title: Unveiling the genes responsible for the unique Pseudomonas aeruginosa oleate-diol synthase activity.
abstract: Pseudomonas aeruginosa displays the ability to perform bioconversion of oleic acid into a class of hydroxylated fatty acids known as oxylipins. A diol synthase activity is responsible for such a conversion, which proceeds through the dioxygenation of oleic acid to release hydroperoxide 10-H(P)OME ((10S)-hydroxy-(8E)-octadecenoic acid), followed by conversion of the hydroperoxide intermediate into 7,10-DiHOME ((7S,10S)-dihydroxy-(8E)-octadecenoic acid), both of which accumulate in the culture supernatant. Several mutants of P. aeruginosa PAO1 were analyzed for the production of 10-H(P)OME and 7,10-DiHOME and two of them (ORFs PA2077 and PA2078), unable to release hydroxylated fatty acids, were detected and selected for further analysis. Involvement of ORFs PA2077 and PA2078 in oleate-diol synthase activity was confirmed, and their respective role in the conversion of oleic acid was analyzed by mutation complementation. Activity restoration revealed that gene PA2077 codes for the 10S-dioxygenase activity (10S-DOX) responsible for the first step of the reaction, whereas PA2078 encodes for the (7S,10S)-hydroperoxide diol synthase enzyme (7,10-DS) which allows the conversion of 10-H(P)OME into 7,10-DiHOME. Heterologous expression of both enzymes separately showed that no hetero-complex formation is required for enzymatic activity. Bioinformatics and RT-PCR analysis revealed that both genes constitute a new fine regulated oleate-diol synthase operon, originated by a gene duplication event followed by neofunctionalization for environmental adaptation, being unprecedented in prokaryotes.
authors: Department of Microbiology, Faculty of Biology, University of Barcelona, Barcelona, Spain; Unitat de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, University of Barcelona, Barcelona, Spain.
authors: Mónica
authors: M
authors: Estupiñán
authors: Department of Microbiology, Faculty of Biology, University of Barcelona, Barcelona, Spain. Electronic address: pdiaz@ub.edu.
authors: Pilar
authors: P
authors: Diaz
authors: Unitat de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, University of Barcelona, Barcelona, Spain.
authors: Angels
authors: A
authors: Manresa
keywords: Oleate-diol synthase activity; Oleic acid metabolism; Oxylipin; P. aeruginosa