Abstract
Fatty acid desaturase enzymes perform dehydrogenation reactions leading to the insertion of double bonds in fatty acids, and are divided into soluble and integral membrane classes. Crystal structures of soluble desaturases are available; however, membrane desaturases have defied decades of efforts due largely to the difficulty of generating recombinant desaturase proteins for crystallographic analysis. Mortierella alpina is an oleaginous fungus which possesses eight membrane desaturases involved in the synthesis of saturated, monounsaturated and polyunsaturated fatty acids. Here, we describe the successful expression, purification and enzymatic assay of three M. alpina desaturases (FADS15, FADS12, and FADS9-I). Estimated yields of desaturases with purity >95% are approximately 3.5% (Ca. 4.6 mg/L of culture) for FADS15, 2.3% (Ca. 2.5 mg/L of culture) for FADS12 and 10.7% (Ca. 37.5 mg/L of culture) for FADS9-I. Successful expression of high amounts of recombinant proteins represents a critical step towards the structural elucidation of membrane fatty acid desaturases.
Highlights
Lipids are first synthesized as saturated fatty acids, and double bonds are introduced post-synthetically by oxygen-dependent enzymes known as fatty acid desaturases, in a process that is initiated by abstraction of hydrogen from a methylene group
The high similarity of FADS12 and FADS9-I genes between two strains indicates that these genes are highly conserved in M. alpina
There was a 5% increase in C18:3D9,12,15 in cells expressing FADS15, suggesting that FADS15 can desaturate C18:2 D9,12 at the c15-position to produce C18:3D9,12,15. These results suggest that the recombinant desaturases, FADS9-I, FADS12 and FADS15, were active in P. pastoris
Summary
Lipids are first synthesized as saturated fatty acids, and double bonds are introduced post-synthetically by oxygen-dependent enzymes known as fatty acid desaturases, in a process that is initiated by abstraction of hydrogen from a methylene group. Fatty acid desaturases are divided into soluble and integral membrane classes, which may have evolved independently [1]. Fatty acid desaturases in each class are closely related homologs based on their amino acid sequences, and yet perform highly regio- and stereo-selective reactions on long-chain fatty acids composed of essentially equivalent methylene chains that lack distinguishing landmarks close to the site of desaturation. The membrane class of desaturases consists of enzymes with c5, c6, c9, c12 or v3 regio-selectivity. M. alpina has all known regioselective groups of membrane desaturases
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