# Patent

## Title
Yeast strains and their uses in the production of lipids

## Abstract

The invention is directed to isolated microorganisms, as well as biomasses, cultures, microbial oils, and compositions thereof. The invention also provides methods of producing the microbial oils and methods of using the isolated microorganisms, biomasses, and microbial oils.

## Background
The present application claims benefit of U.S. provisional application Nos. 61/445,469 and 61/313,055, filed Feb. 22, 2011 and Mar. 11, 2010, respectively.


REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The content of the electronically submitted sequence listing (Name: 2715.2390001_SequenceListing_ascii.txt; Size: 5,788 bytes; and Date of Creation: Oct. 19, 2011) filed with the application is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention is directed to isolated microorganisms, as well as biomasses, cultures, microbial oils, and compositions thereof. The invention also provides methods of producing the microbial oils and methods of using the isolated microorganisms, biomasses, cultures, and microbial oils.
2. Background Art
Fatty acids are classified based on the length and saturation characteristics of the carbon chain. Fatty acids are termed saturated fatty acids when no double bonds are present between the carbon atoms and are termed unsaturated fatty acids when double bonds are present. Unsaturated long chain fatty acids are monounsaturated when only one double bond is present and are polyunsaturated when more than one double bond is present.
Polyunsaturated fatty acids (“PUFAs”) are classified based on the position of the first double bond from the methyl end of the fatty acid: omega-3 (n-3) fatty acids contain a first double bond at the third carbon, while omega-6 (n-6) fatty acids contain a first double bond at the sixth carbon. For example, docosahexaenoic acid (“DHA”) is an omega-3 long chain polyunsaturated fatty acid (“LC-PUFA”) with a chain length of 22 carbons and 6 double bonds, often designated as “22:6 n-3.” Other omega-3 LC-PUFAs include eicosapentaenoic acid (“EPA”), designated as “20:5 n-3,” and omega-3 docosapentaenoic acid (“DPA n-3”), designated as “22:5 n-3.” DHA and EPA have been termed “essential” fatty acids. Omega-6 LC-PUFAs include arachidonic acid (“ARA”), designated as “20:4 n-6,” and omega-6 docosapentaenoic acid (“DPA n-6”), designated as “22:5 n-6.”
The production of biological oils from sources such as plants (including oilseeds), microorganisms, and animals is essential for various purposes. For example, it is desirable to increase the dietary intake of many beneficial nutrients found in biological oils. Particularly beneficial nutrients include fatty acids such as omega-3 and omega-6 fatty acids and esters thereof. Omega-3 fatty acids are recognized as important dietary compounds for preventing arteriosclerosis and coronary heart disease, for alleviating inflammatory conditions and for retarding the growth of tumor cells. Omega-6 fatty acids serve not only as structural lipids in the human body, but also as precursors for a number of factors in inflammation, such as prostaglandins, leukotrienes, and oxylipins.
Because humans and many other animals cannot directly synthesize omega-3 and omega-6 essential fatty acids, they must be obtained in the diet. Traditional dietary sources of essential fatty acids include vegetable oils, marine animal oils, fish oils and oilseeds. In addition, oils produced by certain microorganisms have been found to be rich in essential fatty acids.
Oleic acid is another important beneficial fatty acid. Oleic acid is an omega-9 fatty acid that has been associated with health benefits such as slowing the development of heart disease and promoting the production of antioxidants. It is also used as an ingredient in Lorenzo's oil, a medication developed to prevent the onset of adrenoleukodystrophy (ALD). Oleic acid has also been used as a cosmetic ingredient due to its moisturizing effect.
Linoleic acid is also an example of an important fatty acid. It is an unsaturated omega-6 fatty acid that is essential to various biological processes, such as those involved in hair loss, wound healing, cystic fibrosis, dermatitis, and diabetes. Linoleic acid is also used as a cosmetic ingredient due to its beneficial effects on the skin, and in the making of soaps and emulsifiers.
In order to reduce the costs associated with the production of beneficial fatty acids for dietary, pharmaceutical, and cosmetic uses, there exists a need for a low-cost and efficient method of producing biological oils containing these fatty acids.
BRIEF SUMMARY OF THE INVENTION
The present invention is directed to an isolated microorganism of the species selected from the group consisting of Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, and Rhodotorula ingeniosa, wherein the microorganism is capable of producing a microbial oil comprising less than 30% by weight saturated fatty acids.
In some embodiments, the isolated microorganisms are capable of producing fatty acids in an amount that is at least 30% by weight of the dry cell weight. In some embodiments, the isolated microorganisms are capable of producing fatty acids in an amount that is at least 40%, at least 50%, or at least 60% by weight of the dry cell weight.
In some embodiments, the isolated microorganisms of the invention are capable of producing a microbial oil comprising greater than 30% by weight, greater than 40% by weight, greater than 50% by weight, or greater than 60% by weight oleic acid.
In some embodiments, the isolated microorganisms of the invention are capable of producing a microbial oil comprising greater than 5% by weight, greater than 10% by weight, or greater than 15% by weight linoleic acid.
In some embodiments, the isolated microorganisms of the invention are capable of producing a microbial oil comprising less than 25% by weight saturated fatty acids. In some embodiments, the microorganisms of the invention are capable of producing a microbial oil comprising less than 25% by weight, less than 20% by weight, or less than 15% by weight palmitic acid.
In some embodiments, the isolated microorganisms of the invention are capable of producing a microbial oil comprising less than 10% by weight, or less than 5% by weight long chain polyunsaturated fatty acids of 20 or more carbon chain length.
The present invention is directed to an isolated microorganism of the species selected from the group consisting of Pseudozyma aphidis, Pseudozyma rugulosa, and Rhodotorula ingeniosa, wherein the microorganism is capable of producing fatty acids in an amount that is at least 30% by weight of the dry cell weight. In some embodiments, the isolated microorganisms of the species selected from the group consisting of Pseudozyma aphidis, Pseudozyma rugulosa, and Rhodotorula ingeniosa are capable of producing fatty acids in an amount that is at least 40% or at least 50% by weight of the dry cell weight.
The present invention is further directed to an isolated microorganism of the Sporidiobolus pararoseus species, wherein the microorganism is capable of producing fatty acids in an amount that is at least 50% by weight of the dry cell weight. In some embodiments, the isolated microorganisms of the Sporidiobolus pararoseus species are capable of producing fatty acids in an amount that is at least 55% or at least 60% by weight of the dry cell weight.
The present invention is directed to an isolated microorganism of the species selected from the group consisting of Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, and Rhodotorula ingeniosa, wherein the microorganism is capable of producing a microbial oil comprising 0.5% to 30% by weight saturated fatty acids, and wherein the microorganism is capable of producing fatty acids in an amount that is 30% to 80% by weight of the dry cell weight.
The present invention is also directed to an isolated microorganism of the species selected from the group consisting of Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, and Rhodotorula ingeniosa, wherein the microorganism is capable of producing a microbial oil comprising 30% to 70% by weight oleic acid and 5% to 30% by weight linoleic acid.
The present invention is further directed to an isolated microorganism of the Sporidiobolus pararoseus species, wherein the microorganism is capable of producing fatty acids in an amount that is 50% to 80% by weight of the dry cell weight, and wherein the microorganism is capable of producing a microbial oil comprising 5% to 30% by weight linoleic acid.
The present invention is directed to an isolated microorganism deposited under ATCC Accession No. PTA-11615, an isolated microorganism deposited under ATCC Accession No. PTA-11616, and an isolated microorganism deposited under ATCC Accession No. PTA-11617.
The present invention is also directed to an isolated microorganism having the characteristics of the microorganism deposited under ATCC Accession No. PTA-11615, an isolated microorganism having the characteristics of the microorganism deposited under ATCC Accession No. PTA-11616, and an isolated microorganism having the characteristics of the microorganism deposited under ATCC Accession No. PTA-11617.
The present invention is directed to an isolated biomass comprising an isolated microorganism of the invention or mixtures of the isolated microorganisms of the invention.
In some embodiments, at least 30% by weight of the dry cell weight of the isolated biomass are fatty acids.
The present invention is also directed to a culture comprising an isolated microorganism of invention or mixtures of the isolated microorganisms of the invention.
In some embodiments, the culture comprises at least 5 g/L of biomass of the isolated microorganism.
The present invention is directed to a method for producing a microbial oil, comprising: growing an isolated microorganism of the invention or mixtures of isolated microorganisms of the invention in a culture to produce a microbial oil.
In some embodiments, the method further comprises extracting the microbial oil.
In some embodiments, the isolated microorganisms are grown in the presence of a carbon source selected from the group consisting, of sucrose, glucose, fructose, xylose, glycerol, mannose, arabinose, lactose, galactose, maltose, cellulose, lignocellulose, and combinations thereof.
In some embodiments, the method produces a culture comprising biomass of the isolated microorganism, and the culture comprises at least 5 g/L of the biomass.
The present invention is further directed to microbial oils produced by the methods of the invention, and to the use of the isolated microorganism, biomass, culture, or microbial oil of the invention for the manufacture of a food, supplement, cosmetic, or pharmaceutical composition for a non-human animal or human.



BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
FIG. 1 shows the DNA sequences of strain 28428 (ATCC Accession No. PTA-11615), including the 28428 D1D2 sequence (SEQ ID NO: 1) and the 28428 ITS sequence (SEQ ID NO: 2).
FIG. 2 shows the phylogenetic tree of strain 28428 (ATCC Accession No. PTA-11615) D1/D2 DNA sequences.
FIG. 3 shows the phylogenetic tree of strain 28428 (ATCC Accession No. PTA-11615) ITS DNA sequences.
FIG. 4 shows the DNA sequences of strain 29404 (ATCC Accession No. PTA-11616), including the 29404 D1D2 sequence (SEQ ID NO: 3) and the 29404 ITS sequence (SEQ ID NO: 4).
FIG. 5 shows the phylogenetic tree of strain 29404 (ATCC Accession No. PTA-11616) D1/D2 DNA sequences.
FIG. 6 shows the phylogenetic tree of strain 29404 (ATCC Accession No. PTA-11616) ITS DNA sequences.
FIG. 7 shows the DNA sequences of strain 29794 (ATCC Accession No. PTA-11617), including the 29794 D1D2 sequence (SEQ ID NO: 5) and the 29794 ITS sequence (SEQ ID NO: 6).
FIG. 8 shows the phylogenetic tree of strain 29794 (ATCC Accession No. PTA-11617) D1/D2 DNA sequences.
FIG. 9 shows the phylogenetic tree of strain 29794 (ATCC Accession No. PTA-11617) ITS DNA sequences.



DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to isolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosa species, as well as microorganisms having the characteristics of the isolated microorganisms, and biomasses, microbial oils, compositions, and cultures thereof. The present invention is also directed to methods of producing microbial oils from the isolated microorganisms of the invention, and methods of using the isolated microorganisms, biomasses, cultures, and microbial oils. The isolated microorganisms described herein are highly productive as compared to prior isolates and produce unique fatty acid profiles, characterized in part by high levels of short-chain unsaturated fatty acids, low levels of saturated fatty acids, and low levels of long chain polyunsaturated fatty acids.
In some embodiments, the isolated microorganisms of the invention are capable of producing fatty acids in an amount that is at least 30% by weight of the dry cell weight. In some embodiments, the isolated microorganisms of the invention are capable of producing fatty acids in an amount that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% by weight of the dry cell weight. In some embodiments, the isolated microorganisms of the invention are capable of producing fatty acids in an amount that is 30% to 80% by weight of the dry cell weight, 35% to 80% by weight of the dry cell weight, 40% to 80% by weight of the dry cell weight, 45% to 75% by weight of the dry cell weight, 50% to 75% by weight of the dry cell weight, 55% to 70% by weight of the dry cell weight, or 60% to 70% by weight of the dry cell weight.
In some embodiments, the invention is directed to an isolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, or Rhodotorula ingeniosa species, wherein the microorganism is capable of producing fatty acids in an amount that is at least 30% by weight of the dry cell weight. In some embodiments, an isolated microorganisms of the Pseudozyma aphidis, Pseudozyma rugulosa, or Rhodotorula ingeniosa species are capable of producing fatty acids in an amount that is at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% by weight of the dry cell weight. In some embodiments, the isolated microorganisms of the Pseudozyma aphidis, Pseudozyma rugulosa, or Rhodotorula ingeniosa species are capable of producing fatty acids in an amount that is 30% to 80% by weight of the dry cell weight, 35% to 75% by weight of the dry cell weight, 40% to 70% by weight of the dry cell weight, 45% to 70% by weight of the dry cell weight, 50% to 65% by weight of the dry cell weight, or 55% to 65% by weight of the dry cell weight.
The present invention is also directed to an isolated microorganism of the Sporidiobolus pararoseus species, wherein the microorganism is capable of producing fatty acids in an amount that is greater than 45% by weight of the dry cell weight. In some embodiments, the isolated microorganisms of the Sporidiobolus pararoseus species are capable of producing fatty acids in an amount that is at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% by weight of the dry cell weight. In some embodiments, the isolated microorganisms of the Sporidiobolus pararoseus species are capable of producing fatty acids in an amount that is 30% to 85% by weight of the dry cell weight, 40% to 80% by weight of the dry cell weight, 45% to 80% by weight of the dry cell weight, 46% to 80% by weight of the dry cell weight, 47% to 80% by weight of the dry cell weight, 48% to 80% by weight of the dry cell weight, 49% to 80% by weight of the dry cell weight, 50% to 80% by weight of the dry cell weight, 55% to 75% by weight of the dry cell weight, 60% to 70% by weight of the dry cell weight, or 65% to 70% by weight of the dry cell weight.
In some embodiments, the invention is directed to an isolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosa species, wherein the microorganism is capable of producing a microbial oil comprising greater than 30% by weight oleic acid (18:1 n-9). In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, or greater than 65% by weight of oleic acid. In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising from 30% to 70% by weight of oleic acid, from 35% to 70% by weight of oleic acid, from 40% to 65% by weight of oleic acid, from 45% to 65% by weight of oleic acid, from 50% to 65% by weight of oleic acid, from 55% to 65% by weight of oleic acid, or from 60% to 65% by weight of oleic acid.
In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising greater than 5% by weight linoleic acid (18:2). In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising greater than 7%, greater than 10%, greater than 12%, greater than 15%, greater than 17%, or greater than 20% by weight of linoleic acid. In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising from 5% to 30% by weight of linoleic acid, from 7% to 28% by weight of linoleic acid, from 10% to 25% by weight of linoleic acid, from 12% to 25% by weight of linoleic acid, from 15% to 25% by weight of linoleic acid, from 17% to 25% by weight of linoleic acid, or from 20% to 23% by weight of linoleic acid.
In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising less than 30% by weight saturated fatty acids. In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising less than 25%, less than 20%, less than 15%, less than 10%, less than 8%, or less than 5% by weight of saturated fatty acids. In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising from 0.5% to 30% by weight of saturated fatty acids, from 1% to 25% by weight of saturated fatty acids, from 1% to 20% by weight of saturated fatty acids, from 1% to 15% by weight of saturated fatty acids, from 1% to 10% by weight of saturated fatty acids, from 1% to 8% by weight of saturated fatty acids, or from 1% to 5% by weight of saturated fatty acids.
In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising less than 25% by weight palmitic acid (16:0). In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising less than 23%, less than 20%, less than 18%, or less than 15% by weight of palmitic acid. In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising from 0.5% to 25% by weight of palmitic acid, from 1% to 20% by weight of palmitic acid, from 1% to 18% by weight of palmitic acid, from 1% to 15% by weight of palmitic acid, from 5% to 15% by weight of palmitic acid, or from 10% to 15% by weight of palmitic acid.
In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising less than 10% by weight long chain polyunsaturated fatty acids (LC-PUFAs) of 20 or more carbon chain length. In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising less than 8%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% by weight of LCPUFAs of 20 or more carbon chain length. In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising from 0% to 10% by weight, from 0% to 8% by weight, from 0% to 5% by weight, from 0% to 4% by weight, from 0% to 3% by weight, from 0% to 2% by weight, or from 0% to 1% by weight of LCPUFAs of 20 or more carbon chain length. In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil with no detectable amount of LCPUFAs of 20 or more carbon chain length.
In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising less than 10%, less than 8%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% by weight of docosahexaenoic acid (DHA). In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising from 0% to 10% by weight of DHA, from 0% to 8% by weight of DHA, from 0% to 5% by weight of DHA, from 0% to 4% by weight of DHA, from 0% to 3% by weight of DHA, from 0% to 2% by weight of DHA, or from 0% to 1% by weight of DHA. In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil with no detectable amount of DHA.
In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising less than 10%, less than 8%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% by weight of eicosapentaenoic acid (EPA). In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising from 0% to 10% by weight of EPA, from 0% to 8% by weight of EPA, from 0% to 5% by weight of EPA, from 0% to 4% by weight of EPA, from 0% to 3% by weight of EPA, from 0% to 2% by weight of EPA, or from 0% to 1% by weight of EPA. In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil with no detectable amount of EPA.
In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising less than 10%, less than 8%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% by weight of omega-3 docosapentaenoic acid (DPA n-3). In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising from 0% to 10% by weight of DPA n-3, from 0% to 8% by weight of DPA n-3, from 0% to 5% by weight of DPA n-3, from 0% to 4% by weight of DPA n-3, from 0% to 3% by weight of DPA n-3, from 0% to 2% by weight of DPA n-3, or from 0% to 1% by weight of DPA n-3. In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil with no detectable amount of DPA n-3.
In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising less than 10%, less than 8%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% by weight of omega-6 docosapentaenoic acid (DPA n-6). In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising from 0% to 10% by weight of DPA n-6, from 0% to 8% by weight of DPA n-6, from 0% to 5% by weight of DPA n-6, from 0% to 4% by weight of DPA n-6, from 0% to 3% by weight of DPA n-6, from 0% to 2% by weight of DPA n-6, or from 0% to 1% by weight of DPA n-6. In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil with no detectable amount of DPA n-6.
In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising less than 10%, less than 8%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% by weight of omega-6 arachidonic acid (ARA). In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil comprising from 0% to 10% by weight of ARA, from 0% to 8% by weight of ARA, from 0% to 5% by weight of ARA, from 0% to 4% by weight of ARA, from 0% to 3% by weight of ARA, from 0% to 2% by weight of ARA, or from 0% to 1% by weight of ARA. In some embodiments, the isolated microorganism of the invention is capable of producing a microbial oil with no detectable amount of ARA.
In some embodiments, the invention is directed to an isolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosa species, wherein the microorganism is capable of producing a microbial oil comprising 0.5% to 30% by weight saturated fatty acids, and wherein the microorganism is capable of producing fatty acids in an amount that is 30% to 80% by weight of the dry cell weight. In some embodiments, the isolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosa species is capable of producing a microbial oil comprising 1% to 25% by weight saturated fatty acids, and is capable of producing fatty acids in an amount that is 40% to 80% by weight of the dry cell weight. In some embodiments, the isolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosa species is capable of producing a microbial oil comprising 1% to 20% by weight saturated fatty acids, and is capable of producing fatty acids in an amount that is 50% to 80% by weight of the dry cell weight. In some embodiments, the isolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosa species is capable of producing a microbial oil comprising 1% to 20% by weight saturated fatty acids, and is capable of producing fatty acids in an amount that is 55% to 75% by weight of the dry cell weight. In some embodiments, the isolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosa species is capable of producing a microbial oil comprising 1% to 20% by weight saturated fatty acids, and is capable of producing fatty acids in an amount that is 60% to 75% by weight of the dry cell weight. In some embodiments, the isolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosa species is capable of producing a microbial oil comprising 1% to 20% by weight saturated fatty acids, and is capable of producing fatty acids in an amount that is 60% to 70% by weight of the dry cell weight.
In some embodiments, the invention is directed to an isolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosa species, wherein the microorganism is capable of producing a microbial oil comprising 30% to 70% by weight oleic acid and 5% to 30% by weight linoleic acid. In some embodiments, the isolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosa species is capable of producing a microbial oil comprising 35% to 70% by weight oleic acid and 5% to 25% by weight linoleic acid. In some embodiments, the isolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosa species is capable of producing a microbial oil comprising 40% to 65% by weight oleic acid and 5% to 20% by weight linoleic acid. In some embodiments, the isolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosa species is capable of producing a microbial oil comprising 45% to 65% by weight oleic acid and 5% to 20% by weight linoleic acid. In some embodiments, the isolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosa species is capable of producing a microbial oil comprising 50% to 65% by weight oleic acid and 7% to 20% by weight linoleic acid. In some embodiments, the isolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosa species is capable of producing a microbial oil comprising 55% to 65% by weight oleic acid and 7% to 15% by weight linoleic acid.
