Engineering E. coli for Caffeine Addiction

One of the prominent pathways for bacterial degradation of caffeine is sequential N-demethylation of the substrate to xanthine.  The entire operon containing all the genes, Alx (alkylxanthine operon) gene cluster was cloned into E. coli.  However, this did not provide the desired attribute of caffeine addiction.  An additional homolog of NdmE gene of Alx operon, a glutathione-S-transferase gene from Janthinobacterium sp. had to be engineered into the cluster.  This gene provided a critical protein needed, along with NdmC & D, to achieve N-7 demethylation of 7-methylxanthine to xanthine.  In addition, xanthine deficient strain had to be created to “force” E. coli to utilize caffeine as a source of xanthine.  The engineered strain was able to grow on caffeine, coca-cola, diet coke, but not on caffeine-free diet coke.  The extract prepared from this strain was shown to convert caffeine to xanthine.  In the process of this work, a new oxygenase was discovered, a 7-methylxanthine N-demethylase.  This is the first oxygenase that is completely dependent on NdmE, a glutathione transferase homolog, for catalytic activity.