ABSTRACTThe choline oxidase (CHOA) and betaine aldehyde dehydrogenase (BADH) genes identified inAspergillus fumigatusare present as a cluster specific for fungal genomes. Biochemical and molecular analyses of this cluster showed that it has very specific biochemical and functional features that make it unique and different from its plant and bacterial homologs.A. fumigatusChoAp catalyzed the oxidation of choline to glycine betaine with betaine aldehyde as an intermediate and reduced molecular oxygen to hydrogen peroxide using FAD as a cofactor.A. fumigatusBadhp oxidized betaine aldehyde to glycine betaine with reduction of NAD+to NADH. Analysis of theAfchoAΔ::HPHandAfbadAΔ::HPHsingle mutants and theAfchoAΔAfbadAΔ::HPHdouble mutant showed thatAfChoAp is essential for the use of choline as the sole nitrogen, carbon, or carbon and nitrogen source during the germination process.AfChoAp andAfBadAp were localized in the cytosol of germinating conidia and mycelia but were absent from resting conidia. Characterization of the mutant phenotypes showed that glycine betaine inA. fumigatusfunctions exclusively as a metabolic intermediate in the catabolism of choline and not as a stress protectant. This study inA. fumigatusis the first molecular, cellular, and biochemical characterization of the glycine betaine biosynthetic pathway in the fungal kingdom.