In some embodiments, the invention is directed to an isolated microorganism of the Sporidiobolus pararoseus species, wherein the microorganism is capable of producing fatty acids in an amount that is 50% to 80% by weight of the dry cell weight, and wherein the microorganism is capable of producing a microbial oil comprising 5% to 30% by weight linoleic acid. In some embodiments, the isolated microorganism of the Sporidiobolus pararoseus species is capable of producing fatty acids in an amount that is 55% to 75% by weight of the dry cell weight, and wherein the microorganism is capable of producing a microbial oil comprising 5% to 25% by weight linoleic acid. In some embodiments, the isolated microorganism of the Sporidiobolus pararoseus species is capable of producing fatty acids in an amount that is 60% to 70% by weight of the dry cell weight, and wherein the microorganism is capable of producing a microbial oil comprising 7% to 20% by weight linoleic acid. In some embodiments, the isolated microorganism of the Sporidiobolus pararoseus species is capable of producing fatty acids in an amount that is 65% to 70% by weight of the dry cell weight, and wherein the microorganism is capable of producing a microbial oil comprising 7% to 15% by weight linoleic acid.
In some embodiments, the invention is directed to an isolated microorganism of the species deposited under ATCC Accession No. PTA-11615. The isolated microorganism is also known herein as Pseudozyma aphidis/rugulosa ATCC PTA-11615. The isolated microorganism associated with ATCC Accession No. PTA-11615 was deposited under the Budapest Treaty on Jan. 26, 2011 at the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, Va. 20110-2209.
In some embodiments, the invention is directed to an isolated microorganism of the species deposited under ATCC Accession No. PTA-11616. The isolated microorganism is also known herein as Sporidiobolus pararoseus ATCC PTA-11616. The isolated microorganism associated with ATCC Accession No. PTA-11616 was deposited under the Budapest Treaty on Jan. 26, 2011 at the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, Va. 20110-2209.
In some embodiments, the invention is directed to an isolated microorganism of the species deposited under ATCC Accession No. PTA-11617. The isolated microorganism is also known herein as Rhodotorula ingeniosa ATCC PTA-11617. The isolated microorganism associated with ATCC Accession No. PTA-11617 was deposited under the Budapest Treaty on Jan. 26, 2011 at the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, Va. 20110-2209.
In some embodiments, the invention is directed to an isolated microorganism having the characteristics of the microorganism deposited under ATCC Accession No. PTA-11615, ATCC Accession No. PTA-11616, or ATCC Accession No. PTA-11617. The characteristics of the species deposited under ATCC Accession No. PTA-11615, ATCC Accession No. PTA-11616, or ATCC Accession No. PTA-11617 include its growth and phenotypic properties (examples of phenotypic properties include morphological and reproductive properties), its physical and chemical properties (such as dry weights and lipid profiles), and its gene sequences. In some embodiments, the isolated microorganisms of the invention have substantially identical phenotypic properties of the microorganism deposited under ATCC Accession No. PTA-11615, ATCC Accession No. PTA-11616, or ATCC Accession No. PTA-11617. In some embodiments, the isolated microorganisms of the invention have substantially identical growth properties of the microorganisms deposited under ATCC Accession No. PTA-11615, ATCC Accession No. PTA-11616, or ATCC Accession No. PTA-11617.
The invention is further directed to an isolated biomass comprising the isolated microorganisms of the invention. An isolated biomass of the invention is a harvested cellular biomass obtained by any conventional method for the isolation of a microbial biomass.
The biomass of the invention contains a high level of fatty acids. In some embodiments, at least 30% by weight of the dry cell weight of the biomass of the invention are fatty acids. (In some embodiments, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% by weight of the dry cell weight of the biomass of the invention are fatty acids. In some embodiments, the biomass of the invention comprises from 30% to 75%, from 40% to 75%, from 45% to 70%, from 50% to 70%, from 55% to 65% fatty acids by the dry cell weight of the biomass.
The invention is further directed to a culture comprising one or more isolated microorganisms of the invention. Various fermentation parameters for inoculating, growing, and recovering yeast strains are known in the art. Any conventional medium for growth of yeasts can be used.
The cultures of the invention contain a high amount of biomass per liter of culture broth, indicating efficient growth of the isolated microorganisms. In some embodiments, the cultures comprise at least 1 g/L of biomass of the isolated microorganisms of the invention. In some embodiments, the cultures comprise at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, or at least 10 g/L of biomass of the isolated microorganisms of the invention. In some embodiments, the cultures comprises 1 g/L to 10 g/L, 2 g/L to 10 g/L, 3 g/L to 10 g/L, 4 g/L to 10 g/L, 5 g/L to 10 g/L, 6 g/L to 10 g/L, 7 g/L to 10 g/L, 8 g/L to 10 g/L, or 9 g/L to 10 g/L of biomass of the isolated microorganisms of the invention.
The present invention is further directed to methods of producing microbial oils.
In some embodiments, the method comprises growing an isolated microorganism of the invention or mixtures thereof in a culture to produce a microbial oil. The isolated microorganisms of the invention can be grown in the presence of various carbon sources, including, for example, sucrose, glucose, fructose, xylose, glycerol, mannose, arabinose, lactose, galactose, maltose, cellulose, lignocellulose, or combinations thereof. The method can further comprise extracting the microbial oil. The oil can be extracted from a freshly harvested biomass or can be extracted from a previously harvested biomass that has been stored under conditions that prevent spoilage. Known methods can be used to culture a microorganism of the invention, to isolate a biomass from the culture, to extract a microbial oil from the biomass, and to analyze the fatty acid profile of oils extracted from the biomass.
The invention is further directed to a microbial oil produced by the methods of the invention. In some embodiments, the microbial oil comprises a fatty acid profile having the same characteristics as the fatty acid profile of the isolated microorganisms of the invention. A microbial oil of the invention can be any oil derived from a microorganism, including, for example: a crude oil extracted from the biomass of the microorganism without further processing; a refined oil that is obtained by treating a crude microbial oil with further processing steps such as refining, bleaching, and/or deodorizing; a diluted microbial oil obtained by diluting a crude or refined microbial oil; or an enriched oil that is obtained, for example, by treating a crude or refined microbial oil with further methods of purification to increase the concentration of a fatty acid in the oil.
The invention is also directed to the use of the isolated microorganism, biomass, culture, or microbial oil of the invention for the manufacture of a food, dietary supplement, cosmetic, or pharmaceutical composition for a non-human animal or human.
Compositions
The invention is further directed to compositions comprising an isolated microorganism of the invention, an isolated biomass of the invention, a microbial oil, of the invention, or combinations thereof.
An isolated microorganism, biomass, or microbial oil of the invention can be further chemically or physically modified or processed based on the requirements of the composition by any known technique.
Microorganism cells or biomasses can be dried prior to use in a composition by methods including, but not limited to, freeze drying, air drying, spray drying, tunnel drying, vacuum drying (lyophilization), or a similar process. Alternatively, a harvested and washed biomass can be used directly in a composition without drying.
Microbial oils of the invention can be used as starting material to more efficiently produce a product enriched in a fatty acid. For example, the microbial oils of the invention can be subjected to various purification techniques known in the art, such as distillation or urea adduction, to produce a higher potency product with higher concentrations of a particular fatty acid. The microbial oils of the invention can also be used in chemical reactions to produce compounds derived from fatty acids in the oils, such as esters and salts of a fatty acid.
A composition of the invention can include one or more excipients. As used herein, “excipient” refers to a component, or mixture of components, that is used in a composition of the present invention to give desirable characteristics to the composition, including foods as well as pharmaceutical, cosmetic, and industrial compositions. An excipient of the present invention can be described as a “pharmaceutically acceptable” excipient when added to a pharmaceutical composition, meaning that the excipient is a compound, material, composition, salt, and/or dosage form which is, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problematic complications over the desired duration of contact commensurate with a reasonable benefit/risk ratio. In some embodiments, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized international pharmacopeia for use in animals, and more particularly in humans. Various excipients can be used. In some embodiments, the excipient can be, but is not limited to, an alkaline agent, a stabilizer, an antioxidant, an adhesion agent, a separating agent, a coating agent, an exterior phase component, a controlled-release component, a solvent, a surfactant, a humectant, a buffering agent, a filler, an emollient, or combinations thereof. Excipients in addition to those discussed herein can include excipients listed in, though not limited to, Remington: The Science and Practice of Pharmacy, 21st ed. (2005). Inclusion of an excipient in a particular classification herein (e.g., “solvent”) is intended to illustrate rather than limit the role of the excipient. A particular excipient can fall within multiple classifications.
Compositions of the invention include, but are not limited to, food products, pharmaceutical compositions, cosmetics, and industrial compositions.
In some embodiments, the composition is a food product. A food product is any food for animal or human consumption, and includes both solid and liquid compositions. A food product can be an additive to animal or human foods. Foods include, but are not limited to, common foods; liquid products, including milks, beverages, therapeutic drinks, and nutritional drinks; functional foods; supplements; nutraceuticals; infant formulas, including formulas for pre-mature infants; foods for pregnant or nursing women; foods for adults; geriatric foods; and animal foods.
In some embodiments, an isolated microorganism, biomass, or microbial oil of the invention can be used directly as or included as an additive within one or more of: an oil, shortening, spread, other fatty ingredient, beverage, sauce, dairy-based or soy-based food (such as milk, yogurt, cheese and ice-cream), a baked good, a nutritional product, e.g., as a nutritional supplement (in capsule or tablet form), a vitamin supplement, a diet supplement, a powdered drink, a finished or semi-finished powdered food product, and combinations thereof.
A partial list of food compositions that can include a microbial oil of the invention includes, but is not limited to, soya based products (milks, ice creams, yogurts, drinks, creams, spreads, whiteners); soups and soup mixes; doughs, batters, and baked food items including, for example, fine bakery wares, breakfast cereals, cakes, cheesecakes, pies, cupcakes, cookies, bars, breads, rolls, biscuits, muffins, pastries, scones, croutons, crackers, sweet goods, snack cakes, pies, granola/snack bars, and toaster pastries; candy; hard confectionery; chocolate and other confectionery; chewing gum; liquid food products, for example milks, energy drinks, infant formula, carbonated drinks, teas, liquid meals, fruit juices, fruit-based drinks, vegetable-based drinks; multivitamin syrups, meal replacers, medicinal foods, and syrups; powdered beverage mixes; pasta; processed fish products; processed meat products; processed poultry products; gravies and sauces; condiments (ketchup, mayonnaise, etc.); vegetable oil-based spreads; dairy products; yogurt; butters; frozen dairy products; ice creams; frozen desserts; frozen yogurts; semi-solid food products such as baby food; puddings and gelatin desserts; processed and unprocessed cheese; pancake mixes; food bars including energy bars; waffle mixes; salad dressings; replacement egg mixes; nut and nut-based spreads; salted snacks such as potato chips and other chips or crisps, corn chips, tortilla chips, extruded snacks, popcorn, pretzels, potato crisps, and nuts; specialty snacks such as dips, dried fruit snacks, meat snacks, pork rinds, health food bars and rice/corn cakes.
In some embodiments, a microbial oil of the invention can be used to supplement infant formula. Infant formula can be supplemented with a microbial oil of the invention alone or in combination with a physically refined oil derived from an arachidonic acid (ARA)-producing microorganism.
