WO2019092421A1

WO2019092421A1 - Microorganism

Info

Publication number: WO2019092421A1
Application number: PCT/GB2018/053234
Authority: WO
Inventors: Lydia BROWN; David ALDERMAN; Jane Lesley POLGLASE
Original assignee: Strameno Ltd
Current assignee: Strameno Ltd
Priority date: 2017-11-07
Filing date: 2018-11-07
Publication date: 2019-05-16
Anticipated expiration: 2020-05-07

Abstract

Isolated thraustochytrids are described. The thraustochytrids have an ITS2-28S ribosomal RNA gene sequence which has at least 85% sequence identity to the sequence set forth in SEQ. ID NO: 1.

Description

Microorganism

Introduction

Eukaryotic microorganisms are increasingly being studied for potential

biotechnological applications. These organisms include, for example, stramenopiles such as thraustochytrids, which are heterotrophic marine biflagellate protists.

Summary

The present invention is directed towards an isolated thraustochytrid as defined in the associated independent claims. Preferred embodiments are set out in the sub-claims.

According to a first aspect, an isolated thraustochytrid having an ITS2-28S ribosomal RNA sequence is provided. The ITS2-28S ribosomal RNA sequence has at least 85% sequence identity to the sequence set forth in SEQ. ID NO: 1. The ITS2-28S ribosomal RNA sequence may have at least 86%, 87%, 88%, 89%, or 90% sequence identity to the sequence set forth in SEQ. ID NO: 1. The ITS2-28S ribosomal RNA sequence may even have at least 91% or 92% sequence identity to the sequence set forth in SEQ. ID NO: 1, such as 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ. ID NO: 1.

In some embodiments, an isolated thraustochytrid having an 18S ribosomal RNA sequence is provided. The 18S ribosomal RNA sequence has at least 86% sequence identity to the sequence set forth in SEQ. ID NO: 8. The 18S ribosomal RNA sequence may have at least 87%, 88%, 89%, or 90% sequence identity to the sequence set forth in SEQ. ID NO: 8. The 18S ribosomal RNA sequence may even have at least 91% or 92% sequence identity to the sequence set forth in SEQ. ID NO: 8, such as 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ. ID NO: 8. In some embodiments, an isolated thraustochytrid having an 18S ribosomal RNA sequence and an ITS2-28S ribosomal RNA sequence is provided. The 18S ribosomal RNA sequence has at least 86% sequence identity to the sequence set forth in SEQ. ID NO: 8 and the ITS2-28S ribosomal RNA sequence has at least 85% sequence identity to the sequence set forth in SEQ. ID NO: 1. The 18S ribosomal RNA sequence may have at least 87%, 88%, 89% or 90% sequence identity to the sequence set forth in SEQ. ID NO: 8 and the ITS2-28S ribosomal RNA sequence may have at least 86%, 87%, 88%, 89%, or 90% sequence identity to the sequence set forth in SEQ. ID NO: 1. The 18S ribosomal RNA sequence may even have at least 91% or 92% sequence identity to the sequence set forth in SEQ. ID NO: 8, such as 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ. ID NO: 8 and the ITS2-28S ribosomal RNA sequence 18S ribosomal RNA sequence may have at least 91% or 92% sequence identity to the sequence set forth in SEQ. ID NO: 1, such as 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ. ID NO: 1. When cultured in heterotrophic medium, the culture of the isolated thraustochytrid of the first aspect may comprise a cell having an internal daughter vegetative cell, wherein that daughter cell comprises a vegetative granddaughter cell within it. At least 0.01% or 0.1% of the cells in the thraustochytrid culture may comprise both daughter and granddaughter vegetative cells.

The isolated thraustochytrid may be one of the thraustochytrid strains referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14, or may be a mutant, variant, or recombinant derived from one of these strains. The thraustochytrid strains referred to as CAi, CA2, CA3, CA4, CA5, CA8, and CA14 have been deposited at the CABI depositary institution under Accession Numbers IMI 506775, IMI 506776, IMI 506777, IMI 506778, IMI 506779, IMI 506780, and IMI 507005 respectively, and some of the strains have also been deposited at the CCAP depositary institution under Accession Numbers CCAP 4063/1 (CAi), CCAP 4063/2 (CA2), CCAP 4063/3 (CA8). The isolated thraustochytrid may be capable of producing fatty acids. In some embodiments, 45-75% of the total fatty acids produced comprise omega-3 fatty acids.

The isolated thraustochytrid may be capable of producing fatty acids. In some embodiments, 35-70% of the total fatty acids produced comprise DHA.

In some embodiments, when the thraustochytrid is cultured in heterotrophic medium to the late stationary phase, the concentration of DHA in the culture medium may be 5- 20 mgL ¹. The isolated thraustochytrid may be capable of producing carotenoids when grown in MCBHB medium. The total amount of carotenoid produced may be greater than 80, loo, 200, 300, 400, 500, 6oo, 8oo, ιοοο, 1200, 1500, 1800, 2000, or 2200 g/kg dry weight of sample, such as 50-1100, 100-1000, 150-900, 40-2500, 70-2400, 90-2300, or 120-2250 g/kg dry weight of sample. For example, the isolated thraustochytrid may be a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CA14.

In some embodiments, the isolated thraustochytrid may be capable of producing beta- carotene, and/or astaxanthin and/or canthaxanthin and/or adonirubin and/or echinenone/cis-echinenone when grown in MCBHB medium.

The amount of beta-carotene produced may be greater than 40, 50, 60, 80, 100, 150, 200, 500, 800, 1000, 1200, 1400, or 1600 g/kg dry weight of sample, such as 10-200, 20-150, 30-120, 5-2000, 15-1800, or 25-1700 g/kg dry weight of sample. For example, the isolated thraustochytrid may be a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CA14.

The amount of astaxanthin produced may be greater than 100, 200, 300, or 400 μg/kg dry weight of sample, such as 50-1000, 100-900, 150-850 μg/kg dry weight of sample. For example, the isolated thraustochytrid may be a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CA14.

The amount of canthaxanthin produced may be greater than 4, 5, 6, 8, 10, 15, 20, 50, 100, 200, 250, or 300 μg/kg dry weight of sample, such as 1-30, 2-27, 3-24, 4-400, 7- 350, 12-330, or 25-310 μg/kg dry weight of sample. For example, the isolated thraustochytrid may be a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CA14.

The amount of adonirubin may be greater than 2, 3, 4, 6, 8, 10, 12, 15, 18, 20, 30, 40, 50, or 60 μg/kg dry weight of sample, such as 1-100, 2-80, 3-70 or 5-75 μg/kg dry weight. For example, the isolated thraustochytrid may be a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CA14.

The amount of echinenone/cis-echinenone produced may be greater than 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 20, or 22 μg/kg dry weight of sample, such as 1-30, 2-28, or 3-25 μg/kg dry weight. For example, the isolated thraustochytrid may be a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CA14. When the thraustochytrid is cultured in heterotrophic medium to the late stationary phase (for example in MCBHB (Rosa et al 2011) for approximately 10 days at 2i°C), the concentration of DHA in the culture medium may be 5-20 mgL ¹.

According to a second aspect, a thraustochytrid biomass comprising an isolated thraustochytrid of the first aspect is provided.

According to a third aspect, a microbial oil comprising an isolated thraustochytrid of the first aspect is provided.

According to a fourth aspect, a composition comprising an isolated thraustochytrid of the first aspect, a biomass of the second aspect, and/or a microbial oil of the third aspect, is provided.

The composition of the fourth aspect may be for use in therapy. For example, the composition may be for use in the treatment of inflammation, or in the treatment of an oxidative stress-related condition. In some embodiments, a composition comprising a microbial oil and/or carotenoid, for use in therapy is provided, wherein the microbial oil and/ or carotenoid is obtained or derived from an isolated thraustochytrid of the first aspect.

According to a fifth aspect, a food product, feed additive, nutritional supplement, cosmetic, or pharmaceutical composition for animals or humans, comprising a composition of the fourth aspect is provided.

For example, the feed additive may be a feed additive for use in aquaculture, and may comprise an isolated thraustochytrid of the first aspect, a biomass of the second aspect, and/ or a microbial oil of the third aspect. The feed additive may comprise fatty acids, including omega-3 fatty acids, and/or carotenoids, produced by the isolated

thraustochytrid.

According to a sixth aspect, a method for identifying a strain of thraustochytrid which is capable of producing high levels of fatty acids is provided. The method comprises culturing the thraustochytrid strain in heterotrophic medium and determining whether the thraustochytrid culture comprises a cell having an internal vegetative daughter cell, wherein that daughter cell comprises a vegetative granddaughter cell within it. The presence of a cell comprising internal daughter and granddaughter cells is indicative that the thraustochytrid strain is capable of producing high levels of fatty acids.

According to a seventh aspect, a method for producing a thraustochytrid biomass is provided. The method comprises culturing an isolated thraustochytrid of the first aspect, and producing the thraustochytrid biomass from the cultured thraustochytrids. The method of the seventh aspect may further comprise isolating from the biomass: a) a microbial oil;

b) omega-3 fatty acids;

c) DHA, ARA, EPA, and/or DPA n-6;

d) a carotenoid, which may be beta-carotene;

e) a xanthophyll, which may be astaxanthin, and/ or canthaxanthin;

f) squalene; and/or

g) an exopolysaccharide composition.

According to an eighth aspect, a method for producing a lipid composition comprising omega-3 fatty acids is provided. The method comprises culturing an isolated thraustochytrid of the first aspect to produce a biomass, and isolating the lipid composition comprising omega-3 fatty acids from the biomass.

According to a ninth aspect, a method for producing a lipid composition comprising DHA, ARA, EPA, and/or DPA n-6 is provided. The method comprises culturing an isolated thraustochytrid of the first aspect to produce a biomass and isolating the lipid composition comprising DHA, ARA, EPA, and/or DPA n-6 from the biomass.

According to a tenth aspect, a method for producing a composition comprising an antioxidant comprising a carotenoid and/or a xanthophyll is provided. The method comprises culturing an isolated thraustochytrid of the first aspect to produce a biomass and isolating the antioxidant comprising from the biomass.

According to an eleventh aspect, a method for producing an exopolysaccharide composition is provided. The method comprises culturing an isolated thraustochytrid of the first aspect and isolating the exopolysaccharide from the cultured

thraustochytrids.

According to a twelfth aspect, a method for removal of hydrocarbons from water is provided, the method comprising contacting hydrocarbon-containing water with a culture of an isolated thraustochytrid of the first aspect.

In summary, the present invention is directed to an isolated thraustochytrid as defined in the claims. Further, the present disclosure is directed to deposited thraustochytrid strains, as well as derivatives and mutants thereof, biomasses, microbial oils, compositions, and cultures comprising the thraustochytrids. The present invention is also directed to a method for identifying a strain of thraustochytrid which is capable of producing high levels of fatty acids, and also carotenoids. The invention is also directed to methods of producing biomasses and microbial oils from the thraustochytrids of the disclosure, for example for use in feed additives. The thraustochytrids described herein are highly productive compared to prior isolates particularly for the percentage of DHA amongst the omega-3 fatty acids.

Figures

Embodiments will now be described, by way of example only, with reference to the accompanying figures, in which:

Figure la is a micrograph showing an example of the disclosed thraustochytrids in a mature stage with ectoplasmic net elements (white arrow). (The body at the right-hand edge of the image is a pine pollen grain);

Figure lb is a micrograph of an example of the disclosed thraustochytrids showing major lipid inclusions (phase bright, arrowed);

Figure 2a is a micrograph showing actively growing cultures of the disclosed thraustochytrids showing binary division (b), tetrads (t) and clusters of maturing cells (c). (Dark bodies are pollen grains);

Figure 2b is an electron micrograph of an example of the disclosed thraustochytrids undergoing binary fission;

Figure 3a is a micrograph of an example of the disclosed thraustochytrids showing a flagellated zoospore, including a rear directed whiplash flagellum (Wh) and forward directed flimmer flagellum (arrow) moving too rapidly to be seen clearly;

Figure 3b is an electron micrograph of an example of the disclosed thraustochytrids showing settling zoospores (white arrows); Figure 3c is a micrograph of an example of the disclosed thraustochytrids showing exopolysaccharide (ep);

Figure 4 is a micrograph showing a limax amoeba (ab) and aplanospores (aps);

Figures 5a and b are electron micrographs of a semi thin resin section of the disclosed thraustochytrids, stained in Toluidine Blue showing either single (s) or multiple (M) daughter cells maturing in a vacuole created out of the cytoplasm of the mother cell. Arrow shows example of cell already maturing within a daughter cell, (e) shows evacuating cell, (v) vacuole;

Figures 5c and d are micrographs showing 10 week old cells of CA2 stained with 5 g/ml "nile red" (9-diethylamino-5H-benzo[alpha]phenoxazine-5-one) in filter-sterilised seawater, viewed under ultra-violet illumination to give yellow-gold fluorescence (excitation wavelengths 450-500nm). Arrow indicates brightly fluorescing lipid droplets. Scale = 20μπι. (Images courtesy of James Richard Iremonger)

Figure 6 is an electron micrograph of an example of the disclosed thraustochytrids showing vacuolation beginning in a young cell. (V= vacuole);

Figure 7 is an electron micrograph of an example of the disclosed thraustochytrids showing daughter cells (highlighted with arrows) in the vacuole of a mother cell prior to release (n= nucleus);

Figure 8 is an electron micrograph of an example of the disclosed thraustochytrids showing nucleus (n), paranuclear bodies (pn), and scales (s) enclosed in membranes of golgi body;

Figure 9 is an electron micrograph of an example of the disclosed thraustochytrids showing scales (s) enclosed in golgi apparatus;

Figure 10 is an electron micrograph of an example of the disclosed thraustochytrids showing scales (highlighted with arrows) on external surface of cell membrane;

Figure 11 is an electron micrograph of an example of the disclosed thraustochytrids showing scales (s) external to the cell;

Figures 12 and 13 are electron micrographs of examples of the disclosed

thraustochytrids showing a grazing section through the Sagenogenetosome (arrows and s);

Figure 14 is an electron micrograph of an example of the disclosed thraustochytrids showing a mitochondrion (M) with tubular cristae (cr);

Figures 15-17 are electron micrographs of examples of the disclosed thraustochytrids showing a centriole (c and arrows) in a mother cell (n is nucleus);

Figure 18 shows a phylogenetic tree of the relationship between the ITS2-28S rRNA gene of the disclosed thraustochytrids (SEQ ID No. 1, labelled as "Sequence_i"), together with known thraustochytrid strains. The tree is rooted by the inclusion of the yeasts Candida albicans and Saccharomyces cerevisiae.

Figure 19 shows a phylogenetic tree of the relationship between the 18S rRNA gene of the disclosed thraustochytrids (SEQ ID No. 8, labelled as "novel labyrinthulomycetes"), together with strains of thraustochytrid and other microorganisms. The tree is rooted by the inclusion of Labyrinthula zosteri.

For the avoidance of doubt, all of the electron micrograph images are crown copyright.

