US 20170183669A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0183669 A1 LPPMEER et al. (43) Pub. Date: Jun. 29, 2017

(54) RECOMBINANT THRAUSTOCHYTRIDS Publication Classification THAT GROW ON SUCROSE, AND COMPOSITIONS, METHODS OF MAKING, (51) Int. Cl. AND USES THEREOF CI2N 15/79 (2006.01) A2.3L 2/52 (2006.01) A23K2O/It (2006.01) (71) Applicant: DSM IPASSETS B.V., TE Heerlen A2.3L 33/10 (2006.01) (NL) CI2N I/O (2006.01) (72) Inventors: James Casey LIPPMEIER, Columbia, A23K 50/80 (2006.01) MD (US); Kirk E. Apt, Ellicott City, (52) U.S. Cl. MD (US); Jon Milton Hansen, CPC ...... CI2N 15/79 (2013.01); C12N I/10 Chandler, AZ (US) (2013.01): CI2Y 302/01026 (2013.01); A23K 50/80 (2016.05); A23K 20/10 (2016.05); A23L (21) Appl. No.: 15/400,318 33/10 (2016.08); A23L 2/52 (2013.01); A23V 2002/00 (2013.01) (22) Filed: Jan. 6, 2017 (57) ABSTRACT Related U.S. Application Data The present invention is directed to recombinant thraus (62) Division of application No. 12/980,322, filed on Dec. tochytrids that grow on Sucrose and cell cultures comprising 28, 2010, now abandoned. the recombinant thraustochytrids as well as methods of (60) Provisional application No. 61/290,443, filed on Dec. producing cell cultures, biomasses, microbial oils, compo 28, 2009. sitions, and biofuels using the recombinant thraustochytrids. Patent Application Publication Jun. 29, 2017 Sheet 1 of 15 US 2017/O183669 A1

Schicochytrium Codon Usage Table AAcid Codon Fraction Fraction AmAcid Codon Fraction AmAcid Codon Fraction AA O34 Leg CT 0.16 Ser TCG 0.33 GA 033: Leu TTG 0.02 Ser TCC 0.31 TAG O33 Leg CTG 0.12 Ser AGT 0.03 0.69 Ser 0.01 Gin CAG 0.92 Leg TTA O Ser TCT 0.09 Arg AGA O Giu GAA 0.09 Let CTA OThr ACG 03 Arg CGC 0.8 Giu GAG 0.91 Lys AAA 0.04 Thr ACC 0.54 Arg CGA 0.01 Gly GGA 04 Lys AAG 0.96 Thr ACA 0.02 Arg AGG OGly GGT O2 Met ATG 1 Thr ACT 0.14 Arg CGT 0.17 Gly GGG O Phe TTT 0.45 Trp TGG 1.

034 Val 0.62 0.43 Val GTA O

Cys TGC 0.95 lie ATA Pro CCA 0.02 Val GT 0.14

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Mannose N-acetyl-glucosamine

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R. R. 'sCage 's- Hexs HexNAc -

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Patent Application Publication Jun. 29, 2017. Sheet 15 of 15 US 2017/0183669 A1

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RECOMBINANT THRAUSTOCHYTRIDS 30%-60% of lipids in the retina, is concentrated in the testes THAT GROW ON SUCROSE, AND and sperm, and is an important component of breast milk. COMPOSITIONS, METHODS OF MAKING, Jean-Pascal Berge & Gilles Barnathan, Fatty Acids from AND USES THEREOF Lipids of Marine Organisms. Molecular Biodiversity, Roles as Biomarkers, Biologically Active Compounds, and Eco CROSS-REFERENCE TO RELATED nomical Aspects, in Marine Biotechnology I 49 (T. Scheper, APPLICATIONS ed., 2005). DHA accounts for up to 97% of the omega-3 fatty 0001. This application is a divisional of U.S. patent acids in the brain and up to 93% of the omega-3 fatty acids application Ser. No. 12/980,322 filed Dec. 28, 2010, which in the retina. Moreover, DHA is essential for both fetal and claims the benefit of the filing date of U.S. Appl. No. infant development as well as maintenance of cognitive 61/290,443, filed Dec. 28, 2009, the entire contents of which functions in adults. Id. Omega-3 fatty acids, including DHA are hereby incorporated by reference herein in their entire and EPA, also possess anti-inflammatory properties. See, ties. e.g., id. and Simopoulos, A. P. J. Am. Coll. Nutr. 21: 495-595 (2002). In particular, EPA competes with arachi REFERENCE TO SEQUENCE LISTING donic acid for synthesis of eicosanoids such as prostaglan SUBMITTED ELECTRONICALLY dins and leukotrienes through cyclooxygenases and lipoxy genases. Id. Because omega-3 fatty acids are not synthesized 0002 The content of the electronically submitted de novo in the human body, these fatty acids must be derived sequence listing (“sequence listing ascii.txt, 77,186 bytes, from nutritional sources. created on Dec. 23, 2010) filed with the application is 0009 Thraustochytrids, which are microalgal organisms incorporated herein by reference in its entirety. of the order Thraustochytriales, are recognized as alternative sources for the production of lipids. For example, strains of BACKGROUND OF THE INVENTION thraustrochytrid species have been reported to produce 0003 Field of the Invention omega-3 fatty acids as a high percentage of the total fatty 0004. The present invention is directed to recombinant acids produced by the organisms. See, e.g., U.S. Pat. No. thraustochytrids that grow on Sucrose and cell cultures 5,130,242; Huang, J. et al., J. Am. Oil. Chem. Soc. 78: comprising the recombinant thraustochytrids, as well as 605-610 (2001); and Huang, J. et al., Mar: Biotechnol. 5: methods of producing cell cultures, biomasses, microbial 450-457 (2003), incorporated by reference herein in their oils, compositions, and biofuels using the recombinant entireties. Thraustochytrids have also been recognized as thraustochytrids. sources of lipids for the production of biofuels. See, e.g., 0005 Background U.S. Publ. No. 2009/0064567 and WO 2008/067605, incor 0006 Fatty acids are classified based on the length and porated by reference herein in their entireties. saturation characteristics of the carbon chain. Fatty acids are 0010 While thraustochytrids metabolize glucose and/or termed short chain, medium chain, or long chain fatty acids fructose, which are the two Sugar components of Sucrose, based on the number of carbons present in the chain, are they do not appear to naturally metabolize Sucrose. See, e.g., termed saturated fatty acids when no double bonds are Aki et al., JAOCS 80:789-794 (2003); Yokochi et al., Appl. present between the carbon atoms, and are termed unsatu Microbiol. Biotechnol. 49:72-76 (1998); Honda et al., rated fatty acids when double bonds are present. Unsaturated Mycol. Res. 4:439-448 (1998); Singh, World J. of Microbi long chain fatty acids are monounsaturated when only one ology 12:76-81 (1996); Goldstein, American J. of Botany double bond is present and are polyunsaturated when more 50:271-279 (1963); and U.S. Pat. No. 5,340,742. As such, than one double bond is present. thraustochytrid cultures, including commercial and indus 0007 Polyunsaturated fatty acids (PUFAs) are classified trial cultures, currently require glucose syrups as carbon and based on the position of the first double bond from the energy sources, rather than more inexpensive carbohydrate methyl end of the fatty acid: omega-3 (n-3) fatty acids Sources containing Sucrose. contain a first double bond at the third carbon, while 0011. A continuing need exists for producing thraus omega-6 (n-6) fatty acids contain a first double bond at the tochytrids with the ability to grow on alternative carbon sixth carbon. For example, docosahexaenoic acid (“DHA) Sources such as Sucrose for use in commercial and industrial is an omega-3 long chain polyunsaturated fatty acid (LC applications. PUFA) with a chain length of 22 carbons and 6 double bonds, often designated as “22:6 n-3.” Other omega-3 BRIEF SUMMARY OF THE INVENTION LC-PUFAs include eicosapentaenoic acid ("EPA), desig 0012. The present invention is directed to a method of nated as “20:5 in-3, and omega-3 docosapentaenoic acid producing a thraustochytrid cell culture, comprising: (a) (“DPA n-3), designated as “22:5 n-3.” DHA and EPA have transforming a thraustochytrid cell with a nucleic acid been termed “essential fatty acids. Omega-6 LC-PUFAs molecule comprising a polynucleotide sequence encoding a include arachidonic acid (“ARA”), designated as “20:4n-6. heterologous Sucrase, and (b) growing the transformed and omega-6 docosapentaenoic acid (“DPA n-6”), desig thraustochytrid cell in a culture medium comprising Sucrose nated as “22:5 n-6. as a carbon Source. In some embodiments, the Sucrase is an 0008 Omega-3 fatty acids are biologically important invertase. In some embodiments, the Sucrase is operably molecules that affect cellular physiology due to their pres linked to a signal peptide. In some embodiments, the poly ence in cell membranes, regulate production and gene nucleotide sequence encoding the heterologous Sucrase is at expression of biologically active compounds, and serve as least 90% identical to a sequence selected from the group biosynthetic substrates. Roche, H. M., Proc. Nutr Soc. 58: consisting of a polynucleotide sequence encoding the amino 397-401 (1999). DHA, for example, accounts for approxi acid sequence of Accession No. P00724 or S33920; a mately 15%-20% of lipids in the human cerebral cortex and polynucleotide sequence encoding the amino acid sequence US 2017/O 183669 A1 Jun. 29, 2017

of Accession No. P00724 or S33920, wherein the polynucle the biomass. In some embodiments, the method further otide sequence is codon-optimized for expression in comprises extracting an oil from the biomass and preparing Schizochytrium; and the polynucleotide sequence of SEQID the food product, cosmetic, industrial composition, or phar NO: 1 or SEQID NO: 4. In some embodiments, the method maceutical composition from the oil. In some embodiments, further comprises transforming the thraustochytrid cell with the food product is an infant formula. In some embodiments, a nucleic acid molecule comprising a polynucleotide the infant formula is suitable for premature infants. In some sequence encoding a heterologous Sucrose transporter. In embodiments, the food product is milk, a beverage, a Some embodiments, the polynucleotide sequence encoding therapeutic drink, a nutritional drink, or a combination the heterologous sucrose transporter is at least 90% identical thereof. In some embodiments, the food product is an to a sequence selected from the group consisting of a additive for animal or human food. In some embodiments, polynucleotide sequence of Accession No. L47346 or the food product is a nutritional Supplement. In some X69165; and a polynucleotide sequence of Accession No. embodiments, the food product is an animal feed. In some L47346 or X69165 that is codon-optimized for expression in embodiments, the animal feed is an aquaculture feed. In Schizochytrium. In some embodiments, the thraustochytrid Some embodiments, the animal feed is a domestic animal is a Schizochytrium or a Thraustochytrium. In some embodi feed, a Zoological animal feed, a work animal feed, a ments, the invention is directed to a thraustochytrid cell livestock feed, or a combination thereof. culture produced by the method. 0018. The present invention is also directed to a method 0013 The present invention is also directed to a thraus for producing a biofuel, comprising: (a) growing any of the tochytrid cell comprising a nucleic acid molecule compris thraustochytrid cells described herein in a culture medium ing a polynucleotide sequence encoding a heterologous comprising Sucrose as a carbon Source to produce a biomass, Sucrase. In some embodiments, the Sucrase is an invertase. (b) extracting an oil from the biomass, and (c) producing a In some embodiments, the Sucrase is operably linked to a biofuel by transesterifying the oil, cracking the oil, process signal peptide. In some embodiments, the polynucleotide ing the oil by thermal depolymerization, adding the oil to a sequence encoding the heterologous Sucrase is at least 90% petroleum refining process, or a combination thereof. In identical to a sequence selected from the group consisting some embodiments, the biofuel is produced by transesteri of a polynucleotide sequence encoding the amino acid fying the oil. In some embodiments, the biofuel is a bio sequence of Accession No. P00724 or S33920; a polynucle diesel. In some embodiments, the biofuel is produced by otide sequence encoding the amino acid sequence of Acces cracking the oil. In some embodiments, the biofuel is a jet sion No. P00724 or S33920, wherein the polynucleotide biofuel. In some embodiments, the biofuel is produced by sequence is codon-optimized for expression in Schizochy processing the oil by thermal depolymerization. In some trium; and the polynucleotide sequence of SEQID NO: 1 or embodiments, the biofuel is a renewable diesel. In some SEQ ID NO: 4. In some embodiments, the cell further embodiments, the biofuel is produced by adding the oil to a comprises a polynucleotide sequence encoding a heterolo petroleum refining process. In some embodiments, the bio gous Sucrose transporter. In some embodiments, the poly fuel is a co-processed renewable diesel. nucleotide sequence encoding the heterologous Sucrose transporter is at least 90% identical to a sequence selected BRIEF DESCRIPTION OF THE from the group consisting of a polynucleotide sequence of DRAWINGSFFIGURES Accession No. L47346 or X69165; and a polynucleotide sequence of Accession No. L47346 or X69165 that is 0019 FIG. 1 shows a codon usage table for Schizochy codon-optimized for expression in Schizochytrium. In some trium. embodiments, the thraustochytrid is a Schizochytrium or a 0020 FIG. 2 shows a plasmid map of pSchiz-EPCT(+)- Thraustochytrium. s1Suc2 CL0076, also termed pCL0076 (SEQ ID NO:2). 0014. The present invention is also directed to a thraus 0021 FIG. 3 shows dry weight (g/L) of cell pellets from tochytrid culture comprising: (a) any of the thraustochytrid cultures of Schizochytrium sp. ATCC 20888 transformed cells described herein, and (b) a cell culture medium com with pCL0076 grown on sucrose-SSFM. The transformants prising Sucrose as a carbon Source. are referred to as 1-1, 1-3, 1-24, 3-1, 3-2, 3-5, 3-21, 4-1, 0015 The present invention is also directed to a method 4-24, and 4-31. “Control” refers to wild-type Schizochytrium of producing a thraustochytrid biomass, comprising: (a) sp. ATCC 20888 cells grown on glucose-SSFM. growing any of the thraustochytrid cells described herein in 0022 FIG. 4 shows fat content (expressed as % weight of a culture medium comprising Sucrose as a carbon Source, the dry biomass) in cell pellets from cultures of Schizochy and (b) harvesting the biomass from the culture medium. trium sp. ATCC 20888 transformed with pCL0076 grown on 0016. The present invention is also directed to a method sucrose-SSFM. The transformants are referred to as 1-1, 1-3, of producing a microbial oil, comprising: (a) growing any of 1-24, 3-1, 3-2, 3-5, 3-21, 4-1, 4-24, and 4-31. “Control the thraustochytrid cells described herein in a culture refers to wild-type Schizochytrium sp. ATCC 20888 cells medium comprising Sucrose as a carbon source to produce grown on glucose-SSFM. a biomass, and (b) extracting an oil from the biomass. (0023 FIG. 5 shows dry weight (g/L) of cell pellets 0017. The present invention is also directed to a method measured over time (days) for cultures of Schizochytrium sp. of producing a food product, cosmetic, industrial composi ATCC 20888 transformed with pCL0076 grown on sucrose tion, or pharmaceutical composition for an animal or human, SSFM. The transformants are referred to as 1-3 and 3-5. comprising: (a) growing any of the thraustochytrid cells “Control” refers to wild-type Schizochytrium sp. ATCC described herein in a culture medium comprising Sucrose as 20888 cells grown on glucose-SSFM. a carbon Source, (b) harvesting a biomass from the culture 0024 FIG. 6 shows fat content (expressed as % weight of medium, and (c) preparing the food product, cosmetic, the dry biomass) in cell pellets from cultures of two trans industrial composition, or pharmaceutical composition from formants grown on sucrose-SSFM. The transformants are US 2017/O 183669 A1 Jun. 29, 2017

