Genetic characterization of caffeine degradation by bacteria and its potential applications

Abstract

The ability of bacteria to grow on caffeine as sole carbon and nitrogen source has been known for over 40 years. Extensive research into this subject has revealed two distinct pathways, N-demethylation and C-8 oxidation, for bacterial caffeine degradation. However, the enzymological and genetic basis for bacterial caffeine degradation has only recently been discovered. This review article discusses the recent discoveries of the genes responsible for both N-demethylation and C-8 oxidation. All of the genes for the N-demethylation pathway, encoding enzymes in the Rieske oxygenase family, reside on 13.2-kb genomic DNA fragment found in Pseudomonas putida CBB5. A nearly identical DNA fragment, with homologous genes in similar orientation, is found in Pseudomonas sp. CES. Similarly, genes for C-8 oxidation of caffeine have been located on a 25.2-kb genomic DNA fragment of Pseudomonas sp. CBB1. The C-8 oxidation genes encode enzymes similar to those found in the uric acid metabolic pathway of Klebsiella pneumoniae. Various biotechnological applications of these genes responsible for bacterial caffeine degradation, including bio-decaffeination, remediation of caffeine-contaminated environments, production of chemical and fuels and development of diagnostic tests have also been demonstrated.

Fig 1

Proposed caffeine N-demethylation pathway (A) and map of associated genes (B) in Pseudomonas putida CBB5. The dashed arrows in part (A) represent a minor pathway, accounting for 1–2% of metabolized caffeine. NdmA/ndmA = N₁-demethylase specific for N₁-methyl group of caffeine; NdmD/ndmD = reductase; NdmB/ndmB = N₃-demethylase specific for N₃-methyl group of theobromine; NdmCDE = protein complex containing N₇-demethylase specific for N₇-demethylation of 7-methylxanthine; ndmC = NdmC gene; ndmE = NdmE gene; frmA = glutathione-dependent formaldehyde dehydrogenase; frmB = S-formylglutathione hydrolase. (C) A numbered structure of the caffeine molecule.

Fig 2

Proposed caffeine C-8 oxidation pathway and associated genes in Pseudomonas sp. CBB1. The solid arrows represent enzymatic steps, while the dashed arrow represents the spontaneous degradation of 1,3,7-trimethyl-5-hydroxyisouric acid (TM-HIU) to racemic TMA. Inset: map of the 25.2 kb caffeine gene cluster on the CBB1 genome containing the entire C-8 oxidation pathway genes. Cdh/cdhABC = trimeric caffeine dehydrogenase; TmuM/tmuM = trimethyluric acid monooxygenase; tumH = putative TM-HIU hydrolase; tmuD = putative TM-OHCU decarboxylase; orf1 = putative trimethylallantoinase; orf2, orf3 = putative genes responsible for catabolism of 1,6,8-trimethylallantoic acid to simpler metabolites such as dimethylurea, monomethylurea and glyoxylic acid.

Fig 3

Caffeine dehydrogenase (Cdh)-based caffeine detection. Iodonitrotetrazolium chloride (INT) dye in the presence of Cdh results in shades of red to detect caffeine in test samples such as nursing mothers' milk (left, with 20 ppm caffeine), pharmaceuticals (top, tablets dissolved and diluted to 20 ppm caffeine), soft drinks (right) and brewed coffee (bottom).