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

a biogenesis factor and peroxisome technology, applied in the field of biotechnology, can solve the problems of unsuitable commercial exploitation for any preliminary results demonstrating limited production of linoleic acid, and achieve the effect of increasing or decreasing the total lipid content of the pex-disrupted organism

Inactive Publication Date: 2009-05-07
EI DU PONT DE NEMOURS & CO
View PDF8 Cites 29 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015]In some of the described methods, the total lipid content in the PEX-disrupted organism may be increased or decreased compared with that of an oleaginous eukaryote having no disruption in a native PEX gene.

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.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Peroxisome biogenesis factor protein (PEX) disruptions for altering polyunsaturated fatty acids and total lipid content in oleaginous eukaryotic organisms
  • Peroxisome biogenesis factor protein (PEX) disruptions for altering polyunsaturated fatty acids and total lipid content in oleaginous eukaryotic organisms
  • Peroxisome biogenesis factor protein (PEX) disruptions for altering polyunsaturated fatty acids and total lipid content in oleaginous eukaryotic organisms

Examples

Experimental program
Comparison scheme
Effect test

example 1

Generation of Yarrowia lipolytica Strain Y4086 to Produce about 14% EPA of Total Lipids Via the Δ9 Elongase / Δ8 Desaturase Pathway

[0240]The present Example describes the construction of strain Y4086, derived from Yarrowia lipolytica ATCC #20362, capable of producing about 14% EPA relative to the total lipids via expression of a Δ9 elongase / Δ8 desaturase pathway (FIG. 3A).

[0241]The development of strain Y4086 required the construction of strain Y2224 (a FOA resistant mutant from an autonomous mutation of the Ura3 gene of wildtype Yarrowia strain ATCC #20362), strain Y4001 (producing 17% EDA with a Leu− phenotype), strain Y4001U (Leu− and Ura− phenotype), strain Y4036 (producing 18% DGLA with a Leu− phenotype), strain Y4036U (Leu− and Ura− phenotype) and strain Y4070 (producing 12% ARA with a Ura− phenotype). Further details regarding the construction of strains Y2224, Y4001, Y4001U, Y4036, Y4036U and Y4070 are described in Example 7 of Int'l. App. Pub. No. WO 2008 / 073367, hereby incor...

example 2

Generation of Yarrowia Lipolytica Strain Y4128 to Produce about 37% EPA of Total Lipids Via the Δ9 Elongase / Δ8 Desaturase Pathway

[0247]The present Example describes the construction of strain Y4128, derived from Yarrowia lipolytica ATCC #20362, capable of producing about 37.6% EPA relative to the total lipids (i.e., greater than a 2-fold increase in EPA concentration as percent of total fatty acids with respect to Y4086; FIG. 3A).

[0248]The development of strain Y4128 required the construction of strains Y2224, Y4001, Y4001U, Y4036, Y4036U, Y4070 and Y4086 (described in Example 1), as well as construction of strain Y4086U1 (Ura−).

Generation Of Strain Y4086U1 (Ura−)

[0249]Strain Y4086U1 was created via temporary expression of the Cre recombinase enzyme in construct pY117 (FIG. 4A; SEQ ID NO:29; described in Table 20 of Int'l. App. Pub. No. WO 2008 / 073367, hereby incorporated herein by reference) within strain Y4086 to produce a Ura− phenotype. This released the LoxP sandwiched Ura3 gen...

example 3

Determination of Total Lipid Content of Yarrowia lipolytica Strain Y4128

[0259]The total amount of lipid produced by strain Y4128 and the percentage of each fatty acid species in the lipid were measured by GC analysis. Specifically, total lipids were extracted, and FAMEs were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC, as described in the General Methods.

[0260]Dry cell weight was determined by collecting cells from 10 mL of culture via centrifugation, washing the cells with water once to remove residual medium, drying the cells in a vacuum oven at 80° C. overnight, and weighing the dried cells. The total amount of FAMEs in a sample was determined by comparing the areas of all peaks in the GC profile with the peak area of an added known amount of internal standard C15:0 fatty acid.

[0261]Based on the above analyses, lipid content as a percentage of dry cell weight (DCW) and lipid composition was determined for strains Y4086 and Y4128. Str...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
weight percentaaaaaaaaaa
weightaaaaaaaaaa
fatty acidaaaaaaaaaa
Login to view more

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...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(United States)
IPC IPC(8): A23K1/16C12N15/63C12N1/19
CPCC12N9/0083C12N9/1029C12N15/815C12P7/6472C12P7/6427A61P1/00A61P1/16A61P17/02A61P19/02A61P19/10A61P25/00A61P25/18A61P25/24A61P25/28A61P29/00A61P3/00A61P35/00A61P3/06A61P5/24A61P9/00A61P9/10A61P9/12A61P3/10C12P7/6432C12P7/6458
Inventor HONG, SEUNG-PYOSHARPE, PAMELA L.XUE, ZHIXIONGYADAV, NARENDRA S.ZHU, QUINN QUN
Owner EI DU PONT DE NEMOURS & CO
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap