in the pathway is xanthosine, which already occurs naturally in S. cerevisiae. What our team seeks to do is introduce the genes into yeast that can transform the xanthosine into caffeine, via the xanothosine -> 7-methlyxanothine -> 7-methylxanthine -> theobromine -> caffeine pathway. The two genes we're incorporating into our plasmid are XMT1 and DXMT1. We plan to construct our plasmid de novo from several segments of DNA that we'll ligate using Gibson Assembly. Once construction of the plasmid is complete, we'll transform into yeast and screen for caffeine production.