Detailed Description

Thraustochytrids are single celled protists, which are currently classified as heterokonts or stramenopiles. The standard taxon hierarchy in relation to stramenopiles is in a period of flux, and is not settled, but is subject to ongoing revision. Within the

Stramenopiles, thraustochytrids belong to the Labyrinthulomycetes. The

Thraustochytriidae form one of three main groupings within this taxon. However, due to the current uncertainties over the precise taxonomy of the Labyrinthulomycetes, it is currently common to refer to protists having the defining features of both members of the Thraustochytrids and Aplanochytrids as "thraustochytrids". The terms

"thraustochytrid" and "thraustochytrids" are used throughout the present specification to include both Thraustochytrids and Aplanochytrids.

The present specification discloses a novel group of thraustochytrids, which has been given the proposed name of Caledochytrium aldermanii.

The disclosed thraustochytrids have a number of genotypic and phenotypic features which have not previously been observed in thraustochytrids or related species of microorganisms.

Initial observations showed the disclosed thraustochytrids to contain many cellular inclusions, including highly refractile vacuoles, which stain positively with the dye known as "nile red" (9-diethylamino-5H-benzo[alpha]phenoxazine-5-one) under ultraviolet illumination (as shown in figures 5c and 5d). These features are typically indicative of high levels of oil production, and subsequent analysis has confirmed this. Indeed, the disclosed thraustochytrids have been found to have properties which make them potentially useful in a number of biotechnological applications, in particular, as a source of carotenoids and omega-3 fatty acids, including DHA. Deposited Strains

Examples of the disclosed thraustochytnds have been deposited under the Budapest Treaty prior to the filing date of the present application. Each of the deposited strains is derived from a single thraustochytrid isolate that was obtained as long as 40 years ago. The disclosed thraustochytnds have been sub- cultured approximately every 6 months throughout the intervening period (see Example 1 for details). In the time that the disclosed thraustochytnds have been in culture it is estimated that they have undergone in excess of 30,000 generations (rounds of cell division).

Thraustochytnds, like other living organisms, experience mutations in their genome over time, but they have no known form of sexual reproduction to remix the genetic changes, which, as a result, accumulate in the genome. Thraustochytnds are generally understood not to reproduce sexually and sexual stages have not been observed in the disclosed thraustochytnds. Mutation is therefore essential for thraustochytnds to produce variability in these protists. As a direct result of this extended period of isolated culturing conditions, in which mutations have accumulated, each of the deposited strains may differ to a greater or lesser extent from the isolated strain from which it was originally derived.

Examples of the disclosed thraustochytnds have been deposited under the Budapest Treaty with the following deposit details:

Reference Depositary Institution Date of deposit Accession No.

CAi CABI 1 November 2017 IMI 506775

CA2 CABI 1 November 2017 IMI 506776

CA3 CABI 1 November 2017 IMI 506777

CA4 CABI 1 November 2017 IMI 506778

CA5 CABI 1 November 2017 IMI 506779

CA8 CABI 1 November 2017 IMI 506780

CA14 CABI 16 October 2018 IMI 507005 CABI has the International Depository Authority Designation of CABI Bioscience, UK Centre (International Mycological Institute), Genetic Resources Collection, Bakeham Lane, Englefield Green, Egham, Surrey, TW20 9ΤΎ, UK. The strains listed in the table below have also been deposited under the Budapest Treaty with the following deposit details:

Reference Depositary Institution Date of deposit Accession No.

CAi CCAP 2 November 2017 CCAP 4063/1 CA2 CCAP 2 November 2017 CCAP 4063/2

CA8 CCAP 2 November 2017 CCAP 4063/3

CCAP has the International Depository Authority Designation of the Culture Collection of Algae and Protozoa, SAMS Limited, Scottish Marine Institute, OBAN, Argyll PA37 lQA, Scotland, United Kingdom.

For the avoidance of doubt, the thraustochytrid microorganism with reference CAi has been deposited under the Budapest Treaty at both the CABI and CCAP institutions. Likewise, the thraustochytrid microorganisms with references CA2 and CA8 have also been deposited under the Budapest Treaty at both CABI and CCAP institutions.

Characterisation of the disclosed thraustochytrids

The present disclosure is directed towards a novel group of thraustochytrids, which is exemplified by, but not limited to, the deposited thraustochytrid strains.

The disclosure includes thraustochytrid strains that are, or can be, derived from one of the deposited strains, by any suitable method such as mutation, genetic modification, chemical mutagenesis, fermentative adaptation. The disclosure also includes thraustochytrid strains that have genetic or morphological and functional features substantially the same as those of the deposited

thraustochytrids.

The disclosure also includes thraustochytrid strains that are strains of the same organism as the deposited strains, having the same genotype and/or phenotype as the deposited strains, and which may be co-isolated with the deposited strains, or may be isolated using the same methods as the deposited strains.

The disclosed new group of thraustochytrids can be characterised and identified in a number of ways. The characterising features are listed here and discussed in more detail in the sections below.

Firstly, the disclosed thraustochytrids are thraustochytrids and therefore possess all of the features that are used and known in the art for the identification and

characterisation of thraustochytrids.

Unlike any other known thraustochytrids, however, the disclosed thraustochytrids can be identified, and distinguished from other similar microorganisms, by means of utilising a novel method of cell division that has not previously been observed or reported in thraustochytrids. The method by which the cells of the disclosed thraustochytrids are observed to divide has not been observed in relation to other similar microorganisms, including other thraustochytrid strains.

The disclosed thraustochytrids can also be identified and distinguished by means of having 18S and ITS2-28S ribosomal RNA sequences which differ from those of other thraustochytrids, and indeed, all other known organisms.

The disclosed thraustochytrids also possess a unique profile of fatty acid production, including, in particular, a high percentage of omega-3 fatty acids such as DHA.

Any of these forms of identification and characterisation, such as morphology, reproductive method, genotype, etc, can be used to characterise and identify the disclosed thraustochytrids. The deposited thraustochytrid strains are provided as examples for comparative purposes, in conjunction with the present disclosure, to assist the skilled person to identify the disclosed new group of thraustochytrids.

Thraustochytrids

The disclosed thraustochytrids have all of the features that are conventionally used in the art to uniquely characterise and classify microorganisms as thraustochytrids. That the disclosed thraustochytrids have all of these characterising features can be determined using either or both light and electron microscopic techniques. Various suitable light and electron microscopic methods will be known to the skilled person and example methods are described in Examples 2 and 3.

Morphological features which are commonly used to characterise thraustochytrids, and which are observed in the disclosed thraustochytrids include:

A sagenogenetosome (also known as a bothrosome). This is a sub-cellular organelle unique to the Labyrinthulomycetes, to which both thraustochytrids and aplanochytrids belong, which produces a characteristic "root-like", branching ectoplasmic net (see figures 12 and 13);

A non-cellulosic cell wall coated with circular scales (0.5-ΐμπι in diameter, 2-3 nm thick) produced in the golgi apparatus (see figures 9, 10, and 11);

A single stack of membranes in the golgi apparatus (see figure 9);

· Mitochondria with tubular cristae (see figure 14);

Membranous paranuclear bodies (see figure 8);

Conspicuous centrioles (see figures 15-17); and,

Characteristic biflagellated zoospores (2-5μπι). The longer of the two flagellae is covered in hair-like mastigonemes, while the shorter (Whiplash) flagellum is smooth.

Cell Division

The disclosed thraustochytrids can be identified by the fact that they are capable of reproducing by means of a novel mechanism that has never before been observed in thraustochytrids.

In pollen culture, the disclosed thraustochytrids are most commonly observed to divide by binary fission to form diads, tetrads and clusters of vegetative cells (see figures 2a and 2b). Mature cells often become sporangia, undergoing internal division to form the bi-flagellated, motile zoospores characteristic of thraustochytrids (see figures 3 and 4). Alternatively, they release small immature cells which move slowly away on their ectoplasmic net in the manner of aplanospores (see figure 4).

The disclosed thraustochytrids are also unusual in that addition of low levels of B complex vitamins with Vitamin C to the seawater supplying the microscope chamber triggered the cytoplasm of a number of maturing cells rounding up to produce limax amoebae (see figure 4), which are observed to emerge through the ruptured cell wall of the original (parent) cell and to subsequently glide away. This behaviour is not unique to the disclosed thraustochytrids, but is not regularly observed in thraustochytrids, and may, therefore, be used in conjunction with some or all of the other described identifying features to assist the skilled person in identifying the disclosed

thraustochytrids.

Electron micrographs of examples of the disclosed thraustochytrids are shown in figures 5a, 5b, 6, and 7, and these studies and others have revealed that the disclosed thraustochytrids utilise a method of cell division that no other thraustochytrids have been found to employ.

Specifically, in this novel method of cell division, the cytoplasm of the mother-cell vacuolates (see figure 6) until a large space is created, which, in older cells, can occupy the majority of the cell volume. Within this vacuole, small, new, secondary cells are created from mother cell cytoplasm, producing a cell or cells within a cell (see figure 7). The mother cell retains its structure and nucleus (labelled "n" in figure 7), giving it a signet-ring like shape, and can continue to grow. The secondary (daughter) cells then grow within the mother cell until either the cell wall of the mother cell breaks, releasing the daughter cells, or until the daughter cells are expelled explosively. These rapidly expelled daughter cells are observed under light microscopy to apparently appear "from nowhere", however full details of this method of cell division cannot be observed using light microscopy because the daughter cells are screened by the mother cell cytoplasm.

This form of reproduction is known to occur in Paramixids, in which up to five or even six nested cells can be observed, and in Mixozoans, which commonly contain three generations within a single cell. However, this mode of reproduction has never previously been found to occur in thraustochytrids or other closely related

microorganisms. This finding is significant because although this form of reproduction is known to be used in other groups of protozoa, such as the paramixids and mixozoa, these organisms are not closely related to the thraustochytrids, and no other thraustochytrid has been observed to use this form of reproduction.

The main component of cell membranes and also the ectoplasmic net of the

thraustochytrids (which is essentially a tube made of unit membrane) are

phospholipids. Thraustochytrids are exceptional in the amount of membranes they produce over the course of their lifetime, both within the cell body and the ectoplasmic net. One cell of around 10 μηι diameter can be seen to produce 10s of net elements extending out from the cell for distances of more than 10 times the diameter of the cell (as shown in figure la) and these are constantly renewed. These membrane lipids add to the lipids stored in cytoplasmic vesicles, sometimes occupying more than half the cell (as shown in figure lb) to give the substantial amounts of polyunsaturated fatty acids for which thraustochytrids are now known. In addition, uniquely in the disclosed thraustochytrids, because the tertiary, secondary and occasionally quaternary generations of cells within a mother cell have further extensive membranous structures (although not fully developed ectoplasmic net within the mother cell), this adds to the total lipid content that is available for extraction. Thus, it is believed that the unique mode of reproduction employed by the disclosed thraustochytrids is, at least in part, a contributing factor to the observed high levels of lipids produced by the disclosed thraustochytrids. This mode of reproduction, which has not previously been found to occur in

thraustochytrids, has been informally termed "babushka doll" reproduction, after the sets of dolls commonly made in Russia, in which the dolls are of sequentially decreasing size, such that each doll fits inside another. The ability to undergo cell division using this method has been found to be characteristic of the disclosed thraustochytrids, and it can be used to identify and distinguish the thraustochytrids of the present disclosure from other known thraustochytrids.

The disclosed thraustochytrids are frequently observed to include cells in which the daughter cells themselves contain tertiary (i.e. granddaughter) cells (highlighted using an arrow in figure 5a). Occasionally a fourth generation of cells can also be observed.

The method of reproduction exhibited by the disclosed thraustochytrids is very different to that of other labyrinthulomycetes, even though the Labyrinthulomycetes in general have a number of different methods of reproduction and different

Labyrinthulomycetes exhibit a range of methods of reproduction. These methods include for example, the production of zoospores or aplanospores or the production of an amoeboid stage, such as a limax amoeba. For the avoidance of doubt, the novel thraustochytrids can also produce zoospores, aplanospores and limax amoebae, but only the disclosed thraustochytrids have been observe to reproduce using the methods described above. As an example, Aplanochy rium haliotidis (formerly Labyrinthuloides haliotidis) described in Bower et al ((1987) Can. J. Zool., 65 (8): PP1996-2007) exhibits a method of reproduction that is typical of thraustochytrid-like microorganisms but differs very significantly from the method of reproduction exhibited by the disclosed

thraustochytrids. Neither A. haliotidis, nor indeed any other thraustochytrid-like microorganisms has ever been reported to reproduce by a method resembling that exhibited by the disclosed thraustochytrids, despite an extensive body of literature. Other thraustochytrid-like microorganisms can divide by simple division within the wall of the mother cell, including a binary division of the cell nucleus and the utilisation of all available cytoplasm (as shown in Bower (1987), figures 1, 5, 6). In this form of reproduction, production of the offspring necessarily results in the parent cell no longer being a viable cell. For example in A. haliotidis, Bower observes that the enveloping membranes are breaking down as the daughter cells form. In contrast, the mother cell of the disclosed thraustochytrids survives as a viable nucleated cell throughout the production of a number of daughter vegetative cells within the vacuole, and this can clearly be seen for example in the accompanying figures 5a, 5b, 6 and 7.

Furthermore, in other thraustochytrid-like microorganisms, the rapid sequential or synchronous fission, which results in a number of daughter cells within the remains of the original and daughter cell, occurs when a zoosporoblast, and subsequently zoospores, are formed, and not when further vegetative cells are formed. Although in the Labyrinthulomycetes, zoospores are produced asexually, their production is a separate reproductive process and the resulting cells are specialised zoospores, and cannot be considered to be a subsequent generation of reproductive cells, until spore settlement and further development has occurred.

In other reported thraustochytrid species (such as Thraustochytrium

multirudimentale) the cell from which the sagenogenetosome arises forms a specialised structure known as a basal rudiment, within a sporangium. The basal rudiment can grow to generate a second sporangium within which zoospores are formed but these are not vegetative cells. In the disclosed thraustochytrids, however, the mother cell survives as a viable nucleated cell surrounding its daughter vegetative cells (as shown in the accompanying Figures) and this has not been reported in relation to any other genera of the Labyrinthulomycetes, despite a substantial body of literature on the subject. No other thraustochytrid species exhibiting this method of reproduction has ever been identified or published in what is now an extensive body of literature. This is true even for observations of atypical forms of reproduction in other species. There are other taxa within the Protista that appear to reproduce by this form of internal formation of daughter cells, but these are taxonomically very remote from the

Labyrinthomycetes. The trout parasite Tetracapsuloides bryosalmonae is an example.

The disclosed thraustochytrids, of which the deposited strains are typical examples, are understood to fully meet the requirements for being regarded as a new genus and species. A manuscript is in preparation that will meet the requirements of the appropriate International Code of Nomenclature and which when published will provide the required scientific types for genus and species. The new genus has been given the proposed name of Caledochytrium aldermanii. When formally described, the disclosed thraustochytrids will therefore be described taxonomically as the type genus of a new genus of the Thraustochytriales, and moreover, also as the type species. When the disclosed thraustochytrids are cultured in heterotrophic medium, such as MCBHB medium or Pandey and Bhathena's Broth, cells can be observed (for example, using electron microscopy) which include both daughter and granddaughter vegetative cells. In contrast, when other known thraustochytrids are treated in the same way and visualised by electron microscopy, cells have never been observed which include both daughter and granddaughter vegetative cells. It is important that cells comprising both daughter and granddaughter vegetative cells are considered because various artefacts may give rise to cells which appear to comprise a daughter cell only, such as, for example, zoospores. It must be noted, however, that the observation of internal granddaughter cells is merely a requirement for clarity of identification and in fact other thraustochytrids have not been observed to include internal vegetative daughter cells within a vacuole in the cytoplasm of the mother cell.