In some embodiments, the composition is an animal feed. An “animal” means any non-human organism belonging to the kingdom Animalia, and includes, without limitation, aquatic animals and terrestrial animals. The term “animal feed” or “animal food” refers to any food intended for non-human animals, whether for fish; commercial fish; ornamental fish; fish larvae; bivalves; mollusks; crustaceans; shellfish; shrimp; larval shrimp; artemia; rotifers; brine shrimp; filter feeders; amphibians; reptiles; mammals; domestic animals; farm animals; zoo animals; sport animals; breeding stock; racing animals; show animals; heirloom animals; rare or endangered animals; companion animals; pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, or horses; primates such as monkeys (e.g., cebus, rhesus, African green, patas, cynomolgus, and cercopithecus), apes, orangutans, baboons, gibbons, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, cattle, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. An animal feed includes, but is not limited to, an aquaculture feed, a domestic animal feed including pet feed, a zoological animal feed, a work animal feed, a livestock feed, or a combination thereof.
In some embodiments, the composition is a feed or feed supplement for any animal whose meat or products are consumed by humans, such as any animal from which meat, eggs, or milk is derived for human consumption. When fed to such animals, nutrients such as certain fatty acids can be incorporated into the flesh, milk, eggs or other products of such animals to increase their content of these nutrients.
In some embodiments, the composition is a spray-dried material that can be crumbled to form particles of an appropriate size for consumption by zooplankton, artemia, rotifers, and filter feeders. In some embodiments, the zooplankton, artemia, or rotifers fed by the composition are in turn fed to fish larvae, fish, shellfish, bivalves, or crustaceans.
In some embodiments, the composition is a pharmaceutical composition. Suitable pharmaceutical compositions include, but are not limited to, an anti-inflammatory composition, a drug for treatment of coronary heart disease, a drug for treatment of arteriosclerosis, a chemotherapeutic agent, an active excipient, an osteoporosis drug, an anti-depressant, an anti-convulsant, an anti-Helicobacter pylori drug, a drug for treatment of neurodegenerative disease, a drug for treatment of degenerative liver disease, an antibiotic, a cholesterol lowering composition, and a triglyceride lowering composition. In some embodiments, the composition is a medical food. A medical food includes a food that is in a composition to be consumed or administered externally under the supervision of a physician and that is intended for the specific dietary management of a condition, for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation.
In some embodiments, the microbial oil can be formulated in a dosage form. Dosage forms can include, but are not limited to, tablets, capsules, cachets, pellets, pills, powders and granules, and parenteral dosage forms, which include, but are not limited to, solutions, suspensions, emulsions, and dry powders comprising an effective amount of the microbial oil. It is also known in the art that such formulations can also contain pharmaceutically acceptable diluents, fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers, humectants, moisturizers, solubilizers, preservatives and the like. Administration forms can include, but are not limited to, tablets, dragees, capsules, caplets, and pills, which contain the microbial oil and one or more suitable pharmaceutically acceptable carriers.
For oral administration, the microbial oil can be combined with pharmaceutically acceptable carriers well known in the art. Such carriers enable the microbial oils of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. In some embodiments, the dosage form is a tablet, pill or caplet. Pharmaceutical preparations for oral use can be obtained by adding a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including, but not limited to, lactose, sucrose, mannitol, and sorbitol; cellulose preparations such as, but not limited to, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmehyl cellulose, sodium carboxymethyl cellulose, and polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as, but not limited to, the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Pharmaceutical preparations that can be used orally include, but are not limited to, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
In some embodiments, the composition is a cosmetic. Cosmetics include, but are not limited to, emulsions, creams, lotions, masks, soaps, shampoos, washes, facial creams, conditioners, make-ups, bath agents, and dispersion liquids. Cosmetic agents can be medicinal or non-medicinal.
In some embodiments, the composition is an industrial composition. In some embodiments, the composition is a starting material for one or more manufactures. A manufacture includes, but is not limited to, a polymer; a photographic photosensitive material; a detergent; an industrial oil; or an industrial detergent. For example, U.S. Pat. No. 7,259,006 describes use of DHA-containing fat and oil for production of behenic acid and production of photographic sensitive materials using behenic acid.
In some embodiments, the composition is a lipid-based biofuel manufactured by converting the biological oil of the invention into lipid-based biofuel through known means in the art, such as through transesterifying the biological oil to produce biodiesel. Different uses of the biological oils of the present invention for lipid-based biofuel purposes include, but are not limited to, uses as heating oil, transportation biodiesel, jet fuel, fuel additives, specialty fuels and lubricants. In some embodiments, the conversion of biological oils into lipid-based biofuels involves chemical processes and refining techniques known in the art which may also produce or be used to produce specialty chemical compounds similar to petroleum distillates.
Kits Comprising the Compositions
The invention is further directed to kits or packages containing one or more units of a composition of the invention. Kits or packages can include units of a food product, pharmaceutical composition, cosmetic, or industrial composition comprising the isolated microorganism, biomass, or microbial oil of the invention, or combinations thereof. Kits or packages can also include an additive comprising the isolated microorganism, biomass, or microbial oil of the invention, or combinations thereof for preparation of a food, cosmetic, pharmaceutical composition, or industrial composition.
In some embodiments, the kit or package contains one or more units of a pharmaceutical composition to be administered according to the methods of the present invention. The kit or package can contain one dosage unit, or more than one dosage unit (i.e., multiple dosage units). If multiple dosage units are present in the kit or package, the multiple dosage units can be optionally arranged for sequential administration.
The kits of the present invention can optionally contain instructions associated with the units or dosage forms of the kits. Such instructions can be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of the manufacture, use or sale for human administration to treat a condition or disorder. The instructions can be in any form which conveys information on the use of the units or dosage forms in the kit according to the methods of the invention. For example, the instructions can be in the form of printed matter, or in the form of a pre-recorded media device.
In the course of examination of a patient, a medical professional can determine that administration of one of the methods of the present invention is appropriate for the patient, or the physician can determine that the patient's condition can be improved by the administration of one of the methods of the present invention. Prior to prescribing any regimen, the physician can counsel the patient, for example, on the various risks and benefits associated with the regimen. The patient can be provided full disclosure of all known and suspected risks associated with the regimen. Such counseling can be provided verbally, as well as in written form. In some embodiments, the physician can provide the patient with literature materials on the regimen, such as product information, educational materials, and the like.
The present invention is also directed to methods of educating consumers about the methods of treatment, the method comprising distributing the dosage forms with consumer information at a point of sale. In some embodiments, the distribution will occur at a point of sale having a pharmacist or healthcare provider.
The term “consumer information” can include, but is not limited to, an English language text, non-English language text, visual image, chart, telephone recording, website, and access to a live customer service representative. In some embodiments, consumer information will provide directions for use of the dosage forms according to the methods of the present invention, appropriate age use, indication, contraindications, appropriate dosing, warnings, telephone number of website address. In some embodiments, the method further comprises providing professional information to relevant persons in a position to answer consumer questions regarding use of the disclosed regimens according to the methods of the present invention. The term “professional information” includes, but is not limited to, information concerning the regimen when administered according to the methods of the present invention that is designed to enable a medical professional to answer customer questions.
A “medical professional,” includes, for example, a physician, physician assistant, nurse practitioner, pharmacist and customer service representative.
EXAMPLES
Example 1
Identifications were determined for three yeast strains: ATCC Accession No. PTA-11615 (Strain 28428), ATCC Accession No. PTA-11616 (Strain 29404) and ATCC Accession No. PTA-11617 (Strain 29794). The yeasts were plated on Malt Agar. DNA extractions were conducted and ribosomal gene sequence analysis performed. Sequence homology comparisons were done between the D1D2 and ITS regions and known DNA sequences from yeast strains in the public databases. Morphological examination was also conducted for comparisons to known yeast species.
Strain 28428 DNA sequences (FIG. 1) matched 100% with the Genbank sequence for the Type strain of Pseudozyma aphidis in the first region analyzed (D1/D2 domains of the Large subunit) and differed by 1 mismatch from the Type strain of Pseudozyma rugulosa (FIG. 2). In the second region (ITS) out of 545 possible matches, the strain showed 2 mismatches from the Type strain of P. aphidis and again 1 from the Type strain of P. rugulosa (FIG. 3). The two species appear to be closely related showing genetic variability within and between them. Morphological characteristics are also very similar between P. aphidis and P. rugulosa and matched the morphology observations in strain 28428, which showed flat, dull, yellowish-cream colonies with fringed margin and fusiform cells, variable in size, with polar budding on short denticles. Based on the data in hand, it is not possible to distinguish the identification more specifically than as either P. aphidis or P rugulosa. 
Strain 29404 DNA sequences (FIG. 4) matched perfectly in both regions with the Genbank sequences for Sporidiobolus pararoseus CBS 484. Comparison to the Type strain of the species CBS 491 had 2 mismatches in D1D2 and 2 in ITS (FIGS. 5 & 6). Morphological characteristics (ovoid cells, single or in short chains, and shiny coral red colonies with a smooth surface and an entire margin) confirmed the identification. The identification of this strain is Sporidiobolus pararoseus. 
Strain 29794 DNA sequences (FIG. 7) showed an identical sequence match to the type strain of Rhodotorula ingeniosa (CBS 4240) in the D1D2 domains (FIG. 8) and only one mismatch out of 590 possible matches in the ITS regions (FIG. 9), which is possibly due to intra-specific variability. Cell and colony morphology (ovoid to cylindrical cells, single or in pairs, a thin capsule and yellowish, glistening, highly mucoid colonies) confirmed the identification. The identification of this strain is R ingeniosa. 
Example 2
Isolated microorganisms of Sporidiobolus pararoseus and Rhodotorula ingeniosa were grown in 1⁄4×BFGM medium (Table 1). Each strain was picked from an agar plate, and inoculated into a shake flask. The shake flask was then used to inoculate another flask (250 ml Erlenmeyer flask containing 50 ml of medium) that was then grown for 7 days. After 7 days the flask was harvested by centrifugation, the pellet was washed with water and centrifuged again. The final pellet was freeze dried, the weight was measured and then total fat (total fatty acids) and fatty acid profile were determined by FAME procedure.
The isolated microorganism of Pseudozyma aphidis/rugulosa was grown in the media below (Media 2) and the fatty acid profile was determined. Results of the fatty acid profile analysis for all three strains are shown in Table 2.
Media 2:
15 g/L Reef Crystal Artificial Sea salts
1 g/L glucose
1 g/L monosodium glutamate
0.2 g/L yeast extract
1 mL/L vitamin mix* *vitamin mix contains 100 mg/L thiamine, 0.5 mg/L biotin, 0.5 g/L cyanocobalmin
5 mL/L PII trace metal mix** **PII trace metal mix contains:
0.1 g/L penicillin G
0.1 g/L streptomycin sulfate

