Characterization of two long-chain fatty acid CoA ligases in the Gram-positive bacterium Geobacillus thermodenitrificans NG80-2.

The functions of two long-chain fatty acid CoA ligase genes (facl) in crude oil-degrading Geobacillus thermodenitrificans NG80-2 were characterized. Facl1 and Facl2 encoded by GTNG_0892 and GTNG_1447 were expressed in Escherichia coli and purified as His-tagged fusion proteins. Both enzymes utilized a broad range of fatty acids ranging from acetic acid (C(2)) to melissic acid (C(30)). The most preferred substrates were capric acid (C(10)) for Facl1 and palmitic acid (C(16)) for Facl2, respectively. Both enzymes had an optimal temperature of 60°C, an optimal pH of 7.5, and required ATP as a cofactor. Thermostability of the enzymes and effects of metal ions, EDTA, SDS and Triton X-100 on the enzyme activity were also investigated. When NG80-2 was cultured with crude oil rather than sucrose as the sole carbon source, upregulation of facl1 and facl2 mRNA was observed by real time RT-PCR. This is the first time that the activity of fatty acid CoA ligases toward long-chain fatty acids up to at least C(30) has been demonstrated in bacteria.