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Delta15 desaturases suitable for altering levels of polyunsaturated fatty acids in oilseed plants and oleaginous yeast

a technology of oleaginous yeast and desaturases, applied in the field of biotechnology, can solve the problems of high heterogeneous oil composition of natural sources such as fish and plants, uncontrollable fluctuations in availability of natural sources, and poor economic competitiveness of crops producing pufas with hybrid crops developed, and achieve the effect of increasing the ratio of -3 fatty acids to -6 fatty acids

Inactive Publication Date: 2005-06-16
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes how scientists have introduced a new gene that produces a protein with high similarity to another known protein. This introduction results in higher levels of omega-3 fats which are beneficial for human health.

Problems solved by technology

The technical problem addressed in this patent text is finding new ways to express genes responsible for producing commercially important polyunsaturated fats (PUFAs) in oleaginous yeast and other host systems such as plants and mammals. Specifically, the text discusses identifying and isolating genes coding for Δ-15 desaturases, which play a crucial role in converting linoleic acid to α-linolenic acid. While some researchers have attempted to solve this issue through various means, there still remain uncertainties regarding the effectiveness of current solutions.

Method used

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  • Delta15 desaturases suitable for altering levels of polyunsaturated fatty acids in oilseed plants and oleaginous yeast
  • Delta15 desaturases suitable for altering levels of polyunsaturated fatty acids in oilseed plants and oleaginous yeast
  • Delta15 desaturases suitable for altering levels of polyunsaturated fatty acids in oilseed plants and oleaginous yeast

Examples

Experimental program
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Effect test

example 1

Construction of Yarrowia Expression Vectors

[0426] The present Example describes the construction of pY5-13 (comprising a chimeric TEF promoter::XPR terminator gene), pY5-13GPDN (comprising a chimeric GPD promoter::XPR terminator gene), and pY5-20 (comprising a chimeric hygromycin resistance gene).

Construction of Plasmid PY5-13

[0427] The plasmid pY5, a derivative of pINA532 (a gift from Dr. Claude Gaillardin, Insitut National Agronomics, Centre de biotechnologie Agro-Industrielle, laboratoire de Genetique Moleculaire et Cellularie INRA-CNRS, F-78850 Thiverval-Grignon, France), was constructed for expression of heterologous genes in Yarrowia lipolytica (FIG. 3). First, the the partially-digested 3598 bp EcoRI fragment containing the ARS18 sequence and LEU2 gene of pINA532 was subcloned into the EcoRI site of pBluescript (Strategene, San Diego, Calif.) to generate pY2. The TEF promoter (Muller S., et al., Yeast, 14: 12671283 (1998)) was amplified from Yarrowia lipolytica genomic DNA...

example 2

Cloning of the Yarrowia Lipolytica Δ12 Desaturase and Disruption of the Endogenous Δ12 Desaturase Gene

[0441] Based on the fatty acid composition of Yarrowia lipolytica (ATCC #76982) which demonstrated that the organism could make LA (18:2) but not ALA (18:3), it was assumed that Y. lipolytica would likely contain gene(s) having Δ12 desaturase activity but not Δ15 desaturase activity. Thus, the present Example describes the use of degenerate PCR primers to isolate a partial coding sequence of the Yarrowia lipolytica Δ12 desaturase, the use of the partial sequence to disrupt the native gene in Yarrowia lipolytica, and subsequent cloning of the full-length gene.

Cloning of a Partial Putative Δ12 Desaturase Sequence from Yarrowia lipolytica by PCR Using Degenerate PCR Primers

[0442] Genomic DNA was isolated from Yarrowia lipolytica (ATCC #76982) using DNeasy Tissue Kit (Qiagen, Catalog #69504) and resuspended in kit buffer AE at a DNA concentration of 0.5 μg / μl. PCR amplifications were...

example 3

Identification of Δ15 Desaturases from Filamentous Fungi

[0458] The present Example describes the identification of Δ15 desaturases in various filamentous fungi. These sequences were identified based on their homology to the Yarrowia lipolytica Δ12 desaturase (Example 2); and, the sequences from each species fell into one of two “sub-families” based on phylogenetic analyses.

Homology Searches With Synechochytis Δ15 Desaturase

[0459] First, public databases of the filamentous fungi Neurospora crassa and Magnaporthe grisea sequences were subjected to BLAST searches (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993)) using the Synechochytis Δ15 desaturase protein sequence (gene desB; GenBank Accession No. D90913) as the query sequence. Unexpectedly, these searches failed to identify any homologous sequence.

Homology Searches with Yarrowia lipolytica Δ12 Desaturase

[0460] Applicants then performed BLAST searches of the same databases with the Y...

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Abstract

The present invention relates to fungal Δ-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 Δ-15 fatty acid desaturases are also described herein.

Description

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Claims

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Application Information

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Owner EI DU PONT DE NEMOURS & CO
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