(177s) Engineering Yarrowia Lipolytica for Efficient Production of Plant-Derived Very Long-Chain Monounsaturated Fatty Acid-Nervonic Acid

Nervonic acid (24:1, ∆15, NA) is a natural omega-9 long chain monounsaturated fatty acid, the only specific substance that can promote the repair and regeneration of damaged brain nerve tissue. At present, NA was mainly extracted from the plant Malania Oleifera. However, the yield is significantly affected by the season, and thus hinders its further application. Yarrowia lipolytica, a FDA-approved non-conventional yeast, has attracted increasing interest for producing value-added products due to their oleaginous characteristics.

Herein, Y. lipolytica was engineered for NA biosynthesis. A URA-blast method for cycling selection marker was developed into this yeast. In addition, NA is synthetized by a membrane-bound fatty acid elongation (FAE) complex using C18:1 as the substrates. 3-ketoacyl-CoA synthases (KCS) is the rate-limiting enzyme in the FAE complex and it is the substrate specificity of this enzyme which determines the chain length produced. Thus, four exogenous NA biosynthetic genes (KCS1-4) from Brassica napus, Crambe abyssinica, Lunaria annua, Cardamine graeca and an endogenous fatty acid elongation gene (YlELO2) were individually integrated into the genome to explore the influence of NA biosynthesis. Under shake-flask fermentation, the engineered strain YL3-1 which harboring the KCS3 gene accumulated 0.7% fatty acid NA and YL4-1 which harboring the KCS4 gene accumulated 1.1% NA. Subsequently, the Acetyl-CoA carboxylase and Diacylglyceride acyl-transferase were overexpressed, the lipid yield improved 3-fold compared to the control. Furthermore, the endogenous delta-9 stearoyl-CoA desaturase (SCD) gene, which can both catalyse C16:0 to C16:1 and C18:0 to C18:1, was substituted by pgSCD from Puccinia graminis which perfer to transfer the C18:0 to C18:1. The FAD2 gene which catalyse C18:1 to C18:2 was knocked out to further improve the C18:1 yield. The titer of NA in the engineered strain YL4-1-1 was improved by 210%. Altogether, the methods developed here could be applied for synthesizing other monounsaturated fatty acids.