Schizochytrium PKS genes

Details Schizochytrium PKS genes Schizochytrium PKS genes

1999-01-14

2003-05-20

McElwain, Elizabeth F. (Department: 1649)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide alters fat, fatty oil, ester-type wax, or...

C536S023200, C435S419000, C435S252300, C435S320100

Reexamination Certificate

active

06566583

ABSTRACT:

INTRODUCTION
1. Field of the Invention
This invention relates to modulating levels of enzymes and/or enzyme components capable of modifying long chain poly-unsaturated fatty acids (PUFAs) in a host cell, and constructs and methods for producing PUFAs in a host cell. The invention is exemplified by production of eicosapentenoic acid (EPA) using genes derived from
Shewanella putrefaciens
and
Vibrio marinus.
2. Background
Two main families of poly-unsaturated fatty acids (PUFAs) are the &ohgr;3 fatty acids, exemplified by eicosapentenoic acid, and the &ohgr;6 fatty acids, exemplified by arachidonic acid. PUFAs are important components of the plasma membrane of the cell, where they can be found in such forms as phospholipids, and also can be found in triglycerides. PUFAs also serve as precursors to other molecules of importance in human beings and animals, including the prostacyclins, leukotrienes and prostaglandins. Long chain PUFAs of importance include docosahexenoic acid (DHA) and eicosapentenoic acid (EPA), which are found primarily in different types of fish oil, gamma-linolenic acid (GLA), which is found in the seeds of a number of plants, including evening primrose (
Oenothera biennis
), borage (
Borago officinalis
) and black currants (
Ribes nigrum
), stearidonic acid (SDA), which is found in marine oils and plant seeds, and arachidonic acid (ARA), which along with GLA is found in filamentous fungi. ARA can be purified from animal tissues including liver and adrenal gland. Several genera of marine bacteria are known which synthesize either EPA or DHA. DHA is present in human milk along with ARA.
PUFAs are necessary for proper development, particularly in the developing infant brain, and for tissue formation and repair. As an example, DHA, is an important constituent of many human cell membranes, in particular nervous cells (gray matter), muscle cells, and spermatozoa and believed to affect the development of brain functions in general and to be essential for the development of eyesight. EPA and DHA have a number of nutritional and pharmacological uses. As an example adults affected by diabetes (especially non insulin-dependent) show deficiencies and imbalances in their levels of DHA which are believed to contribute to later coronary conditions. Therefore a diet balanced in DHA may be beneficial to diabetics.
For DHA, a number of sources exist for commercial production including a variety of marine organisms, oils obtained from cold water marine fish, and egg yolk fractions. The purification of DHA from fish sources is relatively expensive due to technical difficulties, making DHA expensive and in short supply. In algae such as Amphidinium and Schizochytrium and marine fungi such as Thraustochytrium DHA may represent up to 48% of the fatty acid content of the cell. A few bacteria also are reported to produce DHA. These are generally deep sea bacteria such as
Vibrio marinus
. For ARA, microorganisms including the genera Mortierella, Entomophthora, Phytium and Porphyridium can be used for commercial production. Commercial sources of SDA include the genera Trichodesma and Echium. Commercial sources of GLA include evening primrose, black currants and borage. However, there are several disadvantages associated with commercial production of PUFAs from natural sources. Natural sources of PUFA, such as animals and plants, tend to have highly heterogeneous oil compositions. The oils obtained from these sources can require extensive purification to separate out one or more desired PUFA or to produce an oil which is enriched in one or more desired PUFA.