    
    
        6.0 g/L Na2EDTA
        0.29 g/L FeCl3.6H2O
        6.84 g/L H3BO3 
        0.86 g/L MnCl2.4H2O
        0.06 g/L ZnCl2 
        0.026 g/L CoCl2.6H2O
        0.052 g/L NiSO4.6H2O
        0.002 g/L CuSO4.5H2O
        0.005 g/L Na2MoO4.2H2O
    
    









TABLE 1







1⁄4 × BFGM Medium
















mL of






Amount

stock to



per liter
[Stock]
use per

g/l
mg/l





















Component
(g)
(g/l)
liter

Na
K
Mg
Ca
Cl
Fe
Cu
Mn
Co
Zn

























NaCl
0.625

dry


0.25



0.38







KCl
1

50
   20 ml


0.52


0.48


MgSO4•7H2O
5

227
   22 ml



0.47


(NH4)2SO4
0.05

190
0.2625 ml


CaCl2 2H2O
0.29

dry





0.08
0.14


MSG
0.5

dry


monohydrate


Tastone 154
0.5

dry


HEPES (100 mM)
23.8

dry


pH 7


KH2PO4
0.025

56.5
0.4425 ml
add after autoclaving


Sucrose
50

500
  100 ml
add after autoclaving


Trace Metals


see
   2 ml
add after autoclaving





below


Vitamins


see
   1 ml
add after autoclaving





below


Trace Metal


Solution


Citric Acid
1.0
g
dry


FeSO4•7H2O
10.3
mg
5.15







2.07


MnCl2•4H2O
3.1
mg
1.55









0.86


ZnSO4•7H2O
1.93
mg
0.965











0.44


CoCl2•6H2O
0.04
mg
0.02










0.01


Na2MoO4•2H2O
0.04
mg
0.02


CuSO4•5H2O
2.07
mg
1.035








0.53


NiSO4•6H2O
2.07
mg
1.035


pH to 2.5 with


HCl


Vitamin Solution


Vitamin B12
0.16
mg
0.16


Thiamine
9.75
mg
9.75


CaPantothenate
3.33
mg
3.33


Ion Totals (ppm)


995.8536


















TABLE 2









Media










1⁄4 BFGM
Media 2









Microorganism













Pseudozyma



Rhodotorula
Sporidiobolus
aphidis/



ingeniosa
pararoseus
rugulosa









Sugar












Sucrose
Xylose
Sucrose
Glucose















Temperature
27
27
22.5
30° C.


(° C.)