referred to as 1-3 and 3-5. “Control” refers to wild-type tochytriales, which includes the family Thraustochytriaceae. Schizochytrium sp. ATCC 20888 cells grown on glucose The current taxonomic placement of the thraustochytrids can SSFM. be summarized as follows: 0025 FIG. 7 shows dry weight (g/L) of cell pellets from cultures of Schizochytrium strain B76-32 transformed with Realm: Stramenopila (Chromista) pCL0076 and harvested after either 2 days or 7 days of growth in sucrose-SSFM. 2118 refers to a sub-isolate of 0038 Phylum: Labyrinthulomycota (Heterokonta) wild-type Schizochytrium sp. ATCC 20888 cells grown on 0039 Class: Labyrinthulomycetes (Labyrinthulae) glucose-SSFM. B76-32** refers to the B76-32 parent strain 0040 Order: Labyrinthulales grown on glucose-SSFM. 0041 Family: Labyrinthulaceae 0026 FIG. 8 shows fat content of cell pellets from 0042. Order: Thraustochytriales cultures of Schizochytrium strain B76-32 transformed with 0043 Family: Thraustochytriaceae pCL0076 and harvested after either 2 days or 7 days of 0044) For purposes of the present invention, strains growth in sucrose-SSFM. The rightmost column for each described as thraustochytrids include the following organ sample shows fat content as % weight of the dry biomass. isms: Order: Thraustochytriales; Family: Thraustochytri The leftmost column for each sample shows 9% of total fat aceae; Genera: Thraustochytrium (Species: sp., arudimen composed of acyl groups with 18 or fewer carbons (light tale, aureum, benthicola, globosum, kinnei, motivum, grey) or 20 or more carbons (medium grey). 2118 refers to multirudimentale, pachydermum, proliferum, roseum, stria a sub-isolate of wild-type Schizochytrium sp. ATCC 20888 tum), Ulkenia (Species: sp., amoeboidea, kerguelensis, cells grown on glucose-SSFM. B76-32** refers to the minuta, profunda, radiata, sailens, Sarkariana, B76-32 parent strain grown on glucose-SSFM. schizochytrops, visurgensis, yorkensis), Schizochytrium 0027 FIG.9A shows a Western blot for invertase protein. (Species: sp., aggregatum, limnaceum, mangrovei, minu S. cerevisiae invertase control and Supernatants of a 3-day turn, Octosporum), Japonochytrium (Species: sp., marinum), culture of pCL0076 transformant 1-3 were loaded in Aplanochytrium (Species: sp., haliotidis, kerguelensis, pro amounts of 5ug, 2.5 Lig, 1.25 ug, and 0.625 ug, respectively, finda, stocchinoi), Althornia (Species: sp., crouchii), or as indicated at the top of the Western blot. Elina (Species: sp., marisalba, Sinorifica). For the purposes 0028 FIG.9B shows a corresponding Coomassie-stained of this invention, species described within Ulkenia will be SDS-PAGE gel. S. cerevisiae invertase control and super considered to be members of the genus Thraustochytrium. natants of a 3-day culture of pCL0076 transformant 1-3 were Aurantiochytrium, Oblongichytrium, Botryochytrium, Pari loaded in amounts of 5 ug. 2.5 Lig, 1.25 ug, and 0.625 ug, etichytrium, and Sicyoidochytrium are additional genuses respectively, as indicated at the top of the Western blot. encompassed by the present invention. 0029 FIG. 10A shows an invertase activity assay illus 0045 Strains of Thraustochytriales include, but are not trated by the reaction rate as a function of Sucrose concen limited to, deposited strains PTA-10212, PTA-10213, PTA tration. 10214, PTA-10215, PTA-9695, PTA-9696, PTA-9697, PTA 0030 FIG. 10B shows a standard Lineweaver-Burk plot 9698, PTA-10208, PTA-10209, PTA-10210, PTA-10211, used to calculate the K and V. Thraustochytrium sp. (23B) (ATCC 20891); Thraustochy 0031 FIG. 11 shows N-glycan structures detected on trium striatum (Schneider) (ATCC 24473); Thraustochy Schizochytrium secreted proteins as determined by NSI trium aureum (Goldstein) (ATCC 34304); Thraustochytrium Total Ion Mapping of permethylated N-glycans. roseum (Goldstein) (ATCC 28210); and Japonochytrium sp. (L1) (ATCC 28207). Schizochytrium include, but are not 0032 FIG. 12 shows a table of glycan species obtained limited to Schizochytrium aggregatum, Schizochytrium by NSI-Total Ion Mapping of permethylated N-glycans. limacinum, Schizochytrium sp. (S31) (ATCC 20888), 0033 FIG. 13 shows a plasmid map of pCL0120 (SEQ Schizochytrium sp. (S8) (ATCC 20889), Schizochytrium sp. ID NO:5). (LC-RM) (ATCC 18915), Schizochytrium sp. (SR 21), 0034 FIG. 14 shows a codon-optimized nucleic acid deposited strain ATCC 28209 and deposited Schizochytrium sequence (SEQ ID NO:4) encoding the Sec1 signal peptide limacinum Strain IFO 32693. from Schizochytrium fused to the mature Suc1 invertase 0046. In some embodiments, the thraustochytrid cell of from Aspergillus niger (GenBank Accession No. S33920). the invention is a Schizochytrium or a Thraustochytrium 0035 FIG. 15 shows a plasmid map of pCL0137 EPCT cell. In some embodiments, the thraustochytrid is from a (+)-s1Suc1, also termed pCL0137. species selected from Schizochytrium sp., Schizochytrium aggregatum, Schizochytrium limacinum, Schizochytrium DETAILED DESCRIPTION OF THE minutum, Thraustochytrium sp., Thraustochytrium striatum, INVENTION Thraustochytrium aureum, Thraustochytrium roseum, 0036. The present invention is directed to recombinant Japonochytrium sp., and strains derived therefrom. thraustochytrids that grow on Sucrose and cell cultures 0047 According to the present invention, the term “trans comprising the recombinant thraustochytrids, as well as formation' is used to refer to any method by which a nucleic methods of producing cell cultures, biomasses, microbial acid molecule (i.e., a recombinant nucleic acid molecule) oils, compositions, and biofuels using the recombinant can be inserted into a thraustochytrid cell of the invention. thraustochytrids. In microbial systems, the term “transformation' is used to describe an inherited change due to the acquisition of Thraustochytrids exogenous nucleic acids by the microorganism and is essen tially synonymous with the term “transfection.” Suitable 0037 According to the present invention, the term transformation techniques for introducing nucleic acid mol “thraustochytrid' refers to any member of the order Thraus ecules into thraustochytrid cells include, but are not limited US 2017/O 183669 A1 Jun. 29, 2017 to, particle bombardment, electroporation, microinjection, one or more heterologous Sucrases, a heterologous Sucrose lipofection, adsorption, infection, and protoplast fusion. transporter, or a combination thereof. In some embodiments, 0048 Although the phrase “nucleic acid molecule' pri the thraustochytrid cell comprises a nucleic acid molecule marily refers to the physical nucleic acid molecule and the comprising a polynucleotide sequence encoding a secreted phrases “nucleic acid sequence' or “polynucleotide Sucrase. In some embodiments, the thraustochytrid cell sequence' primarily refers to the sequence of nucleotides in comprises a nucleic acid molecule comprising a polynucle the nucleic acid molecule, the phrases are used interchange otide sequence encoding a cytoplasmic Sucrase. In some ably, especially with respect to a nucleic acid molecule, embodiments, the thraustochytrid cell comprises a nucleic polynucleotide sequence, or a nucleic acid sequence that is acid molecule comprising a polynucleotide sequence encod capable of encoding a protein. In some embodiments, iso ing a Sucrose transporter. In some embodiments, the thraus lated nucleic acid molecules as described herein are pro tochytrid cell comprises one or more nucleic acid molecules duced using recombinant DNA technology (e.g., polymerase comprising one or more polynucleotides encoding one or chain reaction (PCR) amplification or cloning) or chemical synthesis. In accordance with the present invention, an more heterologous Sucrases and a heterologous Sucrose "isolated nucleic acid molecule is a nucleic acid molecule transporter. In some embodiments, the thraustochytrid cell that has been removed from its natural milieu (i.e., that has comprises one or more nucleic acid molecules comprising been subject to human manipulation), its natural milieu one or more polynucleotides encoding a heterologous cyto being the genome or chromosome in which the nucleic acid plasmic invertase and a heterologous Sucrose transporter. In molecule is found in nature. As such, "isolated” does not Some embodiments, the thraustochytrid cell comprises one necessarily reflect the extent to which the nucleic acid or more nucleic acid molecules comprising one or more molecule has been purified, but indicates that the molecule polynucleotides encoding a heterologous secreted invertase does not include an entire genome or an entire chromosome and a heterologous Sucrose transporter. In some embodi in which the nucleic acid molecule is found in nature. An ments, the thraustochytrid cell comprises one or more isolated nucleic acid molecule can include DNA, RNA (e.g., nucleic acid molecules comprising one or more polynucle mRNA), or derivatives of either DNA or RNA (e.g., cDNA). otides encoding a heterologous cytoplasmic invertase, a Isolated nucleic acid molecules include natural nucleic acid heterologous secreted invertase, and a heterologous Sucrose molecules and homologues thereof, including, but not lim transporter. In some embodiments, the polynucleotide ited to, natural allelic variants and modified nucleic acid sequence is codon-optimized to maximize translation effi molecules in which nucleotides have been inserted, deleted, ciency in the thraustochytrid cell. In some embodiments, the Substituted, and/or inverted in Such a manner that Such polynucleotide sequence encoding the Sucrase, Sucrose modifications provide the desired effect on sequence, func transporter, or combination thereof is integrated into the tion, and/or the biological activity of the encoded peptide or genome of the thraustochytrid cell. In some embodiments, protein. the polynucleotide sequence encoding the Sucrase, Sucrose 0049. The term “protein' includes single-chain polypep transporter, or combination thereof is stably integrated into tide molecules as well as multiple-polypeptide complexes the genome of the thraustochytrid cell. Sucrases are part of where individual constituent polypeptides are linked by a family of enzymes known as glycoside hydrolases (also covalent or non-covalent means. The term “polypeptide' termed glycosidases) that catalyze the hydrolysis of glyco includes peptides of two or more amino acids in length, sidic linkages to generate two smaller Sugars. As described typically having more than 5, 10, or 20 amino acids. In some herein, a “Sucrase' is any enzyme that catalyzes the hydro embodiments, a polypeptide as described herein is a heter lysis of Sucrose (also known as saccharose or table Sugar) to ologous polypeptide. The term "heterologous' as used fructose and glucose. In certain embodiments, the Sucrase is herein refers to a polypeptide (or polynucleotide) sequence, an invertase, a Sucrase-isomaltase, a levanase, sacridase, for example, that is not naturally found in the thraustochytrid beta-fructosidase, beta-fructofuranosidases, fructohydro cell of the invention. lase, Saccharase, inulinase, Sucrose alpha-glucohydrolase, 0050 Schizochytrium naturally uses hexose sugars and alpha-D-glucosidase, or a combination thereof. In some glycerol as a carbon source instead of Sucrose. Examination embodiments the sucrase is the invertase SUC2 from Sac of the genome database of Schizochytrium did not reveal charomyces cerevisiae; the invertase SacC from Zymomo genes required for the initial steps of Sucrose catabolism, nas; the invertase INV1 from Pichia; the invertase INV from Supporting the general finding that thraustochytrids metabo Debaryomyces; the Sucrase Suc1 from Aspergillus; a Sucrase lize glucose and/or fructose, which are the two Sugar com from other fungal, protist, algal, plant, or eukaryotic sources; ponents of Sucrose, but do not naturally metabolize Sucrose. or any combinations thereof. In certain embodiments, the See also, e.g., Aki et al., JAOCS80:789-794 (2003); Yokochi Sucrase is from a prokaryotic source Such as coryneform et al., Appl. Microbiol. Biotechnol. 49:72-76 (1998); Honda bacteria or Escherichia coli. In some embodiments, a et al., Mycol. Res. 4:439-448 (1998); Singh, World J. of Sucrase expressed in a thraustochytrid cell of the invention Microbiology 12:76-81 (1996); Goldstein, American J. of comprises a different glycosylation pattern than when Botany 50:271-279 (1963); and U.S. Pat. No. 5,340,742. expressed in the organism from which the Sucrase is derived. 0051. The present invention is directed to a thraustochy In some embodiments, the Sucrase that is expressed in the trid cell transformed with a nucleic acid molecule compris thraustochytrid cell is a glycoprotein comprising high-man ing a polynucleotide sequence encoding a heterologous nose oligosaccharides. In some embodiments, the thraus polypeptide associated with Sucrose metabolism. In some tochytrid cell also comprises a polynucleotide sequence embodiments, the polypeptide is a heterologous polyp encoding an enzyme required for saccharification of a cel eptide. In some embodiments, a thraustochytrid cell of the lulose feedstock (e.g., Sugar cane). See, e.g., U.S. 2009/ invention comprises one or more nucleic acid molecules 0064567, incorporated by reference herein in its entirety. In comprising one or more polynucleotide sequences encoding Some embodiments, the thraustochytrid cell also comprises US 2017/O 183669 A1 Jun. 29, 2017

