E.i. Dupont De Nemours and Company

Dana M Walters Pollak

Quinn Qun Zhu

Zhixiong Xue

Howard Glenn Damude

Narendra S Yadav

Hongxiang Zhang

Stephen K Picataggio

Daniel Joseph Macool

Peter John Gillies

James John Ragghianti

Pierre E Rouviere

Hongxian He

Seung-Pyo Hong

Pamela L Sharpe

Patricia C Brzostowicz

Michael W Bostick

Melissa D Bosak

Qun Zhu

Yehong Jamie Wang

Engineered strains of the oleaginous yeastcapable of producing greater than 25% eicosapentaenoic acid (EPA, an &#x3c9;-3 polyunsaturated fatty acid) in the total oil fraction are described. These strains comprise various chimeric genes expressing heterologous desaturases, elongases and acyltransferases and optionally comprise various native desaturase and acyltransferase knockouts to enable synthesis and high accumulation of EPA. Production host cells are claimed, as are methods for producing EPA within said host cells.

[object Object]

Mutant delta-5 desaturases, having the ability to convert dihomo-gamma-linolenic acid [DGLA; 20:3 omega-6] to arachidonic acid [ARA; 20:4 omega-6] and/or eicosatetraenoic acid [ETA; 20:4 omega-3] to eicosapentaenoic acid [EPA; 20:5 omega-3] and possessing at least one mutation within the HPGG (SEQ ID NO:7) motif of the cytochome b-like domain and at least one mutation within the HDASH (SEQ ID NO:8) motif are disclosed. Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding delta-5 desaturases, along with a method of making long chain polyunsaturated fatty acids ['PUFAs'], are also disclosed.

Mutant HPGG motif and HDASH motif delta-5 desaturases and their use in making polyunsaturated fatty acids

The present invention relates to mutant &#x394;5 desaturases, which have the ability to convert dihomo-&#x3b3;-linolenic acid [DGLA; 20:3 &#x3c9;-6] to arachidonic acid [ARA; 20:4 &#x3c9;-6] and/or eicosatetraenoic acid [ETA; 20:4 &#x3c9;-3] to eicosapentaenoic acid [EPA; 20:5 &#x3c9;-3] and which possess at least one mutation within the HPGG motif of the cytochome b-like domain. Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding &#x394;5 desaturases, along with a method of making long chain polyunsaturated fatty acids ['PUFAs'] using these mutant &#x394;5 desaturases in oleaginous yeast, are disclosed.

Mutant &#x394;-5 desaturases mutated in the heme-binding motif (HPGG)

Mutant &#x394;-5 desaturases mutated in the heme-binding motif (HPGG) and their use in making polyunsaturated fatty acids

The present invention relates to mutant &#x394;5 desaturases, which have the ability to convert dihomo-&#x3b3;-linolenic acid [DGLA; 20:3 &#x3c9;-6] to arachidonic acid [ARA; 20:4 &#x3c9;-6] and/or eicosatetraenoic acid [ETA; 20:4 &#x3c9;-3] to eicosapentaenoic acid [EPA; 20:5 &#x3c9;-3] and which possess at least one mutation within the HPGG motif of the cytochrome b-like domain. Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding &#x394;5 desaturases, along with a method of making long chain polyunsaturated fatty acids ['PUFAs'] using these mutant &#x394;5 desaturases in oleaginous yeast, are disclosed.

Mutant  &#x394;5 desaturases mutated in the heme-binding motif (HPGG) and their use in making polyunsaturated fatty acids

The present invention relates to a &#x394;5 desaturase, which has the ability to convert dihomo-&#x3b3;-linolenic acid (DGLA; 20:3 &#x3c9;-6) to arachidonic acid (ARA; 20:4 &#x3c9;-6) and/or eicosatetraenoic acid (ETA; 20:4 &#x3c9;-3) to eicosapentaenoic acid (EPA; 20:5 &#x3c9;-3). Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding &#x394;5 desaturase along with a method of making long chain polyunsaturated fatty acids (PUFAs) using this &#x394;5 desaturase in oleaginous yeast are disclosed.

&#x394;5 desaturase and its use in making polyunsaturated fatty acids

&#x394;  5 desaturase and its use in making polyunsaturated fatty acids

Engineered strains of the oleaginous yeastcapable of producing greater than 10% arachidonic acid (ARA, an &#x3c9;-6 polyunsaturated fatty acid) in the total oil fraction are described. These strains comprise various chimeric genes expressing heterologous desaturases, elongases and acyltransferases, and optionally comprise various native desaturase and acyltransferase knockouts to enable synthesis and high accumulation of ARA. Production host cells are claimed, as are methods for producing ARA within said host cells.

The present invention relates to methods for the production of &#x3b3;-linolenic acid (GLA) in oleaginous yeast. Thus, &#x394;12 and &#x394;6 desaturases able to catalyze the conversion of oleic acid to GLA have been introduced into the genome of, using zeta-directed integration. Transformed strains produced over 25% GLA in the total lipids, as opposed to wildtypethat is unable to synthesize any GLA. Metabolic engineering and fermentation methods are provided to further enhance GLA productivity in oleaginous yeast.

Production of &#x3b3;-linolenic acid in oleaginous yeast

The regulatory sequences (i.e., promoter regions, introns and enhancers) associated with thegene encoding fructose bis-phospate aldolase (FBA1) have been found to be particularly effective for the expression of heterologus genes in oleaginous yeast. The promoter regions of the invention have been shown to drive high-level expression of genes involved in the production of &#x3c9;-3 and &#x3c9;-6 fatty acids.

Fructose-bisphosphate aldolase regulatory sequences for gene expression in oleaginous yeast

Mutant phytoene desaturase genes are provided encoding polypeptides having the ability to convert a phytoene desaturase substrate to tetradehydrolycopene. Both in vivo and in vitro methods are provided using the present phytoene desaturases for tetradehydrolycopene production.

Growing community of inventors

Media, PA, United States of America

Dana M Walters Pollak