High-level production of nervonic acid in the oleaginous yeastYarrowia lipolyticaby systematic metabolic engineering

Abstract

AbstractBrain and neurological diseases are influencing more than one billion world’s people. Nervonic acid (cis-15-tetracosenoic acid, C24:1 Δ15) benefits the treatment of neurological diseases and the health of brain. Currently, the sources of nervonic acid are limited to the seeds of a couple of plants. In this study, we employed the oleaginous yeastYarrowia lipolyticato overproduce nervonic acid oil by systematic metabolic engineering. First, engineering the fatty acid elongation (FAE) pathway by expressing a heterologous β-ketoacyl-CoA synthase geneCgKCSenabled the production of nervonic acid inY. lipolytica.Second, modulation of endogenous pathways by expressing a C16:0-acyl-CoA preferred fatty acid elongase gELOVL6 together with a C18:0-acyl-CoA preferred fatty acid desaturase MaOLE2 increased the content of nervonic acid in total fatty acids (TFA). Third, iterative expression ofCgKCS,gELOVL6andMaOLE2at the genomic loci ofrDNA,FAD2,TGL4,GSY1andSNF1dramatically improved the production of nervonic acid. Fourth, the biosynthesis of both nervonic acid and lipids were further enhanced by expression of the MaOLE2-CgKCS fusion protein and glycerol-3-phosphate acyltransferases (GPAT) and diacylglycerol acyltransferases (DGAT) fromMalania oleiferain the endoplasmic reticulum (ER) membrane. Fifth, an ER structure regulator YlINO2 was identified inY. lipolyticaand the overexpression of YlINO2 led to a 39.3% increase in lipid production. Next, pilot-scale fermentation in 50-L reactor using the strain YLNA9 exhibited a lipid titer of 96.7 g/L and a nervonic acid titer of 17.3 g/L, the highest reported titer to date forde novonervonic acid production. We also found that disruption of the AMP-activated S/T protein kinaseSNF1increased the ratio of nervonic acid (C24:1) to lignoceric acid (C24:0) by 61.6% and a ratio of 3.5:1 (nervonic acid to lignoceric acid) was achieved in the strain YLNA10. Finally, a proof-of-concept purification and separation of nervonic acid were performed and the purity of it reached 98.7%. This study suggested that oleaginous yeasts are attractive hosts for the cost-efficient production of nervonic acid and possibly other very long-chain fatty acids (VLCFAs).