a polynucleotide sequence encoding a fructokinase, a glu although the activity can be increased, decreased, or made cokinase, a hexokinase, or a combination thereof. dependent upon certain stimuli. In some embodiments, the 0052. In some embodiments, the expression system used signal peptide sequence includes, but is not limited to, a for the production of a Sucrase, a Sucrose transporter, or a Schizochytrium sequence described in U.S. Publ. No. 2010/ combination thereof in a thraustochytrid cell comprises 0233760, such as: a Na/Pi transporter signal peptide, a regulatory elements that are derived from thraustochytrid shortened Na/Pi transporter signal peptide, an alpha-1,6- sequences. In some embodiments, the expression system mannosyltransferase (ALG12) signal peptide, a binding used for the production of a Sucrase, a Sucrose transporter, or immunoglobulin protein (BiP) signal peptide, an alpha-1,3- a combination thereof in a thraustochytrid cell comprises glucosidase (GLS2) Signal peptide, an alpha-1,3-1,6-man regulatory elements that are derived from non-thraustochy nosidase-like signal peptide, an alpha-1,3-1,6-mannosidase trid sequences, including sequences derived from non like #1 signal peptide, an alpha-1,2-mannosidase-like signal thraustochytrid algal sequences. In some embodiments, the peptide, a beta-xylosdiase-like signal peptide, a carotene expression system comprises a polynucleotide sequence synthase signal peptide, and a Sec1 signal peptide. encoding a Sucrase, a Sucrose transporter, or the combination 0054. In some embodiments, the sucrase is expressed of the two wherein the polynucleotide sequence is operably cytoplasmically along with expression of a Sucrose trans linked to any promoter sequence, any terminator sequence, porter. In some embodiments, the Sucrase is membrane and/or any other regulatory sequence that is functional in a bound, e.g., Sucrase-isomaltase, which has an N-terminal thraustochytrid cell. In certain embodiments, the expression membrane spanning domain (signal anchor). In certain system comprises polynucleotide sequences encoding a embodiments, the sucrase is localized to the exterior of the cytoplasmically expressed Sucrase and a Sucrose transporter. plasma membrane. In some embodiments, the Sucrase is Inducible or constitutively active regulatory sequences can fused to a membrane spanning domain. be used. Regulatory sequences include but are not limited to 0055. In some embodiments, an expression cassette is the Schizochytrium regulatory sequences described in U.S. used for the expression of a Sucrase, a Sucrose transporter, or Publ. No. 2010/0233760, U.S. Pat. No. 7,001,772, and U.S. a combination thereof in a thraustochytrid cell. The design Publ. Nos. 2006/0275904 and 2006/0286650, incorporated and construction of expression cassettes use standard by reference herein in their entireties, such as: an Orf molecular biology techniques known to persons skilled in promoter, an Orfor terminator, an EF1 short promoter, EF1 the art. See, e.g., Sambrook et al., 2001, Molecular Cloning: long promoter, a Sec1 promoter, 60S short promoter, 60S A Laboratory Manual, 3" edition. In some embodiments, the long promoter, an acetolactate Synthase promoter, an aceto thraustochytrid cell comprises an expression cassette con lactate synthase terminator, an O-tubulin promoter, a pro taining genetic elements, such as at least the following moter from a polyketide synthase (PKS) system, a fatty acid operably linked in such a way that they are functional in the desaturase promoter, an actin promoter, an actin terminator, thraustochytrid cell: a promoter, a coding sequence com an elongation factor 1 alpha (eflC) promoter, an eflC. prising a Sucrase, a Sucrose transporter, or a combination terminator, a glyceraldehyde 3-phosphate dehydrogenase thereof, and a terminator region. In some embodiments, the (gapdh) promoter, a gapdh terminator, and combinations expression cassette further comprises a polynucleotide thereof. sequence encoding a signal peptide or a membrane domain 0053. In some embodiments, the sucrase is secreted into operably linked to the Sucrase or Sucrose transporter. The the fermentation medium by a recombinant thraustochytrid term “membrane domain” as used herein refers to any transformed with a sucrase gene. In some embodiments, the domain within a polypeptide that targets the polypeptide to Sucrase is a secreted protein comprising a signal peptide. In a membrane and/or allows the polypeptide to maintain Some embodiments, the signal peptide is naturally associ association with a membrane and includes, but is not limited ated with the exogenous Sucrase (i.e., the signal peptide is to, a transmembrane domain (e.g., a single or multiple the native signal sequence of the exogenous Sucrase). In membrane spanning region), an integral monotopic domain, Some embodiments, the signal peptide is not naturally asso a signal anchor sequence, an ER signal sequence, an N-ter ciated with the exogenous sucrase but is instead a heterolo minal or internal or C-terminal stop transfer signal, a gly gous signal peptide, Such as a signal peptide from a micro cosylphosophatidylinositol anchor, and combinations organism of the phylum Labyrinthulomycota. In some thereof. A membrane domain can be located at any position embodiments, the signal peptide is from a thraustochytrid. In in the polypeptide, including the N-terminal, C-terminal, or Some embodiments, the signal peptide is from a Schizochy middle of the polypeptide. A membrane domain can be trium or a Thraustochytrium. In some embodiments, the associated with permanent or temporary attachment of a signal peptide is the Schizochytrium Sec1 signal peptide polypeptide to a membrane. In some embodiments, a recom (SEQ ID NO:3). A “signal peptide' includes full-length binant vector comprises the expression cassette. Recombi peptides and functional fragments thereof, fusion peptides, nant vectors include, but are not limited to, plasmids, and homologues of a naturally occurring signal peptide. A phages, and viruses. In some embodiments, the recombinant homologue of a signal peptide differs from a naturally vector is a linearized vector. In some embodiments, the occurring signal peptide in that at least one or a few, but not recombinant vector is an expression vector. As used herein, limited to one or a few, amino acids have been deleted (e.g., the phrase “expression vector” refers to a vector that is a truncated version of the protein, Such as a peptide or Suitable for production of an encoded product (e.g., a fragment), inserted, inverted, Substituted, and/or derivatized Sucrase, a Sucrose transporter, or a combination thereof). In (e.g., by glycosylation, phosphorylation, acetylation, acy Some embodiments, a polynucleotide sequence encoding the lation (e.g., myristoylation and palmitoylation), prenylation, Sucrase. Sucrose transporter, or combination thereof is amidation, and/or addition of glycosylphosphatidyl inosi inserted into the recombinant vector to produce a recombi tol). In some embodiments, homologues of a signal peptide nant nucleic acid molecule. In some embodiments, the retain activity as a signal at least in a thraustochytrid, polynucleotide sequence encoding the Sucrase, Sucrose US 2017/O 183669 A1 Jun. 29, 2017

transporter, or combination thereof is inserted into the vector of any of the methods of the invention encodes a polypeptide in a manner that operatively links the nucleic acid sequence comprising an amino acid sequence that is at least 90%, at to regulatory sequences in the vector, which enables the least 91%, at least 92%, at least 93%, at least 94%, at least transcription and translation of the nucleic acid sequence 95%, at least 96%, at least 97%, at least 98%, at least 99%, within the recombinant microorganism. In some embodi or that is identical to the amino acid sequence encoded by ments, a selectable marker enables the selection of a recom SEQ ID NO:1 or SEQ ID NO:4. In some embodiments, a binant microorganism into which a recombinant nucleic acid polynucleotide sequence of any of the methods of the molecule of the present invention has successfully been invention is at least 90%, at least 91%, at least 92%, at least introduced. Selectable markers include but are not limited to 93%, at least 94%, at least 95%, at least 96%, at least 97%, the Selectable markers described in U.S. Publ. No. 2010/ at least 98%, at least 99%, or that is identical to: the 0233760 and U.S. Pat. No. 7,001,772, such as Schizochy polynucleotide sequence of Accession No. L47346 or trium acetolactate synthase or bacterial ble. In some embodi X69165; a polynucleotide sequence of Accession No. ments, the selection marker is an auxotrophic marker, a L47346 or X69165 that is codon-optimized for expression in dominant selection marker (such as, for example, an enzyme Schizochytrium; a polynucleotide sequence encoding the that degrades antibiotic activity), or another protein involved amino acid sequence of Accession No. P00724 or S33920; in transformation selection. a polynucleotide sequence encoding the amino acid 0056. In some embodiments, the thraustochytrid cell is sequence of Accession No. P00724 or S33920, wherein the genetically modified to introduce or delete genes involved in polynucleotide sequence is codon-optimized for expression biosynthetic pathways associated with the transport and/or in Schizochytrium; or the polynucleotide sequence of SEQ synthesis of carbohydrates, including those involved in ID NO:1 or SEQ ID NO:4. glycosylation. For example, the thraustochytrid cell can be modified by deleting endogenous glycosylation genes and/or Thraustochytrid Cultures, Biomasses, and Microbial Oils inserting human or animal glycosylation genes to allow for 0058. The present invention is also directed to a thraus glycosylation patterns that more closely resemble those of tochytrid cell culture comprising any of the thraustochytrid humans. Modification of glycosylation in yeast is exempli cells of the invention as described herein and a cell culture fied in, for example, U.S. Pat. No. 7,029,872 and U.S. Publ. medium comprising Sucrose as a carbon Source. Nos. 2004/0171826, 2004/0230042, 2006/0257399, 2006/ 0059. The present invention is also directed to a method 0029604, and 2006/0040353. In some embodiments, the of producing a thraustochytrid cell culture, comprising thraustochytrid cell includes a cell in which RNA viral transforming a thraustochytrid cell with a nucleic acid elements are employed to increase or regulate gene expres molecule comprising a polynucleotide sequence encoding a S1O. polypeptide associated with Sucrose metabolism as 0057. In some embodiments, a polynucleotide sequence described herein, selecting the transformed thraustochytrid of any of the methods of the invention encodes a heterolo cell, and growing the transformed thraustochytrid cell in a gous polypeptide comprising an amino acid sequence that is culture medium comprising Sucrose as a carbon Source, at least 90%, at least 91%, at least 92%, at least 93%, at least wherein a thraustochytrid cell culture is produced. The 94%, at least 95%, at least 96%, at least 97%, at least 98%, present invention is also directed to a thraustochytrid cell at least 99%, or that is identical to the amino acid sequence culture produced by the method. of a polypeptide associated with Sucrose metabolism includ 0060. In some embodiments, sucrose is the primary car ing, but not limited to, a polypeptide associated with a bon source in the cell culture medium. In some embodi Sucrase (such as, for example, an invertase, Sucrase-isomal ments, sucrose is the only carbon Source in the cell culture tase, levanase, sacridase, beta-fructosidase, beta-fructofura medium. Sucrose sources include but are not limited to Sugar nosidases, fructohydrolase, Saccharase, inulinase. Sucrose cane, Sugar beets, and other plants. The composition of the alpha-glucohydrolase, alpha-D-glucosidase) or a Sucrose purified or partially purified Sucrose can be predominantly transporter. In some embodiments, a polynucleotide Sucrose, i.e., purified/refined Sucrose, crude Sucrose extracts, sequence of any of the methods of the invention encodes a or a mixture of Sucrose, glucose, and fructose (e.g., molas heterologous polypeptide comprising an amino acid ses). sequence that is at least 90%, at least 91%, at least 92%, at 0061. In some embodiments, the culture medium com least 93%, at least 94%, at least 95%, at least 96%, at least prises molasses, a syrup, or juice from any Sucrose-produc 97%, at least 98%, at least 99%, or that is identical to: the ing vegetable (e.g., Sugar cane juice or Sugar beet juice), or amino acid sequence of the invertase SUC2 from Saccha combinations thereof. In some embodiments, the culture romyces cerevisiae; the invertase SacC from Zymomonas; medium comprises a cellulose-containing feedstock, Such as the invertase INV1 from Pichia; the invertase INV from Sugar cane bagasse. In some embodiments, the cell culture Debaryomyces; the Sucrase Suc1 from Aspergillus; a Sucrase medium comprises a Sucrase. In some embodiments, the from other fungal, protist, algal, plant, or eukaryotic sources; thraustochytrid cell secretes a heterologous Sucrase into the or a Sucrase from a prokaryotic source Such as coryneform culture medium. bacteria or Escherichia coli. In some embodiments, a poly 0062 Culture conditions for thraustochytrid cells nucleotide sequence of any of the methods of the invention include, but are not limited to, effective media, bioreactor, encodes a polypeptide comprising an amino acid sequence temperature, pH, and oxygen conditions that permit protein that is at least 90%, at least 91%, at least 92%, at least 93%, production and/or recombination. An effective medium at least 94%, at least 95%, at least 96%, at least 97%, at least refers to any medium in which a thraustochytrid cell is 98%, at least 99%, or that is identical to: the amino acid typically cultured. Such medium typically comprises an sequence encoded by Accession No. L47346 or X69165; or aqueous medium having assimilable carbon, nitrogen, and the amino acid sequence of Accession No. P00724 or phosphate sources, as well as appropriate salts, minerals, S33920. In some embodiments, a polynucleotide sequence metals, and other nutrients, such as vitamins. Nitrogen US 2017/O 183669 A1 Jun. 29, 2017

Sources include, but are not limited to, peptone, yeast 0067. In some embodiments, the crude oil can be isolated extract, polypeptone, malt extract, meat extract, casamino from a thraustochytrid using standard techniques, without acid, corn steep liquor, organic nitrogen Sources, sodium being subjected to further refinement or purification. For glutamate, urea, inorganic nitrogen sources, ammonium example, the crude oil can be isolated using solvent extrac acetate, ammonium Sulfate, ammonium chloride, ammo tion, such as, but not limited to, hexane extraction or nium nitrate, and sodium sulfate. Non-limiting culture con isopropanol extraction. In some embodiments, the crude oil ditions suitable for thraustochytrids are described, for can be isolated using physical and/or mechanical extraction example, in U.S. Pat. No. 5,340.742. Various fermentation methods such as, but not limited to, extraction through the parameters for inoculating, growing, and recovering micro use of a homogenizer or by pressing. flora are also described, for example, in U.S. Pat. No. 0068. In some embodiments, the crude oil can be sub 5,130,242. Liquid or solid media can contain natural or jected to further processing, such as refining, desolventiza artificial sea water. Thraustochytrid cells of the present tion, deodorization, winterization, chill filtration, and/or invention can be cultured in fermentation bioreactors, shake bleaching. Such “processed oils include microbial oils that flasks, test tubes, microtiter dishes, and petri plates. have been Subjected to solvent extraction and one or more 0063. The fermentation volume can be any volume used further processing. In some embodiments, oils are minimally for the growth of thraustochytrids, including commercial processed. “Minimally processed oils include microbial and industrial volumes. In some embodiments, the fermen oils that have been subjected to solvent extraction and tation volume (volume of culture) is at least 2 L, at least 10 filtration. In certain embodiments, minimally processed oils L., at least 50 L, at least 100 L, at least 200 L, at least 500 are further subjected to winterization. L., at least 1000 L, at least 10,000 L, at least 20,000 L, at least 0069. In some embodiments, a method similar to the 50,000 L, at least 100,000 L, at least 150,000 L, at least FRIOLEX(R) process (Westfalia Separator Industry GmbH, 200,000 L, or at least 250,000 L, at least 300,000 L, at least Germany) is used to extract the microbial oils produced by 350,000 L, at least 400,000 L, or at least 500,000 L. In some the thraustochytrids. The FRIOLEXOR) process is a water embodiments, the fermentation volume is 2 L to 2,000,000 based physical oil extraction process, whereby raw material L., 2 L to 1,000,000 L, 2 L to 500,000 L, 2 L to 200,000 L, containing oil can be used directly for extracting oil without 2 L to 100,000 L, 2 L to 50,000 L, 2 L to 25,000 L, 2 L to using any conventional solvent extraction methods. In this 20,000 L, 2 L to 15,000 L, 2 L to 10,000 L, 2 L to 5,000 L, process, a water-soluble organic solvent can be used as a 2 L to 1,000 L, 2 L to 500 L, or 2 L to 100 L. process aid and the oil is separated from the raw material broth by density separation using gravity or centrifugal 0064. The present invention is also directed to a method forces. WO96/05278 discloses such extraction methods and of producing a thraustochytrid biomass comprising growing is herein incorporated by reference in its entirety. a thraustochytrid cell of the invention in a culture medium 0070. After the oil has been extracted, the oil can be comprising Sucrose as a carbon source, and harvesting a recovered or separated from non-lipid components by any biomass from the culture medium. A thraustochytrid bio Suitable means known in the art. In some embodiments, mass is a harvested cellular biomass obtained by any con low-cost physical and/or mechanical techniques are used to ventional method for the isolation of a thraustochytrid separate the lipid-containing compositions from non-lipid biomass, such as described in U.S. Pat. No. 5,130,242 and compositions. For example, if multiple phases or fractions U.S. Publ. No. 2002/0001833, incorporated by reference are created by the extraction method used to extract the oil, herein in their entireties. A harvested biomass can contain where one or more phases or fractions contain lipids, a thraustochytrid cells as well as thraustochytrid cellular frag method for recovering the lipid-containing phases or frac mentS. tions can involve physically removing the lipid-containing 0065. The present invention is also directed to a method phases or fractions from the non-lipid phases or fractions, or of producing a microbial oil comprising growing a thraus vice versa. In some embodiments, a FRIOLEXR) type tochytrid cell of the invention in a culture medium compris method is used to extract the lipids produced by the micro ing Sucrose as a carbon source to produce a biomass, and organisms, and the lipid-rich light phase is then physically extracting an oil from the biomass. The oil can be extracted separated from the protein-rich heavy phase (such as by from a freshly harvested biomass or can be extracted from skimming off the lipid-rich phase that is on top of the a previously harvested biomass that has been stored under protein-rich heavy phase after density separation). conditions that prevent spoilage. Known methods can be 0071. The microbial oils produced by thraustochytrids of used to culture a thraustochytrid of the invention, to isolate the present invention can be recovered from autolysis or a biomass from the culture, to extract a microbial oil from induced lysis by exposing thraustochytrid cells to a condi the biomass, and to analyze the fatty acid profile of oils tion including, but not limited to, a certain pH, a certain extracted from the biomass. See, e.g., U.S. Pat. No. 5,130. temperature, the presence of an enzyme, the presence of a 242. detergent, physical disruptions, or combinations thereof. In 0066. A microbial oil can be any oil derived from any Some embodiments, lysis or autolysis of the thraustochytrid thraustochytrid, including, for example: a crude oil extracted cells is performed by the use of mechanical forces. In further from the biomass of a thraustochytrid without further pro embodiments of the present invention, the lysis or autolysis cessing: a refined oil that is obtained by treating a crude oil of thraustochytrid cells is followed by mechanical separation with further processing Such as refining, bleaching, and/or of the lipids from the non-lipid compositions. deodorizing; a diluted oil obtained by diluting a crude or 0072 Suitable enzymes that can be used to induce lysis refined oil; or an enriched oil that is obtained, for example, of the thraustochytrid cells include, but are not limited to, by treating a crude or refined oil with further methods of commercially available enzymes or enzyme mixtures Such purification to increase the concentration of a fatty acid in as Proteinase K or ALCALASER). In some embodiments, the oil. the oil-producing thraustochytrids undergo induced lysis in US 2017/O 183669 A1 Jun. 29, 2017