Thus, one method by which the disclosed thraustochytrids can be unambiguously distinguished from other thraustochytrids, is that the disclosed thraustochytrids, when cultured in heterotrophic medium, such as MCBHB medium or what is informally known as "Mumbai Broth", devised by Pandey and Bhathena (Journal of Food and Nutrition Research, 2014, Vol. 2, No. 12, 993-999), can be observed (for example, using electron microscopy) to comprise cells including an internal daughter cell, wherein that daughter cell also comprises an internal daughter cell (i.e. granddaughter cell) within it. The proportion of the disclosed thraustochytrids, when cultured in heterotrophic medium, which may be observed (for example by electron microscopy) to include both daughter and granddaughter cells maybe at least 0.01%, 0.05% or 0.1% of the total cell population (or in any given electron micrographic study or field of view comprising more than 10,000 cells, such as more than 5000 or more than 1000 cells). In some cases, at least 0.2%, 0.3%, 0.5%, or 1% of the cells may be seen to include both daughter and granddaughter cells.

Due to this unique mode of reproduction, the disclosure includes a method for identifying a strain of thraustochytrid which is capable of producing high levels of fatty acids. The method comprises culturing the thraustochytrid strain in heterotrophic medium and determining whether the thraustochytrid culture comprises a cell having an internal daughter cell, wherein that daughter cell comprises a granddaughter cell within it. The presence of a cell comprising internal daughter and granddaughter cells is indicative that the thraustochytrid strain is capable of producing high levels of fatty acids.

Sequence Identity

The disclosed thraustochytrids can be identified by means of having above a specific level of sequence identity to a defined 18S ribosome RNA genetic sequence and/or ITS2-28S ribosomal RNA genetic sequence.

SEQ ID NO. 1 is an approximately 600 base pair sequence encompassing a portion of the 3' end of the ITS2 sequence and a portion of the 5' end of the 28S ribosome RNA genetic sequence of the disclosed thraustochytrids, specifically, of the strain CAi. For clarity, the region of genetic sequence encompassed by SEQ ID NO. 1 is referred to herein as an ITS2-28S ribosome RNA sequence.

This portion of the ribosome RNA genetic sequence to which the sequence of SEQ ID NO. 1 corresponds is known to be highly variable, and has, therefore, been selected in order to provide the maximum degree of discrimination between thraustochytrid strains, allowing the skilled person to determine whether or not a given strain is a thraustochytrid strain of the present disclosure.

In eukaryotes, the nuclear 18S rRNA gene (18S rRNA) is popularly used in scientific research as a marker for the taxonomic identification of microorganisms. SEQ ID NO. 8 is a 1628 base pair sequence corresponding to the 18S ribosomal RNA genetic sequence of the disclosed thraustochytrids. Details of PCR primers and conditions that may be used to amplify the 18S and/ or ITS2-28S ribosomal RNA sequences are described below. The skilled person will be able to use knowledge of the forward and reverse primer sequences to determine the exact region of genetic sequence that corresponds to SEQ ID NO. 1 and/ or SEQ ID NO. 8.

References in the present disclosure to the level of sequence identity that a given sequence has to SEQ ID NO. 1 or SEQ ID NO. 8 are to be interpreted specifically as referring to the level of sequence identity between SEQ ID NO. 1 or SEQ ID NO. 8 and the corresponding portion of the ITS2-28S or 18S ribosomal RNA sequence in question, respectively.

The disclosed thraustochytrids may have at least 86%, 87%, 88%, 89%, 90%, 91%, 93%, or 94% sequence identity to SEQ ID NO:8. Specifically, the disclosed thraustochytrids may have at least 86%, 92%, or 95% sequence identity to SEQ ID NO:8.

For example, the disclosed thraustochytrids may have at least 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, or 99% sequence identity to SEQ ID NO:8. The disclosed thraustochytrids may have more than 99% sequence identity to SEQ ID NO: 8, such as at least 99.1%, 99.2%, 99.4%, 99-5%, 99.6%, 99.8% sequence identity to SEQ ID NO:8.

The disclosed thraustochytrids may have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 93%, or 94% sequence identity to SEQ ID NO:i. Specifically, the disclosed

thraustochytrids may have at least 85%, 92%, or 95% sequence identity to SEQ ID NO:l.

For example, the disclosed thraustochytrids may have at least 95.5%, 96%, 96.5%, 97%, 97-5%, 98%, 98.5%, or 99% sequence identity to SEQ ID NO:i. The disclosed thraustochytrid may have more than 99% sequence identity to SEQ ID NO:i, such as at least 99.1%, 99.2%, 99.4%, 99-5%, 99.6%, 99.8% sequence identity to SEQ ID NO:i.

In some embodiments, the disclosed thraustochytrids have a corresponding 18S ribosome RNA gene sequence which differs from the sequence set out in SEQ ID NO:8 in less than 200, 175, 150, 125, 100, 80, 60, 50, 40, or 30 positions (i.e. nucleotides, bases, etc), such as less than 25, 20, 15, 12, 10, 7, or 5, positions. In some embodiments, the disclosed thraustochytrids have a corresponding ITS2-28S ribosome RNA gene sequence which differs from the sequence set out in SEQ ID NO:i in less than 60, 50, 40, or 30 positions (i.e. nucleotides, bases, etc), such as less than 25, 20, 15, 12, 10, 7, or 5, positions.

Suitable methods of extracting DNA from thraustochytrids and determining the ITS2- 28S and/or 18S rRNA sequence will be known to the skilled person. Examples of suitable methods are set out in Examples 6 and 7.

For the purposes of the present specification, the terms "sequence identity", "sequence homology", "sequence similarity", and similar terms, are to be interpreted as referring to the degree of similarity or relatedness between two nucleic acid sequences. Thus, for example, if the use of the word "homology" is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their nucleic acid sequences. Many of the methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related or not.

The terms "% sequence identity", "% identical", "% identity", "% homology", "% similarity" and similar terms, are to be understood to refer to the percentage of nucleotides that two or more sequences contain that are the same in corresponding positions when compared and aligned for maximum correspondence. A specified percentage of nucleotides can be referred to as having, for example, at least 70%, 80%, 85%, 90%, 95%, 99% sequence identity or homology over a specified region when compared and aligned for maximum correspondence. Unless otherwise stated, the determination of % sequence identity/homology is based on a comparison of the sequence in question with the entire sequence of SEQ ID NO. 8 or SEQ ID NO. 1 as appropriate, and not on a selected portion of SEQ ID NO. 8 or SEQ ID NO. 1. Thus, in the case that a given sequence has only 50% of the length of SEQ ID NO. 8 or SEQ ID NO. 1, the maximum possible percentage of sequence identity is 50%. The skilled person will understand that various means for comparing sequences are available. For example, one non-limiting example of a computer sequence alignment program which may be used for determining the percent sequence identity/ homology between sequences is the Basic Local Alignment Search Tool (BLAST).

Fatty Acids

Thraustochytrids are known to be able to produce compounds that are potentially valuable for use in biotechnology. In particular, thraustochytrids have been found to produce polyunsaturated fatty acids (PUFAs).

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."

Thraustochytrids are known to produce all of the above listed fatty acids.

Omega-3 fatty acids are biologically important molecules that have been found to affect cellular physiology, due to their presence in cell membranes, regulate production and gene expression of biologically active compounds, and serve as biosynthetic substrates. DHA accounts for approximately i5%-20% of lipids in the human cerebral cortex, 30%- 60% of lipids in the retina. DHA also accounts for about 97% of the omega-3 fatty acids in the brain and up to 93% of the omega-3 fatty acids in the retina, and is an important component of breast milk. DHA is essential for both foetal and infant development as well as maintenance of cognitive functions in adults.

Omega-3 fatty acids, including DHA, have also been found to possess antiinflammatory properties (as described, for example, in Hidalgo-Lucas et al.

Antiinflamm. Antiallergy. Agents Med. Chem. 13, 154-164). Omega-3 fatty acids are not synthesized in the human (or mammalian) body, however, and must, therefore, be obtained from nutritional sources. Fish oils, for example, are considered to be a good dietary source of omega-3 fatty acids. However, fish oils vary considerably in the type and level of fatty acid composition depending on the particular fish species and their diets. For example, fish raised by aquaculture tend to have a lower level of omega-3 fatty acids than those in the wild. The supply of wild fish is becoming increasingly limited. Furthermore, fish oils carry the risk of containing environmental contaminants and can be associated with stability problems and a fishy taste. Thus, there is a need for alternative, sustainable sources of omega-3 fatty acids. The disclosed thraustochytrids can clearly be seen using light or electron microscopy to comprise major lipid inclusions, as shown in figure lb.

Subsequent analysis of the lipids has shown that the disclosed thraustochytrids produce fatty acids, including omega-3 fatty acids, and in particular, DHA in significant quantities. In particular, the disclosed thraustochytrids have been found to produce significant quantities of fatty acids when cultured using heterotrophic medium, such as, for example, MCBHB medium or Pandey and Bhathena's Broth.

The disclosed thraustochytrids are capable of producing fatty acids, of which at least 30%, 35%, 40%, 43%, 48%, 53%, 57%, or even 60%, such as 45-75%, such as 50-70%, or 55-65% comprises omega-3 fatty acids.

In addition, the disclosed thraustochytrids are capable of producing fatty acids, of which at least 20%, 25%, 30%, 33%, 38%, 40%, 43%, 47%, 50%, 53%, 57% or even 60%, such as 35-70%, such as 45-67%, or 55-65% comprises DHA.

When the disclosed thraustochytrids are cultured in heterotrophic media such as MCBHB medium or Pandey and Bhathena's Broth, the concentrations of DHA in the culture medium may be at least 1, 2, 3, 4, 6, 8, 9, 11, 12, 13, 14, or 15 mgL ¹, such as 5-20 mgL ¹, such as 7-18 mgL-i, or 10-17 mgL-i.

Carotenoids

Oxygen is required for metabolic functions, but it also presents challenges to cells. This oxidative stress is believed to be a contributing factor in conditions such as rheumatoid arthritis, ischemic heart disease and stroke, Alzheimer's dementia, cancer and ageing. Therefore, compounds with antioxidant properties have the potential to protect against a wide spectrum of diseases (as described, for example, in V. Hajhashemi et al. Res Pharm Sci. 2010 Jan-Jun; 5(1): 1-8).

Several antioxidant compounds are known to be produced by thraustochytrids, and these include astaxanthin, beta-carotene and other carotenoids. The disclosed thraustochytrids have been found to produce carotenoids (see Example 2). Specifically, when grown in MCBHB medium the disclosed thraustochytrids have been found to produce carotenoids in a total amount of greater than 80, 100, 200, 300, 400, 500, 600, 800, 1000, 1200, 1500, 1800, 2000, or 2200 g/kg dry weight of sample, such as 50-2500, 80-2400, 100-2300, or l50-2250 g/kg dry weight of sample.

In particular, the disclosed thraustochytrids have been found to produce beta-carotene, astaxanthin, canthaxanthin, adonirubin and/or echinenone/cis-echinenone when grown in MCBHB medium.

The amount of beta-carotene produced maybe greater than 40, 50, 60, 80, 100, 150, 200, 500, 800, 1000, 1200, 1400, or 1600 g/kg dry weight of sample, such as 10- 2000, 20-1800, or 30-1700 g/kg dry weight of sample. The amount of astaxanthin produced may be greater than 100, 200, 300, or 400 μg/kg dry weight of sample, such as 50-1000, 100-900, or 150-850 μg/kg dry weight of sample.

The amount of canthaxanthin produced may be greater than 4, 5, 6, 8, 10, 15, 20, 50, 100, 200, 250, or 300 μg/kg dry weight of sample, such as 1-400, 2-350, 3-330, or 25- 310 μg/kg dry weight of sample.

The amount of adonirubin produced may be greater than 2, 3, 4, 6, 8, 10, 12, 15, 18, 20, 30, 40, 50, or 60 μg/kg dry weight of sample, such as 1-100, 2-80, 3-70 or 5-75 μg/kg dry weight of sample.

The amount of echinenone/cis-echinenone produced may be greater than 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 20, or 22 μg/kg dry weight of sample, such as 1-30, 2-28, or 3-25 μg/kg dry weight of sample. The disclosed thraustochytrids may produce carotenoids in a total amount of greater than 2200 g/kg dry weight of sample, including beta-carotene in an amount of greater than 1600 g/kg dry weight of sample, astaxanthin in an amount of greater than 150 g/kg dry weight of sample, canthaxanthin in an amount of greater than 300 g/kg dry weight of sample, adonirubin in an amount of greater than 60 μg/kg dry weight of sample, and/or echinenone/cis-echinenone in an amount of greater than 22 μg/kg dry weight of sample, when grown in MCBHB medium. For example, the disclosed thraustochytrid may be a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CA14.

Carotenoids are important natural pigments which are usually red, orange or yellow in colour. Traditionally, carotenoids have been used in the feed, food and nutraceutical industries. They are known to be essential for plant growth and photosynthesis, and are a main dietary source of vitamin A in humans. Carotenoids are potent biological antioxidants that can absorb the excited energy of singlet oxygen onto the carotenoid chain, leading to the degradation of the carotenoid molecule but preventing other molecules or tissues from being damaged.

Carotenoids are a widely distributed group of naturally occurring pigments, with astaxanthin and its derivatives being of particular interest commercially. Astaxanthin is an extremely effective antioxidant protector. Yet, unlike beta-carotene, astaxanthin readily crosses the blood-brain/retina barrier, and therefore also has potential to protect from diseases of the brain and the eyes. The disclosed thraustochytrids may be used in the production of carotenoids including carotene (such as beta-carotene), the xanthophyll compounds astaxanthin, zeaxanthin, canthaxanthin, and echinenone, lycopene, and others, including phoenicoxanthin, capsanthin, lutein, annatto, beta-apo-8-carotenal, and beta-apo-8-carotenal- ester. The carotenoids may be used in the treatment of any oxidative stress-related conditions, including those discussed above.

Squalene

Squalene is most commonly obtained from the livers of sharks as the concentration of the material is highest in those animals. Estimated demand for squalene is between 1,000-2,000 tons per annum. The livers of approximately 3,000 sharks are typically required to produce just 1 ton. Due to the excessive killing of sharks, growing environmental concerns and government regulation have created difficulties in getting raw material.

Squalene is also extracted from vegetable sources such as olive oil, sugarcanes, and from other sources such as wheat germ oil, and rice bran oil. The vegetable sources have low concentration of squalene and large acreage sites are required to make the whole operation feasible.

