Appl Microbiol Biotechnol (2014) 98:251–262 DOI 10.1007/s00253-013-5295-x
APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica A. Beopoulos & J. Verbeke & F. Bordes & M. Guicherd & M. Bressy & A. Marty & Jean-Marc Nicaud
Received: 13 June 2013 / Revised: 23 September 2013 / Accepted: 26 September 2013 / Published online: 18 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract 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 βoxidation and is invalidated for the native triacylglycerol (TAG) acyltransferases (Dga1p, Dga2p, and Lro1p) and the Δ12 desaturase (Fad2p). We also expressed the Ricinus communis Δ12 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. Keywords Ricinoleic acid . Oleaginous yeast . Yarrowia lipolytica . Lipid metabolism . Metabolic engineering . Acyltransferase
A. Beopoulos, J. Verbeke, and F. Bordes contributed equally to this work. A. Beopoulos : J. Verbeke INRA, UMR1319, Micalis, 78352 Jouy-en-Josas, France F. Bordes : M. Guicherd : M. Bressy : A. Marty Université de Toulouse; INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, 31077 Toulouse, France F. Bordes : M. Guicherd : M. Bressy : A. Marty CNRS, UMR5504, 31400 Toulouse, France F. Bordes : M. Guicherd : M. Bressy : A. Marty INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, 31400 Toulouse, France J.
APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica A. Beopoulos & J. Verbeke & F. Bordes & M. Guicherd & M. Bressy & A. Marty & Jean-Marc Nicaud
Received: 13 June 2013 / Revised: 23 September 2013 / Accepted: 26 September 2013 / Published online: 18 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract 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 βoxidation and is invalidated for the native triacylglycerol (TAG) acyltransferases (Dga1p, Dga2p, and Lro1p) and the Δ12 desaturase (Fad2p). We also expressed the Ricinus communis Δ12 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. Keywords Ricinoleic acid . Oleaginous yeast . Yarrowia lipolytica . Lipid metabolism . Metabolic engineering . Acyltransferase
A. Beopoulos, J. Verbeke, and F. Bordes contributed equally to this work. A. Beopoulos : J. Verbeke INRA, UMR1319, Micalis, 78352 Jouy-en-Josas, France F. Bordes : M. Guicherd : M. Bressy : A. Marty Université de Toulouse; INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, 31077 Toulouse, France F. Bordes : M. Guicherd : M. Bressy : A. Marty CNRS, UMR5504, 31400 Toulouse, France F. Bordes : M. Guicherd : M. Bressy : A. Marty INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, 31400 Toulouse, France J.