the presence of a detergent such as ionic (cationic oranionic) desirable attribute of the thraustochytrid, such as but not detergents, nonionic detergents, Zwitterionic detergents, or limited to the cell density of a cell culture of the thraus combinations thereof. In further embodiments of the present tochytrid, the percentages and types of fatty acids associated invention, physical disruption methods such as mechanical with the dry cell of a biomass of the thraustochytrid, the fatty grinding, liquid homogenization, use of high frequency acid profile associated with the biomass and/or the microbial Sound waves in Sonication, freeze? thaw cycles methods, oil of the thraustochytrid, or a combination thereof. In some pressing, extruding, or milling can be used to induce lysis of embodiments, the thraustochytrid biomass or microbial oil the oil-producing thraustochytrids. The extraction of the oils comprises a greater percentage of polyunsaturated fatty can take place in the fermentors at the end of the fermen acids than monounsaturated fatty acids. In some embodi tation by in-tank lysis of the thraustochytrid cells. ments, the thraustochytrid biomass or microbial oil com 0073. In some embodiments, the cell cultures, biomasses, prises a greater percentage of omega-3 fatty acids than and microbial oils produced from the thraustochytrid cells of omega-6 fatty acids. In some embodiments, the thraustochy the invention grown on Sucrose have the same or Substan trid biomass or microbial oil comprises a greater percentage tially similar characteristics (e.g., cell densities, dry cell of DHA than other omega-3 fatty acids. In some embodi weights, fatty acid productivities, and fatty acid profiles) ments, the thraustochytrid biomass comprises a greater associated with cultures, biomasses, and microbial oils pro percentage of DHA than other polyunsaturated fatty acids. duced from the corresponding untransformed thraustochy In some embodiments, the thraustochytrid biomass or trid cells grown on glucose and/or fructose. In some embodi microbial oil comprises a greater percentage of EPA than ments, the dry cell weight of a biomass produced from a other omega-3 fatty acids. In some embodiments, the thraus thraustochytrid culture of the invention is higher after tochytrid biomass or microbial oil comprises a greater growth on Sucrose than the dry cell weight obtained under percentage of EPA than other polyunsaturated fatty acids. identical conditions from growth of a culture of the corre 0078. A composition can include one or more excipients. sponding untransformed thraustochytrid cells on glucose As used herein, "excipient” refers to a component, or and/or fructose. In some embodiments, the total amount of mixture of components, that is used in a composition to give fatty acids as a percentage of the dry cell weight of the desirable characteristics to the composition, including foods biomass is higher after growth of a thraustochytrid of the as well as pharmaceutical, cosmetic, and industrial compo invention on Sucrose than the amount obtained under iden sitions. An excipient can be described as a “pharmaceuti tical conditions from growth of a corresponding untrans cally acceptable' excipient when added to a pharmaceutical formed thraustochytrid cell on glucose and/or fructose. composition, meaning that the excipient is a compound, material, composition, salt, and/or dosage form which is, Methods of Producing Compositions within the scope of Sound medical judgment, Suitable for 0074 The present invention is also directed to a method contact with the tissues of human beings and animals of preparing a food product, cosmetic, industrial composi without excessive toxicity, irritation, allergic response, or tion, or pharmaceutical composition for animals or humans, other problematic complications over the desired duration of comprising growing a thraustochytrid cell of the invention in contact commensurate with a reasonable benefit/risk ratio. a culture medium comprising Sucrose as a carbon Source to In some embodiments, the term “pharmaceutically accept produce a biomass, harvesting the biomass, and preparing able' means approved by a regulatory agency of the Federal the food product, cosmetic, industrial composition, or phar or a state government or listed in the U.S. Pharmacopeia or maceutical composition for animals or humans from the other generally recognized international pharmacopeia for biomass. use in animals, and more particularly in humans. Various 0075. Thraustochytrid biomasses can be dried prior to use excipients can be used. In some embodiments, the excipient in a composition by methods including, but not limited to, can be, but is not limited to, an alkaline agent, a stabilizer, freeze drying, air drying, spray drying, tunnel drying, an antioxidant, an adhesion agent, a separating agent, a vacuum drying (lyophilization), or a similar process. Alter coating agent, an exterior phase component, a controlled natively, a harvested and washed biomass can be used release component, a solvent, a Surfactant, a humectant, a directly in a composition without drying. See, e.g., U.S. Pat. buffering agent, a filler, an emollient, or combinations Nos. 5,130,242 and 6,812,009, incorporated by reference thereof. Excipients in addition to those discussed herein can herein in their entireties. include excipients listed in, though not limited to, Reming 0076. In some embodiments, the method of preparing a ton: The Science and Practice of Pharmacy, 21 ed. (2005). food product, cosmetic, industrial composition, or pharma 0079. In some embodiments, the composition is a food ceutical composition for animals or humans, further com product. A food product is any food for animal or human prises extracting an oil from the biomass. Microbial oils can consumption, and includes both solid and liquid composi be used as starting material to more efficiently produce a tions. A food product can be an additive to animal or human product enriched in a fatty acid such as DHA or EPA. For foods. Foods include, but are not limited to, common foods; example, the microbial oils can be subjected to various liquid products, including milks, beverages, therapeutic purification techniques known in the art, such as distillation drinks, and nutritional drinks; functional foods; Supple or urea adduction, to produce a higher potency product with ments; nutraceuticals; infant formulas, including formulas higher concentrations of DHA, EPA, or another fatty acid. for pre-mature infants; foods for pregnant or nursing The microbial oils can also be used in chemical reactions to women; foods for adults; geriatric foods; and animal foods. produce compounds derived from fatty acids in the oils, such 0080. In some embodiments, a thraustochytrid biomass as esters and salts of DHA, EPA, or another fatty acid. or microbial oil can be used directly as or included as an 0077. Any thraustochytrid that has been transformed as additive within one or more of an oil, shortening, spread, described herein to grow on sucrose can be selected for other fatty ingredient, beverage, sauce, dairy-based or Soy preparing any of the described compositions based on a based food (such as milk, yogurt, cheese and ice-cream), a US 2017/O 183669 A1 Jun. 29, 2017

baked good, a nutritional product, e.g., as a nutritional but is not limited to, an aquaculture feed, a domestic animal Supplement (in capsule or tablet form), a vitamin Supple feed including pet feed, a Zoological animal feed, a work ment, a diet Supplement, a powdered drink, a finished or animal feed, a livestock feed, or a combination thereof. semi-finished powdered food product, and combinations I0084. In some embodiments, the composition is a feed or thereof. feed Supplement for any animal whose meat or products are 0081. A partial list of food compositions that can include consumed by humans, such as any animal from which meat, a microbial oil includes, but is not limited to, soya based eggs, or milk is derived for human consumption. When fed products (milks, ice creams, yogurts, drinks, creams, to such animals, nutrients such as LC-PUFAs can be incor spreads, whiteners); Soups and Soup mixes; doughs, batters, porated into the flesh, milk, eggs or other products of Such and baked food items including, for example, fine bakery animals to increase their content of these nutrients. wares, breakfast cereals, cakes, cheesecakes, pies, cupcakes, I0085. In some embodiments, the composition is a spray cookies, bars, breads, rolls, biscuits, muffins, pastries, dried material that can be crumbled to form particles of an Scones, croutons, crackers, Sweet goods, Snack cakes, pies, appropriate size for consumption by Zooplankton, artemia, granola/snack bars, and toaster pastries; candy; hard con rotifers, and filter feeders. In some embodiments, the Zoo fectionery; chocolate and other confectionery; chewing plankton, artemia, or rotifers fed by the composition are in gum, liquid food products, for example milks, energy drinks, turn fed to fish larvae, fish, shellfish, bivalves, or crusta infant formula, carbonated drinks, teas, liquid meals, fruit CCaS. juices, fruit-based drinks, vegetable-based drinks; multivi I0086. In some embodiments, the composition is a phar tamin syrups, meal replacers, medicinal foods, and syrups; maceutical composition. Suitable pharmaceutical composi powdered beverage mixes; pasta; processed fish products; tions include, but are not limited to, an anti-inflammatory processed meat products; processed poultry products; gra composition, a drug for treatment of coronary heart disease, vies and sauces; condiments (ketchup, mayonnaise, etc.); a drug for treatment of arteriosclerosis, a chemotherapeutic vegetable oil-based spreads; dairy products; yogurt, butters; agent, an active excipient, an osteoporosis drug, an anti frozen dairy products; ice creams; frozen desserts; frozen depressant, an anti-convulsant, an anti-Helicobacter pylori yogurts; semi-solid food products such as baby food; pud drug, a drug for treatment of neurodegenerative disease, a dings and gelatin desserts; processed and unprocessed drug for treatment of degenerative liver disease, an antibi cheese; pancake mixes; food bars including energy bars; otic, a cholesterol lowering composition, and a triglyceride waffle mixes; Salad dressings; replacement egg mixes; nut lowering composition. In some embodiments, the composi and nut-based spreads; salted Snacks such as potato chips tion is a medical food. A medical food includes a food that and other chips or crisps, corn chips, tortilla chips, extruded is in a composition to be consumed or administered exter Snacks, popcorn, pretzels, potato crisps, and nuts; specialty nally under the Supervision of a physician and that is Snacks Such as dips, dried fruit Snacks, meat Snacks, pork intended for the specific dietary management of a condition, rinds, health food bars and rice/corn cakes. for which distinctive nutritional requirements, based on 0082 In some embodiments, a microbial oil can be used recognized scientific principles, are established by medical to Supplement infant formula. Infant formula can be supple evaluation. mented with a microbial oil alone or in combination with a 0087. In some embodiments, the microbial oil can be physically refined oil derived from an arachidonic acid formulated in a dosage form. Dosage forms can include, but (ARA)-producing microorganism. An ARA-producing are not limited to, tablets, capsules, cachets, pellets, pills, microorganism, for example, is Mortierella alpina or Mor powders and granules, and parenteral dosage forms, which tierella sect. Schmuckeri. Alternatively, infant formulas can include, but are not limited to, solutions, Suspensions, emul be supplemented with a microbial oil in combination with an sions, and dry powders comprising an effective amount of oil rich in ARA, including ARASCOR) (Martek Biosciences, the microbial oil. It is also known in the art that such Columbia, Md.). formulations can also contain pharmaceutically acceptable 0083. In some embodiments, the composition is an ani diluents, fillers, disintegrants, binders, lubricants, Surfac mal feed. An "animal' means any non-human organism tants, hydrophobic vehicles, water soluble vehicles, emul belonging to the kingdom Animalia, and includes, without sifiers, buffers, humectants, moisturizers, solubilizers, pre limitation, aquatic animals and terrestrial animals. The term servatives, and the like. Administration forms can include, “animal feed” or “animal food” refers to any food intended but are not limited to, tablets, dragees, capsules, caplets, and for non-human animals, whether for fish; commercial fish; pills, which contain the microbial oil and one or more ornamental fish; fish larvae; bivalves; mollusks; crustaceans; Suitable pharmaceutically acceptable carriers. shellfish; shrimp; larval shrimp; artemia; rotifers; brine 0088. For oral administration, the microbial oil can be shrimp; filter feeders; amphibians; reptiles; mammals; combined with pharmaceutically acceptable carriers well domestic animals; farm animals; Zoo animals; sport animals; known in the art. Such carriers enable the microbial oils to breeding Stock; racing animals; show animals; heirloom be formulated as tablets, pills, dragees, capsules, liquids, animals; rare or endangered animals; companion animals; gels, syrups, slurries, Suspensions, and the like, for oral pet animals such as dogs, cats, guinea pigs, rabbits, rats, ingestion by a Subject to be treated. In some embodiments, mice, or horses; primates Such as monkeys (e.g., cebus, the dosage form is a tablet, pill or caplet. Pharmaceutical rhesus, African green, patas, cynomolgus, and cercopith preparations for oral use can be obtained by adding a solid ecus), apes, orangutans, baboons, gibbons, and chimpan excipient, optionally grinding the resulting mixture, and Zees; canids such as dogs and wolves; felids Such as cats, processing the mixture of granules, after adding Suitable lions, and tigers; equids such as horses, donkeys, and Zebras; auxiliaries, if desired, to obtain tablets or dragee cores. food animals such as cows, cattle, pigs, and sheep; ungulates Suitable excipients include, but are not limited to, fillers Such as deer and giraffes; rodents such as mice, rats, ham Such as Sugars, including, but not limited to, lactose. Sucrose, sters and guinea pigs; and so on. An animal feed includes, mannitol, and Sorbitol; cellulose preparations such as, but US 2017/0183669 A1 Jun. 29, 2017 not limited to, maize starch, wheat starch, rice starch, potato 0094. In some embodiments, the biofuel is produced by starch, gelatin, gum tragacanth, methyl cellulose, hydroxy transesterifying the oil. In some embodiments, the biofuel is propylmethyl cellulose, sodium carboxymethyl cellulose. a biodiesel. Various forms of biodiesel are known and and polyvinylpyrrolidone (PVP). If desired, disintegrating include, but are not limited to, biodiesels described in WO agents can be added, such as, but not limited to, the 07/061903, U.S. Pat. No. 7,172,635, EP Pat. No. 1 227 143, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a WO 02/38709, WO 02/38707, and U.S. Publ. No. 2007/ salt thereof such as sodium alginate. Pharmaceutical prepa 0113467. Transesterification of triglycerides in the oil pro rations that can be used orally include, but are not limited to, duces long chain fatty acid esters, i.e., alkyl esters, and can push-fit capsules made of gelatin, as well as soft, sealed be performed by any method known in the art for the capsules made of gelatin and a plasticizer, such as glycerol production of biofuels. In some embodiments, the alkyl or sorbitol. ether is a methyl ester or an ethyl ester. In some embodi 0089. In some embodiments, the composition is a cos ments, the extracting the oil from the thraustochytrid bio metic. Cosmetics include, but are not limited to, emulsions, mass and transesterifying the oil can be performed simul creams, lotions, masks, soaps, shampoos, washes, facial taneously. In some embodiments, the extracting the oil from creams, conditioners, make-ups, bath agents, and dispersion the thraustochytrid biomass and transesterifying the oil are liquids. Cosmetic agents can be medicinal or non-medicinal. performed separately. See, e.g., U.S. Publ. No. 2009/ 0090. In some embodiments, the composition is an indus OO64567. trial composition. In some embodiments, the composition is a starting material for one or more manufactures. A manu 0095. In some embodiments, transesterification is per facture includes, but is not limited to, a polymer; a photo formed using a lower alkyl alcohol and an acid or base graphic photosensitive material; a detergent; an industrial catalyst. Alcohols suitable for use in the present invention oil; or an industrial detergent. For example, U.S. Pat. No. include any lower alkyl alcohol containing from 1 to 6 7.259,006 describes use of DHA-containing fat and oil for carbon atoms (i.e., a C- alkyl alcohol, such as methyl. production of behenic acid and production of photographic ethyl, isopropyl, butyl, pentyl, hexyl alcohols and isomers thereof). In some embodiments, the alcohol comprises from sensitive materials using behenic acid. 5 wt.% to 70 wt.%, 5 wt.% to 60 wt.%, 5 wt.% to 50 wt. Methods of Producing Biofuels %, 7 wt.% to 40 wt.%, 9 wt.% to 30 wt.%, or 10 wt.% to 25 wt.% of the mixture of the oil composition, the (0091. The present invention is also directed to a method alcohol, and a catalytic base. In some embodiments, the oil for producing a biofuel from a recombinant thraustochytrid composition and the base can be added to either pure ethanol of the invention. or pure methanol. The amount of alcohol used can vary with 0092. As used herein, “biofuel refers to any fuel that is the solubility of the oil in the alcohol. Any base known in the produced from or using a biomass or a microbial oil of a art to be suitable for use as a reactant can be used, including thraustochytrid of the invention, including, but not limited bases of the formula RO-M, wherein M is a monovalent to, a biodiesel, a renewable diesel, a co-processed renewable cation and RO is an alkoxide of a C- alkyl alcohol. diesel, a jet biofuel, a heating oil, and a fuel additive. Any Examples of suitable bases include, but are not limited to. method known in the art can be used to generate a biofuel elemental sodium, sodium methoxide, sodium ethoxide, from the biomasses and microbial oils of the thraus potassium methoxide, and potassium ethoxide. In some tochytrids of the present invention, including but not limited embodiments, the base is sodium ethoxide. In some embodi to any methods described in WO 2005/035693, U.S. Publ. ments, the base is added to the reaction with the oil com No. 2005/0112735, 2007/027633, WO 2006/127512, U.S. position and the alcohol in an amount of from 0.05 to 2.0 Publ. No. 2007/0099278, U.S. Publ. No. 2007/0089356, WO molar equivalents of triglycerides in the oil, 0.05 to 1.5 2008/067605, U.S. Publ. No. 2009/0035842, and U.S. Publ. molar equivalents, 0.1 to 1.4 molar equivalents, 0.2 to 1.3 No. 2009/0064567, incorporated by reference herein in their molar equivalents, or 0.25 to 1.2 molar equivalents. The oil entireties. Any of the biofuels described herein can be comprising triglycerides, the alcohol, and the base are blended with a fossil fuel. For example, a biodiesel can be reacted together at a temperature and for an amount of time blended with a fossil diesel at ratios of 1%-99%. In some that allows the production of an ester from the fatty acid embodiments, a microbial oil of a thraustochytrid of the residues and the alcohol. In some embodiments, the reacting invention is used as a starting point for production of a of the composition in the presence of an alcohol and a base biofuel even though it has not been subjected to conven is performed at a temperature from 20° C. to 140°C., from tional processing. Examples of conventional processing that 20° C. to 120° C., from 20° C. to 110° C., from 20° C. to can be avoided include refining (e.g., physical refining, silica 100° C., or from 20° C. to 90° C. In some embodiments, the refining or caustic refining), desolventization, deodorization, reacting of the composition in the presence of an alcohol and winterization, chill filtration, and/or bleaching. Thus, in a base is performed at a temperature of at least 20° C., at certain embodiments, the oils have not been subjected to least 75° C., at least 80° C., at least 85°C., at least 90° C. refining, desolventization, deodorization, winterization, at least 95° C., at least 105° C., or at least 120° C. In some chill filtration, bleaching, or a combination thereof. embodiments, the reacting of the composition in the pres 0093. In some embodiments, the method of producing a ence of an alcohol and a base is performed at a temperature biofuel comprises growing a thraustochytrid of the invention of 20° C., 75° C., 80° C., 85°C., 90° C., 95° C., 1059 C., or in a culture medium comprising sucrose as a carbon source 120° C. In some embodiments, the reacting of the compo to produce a biomass, extracting an oil from the biomass, sition in the presence of an alcohol and a base is performed and producing a biofuel by transesterifying the oil, cracking for a time from 2 hours to 36 hours, from 3 hours to 36 the oil, processing the oil by thermal depolymerization, hours, from 4 hours to 36 hours, from 5 hours to 36 hours, adding the oil to a traditional petroleum refining process, or or from 6 hours to 36 hours. In some embodiments, the a combination thereof. reacting of the composition in the presence of an alcohol and US 2017/O 183669 A1 Jun. 29, 2017

