Fungal delta 12 desaturase and delta 15 desaturase motifs

Inactive Publication Date: 2007-11-01
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]The invention relates to the discovery of motifs present in desaturases enzymes having either Δ12 or Δ15 desaturase activity. The Δ12 desaturase motifs are diagnostic for Δ12 desaturase activity; and conversely, th

Problems solved by technology

Additionally, no facile sequence-based method is available to fac

Method used

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  • Fungal delta 12 desaturase and delta 15 desaturase motifs
  • Fungal delta 12 desaturase and delta 15 desaturase motifs
  • Fungal delta 12 desaturase and delta 15 desaturase motifs

Examples

Experimental program
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Example

Example 1

Description Of Δ12 Knockout Yarrowia Strain L38

[0266]Strain L38 is a Δ12 desaturase-disrupted strain of Yarrowia lipolytica similar to the d12KO strain described as Q-d12D (PCT Publication No. WO 2004 / 10416). It was also derived from Y. lipolytica ATCC #76982 following disruption of its only native Δ12 desaturase gene by replacement with a disrupted version via homologous recombination.

Selection Method Theory

[0267]The methodology used to create the d12KO strain identified herein as L38 relied on site-specific recombinase systems. Briefly, the site-specific recombination system consists of two elements: (1) a recombination site having a characteristic DNA sequence [e.g., LoxP]; and, (2) a recombinase enzyme that binds to the DNA sequence specifically and catalyzes recombination (i.e., excision) between DNA sequences when two or more of the recombination sites are oriented in the same direction at a given interval on the same DNA molecule [e.g., Cre]. For the purposes herein,...

Example

Example 2

Identification Of Fungal Sequences Encoding Δ15 Desaturases

[0274]“Pairs” of Δ12 / Δ15 desaturase-like polypeptides have previously been identified in the following filamentous fungi: Fusarium moniliforme, Fusarium graminearum, Magneporthe grisea, Neurospora crassa, Aspergillus nidulans, Mortirerella alpina and Saccharomyces kluveromyces. In each case, one protein was subsequently determined (or predicted) to possess Δ12 desaturase activity while the other protein was determined to possess Δ15 desaturase activity (PCT Publication No. WO 2005 / 047480; PCT Publication No. WO 2005 / 047485; Sakuradani et al., Appl. Microbiol. Biotechnol., 66:648-654 (2005); Oura, T. and Kajiwara, S., Microbiology (Reading, Engl.), 150(6):1983-1990 (2004); Sakuradani, E., et al., Eur. J. Biochem., 261(3):812-820 (1999); Watanabe, K., et al., Biosci. Biotechnol. Biochem., 68(3):721-727 (2004)). More specifically, the following proteins were previously characterized as Δ15 desaturases: Fm.d15 (SEQ ID N...

Example

Example 3

Amino Acids Motifs For Identification Of Fungal Δ12 And Δ15 Desaturases

[0290]Comparison of the different fungal Δ12 / Δ15 desaturase-like polypeptides (i.e., including both known and putative Δ12 and Δ15 desaturase sequences) from sixteen different fungal species (Table 4, supra) enabled the identification of regions of significant homology between the genes, such as the 15 amino acids surrounding and including the conserved His Box I (“HE[C / A]GH”; SEQ ID NO:6). More specifically, a total of 7 different sequence variants within this region (i.e., from 6 residues upstream of the His Box I to 4 residues downstream of the His Box I) were identified within the Δ12 desaturases (i.e., SEQ ID NOs:7, 8, 9, 10, 11, 12 and 13), while a total of 9 different sequence variants within this region were identified within the Δ15 desaturases (i.e., SEQ ID NOs:22, 23, 24, 25, 26, 27, 28, 29 and 30). These conserved sequences are summarized below in Tables 12 and 13 (wherein the shaded portion ...

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Abstract

The present invention relates to fungal Δ12 desaturases (responsible for conversion of oleic acid to linoleic acid (18:2, LA)) and Δ15 fatty acid desaturases (responsible for conversion of LA to α-linolenic acid (18:3, ALA)). Amino acid motifs diagnostic of Δ12 desaturases and Δ15 desaturases are also provided. Methods of altering enzyme specificity towards Δ12 desaturation or towards Δ15 desaturation and/or increasing production of specific ω-3 and ω-6 fatty acids by over-expression of the fungal desaturases are also described.

Description

[0001]This application claims the benefit of U.S. Provisional Application No. 60 / 796637, filed May 1, 2006.FIELD OF THE INVENTION[0002]This invention is in the field of biotechnology. More specifically, this invention pertains to the identification of nucleic acid fragments encoding fungal motifs indicative of Δ15 fatty acid desaturase enzymes and Δ12 fatty acid desaturase enzymes.BACKGROUND OF THE INVENTION[0003]The importance of long chain polyunsaturated fatty acids (PUFAs) is undisputed. For example, certain PUFAs are important biological components of healthy cells and are recognized as: “essential” fatty acids that cannot be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further desaturation and elongation of linoleic acid (LA;18:2 ω-6) or α-linolenic acid (ALA; 18:3 ω-3); constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or triacylglycerols; necessary for proper development (particul...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C07H21/04C12P7/64C12N9/04C12N1/18
CPCC12N9/001C12N9/0083C12Q1/6895C12P7/6472C12P7/6427C12P7/6431
Inventor YADAV, NARENDRA S.ZHANG, HONGXIANG
Owner EI DU PONT DE NEMOURS & CO
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