Biomass g/L
9.506
5.338
8.79



pH
6.9
6.78
6.68


% 08:0
0.00
0.00
0.00


% 09:0
0.00
0.00
0.00


% 10:0
0.00
0.00
0.00


% 11:0
0.00
0.00
0.00


% 11:1
0.00
0.00
0.00


% 12:0
0.00
0.00
0.00


% 12:1
0.00
0.00
0.00


% 13:0
0.17
0.18
0.00


% 13:1
0.00
0.00
0.00


% 14:0
0.58
0.73
0.00
0.8


% 14:1
0.00
0.00
0.00
Trace (<1.0%)


% 15:1
0.00
0.00
0.00


% 16:0
18.14
19.62
16.02
21.9


% 16:1
0.64
0.69
0.29
6.6


% 16:2
0.00
0.00
0.00


% 16:3
0.00
0.00
0.00


% 17:0
0.00
0.00
0.11


% 18:0
3.88
4.13
4.86
2.1


% 18:1 n-9
61.79
59.54
60.78
40.4


% 18:1 n-7
0.43
0.19
0.00


% 18:2
8.83
9.76
9.75
24.7


% 18:3 n-6
0.06
0.00
0.00


% 18:3 n-3
2.71
2.54
4.72


% 18:4 n-3
0.00
0.00
0.00


% 20:0
1.18
0.92
0.00
0.8


% 20:1 n-9
0.29
0.30
0.00
0.5


% 20:2
0.00
0.00
0.00


% 20:3 n-9
0.00
0.00
0.00


% 20:3 n-6
0.00
0.00
0.00


% 20:3 n-3
0.00
0.00
0.00


% 20:4 ARA
0.00
0.14
0.00


% 20:5 n-3
0.00
0.00
0.00


EPA


% 22:0
0.54
0.55
0.00
1.1


% 22:1
0.00
0.00
0.00


% 22:2
0.17
0.21
0.00


% 22:3
0.00
0.00
0.00


% 22:4 n-6
0.00
0.00
0.00


% 22:5 n-6
0.00
0.00
0.00


% 22:5 n-3
0.00
0.00
0.00


% 22:6 n-3
0.00
0.00
0.00


DHA


% 24:0
0.21
0.20
0.00
1.1


% 24:1
0.00
0.00
0.00


% Fat
55.19
45.79
65.17


% Unknown
0.38
0.31
3.46


08:0
0.00
0.00
0


09:0
0.00
0.00
0


10:0
0.00
0.00
0


11:0
0.00
0.00
0


11:1
0.00
0.00


12:0
0.00
0.00
0


12:1


0


13:0
6.04
5.70
0


13:1
0.00
0.00
0


14:0
20.92
22.93
0


14:1
0.00
0.00
0


15:0 INT
681.22
684.35
112753


STD


15:1
0.00
0.00
0


16:0
654.11
617.77
110650


16:1
23.02
21.84
2025


16:2


16:3


17:0
0.00
0.00
759


18:0
140.00
129.99
33583


18:1 n-9
2227.57
1874.95
419870


18:1 n-7
15.60
6.08
0


18:2
318.37
307.24
67375


18:3 n-6
2.23
0.00
0


18:3 n-3
97.78
79.91
32596


18:4 n-3


20:0
42.43
28.86
0


20:1 n-9
10.38
9.59
0


20:2
0.00
0.00
0


20:3 n-9


20:3 n-6
0.00
0.00
0


20:3 n-3
0.00
0.00
0


20:4 ARA
0.00
4.40
0


20:5 n-3 EPA
0.00
0.00
0


22:0
19.48
17.18
0


22:1
0.00
0.00
0


22:2
6.08
6.67
0


22:3
0.00
0.00
0


22:4 n-6
0.00
0.00
0


22:5 n-6
0.00
0.00
0


22:5 n-3
0.00
0.00
0


22:6 n-3
0.00
0.00
0


DHA


23:0 INT
0.00
0.00
0


STD


24:0
7.55
6.45
0


24:1
0.00
0.00
0


Total Area
4286.43
3833.56
803514


Total
3605.22
3149.22
690761









Example 3
Isolated microorganisms having matching DNA sequences to that of ATCC Accession No. PTA-11615 (Pseudozyma aphidis/rugulosa; Strain 28428), ATCC Accession No. PTA-11616 (Sporidiobolus pararoseus; Strain 29404) and ATCC Accession No. PTA-11617 (Rhodotorula ingeniosa; Strain 29794) were grown and the total fat (total fatty acids) and fatty acid profiles were determined. Results are shown in Tables 3 and 4.









TABLE 3









ID










Rhodotorula




ingeniosa
Sporidiobolus pararoseus









Strain #


























29764
29764
29664
29405
29412
29459
29556
29568
29573
29577
29588
29591
29594
29631
29655
29596
29631
29655





























Dry Weight (g/l)
4.23
11.054
6.44
9.89
6.23
6.86
4.85
1.3
8.46
3.87
5.46
6.31
7.3
4.73
9.41
8.12
4.73
9.41


Medium
1⁄4 BFGM
1⁄4 BFGM
1⁄4 BFGM
1⁄4 BFGM
1⁄4 BFGM
1⁄4
1⁄4
1⁄4
1⁄4
1⁄4
1⁄4
1⁄4
1⁄4
1⁄4
1⁄4
1⁄4
1⁄4
1⁄4








BFGM
BFGM
BFGM
BFGM
BFGM
BFGM
BFGM
BFGM
BFGM
BFGM
BFGM
BFGM
BFGM


Sugar
Xylose
Sucrose
sucrose
sucrose
sucrose
sucrose
sucrose
sucrose
sucrose
sucrose
sucrose
sucrose
sucrose
sucrose
sucrose
sucrose
sucrose
sucrose


 8:0
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


 9:0
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


10:0
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


11:0
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


11:1
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


12:0
0.00
0.07
0.00
0.00
0.00
0.00
0.03
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


12:1
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


13:0
0.22
0.15
0.09
0.12
0.13
0.09
0.13
0.18
0.15
0.27
0.18
0.16
0.14
0.21
0.15
0.38
0.21
0.15


13:1
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


14:0
0.81
0.71
0.00
0.61
0.85
0.65
0.86
0.80
0.88
0.55
0.63
0.57
0.94
0.53
0.86
0.57
0.53
0.86


14:1
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


15:1
0.00
0.00
0.03
0.00
0.00
0.00
0.00
0.11
0.00
0.14
0.07
0.00
0.04
0.12
0.00
0.11
0.12
0.00


16:0
21.71
22.03
16.96
16.52
18.79
16.01
19.53
23.82
19.22
20.04
18.50
16.84
19.22
18.33
19.98
17.98
18.33
19.98


16:1
0.57
0.66
0.44
0.32
0.20
0.21
0.34
0.50
0.41
0.33
0.41
0.34
0.60
0.48
0.41
0.31
0.48
0.41


16:2
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


16:3
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


17:0
0.00
0.00
0.17
0.19
0.09
0.17
0.13
0.18
0.14
0.33
0.21
0.22
0.16
0.20
0.16
0.26
0.20
0.16


18:0
6.58
6.27
4.04
4.67
7.70
5.83
5.72
10.66
4.30
5.14
3.67
4.42
3.34
2.37
5.06
5.48
2.37
5.06


18:1n9
52.63
53.65
60.31
59.97
0.00
62.94
56.56
40.16
59.19
42.35
57.13
57.60
60.19
56.53
54.71
46.89
56.53
54.71


18:1n7
0.28
0.59
0.00
0.00
61.36
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


18:2
10.99
10.52
11.53
9.96
5.84
8.84
11.03
16.40
9.89
25.32
14.17
14.55
10.72
15.84
13.05
21.79
15.84
13.05


18:3n6
0.08
0.08
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


18:3n3
3.73
2.76
2.15
5.24
1.97
1.59
2.78
4.19
2.86
2.67
1.59
2.87
1.55
2.03
3.52
3.02
2.03
3.52


18:4n3
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


20:0
0.84
1.14
0.79
0.68
0.83
0.78
0.87
0.38
0.68
0.42
0.55
0.36
0.57
0.56
0.72
0.72
0.56
0.72


20:1n9
0.26
0.23
0.31
0.06
0.29
0.35
0.22
0.08
0.09
0.16
0.09
0.09
0.08
0.27
0.18
0.18
0.27
0.18


20:2
0.00
0.00
0.05
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


20:3n9
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


20:3n6
0.00
0.00
0.12
0.11
0.00
0.13
0.10
0.14
0.11
0.21
0.17
0.13
0.12
0.20
0.00
0.00
0.20
0.00


20:3n3
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


20:4n6 ARA
0.20
0.11
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


20:5n3 EPA
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


22:0
0.64
0.50
1.14
0.64
0.94
1.27
0.88
0.69
0.91
0.63
0.96
0.52
0.88
0.90
0.73
0.78
0.90
0.73