a base is performed for 0.25, 0.5, 1, 2, 4, 5, 5.5, 6, 6.5, 7, 7.5, trid cell of the invention also comprises a polynucleotide 8, 8.5, 10, 12, 16, 20, 24, 28, 32, or 36 hours. In some sequence encoding a lipase for catalysis of transesterifica embodiments, the reacting of the oil composition, alcohol, tion of triglycerides within the cytoplasm. See, for example, and base can be conducted by refluxing the components to U.S. Pat. No. 7,226,771, U.S. Publ. No. 2004/0005604, WO produce the fatty acid esters, such as PUFA esters. In some 03/089620, WO 05/086900, U.S. Publ. No. 2005/0108789, embodiments, the reacting of the oil composition can be WO 05/032496, WO 05/108533, U.S. Pat. No. 6,982,155, carried out at a temperature that does not result in the WO 06/009676, WO 06/133698, WO 06/037334, WO refluxing of the reaction components. For example, carrying 07/076163, WO 07/056786, and WO 06/124818, herein out the reacting the oil composition under pressures greater incorporated by reference in their entireties. than atmospheric pressure can increase the boiling point of 0098. In some embodiments, the biofuel is produced by the solvents present in the reaction mixture. Under such cracking the oil. In some embodiments, the biofuel is a jet conditions, the reaction can occur at a temperature at which biofuel. “Cracking is understood in the art to describe the the solvents would boil at atmospheric pressure, but would reduction of the chain length of fatty acids in an oil by not result in the refluxing of the reaction components. In methods such as those used in the oil industry. In some Some embodiments, the reaction is conducted at a pressure embodiments, the presence of a significant amount of poly from 5 pounds per square inch (psi) to 20 psi; from 7 psi to unsaturated fatty acid in the oil will provide greater flex 15 psi; or from 9 psi to 12 psi. In some embodiments, the ibility and variety for the production of hydrocarbons, since reaction is conducted at a pressure of 7, 8, 9, 10, 11, or 12 the multiple sites of unsaturation in a polyunsaturated fatty psi. Reactions conducted under pressure can be carried out acid provide multiple sites for cleavage to make hydrocar at the reaction temperatures listed above. In some embodi bons. For example, certain jet fuels require hydrocarbons ments, reactions conducted under pressure can be carried out with two to eight carbons. Polyunsaturated fatty acids can be at least from 20° C. to 140°C., from 20° C. to 120° C., from cleaved through known processes in the art, such as crack 20° C. to 110° C., from 20° C. to 100° C., or from 20° C. to ing, to produce shorter hydrocarbons of various chain 90° C. In some embodiments, reactions conducted under lengths. pressure can be carried out at least 70° C., at least 75°C., at 0099. In some embodiments, the biofuel is produced by least 80° C., at least 85° C., or at least 90° C. In some thermal depolymerization. In some embodiments, the bio embodiments, reactions conducted under pressure can be fuel is a renewable diesel. As used herein, thermal depo carried out at 70° C., 75° C., 80° C., 85°C., or 90° C. lymerization includes any process for the production of 0096 Reduced amounts of PUFA in an oil used for renewable diesel using Superheated water. producing a biofuel. Such as a biodiesel, can increase the 0100. In some embodiments, the biofuel is produced by energy density of the biofuel and can reduce the number of adding the oil to a petroleum refining process during the sites for potential oxidation or Sulfation. In some embodi production of diesel fuel. In some embodiments, the biofuel ments, the thraustochytrid biomass or microbial oil, or is a co-processed renewable diesel. triglyceride fraction thereof, comprises a greater percentage 0101 Having generally described this invention, a further of monounsaturated fatty acids than polyunsaturated fatty understanding can be obtained by reference to the examples acids. In some embodiments, the microbial oil is fraction provided herein. These examples are for purposes of illus ated to remove polyunsaturated fatty acids. In some embodi tration only and are not intended to be limiting. ments, the oil is hydrogenated in order to convert polyun saturated fatty acids to monounsaturated fatty acids. To EXAMPLE 1. ensure that greater percentages of microalgal oil-derived 0102 Construction of the pCL0076 Expression Vector biodiesel can be burned, any method of partial or total oil (0103) The vector pAB0018 (ATCC Accession No. PTA hydrogenation, as is routine in the manufacture of marga 9616) was digested with BamHI and Ndel resulting in two rines, can be used. In some embodiments, thraustochytrids fragments of 838 bp and 9879 bp in length. The 9879 bp that produce low levels of PUFAs are used to produce the fragment was fractionated by standard electrophoretic tech microbial oils. In some embodiments, thraustochytrids of the niques in an agar gel, purified using commercial DNA invention are selected that produce greater amounts of purification kits, and ligated to a sequence (SEQ ID NO:1) monounsaturated fatty acids than polyunsaturated fatty comprising a polynucleotide sequence encoding the native acids. In some embodiments, less than 50% of unsaturated secretion signal of the Sec1 protein of Schizochytrium sp. fatty acids in the biological oil are PUFAs. In some embodi ATCC number 20888 followed by a synthetic sequence ments, the unsaturated fatty acids in the biological oil encoding the mature invertase protein (SUC2) of Saccha contain less than 40%, less than 30%, less than 20%, less romyces cerevisiae, codon-optimized using the Schizochy than 10%, or less than 5% PUFAs. In some embodiments, trium codon usage table in FIG. 1 (codon optimization was the microbial oil comprises less than 50%, less than 40%, performed by Blue Heron Biotechnology, Bothell, Wash.). A less than 30%, less than 20%, less than 10%, or less than 5% fusion sequence containing the sequences encoding the by weight of PUFAs. Schizochytrium Sec1 signal peptide and the Saccharomyces 0097. In addition to the transesterification methods cerevisiae SUC2 secreted invertase protein (GenBank described above, other techniques of reducing the Viscosity Accession No. P00724) was inserted into the Schizochy of the microbial oils can also be used to produce biofuels. trium vector pSchiz, followed by digestion with BamHI and These techniques include, but are not limited to, the use of Ndel to yield SEQID NO:1. lipases, Supercritical methanol catalysis, and the use of 0104. The ligation product was then used to transform a whole-cell systems involving cytoplasmic overexpression of commercially supplied strain of competent DH5C. E. coli lipases in a host cell followed by permeabilization of the cells (Invitrogen) using the manufacturer's protocols. Sev host to allow catalysis of transesterification of triglycerides eral of the resulting clones were propagated and their within the cytoplasm. In some embodiments, a thraustochy plasmids were extracted and purified. These plasmids were US 2017/O 183669 A1 Jun. 29, 2017

