Single cell oil production on molasses by Yarrowia lipolytica strains overexpressing DGA2 in multicopy

Abstract

Yarrowia lipolytica is a promising platform for single cell oil production. It is well-known for its metabolism oriented toward utilization of hydrophobic substrates and accumulation of storage lipids. Multiple copies of DGA2 under constitutive promoter were introduced into the Q4 strain, a quadruple mutant deleted for the four acyltransferases (Δdga1, Δdga2, Δlro1, and Δare1) to improve lipid accumulation. The Q4-DGA2 x3 strain contains three copies of DGA2. Further increase in accumulation was accomplished by blocking the β-oxidation pathway through MFE1 gene deletion yielding Q4-Δmfe DGA2 x3. In order to use molasses as a substrate for single cell oil production, sucrose utilization was established by expressing the Saccharomyces cerevisiae SUC2 gene yielding Q4-SUC2 DGA2 x3 and Q4-Δmfe SUC2 DGA2 x3. During cultivation on sucrose medium with a carbon to nitrogen ratio of 80, both strains accumulated more than 40 % of lipids, which was a 2-fold increase in lipid storage. Q4-Δmfe SUC2 DGA2 x3 accumulated more lipids than Q4-SUC2 DGA2 x3 (49 vs. 43 %) but yielded less biomass (13.7 vs. 15 g/L). When grown on 8 % (v/v) molasses, both strains accumulated more than 30 % of lipids after 3 days, while biomass yield was higher in Q4-SUC2 DGA2 x3 (16.4 vs. 14.4 g/L). Further addition of molasses at 72 h resulted in higher biomass yield, 26.6 g/L for Q4-SUC2 DGA2 x3, without modification of lipid content. This work presents genetically modified strains of Y. lipolytica as suitable tools for direct conversion of industrial molasses into value added products based on single cell oils.

Engineering Yarrowia lipolytica for Enhanced Production of Lipid and Citric Acid

“…Similarly, the overexpression of diacylglycerol acyltransferase increased the lipid content from 13% to the range of 39-53% of the DCW in the strain without active PEX10 [58]. Other studies have attempted DGA1 or DGA2 overexpression strategy to enhance lipid content in this yeast [3,[54][55][56][57]. Overall, overexpression of DGA genes increased lipid content while decreasing biomass formation in the HPDD strain due to LEU2 deletion.…”

“…In the final step of the TAG synthesis pathway, DGA1, YALI0E32769g on the lipid droplet (LD) membrane, and DGA2, YALI0D07986g in the ER, play prominent roles in acylation of diacylglycerol to produce TAG, which is stored in LDs especially during the stationary phase [49][50][51]. Therefore, the overexpression of DGA1 and DGA2 results in enhanced lipid accumulation [3,[52][53][54][55][56][57][58][59]. TAG synthesis and remobilization is a dynamic process.…”

“…Our best-engineered strain, HPDDS, produced lipid at a titer of 3.15 g/L in the shake flask, rising to 4.8 g/L in the batch bioreactor cultivation. A lipid titer of 2.6 to 6.5 g/L was obtained by engineered Y. lipolytica from 60 grams of sugar using batch fermentation [51,56]. The coupling of DGA1,2 overexpression and SNF1 deletion enabled a maximum lipid content of nearly 44% in the best lipid producing strain, HPDDS.…”

Microbial oils as food additives: recent approaches for improving microbial oil production and its polyunsaturated fatty acid content

“…One acyl-CoA oxidase has been found in S. cerevisiae, whereas in Y. lipolytica six different acyl-CoA oxidases (encoded by the POX1 to POX6 genes) have been identified (see review [47 ]). In Y. lipolytica inactivation of POX1-6 genes coupled with G3P shuttle defects increased overall lipid accumulation (65-75%) and TAG storage [85], while deletion of YLSNF1 (encoding for SNF1 protein kinase) [92] or MFE1 (encoding for a multifunctional enzyme catalyzing the second step of the b-oxidation pathway) [93 ] significantly enhanced lipid content. Repression of FA oxidation may be also achieved by disturbing peroxisome biogenesis via PEX10 deletion [28 ,63 ].…”

Metabolic Engineering for Expanding the Substrate Range of Yarrowia lipolytica

“…Some of these complex substrates are already being used and even when the carbon sources are only partially used, promising amounts of chemicals and fuels have been produced. Such substrates include glycerol-containing waste [63,64], cooking oil [65], rice bran hydrolysate [66], wheat straw hydrolysate [67,68], sugarcane bagasse hydrolysate [69], bioethanol waste effluent containing starch [49], and molasses [17,70]. Therefore, any improvements that result in more efficient use of these substrates is expected to boost yields.…”