Thraustochytrids are known to produce squalene, and the disclosed thraustochytrids may be used as an eco-friendly alternative source of squalene.

Exopolysaccharides

Thraustochytrids are known to produce an extracellular composition comprising polysaccharides (up to about 53% of the composition), proteins, lipids, uronic acids, and sulphates. These compositions are known as "exopolysaccharides" (EPS) and are produced around the thraustochytrid cells, particularly as the cells age. The major polysaccharide component of the EPS is glucose, with some galactose, mannose, and arabinose. EPS are believed to protect the cells from desiccation and other environmental factors and to assist with adherence. Thraustochytrid-derived EPS has also been found to exhibit a broad spectrum of anti-viral activities.

The disclosed thraustochytrid has been found to produce large quantities of

exopolysaccharide, as shown in figure 3c.

Enzymes for Oil Breakdown

Thraustochytrids are heterotrophic microorganisms and are, therefore, capable of utilising organic compounds in their environment including hydrocarbons for their nutrition, and converting these to their cell biomass and to carbon dioxide through respiration. Thraustochytrids have been found to be capable of degrading and removing organic pollutants.

Hydrocarbons are one of the major pollutants in the marine environment. Sources of this pollutant are crude oils, as well as products of their distillation, such as gasolines, heating fuels, diesels, and heavy fuel oils. Once introduced into the water, hydrocarbons undergo a rapid process of weathering and biodegradation, whereby the volatile aromatics and small molecular aliphatics are removed. More complex components of the hydrocarbons, in the form of tar balls, persist for a longer time and continue to induce toxic effects. Such tar balls consist of long chain aliphatics, complex

polyaromatics and asphaltenes.

Thraustochytrids have been found to be capable of degrading and thereby removing hydrocarbon pollutants, including tar balls, in marine sediments. The disclosed thraustochytrids may be used in a method of removal of hydrocarbon pollutants, including tar ball pollutants. For use in the disclosed method,

thraustochytrids as disclosed can be grown in large quantities in any suitable nutrient medium. Thraustochytrids for use in the process can be obtained by culture in seawater/peptone broth containing autoclaved crude oil, but may be obtained by growing thraustochytrids in any standard nutrient broth containing seawater.

Autoclaved crude oil has been found to support excellent growth of thraustochytrids. The thraustochytrids can be applied directly to the polluted sediments. No

supplementary addition of nutrients is necessary; the thraustochytrids degrade the tar ball components.

For analytical purposes, tar balls may be dissolved in a small quantity of hexane and mixed with a marine sediment sample. After addition of thraustochytrids as disclosed to such a sediment, degradation of tar balls can be monitored periodically using gas chromatography.

Thus, the disclosed thraustochytrids may be used in a method for removal of hydrocarbon pollutants, including tar ball pollutants. For example, a method may comprise contacting hydrocarbon (such as tar ball)-polluted sediments with the disclosed thraustochytrid for an incubation period, and separating the thraustochytrids and sediments from the residual pollutants. The incubation period may be a period until the desired level of degradation of the pollutant (such as the tar ball) has been achieved, or it may be a fixed time period, such as for example, 5-200 days, such as, for example, 10-100 or 20-30 days. Extracting the sediment grown culture may comprise using hexane to remove the residual tar ball. Disclosed Thraustochytrids

In view of this, the present disclosure is directed to a newly identified group of isolated thraustochytrids, exemplified by the deposited thraustochytrid strains, and also including mutants, recombinants, and variants of the deposited thraustochytrid strains, together with thraustochytrid strains that have genetic and/ or morphological and functional features that are substantially the same as those of the deposited

thraustochytrids.

It is to be understood that the disclosed products and/ or methods are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, because such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Those skilled in the art will recognise, or be able to ascertain' using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the present disclosure and claims.

A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

Terms such as "isolated", and any form such as "isolate", refer to a situation where something is in a form wherein it can be manipulated or further purified. The term "isolated", and its forms, indicates that something is in a current state which is different than a previous state. For example, a compound can be "isolated" if it is, for example removed from an organism, synthesised or produced recombinantly. Often, the

"isolation" of one thing is in relation to something else. For example, the disclosed thraustochytrids may be isolated, for example, by being cultured in the absence of appreciable (detectable) amounts of other organisms. It is understood that unless specifically indicated otherwise, any of the disclosed compositions can be isolated as disclosed.

In some embodiments, the disclosure is directed to an isolated thraustochytrid strain, referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14, deposited under the Budapest Treaty on 1 November 2017 under Accession Numbers IMI 506775, IMI 506776, IMI 506777, IMI 506778, IMI 506779, IMI 506780, or on 16 October 2018 under Accession Number IMI 507005, respectively, at CABI, the International

Mycological Institute, Genetic Resources Collection, Bakeham Lane, Egham, Surrey, TW20 9ΤΎ, UK. In some embodiments, the disclosure is directed to an isolated thraustochytrid strain, referred to herein as CAi, CA2, or CA8, deposited under the Budapest Treaty on 2 November 2017 under Accession Numbers CCAP 4063/1, CCAP 4063/2, CCAP 4063/3, respectively, at the Culture Collection of Algae and Protozoa (CCAP), SAMS Limited, Scottish Marine Institute, OBAN, Argyll PA37 lQA, Scotland, United Kingdom.

In some embodiments, the disclosure is directed to an isolated thraustochytrid of the same species as the thraustochytrid strains referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, and CA14 (corresponding Accession Numbers as set out above). In some embodiments, the disclosure is directed to a mutant, variant, or recombinant strain derived from one of the isolated thraustochytrid strains referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, and CA14 (corresponding Accession Numbers as set out above). Mutant strains can be produced by well-known methods, and the skilled person will be familiar with various suitable methods. For example, common methods include irradiation, treatment at high temperatures, and treatment with a mutagen.

Variant strains can be other naturally occurring isolates and/ or sub-isolates of the species described in the present disclosure, such as those referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, and CA14 (corresponding Accession Numbers as set out above).

Recombinant strains can be produced by any well-known methods for the expression of exogenous genes or the alteration of endogenous gene function or expression, and the skilled person will be familiar with various suitable methods.

In some embodiments, the disclosure is directed to an isolated thraustochytrid strain having the characteristics of the strains referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, and CA14 (corresponding Accession Numbers as set out above). The

characteristics of the deposited thraustochytrid strain 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 particular, in some embodiments, the disclosure is directed to an isolated

thraustochytrid strain having the reproductive properties of the strains referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, and CA14 (corresponding Accession Numbers as set out above). Thus, in some embodiments, the disclosure is directed to an isolated thraustochytrid strain wherein, when the thraustochytrid strain is cultured in heterotrophic medium and visualised using electron microscopy, cells can be observed which include an internal daughter cell, wherein that daughter cell also comprises an internal daughter cell (i.e. granddaughter cell) within it.

In some embodiments, the proportion of cells which include both daughter and granddaughter cells may be at least 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, or 1% of the total cell population (or in any given electron micrographic study or field of view comprising more than 1000 cells).

In some embodiments, the isolated thraustochytrid strains of the disclosure have substantially identical phenotypic properties to the thraustochytrid strains referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, and CA14 (corresponding Accession Numbers as set out above).

In some embodiments, the isolated thraustochytrid strains of the disclosure have substantially identical growth properties to the thraustochytrid strains referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, and CA14 (corresponding Accession Numbers as set out above).

The disclosure is directed to an isolated thraustochytrid strain having an ITS2-28S ribosome RNA sequence which has at least 85% sequence identity to SEQ ID NO:i. In some embodiments, the disclosure is directed to an isolated thraustochytrid strain having an ITS2-28S ribosome RNA sequence which has at least 92% or 95% sequence identity to SEQ ID NO:i. In some embodiments, the disclosure is directed to an isolated thraustochytrid strain having an 18S ribosome RNA sequence which has at least 86%, 92%, or 95% sequence identity to SEQ ID NO:8. In some embodiments, the disclosure is directed to an isolated thraustochytrid strain having an ITS2-28S ribosome RNA sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 93%, 94%, 95-5%, 96%, 96.5%, 97%, 97-5%, 98%, 98.5%, 99%, 99-1%, 99.2%, 99.4%, 99-5%, 99.6%, or 99.8% sequence identity to SEQ ID NO:i. In some embodiments, the disclosure is directed to an isolated thraustochytrid strain having an 18S ribosome RNA sequence which has at least, 87%, 88%, 89%, 90%, 91%,

93%, 94%, 95-5%, 96%, 96.5%, 97%, 97-5%, 98%, 98.5%, 99%, 99-1%, 99-2%, 99-4%, 99.5%, 99.6%, or 99.8% sequence identity to SEQ ID NO:8. In some embodiments, the disclosure is directed to an isolated thraustochytrid strain having an ITS2-28S ribosome RNA sequence which differs from the sequence set out in SEQ ID NO:i in less than 80, 70, 60, 50, 40, or 30 positions, such as less than 25, 20, 15, 12, 10, 7, or 5, positions. In some embodiments, the disclosure is directed to an isolated thraustochytrid strain having an 18S ribosome RNA sequence which differs from the sequence set out in SEQ ID NO:8 in less than 200, 175, 150, 125, 100, 80, 60, 50 positions, such as less than 40, 35, 30, 25, 20, 15, 12, 10, 7, or 5, 4, 3, or 2 positions. The disclosure is directed to an isolated thraustochytrid strain having an ITS2-28S ribosomal RNA sequence, wherein the portion of the ITS2-28S ribosomal RNA sequence that corresponds to SEQ ID NO. 1 has at least 85%sequence identity to the sequence set forth in SEQ. ID NO: 1. In some embodiments, the disclosure is directed to an isolated thraustochytrid strain having an ITS2-28S ribosomal RNA sequence, wherein the portion of the ITS2-28S ribosomal RNA sequence that corresponds to SEQ ID NO. 1 has at least 86%, 87%, 88%, 89%, 90%, 91%, 93%, 94%, or 95% sequence identity to the sequence set forth in SEQ. ID NO: 1.

In some embodiments, the disclosure is directed to an isolated thraustochytrid strain having an ITS2-28S ribosomal RNA sequence, wherein the portion of the ITS2-28S ribosomal RNA sequence that corresponds to SEQ ID NO. 1 has at least 95.5%, 96%, 96.5%, 97%, 97-5%, 98%, 98.5%, 99%, 99·ΐ%, 99-2%, 99-4%, 99-5%, 99-6%, or 99.8% sequence identity to SEQ ID NO:i.

In some embodiments, the disclosure is directed to an isolated thraustochytrid strain having an ITS2-28S ribosomal RNA sequence, wherein the portion of the ITS2-28S ribosomal RNA sequence that corresponds to SEQ ID NO. 1 differs from the sequence set out in SEQ ID NO:i in less than 80, 70, 60, 50, 40, or 30 positions (i.e. bases), such as less than 25, 20, 15, 12, 10, 7, or 5, positions. The disclosure is directed to an isolated thraustochytrid strain having an ITS2-28S ribosome RNA sequence which has at least 85% sequence identity to the ITS2-28S ribosome RNA sequence of an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above). In some embodiments, the disclosure is directed to an isolated thraustochytrid strain having an ITS2-28S ribosome RNA sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 93%, 94%, or 95% sequence identity to the ITS2-28S ribosome RNA sequence of an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above). In some embodiments, the disclosure is directed to an isolated thraustochytrid strain having an 18S ribosome RNA sequence which has at least 86%, 90%, 92%, or 95% sequence identity to the 18S ribosome RNA sequence of an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above).

In some embodiments, the disclosure is directed to an isolated thraustochytrid strain having an ITS2-28S ribosome RNA sequence which has at least 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.4%, 99.5%, 99.6%, or 99.8% sequence identity to the ITS2-28S ribosome RNA sequence of an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above).

In some embodiments, the disclosure is directed to an isolated thraustochytrid strain having an 18S ribosome RNA sequence which has at least 95.5%, 96%, 96.5%, 97%, 97-5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.4%, 99-5%, 99.6%, or 99.8% sequence identity to the 18S ribosome RNA sequence of an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above).

The disclosed thraustochytrids may be used to produce lipids, such as fatty acids, including omega-3 fatty acids, DHA, ARA, EPA, and DPA n-6.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the mutant, variant, or recombinant is capable of producing fatty acids, of which 45- 75%, 50-70%, or 55-65% of the total fatty acids produced comprises omega-3 fatty acids.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the mutant, variant, or recombinant is capable of producing fatty acids, of which 35- 70%, 45-67%, or 55-65% comprises DHA. In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the mutant, variant, or recombinant is capable of producing fatty acids comprising ARA, EPA, and/or DPA n-6.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, CA14 (corresponding Accession Numbers as set out above), wherein, when cultured in heterotrophic medium, the mutant, variant, or recombinant is capable of producing DHA in the culture medium at a concentration of 5-20 mgL-i, such as 7- 18 mgL-i, or 10-17 mgL-i.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the total level of fatty acids produced by the mutant, variant, or recombinant is within 10%, such as within 7%, 5%, 3%, 2%, or 1% of the total level of fatty acids produced by one of the deposited strains.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, and CA14 (corresponding Accession Numbers as set out above), wherein the total level of DHA produced by the mutant, variant, or recombinant is within 10%, such as within 7%, 5%, 3%, 2%, or 1% of the total level of DHA produced by one of the deposited strains.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the total level of ARA, EPA, and/ or DPA n-6 produced by the mutant, variant, or recombinant is within 10%, such as within 7%, 5%, 3%, 2%, or 1% of the total level of ARA, EPA, and/or DPA n-6 produced by one of the deposited strains.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein when the mutant, variant, or recombinant is cultured in heterotrophic medium, the concentration of DHA in the culture medium is within 10%, such as within 7%, 5%, 3%, 2%, or 1% of the concentration of DHA in the culture medium produced by one of the deposited strains.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein when the mutant, variant, or recombinant is cultured in heterotrophic medium, the concentration of ARA, EPA, and/ or DPA n-6 in the culture medium is within 10%, such as within 7%, 5%, 3%, 2%, or 1% of the concentration of ARA, EPA, and DPA n-6 in the culture medium produced by one of the deposited strains.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein when the mutant, variant, or recombinant is used to produce a biomass comprising a fatty acid profile substantially the same as the fatty acid profile produced by one of the deposited strains. Methods of determining fatty acid profiles are known. In some embodiments, the disclosure is directed to an isolated thraustochytrid biomass produced by an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), or a mutant, variant, or recombinant derived from one of the deposited strains. An isolated thraustochytrid biomass of the disclosure is a harvested cellular biomass obtained by any conventional method for the isolation of a thraustochytrid biomass.

In some embodiments, the disclosure is directed to a culture comprising one or more isolated thraustochytrid strains selected from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), a mutant, variant, or recombinant derived from one of the deposited strains. Various parameters for inoculating, growing, and recovering thraustochytrids are set out in the Examples below, and are known in the art.

The disclosed thraustochytrids may be used to produce antioxidants, such as but not limited to the carotenoid compound carotene, (for example β-carotene) and the xanthophylls compounds astaxanthin, and canthaxanthin.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the mutant, variant, or recombinant is capable of producing carotenoids, wherein carotenoids comprise greater than 80, 100, 200, 300, 400, 500, 600, 800, 1000, 1200, 1500, 1800, 2000, or 2200 g/kg dry weight of sample, such as 50-2500, 80-2400, 100-2300, or 150-2250 g/kg dry weight of sample. For example, the isolated thraustochytrid may be a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CA14, and may be capable of producing carotenoids in an amount of greater than 2200 g/kg dry weight of sample.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein CAi, CA2, CA3, CA4, CA5, CA8, CA14 (corresponding Accession Numbers as set out above), wherein the mutant, variant, or recombinant is capable of producing beta-carotene, wherein beta- carotene comprises greater than 40, 50, 60, 80, 100, 150, 200, 500, 800, 1000, 1200, 1400, or 1600 g/kg dry weight of sample, such as 10-2000, 20-1800, or 30-1700 g/kg dry weight of sample. For example, the isolated thraustochytrid may be a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CA14, and may be capable of producing beta-carotene in an amount of greater than 1600 g/kg dry weight of sample.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the mutant, variant, or recombinant is capable of producing astaxanthin, wherein astaxanthin comprises greater than 100, 200, 300, or 400 g/kg dry weight of sample, such as 50-1000, 100-900, or 150-850 μg/kg dry weight of sample. For example, the isolated thraustochytrid may be a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CA3, and may be capable of producing astaxanthin in an amount of greater than 580 μg/kg dry weight of sample.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the mutant, variant, or recombinant is capable of producing canthaxanthin, wherein canthaxanthin comprises greater than 4, 5, 6, 8, 10, 15, 20, 50, 100, 200, 250, or 300 μg/kg dry weight of sample, such as 1-400, 2-350, 3-330, or 25-310 μg/kg dry weight of sample. For example, the isolated thraustochytrid may be a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CA14, and may be capable of producing canthaxanthin in an amount of greater than 300 μg/kg dry weight of sample. In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the mutant, variant, or recombinant is capable of producing adonirubin, wherein adonirubin comprises greater than 2, 3, 4, 6, 8, 10, 12, 15, 18, 20, 30, 40, 50, or 60 μg/kg dry weight of sample, such as 1-100, 2-80, 3-70 or 5-75 μg/kg dry weight. For example, the isolated thraustochytrid may be a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CA14, and may be capable of producing adonirubin in an amount of greater than 60 g/kg dry weight of sample.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the mutant, variant, or recombinant is capable of producing echinenone/cis-echinenone, wherein echinenone/cis-echinenone produced is greater than 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 20, or 22 μg/kg dry weight of sample, such as 1-30, 2-28, or 3-25 μg/kg dry weight. For example, the isolated thraustochytrid may be a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CA14, and may be capable of producing echinenone/cis-echinenone in an amount of greater than 22 g/kg dry weight of sample. In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the total level of carotenoid produced by the mutant, variant, or recombinant is greater than the total level of carotenoid produced by one of the deposited strains or within 10%, such as within 7%, 5%, 3%, 2%, or 1% of the total level of carotenoid produced by one of the deposited strains.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the total level of beta-carotene, xanthophyll, astaxanthin, zeaxanthin, canthaxanthin, echinenone, lycopene, and others, including phoenicoxanthin, capsanthin, lutein, annatto, beta-apo-8-carotenal, and/ or beta-apo-8-carotenal- ester produced by the mutant, variant, or recombinant is greater than the total level of the corresponding compound produced by one of the deposited strains or within 10%, such as within 7%, 5%, 3%, 2%, or 1% of the total level of the corresponding compound produced by one of the deposited strains.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the total level of squalene produced by the mutant, variant, or recombinant is greater than the total level of squalene produced by one of the deposited strains or within 10%, such as within 7%, 5%, 3%, 2%, or 1% of the total level of squalene produced by one of the deposited strains.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the profile of exopolysaccharides produced by the mutant, variant, or recombinant is substantially the same as that produced by one of the deposited strains.

In some embodiments, the disclosure is directed to a mutant, variant, or recombinant derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the dry cell weight per litre of culture produced by the mutant, variant, or recombinant comprise is great than the dry cell weight per litre of culture produced by one of the deposited strains or within 10%, such as within 7%, 5%, 3%, 2%, or 1% of the dry cell weight per litre of culture produced by one of the deposited strains. In some embodiments, any clones, modified organisms or genes isolated from any of the thraustochytrid strains referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above) are also disclosed.

Genetics

A new approach for the production of oils has been to transfer a set of genes involved in omega-3 and/or omega-6 synthesis into plants in order to produce high levels of PUFA in oilseed. Thus, the unique ability of thraustochytrids to produce high amounts of DHA and other LC-PUFA has generated interest in metabolic engineering using thraustochytrid enzymes involved in PUFA synthesis. The first example of transfer of thraustochytrid genes into plants for the production of PUFA was described in WO

2004/071467, using an elongase from Thraustochytrium aureum, among other genes. The authors showed maximum level of 19.6% of EPA and 3.3% of DHA in genetically modified Glycine max embryos and seeds. Similarly, Wu et al. (Nat. Biotechnol. 2005 23, 1013-1017) engineered a 9-gene construct, of which 4 genes came from

Thraustochytrium sp. 26185, and showed a maximum level (wt%) of ARA, EPA and DHA (7.3%, 15% and 1.5%, respectively) in seeds of the transgenic Brassicajuncea breeding line 1424. The Rothamsted Institute in the UK carried out extensive work on EPA and DHA biosynthesis mArabidopsis thaliana and Camelina saliva using delta-4 and/or delta-5 desaturases from Thraustochytrium sp., in combination with genes from other microorganisms (Ruiz-Lopez et al, Metab. Eng. 2014 17, 30-41, and Ruiz-Lopez et al. Plant J. 2013 77, 198-208).

In some embodiments, the present disclosure is directed to genes obtained or derived from the disclosed thraustochytrids. Such genes may include, in particular, genes involved in the synthesis of PUFAs, which may include any genes corresponding to the thraustochytrid-derived genes used in the studies discussed above.

In some embodiments, the disclosure is also directed to a recombinant thraustochytrid derived from an isolated thraustochytrid strain referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, or CA14 (corresponding Accession Numbers as set out above), wherein the recombinant thraustochytrid expresses an exogenous gene, or has altered levels of expression or activity of an endogenous gene.

Culturing

The disclosed thraustochytrids are able to utilize various nutritional components.

Preferably, the disclosed thraustochytrids may be cultured using heterotrophic medium. As the skilled person will be aware, suitable heterotrophic medium for the culturing of thraustochytrids can comprise sea salt (artificial or natural), one or more carbon sources, and one or more nitrogen sources.

Sea salt can be present in the heterotrophic medium in an amount of from about 2.0 to about 40.0 g L ¹.

The carbon source may be used in the heterotrophic medium at a concentration of 5- 6ogL^_1, such as 0.5-50gL^_1 or i-25gL^_1.

The nitrogen source may be used in the heterotrophic medium at a concentration of 1- 20gL^_1, such as i.5-i5gL^_1, or 2-iogL ¹. The nitrogen source may comprise, for example, peptone, yeast extract, malt extract, sodium glutamate, corn steep liquor and/or cotton seed extract. The yeast extract may be present in an amount of about o.2-2gL ¹, such as 0.5-igL ¹. The monosodium glutamate may be present in an amount of about 0.1-iogL ¹, such as 0.5-8 gL ¹.

The carbon source may comprise D-glucose, D-trehalose, glycerol, D-gluconic acid, L- lactic acid, D,L-malic acid, D-ribose, Tween 20, D-fructose, acetate, acetic acid, maltose, thymidine, L-asparagine, D-xylose, Tween 40,a-keto-glutaric acid, sucrose, L- glutamine, Tween 80, beta-methyl-D-glucoside, maltotriose, adenosinine, fumaric acid, bromo succinic acid, L-serine, D-cellobiose, L-alanyl-glycine, methyl pyruvate, L-malic acid, glycyl-L-proline, D-palcose, L-lyxose, pyruvic acid, alpha-D-lactose, dextrin, D- arabinose, 2-deoxy-D-ribose, gelatin, dextrose, starch, 3-0-beta-D-galactopyranosyl-D- arabinose, D-tagatose, 5-keto-D-gluconic acid, oxalomalic acid, sorbic acid, L-omithine, and dihydroxy acetate.

The medium may further comprise other additives, including phosphates (such as potassium phosphate and sodium phosphates), inorganic salts (such as ammonium sulfate, sodium bicarbonate, sodium orthovanadate, potassium chromate, sodium molybdate, selenous acid, nickel sulfate, copper sulfate, zinc sulfate, cobalt chloride, iron chloride, manganese chloride), a chelating agent (such as EDTA), vitamins (such as pyridoxine hydrochloride, thiamin hydrochloride, calcium pantothenate, p- aminobenzoic acid, riboflavin, nicotinic acid, biotin, folic acid, and vitamin B12). The medium can be at a pH of from about 4.0 to about 6.5.

Incubation can be from about 1 to about 9 days (e.g., from about 3 to about 5 days). Incubation can be at from about 18 to about 30°C (e.g., from about i8-25°C).

Incubation can further comprise shaking or aeration.

Examples of heterotrophic media that will be familiar to the skilled person include those disclosed by Rosa et al., and Pandey et al., which are discussed below: MCBHB medium (see Rosa, S.M., Galvagno, M.A., Velez, C.G., 2011. Adjusting culture conditions to isolate thraustochytrids from temperate and cold environments in southern Argentina. Mycoscience 52, 242-252. doi:io.ioo7/si0207-oio-oo9i-2) (Quantities given in gL ¹, except where noted)

D-Glucose 1.0 gL ¹;

Peptone 0.5 gL ¹;

Yeast Extract 0.5 gL ¹; Monosodium Glutamate 0.5 gL ¹;

Gelatine hydrolysate 1.0 gL ¹;

Brain Heart Broth 17.5 gL ¹;

Corn Steep Liquor 0.5ml per litre.

Ingredients made up in 1 litre of 75% double filtered natural seawater; 25 % distilled water.

To make MCBHB agar plates, 20g of agar is added to the mixture above. Pandey and Bhathena's Broth (see Pandey, A., Bhathena, Z., 2014. Prevalence of PUFA rich thraustochytrids sps. along the coast of Mumbai for production of bio oil. J. Food Nutr. Res. 2, 993-999. doi:io.i209i/jfnr-2-i2-2i)

(Quantities given in g except when specified)

Peptone 5 g;

Yeast Extract 3 g;

Glycerol 3 ml;

1 litre of 75% double filtered natural seawater, made up to 1 litre with distilled water. Plus, after autoclaving;

Ampicillin 20 mgL ¹;

Tetracycline 2 mgL ¹;

Fluconazole 100 mgL ¹;

Rifampicin 25 mgL ¹;

Penicillin 300 mgL ¹

Streptomycin 500 mgL ¹

Methods of Obtaining Products

Methods of obtaining the products disclosed above, including fatty acids, carotenoids, squalene, etc from the disclosed thraustochytrids comprise culturing a thraustochytrid as disclosed and isolating the desired product.

Various suitable procedures can be employed in the recovery of the cellular biomass from the culture media, such as by filtration or centrifugation, and these methods will be familiar to the skilled person. The cells and/or media can then be prepared for further use or for storage as appropriate. For example, the cells can be washed, frozen, lyophilized, or spray dried, as appropriate, using any suitable known method. For the production of fatty acids specifically, thraustochytrids as disclosed may be grown in a heterotrophic culture medium (as described above). The supply of nitrogen is limited after about 24 to about 48 hours, while the supply of carbon remains in abundance. The thraustochytrid continues to assimilate the carbon but the lack of nitrogen prevents protein and nucleic acid synthesis, causing the organisms to cease cell division. The result is that the sugars within the medium are converted to lipids by the thraustochytrids.

Lipids, for example, containing DHA and other PUFAs, can be extracted from the cellular biomass produced from the disclosed thraustochytrids using methods known to the skilled person. These include supercritical fluid extraction, or extraction with solvents such as chloroform, hexane, methylene chloride, or methanol. The resulting extract can then be evaporated under negative pressure to produce a sample of concentrated lipid material.

The omega-3 PUFAs, for example, may be further concentrated by hydrolyzing the lipids and concentrating the highly unsaturated fraction by employing traditional methods such as urea adduction or fractional distillation, column chromatography, or by supercritical fluid fractionation. The cells can also be broken or lysed and the lipids extracted into vegetable or animal oils. The extracted oils can be refined by well-known processes routinely employed to refine vegetable oils (e.g. by chemical or physical refining). These refining processes remove impurities from extracted oils. After refining, the oils can be used directly, for example, as a feed or food additive to produce omega-3 enriched products. Alternatively, the oil can be further processed and purified and then used in the above applications and also in pharmaceutical applications.

Concentrated omega-3 may also be produced from the disclosed thraustochytrids by rupturing or permeabilising the harvested cellular biomass by well-known techniques such as sonication, liquid-shear disruption methods, bead milling, pressing under high pressure, freeze-thawing, or enzymatic digestion of the cell wall. The lipids from the ruptured cells may be extracted using a solvent or mixture of solvents such as hexane, chloroform, ether, or methanol. The solvent may be removed and the lipids hydrolyzed using any of the well-known methods. After hydrolysis, the nonsaponifiable compounds may be extracted into a solvent such as ether, hexane or chloroform and removed. The remaining solution may then be acidified by addition of an acid, and the free fatty acid extracted into a solvent such as hexane, ether or chloroform. The solvent solution containing the free fatty acids may then be cooled to a temperature low enough for crystallization of the non-PUFA compounds, which can then be removed via filtration, centrifugation or settling. This results in a concentrated preparation of the remaining PUFA compounds which may be used, for example, as nutritional supplements for humans, as food additive, or in pharmaceutical applications.

Microbial Oils

The present disclosure is further directed to methods of producing microbial oils. In some embodiments, the method comprises growing a thraustochytrid of the disclosure in a culture to produce a biomass and extracting an oil comprising omega-3 fatty acids from the biomass.

In some embodiments the oil is extracted from a freshly harvested biomass and in other embodiments the oil is extracted from a previously harvested biomass that has been stored under suitable preservative conditions. Methods of isolating a biomass from a culture of the disclosed thraustochytrids, of extracting a microbial oil from the biomass, and of analysing the fatty acid profile of oils extracted from the biomass, are known. The disclosure is further directed to a microbial oil produced by a thraustochytrid of the disclosure. A microbial oil of the invention can be any oil derived from a

thraustochytrid, including, for example: a crude oil extracted from the thraustochytrid biomass 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 (such as DHA) in the oil. The lipid classes present in the microbial oil can be separated and analysed by methods known in the art. For example, flash chromatography can be used for separation, and thin layer chromatography (TLC) can be used for analysis.

In some embodiments, the microbial oil comprises a sterol esters fraction, a triglyceride fraction, a free fatty acid fraction, a sterol fraction, and/ or a diglyceride fraction. Compositions

In some embodiments, compositions are provided comprising a thraustochytrid as disclosed, an isolated biomass of a thraustochytrid as disclosed, a microbial oil of a thraustochytrid as disclosed, or combinations thereof.