then screened by restriction digests and/or PCR to confirm equal to a 10 LIL Volume) was added to the cuvette, mixed that the ligation generated the expected plasmid vectors. gently with a pipette tip, and placed on ice for 5 min. Cells One Such plasmid vector resulting from the ligation of the were electroporated at 200 ohms (resistance), 25 uF (capaci 9879 bp fragment and SEQID NO:1 was verified by Sanger tance), and either 250V (for 0.1 cm gap) or 500V (0.2 cm sequencing and designated pCL0076 (SEQ ID NO:2). See gap). 0.5 mL of M50-20 media was added immediately to FIG 2. the cuvette. The cells were then transferred to 4.5 mL of M50-20 media in a 25 mL shaker flask and incubated for 2-3 EXAMPLE 2 h at 30° C. and about 100 rpm on a shaker. The cells were centrifuged for 5 min at about 2500xg in round bottom Growth of Schizochytrium on Sucrose tubes. The supernatant was removed and the cell pellet was 0105. Cultures of Schizochytrium sp. ATCC 20888 and a resuspended in 0.5 mL of M50-20 media. Cells were plated genetically modified Schizochytrium derivative, designated onto an appropriate number (2 to 5) of M2B plates with B76-32, were grown in M2B medium consisting of 10 g/L appropriate selection (if needed) and incubated at 30° C. glucose, 0.8 g/L (NH4)2SO4, 5 g/L Na2SO4, 2 g/L MgSO4. 0.108 Transformants were selected for growth in either 7HO, 0.5 g/L KHPO, 0.5 g/L KC1, 0.1 g/L CaC12H2O, M2B+SMM media or directly selected for growth on 0.1 M MES (pH 6.0) 0.1% PB26 metals, and 0.1% PB26 sucrose by plating onto MSFM--sucrose. For MSFM--su Vitamins (v/v). PB26 vitamins consisted of 50 mg/mL crose selection, after 1-2 weeks colonies were replated with vitamin B12, 100 ug/mL thiamine, and 100 ug/mL Ca several passes onto fresh Sucrose-containing media. It was pantothenate. PB26 metals were adjusted to pH 4.5 and determined that expression of invertase can be used as a consisted of 3 g/L FeSO.7H2O, 1 g/L MnClFIO, 800 selectable marker for thraustochytrid colonies grown on mg/mL ZnSO.7H2O, 20 mg/mL CoCl2.6H2O. 10 mg/mL Sucrose as a sole carbon source. NaMoO2HO, 600 mg/mL CuSO4.5HO, and 800 0109 For the following experiments, primary transfor mg/mL NiSO4.6H2O. PB26 stock solutions were filter ster mants were selected for growth on solid M2B media con ilized separately and added to the broth after autoclaving. taining 20 g/L agar (VWR, West Chester, Pa.) and 10 ug/mL Glucose, KHPO, and CaCl2.H2O were each autoclaved SMM (Chem Service, Westchester, Pa.) after 2-6 days of separately from the remainder of the broth ingredients incubation at 27°C. All primary transformants were manu before mixing to prevent salt precipitation and carbohydrate ally transferred to fresh M2B plates with SMM. After 1 caramelizing. All medium ingredients were purchased from week the colonies were transferred to MSFM and 5 g/L Sigma Chemical (St. Louis, Mo.). Strain B76-32 is a deriva sucrose without SMM. After 1 week the largest colonies tive of Schizochytrium sp. ATCC 20888 engineered accord were transferred to fresh MSFM/sucrose media plates. Ten ing to U.S. Pat. No. 7,211,418 and U.S. Publ. Nos. 2008/ of the Schizochytrium sp. ATCC 20888 transformants grow 0022422 and 2008/0026434. ing on Sucrose were selected for further characterization and 0106 Cultures of Schizochytrium sp. ATCC 20888 and were designated as 1-1, 1-3, 1-24, 3-1, 3-2, 3-5, 3-21, 4-1, B76-32 were grown to log phase and were transformed with 4-24, and 4-31, respectively. Nine of the B76-32 transfor the vector pCL0076 using electroporation with enzyme mants growing on Sucrose were selected for further charac pretreatment as described below. terization and were designated as B76-32 #2, #12, #19, #26, 0107 Electroporation with enzyme pretreatment Cells #30, #39, #42, #56, and #61. were grown in 50 mL of M50-20 media (see U.S. Publ. No. 2008/0022422) on a shaker at 200 rpm for 2 days at 30° C. 0110 Colonies growing on sucrose (1-1, 1-3, 1-24, 3-1, The cells were diluted at 1:100 into M2B media (see 3-2, 3-5, 3-21, 4-1, 4-24, 4-31) were removed from plates following paragraph) and grown overnight (16-24 h), using an inoculation loop and transferred into culture tubes attempting to reach mid-log phase growth (OD600 of 1.5- containing 5 mL of Sucrose media and grown for 4 days at 2.5). The cells were centrifuged in a 50 mL conical tube for 29°C. on a shaker. 2 mL of this culture was used to inoculate 5 min at about 3000xg. The supernatant was removed and 50 mL of media (MSFM or SSFM) in 250 ml flasks and the cells were resuspended in 1 M mannitol, pH 5.5, in a grown at 29° C. on a shaker at 200 rpm. suitable volume to reach a final concentration of 2 ODoo 0111 Control flasks of the parental strain Schizochytrium units. 5 mL of cells were aliquoted into a 25 mL shaker flask sp. ATCC 20888 were grown the same way but using and amended with 10 mM CaCl2 (1.0 M stock, filter steril glucose containing media. Cells were harvested after 7 days. ized) and 0.25 mg/mL Protease XIV (10 mg/mL stock, filter Cells were centrifuged and washed with a 50% isopropanol: sterilized; Sigma-Aldrich, St. Louis, Mo.). Flasks were distilled water mixture. The pelleted cells were freeze-dried, incubated on a shaker at 30° C. and about 100 rpm for 4 h. weighed, and a fatty acid methyl esters (FAME) analysis Cells were monitored under the microscope to determine the was performed. For FAME analysis, oils were extracted degree of protoplasting, with single cells desired. The cells from cell cultures using standard procedures and analyzed were centrifuged for 5 min at about 2500xg in round-bottom for fatty acid composition as a percent of total fatty acid tubes (i.e., 14 mL FalconTM tubes, BD Biosciences, San methyl esters (FAMEs). See, e.g., U.S. Publ. No. 2010/ Jose, Calif.). The supernatant was removed and the cells 0239533, incorporated by reference in its entirety. Growth were gently resuspended with 5 mL of ice cold 10% glyc and fat content of CL0076 transformants of Schizochytrium erol. The cells were re-centrifuged for 5 min at about 2500xg sp. ATCC 20888 or B76-32 were assayed gravimetrically in round-bottom tubes. The Supernatant was removed and and by gas chromatography of derivatized oils as previously the cells were gently resuspended with 500 uL of ice cold described in U.S. Publ. No. 2008/0022422, incorporated 10% glycerol, using wide-bore pipette tips. 90 uL of cells herein by reference in its entirety. Results are shown in were aliquoted into a prechilled electro-cuvette (Gene Tables 5-8. Dry weights and fat content of pellets from shake Pulser R cuvette—0.1 cm gap or 0.2 cm gap, Bio-Rad, flask cultures of transformants as well as parent strains are Hercules, Calif.). One ug to 5 lug of DNA (in less than or shown in FIGS. 3-8. US 2017/O 183669 A1 Jun. 29, 2017

0112 SSFM media: 50 g/L glucose or sucrose, 13.6 g/L with glucose (control) and Schizochytrium sp. ATCC 20888 NaSO, 0.7 g/L KSO, 0.36 g/L KC1, 2.3 g/L MgSO. transformed cell lines grown in SSFM media with sucrose. 7H2O, 0.1M MES (pH 6.0), 1.2 g/L (NH)SO, 0.13 g/L monosodium glutamate, 0.056 g/L KHPO, and 0.2 g/L 0117 Table 8 shows the dry weight and % fatty acid for CaCl2.H2O. Vitamins were added at 1 mL/L from a stock Schizochytrium B76-32 grown in SSFM media with glucose consisting of 0.16 g/L vitamin B12, 9.7 g/L thiamine, and (control) and Schizochytrium B76-32 transformed cell lines 3.3 g/L Ca-pantothenate. Trace metals were added at 2 mL/L grown in SSFM media with sucrose. from a stock consisting of 1 g/L citric acid, 5.2 g/L FeSO4. 7H2O, 1.5 g/L MnClFIO, 1.5 g/L ZnSO.7HO, 0.02 g/L TABLE 5 CoCl2.6H2O, 0.02 g/L NaMoC).2H2O, 1.0 g/L CuSO4. 5HO, and 1.0 g/L NiSO4.6HO, adjusted to pH 2.5. Growth and fat levels of Schizochytrium sp. ATCC 20888 grown in 0113 Modified SFM (MSFM) media: 10 g/L glucose or MSFM with glucose, fructose, sucrose or no added carbon source. sucrose, 25.0 g/L NaCl, 1.0 g/L KC1, 0.2 g/L (NH4)2SO4, 5 Glucose Fructose Sucrose No added carbon g/L, 5.0 g/L MgSO4.7H2O, 0.1 g/L KH2PO4, 0.3 g/L CaCl2.2H2O, 0.1 M HEPES (pH 7.0), 0.1% PB26 metals, DW (g/L) 2.84 2.65 O16 O.11 and 0.1% PB26 Vitamins (v/v). Vitamins were added at 2 % FA 66.5 65.3 ND ND mL/L from a stock consisting of 0.16 g/L vitamin B12, 9.7 DW = Dry Weight g/L thiamine, and 3.3 g/L Ca-pantothenate. Trace metals FA = Fatty Acids were added at 2 mL/L from a stock consisting of 1 g/L citric acid, 5.2 g/L FeSO4.7H2O, 1.5 g/L MnCl2.4H2O, 1.5 g/L ZnSO4.7H2O, 0.02 g/L CoCl2.6H2O, 0.02 g/L Na2MoCl4. TABLE 6 2H2O, 1.0 g/L CuSO4.5H2O, and 1.0 g/L NiSO4.6H2O, Schizochytrium sp. ATCC 20888 transformed cell lines grown adjusted to pH 2.5. in MSFM media with Sucrose. 0114 Table 5 shows the growth and fat levels of Schizochytrium sp. ATCC 20888 grown in MSFM with glucose, fructose, Sucrose, or no added carbon source. control 1-1 1-3 3-2 3-5 3-21 4-1 4-24 4-31 0115 Table 6 shows the dry weight and % fatty acid for DW 2.94 2.49 2.79 2.21 2.6O 2.64 2.44 3.05 2.24 Schizochytrium sp. ATCC 20888 grown in MSFM media (gL) with glucose (control) and Schizochytrium sp. ATCC 20888 % FA 70.87 70.79 72.36 67.97 69.78 71.OS 68.84 73.8S 73.66 transformed cell lines grown in MSFM media with sucrose. DW = Dry Weight 0116 Table 7 shows the dry weight and % fatty acid for FA = Fatty Acids Schizochytrium sp. ATCC 20888 grown in SSFM media TABLE 7

Schizochytrium sp. ATCC 20888 transformed cell lines grown in SSFM media with sucrose.

2O888 control 1-1 1-3 1-24 3-1 3-2 3-5 3-21 4-1 4-24 4-31

DW 11.24 10.04 10.51 999 8.40 10.29 9.03 8.34 8.16 10.63 10.92 (gL) % FA 78.22 78.20 76.29 77.10 77.37 77.71 74.97 73.44. 73.65 80.05 79.82

DW = Dry Weight FA = Fatty Acids

TABLE 8 B76-32 transformed cell lines grown in SSFM media with sucrose.

B76-32 control #2 #12 #19 #26 #30 #39 #42 #56 #61

7-day 1O.S6 13.37 10.21 13.26 7.88 10.26 11.81 10.47 12.84 8.97 DW (gL) % FA 62.8 74.3 75.2 65.4 66.9 65.1 64.8 71.4 77.9 73.7

