Biochemical characterization of three new α-olefin-producing P450 fatty acid decarboxylases with a halophilic property

BACKGROUND: The CYP152 family member OleT(JE) from Jeotgalicoccus sp. ATCC 8456 has been well-known to catalyze the unusual one-step decarboxylation of free fatty acids towards the formation of terminal alkenes. Efforts to tune up its decarboxylation activity for better production of biological alkenes have been extensively explored via approaches such as site-directed mutagenesis and electron source engineering, but with limited success. To gain more insights into the decarboxylation mechanism and reaction bifurcation (decarboxylation versus hydroxylation), we turned to an alternative approach to explore the natural CYP152 resources for a better variety of enzyme candidates. RESULTS: We biochemically characterized three new P450 fatty acid decarboxylases including OleT(JH), OleT(SQ) and OleT(SA), with respect to their substrate specificity, steady-state kinetics, and salt effects. These enzymes all act as an OleT(JE)-like fatty acid decarboxylase being able to decarboxylate a range of straight-chain saturated fatty acids (C(8)–C(20)) to various degrees. Site-directed mutagenesis analysis to the lower activity P450 enzyme OleT(SA) revealed a number of key amino acid residues within the substrate-binding pocket (T47F, I177L, V319A and L405I) that are important for delicate substrate positioning of different chain-length fatty acids and thus the decarboxylation versus hydroxylation chemoselectivity, in particular for the mid-chain fatty acids (C(8)–C(12)). In addition, the three new decarboxylases exhibited optimal catalytic activity and stability at a salt concentration of 0.5 M, and were thus classified as moderate halophilic enzymes. CONCLUSION: The P450 fatty acid decarboxylases OleT(JE), OleT(JH), OleT(SQ) and OleT(SA) belong to a novel group of moderate halophilic P450 enzymes. OleT(JH) from Jeotgalicoccus halophilus shows the decarboxylation activity, kinetic parameters, as well as salt tolerance and stability that are comparable to OleT(JE). Site-directed mutagenesis of several key amino acid residues near substrate-binding pocket provides important guidance for further engineering of these P450 fatty acid decarboxylases that hold promising application potential for production of α-olefin biohydrocarbons. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13068-019-1419-6) contains supplementary material, which is available to authorized users.