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

Abstract

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.

Description

[0001]This application claims the benefit of U.S. Provisional Applications No. 60 / 977,174 and No. 60 / 977,177, both filed Oct. 3, 2007 and both hereby incorporated herein by reference in their entirety.FIELD OF THE INVENTION[0002]This invention is in the field of biotechnology. More specifically, this invention pertains to methods useful for manipulating the polyunsaturated fatty acid (PUFA) composition and lipid content of eukaryotic organisms, based on disruption of peroxisome biogenesis factor (Pex) proteins.BACKGROUND OF THE INVENTION[0003]The health benefits associated with polyunsaturated fatty acids [“PUFAs”], especially ω-3 and ω-6 PUFAs, have been well documented. In order to find ways to produce large-scale quantities of ω-3 and ω-6 PUFAs, researchers have directed their work toward the discovery of genes and the understanding of the encoded biosynthetic pathways that result in lipids and fatty acids.[0004]One effort to produce these PUFAs has introduced ω-3 / ω-6 PUFA biosyn...

AI Technical Summary

Benefits of technology

The patent describes a method for increasing the amount of polyunsaturated fatty acids (PUFAs) in an oleaginous eukaryotic organism. This is achieved by disrupting a native gene encoding a peroxisome biogenesis factor protein, which results in the organism having a higher weight percent of PUFAs compared to a control organism. The method can be applied to various oleaginous eukaryotic organisms, such as Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Saccharomyces. The PUFAs can include linoleic acid, conjugated linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, docosatetraenoic acid, and others. The increased PUFAs can be in the form of single or a combination of PUFAs. The method can also involve increasing the total lipid content or oil fraction of the organism.

Problems solved by technology

However, none of the preliminary results demonstrating limited production of linoleic acid [“LA”], γ-linolenic acid [“GLA”], α-linolenic acid [“ALA”], stearidonic acid [“STA”] and / or eicosapentaenoic acid [“EPA”] are suitable for commercial exploitation.

All these efforts suffer from an inability to substantially improve the yield of oil or to control the characteristics of the oil composition produced, since the fermentations rely on the natural abilities of the microbes themselves.

But, previous studies of Pex knockouts have not been performed in a PUFA-producing organism.

Applicants have solved the stated problem of optimizing host cells for commercial production of PUFAs by the unpredictable mechanism of disruption of peroxisome biogenesis factor proteins in a PUFA-producing organism, which leads to the unpredictable result of an increase in the amount of PUFAs, as a percent of total fatty acids, in a recombinant PUFA-producing strain of Y. lipolytica.

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