DW = Dry Weight FA = Fatty Acids US 2017/O 183669 A1 Jun. 29, 2017

EXAMPLE 3 calibration. The reaction rate as a function of Sucrose concentration is shown in FIG. 10A. The Km (33.4 mM) and Expression of Invertase in Schizochytrium Vmax (6.8 mM glucose/min) were calculated from a stan dard Lineweaver-Burk plot. See FIG. 10B. 0118 Cell-free supernatants of 50 mL shake-flask cul tures of Schizochytrium sp. ATCC 20888 clone 1-3 trans EXAMPLE 5 formed with pCL0076 (Example 2) grown in SSFM for 3 days (see U.S. Publ. No. 2008/0022422) were collected after Glycosylation of Invertase in Schizochytrium cultures were centrifuged at 5000xg. Culture supernatants were used either directly for SDS-PAGE or were concen I0121 Supernatant proteins from Example 4 were sepa trated 50 to 100-fold using commercially available concen rated by SDS-PAGE on a 4-12% bis-tris gel (Invitrogen). trators equipped with permeable membranes permitting con The proteins were then stained with Coomassie blue (Sim centration of all components heavier than 10 kDa. Total plyBlueTM Safe Stain, Invitrogen, Carlsbad, Calif.). The protein concentration was measured by Bradford assay stained proteins of interest were cut from the gel and slices (Biorad). The expression of invertase was then verified by cut into smaller pieces (~ 1 mm) and destained alternately immunoblot analysis following standard immunoblotting with 40 mM Ammonium bicarbonate (AmBic) and 100% procedure (Sambrook et al.). Briefly, the proteins (0.625 ug acetonitrile until the color turned clear. Destained gel was to 5 lug) were separated by SDS-PAGE on a bis-tris gel reswelled in 10 mM DTT in 40 mM Ambic at 55° C. for 1 (Invitrogen, Carlsbad, Calif., USA). The proteins were then h. The DTT solution was exchanged with 55 mM Iodoac stained with Coomassie blue (SimplyBlue Safe Stain, Invit etamide (IAM) and incubated in the dark for 45 min. rogen, Carlsbad, Calif., USA) or transferred onto polyvi Incubation was followed by washing alternately with 40 mM nylidene fluoride membrane and probed for the presence of AmBic and 100% acetonitrile twice. Dehydrated gel was invertase protein with an invertase antisera (Open Biosys reswelled with trypsin solution (trypsin in 40 mMAmbic) on tems, Huntsville, Ala.) derived from rabbits that had been ice for 45 min initially, and protein digestion was carried out injected with a pure preparation of Saccharomyces cerevi at 37° C. overnight. The supernatant was transferred into siae invertase (Sigma, St. Louis, Mo.). The membrane was another tube. Peptides and glycopeptides were extracted Subsequently incubated with a mouse anti-rabbit secondary from the gel in series with 20% acetonitrile in 5% formic antibody coupled to alkaline phosphatase (Promega Corpo acid, 50% acetonitrile in 5% formic acid, and then 80% ration, Madison, Wis.). The membrane was then treated with acetonitrile in 5% formic acid. The sample solutions were 5-bromo-4-chloro-3-indoyl-phosphate/nitroblue tetrazolium dried and combined into one tube. Extracted tryptic digest solution (BCIP/NBT) according to the manufacturers was passed through a C18 Sep-PakR) cartridge (Waters instructions (KPL, Gaithersburg, Md.). An example is pre Corporation, Milford, Mass.) and washed with 5% acetic sented in FIG. 9. Anti-invertase immunoblot and corre acid to remove contaminants (such as salts and SDS). sponding Coomassie blue-stained gel are presented in panels Peptides and glycopeptides were eluted in series with 20% 9A and 9B, respectively. Of the four major bands seen in isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic culture Supernatants of clone 1-3, only one was shown to acid, and 100% isopropanol and were dried in a speed react with anti-invertase antisera. The identity of the protein vacuum concentrator. The dried samples were combined and was confirmed by peptide sequence analysis. then reconstituted with 50 mM sodium phosphate buffer (pH 7.5) and heated at 100° C. for 5 min to inactivate trypsin. EXAMPLE 4 The tryptic digest was incubated with PNGase F at 37° C. overnight to release N-glycans. After digestion, the sample Invertase Activity in Schizochytrium was passed through a C18 Sep-PakR) cartridge and the carbohydrate fraction was eluted with 5% acetic acid and 0119 Sucrase activity was measured by the rate of lib dried by lyophilization. Released N-linked oligosaccharides eration of fructose and glucose from Sucrose. The assay was were permethylated based on the method of Anumula and performed by adding Sucrose to fermentation broth Super Taylor (Anumula and Taylor, 1992) and profiled by mass natant and the glucose/fructose content was measured by spectrometry. Mass spectrometric analysis was performed HPLC. following the method developed at the Complex Carbohy 0120 Schizochytrium sp. B76-32 clone #2 transformed drates Research Center (Aoki K et al., J. Biol. Chem. with pCL0076 (Example 2) was grown in MSFM (with 282.9127-42 (2007). Mass analysis was determined by using Sucrose) until the ODoo, reached about 4 in 50 mL shake NSI-LTQ/MS. Briefly, permethylated glycans were dis flasks at 29° C. Cells were spun down for 15 min at 4500xg solved in 1 mM NaOH in 50% methanol and infused directly and invertase activity was measured in the Supernatant. into the instrument (FinniganTM LTQTM Linear Ion Trap Invertase was assayed by adding 0.1 M Sucrose to varying Mass Spectrometer. Thermo Electron Corporation, volumes of fermentation broth and adjusting the final vol Waltham, Mass.) at a constant flow rate of 0.4 uL/min. The ume to 1 mL. The reaction was incubated at 55° C. for 3 min. MS analysis was performed in the positive ion mode. For Termination of the reaction was done at 100° C. for 10 min, total ion mapping, automated MS/MS analysis (at 35 colli and then frozen prior to quantification of glucose, fructose, sion energy), m/z range from 500 to 2000 was scanned in and sucrose by HPLC. HPLC was performed using a modi Successive 2.8 mass unit windows that overlapped the pre fied version of the process described in Liu et al., Food Sci. ceding window by 2 mass units. 28:293-296 (2007). Briefly, mono- and di-saccharides were 0.122 Total ion mapping was performed to examine the separated using an HPLC with a Luna NH column and presence of fragment ions indicative of glycans. All MS/MS detected using an RID (refractive index detector). Identifi data from m/z 500 through m/Z 2000 were taken and the raw cation was carried out by comparing retention times to those data were analyzed manually. The chromatogram and table of standards. Quantitation was by an external standard of species obtained by NSI-total ion mapping are shown in US 2017/O 183669 A1 Jun. 29, 2017

FIG. 11 and FIG. 12. This chromatogram was processed by 0.126 The Solanum tuberosum (potato) SUT1 sucrose the scan filter, neutral loss of m/z, 139, which is the one of transporter gene (GenBank Accession No. X69165) was characteristic neutral loss of high-mannose type glycans. codon-optimized for expression in Schizochytrium and was Total ion mapping revealed that this sample contains a series inserted into the vector pCL0121 (SEQID NO:6), compris of high-mannose type glycans with long mannose chains. ing a ZEOCINTM selectable marker, to produce the vector pCL0126 (SEQ ID NO:8). EXAMPLE 6 I0127. The polynucleotide sequence encoding the Saccha Expression of Aspergillus Niger Invertase in Schizochytrium romyces cerevisiae cytoplasmic SUC2 invertase protein (GenBank Accession No. P00724) was codon-optimized for (0123. The vector p AB0018 (ATCC Accession No. PTA expression in Schizochytrium and was inserted into the 9616) was digested with HindIII, treated with mung bean vector pCLO 122 (SEQ ID NO:9), comprising a paromomy nuclease, purified, and then further digested with KpnI cin selectable marker, to produce the vector pCLO127 (SEQ generating four fragments of various sizes. A fragment of ID NO:10). 2552 bp was isolated by standard electrophoretic techniques in an agar gel and purified using commercial DNA purifi I0128 Schizochytrium sp. B76-32 clone #19 transformed cation kits. A second digest of p AB0018 with PmeI and Kpn with pCL0076 was further transformed with either pCL0125 was then performed. A fragment of 6732 bp was isolated and or pCL0126, comprising the AGT1 alpha-glucoside trans purified from this digest and ligated to the 2552 bp fragment. porter and SUT1 sucrose transporter, respectively. Transfor The ligation product was then used to transform commer mants were selected on antibiotics and the presence of the cially supplied strains of competent DH5-C. E. coli cells transporter genes was confirmed by PCR. Transformants (Invitrogen) using the manufacturer's protocol. Plasmids were further transformed with pCLO127 comprising cyto from amplicillin-resistant clones were propagated, purified, plasmic SUC2, were selected on antibiotic, and were ana and then screened by restriction digests or PCR to confirm lyzed for growth on sucrose based on dry weight and FAME that the ligation generated the expected plasmid structures. content as described in Example 2. Growth on Sucrose and One verified plasmid was designated pCLO120. See FIG. 13 production of fatty acids were observed. and SEQ ID NO:5. I0129 Wild type Schizochytrium sp. ATCC 20888 cells 0124. The polynucleotide sequence encoding the mature were transformed with either: (1) pCLO125, comprising the form of the Suc1 secreted invertase protein from the fungus AGT1 alpha-glucoside transporter, in combination with Aspergillus niger (GenBank Accession No. S33920) was codon-optimized for expression in Schizochytrium using the pCLO127, comprising cytoplasmic SUC2; or (2) pCLO 126, Schizochytrium codon usage table of FIG. 1 (codon optimi comprising the SUT1 Sucrose transporter, in combination zation was performed by Blue Heron Biotechnology, Both with pCLO127, comprising cytoplasmic SUC2. Transfor ell, Wash.). The codon-optimized sequence was synthesized mants were grown and selected on Sucrose. Each of the and the resulting polynucleotide sequence was fused to a combinations of pCL0125/pCL0127 and pCL0126/ polynucleotide sequence encoding the Schizochytrium Sec1 pCLO127 produced transformants capable of growth on signal peptide (“Sec1 ss') as an N-terminal leader in place sucrose. Positive transformants were identified and frozen. of the endogenous signal peptide. The resulting coding At a Subsequent date, the positive transformants are thawed, region of the “s 1Suc1 nucleic acid sequence (SEQ ID are further transformed with pCL0076 comprising secreted NO:4) is shown in FIG. 14. This codon-optimized s1Suc1 invertase, are selected on antibiotic, and are analyzed for polynucleotide was cloned to the vector pCL0120 using the growth levels and FAME as described in Example 2. 5' and 3' restriction sites BamHI and Ndel for insertion and ligation according to standard techniques. A plasmid map of I0130 Schizochytrium sp. ATCC 20888 transformed with the resulting vector, pCL0137, is shown in FIG. 15. Wild pCL0076 comprising the SUC2 secreted invertase (clone type strain Schizochytrium sp. ATCC 20888 was trans 1-3. Example 2) was further transformed with either: (1) formed with this vector and the resulting clones were pCLO125, comprising the AGT1 alpha-glucoside trans selected on solid SSFM media containing SMM. SMM porter, in combination with pCL0127, comprising cytoplas resistant clones were then re-plated to SSFM solid media mic SUC2; or (2) pCL0126, comprising the SUT1 sucrose containing Sucrose as a sole carbon Source to assay for transporter, in combination with pCL0127, comprising cyto growth. Depending on the transformation experiment, plasmic SUC2. Transformants were grown and selected on between 50% and 90% of the SMM-resistant primary trans ZEOCINTM and paromomycin. Positive transformants were formants were capable of growth on Sucrose media. identified and frozen. At a Subsequent date, the positive transformants are thawed and are analyzed for growth on EXAMPLE 7 sucrose and for fatty acid methyl esters as described in Example 2. Expression of Secreted Invertase, Cytoplasmic Invertase, I0131 All of the various aspects, embodiments, and and Sucrose Transporter in Schizochytrium options described herein can be combined in any and all 0.125. The Saccharomyces cerevisiae AGT1 alpha-gluco variations. side transporter gene (GenBank Accession No. L47346) was 0132 All publications, patents, and patent applications codon-optimized for expression in Schizochytrium and was mentioned in this specification are herein incorporated by inserted into the vector pCL0121 (SEQID NO:6), compris reference to the same extent as if each individual publica ing a ZEOCINTM selectable marker, to produce the vector tion, patent, or patent application was specifically and indi pCL0125 (SEQ ID NO:7). vidually indicated to be incorporated by reference.

US 2017/O 183669 A1 Jun. 29, 2017

- Continued aaattt caca aataaag.cat ttttitt cact gcattctagt tdtggitttgt ccaaact cat 726 O caatgitat ct tat catgtct gaatticcc.gg ggtac

What is claimed is: 36. A thraustochytrid culture comprising: 1-28. (canceled) (a) the thraustochytrid cell according to claim 29 or claim 29. A thraustochytrid cell comprising a nucleic acid 30, and molecule comprising a polynucleotide sequence encoding a (b) a cell culture medium comprising Sucrose as a carbon heterologous Sucrase. SOUC. 37. A food product comprising the thraustochytrid cell of 30. The cell of claim 29, wherein the sucrase is an claim 29, wherein the food product is milk, a beverage, a invertase. therapeutic drink, a nutritional drink, or a combination 31. The cell according to claim 29 or claim 30, wherein thereof. the Sucrase is operably linked to a signal peptide. 38. The food product of claim 37, wherein the food 32. The cell of claim 31, wherein the polynucleotide product is an additive for animal or human food. sequence encoding the heterologous Sucrase is at least 90% 39. The food product of claim 37, wherein the food identical to a sequence selected from the group consisting product is a nutritional Supplement. of a polynucleotide sequence encoding the amino acid 40. The food product of claim 37, wherein the food sequence of Accession No. P00724 or S33920; a polynucle product is an animal feed. otide sequence encoding the amino acid sequence of Acces 41. The food product of claim 40, wherein the animal feed sion No. P00724 or S33920, wherein the polynucleotide is an aquaculture feed. sequence is codon-optimized for expression in Schizochy 42. The food product of claim 40, wherein the animal feed trium; and the polynucleotide sequence of SEQID NO: 1 or is a domestic animal feed, a Zoological animal feed, a work SEQ ID NO: 4. animal feed, a livestock feed, or a combination thereof. 33. The cell according to claim 29 or claim 30, wherein 43. A method of producing a thraustochytrid cell culture, the cell further comprises a nucleic acid molecule compris comprising: ing a polynucleotide sequence encoding a heterologous (a) transforming a thraustochytrid cell with a nucleic acid Sucrose transporter. molecule comprising a polynucleotide sequence encod 34. The cell of claim 33, wherein the polynucleotide ing a heterologous Sucrase, wherein the Sucrase is sequence encoding the heterologous Sucrose transporter is at bound to the plasma membrane of the thraustochytrid; least 90% identical to a sequence selected from the group and consisting of a polynucleotide sequence of Accession No. (b) growing the transformed thraustochytrid cell in a L47346 or X69165; and a polynucleotide sequence of culture medium comprising Sucrose as a carbon source. Accession No. L47346 or X69165 that is codon-optimized 44. The method of claim 43, wherein the sucrase is an for expression in Schizochytrium. invertase. 35. The cell according to claim 29 or claim 30, wherein 45. The method of claim 43 or claim 44, wherein the the thraustochytrid is a Schizochytrium or a Thraustochy SCaS trium.