Understanding and exploiting the fatty acid desaturation system in Rhodotorula toruloides

Rhodotorula toruloides is a robust producer of triacylglycerol owing to its fast growth rate and strong metabolic flux under conditions of high cell density fermentation. However, the molecular basis of fatty acid biosynthesis, desaturation and regulation remains elusive. We present the molecular characterization of four fatty acid desaturase (FAD) genes in R. toruloides. Biosynthesis of oleic acid (OA) and palmitoleic acid (POA) was conferred by a single-copy ∆9 Fad (Ole1) as targeted deletion of which abolished the biosynthesis of all unsaturated fatty acids. Conversion of OA to linoleic acid (LA) and α-linolenic acid (ALA) was predominantly catalyzed by the bifunctional ∆12/∆15 Fad2. FAD4 was found to encode a trifunctional ∆9/∆12/∆15 FAD, playing important roles in lipid and biomass production as well as stress resistance. Furthermore, an abundantly transcribed OLE1-related gene, OLE2 encoding a 149-aa protein, was shown to regulate Ole1 regioselectivity. Like other fungi, the transcription of FAD genes was controlled by nitrogen levels and fatty acids in the medium. A conserved DNA motif, (T/C)(G/A)TTGCAGA(T/C)CCCAG, was demonstrated to mediate the transcription of OLE1 by POA/OA. The applications of these FAD genes were illustrated by engineering high-level production of OA and γ-linolenic acid (GLA). Our work has gained novel insights on the transcriptional regulation of FAD genes, evolution of FAD enzymes and their roles in UFA biosynthesis, membrane stress resistance and, cell mass and total fatty acid production. Our findings should illuminate fatty acid metabolic engineering in R. toruloides and beyond.