Single cell oil production on molasses by Yarrowia lipolytica strains overexpressing DGA2 in multicopy
Gajdos, Peter | Nicaud, Jean-Marc | Rossignol, Tristan | Certik, Milan | Department of Biochemical Technology, Faculty of Chemical and Food Technology ; Slovak University of Technology | MICrobiologie de l'ALImentation au Service de la Santé (MICALIS) ; Institut National de la Recherche Agronomique (INRA)-AgroParisTech | Grant Agency of the Ministry of Education, Slovak Republic VEGA 1/0574/15 ; Slovak Research and Development Agency, Slovak Republic APVV-0662-11 ; French National Institute for Agricultural Research (Institut National de la Recherche Agronomique)
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 (Delta dga1, Delta dga2, Delta lro1, and Delta are1) to improve lipid accumulation. The Q4-DGA2 x3 strain contains three copies of DGA2. Further increase in accumulation was accomplished by blocking the beta-oxidation pathway through MFE1 gene deletion yielding Q4-Delta mfe 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-Delta mfe 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-Delta mfe 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.
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