Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica

The availability of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is currently limited because they are produced mainly by marine fisheries that cannot keep pace with the demands of the growing market for these products. A sustainable non-animal source of EPA and DHA is needed. Metabolic engineering of the oleaginous yeast Yarrowia lipolytica resulted in a strain that produced EPA at 15% of dry cell weight. The engineered yeast lipid comprises EPA at 56.6% and saturated fatty acids at less than 5% by weight, which are the highest and the lowest percentages, respectively, among known EPA sources. Inactivation of the peroxisome biogenesis gene PEX10 was crucial in obtaining high EPA yields and may increase the yields of other commercially desirable lipid-related products. This technology platform enables the production of lipids with tailored fatty acid compositions and provides a sustainable source of EPA.

[1]  J. Napier,et al.  Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants , 2004, Nature Biotechnology.

[2]  J. Browse,et al.  Production of Polyunsaturated Fatty Acids by Polyketide Synthases in Both Prokaryotes and Eukaryotes , 2001, Science.

[3]  J. Nicaud.  Yarrowia lipolytica , 2012, Yeast.

[4]  J. Browse,et al.  The Δ8-Desaturase ofEuglena gracilis:An Alternate Pathway for Synthesis of 20-Carbon Polyunsaturated Fatty Acids☆☆☆★ , 1999 .

[5]  A. Steinbüchel,et al.  Yarrowia lipolytica , 2013, Microbiology Monographs.

[6]  S. Stymne,et al.  Delta 6- and delta 12-desaturase activities and phosphatidic acid formation in microsomal preparations from the developing cotyledons of common borage (Borago officinalis). , 1988, The Biochemical journal.

[7]  J. Tomb,et al.  Identification of bifunctional Δ12/ω3 fatty acid desaturases for improving the ratio of ω3 to ω6 fatty acids in microbes and plants , 2006 .

[8]  J. Napier,et al.  The production of very-long-chain PUFA biosynthesis in transgenic plants: towards a sustainable source of fish oils , 2005, The Proceedings of the Nutrition Society.

[9]  T. Zank,et al.  Acyl Carriers Used as Substrates by the Desaturases and Elongases Involved in Very Long-chain Polyunsaturated Fatty Acids Biosynthesis Reconstituted in Yeast* , 2003, Journal of Biological Chemistry.

[10]  Carlos Gancedo,et al.  Yarrowia lipolytica Mutants Devoid of Pyruvate Carboxylase Activity Show an Unusual Growth Phenotype , 2005, Eukaryotic Cell.

[11]  W. Harris,et al.  Omega-3 Fatty Acids in Food and Pharma: The Enabling Role of Biotechnology , 2011, Current atherosclerosis reports.

[12]  P. Thonart,et al.  New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. , 2003, Journal of microbiological methods.

[13]  H. Damude,et al.  Three diacylglycerol acyltransferases contribute to oil biosynthesis and normal growth in Yarrowia lipolytica , 2012, Yeast.

[14]  Marit Aursand,et al.  High-resolution (13)C nuclear magnetic resonance spectroscopy pattern recognition of fish oil capsules. , 2007, Journal of agricultural and food chemistry.

[15]  A. Kinney,et al.  Formation of conjugated delta8,delta10-double bonds by delta12-oleic-acid desaturase-related enzymes: biosynthetic origin of calendic acid. , 2001, The Journal of biological chemistry.

[16]  A. Kinney,et al.  Formation of Conjugated Δ8,Δ10-Double Bonds by Δ12-Oleic-acid Desaturase-related Enzymes , 2001, The Journal of Biological Chemistry.

[17]  J. Beckerich,et al.  One-step transformation of the dimorphic yeast Yarrowia lipolytica , 1997, Applied Microbiology and Biotechnology.

[18]  B. Dujon,et al.  Genome evolution in yeasts , 2004, Nature.

[19]  X. Qiu,et al.  The Front-end Desaturase: Structure, Function, Evolution and Biotechnological Use , 2012, Lipids.

[20]  C. Schwechheimer,et al.  Different functions of the C3HC4 zinc RING finger peroxins PEX10, PEX2, and PEX12 in peroxisome formation and matrix protein import , 2010, Proceedings of the National Academy of Sciences.

[21]  Z. Xue,et al.  Engineering Yarrowia lipolytica to express secretory invertase with strong FBA1IN promoter , 2012, Yeast.

[22]  Jennifer J. Smith,et al.  Peroxisome biogenesis in the yeast Yarrowia lipolytica , 2007, Cell Biochemistry and Biophysics.

