Delta-9 elongases and their use in making polyunsaturated fatty acids

a technology of delta9 and elongases, which is applied in the field of polyunsaturated fatty acids encoding delta9 elongases, can solve the problems of insufficient commercial needs, insufficient study of pufas from natural sources and chemical synthesis, and inability to efficiently synthesize by the human body

Inactive Publication Date: 2007-05-24
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, eicosapentaenoic acid (EPA; 20:5, delta-5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6, delta-4,7,10,13,16,19) cannot be synthesized efficiently by the human body and thus must be supplied through the diet.
Humans can synthesize long-chain PUFAs from the essential fatty acids, LA and ALA; however biosynthesis of long-chain PUFAs is somewhat limited (they are regulated by dietary and hormonal changes), and LA and ALA must be obtained from the diet.
An expansive study of PUFAs from natural sources and from chemical synthesis are not sufficient for commercial needs.
However, these efforts have proved largely unsuccessful in improving yield of oil or the ability to control the characteristics of the oil composition produced.

Method used

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  • Delta-9 elongases and their use in making polyunsaturated fatty acids
  • Delta-9 elongases and their use in making polyunsaturated fatty acids
  • Delta-9 elongases and their use in making polyunsaturated fatty acids

Examples

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example 1

Euglena gracilis Growth Conditions, Lipid Profile and mRNA Isolation

[0410]Euglena gracilis was obtained from Dr. Richard Triemer's lab at Michigan State University (East Lansing, Mich.). From 10 mL of actively growing culture, a 1 mL aliquot was transferred into 250 mL of Euglena gracilis (Eg) Medium in a 500 mL glass bottle. Eg medium was made by combining 1 g of sodium acetate, 1 g of beef extract (U126-01, Difco Laboratories, Detroit, Mich.), 2 g of Bacto® tryptone (0123-17-3, Difco Laboratories), 2 g of Bacto®) yeast extract (0127-17-9, Difco Laboratories) in 970 mL of water. After filter sterilizing, 30 mL of soil-water supernatant (15-3790, Carolina Biological Supply Company, Burlington, N.C.,) was aseptically added to give the final Eg medium. Euglena gracilis cultures were grown at 23° C. with a 16 h light, 8 h dark cycle for 2 weeks with no agitation.

[0411] After 2 weeks, 10 mL of culture was removed for lipid analysis and centrifuged at 1,800×g for 5 min. The pellet was ...

example 2

Euglena gracilis cDNA Synthesis, Library Construction and Sequencing

[0413] A cDNA library was generated using the Cloneminer™ CDNA Library Construction Kit (Cat. No. 18249-029, Invitrogen Corporation, Carlsbad, Calif.) and following the manufacturer's protocol provided (Version B, 25-0608). Using the non-radiolabeling method, cDNA was synthesized from 3.2 μg of mRNA (described above) using the Biotin-attB2-Oligo(dT) primer. After synthesis of the first and second strand, the attb1 adapter was added, ligated and the cDNA was size fractionated using column chromatography. DNA from fractions 7 and 8 (size ranging from ˜800-1500 bp) were concentrated, recombined into PDONR T222 and transformed into E. coli ElectroMAX™ DH10B™ T1 Phage-Resistant cells (Invitrogen Corporation). The Euglena gracilis library was named eeg1 c.

[0414] For sequencing, clones first were recovered from archived glycerol cultures grown / frozen in 384-well freezing media plates, and replicated with a sterile 384 pi...

example 3

Identification of Long-Chain Polyunsaturated Fatty Acid Elongation Enzyme Homologs from Euglena gracilis cDNA Library eeg1c

[0416] cDNA clones encoding long-chain polyunsaturated fatty acid elongation enzyme homologs (LC-PUFA ELO homologs or delta-9 elongases) were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol. 215:403-410 (1993)) searches for similarity to sequences contained in the BLAST “nr” database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences obtained in Example 2 were analyzed for similarity to all publicly available DNA sequences contained in the “nr” database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared ...

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Abstract

Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding delta-9 elongases along with a method of making long-chain polyunsaturated fatty acids (PUFAs) using these delta-9 elongases in plants.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 60 / 739,989, filed Nov. 23, 2005, the entire content of which is herein incorporated by reference.FIELD OF THE INVENTION [0002] This invention pertains to polynucleotide sequences encoding delta-9 elongases and the use of these elongase in making long-chain polyunsaturated fatty acids (PUFAs). BACKGROUND OF THE INVENTION [0003] Fatty acids (lipids) are water-insoluble organic biomolecules that can be extracted from cells and tissues by nonpolar solvents such as chloroform, ether or benzene. Lipids have several important biological functions, serving as (1) structural components of membranes; (2) storage and transport forms of metabolic fuels; (3) a protective coating on the surface of many organisms; and, (4) cell-surface components concerned in cell recognition, species specificity and tissue immunity. More specifically, polyunsaturated fatty acids (PUFAs) are important components of the plasma membrane o...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01H5/00C11B1/00C07H21/04C12N9/10C12N15/82C12N5/04
CPCC11B1/025C12N9/1029C12P7/6472C12P7/6427C12P7/6463C12N15/8247C12N9/10C12N15/82C12P7/6458
Inventor DAMUDE, HOWARD GLENNMCGONIGLE, BRIANZHU, QUINN QUNXUE, ZHIXIONG
Owner EI DU PONT DE NEMOURS & CO
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