22:1
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


22:2
0.18
0.13
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


22:3
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


22:4n6
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


22:5n6
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


22:5n3
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


22:6n3
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


24:0
0.29
0.19
0.00
0.43
0.49
0.84
0.41
1.72
0.58
1.02
0.76
0.52
0.76
0.68
0.34
0.85
0.68
0.34


24:1
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00


Fat
39.04
52.86
67.01
56.02
59.60
63.32
64.44
40.38
64.00
29.39
43.32
52.30
62.44
33.57
53.57
26.03
33.57
53.57


Unknown
0.00
0.23
1.88
0.48
0.51
0.30
0.43
0.00
0.58
0.41
0.92
0.79
0.69
0.75
0.14
0.67
0.75
0.14



















TABLE 4










Pseudozyma aphidis/rugulosa







Strain #:
Strain 28426



Media:
Media 2



Sugar:
Glucose















% total fatty acids



Fatty acid
(area %)







14:0



14:1



16:0
27.4



16:1
1.4



18:0
9.6



18.1
31.5



18:2
28.0



20:0
0.4



20:1
1.6



22:0
0.2



24:0










All of the various aspects, embodiments, and options described herein can be combined in any and all variations.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

## Claims

1. An isolated microorganism of the species selected from the group consisting of Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, and Rhodotorula ingeniosa, wherein the microorganism is capable of producing a microbial oil comprising less than 30% by weight saturated fatty acids.

2. The isolated microorganism of claim 1, wherein the microorganism is capable of producing fatty acids in an amount that is at least 30% by weight of the dry cell weight.

3. The isolated microorganism of claim 1, wherein the microorganism is capable of producing fatty acids in an amount that is at least 40% by weight of the dry cell weight.

4. The isolated microorganism of claim 1, wherein the microorganism is capable of producing fatty acids in an amount that is at least 50% by weight of the dry cell weight.

5. The isolated microorganism of claim 1, wherein the microorganism is capable of producing fatty acids in an amount that is at least 60% by weight of the dry cell weight.

6. The isolated microorganism of claim 1, wherein the microorganism is capable of producing a microbial oil comprising greater than 30% by weight oleic acid.

7. The isolated microorganism of claim 1, wherein the microorganism is capable of producing a microbial oil comprising greater than 40% by weight oleic acid.

8. The isolated microorganism of claim 1, wherein the microorganism is capable of producing a microbial oil comprising greater than 50% by weight oleic acid.

9. The isolated microorganism of claim 1, wherein the microorganism is capable of producing a microbial oil comprising greater than 60% by weight oleic acid.

10. The isolated microorganism of claim 1, wherein the microorganism is capable of producing a microbial oil comprising greater than 5% by weight linoleic acid.

11. The isolated microorganism of claim 1, wherein the microorganism is capable of producing a microbial oil comprising greater than 10% by weight linoleic acid.

12. The isolated microorganism of claim 1, wherein the microorganism is capable of producing a microbial oil comprising greater than 15% by weight linoleic acid.

13. The isolated microorganism of claim 1, wherein the microorganism is capable of producing a microbial oil comprising less than 25% by weight saturated fatty acids.

14. The isolated microorganism of claim 1, wherein the microorganism is capable of producing a microbial oil comprising less than 25% by weight palmitic acid.

15. The isolated microorganism of claim 1, wherein the microorganism is capable of producing a microbial oil comprising less than 20% by weight palmitic acid.

16. The isolated microorganism of claim 1, wherein the microorganism is capable of producing a microbial oil comprising less than 15% by weight palmitic acid.

17. The isolated microorganism of claim 1, wherein the microorganism is capable of producing a microbial oil comprising less than 10% by weight long chain polyunsaturated fatty acids of 20 or more carbon chain length.

18. The isolated microorganism of claim 1, wherein the microorganism is capable of producing a microbial oil comprising less than 5% by weight long chain polyunsaturated fatty acids of 20 or more carbon chain length.

19. An isolated microorganism of the species selected from the group consisting of Pseudozyma aphidis, Pseudozyma rugulosa, and Rhodotorula ingeniosa, wherein the microorganism is capable of producing fatty acids in an amount that is at least 30% by weight of the dry cell weight.

20. The isolated microorganism of claim 19, wherein the microorganism is capable of producing fatty acids in an amount that is at least 40% by weight of the dry cell weight.

21. The isolated microorganism of claim 19, wherein the microorganism is capable of producing fatty acids in an amount that is at least 50% by weight of the dry cell weight.

22. An isolated microorganism of the Sporidiobolus pararoseus species, wherein the microorganism is capable of producing fatty acids in an amount that is at least 50% by weight of the dry cell weight.

23. The isolated microorganism of claim 22, wherein the microorganism is capable of producing fatty acids in an amount that is at least 55% by weight of the dry cell weight.

24. The isolated microorganism of claim 22, wherein the microorganism is capable of producing fatty acids in an amount that is at least 60% by weight of the dry cell weight.

25. An isolated microorganism of the species selected from the group consisting of Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, and Rhodotorula ingeniosa, wherein the microorganism is capable of producing a microbial oil comprising 0.5% to 30% by weight saturated fatty acids, and wherein the microorganism is capable of producing fatty acids in an amount that is 30% to 80% by weight of the dry cell weight.

26. An isolated microorganism of the species selected from the group consisting of Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, and Rhodotorula ingeniosa, wherein the microorganism is capable of producing a microbial oil comprising 30% to 70% by weight oleic acid and 5% to 30% by weight linoleic acid.

27. An isolated microorganism of the Sporidiobolus pararoseus species, wherein the microorganism is capable of producing fatty acids in an amount that is 50% to 80% by weight of the dry cell weight, and wherein the microorganism is capable of producing a microbial oil comprising 5% to 30% by weight linoleic acid.

28. An isolated microorganism deposited under ATCC Accession No. PTA-11615.

29. An isolated microorganism deposited under ATCC Accession No. PTA-11616.

30. An isolated microorganism deposited under ATCC Accession No. PTA-11617.

31. An isolated microorganism having all the characteristics of the microorganism deposited under ATCC Accession No. PTA-11615.

32. An isolated microorganism having all the characteristics of the microorganism deposited under ATCC Accession No. PTA-11616.

33. An isolated microorganism having all the characteristics of the microorganism deposited under ATCC Accession No. PTA-11617.

34. An isolated biomass comprising the isolated microorganism of claim 1 or mixtures thereof.

35. The isolated biomass of claim 34, wherein at least 30% by weight of the dry cell weight of the biomass are fatty acids.

36. A culture comprising the isolated microorganism of any one of claims 28-33 or mixtures thereof.

37. The culture of claim 36, wherein the culture comprises at least 5 g/L of biomass of the isolated microorganism.

38. A method for producing a microbial oil, comprising:
growing an isolated microorganism of claim 1 in a culture to produce a microbial oil.

39. The method of claim 38, wherein the isolated microorganism is grown in the presence of a carbon source selected from the group consisting of sucrose, glucose, fructose, xylose, glycerol, mannose, arabinose, lactose, galactose, maltose, cellulose, lignocellulose, and combinations thereof.

40. The method of claim 38, further comprising extracting the microbial oil.

41. A method for producing a microbial oil, comprising extracting the microbial oil from the isolated biomass of claim 34.

42. The method of claim 38, wherein a culture comprising biomass of the isolated microorganism is produced and the culture comprises at least 5 g/L of the biomass.

43. A microbial oil produced by the method of claim 38.

44. A method of making a food, supplement, cosmetic, or pharmaceutical composition for a non-human animal or human comprising admixing the isolated microorganism, biomass, culture, or microbial oil, or a byproduct thereof of claim 1 in a food, supplement, cosmetic, or pharmaceutical composition for a non-human animal or human.