E.i. Dupont De Nemours and Company

Hongxiang Zhang

Narendra S Yadav

Quinn Qun Zhu

Zhixiong Xue

Stephen K Picataggio

Howard Glenn Damude

Daniel Joseph Macool

James John Ragghianti

Dana M Walters Pollak

Peter John Gillies

Michael W Bostick

Qun Zhu

Seung-Pyo Hong

Pamela L Sharpe

John E Seip

Hongxian He

Melissa D Bosak

Yehong Jamie Wang

Dana Walters Pollak

Dana M Pollak

Dana Walters M Pollak

Acyl-CoA:lysophosphatidylcholine acyltransferase ['LPCAT'] having the ability to convert acyl-CoA+1-acyl-sn-glycero-3-phosphocholine to CoA+1,2-diacyl-sn-glycero-3-phosphocholine (EC 2.3.1.23) is disclosed herein to be over-expressed along with the over-expression of phospholipid:diacylglycerol acyltransferase ['PDAT'] having the ability to transfer a fatty acyl group from the sn-2 position of a phospholipid (e.g., phosphatidylcholine) to the sn-3 position of 1,2-diacylglycerol [E.C.2.3.1.158], thus resulting in a lysophospholipid and TAG. Co-expression of these enzymes in a recombinant microbial host cell resulted in increased production of long chain polyunsaturated fatty acids [&#x201c;PUFAs&#x201d;].

Production of polyunsaturated fatty acids by coexpression of acyl-CoA:lysophosphatidylcholine acyltransferases and phospholipid:diacylglycerol acyltransferases

Mutant acyl-CoA:lysophosphatidylcholine acyltransferases ['LPCATs'] having the ability to convert acyl-CoA+1-acyl-sn-glycero-3-phosphocholine to CoA+1,2-diacyl-sn-glycero-3-phosphocholine (EC 2.3.1.23) are disclosed herein. Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding mutant LPCATs, along with a method of making long chain polyunsaturated fatty acids ['PUFAs'] using these mutant LPCATs in oleaginous yeast, are also disclosed.

Nucleic acids encoding mutant acyl-CoA:lysophosphatidylcholine acyltransferases

The present invention relates to fungal &#x394;12 desaturases (responsible for conversion of oleic acid to linoleic acid (18:2, LA)) and &#x394;15 fatty acid desaturases (responsible for conversion of LA to &#x3b1;-linolenic acid (18:3, ALA)). Amino acid motifs diagnostic of &#x394;12 desaturases and &#x394;15 desaturases are also provided. Methods of altering enzyme specificity towards &#x394;12 desaturation or towards &#x394;15 desaturation and/or increasing production of specific &#x3c9;-3 and &#x3c9;-6 fatty acids by over-expression of the fungal desaturases are also described.

Fungal &#x394;-12 desaturase and &#x394;-15 desaturase motifs

Methods for increasing Cto Celongation conversion efficiency and/or &#x394;4 desaturation conversion efficiency in long-chain polyunsaturated fatty acid ['LC-PUFA']-producing recombinant oleaginous microbial host cells are provided herein, based on over-expression of acyl-CoA:lysophospholipid acyltransferases ['LPLATs'] (e.g., Ale1, LPAAT, LPCAT). Production host cells and oils produced by the methods of the invention are also claimed.

Long chain omega-3 and omega-6 polyunsaturated fatty acid biosynthesis by expression of acyl-CoA lysophospholipid acyltransferases

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.

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Engineered strains of the oleaginous yeastare disclosed herein that are capable of producing microbial oil comprising greater than 25 weight percent of eicosapentaenoic acid ['EPA'], an omega-3 polyunsaturated fatty acid, measured as a weight percent of dry cell weight.

Recombinant microbial host cells for high eicosapentaenoic acid production

Methods to increase the percent of polyunsaturated fatty acids (PUFAs) within the total lipids and oils of PUFA-producing oleaginous organisms are provided herein, by regulating the activity of specific acyltransferases. Specifically, since oil biosynthesis is expected to compete with polyunsaturation during oleaginy, it is possible to reduce or inactivate the activity of an organism's DAG ATs (e.g., phospholipid:diacylglycerol acyltransferase (PDAT) and/or diacylglycerol acyltransferase 1 (DGAT1) and/or diacylglycerol acyltransferase 2 (DGAT2)) to thereby reduce the overall rate of oil biosynthesis while concomitantly increasing the percent of PUFAs that are incorporated into the lipid and oil fractions. The teachings herein will thereby enable one to engineer a wide variety of oleaginous organisms to produce oils with very specific fatty acid compositions.

Acyltransferase regulation to increase the percent of polyunsaturated fatty acids in total lipids and oils of oleaginous organisms

Acyltransferases are provided, suitable for use in the manufacture of microbial oils enriched in omega fatty acids in oleaginous yeast (e.g.,). Specifically, genes encoding diacylglycerol acyltransferase (DGAT1) have been isolated fromand. These genes encode enzymes that participate in the terminal step in oil biosynthesis in yeast. Each is expected to play a key role in altering the quantity of polyunsaturated fatty acids produced in oils of oleaginous yeasts.

Diacylglycerol acyltransferases for alteration of polyunsaturated fatty acids and oil content in oleaginous organisms

An engineered strain of the oleaginous yeastcapable of producing greater than 5.6% docosahexaenoic acid acid (DHA, an w-3 polyunsaturated fatty acid) in the total oil fraction is described. This strain comprises various chimeric genes expressing heterologous desaturases, elongases and acyltransferases and optionally comprises various native desaturase and acyltransferase knockouts to enable synthesis and high accumulation of DHA. Production host cells are claimed, as are methods for producing DHA within said host cells.

Docosahexaenoic acid producing strains of yarrowia lipolytica

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 &#x394;6 desaturases, which have the ability to convert linoleic acid ['LA'; 18:2 &#x3c9;-6] to &#x3b3;-linolenic acid ['GLA'; 18:3 &#x3c9;-6] and/or &#x3b1;-linolenic acid [&#x201c;ALA&#x201d;; 18:3 &#x3c9;-3] to stearidonic acid [&#x201c;STA&#x201d;; 18:4 &#x3c9;-3]. Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding &#x394;6 desaturases, along with methods of making long-chain polyunsaturated fatty acids [&#x201c;PUFAs&#x201d;] using these &#x394;6 desaturases in oleaginous yeast, are disclosed.

&#x394;6 desaturases and their use in making polyunsaturated fatty acids

Two acyltransferases are provided, suitable for use in the manufacture of microbial oils enriched in omega fatty acids in oleaginous yeast (e.g.,). Specifically, the genes encoding phophatidylcholine-diacylglycerol acyltransferase (PDAT) and diacylglycerol acyltransferase (DGAT2) have been isolated from. These genes encode enzymes that participate in the terminal step in oil biosynthesis in yeast. Each is expected to play a key role in altering the quantity of polyunsaturated fatty acids produced in oils of oleaginous yeasts.

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Chadds Ford, PA, United States of America

Hongxiang Zhang