In some embodiments, the thraustochytrid, biomass, or microbial oil may be further chemically or physically modified or processed based on the requirements of the composition by any known technique.

In some embodiments, the thraustochytrid cells or biomasses may 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 may be used directly in a composition without drying.

In some embodiments, microbial oils may be used as starting material to more efficiently produce a product enriched in a fatty acid such as DHA. For example, the microbial oils maybe 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 DHA or another fatty acid. The microbial oils may also be used in chemical reactions to produce compounds derived from fatty acids in the oils, such as esters and salts of DHA or another fatty acid.

In some embodiments, the composition may include one or more excipients. For the purposes of the present disclosure, "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 may 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. Various excipients may be used. In some embodiments, for example, the excipient may be 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.

In some embodiments, compositions may include food products, pharmaceutical compositions, cosmetics, and industrial compositions. In some embodiments, the composition may be a food product. By "food product" is meant any article that may be consumed (e.g., eaten, drank, or ingested) by a subject, and includes both solid and liquid compositions. A food product may be an additive to animal or human foods. Also disclosed are food products comprising any of the disclosed compositions. In some embodiments, the composition may be used in feed enrichment (for example through adding the whole freeze-dried organism to animal feed).

In some embodiments, the composition may be a feed or feed supplement for use in different aspects of aquaculture, including the farming of shellfish and fish. For example, use of the compositions may comprise feeding directly with thraustochytrid cells, or may comprise the use of thraustochytrid-derived material, such as oils, incorporated into formulated food. In other examples, use of the compositions may comprise the enrichment of larval brine shrimps or rotifers with live thraustochytrid cells, prior to feeding them to larval fish.

In some embodiments, the composition may be used as a nutritional supplement to a food product. In some embodiments, the compositions may be included in a nutritional supplement, and nutritional supplements are provided comprising any of the disclosed compounds that may be produced by the disclosed thraustochytrids. A nutritional supplement is any compound or composition that may be administered to or taken by a subject to provide, supply, or increase a nutrient(s). For example, a nutritional supplement may comprise any of the disclosed lipids or antioxidants. The nutritional supplement may comprise any amount of the disclosed compounds, and an appropriate amount may be determined by the skilled person using known methods. In one specific example, a nutritional supplement may comprise from about 0.05 to about 20%, from about 1 to about 7.5%, or from about 3 to about 5% by weight of the compound. The nutritional supplement may also comprise other nutrient(s) such as vitamins trace elements, minerals, and the like. Further, the nutritional supplement may comprise other components such as preservatives, antimicrobials, anti-oxidants, chelating agents, thickeners, flavorings, diluents, emulsifiers, dispersing aids, and/or binders.

In some embodiments, the composition may be a pharmaceutical composition. The composition may be combined with pharmaceutically acceptable carriers, such as any suitable carrier known in the art. In some embodiments, the composition may be formulated in a dosage form.

In some embodiments, the composition may be a cosmetic, which may, for example, include emulsions, creams, lotions, masks, soaps, shampoos, washes, facial creams, conditioners, make-ups, bath agents, and dispersion liquids. The cosmetic may be medicinal or non-medicinal.

In some embodiments, the composition may be an industrial composition, such as, for example, a starting material for the production of a polymer, a detergent, an industrial oil, or an industrial detergent.

In some embodiments, the compositions maybe for use in the treatment of a condition in humans or animals.

In some embodiments, the compositions maybe for use as an antiviral. For example, compositions comprising EPS, or a specific EPS constituent, produced from a thraustochytrid as disclosed may be used to treat a variety of viral infections including hepatitis viruses, herpes viruses, enteroviruses, retroviruses, cytomegaloviruses, adenoviruses, etc. The antiviral EPS may be used in a wide variety of formulations to treat viral infections. They may be incorporated in formulations for topical applications against certain viral infections. The purified antiviral EPS may be further fractionated to obtain the active moiety of the EPS that has antiviral activity. The active moiety may be used as a drug for internal use in treating viral infections.

Various dosage amounts of the composition may be administered to a subject, based on the amount of DHA, carotenoid, squalene, EPS, or other fatty acid or organic component of the thraustochytrid, biomass, or microbial oil to be administered to the subject.

In some embodiments, one or more units of the composition may be contained within a kit or package. Kits or packages may include, for example, units of a food product, pharmaceutical composition, cosmetic, or industrial composition comprising the disclosed thraustochytrid, biomass, or microbial oil, or combinations thereof. Kits or packages may also include, for example, an additive comprising the disclosed thraustochytrid, biomass, or microbial oil, or combinations thereof for preparation of a food, cosmetic, pharmaceutical composition, or industrial composition.

In addition, disclosed are the fatty acids and carotenoids produced by the disclosed eukaryotic microorganism and any progeny (genetically modified or otherwise), various feedstuffs, nutraceuticals, pharmaceutical and food supplemented with the lipids and antioxidants, as well as a process for utilizing these compounds as an additive for various feedstuffs and foods.

Examples

The invention will now be explained in further detail in the following Examples. l. Isolation and Culture of the Disclosed Thraustochytrids

The thraustochytrid strains referred to herein as CAi, CA2, CA3, CA4, CA5, CA8, and CA14 (corresponding Accession Numbers as set out above) are part of a large collection of thraustochytrid strains which have been isolated and accumulated in the UK over the last 40 years. The thraustochytrid strains in the collection, including the deposited strains, have never been cryop reserved, but have been maintained in slow-growing, live, pure culture since their original isolation.

The isolates were grown at either 7°C, io°C, or 15°C in flasks containing 20ml of sterile seawater, with 80ml of air space above to provide an oxygen supply. After inoculating the seawater with three standard bacteriology loops of an actively growing culture, approximately o.25gms of pine pollen, which had previously been twice dry air heated at 103-106⁰ for 8 hours in an oven to sterilise it, was added. The pine pollen provides a rich supply of nutrients for the thraustochytrids which can penetrate the tough wall of the pollen grain using their ectoplasmic net. Pollen grains which are not colonised by the thraustochytrids retain their nutrient value for many months. Therefore, the cultures only require to be subbed every three to six months.

When required for further study, colonized pollen grains were plated on to agar produced using heterotrophic medium, such as, for example, MCBHB agar or Pandey and Bhathena's ("Mumbai") agar, and single colonies were isolated and transferred into heterotrophic medium, such as MCBHB or Pandey and Bhathena's ("Mumbai") Broth in flask culture. Each thraustochytrid isolate in the collection, including the deposited strains, was created by inoculating an original culture with one pollen grain, colonised by one thraustochytrid from a culture which had itself been inoculated with a single colony from an agar plate (0.03% peptone, 0.003% yeast extract (Difco Germany) 0.3% malt extract (Biomaltz, Germany) 2% pre-soaked Fadenagar in seawater to make a moist agar, 0.2% bacteriological agar (Difco, Germany)). The agar plates had been inoculated with pollen grains, which came from seawater pollen flasks, which had previously been inoculated with small amounts of sample material from which thraustochytrids were observed to have grown. Each isolate was therefore originally an axenic culture of one thraustochytrid.

2. Light Microscopy and Carotenoid Production

The disclosed thraustochytrids were investigated using light microscopy.

Individual axenic cultures, grown on pollen, were examined in a specially adapted chamber for use with a high-powered Leitz or Zeiss microscope equipped with a camera and/ or video capacity. The adapted chamber was arranged to provide slowly flowing, sterile sea water, which allowed individual cells to be viewed at a range of

magnifications (up to ιοοοχ) and followed over a complete cycle(s) of growth and division over 2-4 days.

A mature cell, which is a typical example of the disclosed thraustochytrids, is shown in figure 1. Ectoplasmic net elements can be seen extending from the cell. The body at the edge of the image is a pine pollen grain. In addition, when observed as colonies grown on agar plates, the disclosed

thraustochytrids demonstrate variations in colour from translucent to white, cream, or pale yellow. Subsequent analysis has shown that the disclosed thraustochytrids produce significant quantities of carotenoids and the observed colours correlate with the levels and form of carotenoids produced by the disclosed thraustochytrids. Analysis of the strains for the production of carotenoids was carried out by the commercial Nutrition Analytical Service (NAS) of the Institute of Aquaculture, the University of Stirling, Stirling FK9 4LA. The Nutrition Analytical Service (NAS) were supplied with a wet pellet of the total cells from mature cultures, grown in MCBHB medium. The cells were centrifuged gently (3000 rpm) and then re-suspended twice in filtered sterile seawater to remove the medium in which they had been grown. All samples were then freeze-dried, prior to carotenoid analysis.

The following methodology was then followed:

1) Accurate weights of the freeze-dried samples were obtained.

2) 5mls of acetone was added directly to the freeze-dried samples, mixed thoroughly and homogenised using a small ultra-turrax.

3) The sample tubes were centrifuged at 4000 rpm for 5 minutes and the supernatants transferred to a clean tube.

4) 5mls of dichloromethane was added to the pelleted materials from step 3. The tubes were mixed thoroughly, centrifuged at 4000rpm for 5 minutes and the supernatants combined with that from step 3.

5) lml of DMSO was added to the pelleted materials from step 4) and the sample tubes were placed in a sonicating water bath for 10 minutes to ensure that the cells were fully disrupted for complete extraction of the carotenoids.

6) Following removal from the water bath, lml of 20% sodium sulphate anhydrous was added and the tubes mixed thoroughly. The sample volume was made up to 7ml with distilled water and mixed again before adding a further 5mls of dichloromethane followed by thorough mixing for 2 minutes in order to disperse the carotenoids into the lower DCM layer.

7) The sample tubes were centrifuged at 40001pm for 1 minute and the volume of the upper aqueous layer was reduced to the 7ml mark by aspiration.

8) Sufficient distilled water was added to the sample tubes to bring the volume back to the 12ml mark before mixing thoroughly, centrifuging at 4000rpm for 1 minute and reducing the aqueous layer back to 7ml by aspiration.

9) Step 8 was repeated a further twice to give a total of 4 washes in order to completely remove the DMSO. 10) After the final wash, the entire aqueous layer was removed by aspiration and the remaining solvent layer, containing the carotenoids, combined with that from step 3.

11) The combined supernatants were then evaporated under oxygen free nitrogen to leave the carotenoid residues which were then re-suspended in known volumes of iso- hexane: acetone (82:18) ready for HPLC analysis

The following are the details of the HPLC methodology used:

Column: stainless steel, length 125 mm, diameter 4.0 mm, stationary phase ROC 5 Silica.

Mobile phase: iso-hexane: acetone (82:18, v/v), flow rate 1.2 ml/min, at room temperature, maximum pressure 5000 psi.

Injection by: auto sampler, injection volume 50 ul.

Detection: absorbance at 47onm or 450nm, as detailed below. The carotenoid concentrations were calculated against the appropriate standards, detailed below, which were injected externally to the samples. Where a specific carotenoid standard was unavailable (e.g. adonirubin) the astaxanthin standard was used for calculation. The following formula was applied:

Cone g/g sample =

(cone of standard ^g/ml) x sample area x volume) / (standard area x sample wt (g))

Carotenoid standards:

The following carotenoid standards were used:

Astaxanthin (all E, >98% reference material)

Canthaxanthin (Sigma 11775 - 1 mg)

Xanthophyll (Lutein) (Sigma 95507 - 1 mg)

Beta carotene (Sigma C9750 - 25G)

Preparation of Astaxanthin Standard:

1) Approximately 2 mg of crystalline astaxanthin (all E, reference material >98%) were added to a 100ml volumetric flask.

2) lg butylhydroxytoluene and 25 ml chloroform were added to the flask and the contents sonicated in an ultrasonic water bath for 15 minutes. The flask was then made up to full volume with chloroform. 3) An aliquot of 10 ml of the resulting solution was transferred into a 100 ml volumetric flask and combined with approximately 85 ml of iso-hexane.

4) The solution was allowed to come to room temperature and the flask was then filled to volume with iso-hexane. 3.5 ml of this stock astaxanthin solution was transferred 7 ml glass vials, to which 3.5 ml of iso-hexane were then added. The resulting mixture was stored refrigerated until required.

5) The concentration of astaxanthin was calculated according to the following equation:

C = (absorption (at 470nm) x 10000) / 2100

Preparation of Canthaxanthin Standard:

The methodology above was repeated, except using canthaxanthin crystals.

The concentration of canthaxanthin is calculated according to the following equation:

C = (absorption (at 470nm) x 10000) / 2200

Preparation of Lutein Standard:

1) 5 ml of chloroform and 1 g butylhydroxytoluene were added to a standard vial containing 1 mg of xanthophyll standard, and mixed well on the whirlimixer. The mixture was transferred to a 100 ml volumetric flask and made to the mark with iso- hexane.

2) Approximately 1 ml aliquots of this solution were measured into individual 7 ml glass vials, to which 5ml of iso-hexane were then added.

3) The concentration of lutein was then calculated according to the following equation:

C = (absorption (at 450nm) x 10000) / 2450 Preparation of BetaCarotene Standard:

1) 5 ml of chloroform and lg butylhydroxytoluene were added to approximately img of beta carotene standard, and mixed well on the whirlimixer. The mixture was then transferred to a 100 ml volumetric flask and made to the mark with iso-hexane.

2) Approximately 1 ml aliquots of the mixture were then decanted into individual 7 ml glass vials to which 5ml of iso-hexane were also added.

3) The concentration of beta carotene is calculated according to the following equation:

C = (absorption (at 450nm) x 10000) / 2300 Results:

Dry weight of sample (values expressed as g/kg dry weight of sample, n.d. = not detected)

CAi CA2 CA3 CA4 CA5 CA14

Beta-carotene 68.62 63.11 59-23 56.66 117-35 1681.46

Echinenone/ Cis-echinenone n.d. 6.08 5-71 3-32 10.27 22.62

3-Hydroxyechinenone n.d. n.d. n.d. n.d. n.d. n.d.

Canthaxanthin 12.81 0.70 4-98 6.17 23.80 308.05

Adonirubin 3-57 2.15 6-37 n.d. 11.19 60.79

Unidentified n.d. n.d. 4.28 n.d. 16.49 n.d.

Astaxanthin (All E+ 9Z + 13Z) 1.96 821.10 598.88 315-53 372.09 154-49

Adonixanthin n.d. n.d. n.d. n.d. n.d. n.d.

Lutein n.d. n.d. n.d. n.d. n.d. n.d.

Total Carotenoids 86.96 893.14 679-45 381.68 551-19 2227.41

All of the isolates tested were found to have the potential to produce carotenoids, including beta-carotene, echinenone/cis-echinenone, canthaxanthin, adonirubin, and astaxanthin, when grown in MCBHB medium.

3. Transmission Electron Microscopy

The disclosed thraustochytrids were investigated using light microscopy.

One isolate, CA2 (corresponding Accession Number as set out above), was grown in a rich nutrient medium at 20°C for 3-4 or longer if observations showed this to be required days, with the aim of harvesting the cultures in their most actively growing phase, when all life stages should be present.