[23]  Z. Wen,et al.  Prospects for Eicosapentaenoic Acid Production Using Microorganisms , 2005 .

[24]  C. Ballantyne,et al.  Eicosapentaenoic acid ethyl ester (AMR101) therapy in patients with very high triglyceride levels (from the Multi-center, plAcebo-controlled, Randomized, double-blINd, 12-week study with an open-label Extension [MARINE] trial). , 2011, The American journal of cardiology.

[25]  A. Ohta,et al.  Peroxisome deficiency represses the expression of n-alkane-inducible YlALK1 encoding cytochrome P450ALK1 in Yarrowia lipolytica. , 2002, FEMS microbiology letters.

[26]  W. Barclay,et al.  Development of a Docosahexaenoic Acid Production Technology Using Schizochytrium: A Historical Perspective , 2005 .

[27]  Z. Xue,et al.  BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Identification and characterization of new Δ-17 fatty acid desaturases , 2013 .

[28]  H. Zhang,et al.  ω3 fatty acid desaturases from microorganisms: structure, function, evolution, and biotechnological use , 2013, Applied Microbiology and Biotechnology.

[29]  B. Tyreus,et al.  Metabolic engineering of an oleaginous yeast for the production of omega-3 fatty acids. , 2010 .

[30]  C. Funk,et al.  Prostaglandins and leukotrienes: advances in eicosanoid biology. , 2001, Science.

[31]  G. Stephanopoulos,et al.  Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. , 2013, Metabolic engineering.

[32]  Qing Liu,et al.  Metabolic engineering of omega-3 long-chain polyunsaturated fatty acids in plants using an acyl-CoA Delta6-desaturase with omega3-preference from the marine microalga Micromonas pusilla. , 2010, Metabolic engineering.

[33]  R. Deckelbaum,et al.  The omega-3 fatty acid nutritional landscape: health benefits and sources. , 2012, The Journal of nutrition.

[34]  Hal Alper,et al.  Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach , 2011, Applied and Environmental Microbiology.

[35]  David J. Chen,et al.  The endless tale of non-homologous end-joining , 2008, Cell Research.

[36]  M. Wyss,et al.  Yarrowia lipolytica: Safety assessment of an oleaginous yeast with a great industrial potential , 2014, Critical reviews in microbiology.

[37]  A. Kinney,et al.  Engineering oilseeds to produce nutritional fatty acids. , 2007, Physiologia plantarum.

[38]  E. Schaefer,et al.  Abstract 20007: Effects of Eicosapentaenoic Acid, Docosahexaenoic Acid, and Olive Oil on Cardiovascular Disease Risk Factors , 2010 .

[39]  F. Sherman.  Getting started with yeast. , 1991, Methods in enzymology.

[40]  P. Schenk,et al.  Microalgal biofactories: a promising approach towards sustainable omega-3 fatty acid production , 2012, Microbial Cell Factories.

[41]  J. Nicaud,et al.  An overview of lipid metabolism in yeasts and its impact on biotechnological processes , 2011, Applied Microbiology and Biotechnology.

[42]  Guohai Wu,et al.  Towards the production of high levels of eicosapentaenoic acid in transgenic plants: the effects of different host species, genes and promoters , 2010, Transgenic Research.

[43]  S. Troëng.  Oil determination of oilseed. Gravimetric routine method , 1955 .

[44]  H. Damude,et al.  Isolation of a Δ5 Desaturase Gene from Euglena gracilis and Functional Dissection of Its HPGG and HDASH Motifs , 2012, Lipids.

[45]  C. Ratledge.  Single Cell Oils for the 21st Century , 2010 .

[46]  A. Dominguez,et al.  Functional genetics of Yarrowia lipolytica , 2003 .

[47]  W. Barclay,et al.  Heterotrophic production of long chain omega-3 fatty acids utilizing algae and algae-like microorganisms , 1994, Journal of Applied Phycology.

[48]  Y. Matsuzawa,et al.  Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis , 2007, The Lancet.

[49]  P. Walther,et al.  Freeze substitution of high‐pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water , 2002, Journal of microscopy.

[50]  J. Napier,et al.  Metabolic Engineering of Saccharomyces cerevisiae for Production of Eicosapentaenoic Acid, Using a Novel Δ5-Desaturase from Paramecium tetraurelia , 2010, Applied and Environmental Microbiology.

[51]  J. Lupton,et al.  n-3 PUFA and membrane microdomains: a new frontier in bioactive lipid research. , 2004, The Journal of nutritional biochemistry.