E.i. Dupont De Nemours and Company

Pioneer Hi-bred International, Inc.

Narendra S Yadav

Quinn Qun Zhu

Hongxiang Zhang

Zhixiong Xue

Howard Glenn Damude

William Dean Hitz

Stephen K Picataggio

Seung-Pyo Hong

Pamela L Sharpe

Daniel Joseph Macool

Anthony John Kinney

James John Ragghianti

Dana M Walters Pollak

Peter John Gillies

Luis Perez-Grau

Qun Zhu

Saverio Carl Falco

Jonathan E Lightner

John R Bedbrook

Roy S Chaleff

Barbara J Mazur

John Joseph Okuley

Knut Meyer

Christopher R Somerville

Michael W Bostick

John E Seip

Luis Perez Grau

Rebecca E Cahoon

John W Pierce

Hongxian He

Michael Dauner

Jianjun Yang

Xiaochun Fan

Melissa D Bosak

Cristopher R Somerville

Anna M Wierzbicki

John Browse

Yehong Jamie Wang

John R Booth

S Carl Falco

Dana Walters Pollak

Dana M Pollak

Luis P Grau

Dana Walters M Pollak

Transgenic soybean seed having increased total fatty acid content of at least 10% and altered fatty acid profiles when compared to the total fatty acid content of non-transgenic, null segregant soybean seed are described. DGAT genes fromare used to achieve the increase in seed storage lipids.

DGAT genes from yarrowia lipolytica for increased seed storage lipid production and altered fatty acid profiles in soybean

Recombinant microbial cells are disclosed herein that produce an oil comprising at least 28 percent eicosapentaenoic acid (EPA) measured as a weight percent of dry cell weight. These cells may comprise a polynucleotide sequence encoding an active acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) comprising at least one amino acid mutation in a membrane-bound O-acyltransferase motif. In addition, the cells may comprise a down-regulation of an endogenous polynucleotide sequence encoding Sou2 sorbitol utilization protein, and/or one or more polynucleotides encoding phospholipid:diacylglycerol acyltransferase (PDAT), delta-12 desaturase, a dihomo-gamma-linolenic acid (DGLA) synthase multizyme, delta-8 desaturase, malonyl-CoA synthetase (MCS), or acyl-CoA:lysophosphatidic acid acyltransferase (LPAAT). Also disclosed are methods of using the recombinant microbial cells to produce oil containing omega-3 polyunsaturated fatty acids such as EPA.

Recombinant microbial cells that produce at least 28% eicosapentaenoic acid as dry cell weight

Methods of increasing the amount of polyunsaturated fatty acids (PUFAs) in the total lipid fraction and in the oil fraction of PUFA-producing, oleaginous eukaryotes, accomplished by modifying the activity of peroxisome biogenesis factor (Pex) proteins. Disruptions of a chromosomal Pex3 gene, Pex10p gene or Pex16p gene in a PUFA-producing, oleaginous eukaryotic strain resulted in an increased amount of PUFAs, as a percent of total fatty acids and as a percent of dry cell weight, in the total lipid fraction and in the oil fraction of the strain, as compared to the parental strain whose native Pex protein was not disrupted.

Peroxisome biogenesis factor protein (Pex) disruptions for altering polyunsaturated fatty acids and total lipid content in oleaginous eukaryotic organisms

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;-15 fatty acid desaturases that are able to catalyze the conversion of linoleic acid (18:2, LA) to alpha-linolenic acid (18:3, ALA). Nucleic acid sequences encoding the desaturases, nucleic acid sequences which hybridize thereto, DNA constructs comprising the desaturase genes, and recombinant host plants and microorganisms expressing increased levels of the desaturases are described. Methods of increasing production of specific omega-3 and omega-6 fatty acids by over-expression of the &#x394;-15 fatty acid desaturases are also described herein.

Delta-15 desaturases suitable for altering levels of polyunsaturated fatty acids in oilseed plants and oleaginous yeast

The present invention relates to methods for the production of &#x3c9;-3 and/or &#x3c9;-6 fatty acids in oleaginous yeast. Thus, desaturases and elongases able to catalyze the conversion of linoleic acid (LA) to &#x3b3;-linolenic acid (GLA); &#x3b1;-linoleic acid (ALA) to stearidonic acid (STA); GLA to dihomo-&#x3b3;-linoleic acid (DGLA); STA to eicosatetraenoic acid (ETA); DGLA to arachidonic acid (ARA); ETA to eicosapentaenoic acid (EPA); DGLA to ETA; EPA to docosapentaenoic acid (DPA); and ARA to EPA have been introduced into the genome offor synthesis of ARA and EPA.

Production of polyunsaturated fatty acids in oleaginous yeasts

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

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Methods of increasing the amount of polyunsaturated fatty acids (PUFAs) in the total lipid fraction and in the oil fraction of PUFA-producing, oleaginous eukaryotes, accomplished by modifying the activity of peroxisome biogenesis factor (Pex) proteins, are disclosed. Disruptions of a chromosomal Pex3 gene, Pex10p gene or Pex16p gene in a PUFA-producing, oleaginous eukaryotic strain resulted in an increased amount of PUFAs, as a percent of total fatty acids and as a percent of dry cell weight, in the total lipid fraction and in the oil fraction of the strain, as compared to the parental strain whose native Pex protein was not disrupted.

Peroxisome biogenesis factor protein (PEX) disruptions for altering polyunsaturated fatty acids and total lipid content in oleaginous eukaryotic organisms

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.

Engineered strains of the oleaginous yeastcapable of producing greater than 50 weight percent of eicosapentaenoic acid in the total oil fraction are described. These strains over-express heterologous &#x394;9 elongases, &#x394;8 desaturases, &#x394;5 desaturases, &#x394;17 desaturases, &#x394;12 desaturases and Celongases, and optionally over-express diacylglycerol cholinephosphotransferases. Gene knockouts are also described, as are methods for producing EPA within host cells, and products comprising EPA from the optimizedstrains.

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

Growing community of inventors

Wilmington, DE, United States of America

Narendra S Yadav