Unveiling the genes responsible for the unique Pseudomonas aeruginosa oleate-diol synthase activity

作者：Estupinan Monica; Diaz Pilar^*; Manresa Angels

来源：Biochimica et Biophysica Acta-Molecular and Cell Biology of Lipids, 2014, 1841(10): 1360-1371.

DOI：10.1016/j.bbalip.2014.06.010

• P. aeruginosa
• Oleate-diol synthase activity
• Oxylipin
• Oleic acid metabolism

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 ((75,10S)-dihydroxy-(8E)-octadecenoic acid), both of which accumulate in the culture supernatant. Several mutants of P. aeruginosa PAW 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 adds, 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 anew fine regulated oleate-diol synthase operon, originated by a gene duplication event followed by neofunctionalization for environmental adaptation, being unprecedented in prokaryotes.

• 出版日期2014-10