Natural sources also are subject to uncontrollable fluctuations in availability. Fish stocks may undergo natural variation or may be depleted by overfishing. Animal oils, and particularly fish oils, can accumulate environmental pollutants. Weather and disease can cause fluctuation in yields from both fish and plant sources. Cropland available for production of alternate oil-producing crops is subject to competition from the steady expansion of human populations and the associated increased need for food production on the remaining arable land. Crops which do produce PUFAs, such as borage, have not been adapted to commercial growth and may not perform well in monoculture. Growth of such crops is thus not economically competitive where more profitable and better established crops can be grown. Large -scale fermentation of organisms such as Shewanella also is expensive. Natural animal tissues contain low amounts of ARA and are difficult to process. Microorganisms such as Porphyridium and Shewanella are difficult to cultivate on a commercial scale.
Dietary supplements and pharmaceutical formulations containing PUFAs can retain the disadvantages of the PUFA source. Supplements such as fish oil capsules can contain low levels of the particular desired component and thus require large dosages. High dosages result in ingestion of high levels of undesired components, including contaminants. Care must be taken in providing fatty acid supplements, as overaddition may result in suppression of endogenous biosynthetic pathways and lead to competition with other necessary fatty acids in various lipid fractions in vivo, leading to undesirable results. For example, Eskimos having a diet high in &ohgr;3 fatty acids have an increased tendency to bleed (U.S. Pat. No. 4,874,603). Fish oils have unpleasant tastes and odors, which may be impossible to economically separate from the desired product, such as a food supplements. Unpleasant tastes and odors of the supplements can make such regimens involving the supplement undesirable and may inhibit compliance by the patient.
A number of enzymes have been identified as being involved in PUFA biosynthesis. Linoleic acid (LA, 18:2 &Dgr;9, 12) is produced from oleic acid (18:1 &Dgr;9) by a &Dgr;12-desaturase. GLA (18:3 &Dgr;9, 12) is produced from linoleic acid (LA, 18:2 &Dgr;9, 12) by a A6-desaturase. ARA (20:4 &Dgr;5, 8, 11, 14) is produced from DGLA (20:3 &Dgr;8, 11, 14), catalyzed by a &Dgr;5-desaturase. Eicosapentenoic acid (EPA) is a 20 carbon, omega 3 fatty acid containing 5 double bonds (&Dgr;5, 8, 11, 14, 17), all in the cis configuration. EPA, and the related DHA (&Dgr;4, 7, 10, 13, 16, 19, C22:6) are produced from oleic acid by a series of elongation and desaturation reactions. Additionally, an elongase (or elongases) is required to extend the 18 carbon PUFAs out to 20 and 22 carbon chain lengths. However, animals cannot convert oleic acid (18:1 &Dgr;9) into linoleic acid (18:2 &Dgr;9, 12). Likewise, &mgr;-linolenic acid (ALA, 18:3 &Dgr;9, 12, 15) cannot be synthesized by mammals. Other eukaryotes, including fungi and plants, have enzymes which desaturate at positions &Dgr;12 and &Dgr;15. The major poly-unsaturated fatty acids of animals therefore are either derived from diet and/or from desaturation and elongation of linoleic acid (18:2 &Dgr;9, 12) or &mgr;-linolenic acid (18:3 &Dgr;9, 12, 15).
Poly-unsaturated fatty acids are considered to be useful for nutritional, pharmaceutical, industrial, and other purposes. An expansive supply of poly-unsaturated fatty acids from natural sources and from chemical synthesis are not sufficient for commercial needs. Because a number of separate desaturase and elongase enzymes are required for fatty acid synthesis from linoleic acid (LA, 18:2 &Dgr;9, 12), common in most plant species, to the more saturated and longer chain PUFAs, engineering plant host cells for the expression of EPA and DHA may require expression of five or six separate enzyme activities to achieve expression, at least for EPA and DHA, and for production of quantities of such PUFAs additional engineering efforts may be required, for instance the down regulation of enzymes competing for substrate, engineering of higher enzyme activities such as by mutagenesis or targeting of enzymes to plastid organelles. Therefore it is of interest to obtain genetic material involved in PUFA biosynthesis from species that naturally produce these fatty acids and to express the isolated material alone or in c

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