Acyl-CoA oxidase, a key step for lipid accumulation in the yeast Yarrowia lipolytica

Abstract

The yeast Yarrowia lipolytica is able to use fatty acids as carbon source via β-oxidation. This yeast contains five acyl-CoA oxidases (Aoxps) encoded by POX genes. Two Aoxps are of particular interest: Aox2p, a medium chain oxidase, and Aox3p, an oxidase specific for short chain substrates. To date, no 3D structure of these enzymes is available. To better understand their specificity, we have expressed 10 enzymes in Escherichia coli; Aox2p, Aox3p, a mutated Aox3 (Aox3mp), and seven chimera obtained by shuffling fragments of Aox2p and Aox3p (M1 to M4 and M6 to M8). Aox3mp and one chimeric protein (M6) were active, Aox3mp exhibited broad chain length specificity. We tested complementation of growth for strain MTLY36 (Δpox2,3,5; reduced growth on oleic acid media) and MTLY40 (Δpox2,3,4,5; not growing on oleic acid media) by expressing either Aox2p, Aox3p, Aox3mp, and M6 chimera encoding genes. We demonstrate that the POX genomic content affects both γ-lactone production [J. Mol. Catal. B, this issue] and lipid storage. Strain MTLY36 having Aox4p could still use oleic acid but grew slower than the wild type strain and did not accumulate lipids. The strains containing Aox3p, Aox3mp (alone or with Aox4p) grew on oleic acid and stored lipids as well as did the wild type. Strains expressing Aox2p (MTLY40-2P) and Aox2p, Aox4p (MTL36-2P) stored more lipids than the wild type. Mastering POX genotype allowed us to construct strains, able to accumulate high lipid content, which could be used for single cell oil (SCO) production.