Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica
2014
Béopoulos, Athanasios | Verbeke, Jonathan | Bordes, Florence | Guicherd, Marie | Boubila-Bressy, Mélusine | Marty, Aurélie | Nicaud, Jean-Marc | MICrobiologie de l'ALImentation au Service de la Santé (MICALIS) ; Institut National de la Recherche Agronomique (INRA)-AgroParisTech | Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP) ; Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse) ; Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS) | INRA; INSA; DGCIS; Conseil Regional Midi-Pyrenees; FEDER within the frame of the French national FUI project OLEOVISION
Although there are numerous oleochemical applications for ricinoleic acid (RA) and its derivatives, their production is limited and subject to various safety legislations. In an effort to produce RA from alternative sources, we constructed a genetically modified strain of the oleaginous yeast Yarrowia lipolytica. This strain is unable to perform beta-oxidation and is invalidated for the native triacylglycerol (TAG) acyltransferases (Dga1p, Dga2p, and Lro1p) and the a dagger 12 desaturase (Fad2p). We also expressed the Ricinus communis a dagger 12 hydroxylase (RcFAH12) under the control of the TEF constitutive promoter in this strain. However, RA constituted only 7 % of the total lipids produced by this modified strain. By contrast, expression of the Claviceps purpurea hydroxylase CpFAH12 in this background resulted in a strain able to accumulate RA to 29 % of total lipids, and expression of an additional copy of CpFAH12 drove RA accumulation up to 35 % of total lipids. The co-expression of the C. purpurea or R. communis type II diacylglycerol acyltransferase (RcDGAT2 or CpDGAT2) had negative effects on RA accumulation in this yeast, with RA levels dropping to below 14 % of total lipids. Overexpression of the native Y. lipolytica PDAT acyltransferase (Lro1p) restored both TAG accumulation and RA levels. Thus, we describe the consequences of rerouting lipid metabolism in this yeast so as to develop a cell factory for RA production. The engineered strain is capable of accumulating RA to 43 % of its total lipids and over 60 mg/g of cell dry weight; this is the most efficient production of RA described to date.
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