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(12) Patent Application Publication (10) Pub. No.: US 2017/0183669 A1 LPPMEER Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2017/0183669 A1 LPPMEER Et Al 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 FG, Patent Application Publication Jun. 29, 2017 Sheet 2 of 15 US 2017/O183669 A1 pScitie-SFC+-stSea Coofs $3.5 ki FG, 2 Patent Application Publication Jun. 29, 2017. Sheet 3 of 15 US 2017/O183669 A1 DW in SSFM + Sucrose ~~~~~~ s • S Strains FIG 3 Patent Application Publication Jun. 29, 2017. Sheet 4 of 15 US 2017/O183669 A1 %. Fat in SSFM )}-- Strains FG, 4 Patent Application Publication Jun. 29, 2017 Sheet 5 of 15 US 2017/O183669 A1 Dry Weight in SSFM s s SSSSS- X ss'sS S S. xmCO rt O 8 s s S S& 1 a 3 S S S XS S- 3 -5 F.G. 5 Patent Application Publication Jun. 29, 2017 Sheet 6 of 15 US 2017/O183669 A1 X at if SSFV s s SS --------, -oc -------, -o- is co-o-o-o: s SS -:xx sex sessex, coxscoso-sock -- r--- & s : f* : . S& Šy.:-Sks: s\f. s : S^& S s s g X - - - - - - - - - SYY --...- ...-...-- - - - - -2 s:S W. W. a 1 m - - - - W. W. a sm s a - - - - - - - - - - - w w w x a W w n w a 1 m - - - - - - - - - - - w w a W win a a - - - - S & rts S : s in SS & 63 - - - - - s S. s . ........... .......... .......... S.S 3. - - - - - - - , , . s SS : 8 s a. S. Patent Application Publication Jun. 29, 2017. Sheet 7 of 15 US 2017/O183669 A1 B76-32 invertase transformants; growth on SiOS |--~~~~ ? FG, 7 Patent Application Publication Jun. 29, 2017. Sheet 8 of 15 US 2017/O183669 A1 B76-32 invertase transformants; fat on sucrose |:=l ae --~~~~=', F.G. 8 Patent Application Publication Jun. 29, 2017 Sheet 9 of 15 US 2017/O183669 A1 FIG 9A ca 25 5 s SO 3& Patent Application Publication Jun. 29, 2017. Sheet 10 of 15 US 2017/0183669 A1 FIG 10 A w Reactionst as a rate as a function of & s W. S 3 &. S : & s s : S : s : s S. s s : S Šw s 8.s FIG 10B . .". s - - - , sww Y, a - - - - - - - - - - - - - - -.'...'... - - - -- www YW - a ...--...- . --- . swww.s a M. - ... .''...'. ---. sww Y \,S s 8 8 8 8 8 & s 8 a. Š 3sks:::::::w s 8 : 8 w w w 3fs essess w w w w w Kin) is 33.4nM Patent Application Publication Jun. 29, 2017. Sheet 11 of 15 US 2017/0183669 A1 Mannose N-acetyl-glucosamine sigresses:is 3 $8.3% F.G. Patent Application Publication Jun. 29, 2017. Sheet 12 of 15 US 2017/0183669 A1 R. R. 'sCage 's- Hexs HexNAc - exexNAc w HexexNAc R y HexsexNAc 8 2 w HexohexNAc 20 2 s HexexNAc 32 2 w Hex HexNAc 47 2 y HexexNAc S6 2 y HeX13 HexNAc 618 2 w ti........Detected n.d......Not detected FG, 2. Patent Application Publication Jun. 29, 2017. Sheet 13 of 15 US 2017/0183669 A1 FG, 3 Patent Application Publication Jun. 29, 2017. Sheet 15 of 15 US 2017/0183669 A1 pCLO 137 EPCT(+)-s1Suc1 .245 ky F.G. 5 US 2017/O 183669 A1 Jun. 29, 2017 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.
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