Cells were prepared by the methodology recommended by the Centre for Environment, Fisheries and Aquaculture Science Laboratory (CEFAS), Weymouth, UK. Briefly, the method used involved:

1. concentrating the cells by gentle centrifugation (i28g for 3 minutes);

alternatively, to improve the condition of the zoospores and other cells at fixation, cultures containing high numbers of zoospores were centrifuged at 70g for 25 minutes including a slow acceleration and deceleration phase 2. washing the cells twice with phosphate buffered saline pH 7.0 (137Π1Μ NaCl, 3mM KCl,iomM Na2HP04, 2mM KH2PO4) to remove any adhering medium, or 0.2M sodium cacodylate buffer; and

3. fixing the cells in 2.0 or 2.5% glutaraldehyde in 0.2M sodium cacodylate buffer.

Samples were stored at 4°C and sent to CEFAS for further processing.

In all cases, the cells were subsequently washed twice in 0.2M sodium cacodylate buffer before being post-fixed in 1% osmium tetroxide in 0.2M sodium cacodylate buffer for 1 hr. A final rinse in 0.2M sodium cacodylate buffer then preceded dehydration in a graded acetone series (10%, 30%, 50%, 70%, 90% and 100%) with each step taking 10 minutes.

The rinse in 100% acetone was performed three times to ensure complete dehydration before the sample went through a graded resin series over 2 hours to replace the acetone. The resin was then polymerised in an oven in a fume cupboard overnight.

The resulting resin blocks were trimmed and sectioned using a glass knife in an ultramicrotome, to produce both semi-thin (approximately ΐμιτι) and thin (70-90nm) sections. The semi-thin sections were stained with 1% aqueous toluidine blue with 1% carmine and 1% borax for 1 minute. The thin sections, taken from areas of interest, were stained with 2% uranyl acetate solution and then in Reynolds lead citrate solution (in a carbon dioxide free container), with two rinse steps in distilled water between each stain.

After drying, the sections were examined with a JOEL 1210 transmission electron microscope at the CEFAS Laboratory.

4. Morphological Analysis

No sexual reproduction has been observed in the disclosed thraustochytrids, as is also typical of thraustochytrids.

However, in heterotrophic culture medium, such as MCBHB culture medium, the disclosed thraustochytrids are capable of a mode of reproduction which has not previously been reported in a thraustochytrid. This form of reproduction is known in other groups of protozoa, such as the paramixids and mixozoa, however these groups of protozoa are not closely related to the thraustochytrids.

In order to observe this novel mechanism of cell division, the disclosed

thraustochytrids may be cultured in heterotrophic culture medium, such as MCBHB medium, as described above, and then prepared in semi-thin sections and visualised by electron microscopy, as described above.

Examples of the resulting electron micrographs are shown in figures 5a, 5b, 6, 7. In any given field of view a proportion of the cells (generally in the region of 0.1-1% of the total number of cells visualised) were observed to include both daughter and granddaughter cells (as indicated, for example, with an arrow in figure 5a). When other known thraustochytrid strains were cultured and visualised in the same way, no cells were observed to include both daughter and granddaughter cells. >. Fatty Acid Composition

In order to investigate the production of fatty acids by the disclosed thraustochytrid microorganisms, colonised pollen grains were plated on agar produced from

heterotrophic medium (in this case, MCBHB Agar) and single colonies were then transferred into heterotrophic medium (in this case, MCBHB medium) in flask culture. 50% natural seawater was used for both agar and medium, diluted with distilled water, rather than artificial sea salts made up in distilled water.

Each isolate to be tested was then innoculated at 3% v/v into conical flasks and incubated on a rotary shaker at 2i°C, loorpm. At the end of the exponential phase of growth, one half of the flasks were processed further, while the remainder were incubated for a further four days, taking the cultures to late stationary phase. Cells were centrifuged, washed twice with phosphate buffered saline at pH 7.2 and the resulting pellets were stored at -20°C and then freeze dried before fatty acid analysis.

For analysis of the fatty acids, samples were prepared and analysed in triplicate. Briefly, cell pellets were transesterified to produce fatty acid methyl esters (FAME), using well known methods. The resulting FAME were analysed using gas chromatography to determine the composition of fatty acids. Confirmation was performed by gas chromatography-mass spectrometry. All of the samples of the disclosed thraustochytnds that were tested, including all of the deposited strains, were found to produce high levels of fatty acids, including omega-3 (n-3) fatty acids, and in particular, docosahexaenoic acid (DHA), relative to levels previously reported to be produced by thraustochytnds.

The mean amount of omega-3 fatty acids produced as a percentage of the total fatty acid production by each strain was 61%. The highest amount of omega-3 fatty acids produced as a percentage of the total fatty acid production was 71% (CA2 - corresponding Accession Number as set out above).

The DHA fatty acid composition of the deposited strains was found to be between 37% (CA5 - corresponding Accession Number as set out above) and 65% (CA2 - corresponding Accession Number as set out above) as a percentage of the total fatty acids produced. This level of DHA production is understood to be the highest level found to be produced by thraustochytnds.

Concentrations of DHA in the culture medium were observed to be in the range of 10- i7mg L ¹., with the highest levels observed to be produced by the strains CAi and CA3 (corresponding Accession Numbers as set out above).

6. DNA Extraction and ITS2-28S Ribosomal RNA Gene Sequencing

Multiple copies of ribosomal RNA gene units, commonly used as molecular identifiers for organisms, are present in clusters in the genome, and each copy of the RNA gene unit comprises the following portions, in order: ETS; 18S; ITSi; 5.8S; ITS2; and 28S. Genomic DNA was used as template for PCR amplification for identification using conserved eukaryotic primers (see below). Specifically, the forward primer was specific for a sequence near the 3' end of the 18S gene, and the reverse primer was specific for a sequence near the 5' end of the 28S gene. For molecular characterisation of the strains, 2 ml of culture was harvested and genomic DNA extracted according to Mo, C. and Rinkevich, B. (2001 "A simple, reliable, and fast protocol for thraustochytrid DNA extraction" Marine Biotechnology 3(2): 100-2, DOI: 10.1007/S101260000069)). Genomic DNA was used as template for PCR amplification of 28S and ITSi and 2 rRNA gene sequences using the conserved eukaryotic primers: UNUP18S42: 5'-GGTAACAAGGTTTCCGTAGGTGAAC-3' (SEQ ID NO. 6); and

UNLO28S576B: 5'-CTCCTTGGTCCGTGTTTCAAGACG-3' (SEQ ID NO. 7).

The PCR primers were as described by Bakkeren, B., Kronstad, J.W. and Levesque, C.A. (2000, "Comparison of AFLP fingerprints and ITS sequences as phylogenetic markers in Ustilaginomycetes", Mycologia, 92, 510-521, DOI: 10.2307/3761510).

The PCR reaction was carried out as follows: 5 μΐ Dreamtaq buffer, 0.2 μΐ Dreamtaq DNA polymerase (ThermoFisher UK), 8 μΐ 1.25 mM dNTPs, 1 μΐ each primer at 10 pMol per microliter, 5 μΐ genomic DNA template were added to water to a final volume of 50 μΐ.

The PCR programme consisted of a 3 minute denaturation at 94°C followed by 35 cycles of 94°C for 30 seconds, 50°C annealing for 30 seconds and 72°C extension for 60 seconds, followed by a final extension at 72 °C for 7 minutes.

The PCR reaction products were purified using a commercial kit from Qiagen (TM) before being sent for Sanger sequencing at Edinburgh Genomics (UK). The reverse primer was used for sequencing. Sequences were obtained (from the deposited strains CAi, CA2, CA3, CA4, CA5, and CA8), each of approximately 600 base pairs, starting from the 5' end of the 28S rRNA gene and extending into the ITS2 sequence. The sequences obtained were as follows:

CAi (SEQ ID NO. 1)

TTGCAGACTTTCGTCTCATCGCGATTTATCATCGAACAAAAAGCCTCTGTTGGGCGGGCCACGC CGAAGTGGCCGTCCTGCTCAAACGAGCCCAGACGAACACACAGTCTGCGGGCCCCGCCAACACA TGGAGACCGACACGGAAAACAGAATAAACCGCTCCTCGAAAAGAAACGACACCCCTGTTCCGCA CACGACACTAGTTGCAAACCCTTCCCTTGTAACGATTTCAGGTCCTTTTCACTCTCTTTTCAAA GTTCTTTTCATCTTTCCTTCACAGTACTTGTTCGCTATCGGTCTCTCGTCGGTATTTAGCTTTA GATGGAATTTACCACCCACTTGGCGCTGCAGTCCCAAGCAACACGACTCGTAAAAAACGGACCG TACGCACACACTAAATGGAAACAATACAGGACTTTCACCTTGCATCGTGCCCCATCCCAAGGGA CTTGTGCCTCCATTGCGTGCTGGCCGCGCCTCTCCATGCTACAATTGGCTTGCGCCATTTTCAA CATGAGCTCTTCCCGCTTCACTCGCCGTTACTGAGGGAATCCCGGTTGGTTTCTTTTGCCTCTG CTTACTTATATGCTTAAATTCAGC CA2 (SEQ ID NO. 2)

AAGCAGTACATTTCGTCTCATCGCGATTTATCATCGAACAAAAAGCCTCTGTTGGGCGGGCCAC GCCGAAGTGGCCGTCCTGCTCAAACGAGCCCAGACGAACACACAGTCTGCGGGCCCCGCCAACA CATGGAGACCGACACGGAAAACAGAATAAACCGCTCCTCAAAAAGAAACGACACCCCTGTTCCG CACACGACACTAGTTGCAAACCCTTCCCTTGTAACGATTTCAGGTCCTTTTCACTCTCTTTTCA AAGTTCTTTTCATCTTTCCTTCACAGTACTTGTTCGCTATCGGTCTCTCGTCGGTATTTAGCTT TAGATGGAATTTACCACCCACTTGGCGCTGCAGTCCCAAGCAACACGACTCGTAAAAAACGGAC CGTACGCACACACTAAATGGAAACAATACAGGACTTTCACCTTGCATCGTGCCCCATCCCAAGG GACTTGTGCCTCCATTGCGTGCTGGCCGCGCCTCTCCATGCTACAATTGGCTTGCGCCATTTTC AACATGAGCTCTTCCCGCTTCACTCGCCGTTACTGAGGGAATCCCGGTTGGTTTCTTTTGCCTC TGCTTACTTATATGCTTAAATTCA

CA3 (SEQ ID NO. 3)

GAAGCAGACTTTCGTCTCATCGCGAGTTATCATCGTACACCCCCTCTGGGGGGGGGGCCACCCC AAAGTGGCCCCCGTGCTCCGCGAGCCCAGACGAACACGTCTGCGGGCCCCCCGCCACTGTGGAG ACCGAGCAGACACAGAATAAACCGCTTCTCGAGAAACGACGCCTCTCTTCACCGCACACGACAC TAGTTGCAAACCCTTCCCTTGTAACGATTTCAGGTCCTTTTCACTCTCTTTTCCAAGTTCTTTT CATCTTTCCTTCCCAGTACTTGTTCGCTATCGGTCTCTCGTCGGTATTTAGCTTTAGATGGAAT TTACCACCCACTTGGCGCTGCAGTCCCAAGCAACACGACTCGTAGAAGACGGACCGTACGCACG CATCATTATGGAAACAATACAGGACTTTCACCTTGCATCGGGCCCCATCCCAAGGGACTGGTGC CTCCATGGCATGCTGGCCGCGCCTCTCCATGCTACAATGGGCTGGCGCCATTTTCAACATGAGC TCT TC CC GCTTCACTCGCCGT TACT GAGGGAATCCCGGTTGGTTTCTTTTGCCCTCTGCTT ACT TATATGCTTAAATTCAGCAGGTCT

CA5 (SEQ ID NO. 4)

TAAGCTGACTTTCGTCTCATCGCGATTTATCATCGAACAAAAAGCCTCTGTTGGGCGGGCCACG CCGAAATGGCCGTCCTGCTCAAACGAGCCCAGACGAACACACAGTCTGCGGGCCCCGCCAACAC ATGGAGACCGACACGGAAAACAGAATAAACCGCTCCTCAAAAAGAAACGACACCCCTGTTCCGC ACACGACACTAGTTGCAAACCCTTCCCTTGTAACGATTTCAGGTCCTTTTCACTCTCTTTTCAA AGTTCTTTTCATCTTTCCTTCACAGTACTTGTTCGCTATCGGTCTCTCGTCGGTATTTAGCTTT AGATGGAATTTACCACCCACTTGGCGCTGCAGTCCCAAGCAACACGACTCGTAAAAGACGGACC GTACGCACACACTATATGGAAACAATACAGGACTTTCACCTTGCATCGTGCCCCATCCCAAGGG ACTTGGGCCTCCCTTGCGTGCTGGCCGCGCCTCTCCATGCTACAATTGGCTTGCGCCATTTTCA ACATGAGCTCTTCCC GCTTCACTCGCCGT TACT GAGGGAATCCCGGGTGGTTTCTTTTGCC TCT GCTTACTTATATGCTTAAATTCAG CA8 (SEQ ID NO. 5)

GAAAGCAGACATTTCGTCTCATCGCGAGTTATCATCGTACACCCCCTCTGGGGGGGGGGCCACC CCAAAGTGGCCGTCGTGCTCCACGAGCCCAGACGAACACACACTCTGCGGGCCCCGCCAACTGT GGAGACCGACACGGAAAACAGAATAAACCGCTCCTCGAGAAAAAACGACACCCCTGTCCCGCAC CCAACACTAGTGGCAAACCCTTCCCTGGAAACGATTTCGGGCCCTTTTCCTCCTCTTTTCAAAG TTCTTTTCTCCTTTCCTTCACAGTATTGGTTCGCTATCGGTCTCTCTCCGGTTTTTACTTTTAA AGGGAATTTCCCACCCCTTGGGGCCTGCAGCCCCAAGCAACACGACTCGTAAAAAACGGACCGT ACGCACACATCTATATGGAAACAATACAGGACTTTCACCTTGCATCGTGCCCCATCCCAAGGGA CTTGTGCCTCCATTGCATGCTGGCCGCGCCTCTCCATGCTACAATTGGCTTGCGCCATTTTCAA CATGAGCTCTTCCCGCTTCACTCGCCGTTACTGAGGGAATCCCGGTTGGTTTCTTTTGCCTCTG CTTACTTATATGCTTAAATTCAGC

The ITS2-28S sequences from strains CAi and CA2 were found to have 99% sequence identity. The sequence from strain CA3 was found to have 93% sequence identity to the sequence from strain CAi. The sequence from strain CA5 was found to have 98% sequence identity to the sequence from strain CAi. The sequence from strain CA8 was found to have 92% sequence identity to the sequence from strain CAi. SEQ ID No. 1 was compared with known 28S rRNA gene sequences in the NCBI databank using the BLAST alignment tool.

SEQ ID No. 1 was found to have 78% sequence identity over 425bp to Japonochytrium (Accession FJ030887), 76% sequence identity over 423bp to Thraustochytrium aureum (FJ030888), and 70% sequence identity over 397bp to Labyrinthula terrestris (KP996006).

