Characterization of methylxanthine production in metabolically engineered Pseudomonas putida

Pseudomonas putida can utilize a wide variety of carbon sources, making it a promising bacterial host for biochemical production. P. putida CBB5 is able to 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 expanded and consolidated the genetic toolkit of P. putida KT2440. This work aims to further develop and optimize the available tools for P. putida KT2440 through the construction and characterization of an expression plasmid and demonstration of its utility using N -demethylase genes. We developed the IPTG-inducible expression plasmid pRC000 for use in P. putida KT2440. Functionality of the inducible system was confirmed using a green fluorescent protein assay and further characterized using a β-galactosidase assay. We observed leaky expression when expressing any gene other than gfp . We also cloned genes for the N 1 -demethylation, N 3 -demethylation, and N 7 -demethylation of methylxanthines into pRC000. Modified KT2440 strains diluted to an OD 600 of 5.0 consumed 4.924 ± 0.158 mM caffeine or 4.430 ± 0.482 mM theobromine in two hours. Strains capable of the two-step conversion of caffeine to 7-methylxanthine experienced relatively little loss of efficiency compared to those performing one-step conversion. N- demethylase mutants ndmA3 and ndmA4 , originally developed in E. coli , were also cloned in pRC000 and successfully expressed in KT2440. Cells expressing ndmA3 converted 0.956 ± 0.044 mM theophylline to 1-methylxanthine in one hour, while cells expressing ndmA4 converted 0.945 ± 0.101 mM theophylline. This study demonstrated the potential of P. putida KT2440 as a microbial host for methylxanthine production. Engineered N 1 - and N 3 -demethylase systems exhibited higher rates and generated higher product yields when expressed in P. putida compared to their E. coli counterparts.