(593f) Production of Caffeine Derived Methylxanthines in Pseudomonas Putida KT2440

Pseudomonas putida has the ability to utilize a wide variety of carbon sources, demonstrating its effectiveness as a bacterial host for biochemical production. P. putida CBB5 can convert caffeine to higher value methylxanthines through a series of N-demethylation reactions. The genes responsible for the N-demethylation of caffeine, ndmABCDE, have been previously isolated and expressed in the model organism Escherichia coli due to its robust set of genetic engineering tools. However, expression in the host of origin is preferred, and recent studies have worked to expand and consolidate the genetic toolkit of P. putida. This work aims to further develop and optimize the available tools for P. putida through the construction and characterization of an expression plasmid. The plasmid pRC000 contains an IPTG-inducible promoter, multiple cloning site, and a broad-host range origin of replication. An N-terminal hexahistidine tag and a thrombin cleavage site are also present within the cloning site for expression of His-tagged proteins. Functionality of the inducible system has been confirmed using green fluorescent protein and characterized using β-galactosidase assays. We cloned genes for the N₁-demethylation (ndmAD), N₃-demethylation (ndmBD), and N₇-demethylation (ndmCDE) of caffeine into pRC000 for expression in P. putida KT2440. The N₁- and N₃-demethylase systems exhibited higher rates and generated higher product yields when expressed in P. putida compared to their E. coli counterparts. Finally, we have constructed P. putida strains expressing ndmABD and ndmA3D for production of the high-value chemicals 7-methylxanthine and 1-methylxanthine.