Homologous sequences were used to construct a phylogenetic tree (shown in figure 18), in which two yeast 28S rRNA sequences (Candida albicans and Saccharomyces cerevisiae) were used to root the tree (www.Phylogeny.fr: Dereeper A.,et al. (2008) "Phylogeny.fr: robust phylogenetic analysis for the non-specialist" Nucleic Acids Res. 36 (Web Server issue) ^465-9).

2- 18S Ribosomal RNA Gene Sequencing

The 18S rRNA sequences of the deposited strains proved to be very technically difficult to obtain. Indeed, 18S rRNA sequence data that was initially obtained was subsequently found to be incorrect. It was because of these technical difficulties in relation to sequencing the 18S rRNA genes that the region starting from the 5' end of the 28S rRNA gene and extending into the ITS2 gene was also sequenced.

However, the improved methodology of Mo et al (Marine Biology (2002) 140: 883-889 (DOI 10.1007/SO0227-002-0778-9)) allowed the subsequent sequencing of the 18S rRNA gene of the deposited strains CAi, CA2, CA5, CA8 and CA14. For the 18S ribosomal RNA gene analysis, the primers described by Mo et al were used to amplify an approximately 1600 bp amplicon. The primers used were:

FAi, 5'-AAAGATTAAGCCATGATGT-3' (SEQ ID No. 9); and

RA3, 5'-CAATCGGTAGGTGCGACGGGCGG-3' (SEQ ID No. 10).

The PCR reaction was carried out as for the 28S sequences, but with an elongation time of 90 seconds. Sequencing was carried out for both strands using the primers FAi and

RA3, and internal primers, as described by Mo et al (2002):

FA2: 5'-GTCTGGTGCCAGCAGCCGCG-3' (SEQ ID No. 11);

FA3: 5'-CTTAAAGGAATTGACGGAAG-3' (SEQ ID No. 12);

RAi: 5'-AGCTTTTTAACTGCAACAAC-3' (SEQ ID No. 13); and

RA2, 5'-CCCGTGTTGAGTCAAATTAAG-3' (SEQ ID No. 14).

A complete 1628 bp sequence was obtained for all 5 strains sequenced (CAi, CA2, CA5, CA8 and CA14). The sequences were 100% identical, and the sequence shared by these deposited isolates is the sequence presented below as SEQ ID No. 8: 18S Ribosomal RNA Gene Sequence (SEQ ID NO. 8):

AAAGATTAAGCCATGCATGTGTTTGTAAATCACACTCAATGTGAGAGACTGCGTACGGCTCATTACAACA GTTATAATCTCCACGACAGTGTCTTTCACGAGTTAAATGGATACTTGGATTAATTCTCGAAATAATACAT GCACTAGAGGCCCGACCGACTCGTTTGGAAGGGCCGCATTTATTAGACTGAAGCCAACATCACTTGGTGA TTCATAGTAACTGAGCAAATCGCATCTGGGCGATGAATCGTTTGAGTTTCT GCCCTATCAGCTGTCGATG CGAGGGTATTGTCCTCGCATGGCCGTCACGGGTACCGGGGATTTAGGGATC GATTCCGGAGAGTTAGCCT GAGAGACGGCTCACACATCCAAGGAAGGCAGCAGGCGCGTAAATTACCCAATGTTAACGCAACGAGGTAG TGACGACCAATAGAATTGGAGGGCGTTTTTGCGTCTTTCTATTTCAATGAGTGCAATGTAAAACTCTCAA CGAGGATCAAGTCGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCGACTAGCGAACTATTA TGTTGTTGCAGCTGAAAAGCTCGTAGTTGAATGTCTGGCCATTGGTGCTTGGGCCTTTCTCGGGCGAATG CCCGTTGACTGTCTGCCTCTTGTGCCTGGCCATCCTTGATTACTTCGGTAGTCATCGTTTACTGTAAACA AAATAGAGTGTTCAAAGCAGGTCGTTCTGACCGGGATATTTCTTATGGGATAATAAGATAGGACTGGCGT GCTATTTTGTTGGTTTGTACATGTCAGTAATGGTTGATAGGGACAGTTGGGGGTATTCGTATTCAGCAGC TAGAGGTGAAATTCTTGGATTTGCGGAAGACGAACAACTGCGAAGGCATTTACCAAGGATGCTCTCTTTA ATCAAGAACGAAAGCTAGAGGATCGAAGATGATTAGATACCATCGTAGTTC TAGCCGTAAACGATGCCGA CTTGCAATTCTCGCTCGTTTGATTCAAATGACAGCGGGAGCGGCACATGAGAAGTCAAAGTCTTTGGGTT CCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAATTGACGGAAGGGCACCACCAGGAGTGGAGCC TGCGGCTTAATTTGACTCAACACGGGAAAACTTACCAGGTCCAGACATAGGAAGGATTGACAGATTGAGA GCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGTTAACGAACGAGACCTCGGCCTACTAAATAGTTGCGTTCATGGCGACATGGGCGAGCATACTTCTT AGAGGGACATGTCCGGTTTACGGGCGGGAAGTTCGAGGCAATAACAGGTCT GTGATGCCCTTAGATGTTC TGGGCCGCACGCGCGCTACACTGATGAGTTCAGCGGGTTACTTTCGGTTCGTCCGAAAGTGTCCTTGGCC GGAAGGTCTGGGTAATCCTTTGAAATGCTCATCGTGATGGGGCTAGATTCT TGCAATTATTAATCTCCAA CGAGGAATTCCTAGTAAACGCAAGTCATCAACTTGCATTGATTACGTCCCT GCCCTTTGTACACACCGCC CGTCGCACCTACCGATTG

Sequencing indicated that the 18S rRNA sequences of the deposited strains may include a degree of intra-genomic C/T heterozygosity at position 643. Similar intra-genomic heterozygosity in the 18S Ribosomal RNA gene sequence has previously been observed in other species.

The 18S rRNA sequence (SEQ ID No.8) was compared with known rRNA genetic sequences in the NCBI databank using the BLAST alignment tool.

SEQ ID No.8 was found to be 84% identical over 1628 bp to Aurantiochytrium sp, (Accession AB8109441), and 84% identical over 1628 bp with Thraustochytrium sp. (Accession JX847376). Homologous sequences were used to construct a phylogenetic tree (shown in Figure 19 in which the scale bar indicates 0.1 substitutions per site). The tree is rooted by the inclusion of Labyrinthula zosteri. In addition to SEQ ID No. 8, the tree indicates other closely related labyrinthomycetes and some commercially important thraustochytrid species for which there are suitable 18S rRNA sequences currently available. All of the 18S rRNA sequences used correspond to the full length of SEQ ID No. 8.

The overall % sequence identities to SEQ ID No. 8 are:

Schizochytrium aggregat m: 67%

Thraustochytrium aureum: 70%

Ulkenia visurgensis: 67%

Labyrinthula zosterae: 62% (Used to root the tree).

8. Exopolysaccharide (EPS) production The disclosed thraustochytrid has been observed by microscopy to produce large quantities of exopolysaccharide (see figure 3c).

For the production of EPS, the disclosed thraustochytrids are cultured in MCBHB or other suitable heterotrophic medium for a period of 2-10 days, or until significant quantities of EPS can be seen to have to accumulated, for example by light microscopy of individual cells.

The cell biomass is then separated from the culture medium by any suitable method, such as by means of centrifugation and filtration. If necessary, the level of EPS in the culture medium filtrate may be concentrated by ultrafiltration, and the skilled person will be aware of suitable methods.

The concentrated EPS may be precipitated by any suitable technique, for example, by means of the addition of 70 % isopropyl alcohol, and freezing the culture filtrate to precipitate the EPS.

The precipitated EPS may be further purified to obtain a specific pure fraction of the EPS. For example, a solution of the EPS may be separated using column

chromatography.

The various embodiments described herein are presented only to assist in

understanding and teaching the claimed features. These embodiments are provided as a representative sample of embodiments only, and are not exhaustive and/or exclusive. It is to be understood that advantages, embodiments, examples, functions, features, structures, and/or other aspects described herein are not to be considered limitations on the scope of the invention as defined by the claims or limitations on equivalents to the claims, and that other embodiments may be utilised and modifications may be made without departing from the scope of the claimed invention. Various embodiments of the invention may suitably comprise, consist of, or consist essentially of, appropriate combinations of the disclosed elements, components, features, parts, steps, means, etc, other than those specifically described herein. In addition, this disclosure may include other inventions not presently claimed, but which may be claimed in future.

Claims

- 6θ - Claims

1. An isolated thraustochytrid having an ITS2-28S ribosomal RNA gene sequence, wherein the ITS2-28S ribosomal RNA gene sequence has at least 85% sequence identity to the sequence set forth in SEQ. ID NO: 1.

2. An isolated thraustochytrid as claimed in claim 1, wherein the thraustochytrid additionally has an 18S ribosomal RNA gene sequence, and wherein the 18S ribosomal RNA gene sequence has at least 86% sequence identity to the sequence set forth in SEQ. ID NO: 8.

3. An isolated thraustochytrid as claimed in claim 1 or 2, wherein the ITS2-28S ribosomal RNA gene sequence has at least 92% sequence identity to the sequence set forth in SEQ. ID NO: 1.

4. An isolated thraustochytrid as claimed in either of claims 2 or 3, wherein the 18S ribosomal RNA gene sequence has at least 90% sequence identity to the sequence set forth in SEQ. ID NO: 8.

5. An isolated thraustochytrid as claimed in any one of claims 1-4, wherein when cultured in heterotrophic medium the thraustochytrid culture comprises a cell having an internal daughter vegetative cell, wherein that daughter cell comprises a

granddaughter vegetative cell within it.

6. An isolated thraustochytrid as claimed in claim 5, wherein at least 0.01% of the cells in the thraustochytrid culture comprise both daughter and granddaughter vegetative cells.

7. An isolated thraustochytrid as claimed in any one of claims 1-6, wherein the thraustochytrid is a mutant, variant, or recombinant thraustochytrid derived from one of the strains deposited at the CABI depositary institution under any one of the following Accession No.s: IMI 506775, IMI 506776, IMI 506777, IMI 506778, IMI 506779, IMI 506780, or IMI 507005.

8. An isolated thraustochytrid as claimed in claim 7, wherein the thraustochytrid is one of the strains deposited at the CABI depositary institution under any one of the - 6l - following Accession No.s: IMI 506775, IMI 506776, IMI 506777, IMI 506778, IMI 506779, IMI 506780, or IMI 507005.

9. An isolated thraustochytrid as claimed in any one of claims 1-8, wherein the isolated thraustochytrid is capable of producing fatty acids, and wherein 45-75% of the total fatty acids produced comprise omega-3 fatty acids.

10. An isolated thraustochytrid as claimed in any one of claims 1-9, wherein the isolated thraustochytrid is capable of producing fatty acids, and wherein 35-70% of the total fatty acids produced comprise DHA.

11. An isolated thraustochytrid as claimed in any one of claims 1-10, wherein when the thraustochytrid is cultured in heterotrophic medium to the late stationary phase, the concentration of DHA in the culture medium is 5-20 mgL ¹.

12. An isolated thraustochytrid as claimed in any one of claims 1-11, wherein the isolated thraustochytrid is capable of producing carotenoids, and wherein the total amount of carotenoid produced may be greater than 80 g/kg dry weight of sample.

13. An isolated thraustochytrid as claimed in claim 12, wherein the total amount of carotenoid produced may be greater than 2000 g/kg dry weight of sample.

14. An isolated thraustochytrid as claimed in either of claims 12 or 13, wherein the isolated thraustochytrid is capable of producing beta-carotene in, and wherein the total amount of beta-carotene in produced may be greater than 55 g/kg dry weight of sample.

15. An isolated thraustochytrid as claimed in any one of claims 12-14, wherein the isolated thraustochytrid is capable of producing beta-carotene in, and wherein the total amount of beta-carotene in produced may be greater than 1600 g/kg dry weight of sample.

16. An isolated thraustochytrid as claimed in claim 13 or 15, wherein the isolated thraustochytrid is capable of producing canthaxanthin, and wherein the total amount of canthaxanthin produced may be greater than 300 μg/kg dry weight of sample.

17. A thraustochytrid biomass comprising an isolated thraustochytrid as claimed in any one of claims 1-16.

18. A microbial oil obtained or derived from an isolated thraustochytrid as claimed in any one of claims 1-16.

19. A composition comprising an isolated thraustochytrid as claimed in any one of claims 1-16, a biomass as claimed in claim 17, and/or a microbial oil as claimed in claim 18.

20. A food product, feed additive, nutritional supplement, cosmetic, or

pharmaceutical composition for animals or humans, comprising a composition as claimed in claim 19.

21. A composition comprising a microbial oil and/or carotenoid, for use in therapy, wherein the microbial oil and/ or carotenoid is obtained or derived from an isolated thraustochytrid as claimed in any one of claims 1-16.

22. A composition as claimed in claim 21 for use in the treatment of inflammation, or in the treatment of an oxidative stress-related condition.

23. A method for producing a thraustochytrid biomass, the method comprising culturing an isolated thraustochytrid as claimed in any one of claims 1-16, and producing the thraustochytrid biomass from the cultured thraustochytrids.

24. A method as claimed in claim 23, the method further comprising isolating from the biomass:

a) a microbial oil;

b) omega-3 fatty acids;

c) DHA, ARA, EPA, and/or DPA n-6;

d) a carotenoid;

e) a xanthophyll;

f) squalene; and/or

g) an exopolysaccharide composition.

25. A method for producing a lipid composition comprising omega-3 fatty acids, the method comprising culturing an isolated thraustochytrid as claimed in any one of claims 1-16 to produce a biomass, and isolating the lipid composition comprising omega-3 fatty acids from the biomass.

26. A method for producing a lipid composition comprising DHA, ARA, EPA, and/ or DPA n-6, the method comprising culturing an isolated thraustochytrid as claimed in any one of claims 1-16 to produce a biomass and isolating the lipid composition comprising DHA, ARA, EPA, and/or DPA n-6 from the biomass.

27. A method for producing a composition comprising an antioxidant comprising a carotenoid and/or a xanthophyll, the method comprising culturing an isolated thraustochytrid as claimed in any one of claims 1-16 to produce a biomass and isolating the antioxidant from the biomass.

28. A method for producing a composition comprising a carotenoid, the method comprising culturing an isolated thraustochytrid as claimed in any one of claims 1-16 to produce a biomass and isolating the carotenoid from the biomass.

29. A method for producing an exopolysaccharide composition, the method comprising culturing an isolated thraustochytrid as claimed in any one of claims 1-16 and isolating the exopolysaccharide from the cultured thraustochytrids.

30. A method for removal of hydrocarbons from water, the method comprising contacting hydrocarbon-containing water with a culture of an isolated thraustochytrid as claimed in any one of claims 1-16.

31. A method for identifying a strain of thraustochytrid which is capable of producing high levels of fatty acids, the method comprising culturing the

thraustochytrid strain in heterotrophic medium and determining whether the thraustochytrid culture comprises a cell having an internal vegetative daughter cell, wherein that daughter cell comprises a vegetative granddaughter cell within it, wherein the presence of a cell comprising internal daughter and granddaughter cells is indicative that the thraustochytrid strain is capable of producing high levels of fatty acids.